Embed Size (px)
Citation preview
BIOCHEMICAL CHARACTERIZATION OF
IN VITRO SALT TOLERANT CELL LINES AND REGENERATED PLANTS OF
POTATO (SOLANUM TUBEROSUM L.)
Zahoor Ahmad Sajid
DEPARTMENT OF BOTANY
UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN.
BIOCHEMICAL CHARACTERIZATION OF IN VITRO SALT TOLERANT CELL LINES
AND REGENERATED PLANTS OF POTATO (SOLANUM TUBEROSUM L.)
A Thesis Submitted to the University of the Punjab in Partial
Fulfillment to the Requirements for the Degree of Doctor of
Philosophy in Botany
By
Zahoor Ahmad Sajid
DEPARTMENT OF BOTANY
UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN.
June, 2010
DEDICATED TO
To My Beloved
MOTHER Whose prayers are real source of my success
CONTENTS
Title Page Number
CERTIFICATE i
ABSTRACT ii
ACKNOWLEDGEMENTS v
ABBREVIATIONS/UNIT ABBREVIATIONS vii
LIST OF TABLES ix
LIST OF FIGURES xi
LIST OF ANNEXURES xiii
Chapter 1: INTRODUCTION 1
Chapter 2: LITERATURE REVIEW 8
2.1: Tissue Culture Studies in Potato 9
2.1.1: Micropropagation 9
2.1.2: Callus Induction and Proliferation 11
2.1.3: Cell Suspension Cultures 13
2.1.4: Plant Regeneration 15
2.1.5: Acclimatization 17
2.2: Salt Tolerance 18
2.2.1: Salt Tolerance Studies in In vitro Potato Plants 18
2.2.2: Selection of Salt Tolerant Cell lines 19
2.3: Biochemical Markers of Salt Tolerance 22
2.3.1: Role of Proteins in Salt Tolerance 23
2.3.2: Antioxidants and Salinity Tolerance 26
Chapter 3: MATERIALS AND METHODS 37
3.1: Plant Material 37
3.2: Media Preparation 37
3.2.1: Preparation of Concentrated Stock Solution 37
3.2.2: Stock Solutions of Growth Regulators 38
3.2.3: Preparation of Medium from the Stocks 38
3.3: Sterilization 39
3.3.1 Sterilization of Glassware 39
3.3.2 Sterilization of the Media 39
3.3.3 Sterilization of Working Area and Surgical Tools 39
3.4: Explants Inoculation 40
3.5: Culture Conditions 40
3.6: Biochemical Studies 40
3.6.1: Quantitative Estimation of Soluble Protein Contents 41
3.6.2: Quantitative Estimation of Peroxidase, Catalase and
Superoxide Dismutase 42
3.7: Experimental Plan and Data Collection 43
3.7.1: Standardization of Medium and Maintenance of Germplasm
of the two Potato Cultivars, i.e., Cardinal and Desiree 43
3.7.2: Standardization of Medium for Callus Induction and
Proliferation 44
3.7.3: Optimization of Medium for Regeneration of Callus
Cultures in Solanum tuberosum L 44
3.7.4: Standardization of Medium and Conditions for Cell
Suspension Cultures 45
3.7.5: Optimization of Conditions and Medium for Acclimatization
of Potato Plants 45
3.7.6: Effect of different Concentrations of Salt (NaCl) on In vitro
Plantlets of Solanum tuberosum L. 46
3.7.7: Effect of Different Concentrations of NaCl on Callus Growth
and Development 47
3.7.8: Identification of Sub-lethal Salt Concentration and
Maintenance of Callus Cultures on Respective Salt
Concentration for 6 Sub-cultures
47
3.7.9: Regeneration of Callus Culture of Solanum tuberosum L.
After 30 Days of Salt Treatment 48
3.7.10: Regeneration Potential of Recurrently Selected Callus
Cultures on Salt-free Regeneration Medium 49
3.7.11: Assessment of the Stability of the Acquired Salt Tolerance
after Recurrent Selection of Potato 49
3.7.12: Effect of Ascorbic Acid Pretreatment to In vitro Salinized
Plants and Callus Cultures of Solanum tuberosum L. 50
3.7.13: Effect of Ascorbic Acid Foliar Spray to Salinized Plants of
Solanum tuberosum cv. Cardinal 50
3.7.14: Effect of Salicylic Acid Treatment to In vitro Salinized
Plants of Solanum tuberosum 51
3.7.15: Statistical Analysis 52
3.8: Mechanism of Salinity Tolerance in Thellungiella halophila 52
Chapter 4: Standardization of Conditions for Micropropagation, Callus
Induction, Regeneration, Cell Suspension Culture and
Acclimatization of Regenerated Plants of Solanum tuberosum
L. cvs. Cardinal and Desiree
53
RESULTS 53
4.1: Standardization of Medium and Maintenance of Germplasm of Solanum
tuberosum L. cvs. Cardinal and Desiree 53
4.2: Effect of Different Growth Regulators on Callus Induction and
Proliferation in Potato 57
4.3: Optimization of Conditions for Plant Regeneration through Callus
Cultures in Solanum tuberosum L. cvs. Cardinal and Desiree 61
4.4: Optimization of Conditions for the Initiation of Cell Suspension Cultures 65
4.5: Optimization of Acclimatization Conditions and Medium 69
DISCUSSION 73
Chapter 5: Effect of NaCl Stress on In vitro Plants/Callus Cultures
and Selection of Salt tolerant Cell lines, Regeneration,
Subsequent Establishment under Ex vitro Conditions and
Biochemical Characterization
79
RESULTS 79
5.1: Exposure of In vitro Plants to Different Concentrations of NaCl
(0-140 mM). 79
5.2: Effect of Different Concentrations of NaCl on Callus Proliferation in
Solanum tuberosum L. (cvs. Cardinal and Desiree) 86
5.3: Callus Morphology of Potato (cvs. Cardinal and Desiree), Relative Fresh
Weight Growth and Selection of Sub-lethal Salt Concentration and
Subsequent Maintenance on Respective Salt Concentration for Six Sub-
cultures for Recurrent Selection
90
5.4: Regeneration Potential of Potato (cvs. Cardinal and Desiree) at Different
Concentrations (0-140 mM) of NaCl) 95
5.5: Regeneration of Callus Cultures after Recurrent Selection on Salt-free
Regeneration Medium 96
5.6: Assessment of Stability of Acquired Salt Tolerance in Potato Plants
in Greenhouse 100
DISCUSSION 102
Chapter 6A: Role of Ascorbic Acid in Amelioration of Salt tolerance in
Potato (cvs. Cardinal and Desiree) 109
RESULTS 109
6.1: Effect of Ascorbic Acid Pretreatment to In vitro Salinized Plants and
Callus Cultures of two Cultivars of Solanum tuberosum L. cvs. Cardinal
and Desiree
109
6.2: Regeneration Potential of Ascorbic Acid-pretreated and Non-pretreated
Callus Cultures at Different Concentration of NaCl 109
6.3: Amelioration of Salinity Tolerance by Foliar Application of Ascorbic
Acid in Potato cv. Cardinal 111
DISCUSSION 114
Chapter 6B: Role of Salicylic Acid in Amelioration of Salt Tolerance in
Potato (cvs. Cardinal and Desiree) 117
RESULTS 117
6.4: Salinity Tolerance and Effect of Salicylic Acid 117
DISCUSSION 124
Chapter 7: LITERATURE CITED 127
ANNEXURE 1-8 167
ANNEXURE 9A-E (Published Articles) 175
i
CERTIFICATE
This is to certify that the research work entitled “Biochemical Characterization of
In vitro Salt Tolerant Cell lines and Regenerated Plants of Potato (Solanum tuberosum
L.)” described in this thesis by Mr. Zahoor Ahmad Sajid is an original work of the author
and has been carried out under my direct supervision. I have personally gone through all the
data, results, materials reported in the manuscript and certify their correctness and
authenticity. I further certify that the material included in this thesis has not been used in part
or full in a manuscript already submitted or in the process of submission in partial or
complete fulfillment of the award of any other degree from any institution. I also certify that
the thesis has been prepared under my supervision according to the prescribed format and I
endorse its evaluation for the award of PhD degree through the official procedures of the
University of the Punjab, Lahore.
Supervisor: (Dr. Faheem Aftab) Associate Professor Department of Botany University of the Punjab, Lahore Date: ____________
ii
ABSTRACT
The present investigation deals with the establishment of an efficient in vitro
selection strategy to produce salt-tolerant cell lines and subsequent regeneration protocols in
potato (cvs. Cardinal and Desiree). The activities of antioxidant enzymes (peroxidase,
catalase and superoxide dismutase) and total soluble protein contents of various tissues under
stress were evaluated to understand their possible role in salinity tolerance. Exogenous
application of ascorbic acid and salicylic acid were also tested for salt stress alleviation. In
order to proceed with these objectives, the initial focus was to establish protocols for
micropropagation, callus induction and maintenance, plant regeneration, establishment of
cell suspension cultures and ex vitro acclimatization of regenerated plants. Three different
concentrations of TDZ (10-8, 10-9, or 10-10 M) in MS medium were tested for the purpose of
in vitro clonal propagation. MS basal medium fairly supported micropropagation of both the
tested potato cultivars followed closely by MS medium supplemented with TDZ (10-10 M).
For callus induction and proliferation in dark, internodal segments proved to be a good
explant source whereas MS medium fortified with 2, 4-D (18.09 µM) was the best medium
composition equally effective for both the potato cultivars. A combination of NAA (2.64
µM) and TDZ (1.00 µM) supplemented to MS medium was the best choice for shoot
initiation from callus cultures after 20 and 21 days in Cardinal and Desiree, respectively.
Rooting of regenerated shoots was achieved on MS medium supplemented with 8.87 µM
BAP, 2.64 µM NAA and 0.123 µM IBA. Cell suspension cultures using friable calluses were
developed successfully using MS2 medium for the two cultivars. The best supporting
medium for ex-vitro transplantation of potato plants was vermiculite.
iii
It was observed in this study that different in vitro growth parameters, i.e., shoot/root
length and numbers of roots decreased while number of shoots increased with an increase in
NaCl (20-140 mM) concentration in the medium. In Desiree, rosette-type of shoot
development initiated at 100 mM whereas in Cardinal it was evident at 120 mM NaCl level.
During this investigation, a direct recurrent selection procedure was employed to select salt-
tolerant cell lines in potato (Cvs. Cardinal and Desiree) on the basis of sub-lethal
concentration of salt. Results have shown more than 50% reduction in relative fresh weight
in both the cultivars above 100 mM NaCl. Callus morphology correspondingly changed from
off-white to blackish-brown above 100 mM to acutely-necrotic at 140 mM NaCl.
Regeneration potential of recurrently-selected callus cultures (100 mM NaCl-treated) on salt-
free medium was more pronounced in Desiree as compared to Cardinal. When well-
acclimatized recurrently-selected plants were treated with 100 mM NaCl and compared with
control plants to check their acquired salinity tolerance, it was observed that recurrently-
selected plants showed higher fresh/dry weight and number of tubers in both the cultivars. A
slight decrease in protein contents of in vitro Cardinal cultures was observed as the
concentration of NaCl (20-140 mM) gradually increased in the media. However, there was
an increase in protein contents in Desiree plants when subjected to increasing salt
concentrations. In case of in vitro recurrently-selected plants, protein contents were higher as
compared to control (non-selected ones) in both the cultivars. The peroxidase activity
exhibited a slightly decreasing trend in Cardinal though an increasing one was observed in
Desiree with an increasing NaCl level in the medium. In the present investigation,
recurrently-selected plants had higher POD, CAT and SOD activities as compared to the
control ones in both the cultivars.
iv
Pretreatment with ascorbic acid to salt-treated plants and callus cultures resulted in
significant differences with respect to almost all the studied growth parameters. Protein
contents as well as CAT and SOD activities increased significantly in both the cultivars
although POD activity had a decreasing trend in ascorbic acid pretreated plants and callus
cultures. Regeneration potential correspondingly decreased with an increase in salt level (20-
140 mM) in MS medium. Plant regeneration was completely inhibited above 60 mM NaCl
concentration in Desiree and 80 mM in Cardinal. On the other hand, ascorbic acid-pretreated
salinized callus cultures showed a better regeneration potential as compared to non-
pretreated ones at all the tested salt levels in both the cultivars. In pot experiments involving
foliar application of ascorbic acid, the response of control potato plants to high level of
salinity was reflected by decrease in tuber fresh/dry weight, shoot length and shoot
numbers/plant. On the other hand, foliar application of ascorbic acid to control and salinized
(120 mM of NaCl) plants not only promoted growth parameters but also resulted in an
increase in protein contents and antioxidant enzyme activities as compared to plants without
ascorbic acid treatment. Up-regulation in the activity of POD, CAT and SOD indicated that
these enzymes were somehow involved in the scavenging process of reactive oxygen species
in potato. Exogenously-applied salicylic acid (SA) at 0.125 or 0.25 mM was quite effective
in enhancing growth and biochemical parameters under NaCl stress in Cardinal and Desiree,
respectively. These results hint at a possibility that moderate concentrations of salicylic acid
may, in future, be helpful in improving yield of plants under saline conditions.
v
ACKNOWLEDGEMENTS
All praises to Almighty Allah, most Merciful and most Compassionate, who had
blessed me with the potential to complete this research work and compile this thesis
successfully.
The author feel great pleasure to convey hearty gratitude to his benignant supervisor
Dr. Faheem Aftab, Associate Professor, Department of Botany, University of the Punjab,
Lahore, for his enthusiastic guidance, constructive criticism, keen interest, co-operation and
encouragement throughout the research work. Apart from the subject of research, author
learnt a lot from him.
Special thanks are due to Prof. Dr. Rass Masood Khan, Chairman, Department of
Botany, for his ever appreciating attitude and providing excellent research facilities during
the course of this research work.
Author would like to express his gratitude to Prof. Dr. Shahida Hasnain,
Chairperson DPCC/ Dean Faculties of Life Sciences and Former Chairperson, Department of
Botany, for her very kind behavior and providing conducive environment during her
chairpersonship.
Author further extend his gratitude to Prof. Dr. Javed Iqbal, Professor Emeritus,
Department of Botany and Director School of Biological Sciences for his valuable guidance
throughout the course of this work.
The cooperation and guidance of Dr. Michael V. Mickelbart Assistant Professor,
Center for Environmental Stress Physiology, Department of Horticulture and Landscape
Architecture, Purdue University, West Lafayette Indiana, USA and other lab members are
also gratefully acknowledge.
Thanks are also to Higher Education Commission (HEC) for financial support
during this investigation in the form of a research project (HEC, 20-143) to Dr. Faheem
Aftab and six months scholarship (PIN# IRSIP 7-BMS-06) for short duration training at
vi
Center for Environmental Stress Physiology, Department of Horticulture and Landscape
Architecture, Purdue University, West Lafayette Indiana, USA to the author.
The author feels pleasure to say thanks to his lab fellows M. Akram, Neelma Munir,
Adeela Haroon and Farheen Yameen for their full cooperation and moral support.
The author is also cordially grateful to his ever loving Parents, Brothers and Sisters
for their patience, encouragement, love and countless prayers.
ZAHOOR AHMAD SAJID
vii
ABBREVIATIONS/UNIT ABBREVIATIONS
µm Micrometer
µM Micromolar
2, 4-D 2, 4-Dichlorophenoxyacetic acid
AA Muller and Grafe (1978) liquid medium
BA or BAP 6-Benzyladenine or 6-Benzylaminopurine
ca. In approximately
Cl- Chloride ion
cm Centimeter
Conc. Concentration
cv. Cultivar
cvs. Cultivars
DNA Deoxyribonucleic acid
dS m-1 Decisiemens per meter
EC Electrical conductivity
EDTA Ethylenediaminetetraacetate
Fig. Figure
g L-1 Gram per liter
g Gram
GA3 Gibberellic acid
h Hour
ha Hectare
IBA Indole-3-butyric acid
K+ Potassium ion
kg Kilogram
L Liter
L. Linnaeus
M ha Million hectare
M Molar
mg g-1 Milligram per gram
viii
mg L-1 Milligram per liter
min Minutes
ml L-1 Milliliter per liter
mL Milliliter
mm Millimeter
mM Millimolar
MS Murashige and Skoog (1962) basal medium
N Normal
Na+ Natrium (Sodium) ions
NAA Naphthaleneacetic acid
NaCl Sodium chloride
NBT Nitroblue tetrazolium
nm Nanometer
O2- Ionic oxygen ºC Degree celsius
OH- Hydroxyl ion
pH Hydrogen ion concentration
PVP Polyvinylpolypyrrolidone
ROS Reactive oxygen species
rpm Revolutions per minute
SDS Sodium dodecyl sulphate
SOD Superoxide dismutase
Spp. Species
TDZ Thidiazuron (1-Phenyl-3-(1, 2, 3-Thiadiazol-5-yl) urea
U mg-1 Units per milligarm
U mL-1 Units per milliliter
UV Ultraviolet
v/v Volume/Volume
W Watt
w/v Weight/Volume
ix
LIST OF TABLES
Table Number Title
Page Number
4.1:
Effect of three different TDZ levels supplemented to MS media on in
vitro establishment of shoot apices of potato (Solanum tuberosum L.
cv. Cardinal) at day 30 of the initial culture.
55
4.2:
Effect of three different TDZ levels supplemented to MS media on in
vitro establishment of shoot apices of potato (Solanum tuberosum L.
cv. Desiree) at day 30 of the initial culture.
56
4.3:
Effect of different growth regulators supplemented to MS medium on
callus induction and proliferation in Solanum tuberosum L. cvs.
Cardinal and Desiree.
60
4.4:
Effect of different media on regeneration potential of callus cultures
of Solanum tuberosum cvs. Cardinal and Desiree.
62
4.5:
Optimum conditions for the initiation/establishment of cell
suspension cultures of potato cvs. Cardinal and Desiree.
67
5.1:
Growth parameters and Protein/Peroxidase contents in in vitro plants
of potato (cv. Cardinal) under NaCl stress.
82
5.2:
Growth parameters and Protein/Peroxidase contents in in vitro plants
of potato (cv. Desiree) under NaCl stress.
83
5.3:
Effect of different concentrations of NaCl on callus proliferation
response in Solanum tuberosum L. cv. Cardinal.
88
5.4:
Effect of different concentrations of NaCl on callus proliferation
response in Solanum tuberosum L. cv. Desiree.
89
x
5.5:
Effect of different NaCl levels (0-140 mM) supplemented to
optimized callus proliferation medium on relative fresh weight
growth and callus morphology of potato (cvs. Cardinal and Desiree)
at day 90.
91
5.6:
Growth and biochemical analysis of control and salt-treated plants of
potato cvs. Cardinal and Desiree.
101
6.1:
Effect of foliar application of ascorbic acid on growth and
biochemical parameters of potato plants (cv. Cardinal) with or
without supplemental NaCl treatment of potting mix.
112
6.2:
Effect of salicylic acid on different growth parameters in Solanum
tuberosum L. cv. Cardinal.
119
6.3:
Effect of salicylic acid on different growth parameters in Solanum
tuberosum L. cv. Desiree.
120
xi
LIST OF FIGURES Figure Number
Title
Page Number
4.1-4.4: Morphology of potato callus cultures (cvs. Cardinal and Desiree)
initiated from internodal segments on MS basal medium
supplemented with different growth regulators at day 60.
58
4.5-4.7:
Some selected photographs showing different stages of regeneration
in potato cvs. Cardinal and Desiree.
63
4.8:
A) Elongated and poorly-dividing cell suspension of cv Cardinal
with thick walls. B) Elongated cells in suspension cultures of
Cardinal derived from compact-green callus cultures.
68
4.9:
A) Globular, rounded cells with good division efficiency. B) Clusters
of rapidly-dividing rounded cells with smaller diameter. Both A and
B from the cell suspension cultures of cv. Desiree.
68
4.10:
A comparison of growth and development of potato plants under ex
vitro conditions on different media.
70
4.11:
Well-acclimatized plants of potato growing in pots in glasshouse
conditions, cvs. Cardinal and Desiree are shown growing in plastic
pots.
71
4.12:
Mortality rate of potato plants (Cvs. Cardinal and Desiree) in
different hardening media.
72
5.1-5.8:
In vitro-raised plants of potato cvs. Cardinal/Desiree at different
concentrations of NaCl supplemented to MS medium after 60 days
of culture.
84
5.9-5.15:
Callus morphology of potato cvs. Cardinal and Desiree at different
concentration of salt at day 90.
92
xii
5.16:
Regeneration potential of potato cvs. Cardinal and Desiree at
different concentrations of NaCl.
95
5.17:
Regeneration response of salt-tolerant callus cultures of potato (cv.
Cardinal) on optimized regeneration medium at day 60.
97
5.18:
Regeneration response of salt-tolerant callus cultures of potato (cv.
Desiree) on optimized regeneration medium at day 60.
97
5.19-5.20: Some selected photographs showing regeneration potential of Potato
callus cultures at day 60 of transference to optimized regeneration
medium after recurrent selection.
98
5.21-5.22: Some selected photographs showing regeneration potential of potato
plants at day 120 of transfer of calluses to optimized regeneration
medium after recurrent selection.
98
6.1:
Regeneration potential of ascorbic acid-pretreated and non-pretreated
callus cultures of Solanum tuberosum cvs. Cardinal and Desiree at
day 60 on salt-free regeneration medium.
110
6.2 (A-D): A Comparison of growth of ascorbic acid-treated or non-treated salt-
stressed Cardinal plants.
113
6.3: Comparison of potato shoots (cv. Desiree) at various salicylic acid
levels.
121
6.4: Effect of different SA concentrations on protein contents of in vitro-
grown potato plants (cvs. Cardinal and Desiree).
123
xiii
LIST OF ANNEXURES
Annexure Number Title
Page Number
1: Formulation of MS Medium (Murashige and Skoog, 1962) for
the Preparation of Stock Solutions.
167
2: Preparation of Stock Solutions for MS (Murashige and Skoog,
1962) Medium.
168
3: Preparation of Stock Solutions of Growth Regulators. 169
4: Preparation of 1 liter MS Medium. 169
5:
Preparation of Reagents for the Estimation of Peroxidase,
Catalase and Superoxide dismutase.
170
6:
Culture Media Used for the Establishment of Cell Suspension
Culture in Solanum tuberosum cvs. Cardinal and Desiree.
172
7: Composition of AA (Muller and Grafe, 1978) Medium. 173
8: Composition of Hoagland Solution (Hoagland and Arnon, 1950). 174
9A-E: Published Articles. 175
1
Chapter 1
INTRODUCTION
Potato (Solanum tuberosum L.) is an important commercial food crop of the world
and ranked number fourth after maize, wheat and rice with annual production of 328 M tones
(FAO, 2008). It is an auto-tetraploid (2 = 4x = 48) and belongs to the family Solanaceae
which includes 90 genera and 2800 species. It is a highly productive crop and is reported to
have far greater nutritive value as a food crop and is consumed at the rate of 11.0 kg per
capita per annum (FAO, 2008). Besides being an important vegetable, it also supplies at least
12 essential minerals other than starch (12-20%), protein (1.87%), fiber (1.80%), fats (0.1%),
vitamin C, and high phosphate contents with small amount of calcium and ash (Irfan, 1992).
Potato has been recognized as a crop of high potential after cereals that can meet future food
demands. In the year 2007-2008, a world-over total area under this crop was 19.327 M ha
with the yield of 16.892 tons/ha. Total area under cultivation in Pakistan is 23.63 M ha out of
which potato is grown over 131.90 thousand hectares with an annual production of 19.90
tons/hectare (FAO, 2008). The per hectare yield of potato in Pakistan is yet very low as
compared to developed countries of the world (Malik, 1995; Farhatullah et al., 2002) due to
several reasons like poor agricultural practices, susceptibility to several diseases and pests,
non-availability of healthy certified seeds, and soil salinity. From all of the aforementioned
limiting factors, soil salinity is the major constraint for low potato production not only in
Pakistan but all over the world. In general, Potato plant is vulnerable to salinity (1.7 dS/m,
EC) and characterized as moderately salt-sensitive (Mass, 1985; Katerji et al., 2003).
Experimental evidence shows that soil salinity is one of the most important abiotic
stresses limiting the productivity of agricultural system around the world (Mahajan and
2
Tuteja, 2005). It is considered as a largest soil toxicity problem in tropical Asia (Greenland,
1984). The severity of this problem is gradually being aggravated by the build-up of salts in
soils through common irrigation practices. According to an FAO statistics (2005), of the
current 230 M ha of irrigated land on the globe, 45 M ha (19.5%) are salt-affected. Out of
almost 1500 M ha of dry land, 32 M ha (2.1%) are considered as saline. It is estimated that
there is a deterioration of 2 M ha (about 1%) world agricultural land each year by salinity
(Szabolcs, 1994; Choukr-Allah, 1995). In Pakistan, 16.72 M ha are being irrigated. Of this
irrigated land, 6.3 M ha are affected by salinity. The magnitude of problem can be estimated
from the fact that the area of productive land is being damaged by salinity at an alarming rate
of about 40,000 ha annually (http://www.icid.org/cp_pakistan.html).
Salinity affects plants in several ways such as osmotic stress, specific ion imbalance,
ion toxicity, nutritional disparity and hormonal disturbance or a combination of all these
(Lauchli and Epstein, 1990; Ashraf, 1994; Wahid et al., 2007). All these factors badly affect
plant growth and development at both physiological and biochemical levels (Munns, 2002;
Munns and James, 2003; Tester and Davenport, 2003). The damaging effects are observed at
the whole-plant level as limiting plant growth and productivity. Suppression of growth
occurs almost in all plants, but their tolerance levels and rate of growth reduction at lethal
concentrations of salt understandably vary widely among different plant species (Hasegawa
et al., 2000). The immediate response of salt stress is to reduce the ability in uptake of water
by plants and this may lead to cessation of leaf expansion as salt concentration increases
(Wang and Nil, 2000). Salt stress affects all the major processes; viz., growth,
photosynthesis, protein synthesis, and lipid metabolism. Investigations on tolerance of saline
environments frequently point to restricted ion accumulation and synthesis of organic solutes
3
as major adaptations leading to salt-resistance in glycophytes (Delauney and Verma, 1993;
Ashraf and Foolad, 2007). Moreover, salt tolerance is a multigenic trait and after exposure to
stress, changes occur at cellular level that alters the expression of genes and accumulation of
stress-related proteins involved in stress tolerance (Bohnert and Jensen, 1996; Iba, 2002). In
addition to these interrelated and co-existing impacts, salinity also produces an oxidative
stress (Panda and Upadhyay, 2004) due to rapid and transient accumulation of reactive
oxygen species (ROS) like superoxide radical, hydroxyl radical and singlet oxygen. These
ROS cause pigment co-oxidation, lipid peroxidation, membrane destruction, protein
denaturation and/or DNA mutation (Mittler, 2002). Plants have to opt for a specific
protective mechanism to lessen the harm initiated by these ROS. Antioxidant molecules that
are produced in response to above-mentioned factors are thus of great significance.
Antioxidants are divided into two classes including non-enzymatic (ascorbic acid, salicylic
acid, α-tocopherol, carotenoids etc) and enzymatic, such as superoxide dismutase, catalase
and peroxidase (Sairam and Srivastava, 2002). Superoxide dismutase is a major scavenger of
O2- and its enzymatic action results in the formation of H2O2 and O2. Peroxidase decomposes
H2O2 by oxidation of co-substrates, such as phenolic compounds and/or antioxidants,
whereas catalase breaks down H2O2 into water and molecular oxygen (Mittler, 2002).
Ascorbate also known as vitamin C is an important antioxidant molecule that acts as primary
substrate in the cyclic pathway for enzymatic detoxification of not only hydrogen peroxide
(H2O2) but also superoxide (O2•-), hydroxyl radical (OH•) and lipid hydroperoxides (Yu,
1994). Its role as an ascorbate peroxidase (APX) substrate that scavenges hydrogen peroxide
in the chloroplast stroma has well been documented by Nakano and Asada (1981), Gadallah
(2000) and Shigeoka et al. (2002). Ascorbic acid is water-soluble, so it has an additional role
4
on thylakoid surface in protecting or regenerating oxidized carotenes and α-tocopherols, a
lipophilic antioxidant molecule (Noctor and Foyer, 1998). Similarly, salicylic acid (SA) has
long been considered as signal molecule (Horvath et al., 2007a) and may promote the
generation of reactive oxygen species during salt stress thus playing an important role in
stress tolerance (Borsani et al., 2001). SA treatments enhance the production of H2O2 which
intern reduces the oxidative damage caused by salinity stress in wheat plants (Wahid et al.,
2007). Several developmental, physiological and biochemical functions of exogenously-
applied salicylic acid in plants have been reported, e.g., enhancing the drought and salt stress
resistance of plants (Senaratna et al., 2000; Tari et al., 2002), influencing seed germination
and fruit yield (Cutt and Klessing, 1992; Raskin, 1992), transpiration rate, stomatal closure
(Rai et al., 1986), membrane permeability (Barkosky and Einheling, 1993), growth and
photosynthesis (Khan et al., 2003; Khodary, 2004; El-Tayeb, 2005).
Several strategies have been worked out to improve abiotic or biotic stress-resistance
in crops involving pre-sowing seed treatments, exogenous application of different
compounds, breeding, mutation, pooling physiological traits, interspecific hybridization, the
use of marker-aided selection transformation (Ashraf et al., 2008), and in vitro selection
(Ochatt et al., 1999; Queiros et al., 2007). In vitro culture techniques are an excellent tool to
study the behavior of undifferentiated cells and whole plants in ambient stress under
controlled conditions. The exploitation of somaclonal variation is also potentially quite
helpful for in vitro selection of cells and tissues against several stresses (Bajaj, 1987; Tal,
1996). However, this is only possible when a trait is highly amenable to in vitro selection,
and is expressed and transmitted in the regenerated plants thus being inheritable. Earlier,
plant tissue culture techniques have been used to produce salt-tolerant cell lines and plants in
5
a range of plant species for instance tomato (Hassan and Wilkins, 1988) wheat, (Barakat and
Abdel-Latif, 1996), rice (Lutts et al., 1999), barley (Sibi and Fakiri, 2000), potato (Sabbah
and Tal, 1990; Burgutin et al., 1996; Ochatt et al., 1999; Benavides et al., 2000; Queiros et
al., 2007), sunflower (Alvarez et al., 2003), and sugarcane (Gandonou et al., 2006). Plant
tissue culture is generally considered to be an important technique to select tolerant-clones
from overall non-tolerant populations (Gandonou et al., 2006). It also allows understanding
the mechanisms of salinity tolerance operating at cellular level during stress episodes (Bajji
et al., 1998; Niknam et al., 2006). Potato is highly amenable to tissue culture and several
attempts have been made to get salt-tolerant cell lines, for instance, successful regeneration
of salt-tolerant plants from stable salt-tolerant cell lines was reported by Ochatt et al. (1999).
However, the most prominent of the problems seems to be the reproducibility of protocols
thus limiting sustainability of acquisition of salt tolerance in potato. Sustainable salt
tolerance in potato has thus seldom been achieved (Sabbah and Tal, 1990). A reproducible as
well as sustainable production of salt tolerant potato plants in particular through tissue
culture means for the same reason still remains elusive. Generally two methods have been
adopted for the selection of salt-tolerant cell lines. Selection of salt-tolerant cell lines by
direct selection method is considered as more effective as compared to stepwise method of
selection (Mc-Hughen and Swartz, 1984; Sabbah and Tal, 1990; Aghaei et al., 2008) as it
more closely resembles field conditions. On the contrary, gradual exposure of plants or
tissues is generally considered inefficient since several non-tolerant cells with a labile
metabolism have enough time to adapt to the gradual rise of salt (Ochatt et al., 1999; Miki et
al., 2001; Queiros et al., 2007). It is accepted that long term selection of cell lines is not only
responsible for the necrosis of more than 50-95% cells but also the cause of genetic
6
abnormalities that are usually retained by the cell population (Nabors, 1990). Considering the
above-mentioned facts, it becomes quite obvious that though methods for the production of
salt-tolerant cell lines in potato have been reported, lack of sustainability and even extension
of protocols to most potato cultivars is a major limiting factor in the way of harnessing the
ultimate benefits this technology may render towards sustainable potato production. The
literature survey also indicates that the information regarding mechanism of salt tolerance in
general and in terms of role of proteins and antioxidant enzymes in the selected cell lines of
potato in particular is quite scanty. Interestingly, it is yet debatable as which method of
selection is better since evidence for both direct as well as indirect selection strategies exist
in the literature. Though some really encouraging results have already been achieved using
strategies like exogenous application of antioxidant molecules and compounds for the
amelioration of salinity tolerance but such studies are still in their infancy. The next couple
of years may be quite crucial to determine the truer impact of these approaches towards the
goal of sustainable agriculture.
With this background information in view, present piece of work was undertaken to
establish efficient in vitro selection strategy to produce salt-tolerant cell lines and subsequent
regeneration protocols in potato cvs. Cardinal and Desiree. Emphasis was given on
understanding the mechanism of salinity stress with special reference to total soluble
proteins, and enzymatic antioxidants (peroxidase, catalase and superoxide dismutase).
Another objective of the present study was to investigate the conditions influencing the
establishment of plants in saline soil under glasshouse and/or greenhouse environment. With
an objective to working out other suitable and relatively newer approaches for improving salt
tolerance in potato, two non-enzymatic antioxidants (ascorbic and salicylic acid) were also
7
tested in independent experimentation involving exogenous pretreatments to in vitro plants
as well as callus cultures. It is anticipated that a partial biochemical characterization and
related information derived from this study may contribute towards a broader understanding
of salinity tolerance in potato. This may indirectly facilitate our endeavor to better utilize
moderately saline areas for potato cultivation.
8
Chapter 2
LITERATURE REVIEW
Potato (Solanum tuberosum L.) is an important widely-grown food crop all over the
world (Jones, 1994; Spooner and Salas, 2006). In the year 2007, potatoes were grown in
more than 150 countries on an area of 19.327 M ha with 16.892 tons/ha yield globally (FAO,
2008). Potato produces large quantities of nutritious food which is due to its growth in a very
wide range of ecological conditions (Horton and Sawyer, 1985). Potatoes are grown in
different climatic zones including tropical, subtropical and almost one third of the crop is
produced in the developing countries mostly in Asia (Benkema and Vanderzaag, 1990;
Struik and Wiersema, 1999). Abiotic stress, especially soil salinity is considered to be the
most serious growth-limiting factor for potato crop (Boyer, 1982; Vinocur and Altman,
2005). Soil salinity is a major environmental constraint that not only influences the growth
and development but also reduces the productivity of the crop (Caldiz, 1994; Munns, 2002).
The total area of salt-affected soils including saline and sodic is more than 20% of the
world’s irrigated agricultural land (Flowers and Yeo, 1995). Potato as a glycophyte plant has
been classified as moderately salt-tolerant to relatively salt-sensitive (Katerji et al., 2003).
Several studies have revealed that Solanum species possess genetic variation in stress
tolerance that makes it a good candidate for studies pertaining to abiotic stress tolerance
mechanism (Martinez et al., 1996). However, little research has been conducted on salt stress
resistance mechanisms in this crop since it is a complex phenomenon (Queiros et al., 2007).
Various biotechnological techniques have been developed for the improvement of crop
against biotic and abiotic stresses. One of them is plant tissue culture, a biological tool that
involves exciting prospects for crop productivity and improvement under aseptic conditions
9
(Jain, 2001). Current research in this area extends across many interests including attempts to
select salt tolerant lines, freezing resistant plants, herbicide resistant agronomic crops, and
developing resistance to heavy metals (Davenport, 2003; Gu et al., 2004). NaCl-tolerant cell
lines have been isolated from different plant species and it was observed that various
biochemical processes contribute to the adaptation of cells to salinity (Davenport, 2003;
Lutts et al., 2004). A brief review regarding tissue culture studies in potato and selection of
salt-tolerant cell lines and acclimatization of regenerated plants in potato is given below.
2.1: Tissue Culture Studies in Potato
2.1.1: Micropropagation
Micropropagation is one of the techniques included under the umbrella term of tissue
culture. It is defined as in vitro regeneration of plants from organs, tissues, cells or
protoplasts (Beversdorf, 1990) and the true-to-type propagation of selected genotypes using
in vitro culture techniques (Debergh and Read, 1991). True-to-type propagation has
important benefits for highly heterozygous plants (carrying out one dominant and one
recessive trait). It also provides a means of germplasm for maintenance of disease-free
stocks. Potatoes can be micropropagated very efficiently on a large scale by meristem and
shoot-tip cultures (Murashige, 1974; Roca et al., 1978; Goodwin et al., 1980). Nodal cuttings
were also used for axillary shoot development and suggested to be the best explant source by
several researchers (Roca et al., 1978; Hussey and Stacey, 1981) on either liquid or agar-
solidified medium. In one study, Badawi et al. (1995) explored factors affecting production
of potato plantlets via nodal explants. They reported that when liquid MS medium was
compared with solid one for nodal cuttings, it did not affect the percentage survival but
reduced the percentage of cuttings producing roots and length of shoots. Similarly, Ranalli
10
(1997) used single nodal cutting of 5-10 mm long on solid culture media and recommended
it to be a good explant source. Vreugdenhil et al. (1998) also used nodal cutting to study the
development of axillary bud of potato. They observed that depending on the composition of
culture medium, the buds developed into either tubers (MS medium with 8% sucrose), shoots
(1% sucrose), or stolons (8% sucrose and 0.5 µM gibberellins).
For micropropagation, MS basal medium has proven to be quite effective in several
studies (Gopal et al., 1980; Hussey and Stacey, 1981; Aburkhes et al., 1984; Rosell et al.,
1987; Ozkaynak and Samanci, 2005; Ostroshy et al., 2009). However, along with MS
medium, growth regulators and sucrose levels have also got particular attention for the
improvement of growth. For instance, Vinterhalter et al. (1996) reported a relationship
between sucrose and cytokinins for the regulation of growth and branching in potato (cv.
Desiree) shoot cultures. In the absence of exogenous cytokinin, branching was shown to have
been regulated by sucrose. Both sucrose and cytokinin decreased the length of the main shoot
of the explants. Thus it seems that in potato sucrose may take control over processes, which
in other species are usually under the domains of cytokinin regulation. In another study,
Shibli et al. (2001) sub-cultured in vitro shoots of Solanum tuberosum L cv. spunta in liquid
MS medium containing 0.0, 0.5, 1.0, 1.5 and 2.0 mgl-1 benzyladenine (BA) or kinetin. They
observed a significant reduction in stem and internodal length by increasing BA and kinetin
concentration in MS medium. BA up to 1.0 and 1.5 mgl-1 resulted in an increase in number
of proliferating shoots and nodes per culture flask. The use of gibberellins (gibberellic acid,
GA3), on the other hand have also shown positive effect on micropropagation in potato as
indicated by the study of Pereira and Fortes (2003). They developed a protocol for in vitro
multiplication of potato in liquid culture medium. The explants of potato (cv. Eliza) with an
11
axillary bud were cultured in six different levels of GA3. They observed that full-strength MS
medium supplemented with 0.25 mgl-1 gibberellic acid, 5.0 mgl-1 pantothenic acid, 1.0 mgl-1
thiamine and 20 gl-1 sucrose under constant agitation was the most suitable one. Shah et al.
(2003) used 0.5 mgl-1 gibberellic acid in MS medium for micropropagation of potato cv.
Cardinal and got good results. Their results also revealed Plantago ovata husk to be an
equally effective gelling material for cheaper micropropagation of potato. In another study,
Farhatullah et al. (2009) reported the affects of different concentrations of GA3 on
micropropagation and suggested that the dosage of 0.248 mgl-1 of GA3 boosted all the
morphological characters over control and other treatments. They suggested that this level
(0.248 mgl-1) could be used as standard dose for micropropagation of potato. Quite recently,
Badoni and Chauhan (2009) studied the effect of various growth regulators on meristem tip
development and in vitro multiplication of potato cultivar “Kufri Himalini”. They tried
different combinations of growth regulators, e.g., GA3, NAA, and KIN. Results showed that
lower concentration of auxin (0.01 mgl-1 NAA) with gibberellic acid (0.25 mgl-1) was the
best one for the development of complete plantlets from meristem tips avoiding callus
formation and with satisfactory root formation. In addition to BA, NAA, 2, 4-D, KIN and
GA3, several other plant growth regulators (IBA, IAA, picloram, TDZ) and compounds like
chloride were also reported in literature for micropropagation of potato. By going through the
literature, it is evident that there are a number of excellent reports on the micropropagation of
potato. Reports describing the use of thiadiazuron are however limited.
2.1.2: Callus Induction and Proliferation
Callus is an unorganized, proliferative mass of predominantly thin-walled
parenchyma cells (Bhojwani and Razdan, 2004). Callus formation is an essential step in the
12
use of tissue culture studies for various physiological phenomena including resistance against
various abiotic stresses. The selection of suitable parent material, explants, choice of culture
media and condition are prerequisite for the successful establishment of callus cultures
(Evans et al., 2003). For callogenesis in potato, different workers (Ahloowalia, 1982; Al-
Wareh et al., 1989) have used various explants (nodes, internodes, leaves and tuber discs)
and media for callus induction and proliferation. They suggested that formation of callus was
not only dependent on plant species but also on type of explants, nutrient medium, light,
temperature and season. For instance, Qureshi and John (1985) studied the callogenic
response from a number of potato cultivars and recommended internodal explants to be the
best for callogenesis in potato. Several other workers have also used internodal segment as
an explant source for callus induction in potato (Zel et al., 1999; Vargas et al., 2005; Shirin
et al., 2007). On the contrary, leaf explants were also suggested to be a good explant source
by Jaya-Sree et al. (2001), Yasmin et al. (2003) and Haque et al. (2009). Studies have
revealed that callus induction in potato requires the presence of appropriate amount and
concentration of different growth regulators in MS basal medium. (Shepard and Totten,
1977; Kuldybear et al. 1995; Jaya-Sree et al., 2001). In general, auxin and cytokinin
combination (1.0 mg l-1 BAP + 0.5 mgl-1 2, 4-D) in MS medium was quite effective for
callus induction and proliferation in potato cv. Nevskii (Esna-Ashari and Villiers, 1998).
Similarly, Yasmin et al. (2003) supplemented MS medium with five levels of NAA (0,
1.25, 2.50, 5.00 or 10.00 mgl-1) and BAP (0, 0.5, 1.0, 2.0 or 4.0 mgl-1). They observed that
highest percentage of callus (95%) was induced with 2.5 mgl-1 NAA + 2.0 mgl-1 BAP and
also minimum time required for callus induction in the same concentration. In another study,
Omidi and Shahpiri (2003) inoculated internodal explants on MS medium supplemented with
13
a combination of 1.0, 2.0 or 3.0 mgl-1 2, 4-D and 0.01 or 0.1 mgl-1 kinetin. Analysis of
variance revealed a significant effect of 2, 4-D on initiation time and volume of calluses.
Edriss et al. (2003) reported that high concentration of kinetin (2.0 mgl-1) or low
concentration of NAA (0.1 mgl-1) produced callus with 100% shooting. High BA or low IBA
levels, on the other hand induced callus with poor shooting. Role of yeast extract along with
growth regulators (2, 4-D, BAP, and kinetin, 2.0 mgl-1 each and yeast extract 1.0 gl-1) was
also reported for callus induction in potato (Ehsanpour et al., 2007). Studies have revealed
that amongst the growth regulators tested for callus induction, 2, 4-D alone was proven to be
the best growth regulator in both mono and dicotyledonous plants (Evens et al., 1981; Ho
and Vasil, 1983; Jaiswal and Naryan, 1985; Chee, 1990; Khatun et al., 2003). Like previous
studies, Shirin et al. (2007) also found highest percentage (80%) of callus on MS medium
containing 3.0 mgl-1 2, 4-D alone as compared to a combination of different growth
regulators in four potato cultivars. Quite recently, Abd-Elaleem et al. (2009) studied the
effect of plant growth regulators on callus induction in potato cv. Diamant. They used MS
medium supplemented with different concentrations of NAA, 2, 4-D, BA and TDZ alone and
2, 4-D in combinations with BA for callus induction. The best callus formation was obtained
on MS medium supplemented with 3.0 mgl-1 2, 4-D. From this review it is clear that although
much work has been carried out on callus induction and growth in potatoes but protocols and
procedures may vary from cultivar to cultivar.
2.1.3: Cell Suspension Cultures
Cell suspension cultures are rapidly-dividing homogeneous population of cells grown
in liquid medium. In general, suspension cultures in liquid media grow much rapidly than
callus cultures on agar-solidified media and are amenable to experimental manipulations in
14
several ways (Evans et al., 2003). Cell suspensions are used as a model system to study the
various factors that affect various responses including growth and differentiation of cells
under biotic or abiotic stress. Plant physiologist and biochemist prefer single cell system over
intact organ or whole plants for studying cell metabolism and adaptive mechanism to long or
short-term stress (Fallon and Phillips, 1989; Leone et al., 1994). Studies have reveals that for
successful establishment of cell suspension cultures, species of plants, starting material,
growth media, cell to medium ratio, agitation speed and the duration of sub-culturing are
considered as prerequisites. In several previous studies, cell suspension cultures were usually
initiated to obtain somatic embryos in different potato genotype by using callus cultures
derived from different plant tissues (Sopory et al., 1978; Petrova and Dedicova, 1992; De-
Garcia and Martinez, 1995; Seabrook and Douglass, 2001; Seabrook et al., 2001; Jaya-Sree
et al., 2001). Zhang and Dai (2000) explored the effect of status of callus, periods of sub-
culturing of the callus and different kinds of media on the quality of suspension cultures. In
another study, Wang and Zhang (2002) investigated suitable explants for the initiation of cell
suspension cultures. They suggested that cotyledon, hypocotyle and internodal segments
were excellent explants to initiate loose and healthy callus and for the development of good
cell suspension cultures. Whatever the plant material selected, in all cases, the callus selected
for initiation of a suspension culture should be healthy, friable and vigorously growing
(Evans et al., 2003; Vargas et al., 2005). From different studies, it is obvious that different
media were used for the establishment of cell suspension cultures. For example, Lindeque et
al. (1991) used a combination of 2, 4-D, NAA, and KIN with TDZ and yeast extract. Similar
combination of growth regulators was also recommended by Torabi et al. (2008). In addition
to this, it was also considered as an important step to set up an appropriate ratio of callus
15
mass to liquid medium. Vargas et al. (2005) inoculated one gram callus tissue into 100 ml
liquid medium in darkness on an orbital shaker at 160 rpm. By repeated filtering and sub-
culturing, the cultures were reduced to a suspension of small aggregates and free-floating
single cells.
2.1.4: Plant Regeneration
Plant regeneration from a tissue culture system is often a more critical step in the
success of various biotechnological techniques of any plant improvement program. In vitro
regeneration of plants from callus cultures in potato is highly dependent on the geneotype,
source of the explants, growth regulators used in the culture medium and culture conditions.
Efficient plant regeneration from range of explant tissues including leaf, stem and tubers
from several genotypes of potato has been reported in several studies (Ahloowalia, 1982;
Hulme et al., 1992). Zel et al. (1999) also reported that shoot regeneration in potato cultivar
Igor and Desiree was most successful on callus derived from internodal explants cultured on
MS medium supplemented with zeatin, NAA and GA3. Additionally, callus formed by leaf
and nodal explants was compact and non-morphogenic with very poor regeneration response.
Generally, it is suggested that process of regeneration varies in different regeneration media
(Yasmin et al., 2003). They observed highest regeneration percentage (80%) in potato with
2.5 mgl-1 NAA + 2 mgl-1 BAP from all the tested combinations. Similarly, this varied
behavior of culture medium on regeneration was observed by Khatun et al. (2003). They
studied the callus induction and regeneration from nodal segments of potato cv. Diamant and
reported that MS medium when supplemented with 5 mgl-1 BAP and 0.1 mgl-1 IBA gave best
results for shoot formation from in vitro callus cultures in Potato cv. Diamant. Anjum and
Ali (2004) also observed this varied effect of culture medium on shoot initiation from
16
calluses of different origin in potato (Solanum tuberosum). They tested regeneration media as
reported by Jarret et al. (1980), Ahloowalia (1982) and Lapichino et al. (1991) on two
cultivar of Solanum tuberosum Desiree and Maris piper. The medium of Lapichino et al.
(1991) took less time for shoot initiation from tuber-derived callus than the other tested
media in both the cultivars. Attempts to regenerate shoots on medium of Jarret et al. (1980)
proved unsuccessful. Shoot regeneration from the leaf-derived calluses was achieved on all
the three tested media. The frequency of callus producing shoot and number of shoots
produced per callus were higher on the medium of Lapichino et al. (1991) and lowest on the
medium of Ahloowalia (1982). In an experiment, Hussain et al. (2005) evaluated the
morphogenic potential of three potato cultivars from diverse explants. They suggested that
explants source (shoot tips, leaf discs, node and internode) had a significant effect on direct
regeneration and in this regard nodal explants had maximum regeneration. The most suitable
medium was MS with 2 mgl-1 BAP and 0.5 mgl-1 IAA. In certain studies, Kinetin in
combination with NAA (4 mgl-1 kinetin and 0.5 mgl-1 NAA) in MS medium was also proven
to be very effective for plant regeneration (Shirin et al., 2007). In another study, Torabi et al.
(2008) reported plant regeneration from cell suspension cultures of potato and observed that
using MS medium supplemented with GA3 (5 mgl-1) and BAP (2.5 mgl-1) more than 80% of
the calluses produced shoot buds and shoots. Recently, the role of TDZ in the MS medium
for plant regeneration has also been investigated. For example, Abd-Elaleem et al. (2009)
tested TDZ alone or in combination with different growth regulators for regeneration. MS
medium containing 5 mgl-1 TDZ resulted in highest regeneration frequency (81%) and
number of shoots per callus (3.4) in potato cv. Diamant. Regenerated shoots were rooted
most effectively using half strength MS medium containing 0.5 mgl-1 IBA.
17
2.1.5: Acclimatization
The ultimate success of commercial micropropagation depends on the ability to
transfer plants out of in vitro culture systems on a large scale. The rooted plantlets are
subsequently acclimatized ex vitro in a glasshouse to produce transplants in the acclimatized
phase before they are transplanted to the field to produce seed tubers (Wattimena et al.,
1983). Mostly, micropropagated plants are difficult to acclimatize to glass-house or field
conditions with lower relative humidity, higher light and septic environment (Hazarika,
2003). Selection of proper potting mix seems to be an important factor in successful
acclimatization of plants under ex vitro conditions. Keeping in view the effectiveness of
potting mix, different workers have used different potting materials in various ratios for
acclimatization of potato. For example, after successful plant regeneration, Ochatt et al.
(1999) transferred in vitro-grown potato plants to a mixture of peat: perlite: soil (1:1:1) and
suggested this combination to be the best one for acclimatization. In another study by
Yasmin et al. (2003), cow-dung was used effectively along with sand and soil (1:1:1) for
hardening of plants. Afterword, these plants were irrigated with fine spray of water and
covered with transparent polythene bags to prevent desiccation. Within 5-7 days, they
established and polythene bags were removed. Vargas et al. (2005) got hardening of potato
plants in a mixture of soil and river sand (3:1). They shifted forty plants (8 cm long) in
earthen pots and placed under high (80-93%) relative humidity with low light conditions (10
µmole m2s-1) in green-house. For ex vitro hardening of plants, Nasir-ud-Din (2006) used one
part sterile garden soil, one part sand and compost mixture. The survival rate of the plants
was more than 80%. Peat, sand, and perlite compost mix (24:2:1) supplemented with
fertilizer and celcote (water retaining gel) was also used for the establishments of in vitro
18
regenerated plants under glass-house (Sharma et al., 2007). Recently, Badoni and Chauhan
(2009) acclimatized potato plants after rooting under in vitro condition. For hardening of
plants, they used mixture of soil sand and vermin compost (2:1:1) and got good results.
2.2: Salt Tolerance
2.2.1: Salt Tolerance in in vitro Potato Plants
Soil salinity is an important environmental constraint for low growth and productivity
of many agricultural crops all over the world (Lauchli and Epstein, 1990; Allakhverdiev et
al., 2000). It affects the plants in different ways such as osmotic stresses, specific ion
toxicity, nutritional imbalance or combination of all these factors (Lauchli and Epstein, 1990;
Ashraf, 1994; Marschner, 1995). Salt stress affects all the major processes such as growth,
photosynthesis, protein synthesis, and energy and lipid metabolism (Hernandez et al., 2000).
There are several studies in literature highlighting the effects of higher concentration of salt
on in vitro growth in potato and several other plant species. For example, under higher
salinity levels, Levy (1992) found that plant canopy expansion was highly reduced in pots
and field-grown potato. In another study, Potluri and Devi-Parsad (1994) investigated the
effect of crude sea salt (2 to 10 gl-1) on the morphology and physiology of axillary bud
cultures of ten commercially used potato cultivars. They observed a normal growth pattern
up to 8 gl-1 salt and above this level reduction in shoot and root length, yellowing of leaves,
distortion in leaf morphology was recorded. Similarly, growth retardation in in vitro-grown
potato was also observed by Martinez et al. (1996) at 200 mM NaCl while studying salt
tolerance and proline accumulation in Andeen potato differing in frost resistance. It is
generally inferred that each Solanum species respond differently to salt stress (Bruns and
19
Caesar, 1990; Jefferies, 1996). On the basis of this varied response in growth, Khrais et al.
(1998) ranked 130 European and North American potato cultivars in 8 groups.
Plant growth reduction is commonly correlated either to ion toxicity or to water
deficit. Heuer and Nadler (1995) observed a significant decline in leaf water and osmotic
potential under intensified salt stress conditions while studying the physiological response of
potato plants to soil salinity and water deficit. It is now well known from several studies that
saline environment results in accumulation of Na+ and Cl- and decrease in Ca+ and K+
(Marconi et al., 2001; Hassan et al., 2004; Shaterian et al., 2005). Studies also have revealed
that salt stress coupled with changes in gene expression leads to an increased synthesis of
osmoprotectant, osmoregulators (Teixeira and Pereira, 2007; Aghaei et al., 2008), stress
induced proteins and several other antioxidant enzymes (Kumar et al., 2008)
2.2.2: Selection of Salt Tolerant Cell lines
Plant tissue culture techniques can be used as an important tool to study the salt stress
response of callus cultures to salinity in controlled and uniform environmental conditions
(Piqueras et al., 1996; Bajji et al., 1998). On the other hand, studies at the whole plant level
reveal physiological and structural variations during improvement for salinity tolerance
under field conditions (Leone et al., 1994; Hawkins and Lips, 1997). Tissue culture studies
now a day’s are successfully used for the isolation and selection of salt-tolerant cell lines to
elucidate the cellular mechanism involved in salt tolerance (Cherian and Reddy, 2003; Gu et
al., 2004; Elkahoui et al., 2005; Niknam et al., 2006). In vitro techniques thus offer an ideal
alternative for selecting variants because of several unique characteristic.
The salt-tolerant cell lines were first isolated from Capsicum annuum L. by Dix and
Street (1975). Since then, production of salt-resistant cultivars of crop plants has gained
20
much attention, and resulted in a number of reports on selection of salt-tolerant cell lines.
Such cell lines have been isolated from several plant species, e.g., tobacco (Nabors et al.,
1980) Oat (Nabors et al., 1982), Sorghum (Bhaskaran et al., 1983), flax (Mc-Hughen and
Swartz, 1984), wheat (Barakat and Abdel-Latif, 1996), Cymbopogan martini (Patnaik and
Debata, 1997), rice (Lutts et al., 1999), Barley (Sibi and Fakiri, 2000), potato (Burgutin et
al., 1996; Ochatt et al., 1999; Benavides et al., 2000; Queiros et al., 2007; Aghaei et al.,
2008), sunflower (Alvarez et al., 2003), and sugarcane (Gandonou et al., 2006). Most of the
plant species belong to the three families, i.e. Solanaceae, Fabaceae and Poaceae. Two
strategies have been used for the selection of salt-tolerant cell lines, i.e., direct or indirect.
According to several authors direct selection is more effective (Mc-Hughen and Swartz,
1984; Chandler and Vasil, 1984; Bowman, 1987; Sabbah and Tal, 1990; Aghaei et al., 2008)
as compared to step-wise selection (Harms and Oertli, 1985; Ochatt et al., 1999; Queiros et
al., 2007). The direct selection strategy is considered to closely resemble the field conditions
since the seeds are planted directly into saline environment while in gradual imposition of
stress, non-tolerant cells also get enough time to acclimatize in that environment (Mc-
Hughen and Swartz, 1984; Chandler and Vasil, 1984). Additionally, several authors
preffered step-wise selection method suggesting that this process allows physiological and
biochemical adjustments that are the basis for a new cellular homeostasis compatible with
the imposed stress (Harms and Oertli, 1985; Leone et al., 1994; Patnaik and Debata, 1997;
Queiros et al., 2007). In general, selected cell lines consisted of mixture of adapted cells,
which lost their tolerance when transferred to salt-free medium and true genetic variants
which retained their tolerance (Hassan and Wilkins, 1988). Regeneration of salt tolerant
plants followed by testing the inheritance at whole plant level is perhaps the only proof of
21
truer genetic variant lines. Thus plants from salt-tolerant cell lines have been regenerated on
NaCl-containing medium in several studies (Heszky et al., 1986; Reddy and Vaidyanath,
1986; Beloualy and Bouharmont, 1992). However, plant regeneration had been exceedingly
difficult in most of the cases. The plants regenerated from salt-tolerant cells or callus lines
were either salt-tolerant but poor grower, sterile and sensitive. It was suggested that the
presence of salt was perhaps inhibitory to plant regeneration process and salt-tolerant cell
lines after selection have therefore been regenerated on salt-free medium (Li and Heszky,
1986; Ben-Hayyim and Goffer, 1989). In rice, selection of salt-tolerant callus lines under
saline conditions were reported by a number of workers (Yoshida et al., 1983; Nabors and
Dykes, 1985; Li and Heszky, 1986; Vajrabhaya et al., 1989; Basu et al., 1997; Lutts et al.,
1999; Shankhdhar et al., 2000; Chauhan et al., 2000; Chowdhury and Mandal, 2001; Shah et
al., 2002) but regeneration of salt-tolerant cell lines has been observed only in few studies.
Mc-Coy (1987) studied the regeneration of stable salt-tolerant callus lines selected by
indirect selection procedure and observed that regenerated plants were morphologically
abnormal and showed poor growth than the parent type in Medicago sativa. Successful
regeneration was also obtained from salt-tolerant cell lines after recurrent selection procedure
in colt cherry by Ochatt and Power (1989). They suggested that direct recurrent selection
procedure was a major source of adaptive variation in physiological traits that had stable
genetic and epigenetic basis. Similarly, in a study by Beloualy and Bouharmont (1992), the
plants regenerated from salt-tolerant cell lines of Poncirus trifoliate showed improved
growth and salt tolerant. In potato, salt-tolerant cell lines were selected by Van-Swaaij et al.
(1986) and Sabbah and Tal (1990) but sal-tolerant plants were rarely recovered. In two
separate studies, Ochatt et al. (1999) and Benavides et al. (2000) selected stable salt-tolerant
22
cell lines of potato by indirect selection procedure. They observed that regenerated plants
from salt-tolerant callus differed phenotypically and also genotypically from the control. In
vitro selection of salt-tolerant cell line by direct or gradual recurrent selection method was
reported by Queiros et al. (2007). They suggested that gradual selection method was most
efficient for the establishment of salt-tolerant potato cell lines that could be used as a model
to understand the mechanism of salinity tolerance.
From this review, it is quite apparent that salt-tolerant cell lines were produced and
studied with different parameters in mind but the information regarding the mechanism
involved in salt tolerance remains elusive.
2.3: Biochemical Markers of Salt Tolerance
Soil salinity is an inevitable problem for agricultural production around the world.
Salt stress imposes very serious effects on several cellular mechanisms in plants. It affects all
the major processes such as growth, photosynthesis, protein synthesis and energy and lipid
metabolism. Plants possess number of biochemical and molecular mechanisms to cope with
salt stress. It generally involves the following strategies; selective accumulation and
exclusion of ions, control of ion uptake by roots and transport into leaves,
compartmentalization of ions at cellular and whole plant level, changes in photosynthetic
pathways, alteration in membrane structure, induction of antioxidant enzyme, induction of
plant hormone and synthesis of compatible solutes. Expression of stress proteins is an
important adaptation to cope with environmental stresses. Most of the stress proteins are
soluble in water and therefore contribute to stress tolerance presumably via hydration of
cellular structures (Wahid and Close, 2007). Understanding the biochemical and
physiological basis of salinity could help selection and improvement of plants. In this regard
23
proteins are considered as an important compatible solute and recognized as biochemical
marker for salinity tolerance (Ashraf and Harris, 2004). Proteins that protect macromolecules
and membranes play an important role in plant abiotic stress tolerance.
2.3.1: Role of Proteins in Salt Tolerance
In salt stress environment, several proteins that specifically respond to stress are
induced in many plants. Although the expression as well as the function of such proteins is
not fully understood, it is suggested that there is a relationship between some forms of plant
adaptations and tolerance to stresses and the expression of stress-induced proteins. There are
several reports in the literature that indicate the effect of salt stress on protein changes in
plants. Soluble protein contents of leaves were shown to have decreased in response to
salinity in Oryza sativa L. (Alamgir and Ali, 1999), Amaranthus trilocular (Wang and Nil,
2000), Raphanus sativus (Muthukumarasamy et al., 2000) and Bruguiera parviflora (Parida
et al., 2002). Shankhdhar et al. (2000) also observed that total protein contents of callus
cultures decreased markedly with an increase in salt concentration after 4 weeks of
inoculation in six cultivars of rice callus cultures. Agastian et al. (2000) reported that soluble
proteins increased at low salinity but decreased at high salinity in mulberry. Similarly, Khedr
et al. (2003) also reported a decrease in growth and protein contents due to salt stress
signaling in the desert plant Pancratium maritimum L. A decrease in intensity of several
protein bands of different molecular weights of 17, 23, 32, 33, and 34 kDa was reported
under salinity stress in Bruguiera parviflora by Parida et al. (2004). They suggested that this
decrease was proportional to the applied NaCl concentration. In another experiment, the
effects of long-term (30 days) NaCl treatments (100-200 mM) on protein contents in potato
leaves were studied by Fidalgo et al. (2004). They observed a significant decrease in protein
24
contents under salt stress. Similarly, Khodary (2004) evaluated the effect of NaCl salinity on
nitrogen assimilation and ion uptake in the seeds of lupine (Lupinus termis L.). According to
them, a significant decrease in protein, amino acid and nucleic acid contents was observed
upon NaCl exposure (0, 500, 1000, 2000 or 3000 ppm). Likewise, Niknam et al. (2004) also
observed NaCl to affect in vitro growth parameters as well as sugars, free proline and
proteins in the seedlings and leaf explants of Nicotiana tabacum cv. Virginia. The fresh and
dry mass of the seedlings decreased under salinity. Free proline content in both seedlings and
leaf explants increased and polysaccharide content decreased continuously with increase in
NaCl concentration. Reducing sugars, oligosaccharides, soluble sugars and total sugar
contents in both seedlings and leaf explants decreased up to 150 mM NaCl and then
increased at higher concentrations of NaCl. Rahnama and Ebrahimzadeh (2004) also
observed a decrease in protein in shoots and calluses with increasing NaCl concentrations
while studied the effect of NaCl on proline accumulation in potato seedling and calluses.
In contrast to above, an increase in protein contents under salinity stress was also
observed by many workers in different plant species. Cano et al. (1998) studied the growth
and physiological responses to salinity of two inter-specific hybrids between the cultivated
tomato (Lycopersicon esculentum Mill.) and its wild salt-tolerant species (Lycopersicon
pennellii) and compared to those of their parents. They concluded that protein contents
increased with salinity in all the genotypes. Bekheet et al. (2000) selected two cultivars of
Asparagus officinalis by culturing shoot segments on callus induction medium supplemented
with salt mixture. The cultivars showed better growth, high protein content, fresh and dry
weight as salt concentration increased up to 6000 ppm. Similarly, in a study by Elavumoottil
et al. (2003), salt-tolerant callus and cell suspension cultures of Brassica oleraceae L. var.
25
Botrytis were obtained by the selection of cells from cultures growing in medium
supplemented with 85, 170 or 255 mM NaCl. It was found that both salt-adapted calluses as
well as cell suspensions differed in their RNA and protein levels. These salt-inductive
proteins were also reported in potato plants by Rahnama and Ebrahimzadeh (2004).
Recently, Queiros et al. (2007) also observed this increasing trend of soluble and insoluble
proteins in potato cultures during the selection of salt-tolerant cell lines. These higher protein
contents might be attributed to the synthesis of stress-induced proteins (Kumar et al., 2008)
that may be helpful for maintaining the osmotic imbalance. Salt-responsive proteins were
also suggested to be quite valuable for further analysis of general cellular adaptive
mechanism to abiotic stress. Biochemical and physiological changes in tissues in response to
several kinds of stresses can thus be verified through alterations in proteins. Kogan et al.
(2000) found that the accumulation of compatible solutes was one of the strategies that plants
had developed to tolerate salt stress. Compatible osmolytes and proteins can therefore be
used as potential biochemical markers useful in the identification and genetic manipulation
of salt-resistant plants and plant cells (Shonjani, 2002). Many reports are available where cell
proteins are used as markers during differentiation of tissues and organs under stress
conditions (Iqbal and Schraudolf, 1977; Ashraf and Harris, 2004). However, not always the
data indicate a positive correlation between the osmolyte accumulation and the adaptation to
stress (Mc-Cue and Hanson, 1990; Ashraf, 1994; Mansour, 2000).
Although there are some reports showing non-significant changes in the levels of
protein, starch, sucrose and α-amino nitrogen in salt-grown callus cultures (Paek et al., 1988)
most of the in vitro studies indicate that salt stress may result in varying levels of proteins
(Lutts et al., 1999; Muthukumarasamy et al., 2000).
26
2.3.2: Antioxidants and Salinity Tolerance
2.3.2.1: Enzymatic Antioxidants
Salt stress results in a number of detrimental processes including an ion imbalance
and toxicity, impairment of mineral nutrition, reduction in water status of the plant tissues
and oxidative stress. This oxidative stress is considered to be one of the major damaging
factors in plants and cells exposed to salinity (Gossett et al., 1994; Hernandez et al., 1995;
Khan and Panda, 2002). The major reactive oxygen species that are produced in response to
salt stress are hydrogen peroxide (H2O2), superoxide (O2•-), hydroxyl radical (OH•)
(Halliwell and Gutteridge, 1985) and singlet oxygen (1O2) (Elstner, 1986). Oxidative stress is
linked to the production of reactive oxygen species (ROS) which cause damage to lipids,
proteins and nucleic acids (Hernandez et al., 2000). Reactive oxygen species (ROS) are
highly reactive because they can control different processes and interact with a number of
other molecule and metabolites such as proteins, lipids, DNA and pigments (Mittler, 2002).
Although a number of harmful effects of ROS at toxic levels on plant growth and
metabolism have been reported in literature but they also play an important role in many
important physiological phenomena at non-toxic levels such as cell signaling, gene
regulation, senescence, programmed cell death, pathogen defense etc (Neill et al., 2002;
Blokhina et al., 2003; Ashraf, 2009). Plant cells have evolved defensive antioxidant
mechanisms to combat the danger posed by the presence of ROS. These include enzymatic
mechanisms involving antioxidant enzymes such as superoxide dismutases, peroxidases, and
catalases (Landberg and Greger, 2002; Meloni et al., 2003) and non-enzymatic compounds
including ascorbate, tocopherol, carotenoids, glutathione, flvonoids etc. In varying degrees,
present day plants possess a number of antioxidant enzymes that protect against potentially
27
cytotoxic effects of ROS. Superoxide dismutase, catalase and peroxidase have collectively
been viewed as a defensive team, whose combined purpose is to protect cells from active
oxygen damage (Fridovich, 1988). Superoxide dismutase (SOD) are metallo-enzymes that
convert O2•- to H2O2 in all aerobic organisms so SOD is considered as a primary defense
against oxygen radicals (Bannister et al., 1987). Reports on the activity of SOD in different
plant species under salinity as well as other stress conditions reflect its important role in the
defense mechanism against ROS. The product of SOD, hydrogen peroxide requires further
detoxification which is achieved by other enzymes such as peroxidase, catalase, glutathione
etc. Thus like SOD, peroxidase and catalase also play a vital role in plant defense against
oxidative stress. In the literature, all of these enzymes have usually been studied together so
the relevant literature regarding these enzymes is reproduced collectively in the present
review.
The effects of salt (NaCl) stress on antioxidant responses have been studied in a
number of plant species. These studies indicate that the degree of oxidative cellular damage
in plants exposed to abiotic stress is controlled by the capacity of antioxidative systems
(Dhindsa, 1991; Perl-Treves and Galun, 1991; Zhang and Kirkham, 1994; Zhu and
Scandalios, 1994; Mc-Kersie et al., 1996; Noctor and Foyer, 1998). It is generally accepted
that the extent of up-regulation of antioxidant enzymes varies not only among plant species
but also between two cultivars of the same species (Gossett et al., 1994; Bartoli et al., 1999).
In rice, Dionisiosese and Tobita (1998) observed the production of antioxidants in some
varieties differing in salt tolerance. They found that under the salt-stress, salt-sensitive rice
varieties (Hitomebore and IR28) showed significant reduction in SOD activity and an
increase in peroxidase activity. However, the pattern of accumulation of antioxidants in salt-
28
tolerant rice varieties (Pokkali and Bankat) was different. Pokkali showed only a slight
increase in SOD and a slight decrease in peroxidase activity under salt stress. In contrast, the
other salt-tolerant cv. Bankat performed similar to the salt-sensitive cultivars in terms of
accumulation of antioxidants. In another experiment, Meloni et al. (2003) observed the effect
of salinity on the activity of antioxidant enzymes (superoxide dismutase, peroxidase and
glutathione reductase), in two cotton cultivars namely Guazuncho and Pora. Plants were
treated with three salt concentrations (50, 100 or 200 mM NaCl) for 21 days. The superoxide
dismutase activity in Pora increased with an increase in the intensity of NaCl stress, but salt
treatment had no significant effect on this enzyme activity in Guazuncho. Similarly, salt
tolerant or sensitive potato cultivars were compared for their capability to produce
antioxidants in response to salt stress by Rahnama and Ebrahimzadeh (2004). They observed
an increased SOD activity at 50 mM NaCl in salt tolerant cultivars (Agria and Kennebec),
but no significant changes were observed in the two salt-sensitive cultivars (Diamant and
Ajax). In contrast, at higher salt levels, SOD activity was reduced in all cultivars. However,
the activities of CAT and POD increased in all cultivars under salt stress. These studies
indicate the cultivar-specific role of antioxidant enzymes under salt stress. Further, it was
also inferred from literature that the effective role of antioxidants in detoxifying ROS
depends on the intensity of stress as well as the growth stage at which the plant was exposed
to stress (Ashraf, 2009). In one such experiment, Swapna (2003) studied the activity of
superoxide dismutase, peroxidase and catalase during different developmental stages of rice
such as embryo, 14-days-old seedling, tillering and flowering stage after exposing to NaCl
stress. It was observed that a 100 mM NaCl stress increased the activities of superoxide
dismutase and peroxidase enzyme at different developmental stages of rice (Oryza sativa L.).
29
Mittova et al. (2003) investigated the response of antioxidant system of leaf cell
mitochondria and peroxisomes of cultivated tomato Lycopersicon esculentum (Lem) and its
wild salt-tolerant species Lycopersicon pennellii (Lpa) to 100 mM NaCl stress. They
observed that mitochondria of Lycopersicon esculentum exhibited decreased activity of SOD
whereas APX and GPX activities remained unchanged. In contrast, mitochondria of L.
pennellii showed an increase in SOD and APX activities. Peroxisomes exhibited an increased
SOD and APX activities in L. esculentum whereas activities of these enzymes remained
unchanged in peroxisomes of L. pennellii.
Most of the studies on activities of antioxidant enzymes under salt stress revealed that
the enhanced activity of these enzymes was directly associated with the increasing salt
tolerance (Shalata and Tal, 1998; Garratt et al., 2002). Association of elevated antioxidant
activity with salt tolerance has thus been observed in several studies. For example, Muscolo
et al. (2003) observed that salinity induced lower activities of catalase, ascorbate-free radical
reductase, and dehydroascorbate reductase enzymes in stressed-plants and the tolerance of
kikuyu grass to salt stress (up to 100 mM) appeared to be related to up-regulation of these
enzymes. In general, the salt-tolerant cultivars had more antioxidant enzyme activities as
compared to salt-sensitive cultivars. In another study, Harinasut et al. (2003) investigated the
salt stress-induced changes of antioxidant enzymes in the leaves of a salt-tolerant mulberry
cultivar. It was found that activities of superoxide dismutase, ascorbate peroxidase and
glutathione reductase slightly increased at 150 mM NaCl. Hence, these enzymes apparently
played an active role in scavenging ROS in this cultivar. In potato, Rahnama et al. (2003)
studied the effect of NaCl stress on antioxidant enzymes of four potato cultivars. According
to their results, peroxidase activity increased at low salt level but decreased at higher NaCl
30
level (100 mM) while catalase activity increased in cv. Agria and Diamant and decreased in
cv. Kennebec and Ajax. On the other hand, changes in SOD and POD isozymic pattern at
100 mM were found quite significant as compared to control. Similarly, Agarwal and Pandey
(2004) also linked the increasing enzymatic activity with salt tolerance in seedlings of Cassia
angustifolia. In potato, Fidalgo et al. (2004) studied antioxidant defense system under long-
term salt stress. They observed that SOD activity in salt-treated plants increased while
catalase activity decreased and peroxidese activity showed no significant change in
comparison with the untreated plants. In another study, Benavides et al. (2000) observed a
relationship between antioxidant defense systems and salt tolerance in two clones of Solanum
tuberosum differing in salt tolerance. The antioxidant defense system of the sensitive clone
responded differently to 100 and 150 mM NaCl. At 100 mM, growth, dehydroascorbate
reductase and catalase activities remained unaltered while increase in superoxide dismutase
activity was observed. The superoxide dismutase increment was higher under 150 mM NaCl
stress while a general decrease in other enzymes was observed. All the antioxidant enzymes
were significantly elevated in salt-tolerant clone as compared to sensitive one when both
were grown on NaCl-free medium. No changes in antioxidant stress parameters were
detected in the tolerant clone at both salt concentrations. Sairam et al. (2005) while studying
the effects of long-term sodium chloride salinity (100 and 200 mM NaCl) in tolerant
(Kharchia 65, KRL 19) and susceptible (HD 2009, HD 2687) wheat genotypes found almost
similar results. It was observed that the salt-tolerant genotypes showed fewer declines in
relative water content, chlorophyll content, and ascorbate peroxidase content and higher
increase in superoxide dismutase and its isozymes. The susceptible genotypes showed the
highest decrease in ascorbic acid content, highest increase in H2O2 and smallest increase in
31
activities of antioxidant enzymes. A similar association was made by Koca et al. (2006) who
observed the effect of salt on lipid peroxidation and superoxide dismutase and peroxidase
activities of Lycopersicon esculentum and L. pennellii. A higher salinity tolerance of L.
pennellii was also correlated with lower lipid peroxidation which might be due to higher
contents of antioxidant enzymes. Liu et al. (2007) reported that the stronger salt tolerance of
grafted eggplant seedlings was related to their higher antioxidant enzyme activities and less
oxidative damage. Kusvuran et al. (2007) studied the changes in ion accumulation and the
possible involvement of the antioxidant system in relation to the tolerance of salt stress in
melon (Cucumis melo L.). They observed that activities of superoxide dismutase and catalase
were inherently higher than in salt-tolerant cultivars of melon. These results possibly
suggested that some cultivars exhibit a better protection mechanism against oxidative
damage by maintaining a higher inherited and induced activity of antioxidant enzymes than
the relatively sensitive plants. In a study conducted by Athar et al. (2007) on wheat, an
increase in the activities of SOD, CAT, and POD was also observed in both the cultivars
under 150 mM NaCl. Jaleel et al. (2007) studied the Phyllanthus amarus plants that were
grown in the presence of NaCl (80 mM) in order to study the effect in induction of oxidative
stress in terms of lipid peroxidation, H2O2 contents, osmolyte concentration, proline and
antioxidant enzyme activities. They observed that under NaCl stress, plants showed an
increase in antioxidant enzymes superoxide, peroxidase and catalase. NaCl strongly induced
activity of antioxidant enzyme in the presence of cellular damage induced by salt in Jatropha
curcus (Kumar et al., 2008). In a recent study in Brassica napus lines differing in salt
tolerance, Ashraf and Ali (2008) have also reported a positive relationship between the
activities of antioxidants enzymes with salt tolerance. Quite recently, Seckin et al. (2009)
32
evaluated the effect of exogenous mannitol on the antioxidant enzyme activities in roots of
wheat under salt stress. According to their observation, antioxidant enzyme activities
increased in mannitol-pretreated plants under 100 mM salt stress. They correlated alleviation
of salt stress with enhanced activity of antioxidant enzymes due to exogenous application of
mannitol.
It is evident from the above cited literature that up-regulation of antioxidant enzymes
under salt stress directly related with the salt tolerance in several plant species. Furthermore,
antioxidant production varies not only between species to species but amongst cultivars of
the same species and even from organ to organ of the same cultivar.
2.3.2.2: Non-Enzymatic Antioxidants
2.3.2.2.1: Ascorbic acid
Environmental stresses (abiotic and biotic), trigger an over-production of reactive
oxygen species (ROS) in plants (Mittler, 2002). ROS are generally produced in mitochondria
and chloroplast under normal metabolic process in all organisms (Fridovich, 1991). However
under stress they are produced in large amount and in the absence of any protective
mechanism they can seriously damage several cellular processes. To normalize the effect of
ROS, plants have to produce both enzymatic and non-enzymatic antioxidants. Non-
enzymatic antioxidants include glutathione, ascorbic acid, α-tocopherol and carotenoids
(Elstner, 1986; Bowler et al., 1992; Menconi et al., 1995; Alscher et al., 1997). Ascorbic
acid has been considered as very important antioxidants because it plays a central role in
plant defense by reacting directly with hydrogen peroxide, superoxide ion and singlet oxygen
(Yu, 1994) as well as by recovering α-tocopherol from its oxidized form (Noctor and Foyer,
1998). Various associations between saline environments and endogenous levels of water-
33
soluble antioxidants and/or antioxidative enzymes have been reported (Smirnoff, 1993;
Gosset et al., 1994; Gueta-Dahan et al., 1997; Lechno et al., 1997; Shalata and Tal, 1998;
Tsugane et al., 1999). Several studied are reported in literature regarding the ameliorative
effect of ascorbic acid against stresses in plants (Arrigoni, 1994; Smirnoff, 1996; Noctor and
Foyer, 1998; Loewus, 1999; Smirnoff and Wheeler, 2000). The role of ascorbic acid as an
antioxidant has been studied by Muller-Moule et al. (2003) who demonstrated that ascorbic
acid deficient mutants of Arabidopsis (vtc mutant) were more sensitive to ozone, sulfur
dioxide, or UV-B light (Veljovic-Jovanovic et al., 2001). Ascorbic acid is one of the most
important antioxidants protecting plants from oxidative stress (Smirnoff, 2005). Similarly,
Jaleel et al. (2007) also observed that the non-enzymatic antioxidants ascorbic acid and
glutathione were affected under NaCl stress in Catharanthus roseus. In contrast, Sairam et
al. (2002) by comparing the ascorbic acid content of two wheat genotypes reported that NaCl
caused decrease in relative water content, chlorophyll, membrane stability index and ascorbic
acid content of both genotypes. However, lesser decline in ascorbic acid contents was
recorded in salt-tolerant genotype. Shalata and Neumann (2001) reported that the addition of
0.5 mM ascorbic acid to the root medium, prior to and during salt-treatment for 9 h,
facilitated subsequent recovery and long-term survival of 50% of the wilted tomato seedlings.
Other organic solutes without known antioxidant activity were not effective. In roots, stems
and leaves, salt-stress increased the accumulation of lipid peroxidation products produced by
interactions with damaging reactive oxygen species. Additional ascorbic acid partially
inhibited this response but did not significantly reduce sodium uptake or plasma membrane
leakiness. Similarly, Al-Hakimi and Hamada (2001) studied the interactive effect of salt (0,
40, 80, 120 or 160 mM NaCl) and ascorbic acid (0.6 mM), thiamine (0.3 mM) or sodium
34
salicylate (0.6 mM) in wheat. They also observed that soaking of wheat grains in ascorbic
acid, thiamine or sodium salicylate could counteract the adverse effects of NaCl salinity on
wheat seedlings. In another study on wheat, Afzal et al. (2005) found that hormonal priming
with salicylic acid and ascorbic acid reduced the severity of the effect of salinity. Highest salt
tolerance was obtained in seeds subjected to salicylic or ascorbic acid treatment (50 ppm
each) as indicated by better shoot and root length and fresh and dry weights of the plants.
Khan et al. (2006) observed the effects of L-ascorbic acid and sea salt solutions on the seed
germination of different halophytes. It was found that increasing concentration of sea salt
inhibited seed germination of all species. Pretreatment of seeds with L-ascorbic acid
alleviated the sea salt effects only in some halophytes while it had no effect on other species.
It was thus concluded that the variability of metabolic responses to salinity depends on a
particular species. Quite recently Sajid and Aftab (2009) studied the effect of ascorbic acid
for the amelioration of salinity tolerance in two cultivar of potato (Cardinal and Desiree).
They observed that by exogenous application of ascorbic acid, activities of antioxidants
enzymes (peroxidase, catalase and superoxide dismutase) increased significantly under NaCl
stress conditions which in turn enhance the plant survival under stressfull environment.
2.3.2.2.2: Salicylic acid
Salicylic acid (SA) has been reported to induce significant effects on various
biological aspects in plants. It is a phenolic compound and plays a vital role in plants
response to adverse environmental conditions such as salt and osmotic stresses (Senaratna et
al., 2000; Borsani et al., 2001; Tasgın et al., 2003, 2006). SA influences in various ways,
inhibiting certain processes and enhancing others (Raskin, 1992) such as application of 100
mgl-1 SA alleviated the adverse effects of salt stress on wheat seedlings by enhancing the
35
accumulation of proline and protein (Al-Hakimi and Hamada, 2001). Studies reveal that
accumulation of salt stressed-induced proteins play a major role in stress tolerance (Bekheet
et al., 2000). In a study on wheat, application of 0.05 mM SA improved the wheat seedling
growth by enhancing cell division and amount of IAA and ABA after salt stress (Shakirova
et al., 2003). In maize, Khodary (2004) found that the application of 10-2 M SA to plants
under saline conditions enhanced their growth and development by activating the
photosynthetic process and sugar level. Recently, it has been observed that SA (1 mM)
applied exogenously to barley plants was effective in ameliorating the adverse effects of salt
stress by enhancing protein contents, photosynthetic pigments and maintaining the
membranes integrity (El-Tayeb, 2005). SA (50 ppm) alleviated the drastic effect of salinity
(15 dS/cm) in seedling growth, fresh/dry weights in wheat (Afzal et al., 2005). In tomato, the
application of 0.1 mM SA to tomato plants as a root drench provided protection against salt,
improving survival, relative growth rate, and photosynthetic capacity (Jason et al., 2006).
Similarly, root drenching with SA protected tomato plants from the adverse effects of salt
due to an increased rate of transpiration, photosynthesis and stomatal conductance against
200 mM NaCl stress (Stevens et al., 2006). The endogenous level of SA increased under salt
stress in rice seedlings and the activity of the SA biosynthesis enzyme, benzoic acid 2-
hydroxylase, was induced (Sawada et al., 2006). SA added to the soil also had an
ameliorating effect on the survival of maize plants during salt stress and decreased the Na+
and Cl- accumulation (Gunes et al., 2007). It was observed in certain studies that SA
treatment was accompanied by a transient increase in the H2O2 level which has an alleviating
effect on the oxidative damage caused by salt stress in wheat plants (Wahid et al., 2007). The
improvement in growth and grain yield of wheat cultivars S-24 under 150 mM salinity stress
36
was considered due to SA application which is associated with improved photosynthetic
capacity and increase in leaf carotenoids contents (Arfan et al., 2007). Quite recently,
Karlidag et al. (2009) observed that SA treatments induced increase in leaf relative water
contents and decrease in electrolyte leakage compared to the control under salt stress. They
suggested that the SA treatments ameliorate the negative effect of salinity on the growth of
strawberries. Mutlu et al. (2009) observed that under salinity, the SA treatments significantly
inhibited CAT activity, whereas increased POX activity. The increases in POX activity
caused by SA were more pronounced in the salt-tolerant than in the salt-sensitive cultivar.
SOD activity was increased by 0.01 mM SA in the salt-tolerant while increased by 0.1 mM
SA treatment in the salt-sensitive cultivar. Palma et al. (2009) suggested that SA application
improved the response to salinity (100 mM NaCl) by increasing plant dry weight and
decreasing the contents of organic solutes like proline and total soluble sugar contents in
Phaseolus vulgaris L.
Overall, on the basis of above cited review, it may be suggested that plant tissue
culture techniques can be effectively used for the selection of salt tolerant cell lines.
Moreover, these lines prove as a model in understanding the biochemical mechanisms
involved in adaptive response in cultured cells of potato. Furthermore, proteins and
antioxidants either enzymatic or non-enzymatic compounds can be used as biochemical
markers to elucidate the mechanism of salinity tolerance.
37
Chapter 3
MATERIALS AND METHODS
3.1: Plant Material Potato germplasm (cvs. Cardinal and Desiree) used in the present research work was
initially obtained from Ayyub Agricultural Research Institute, Faisalabad and Seed Centre,
University of the Punjab, Lahore and then maintained in the greenhouse of Department of
Botany, University of the Punjab, Lahore throughout this study.
3.1.1: Explants Preparation and Disinfestation
Healthy tubers (without disease symptoms) of both the cultivars of potato (Cardinal
and Desiree) were grown in sterile sand in a glasshouse. The plants obtained from these
tubers were used as an explants source. To surface disinfest, the explants were first washed
thoroughly with a household detergent and then placed in a 0.7% sodium hypochlorite
(NaClO) solution containing 0.1% Tween-20 (Polyoxyethylene sorbitan monolaurate) for 15
min on an orbital shaker (120 rpm). After this, the explants were washed three times with
autoclaved distilled water to remove the traces of NaClO. Apical shoot and nodal explants
(ca. 1.0 cm) were used for micropropagation and internode for callus induction from both the
cultivars of potato.
3.2: Media Preparation
3.2.1: Preparation of Concentrated Stock Solutions
Stock solutions of macronutrients (20x), micronutrients (100x), vitamins (200x),
myo-inositol (100x) and iron EDTA (Ethylenediaminetetraacetic acid) (200x) were used for
the preparation of MS basal medium (Murashige and Skoog, 1962; Annexure 1) to raise the
38
germplasm of both the cultivars of potato. All the stock solutions were prepared in advance
by using analytical grade chemicals in double distilled water (Annexure 2).
3.2.2: Stock Solutions of Growth Regulators
Various growth regulators, such as thidiazuron (N-phenyl-N 1, 2, 3-thidiazol-5-
ylurea) (TDZ), 2, 4 dichlorophenoxyacetic acid (2, 4-D), benzylaminopurine (BAP), indole-
3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) were used in this study for callus
induction and maintenance, regeneration and establishment of cell suspension cultures. Stock
solutions of all the above were prepared either in mM or µM concentrations and were used
according to the requirement of the medium (details given in Annexure 3). Stock solutions
were stored in amber-colored bottles to avoid photo-oxidation and placed at 4°C to avoid
biological contamination. Stock solutions were immediately discarded if precipitation or
contamination was observed.
3.2.3: Preparation of Medium from the Stocks
For the preparation of 1 liter MS medium, all the stock solutions of the medium were
shacked well and mixed in an appropriate quantity (Annexure 4). Then sucrose was added
directly as carbohydrate source at a concentration of 3% and the final volume of the solution
was made by the addition of distilled water. The pH of the medium was then adjusted to 5.7-
5.8 with 1N NaOH or 1N HCl. The agar (7 g; Oxoid, Hampshire, England) was added and
the medium was heated till boiling to melt agar. The medium was then poured (10 ml) in pre-
sterilized culture vessels (Pyrex 25 × 150 mm). Culture vessels were wrapped individually
with polypropylene sheets.
39
3.3: Sterilization
3.3.1: Sterilization of Glassware
All the new glassware used during this study was washed using a household detergent
followed by a rinse with tap water and then dipped in chromic acid mixture for 8-10 h.
Chromic acid was prepared by mixing potassium dichromate (10%) and concentrated
sulphuric acid in 2:1 (v/v) ratio. After this treatment, glassware was washed with running tap
water for the removal of all the traces of chromic acid and then rinsed with distilled water
thrice. Routinely used-glassware was washed with household detergent and rinsed thrice with
distilled water before used. Finally, the cleaned glassware was transferred to hot air oven at
180°C for two hours in order to complete glassware sterilization. All sterilized glassware was
stored in dust-proof cupboard till its use.
3.3.2: Sterilization of the Media
Appropriate quantity of medium was poured in each sterilized culture vessel and
opening was wrapped with polypropylene sheet and rubber band. The medium in culture
vessels was sterilized by autoclaving at 121°C and 15 lbs inch-2 for 15-20 min. The sterilized
medium was allowed to cool at room temperature. Thermolabile growth regulators were not
autoclaved but filter-sterilized by using membrane filter (Spritzenfilter steril, Roth) of pore
size 0.22 µm and then added to the cooled autoclaved medium before pouring into the
culture vessels.
3.3.3: Sterilization of Working Area and Surgical Tools
Before inoculation, hands and arms were washed with soap and then sprayed with
70% ethanol. Laminar airflow cabinet, the main working area for aseptic manipulation was
40
first thoroughly scrubbed with 70% ethanol. Ultra-violet (UV) light was switched on one
hour before the inoculation and switched off at least 15 min before inoculation. The surgical
tools (scalpels, forceps, spatulas, needles etc) were sterilized by putting them in a glass-bead
sterilizer (Steri 350 Keller, Burgdorf, Switzerland) at a temperature of 250°C. The hot
forceps and other tools were allowed to cool down for few seconds and then used for culture
manipulation.
3.4: Explants Inoculation
Polypropylene wrapper was removed from each culture vessel for inoculation and
with the help of forceps the explants were transferred to the agar-solidified medium. Each
culture vessel was then wrapped again by polypropylene sheet after briefly heating the
opening of culture vessel.
3.5: Culture Conditions
Different culture conditions were employed as per requirement of the experiment. For
micropropagation, 16 h photoperiod whereas calluses were induced under dark. In case of
regeneration, the cultures were placed under a 16 h photoperiod (35 µmol m-2 s-1) provided
by cool fluorescent tube lights (Philips Pakistan). All cultures were maintained at 27 ± 2°C.
3.6: Biochemical Studies
Quantitative analyses were performed for total soluble proteins and antioxidant
enzymes as detailed below:
41
3.6.1: Quantitative Estimation of Soluble Protein Contents
3.6.1.1: Extraction of Protein
For protein and enzyme assay, one gram fresh plant material (whole plant) was
ground in liquid nitrogen into a very fine powder using an ice-chilled pestle and mortar. The
ground tissue was suspended in 2.0 ml of 0.1 M phosphate buffer, pH 7.2 (13.6 g KH2PO4
and 17.4 g K2HPO4 in 1,000 ml of solution) containing 0.5% (v/v) Triton X-100 and 0.1 g of
polyvinyl-pyrrolidone (PVP). The slurry so obtained was centrifuged at 14,000 rpm at 4°C
for 30 min using Sorval RB-5 refrigerated super-speed centrifuge. The resultant supernatant
was collected and stored at 0°C for further estimation of protein, peroxidase, catalase and
superoxide dismutase activities.
Biuret method of Racusen and Johnstone (1961) was adopted for the estimation of
soluble protein contents. The reaction mixture consisted of 2.0 ml of Biuret reagent (3.8 g
CuSO4.5H2O, 1.0 g KI, 6.7 g Na-EDTA, 200 ml 5N NaOH in 1,000 ml of solution) and 0.2
ml of supernatant. The control consisted of 0.2 ml of distilled water instead of supernatant.
The optical density was measured at 545 nm using Hitachi U-1100 spectrophotometer. The
amount of protein was calculated from standard protein curve, which was prepared from
bovine serum albumin. The following formula was employed for the estimation of protein
contents.
CV= Curve value
TE = Total extract
EU = Extract used
Wt = Fresh weight of sample tissue
3.6.2: Quantitative Estimation of Peroxidase, Catalase and Superoxide
dismutase
42
For the quantitative estimation of peroxidases (E.C 1.11.1.7) ‘Guaiacol-H2O2' method
of Luck (1974) was adopted with certain modifications. The assay mixture consisted of 3.0
ml 0.1 M phosphate buffer (pH 7.2), 0.05 ml of 20 mM guaiacol solution (2-
methoxyphenol), 0.1 ml crude enzyme extract and 0.03 ml of 12.3 mM H2O2 solution
(Annexure 5). Peroxidase activity was calculated by time required to increase the absorbance
by a value of 0.1 (e.g., 0.4-0.5) at 240 nm and expressed as U/ml of enzyme.
Catalase (E.C 1.11.1.6) activity was assayed according to Beers and Sizer (1952) with
certain modification. The reaction was carried out using two buffer solutions (A and B).
Buffer A consisted of 50 mM potassium phosphate (pH 7.0), while buffer B was 0.036%
H2O2 solution in 50 mM potassium phosphate buffer (pH 7.0, Annexure 5). The reaction
mixture consisted of 2.9 ml buffer B and 0.1 ml of enzyme extract while control consisted of
only 3.0 ml of buffer A. The enzyme activity was calculated by time required for the
absorbance (at 240 nm) to decrease from 0.45 to 0.40 and expressed as U/ml of enzyme. The
catalase activity was calculated as follows:
Where,
3.45 correspond to the decomposition of 3.45 micromoles of hydrogen peroxide in a 3.0 ml.
of reaction mixture producing a decrease in the A240nm from 0.45 to 0.40 absorbance units.
df = dilution factor.
Min= Time in minutes required for the A240nm to decrease from 0.45 to 0.40 absorbance units
0.1= Volume of enzyme used (in milliliters).
Superoxide dismutase (E.C 1.15.1.1) activity was assayed spectrophotometrically by
measuring its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT),
according to Maral et al. (1977). Two tubes were taken, each containing 2.0 ml of 1.0 mM
43
sodium cyanide (NaCN), 13 mM methionine, 75 µM NBT, 0.1 mM EDTA and 2.0 µM
riboflavin as a substrate (Annexure 5). One tube was used as sample containing reaction
mixture + 5.0 µl enzyme extract, placed approximately 30 cm below the bank of two 30-W
fluorescent tubes for 15 min. The other tube containing reaction mixture without enzyme
extract was illuminated at the same time. The absorbance of the experimental tube was
compared to control at 560 nm. SOD activity was expressed as U/mg of protein. Superoxide
dismutase activity was determined by calculating the percentage inhibition of NBT as
follows:
The SOD activity was calculated based on the fact that one unit of SOD caused 50 %
inhibition.
3.7: Experimental Plan and Data Collection
3.7.1: Standardization of Medium and Maintenance of Germplasm of the two
Potato Cultivars, i.e., Cardinal and Desiree
To standardize the media for in vitro micropropagation, apical shoot explants (ca. 1.0
cm) from both the cultivars were grown on MS (Murashige and Skoog, 1962: S1) full
strength and MS containing different concentrations of TDZ (10-10M: S2, 10-9M: S3 or 10-
8M: S4). The cultures were incubated under 16 h photoperiod (35 µmol m-2 s-1; cool white
fluorescent lights) at 25 ± 2°C. Ten culture vessels (Pyrex; 25 × 150 mm) were used for each
treatment per experiment and each experiment was repeated thrice for the two cultivars.
Results were recorded for shoot length, shoot number, root length, root number,
number of nodes, fresh and dry weight of plants after 30 days of explants inoculation. Shoot
length was recorded with the help of a graduated scale from the top of the media to the tip of
44
shoot excluding 1.0 cm (size of the original explants) and root length was measured from the
tip of the root up to the shoot. Number of branches, roots and nodes was counted by pulling
out the plantlet from the culture vessels. To estimate dry weight after fresh weight
determination, plant material was placed in small brown envelopes and dried in hot air oven
at 70°C for up to 48 h or until uniform in terms of dry weight.
3.7.2: Standardization of Medium for Callus Induction and Proliferation
To optimize the media and explants type for callus induction/proliferation, Murashige
and Skoog (1962) medium supplemented with different concentration of 2, 4-D alone or in
combination with BAP or NAA (media designated as C1 to C13) was used to study their
effect on callus induction in Solanum tuberosum L. Internodal explants (ca. 1.0 cm) were
used for callus induction. Calluses were sub-cultured after every 15 days. The physical
conditions included darkness at 24 ± 2°C.
3.7.3: Optimization of Medium for Regeneration of Callus Cultures in Solanum
tuberosum L.
MS basal medium supplemented with different concentrations of NAA (2.64-15.91
µM) and TDZ (0.1-1.0 µM), was tested for regeneration of callus cultures of potato. The pH
of the media was adjusted to 5.7 prior to the addition of agar. Ten culture vessels were
inoculated for each combination and the cultures were incubated at 25 ± 2°C in 16 h
photoperiod. Regenerating callus cultures were shifted after 30 days on MS basal medium
supplemented with 8.87 µM BAP, 2.64 µM NAA and 0.123 µM IBA for further proliferation
and rooting of shoots. The regeneration potential was recorded at day 60 of callus inoculation
on regeneration medium.
45
4.7.4: Standardization of Medium and Conditions for Cell Suspension Cultures
A healthy, well-proliferating tissue from different types of callus cultures (compact,
friable, embryogenic or non-embryogenic) was inoculated on different media combinations,
i.e., MS, MS2 (Annexure 6) (Vargas et al., 2005) or AA (Annexure 7) liquid medium
(Muller and Grafe, 1978) containing 18.09 µM 2, 4-D for the establishment of cell
suspension culture. A fixed quantity (0.5-1.0 g) of callus tissue from 60-day-old callus
cultures was transferred to 10-25 ml of liquid medium (as above) in 100 ml Erlenmeyer
flask. Cultures were placed on an orbital shaker and agitated at different speeds (75, 100 or
125 rpm) under 16 h photoperiod at 25 ± 2°C. Medium was changed after every 3 days and
fractionated tissue was filtered after every 6 days through sterile (100-800 µm) mesh to
develop a cell line by transferring resulting suspension to fresh medium under the same
conditions.
3.7.5: Optimization of Conditions and Medium for Acclimatization of Potato
Plants
Potato plants of both the cultivars were extracted from the culture vessels; the leaves
were sprinkled with distilled water and the excess agar on the roots was carefully removed.
Afterwards, the roots were rinsed using sterile water to prevent a possible contamination
during the acclimatization steps; the roots were placed in a solution of fungicide (10.0% w/v)
for 10 min. These plants were then shifted to pots (8 × 8 cm) containing vermiculite, peat
moss, saw dust, sand, soil or a mixture of vermiculite, perlite and soil (1:1:1). The
acclimatized plants were covered with a transparent polyethylene bag thus creating a micro-
environment with a high relative humidity and no light barrier. This was also important from
the view-point of protecting the plants against any damage (during handling or due to
46
insects) or disease. Afterwards, the bags were gradually opened (each week) by making few
small holes (5 mm diameter) every here and there to reduce humidity level until reaching
prevalent greenhouse level. The conditions for growth consisted of 25 ± 2°C temperature and
a 16 h photoperiod. Pots were irrigated with Hoagland solution (Hoagland and Arnon, 1950,
Annexure 8) whenever required. When plants established well-developed root system, they
were transferred to soil with 50% organic matter. Data were recorded for plant mortality rate
(%) after 15 days of plant acclimatization.
3.7.6: Effect of different Concentrations of Salt (NaCl) on In vitro Plantlets of
Solanum tuberosum L.
To observe the effect of NaCl on in vitro micropropagated plants, shoot apices (1.0
cm each) from in vitro-grown plants were shifted on MS basal medium containing different
concentrations of NaCl ranging from 0-140 mM, i.e., 0, 20, 40, 60, 80, 100, 120 or 140 mM
(eight salt treatments; MS basal medium being a control). Ten replicate culture vessels were
inoculated for each treatment and experiment was repeated thrice. At 60 days of initial
culture, data were collected for in vitro shoot length/number, root length/number and the
average number of nodes. Protein contents and peroxidase activity was also determined from
the plant material grown under stress after 60 days. In vitro plants as a whole were used for
the extraction of proteins as well as peroxidases. For the estimation of proteins, Biuret
method of Racusen and Johnstone (1961) was used. The method of Luck (1974) was used for
the estimation of peroxidase activity.
3.7.7: Effect of Different Concentrations of NaCl on Callus Growth and
Development
47
Callus cultures were proliferated for 60 days on MS medium supplemented with
18.09 µM 2, 4-D (C4) to obtain reasonable quantity of callus for further manipulation. Such
well proliferating 60 day-old callus cultures were inoculated on optimized callus
proliferation media (C4) containing different concentrations [0 (control), 20, 40, 60, 80, 100,
120 or 140 mM] of NaCl. Results were recorded from 30 replicate culture vessels for the
effect of salt on percentage increase/decrease in fresh weight of callus cultures and its
morphogenic response after 30 days under salt stress. Less than 20% decrease in fresh weight
of callus cultures was considered to be a ‘Good’ (+ + +) proliferation response, less than 40%
‘Satisfactory’ (+ +) and less than 60% ‘Poor’ (+). The terms for the proliferation response as
used in this study were arbitrarily chosen since it was difficult to express this parameter in
qualitative manner. Its narration however, is based on a combination of two aspects, i. e.,
increase/decrease in fresh weight of callus and by visual observation.
3.7.8: Identification of Sub-lethal Salt Concentration and Maintenance of Callus
Cultures on Respective Salt Concentration for 6 Sub-cultures
Pre-weighed main callus cultures (60-day-old) of both the tested cultivars developed
on MS (Murashige and Skoog, 1962) medium supplemented with 18.09 µM 2, 4-D were sub-
cultured to the same medium but containing different concentrations of NaCl (0, 20, 40, 60,
80, 100, 120 or 140 mM; 8 treatments). Ten culture vessels (25 × 150 mm) were inoculated
for each treatment and the experiment was repeated thrice. Callus cultures were maintained
under dark conditions at 26 ± 2°C. Data were recorded for percentage relative fresh weight
growth (PRFWG) of callus cultures after 90 days of salt treatment. Prior to recording the
data, the calluses were sub-cultured after two weeks to their respective salt concentration.
The PRFWG of callus cultures was calculated by using a formula: W1-W0/W0 × 100 (where
48
W1 was fresh weight after 90 days of salt treatment and W0 being the weight of callus
cultures before salt treatment).
Sub-lethal concentration of salt was selected on the basis of decrease in PRFWG. The
concentration of salt that resulted in 50% decrease in PRFWG was selected as sub-lethal one
for each cultivar (Basu et al., 2002). Furthermore, calluses were sub-cultured and maintained
on this concentration of salt for 4 months. Recurrent selection was done by transferring the
calluses to NaCl-free basal medium (BM) for two successive subcultures, then returned to
their respective MS basal medium plus NaCl. The callus cultures that survived and resumed
growth for at least two further subcultures were picked and inoculated on optimized
regeneration medium (Ochatt et al., 1999).
3.7.9: Regeneration of Callus Culture of Solanum tuberosum L. After 30 Days
of Salt Treatment
Callus cultures after 30 days of salt treatment were shifted to salt free optimized
regeneration medium. Ten culture vessels were inoculated from each salt concentration and
cultures were maintained at 25 ± 2°C in 16 h photoperiod. Regeneration potential was
recorded after 30 days of callus transfer to regeneration medium.
49
3.7.10: Regeneration Potential of Recurrently Selected Callus Cultures on Salt-
free Regeneration Medium
After recurrent selection, callus cultures were picked and transferred to an optimized
callus regeneration medium. After 60 days of callus inoculation on regeneration medium,
different growth parameters (number of days for regeneration, number of shoots per callus
culture and number of nodes) were recorded.
3.7.11: Assessment of the Stability of the Acquired Salt Tolerance after
Recurrent Selection of Potato
Well acclimatized plants of both the cultivars after recurrent selection were
transferred to a mixture of soil and organic matter (50:50) and were irrigated with Hoagland
solution for 30 days. Single plant was planted in each 8 × 8 cm pot in glasshouse under 25/16
± 2°C day/night temperatures and a 16 h photoperiod. After 30 days, pots were irrigated with
Hoagland solution supplemented with or without 100 mM NaCl to experimental and control
ones, respectively whenever required. There were ten replicate for both control and
experimental plants. Different morphological (number of tubers per plant, fresh weight and
dry weight) and biochemical parameters (protein contents, peroxidase, catalse, and
superoxide dismutase activity) were scored at day 30 of salt treatment.
50
In addition to the above-mentioned aspects, the effect of exogenous application of ascorbic
acid and salicylic acid to ameliorate the effect of NaCl in both in vitro plants as well as in
callus cultures of potato was also investigated.
3.7.12: Effect of Ascorbic Acid Pretreatment to In vitro Salinized Plants and
Callus Cultures of Solanum tuberosum L.
3.7.12.1: Methodology regarding these Aspects is given in Paper Published in In vitro
Cellular and Developmental Biology-Plant (Annexure 9D).
3.7.12.2: Regeneration Potential of Ascorbic acid-pretreated and Non-pretreated
Salinized Callus Cultures
Ascorbic acid-pretreated and non-pretreated callus cultures from different (0 - 140
mM) salt treatments after 60-days were picked and transferred to an optimized plant
regeneration medium (MS basal medium supplemented with 2.64 µM NAA and 1.0 µM
TDZ). Regenerated callus cultures were shifted after 30 days on MS basal medium
supplemented with 8.87 µM BAP + 2.64 µM NAA and 0.123 µM IBA for further
proliferation and rooting of shoots. The regeneration potential was recorded at day 60 of
callus inoculation on regeneration medium.
3.7.13: Effect of Ascorbic Acid Foliar Spray to Salinized Plants of Solanum
tuberosum cv. Cardinal
For tissue disinfestation, the tubers were initially sterilized by 0.7% sodium
hypochlorite for 5 min and then thoroughly washed with autoclave distilled water to remove
traces of salt. Potato tubers were cut into pieces so as to have one eye or growing point on
each piece. These pieces were dipped in 5% potassium permanganate solution for 5 min
before sowing. Potato tubers were sown into 8 × 14 earthen pots filled with 10 kg clay, loam
51
soil. The known characteristics of soil were: pH 7.5, EC 6.0 dsm-1, carbonates nil,
bicarbonates 9.5 ml/L, chloride 20 ml/L. The pots were arranged in a randomized complete
block design with 10 replicate.
3.7.13.1: Application of Ascorbic acid
The treatments were (a) without ascorbic acid and salt (b) with ascorbic acid and
without salt (c) without ascorbic acid and with salt (d) with ascorbic acid and salt. Plants
were irrigated with 50% Hoagland solution for one-month with 5 days interval. After 30
days, plants irrigation was done with Hoagland solution supplemented with 120 mM NaCl
for next 60 days (after every 5 days). Simultaneously, 0.5 mM ascorbic acid was sprayed
with Tween-20 to evoke spreading of the applied solution on the plant leaf surface. The
solution was sprayed manually once on the leaves in the early morning after every 3 days
interval.
3.7.13.2: Data Collection and Analysis
After two months of salt and ascorbic acid treatments, following growth parameters
were studied: fresh/dry weight of tubers, shoot length and number of shoots. In addition to
morphological parameter, certain biochemical parameters: total soluble protein contents,
peroxidase, catalase, and superoxide dismutase activities were also analyzed.
3.7.14: Effect of Salicylic Acid Treatment to In vitro Salinized Plants of
Solanum tuberosum L.
Healthy two-month-old potato plants were selected for salicylic acid application.
Single nodal segments of 1.0 cm in length were inoculated on MS medium with or without
60 mM NaCl supplemented with or without different concentrations (0.125 mM, 0.25 mM,
0.50 mM and 0.75 mM) of salicylic acid. The dose and time for pretreatment was based on
52
previous studies on different plant species (Senaratna et al., 2000; Arfan et al., 2007; Gunes
et al., 2007). Three different treatment groups were formed, i.e., 1) control without NaCl and
salicylic acid, 2) with NaCl and without salicylic acid, 3) with NaCl and salicylic acid. Ten
culture vessels (25 × 150 mm) were inoculated for each treatment for both the cultivars.
Experimental design was completely randomized with 10 replicate for each treatment (one
nodal segment for each replicate). The cultures were maintained at 26 ± 2°C in 16 h
photoperiod, 40 µmoles m-2 s-1 light intensity from cool white florescent tube light. After 60
days of inoculation of segments, number of growth (average root, shoot length, number of
root, shoot and nodes, fresh, dry weight) and biochemical parameters (protein contents) were
studied.
3.7.15: Statistical Analysis
The data were analyzed using Univariate analysis of variance (SPSS Version 12.0.0).
3.8: Mechanism of Salinity Tolerance in Thellungiella halophila
Experiments were also conducted to investigate the mechanism of salinity tolerance
in true halophytes (Thellungiella halophila). This research work was carried out at Center for
Environmental Stress Physiology, Department of Horticulture and Landscape Architecture,
Purdue University, West Lafayette, Indiana USA during 6 months scholarship (International
Reasearch Support Initiative Program) awarded by Higher Education Commission of
Pakistan. Details are given in Annexure 9E (Zahoor A. Sajid, Michael J. Gosney and Michael
V. Mickelbart (2010) Effect of salinity on growth and physiology of Thellungiella halophila
ecotypes; unpublished data).
53
Chapter 4
Standardization of Conditions for Micropropagation, Callus Induction,
Regeneration, Cell Suspension Culture and Acclimatization of
Regenerated Plants of Solanum tuberosum L. cvs. Cardinal and Desiree
RESULTS
4.1: Standardization of Medium and Maintenance of Germplasm of
Solanum tuberosum L. cvs. Cardinal and Desiree
For the maintenance of germplasm and standardization of medium for
micropropagation, different media combinations were tested as mentioned in Materials and
Methods section and results were recorded for morphological as well as biochemical
parameters as detailed below:
4.1.1: Shoot and Root length
In potato (cv. Cardinal), highest shoot length (5.74 cm) and root length (5.34 cm) was
observed in MS full-strength medium after 30 days of initial culture (Table 4.1). This was
followed by S2 (MS + TDZ 10-10 M) where the shoot and root length was 5.51 and 5.24 cm,
respectively. Lowest shoot and root length (5.30 and 4.08 cm, respectively) was observed in
S4 (MS + TDZ 10-8 M). However, the mean values for shoot length were not significantly
different from each other. In cv. Desiree, highest shoot and root length was 5.48 cm and 5.32
cm, respectively on MS full-strength medium (Table 4.2). A somewhat similar pattern for
shoot and root data was observed since the lowest values for the two parameters were
observed on MS medium supplemented with 10-8 M TDZ.
54
4.1.2: Number of Shoots, Roots and Nodes
In case of cv. Cardinal, number of shoots, roots and nodes were 2.30, 12.60 and 7.90,
respectively on S1 medium (control). Maximum number of shoots (2.66 and 2.96 for
Cardinal and Desiree, respectively) was observed on MS + TDZ (10-8 M). However, highest
number of roots (12.60 and 14.90) and nodes (7.90 and 7.20) was observed on MS full-
strength medium in Cardinal and Desiree plants. The lowest number of shoot (2.0 and 1.5),
root (7.3 and 4.9) and node (6.3 and 6.3) was obtained on MS + TDZ (10-9 M) in both the
cultivars. The mean values for number of shoots and nodes in cv. Cardinal were not different
from one another statistically. However, the mean values for number of roots on different
media varied in certain cases significantly from one another and also in comparison with the
control (Tables 4.1 and 4.2).
4.1.3: Fresh and Dry weight of Plantlets
In the medium S1, the fresh weights of plantlet were 0.4460 and 0.6040 g for cv.
Cardinal and Desiree, respectively. The maximum fresh and dry weight of the plantlets
(0.5430 g and 0.0524 g) in cv. Cardinal was obtained on MS medium containing 10-9 M
TDZ. In Desiree, the highest fresh and dry weights (1.0560 and 0.0965 g, respectively) on
the other hand, were observed on MS medium containing 10-10 M TDZ (Tables 4.1 and 4.2).
Although the highest shoot and root length (in both the cultivars) was obtained on MS full-
strength medium but highest fresh and dry weights in Desiree and Cardinal were observed
using MS medium with either 10-10 or 10-9 M TDZ, respectively (Published article from this
part of study is attached as Annexure 9A).
55
Table 4.1: Effect of three different TDZ levels supplemented to MS media on in vitro
establishment of shoot apices of potato (Solanum tuberosum L. cv. Cardinal) at day 30
of the initial culture*
Media composition
and Designation
Shoot length**
(cm)
Root length**
(cm)
Number of
shoots**
Number of
roots**
Number of
nodes**
Fresh weight of plantlets**
(g)
Dry weight of plantlets**
(g)
MS full strength (S1)
5.74 ± 0.664a
5.34 ± 0.449a
2.30 ± 0.246a
12.60 ± 1.861a
7.90 ± 0.780a
0.4460 ± 0.0620ab
0.0347 ± 0.008b
MS +10-10M TDZ (S2)
5.51 ± 0.556a
5.24 ± 0.650a
2.28 ± 0.389a
8.42 ± 1.45c
6.71 ± 0.629a
0.4066 ± 0.110b
0.0320 ± 0.006b
MS +10-9M TDZ (S3)
5.36 ± 0.536a
4.09 ± 0.314b
2.00 ± 0.244a
7.30 ± 1.20c
6.30 ±0.0813a
0.5430 ± 0.093a
0.0524 ± 0.008a
MS +10-8M TDZ (S4)
5.30 ± 0.622a
4.08 ± 0.541b
2.66 ± 0.222a
10.50 ± 1.611b
7.55 ± 0.686a
0.3633 ± 0.137b
0.0430 ± 0.010ab
* Results on all parameters are means ± S.E. from thirty replicate cultures.
**Means within a column followed by the same letter do not differ significantly (p< 0.05).
Data are subjected to analysis of variance and the means separated by Duncan’s multiple
range test.
56
Table 4.2: Effect of three different TDZ levels supplemented to MS media on in vitro
establishment of shoot apices of potato (Solanum tuberosum L. cv. Desiree) at day 30 of
the initial culture*
Media composition
and Designation
Shoot length**
(cm)
Root length**
(cm)
Number of
shoots**
Number of
roots**
Number of
nodes**
Fresh weight of plantlets**
(g)
Dry weight of plantlets**
(g)
MS full strength (S1)
5.48 ± 0.664a
5.32 ± 0.399a
1.90 ± 0.223b
14.90 ± 1.151a
7.20 ± 0.394a
0.604 ± 0.0720b
0.074 ± 0.008ab
MS + 10-10M TDZ (S2)
5.27 ± 0.436a
5.12 ± 0.309a
2.90 ± 0.220a
14.72 ± 0.670a
6.81 ± 0.347a
1.056 ± 0.091a
0.096 ± 0.068a
MS + 10-9M TDZ (S3)
5.43 ± 0.436a
4.73 ±0.284ab
1.50 ± 0.259b
4.90 ± 0.538b
6.30 ± 0.691a
0.175 ± 0.043c
0.089 ± 0.070ab
MS + 10-8M TDZ (S4)
5.16 ± 0.594a
4.20 ± 0.254b
2.96 ± 0.386a
13.65 ± 2.081a
6.80 ± 0.796a
0.962 ± 0.130a
0.057 ± 0.090a
*Results on all parameters are mean ± S.E. from thirty replicate cultures.
**Means within a column followed by the same letter do not differ significantly (p< 0.05).
Data are subjected to analysis of variance and the mean separated by Duncan’s multiple
range test.
57
4.2: Effect of Different Growth Regulators on Callus Induction and
Proliferation in Potato
Amongst various concentrations of auxins used in MS medium, 2, 4-D at 18.09 µM
was effective favoring 90% callus induction after 12 days of inoculation. Calluses were
morphologically off-white, friable and granular (Fig. 4.1). Decrease in the concentration of
2, 4-D reduced the rate of callus induction. As evident from the data given in Table 4.3,
when the concentration of 2, 4-D was decreased from 18.09 to about one-half (9.04 µM), rate
of callus formation correspondingly decreased from 90 to 40%. Interestingly, no callus
induction was observed in all the replicated culture vessels using MS medium supplemented
with 4.52 µM 2, 4-D.
Three different combinations of 2, 4-D and BAP were used to find out their effect on
callus induction. Among these combinations, 2, 4-D at a concentration of 13.5 µM with 2.22
µM BAP proved effective with 65% callus induction response after 14 days of inoculation.
Usually the calli were off-white to green, friable and loose (Fig. 4.2). When concentration of
BAP was decreased or increased from 2.22 µM, rate of callus induction was reduced in both
the cultivars.
Of auxin-auxin combinations, 2, 4-D and NAA were also used for callus induction.
MS medium supplemented with 2, 4-D at a concentration of 13.5 µM with 1.07 µM NAA
showed excellent results with 90% callus induction response after 14 days of inoculation
(Fig. 4.3). With further increase in the concentration of NAA, i.e., 2.14 to 3.21 µM with the
same concentration of 2, 4-D (13.5 µM), callus induction response was reduced from 80% to
60%.
58
Best callogenic response was observed by using a combination of BAP (10.0 µM)
and NAA (1.1µM) supplemented to MS medium (Fig. 4.4). At this concentration, 89% callus
induction was obtained after 13 days of explants inoculation. Callus cultures were green with
white patches and compact (Fig. 4.4). By increasing the concentration of NAA to 3.3 µM,
the rate of callus formation was reduced to 70% (Table 4.3).
Figure 4.1-4.4: Morphology of potato callus cultures (cvs. Cardinal and Desiree)
initiated from internodal segments on MS basal medium supplemented with different
growth regulators at day 60
Fig. 4.1: Well-proliferating callus cultures on MS medium supplemented with
18.09 µM 2, 4-D, A; Desiree (1.5x) B; Cardinal (1.2x).
Fig. 4.2: Callus cultures on MS medium supplemented with 13.5 µM 2, 4-D
and 2.22 µM NAA, A; Desiree (1x) B; Cardinal (1.5x).
A B
A B
59
Fig. 4.3: Callus cultures on MS medium supplemented with 13.5 µM 2, 4-D
and 1.07 µM NAA, A; Desiree (1.2x) B; Cardinal (1x).
Fig. 4.4: Callus cultures on MS medium supplemented with 10.0 µM BAP and
1.07 µM NAA, A; Desiree (2x) B; Cardinal (2x).
A B
BA
60
Table 4.3: Effect of different growth regulators supplemented to MS medium on callus
induction and proliferation in Solanum tuberosum L. cvs. Cardinal and Desiree
Data are the means ± S.E. from 30 replicate culture vessels per treatment.
Media Designation
Medium and Growth
regulators
Concentration of growth regulators
(µM)
Time requirement for Callus
induction ( Days)
Callus induction
(%)
C1
C2
C3
C4
MS + 2, 4 –D
4.52 - 0
9.04 19 ± 1.009 40
13.57 19 ± 0.894 60
18.09 12 ± 1.094 90
C5
C6
C7
MS + 2, 4-D
+ BAP
13.5 + 1.11 13 ± 1.394 60
13.5 + 2.22 14 ± 0.304 65
13.5 + 4.43 18 ± 0.794 52
C8
C9
C10
MS + 2, 4-D
+ NAA
13.5 + 1.07 14 ± 0.990 90
13.5 + 2.14 13 ± 1.314 80
13.5 + 3.21 1 ± 1.322 65
C11
C12
C13
MS + BAP
+ NAA
10.0 + 1.07 13 ± 0.994 89
10.0 + 2.20 13 ± 1.334 70
10.0 + 3.30 20 ± 1.340 70
61
4.3: Optimization of Conditions for Plant Regeneration through Callus
Cultures in Solanum tuberosum L. cvs. Cardinal and Desiree
The results given in Table 4.4 illustrate that R3 (2.64 µM NAA and 1.00 µM TDZ)
was the best medium for regeneration of both the cultivars of potato as compared to various
other tested combinations involving NAA and TDZ. By increasing the concentration of TDZ
from 1.00 to 2.00 µM, regeneration response decreased and days for regeneration increased.
Similarly by increasing the concentration of NAA from 2.64 to 15.91 µM, callus
regeneration response decreased and at very high concentration of NAA (13.27 µM),
regeneration phenomenon was completely inhibited. Regeneration of callus cultures took
longer incubation time-period (more days) at higher or lower concentration of NAA than
optimum. Granular, friable and greenish calluses gave better regeneration response than
compact-yellowish calluses (Fig. 4.5 A and B). It was also observed that regeneration
potential of cv. Cardinal was comparatively better as compared to cv. Desiree. For further
proliferation and rooting of regenerated plants, cultures were shifted to MS medium
supplemented with 8.87 µM BAP, 2.64 µM NAA and 0.123 µM IBA (optimized by
preliminary experiments). Figurative depiction of events is given in Fig. 4.5-4.7.
62
Table 4.4: Effect of different media on regeneration potential of callus cultures of
Solanum tuberosum cvs. Cardinal and Desiree
Medium Designation
Media composition MS + NAA + TDZ
(µM)
Regeneration potential (%) **
Number of days required for regeneration
Cultivars* Ds Car Ds Car
R1 2.64 + 0.10 13 15 29 ± 0.349 27 ± 0.230
R2 2.64 + 0.50 23 24 26 ± 0.540 24 ± 0.560
R3 2.64 + 1.00 80 82 21 ± 0.540 20 ± 0.240
R4 2.64 + 1.50 66 77 25 ± 0.340 26 ± 0.343
R5 2.64 + 2.00 58 67 29 ± 0.330 27 ± 0.040
R6 5.35 + 1.00 43 45 25 ± 0.340 28 ± 0.240
R7 7.99 + 1.00 24 27 28 ± 0.140 26 ± 0.339
R8 10.63 + 1.00 11 19 24 ± 0.040 29 ± 0.140
R9 13.27 + 1.00 0 0 0 0
R10 15.91 + 1.00 0 0 0 0
*Cultivar Cardinal; Car and Desiree; Ds.
**Data are the means ± S.E. from 30 replicate culture vessels per treatment.
63
Fig. 4.5-4.7: Some selected photographs showing different stages of regeneration in
potato cvs. Cardinal and Desiree
Fig. 4.5: Shoot initiation (arrows) on MS basal medium
containing 2.64 µM NAA + 1.00 µM TDZ in cv. Cardinal (A &
B; 2.0x) and cv. Desiree (C & D; 2.0x).
Fig. 4.6: Shoot proliferation on MS medium supplemented with
8.87 µM BAP, 2.64 µM NAA and 0.123 µM IBA in cv. Cardinal
(A & B; 1x and 2.0x) and cv. Desiree (C & D; 1.5x).
A B
C D
A B
D C
64
Fig. 4.7: Regenerated plants through callus cultures of potato with well-
developed roots of cv. Cardinal (A & B; 1.2x) and cv. Desiree (C & D; 1.2x).
A B
C D
65
4.4: Optimization of Conditions for the Initiation of Cell Suspension
Cultures
Apart from callus cultures, work on the development of cell suspension cultures was
also initiated to explore the potential benefits it offers. The data reveal that the growth
behavior of the two tested cultivars was quite similar. The summary of conditions determined
for the initiation of cell suspension cultures in both the cultivars of potato is given in Table
4.5.
4.4.1: Source Material
Eight-week-old translucent, friable, off-white callus cultures were an excellent
starting material for the initiation of homogeneous cell suspension cultures as compared to
other tested sources, e.g., compact green, compact white or friable-yellow callus cultures. It
was also noted during this study that elongated cells with thick walls were quite poorly-
dividing (Fig. 4.8 A & B). On the other hand, globular or rounded cell suspensions had better
division efficiency (Fig. 4.9 A & B) in both the cultivars.
4.4.2: Medium
Of the three tested media (MS, MS2 or AA medium containing 18.09 µM 2, 4-D),
MS2 was found to be a better medium for the initiation of cell suspension cultures. The
division efficiency of suspension cells was also comparatively better on this medium.
4.4.3: Physical Conditions
Cell suspension cultures, placed in 16-h photoperiod at 25 ± 2°C and agitated at 120
rpm using a gyratory shaker showed excellent results. It was also observed that filtration
66
through nylon or stainless-steel sieve (450 µm) helped to remove the larger cell aggregates
that could clog smaller pore-size meshes. The cells collected after sieving through 450 µm
mesh proved to be good source material for the establishment of cell suspension cultures in
potato.
67
Table 4.5: Optimum conditions for the initiation/establishment of cell suspension
cultures of potato cvs. Cardinal and Desiree
Parameters Optimum Conditions
Source material Eight-week-old translucent, friable, off-white calluses
Culture medium MS2 medium (Vargas et al. 2005)
Growth regulator/s 18.09 µM 2, 4-D
Source material/medium ratio 0.5 g callus/10 ml medium
Culture vessels Erlenmeyer flasks, capacity 100 ml
Agitation rate (rpm); Optima orbital shaker, OS-752, Japan 120 rpm
Temperature 25 ± 2ºC
Light conditions 16 h photoperiod using fluorescent-white tube-lights (35 µmol m-2 s-1)
Sub-culturing interval Every 3-5 days
Sieving mesh size for the first two subcultures 450 µm
68
Fig. 4.8: A. Elongated and poorly-dividing cell suspension of cv. Cardinal
with thick walls (100x). B. Elongated cells in suspension cultures of Cardinal
derived from compact-green callus cultures (100x).
Fig. 4.9: A. Globular, rounded cells with good division efficiency (100x). B.
Clusters of rapidly-dividing rounded cells with smaller diameter (100x). Both
A and B from the cell suspension cultures of cv. Desiree.
A B
A B
69
4.5: Optimization of Acclimatization Conditions and Medium
In this study, all tested hardening and acclimatization media (sand, soil, peat-moss,
perlite, vermiculite, saw-dust and mixture of vermiculite, perlite and soil; 1:1:1 by volume)
supported the growth of plants more or less equally except for the soil alone. The two
cultivars of potato under investigation in this study showed varied behavior in growth and
acclimatization. Ex vitro acclimation of Cardinal was relatively quicker as compared to
Desiree. Well-acclimatized plants of both the cultivars are shown in Fig. 4.10 & 4.11. The
best supporting medium on the basis of mortality rate for ex vitro transplantation of potato
plants of both the cultivars was vermiculite. Plant mortality rate was up to 42 (in sand) and
43% (in soil) for Cardinal and 40 (in sand) and 45% (in soil) for Desiree. The minimum
mortality rate of 15 and 17% was observed in vermiculite medium in cv. Desiree and
Cardinal respectively (Fig. 4.12).
70
Fig. 4.10: A comparison of growth and development of potato plants under ex vitro
conditions on different media (0.4x)
Vermiculite Mixture of vermiculite, perlite and soil Saw dust
Perlite Sand Soil
Peat moss
71
Fig. 4.11: Well-acclimatized plants of potato growing in pots in glasshouse conditions,
Cvs. Cardinal (A & B; 0.4x) and Desiree (C & D; 0.4 & 0.8x) are shown growing in
plastic pots
A
C D
B
72
Fig. 4.12: Mortality rate of potato plants (Cvs. Cardinal and Desiree) in different
hardening media
Ds: Desiree.
Car: Cardinal.
73
DISCUSSION
To maintain the germplasm for this study, an in vitro clonal propagation technique
was used for producing large number of plants. Micropropagation has an immense advantage
of rapidly generating a large number of genetically identical plants in short time period. For
micropropagation, different growth regulators in MS medium were used earlier in different
potato cultivars, e.g., GA3 with calcium pentothenic acid (Potluri and Devi-Prasad, 1994),
NAA, IAA and kinetin (Merja and Stasa, 1997), GA3 and kinetin (Nagib et al., 2003) or GA3
and NAA (Rahnama and Ebrahimzadeh, 2004). The use of TDZ has never been tested before
in potato for the purpose of in vitro clonal propagation. TDZ, a substituted phenyl-urea as a
plant growth regulator has been reported for many plant species including several recalcitrant
woody plant species like Quercus robur L. (Chalupa, 1988) and Pinus strobes L. (Pijut et al.,
1991). It has been observed during the present investigation that the second highest shoot and
root length was recorded using MS medium supplemented with 10-10 M TDZ which was very
close to medium S1 (MS full strength) in cv. Cardinal and the difference in values was in fact
non-significant in statistical terms. Shoot and root length of in vitro-raised plants decreased
by increasing the concentration of TDZ in the MS medium in both the cultivars used in the
present study. It might have been due to the fact that TDZ has also been reported to modify
the endogenous cytokinin metabolism (Capelle et al., 1983; Hare and Van-Staden, 1994;
Murthy et al., 1995; Hutchinson and Sexena, 1996). Alternatively, it has also been suggested
that TDZ may mimic an auxin response (Visser et al., 1992) or modifies endogenous auxin
metabolism (Murthy et al., 1995; Hutchinson et al., 1996).The effectiveness of TDZ for
micropropagation has also been proven in literature in several studies on different plant
species (Kern and Meyer, 1987; Agarwal et al., 1992; Mondal et al., 1998; Fratini and Ruiz,
74
2002). The results from the present work demonstrated the possibility to micropropagate
potato using TDZ in MS basal medium. This may be so because most studied parameters
were statistically not different or showed better response (fresh/dry weight and number of
shoots) as compared to the control. Its superiority over general basal MS medium, however,
could not be established in this study though it proved to be equally effective for most
parameters in statistical terms in both the cultivars. It also demonstrates that TDZ might be
used at very low concentration (less than a nM level) for potato micropropagation as
compared to other cytokinins (such as BAP) that are usually used at relatively higher (µM)
levels (Shibli et al., 2001; Fengyen and Han, 2002). It is evident from the results that TDZ
does have an influence (though negative in comparison with the control) on
micropropagation of potato even at very low concentrations. Although TDZ is relatively
costly, a very low concentration of TDZ used in this study circumvents its price
consideration and rather seems to be quite cost-effective. Moreover, its influence on all the
growth parameters under study necessitates further work using TDZ as a growth regulator to
better understand its role in potato tissue culture.
During the present investigation, different media combinations were used for callus
induction. Internodal segments (ca. 1cm long) were chosen as explants for the initiation of
callus, since this explant source had shown good callusing potential in many earlier studies
on potato (Svetek et al., 1999; Turhan, 2004; Vargas et al., 2005; Gopal et al., 2008). In this
study, 2, 4-D at a concentration of 18.09 µM was proven to be very effective for callus
induction and proliferation in both the cultivars of potato. This effective behavior of 2, 4-D
in callus induction has been reported in many previous studies on potato (Khatun et al.,
2003; Vargas et al., 2005). It is evident from the literature that 2, 4-D increases the
75
endogenous auxin levels in explants (Michalczuk et al., 1992) which might influence cell
enlargement and cell divisions more efficiently than other growth regulators. On the
contrary, different combinations of growth regulators (auxin and cytokinin) were also used
for callus induction in different potato cultivars (Svetek et al., 1999; Rahnama et al., 2003;
Nasrin, 2003; Rahnama and Ebrahimzadeh, 2004; Queiros et al., 2007). This varied response
for callus induction on different media might be due to genotypic variation.
For plant regeneration from callus cultures, different media combinations were used
and R3 (MS + 2.64 µM NAA and 1.00 µM TDZ) was selected as the best medium for
regeneration of both the tested cultivars of potato as compared to various other tested
combinations involving NAA and TDZ. The use of TDZ has been extensively reported for
regeneration of several recalcitrant woody plant species (Thomas, 2003; Rashid, 2002;
Thomas and Puthur, 2004). Similarly, TDZ was also used in several herbaceous plants
species, i. e., geranium (Pelargonium × hortarum Bailey; Hutchinson et al., 1996), Solanum
melongena L. (Magioli et al., 1998), Carnation (Casanova et al., 2003) for shoot
organogenesis. The use of low concentration of TDZ in combination with NAA for
stimulation of axillary shoots has been recommended as an efficient treatment (Van-
Nieuwkerk et al., 1986; Chalupa, 1988; Yusnita et al., 1990). Chalupa (1987) reported that
cultures of Robinia pseudoacacia L., Sorbus aucuparia L. and Tilia cordata had increased
shoot proliferation and elongation when TDZ was applied in combination with BA, IBA or
NAA. TDZ was also applied for regeneration in sugarcane callus cultures (Jain et al., 2007).
For rooting of the regenerated shoots of potato in this study, cultures were shifted to MS
medium supplemented with 8.87 µM BAP, 2.64 µM NAA and 0.123 µM IBA. It showed
that cytokinin and auxin in combination play an important role in rooting of the regenerated
76
shoots. This need of second medium for rooting of regenerated shoot was also reported in the
literature (Khatun et al., 2003). It was observed that regeneration potential of cv. Cardinal
was better as compared cv. Desiree. This variable response in regeneration potential might be
dependent on cultivar-specific effect of these plant growth regulators.
Cell suspensions cultures are ideal to study various factors that affect growth and
differentiation (Evans et al., 2003). In this study, cell suspension cultures were established in
an attempt to look for an alternative plant source material (other than the callus cultures) that
may be used for an effective selection of salt-tolerant cell lines in potato. During the study, it
was observed that friable callus cultures were an excellent starting material for the
establishment of cell suspension cultures in both the cultivars of potato. The use of friable
callus for the initiation of cell suspension cultures is considered as very important and
primary step (Bhojwani and Razdan, 2004; Liang et al., 2006). Friable callus cultures
segregated into free cells more easily as compared to compact ones. MS2 medium proved to
be quite effective for the initiation of cell suspension cultures. Vargas et al. (2005) also
observed good results on this medium in potato. However, Aftab et al. (1996) selected AA
medium for the establishment of cell suspension cultures in sugarcane. Interestingly, the
same medium in this lab gave good results for the initiation of cell suspension cultures in
potato though less effective than MS2. Likewise, source material to medium ratio was
considered to be an important factor for the establishment of cell suspension cultures (Aftab
and Iqbal, 1999). The only tested ratio in this study (0.5 g fresh weight of callus in 10 ml
liquid medium) gave quite satisfactory results and hence continued to be used throughout the
study period. Liang et al. (2006) had proposed 0.75 g callus mass in 20 ml medium as an
effective ratio of cells to medium for the establishment of cell suspension cultures in
77
Orthosiphon stamineus Benth. Regarding agitation rate, 100-120 rpm was considered
optimum for the establishment of efficient cell suspension cultures (Evans et al., 2003). It
was observed during this study that agitating the suspensions at 120 rpm on a gyratory shaker
yielded good results for the establishment of cell suspensions in both potato cultivars.
For ex vitro establishment of regenerated plants, acclimatization conditions and
media were also optimized in the present study. This is usually a very crucial stage of plant
growth as during in vitro cultures, plantlets grow under strict aseptic conditions in closed
culture vessels and consequently humidity levels are way too higher than the outside. Many
plants consequently perish during the hardening-off or acclimatization process (Sutter et al.,
1988). A gradual transfer to glasshouse or greenhouse environment is thus of great
significance in order to acclimatize in vitro-grown plants to the new growing conditions. The
success of this process is highly dependent on the extent of the morphological and/or
physiological abnormalities acquired during in vitro growth, which may become
incompatible with external life (Preece and Sutter, 1991; Bolar et al., 1998; Pospisilova et al.,
1999; Fila et al., 2006).
The best supporting medium for ex vitro transplantation of potato plants of both the
cultivars was vermiculite during this investigation. Vermiculite does have more moisture-
retaining and aeration capability than various other potting media (Aftab et al., 2005) and
easily removable from the roots (Zimmerman et al., 2007). These results differ from the
results previously reported for acclimatization of Solanum tuberosum where mixture of peat:
perlite and soil (1:1:1) was suggested to be the best acclimatization medium (Ochatt et al.,
1999). So a range of different media may play a role in supporting the initial acclimatization
and different results in different plant groups indicate the specific needs of those plants in
78
question. Sand and soil initially did not help the plant root growth since it was relatively
compact and perhaps damaged roots and that in turn influenced plant growth as well. The
two cultivars of potato under investigation in this study show varied behavior in growth and
acclimatization (shown in Fig. 4.20). Ex vitro establishment of cultivar Cardinal was quicker
as compared to plants of cv. Desiree. This varied response towards acclimatization by two
tested cultivars might be due to their genetic makeup. Plant mortality rate was up to 42 (in
sand) and 43% (in soil) for cv. Cardinal and 40 (in sand) and 45% (in soil) for cv. Desiree.
In conclusion, MS medium was selected as best medium for micropropagation of
both the tested potato cultivars followed closely by MS medium supplemented with TDZ
(10-10 M). The later mentioned medium also supported fair growth of shoot and root. For
callus induction and proliferation in dark, internodal segments proved to be a good explant
source whereas MS medium prepared with 2, 4-D (18.09 µM) was the best medium
composition equally effective for both the cultivars. A combination of NAA (2.64 µM) and
TDZ (1.00 µM) supplemented to MS medium was the best choice for shoot initiation.
Rooting was achievable on MS medium supplemented with 8.87 µM BAP, 2.64 µM NAA
and 0.123 µM IBA. MS2 medium of Vargas et al. (2005) showed quite effective results for
the initiation of cell suspension cultures. The best supporting medium for ex vitro
transplantation of potato plants of both the cultivars was vermiculite during this
investigation.
79
Chapter 5
Effect of NaCl Stress on In vitro Plants/Callus Cultures and Selection of
Salt-tolerant Cell lines, Regeneration, Subsequent Establishment under Ex
vitro Conditions and Biochemical Characterization
RESULTS
5.1: Exposure of In vitro Plants to Different Concentrations of NaCl (0-140
mM)
5.1.1: Effect of different Concentrations of NaCl on In vitro Growth of Potato
A significant difference was observed in in vitro growth of plants when subjected to
different concentrations of NaCl (0-140 mM). The data presented in Table 5.1 show that after
60 days of incubation, an increase in the concentration of NaCl from 0-140 mM (8
treatments) correspondingly resulted in a gradual inhibition of the studied growth parameters.
In case of Cardinal, shoot growth of control plants after 60 days was 11.90 cm whereas it
was 11.77, 8.74, 4.60, 3.9 or 4.03 cm at 20, 40, 60, 80 or 100 mM NaCl level, respectively
(Fig. 5.1-5.2). By further increasing the concentration of NaCl in the growth medium, an
abrupt decline in all the growth parameters was observed except for the fact that the number
of shoots per culture vessel increased. At 120 mM NaCl, rosette type of shoot growth (shoots
more than six in number with small internodal distance growing from single node) was
observed without any root formation (Fig. 5.3). At even higher concentration of salt, i.e., 140
mM, tissue necrosis was observed (Fig. 5.4).
Almost the same trend in growth parameters was observed in case of cv. Desiree.
With an increase in the concentration of salt, there was a gradual decrease both in shoot and
root growth. Data presented in Table 5.2 indicate that the shoot length decreased from 10.70
80
cm (control) to 7.20, 6.60, 1.70 or 2.08 cm at 20, 40, 60 or 80 mM salt concentration. Unlike
the first cultivar, however, tissue necrosis was not observed until a higher salt level (120
mM) was reached (Fig. 5.5-5.8). These observations also suggested that perhaps Cardinal’s
in vitro response was more of a moderately-salt-tolerant cultivar while Desiree was
seemingly comparatively more sensitive to higher salt (NaCl) concentration.
5.1.2: Total Soluble Protein Contents in Potato under NaCl Stress
In Cardinal, there was a gradual decrease in soluble protein contents with an increase
in the concentration of salt in the growth medium (Table 5.1). At 20 mM concentration of
NaCl, protein contents decreased from 2.51 (control) to 0.76 mg/g. The total soluble protein
contents were 0.79, 0.82, 0.29, 0.76 or 0.19 mg/g at 40, 60, 80, 100 or 120 mM NaCl,
respectively. On the other hand, it was observed in Desiree that the protein contents
increased gradually with a corresponding increase in salt concentration (Table 5.2). The total
soluble protein contents were 1.30, 3.98, 2.81, 5.20 or 4.03 mg/g at 20, 40, 60, 80 or 100 mM
NaCl as compared to the control (1.53 mg/g). At 120 mM NaCl concentration, tissue
necrosis of in vitro plants was observed in Desiree whereas necrosis of tissue in Cardinal was
observed at 140 mM.
5.1.3: Peroxidase (POD) Activity in Potato under NaCl Stress
Tables 5.1 and 5.2 depict the POD activity in in vitro plants of Solanum tuberosum
cvs. Cardinal and Desiree after 60 days under various NaCl stress levels. A slight decrease in
POD activity was observed with an increase in the concentration of NaCl in Cardinal. It is
evident from the data given in Table 5.2 that POD activity in control plants of Cardinal was
1.24 units/ml of enzyme whereas in salt-stressed in vitro plants its value was 1.04, 0.72, 0.45,
0.64, 0.46 or 0.56 units/ml of enzyme at 20, 40, 60, 80, 100 or 120 mM NaCl concentration,
81
respectively. In case of Desiree, POD activity values changed from 5.64 units/ml of enzyme
(control) to 6.76, 7.34, 6.94, 3.94 or 1.06 units/ml of enzyme at 20, 40, 60, 80 or 100 mM
concentration of NaCl (For detailed publication, see Annexure 9B).
82
Table 5.1: Growth parameters and Protein/Peroxidase contents in in vitro plants of
potato (cv. Cardinal) under NaCl stressA
Media composition
Shoot lengthB
(cm)
Root lengthB
(cm)
No. of shootsB
No. of rootsB
No. of nodesB
ProteinsB (mg/g)
PODB (units/ml enzyme)
MS (without NaCl)
11.90 ± 0.11
7.10 ± 0.08
4.80 ± 0.03
12.80 ± 0.04
23.10 ± 0.02
2.51 ± 0.10
1.24 ± 0.13
MS + 20 mM NaCl
11.77 ± 0.02
5.40 ± 0.03
5.30 ± 0.02
7.90 ± 0.01
17.10 ± 0.02
0.76 ± 0.12
1.04 ± 0.23
MS + 40 mM NaCl
8.74 ± 0.03
5.24 ± 0.04
5.70 ± 0.12
5.15 ± 0.01
15.50 ± 0.03
0.79 ± 0.21
0.72 ± 0.17
MS + 60 mM NaCl
4.60 ± 0.041
4.10 ± 0.18
6.20 ± 0.08
4.10 ±0.03
12.50 ± 0.07
0.82 ± 0.13
0.45 ± 0.18
MS + 80 mM NaCl
3.90 ± 0.12
4.81 ± 0.06
7.30 ± 0.12
3.00 ± 0.03
7.40 ± 0.03
0.29 ± 0.01
0.64 ± 0.13
MS+100 mM NaCl
4.03 ± 0.003
4.20 ± 0.02
5.25 ± 0.09
3.00 ± 0.03
15.00 ± 0.04
0.76 ± 0.02
0.46 ± 0.16
MS+120 mM NaCl
Shoot bunch
formation NDC
Shoot bunch
formation NDC NDC 0.19
± 0.14 0.56
± 0.13
MS+140 mM NaCl
Tissue necrosis NDC NDC NDC NDC NDC NDC
AShoot apices (1.0 cm long) were used as an explant source. Unless otherwise mentioned, the
same explants size was used throughout this study. BValues are the means ± S.E. from 30 replicate culture vessels per treatment collected after
60 days of salt treatment. CND: Not determined.
83
Table 5.2: Growth parameters and Protein/Peroxidase contents in in vitro plants of
potato (cv. Desiree) under NaCl stressA
AShoot apices (1.0 cm long) were used as an explant source. Unless otherwise mentioned, the
same explants size was used throughout this study. BValues are the means ± S.E. from 30 replicate culture vessels per treatment collected after
60 days of salt treatment. CND: Not determined.
Media composition
Shoot lengthB
(cm)
Root lengthB
(cm)
No. of shootsB
No. of rootsB
No. of nodesB
ProteinsB (mg/g)
PODB (units/ml enzyme)
MS (without NaCl)
10.70 ± 0.03
7.80 ± 0.02
1.60 ± 0.004
17.20 ± 0.02
19.00 ± 0.13
1.54 ± 0.12
5.64 ± 0.15
MS + 20 mM NaCl
7.20 ± 0.12
7.80 ± 0.10
4.00 ± 0.08
10.20 ± 0.002
12.00 ± 0.015
1.37 ± 0.03
6.76 ± 0.16
MS + 40 mM NaCl
6.60 ± 0.04
4.90 ± 0.02
4.50 ± 0.03
6.95 ± 0.08
8.60 ± 0.18
3.98 ± 0.23
7.34 ± 0.28
MS + 60 mM NaCl
1.70 ± 0.18
4.00 ± 0.08
4.60 ± 0.13
6.10 ± 0.03
9.25 ± 0.02
2.81 ± 0.13
6.94 ± 0.13
MS + 80 mM NaCl
2.08 ± 0.018 NDC 7.20
± 0.02 3.00
± 0.13 4.00
± 0.03 5.21
± 0.16 3.94
± 0.11
MS+100 mM NaCl
Shoot bunch
formation NDC
Shoot bunch
formation NDC NDC 4.03
± 0.15 1.06
± 0.03
MS+120 mM NaCl
Tissue necrosis NDC NDC NDC NDC NDC NDC
MS+140 mM NaCl
Tissue necrosis NDC NDC NDC NDC NDC NDC
84
Fig. 5.1-5.8: In vitro-raised plants of potato cvs. Cardinal/Desiree at different
concentrations of NaCl supplemented to MS medium after 60 days of culture
Fig. 5.2: Stunted growth in Cardinal plants leading to rosette formation (arrow) on MS medium containing 100 mM NaCl (0.5x).
Fig. 5.1: In vitro-raised Cardinal plants on MS medium containing 80 mM NaCl (0.8x).
Fig. 5.3: Bunchy appearance (arrows) of Cardinal shoots on MS medium containing 120 mM NaCl (1x).
Fig. 5.4: Tissue necrosis (arrows) of Cardinal plants at 140 mM NaCl (1x).
85
Fig. 5.5: In vitro-raised potato plants of Desiree showing stunted growth on MS medium containing 80 mM NaCl (0.8x).
Fig. 5.6: Shoot bunch (rosette; arrows) without the root formation in Desiree on MS medium containing 100 mM NaCl (1x).
Fig. 5.7: Tissue necrosis (arrows) in Desiree plants on MS medium containing 120 mM NaCl (1x).
Fig. 5.8: Tissue necrosis (arrows) in Desiree plants on MS medium containing 140 mM NaCl at day 60 (1x).
86
5.2: Effect of Different Concentrations of NaCl on Callus Proliferation in
Solanum tuberosum L. (cvs. Cardinal and Desiree)
5.2.1: Callus Proliferation Response of cv. Cardinal under NaCl Stress
It is evident from the data given in Table 5.3 that different treatments of NaCl
affected callus growth and proliferation. At 20 mM NaCl, 3.92% decrease in fresh weight of
callus was observed. By increasing the concentration of salt in the media, a gradual decrease
in fresh weight of callus cultures was observed. At 40, 60, 80, 100, 120 or 140 mM
concentration, the corresponding decrease in fresh weight of callus cultures was more, i.e.,
11.76, 26.41, 37.25, 42.59, 47.16 or 48.15%, respectively. Similarly, salt stress also affected
the proliferation response of callus. It was observed that the proliferation response up to 20
mM NaCl level was ‘good’. By increasing the concentration of salt, the proliferation
response gradually declined. It was also observed that at higher concentration of NaCl in
optimized callus induction medium, i.e., 120 or 140 mM, the callus became brownish and
hence fell in the category of ‘poor’ proliferation response as standardized in this study
(details given in Materials and Methods section).
5.2.2: Callus Proliferation Response of cv. Desiree under NaCl Stress
As in cv. Cardinal, by increasing the concentration of NaCl (20-140 mM) in the
medium, fresh weight of callus cultures decreased correspondingly (Table 5.4). However, the
decrease in fresh weight of callus in Desiree was only of the order of 1.4% at 20 mM NaCl.
The proliferation response was also ‘good’ at this concentration. At 40 and 60 mM salt level,
this decrease in fresh weight of callus was 5.50 and 14.50% respectively and hence regarded
as ‘good’ proliferation response in accordance with the standardized callus proliferation
87
parameters of this study. By further increasing the concentration of salt in the medium, fresh
weight of callus cultures decreased to 16.17, 20.58, 26.08 or 30.88% at 80, 100, 120 or 140
mM NaCl respectively. Since the maximum percent value for the decrease in fresh weight of
callus cultures was 36.00% at 140 mM, the proliferation response though limited to a
reasonable extent fell under the ‘satisfactory’ category.
88
Table 5.3: Effect of different concentrations of NaCl on callus proliferation response in
Solanum tuberosum L. cv. Cardinal
Media composition
Fresh weight of callus at the
time of salt treatment (g)*
Fresh weight of callus after 30 days of salt treatment (g) *
Increase/ decrease in fresh wt. (g) **
Increase/ decrease in fresh
wt. (%) **
Callus proliferation response***
C4**** 0.54 ± 0.02
0.63 ± 0.02
(+) 0.09 ± 0.008 (+) 16.66 + + +
C4 + 20 mM NaCl
0.51 ± 0.03
0.49 ± 0.02
(-) 0.02 ± 0.003 (-) 3.92 + + +
C4 + 40 mM NaCl
0.51 ± 0.01
0.45 ± 0.03
(-) 0.06 ± 0.009 (-) 11.76 + + +
C4 + 60 mM NaCl
0.53 ± 0.02
0.39 ± 0.04
(-) 0.14 ± 0.008 (-) 26.41 + +
C4 + 80 mM NaCl
0.51 ± 0.05
0.32 ± 0.01
(-) 0.19 ± 0.013 (-) 37.25 + +
C4 + 100 mM NaCl
0.54 ± 0.02
0.31 ± 0.01
(-) 0.23 ± 0.012 (-) 42.59 +
C4 + 120 mM NaCl
0.53 ± 0.03
0.28 ± 0.01
(-) 0.25 ± 0.05 (-) 47.16 +
C4 + 140 mM NaCl
0.54 ± 0.02
0.28 ± 0.01
(-) 0.26 ± 0.01 (-) 48.15 +
*Results are means ± S.E. from 30 replicate cultures. **The + and the – signs represent increase or decrease in fresh weight in comparison with the
initial fresh weight in the respective treatment (second column) at day 30. ***Proliferation response: Good (+ + +), Satisfactory (+ +), Poor (+). ****C4: Optimized callus induction medium (MS + 18.09 µM 2, 4-D).
89
Table 5.4: Effect of different concentrations of NaCl on callus proliferation response in
Solanum tuberosum L. cv. Desiree
*Results are means ± S.E. from 30 replicate cultures.
**The + and the – signs represent increase or decrease in fresh weight in comparison with the
initial fresh weight in the respective treatment (second column) at day 30. ***Proliferation response: Good (+ + +), Satisfactory (+ +). ****C4: Optimized callus induction medium (MS + 18.09 µM 2, 4-D).
Media composition
Fresh weight of callus at the time of salt
treatment (g)*
Fresh weight of callus after 30 days of salt treatment (g) *
Increase/ decrease in fresh wt. (g) **
Increase/ decrease in fresh
wt. (%) **
Callus proliferation response***
C4**** 0.71 ± 0.02
0.79 ± 0.03
(+) 0.08 ± 0.014 (+) 11.26 + + +
C4 + 20 mM NaCl
0.71 ± 0.04
0.70 ± 0.02
(-) 0.01 ± 0.002 (-) 1.40 + + +
C4 + 40 mM NaCl
0.72 ± 0.02
0.68 ± 0.03
(-) 0.04 ± 0.009 (-) 5.50 + + +
C4 + 60 mM NaCl
0.69 ± 0.03
0.59 ± 0.03
(-) 0.10 ± 0.025 (-) 14.50 + + +
C4 + 80 mM NaCl
0.68 ± 0.04
0.57 ± 0.02
(-) 0.11 ± 0.015 (-) 16.17 + ++
C4 + 100 mM NaCl
0.68 ± 0.01
0.54 ± 0.02
(-) 0.14 ± 0.020 (-) 20.58 + +
C4 + 120 mM NaCl
0.69 ± 0.01
0.51 ± 0.02
(-) 0.18 ± 0.005 (-) 26.08 ++
C4 + 140 mM NaCl
0.68 ± 0.02
0.47 ± 0.03
(-) 0.21 ± 0.005 (-) 30.88 ++
90
5.3: Callus Morphology of Potato, Relative Fresh Weight Growth and
Selection of Sub-lethal Salt Concentration and Subsequent Maintenance on
Respective Salt Concentration for Six Sub-cultures for Recurrent Selection
Table 5.5 depicts that there was a significant difference with reference to percent
relative fresh weight growth (PRFWG) and callus morphology between different
concentrations of NaCl in callus cultures of both the cultivars. At 0 mM NaCl concentration,
callus cultures from both the cultivars were off-white and granular having efficient
proliferation response (Fig 5.9 A & B). Salt-treated callus cultures showed maximum
PRFWG (72 and 87%) at 20 mM NaCl in Cardinal and Desiree, respectively. As the
concentration of salt was increased in the medium, PRFWG decreased correspondingly and
off-white, green callus cultures turned yellow to brown (Fig. 5.10, 11, 12, 13 A & B). At 100
mM NaCl, relative fresh weight growth was decreased to 54 and 57% in Cardinal and
Desiree, respectively and the morphology of callus cultures changed to greenish-yellow in
both the cultivars (Fig. 5.14 A & B). Callus cultures were completely necrotic above 100
mM NaCl. It was also observed that callus cultures of Desiree had comparatively better
PRFWG as compared to Cardinal at all the tested salt levels. Color of callus cultures in both
the cultivars changed to blackish-brown at 120 mM salt level (Fig. 5.15 A & B). Thus 100
mM NaCl concentration was identified as sub-lethal because above this salt concentration,
calluses turned completely necrotic in both the cultivars. Moreover, calluses were sub-
cultured and maintained on this concentration of salt for 6 sub-cultures (4 months). Recurrent
selection was done by transferring the calluses to NaCl-free basal medium for two successive
subcultures, then returned to their respective MS basal (C4) medium plus NaCl. The callus
cultures that survived and resumed growth for at least two further subcultures were picked
and inoculated on optimized regeneration medium.
91
Table 5.5: Effect of different NaCl levels (0-140 mM) supplemented to optimized callus
proliferation medium* on relative fresh weight growth and callus morphology of potato
(cvs. Cardinal and Desiree) at day 90
Optimized callus proliferation
medium (C4)* + NaCl
concentrations (mM)
Relative fresh weight growth
(%) Callus Morphology***
Car** Ds** Car Ds
C4 + 0 100 100 Off-white with
yellowish portions, granular
Off-white with green portions,
friable
C4 + 20 72 87 Greenish-
yellow, friable Off-white with
yellow portions, friable
C4 + 40 70 89 Greenish with brown patches,
granular
Off-white yellow, granular
C4 + 60 66 73 Greenish with brown patches,
friable
Off-white with brown portions,
translucent
C4 + 80 62 63 Off-white with brown portions,
granular
Off-white with brown portions,
granular
C4 + 100 54 57 Greenish with brown patches,
granular
Greenish- yellow, friable,
granular C4 + 120 43 46 Blackish-brown,
necrotic Blackish, necrotic
C4 + 140 30 37 Necrotic Necrotic
*C4: Optimized callus proliferation medium (MS + 18.09 µM 2, 4-D).
** Car: Cardinal, Ds: Desiree.
***Callus morphology is based on 30 culture vessels per NaCl treatment at day 90 of initial
culturing.
92
Fig. 5.9-5.15: Callus morphology of potato cvs. Cardinal and Desiree at different
concentration of salt at day 90
Fig. 5.9: Off-white callus cultures of potato, cvs. Cardinal (A) and off-
white with green portions in cv. Desiree (B) at 0 mM NaCl (1.4x).
Fig. 5.10: Greenish-yellow callus cultures of potato cv. Cardinal (A,
1.2x) and off-white yellow ones of cv. Desiree (B) at 20 mM NaCl
(1.4x).
Fig. 5.11: Green callus cultures with brown patches of cv. Cardinal (A,
1.2x) and off-white, yellow calluses of cv. Desiree (B) at 40 mM NaCl
(1.4x).
A B
B A
A B
93
Fig. 5.12: Greenish, friable callus cultures of cv. Cardinal (A, 1.4x)
and off-white brown, translucent calluses of cv. Desiree (B) at 60
mM NaCl (1.2x).
Fig. 5.13: Callus cultures of cv. Cardinal (A, 1x) and cv. Desiree (B)
at 80 mM NaCl (1.4x).
Fig. 5.14: Green granular calluses of cv. Cardinal with brown
patches, (A, arrows) and cv. Desiree (B) at 100 mM NaCl (1.4x).
A B
A B
B A
94
Fig. 5.15: Necrotic, blackish-brown callus cultures of cv. Cardinal
(A) and cv. Desiree (B) at 120 mM NaCl (1.2x).
A B
95
5.4: Regeneration Potential of Potato (cvs. Cardinal and Desiree) at
Different Concentrations (0-140 mM) of NaCl
The callus cultures after two subcultures on optimized callus proliferation medium
supplemented with various salt concentrations were shifted to regeneration medium to study
their regeneration potential. Regeneration of non-stressed callus cultures of both the cultivars
of potato was highest (50 and 55% in Desiree and Cardinal, respectively). However, the
regeneration potential of salt-treated or stressed callus cultures decreased correspondingly
with an increase in salt concentration. Regeneration frequency was 7 and 12% in Desiree
and, Cardinal respectively at 60 mM NaCl. Plant regeneration was completely inhibited at
concentration higher than 60 in Desiree and 80 mM NaCl in Cardinal (Fig. 5.16; Annexure
9C as detailed published data).
Fig. 5.16: Regeneration potential of potato cvs. Cardinal and Desiree at different
concentrations of NaCl
Results are means from 30 replicate cultures.
Vertical bars indicate the standard error.
96
5.5: Regeneration of Callus Cultures after Recurrent Selection on Salt-free
Regeneration Medium
The callus cultures of both the cultivars after recurrent selection were transferred to
salt-free optimized callus regeneration medium. It was observed that regeneration potential
of callus cultures without salt was better as compared to NaCl-treated (100 mM) ones. Shoot
formation via organogenesis was observed in both the cultivars. Shoot initiation was noticed
one day earlier in non-treated callus cultures as compared to 100 mM salt-treated callus
cultures in both the cultivars. The difference in number of shoots and nodes between treated
and non-treated calluses was less sharp in both the potato cultivars. The number of shoots in
Cardinal was 9 and 10 and in Desiree 10 and 12 in treated and non-treated callus cultures
respectively. The number of nodes varied from 16 and 17 in Cardinal and 13 and 14 in
Desiree in treated and non-treated callus cultures (Fig. 5.17-5.18). The number of shoots per
culture vessel was high in Desiree as compared to Cardinal. In general, the regeneration
response was more pronounced in Desiree as compared to Cardinal. The overall vigor of
regenerated plants from salt-treated callus cultures in both the cultivars was considerably
lower in comparison with the control. Fig. 5.19-5.22 depict the events of regeneration in
control and salt-treated callus cultures.
97
Fig. 5.17: Regeneration response of salt-tolerant callus cultures of potato (cv. Cardinal)
on optimized regeneration medium at day 60
Values are means from 30 replicate cultures at day 60.
Vertical bars indicate the standard error.
Fig. 5.18: Regeneration response of salt-tolerant callus cultures of potato (cv. Desiree)
on optimized regeneration medium at day 60
Values are means from 30 replicate cultures at day 60.
Vertical bars indicate the standard error.
98
Fig. 5.19-5.20: Some selected photographs showing regeneration potential of potato
callus cultures at day 60 of transference to optimized regeneration medium after
recurrent selection
Fig. 5.19: Regeneration of callus cultures without NaCl treatment in
Cardinal (A) and Desiree (B; both at 1.6x).
Fig. 5.20: Regeneration of callus cultures after 100 mM NaCl treatment of
(A) cv. Cardinal and cv. Desiree (B; both at 1.6x).
A
B
B
A
99
Fig. 5.21-5.22: Some selected photographs showing regeneration potential of potato
plants at day 120 of transfer of calluses to optimized regeneration medium after
recurrent selection
Fig. 5.21: (A) Regeneration of Cardinal callus cultures without NaCl
(control) and Desiree (B; 1.3x).
Fig. 5.22: (A) Regeneration of Cardinal callus cultures after 100 mM NaCl
treatment and Desiree (B; 1.2x).
A B
A B
100
5.6: Assessment of Stability of Acquired Salt Tolerance in Potato Plants in
Greenhouse
To check the stability of acquired salt tolerance of recurrently-selected plants of both
the cultivars, acclimatization was first carried-out in greenhouse and acclimatized plants
were then subjected to salinity stress. A comparison of growth and biochemical features of
control and treated plants is given in Table 5.6. It was observed that number of tubers, fresh
and dry weights were not much different in salt-treated plants as compared to plants without
any salt treatment (control). The tuber numbers as well as fresh/dry weights of salt-treated
Desiree plants were better as compared to Cardinal. Similarly, protein, POD, CAT and SOD
activity also showed an increasing trend in salt-treated plants from both the cultivars.
101
Table 5.6: Growth and biochemical analysis of control and salt-treated plants of potato
cvs. Cardinal and Desiree
Medium Parameter Cultivar*
Car DS
Control
Number of tubers 9.00 ± 1.550 11.00 ± 0.250
Fresh weight of tubers (g) 20.00 ± 0.751 21.00 ± 1.250
Dry weight of tubers (g) 4.68 ± 0.225 4.73 ± 0.225
Protein (mg/g) 3.35 ± 0.750 3.14 ± 0.157
POD activity (units/ml enzyme) 2.09 ± 0.553 1.51 ± 0.250
CAT activity (units/ml enzyme) 5.02 ± 0.625 4.29 ± 0.357
SOD activity (units/mg protein) 9.24 ± 0.205 9.20 ± 0.265
100 mM
NaCl
Number of tubers 10.00 ± 0.252 12.00 ± 1.250
Fresh weight of tubers (g) 24.00 ± 0.115 25.00 ± 0.951
Dry weight of tubers (g) 5.22 ± 0.345 5.50 ± 0.635
Protein (mg/g) 3.98 ± 0.259 3.46 ± 0.346
POD activity (units/ml enzyme) 2.39 ± 0.273 1.93 ± 0.256
CAT activity (units/ml enzyme) 5.80 ± 0.829 5.51 ± 0.252
SOD activity (units/mg protein) 9.82 ± 0.215 9.44 ± 0.285
*Car: cv. Cardinal DS: cv. Desiree.
Data are means ± S.E. from 30 replicate cultures at day 30 of salt treatment.
102
DISCUSSION
Stunted growth of plants is an immediate response to salt stress which apart from
other reasons is likely due to reduction in the rate of leaf surface expansion (Hernandez et al.,
1995). It was observed in this study that different in vitro growth parameters, i.e., shoot/root
length and numbers of roots decreased while number of shoots increased with an increase in
NaCl concentration. The increase in number of shoots though was not in a positive sense as it
resulted in shoot bunch formation or a rosette-type growth pattern. In Desiree, rosette-type of
shoot development initiated at 100 mM whereas in Cardinal it was evident at 120 mM NaCl
level. Higher salt treatments (above 120 mM) caused tissue necrosis or rosette formation in
both the cultivars. These results are in agreement with several previous studies on potato
plants. Potluri and Devi-Prasad (1993) reported similar pattern of in vitro growth in potato
under 0.4 - 0.6% (68.37-102.56 mM) NaCl stress. Similarly, Martinez et al. (1996) reported
a severe growth reduction of Andeen potato cultures at higher NaCl levels (100 to 200 mM).
This decrease in growth at higher salt concentration was also observed by Farhatullah et al.
(2002) in potato (cv. Cardinal). Shaterian et al. (2005) also reported that growth of potato
plants decreased progressively with an increase in salt concentrations. The formation of
shoot bunch might have been due to a severe effect of salt stress on cell division and
elongation (Wang and Nil, 2000).
Short-term effect of different concentrations of NaCl on callus cultures of potato was
also analyzed during this investigation. Results have shown a decrease in fresh weight of
callus cultures of both the cultivars when subjected to increasing concentration of salt in the
medium. These results are also in line with Ochatt et al. (1999). They reported the reduction
in callus growth at higher NaCl concentrations in Solanum tuberosum L. Liu and Staden
103
(1999) also observed similar decrease in the fresh weight of callus tissue within 28 days and
100 mM salt concentration completely inhibited callus growth. Farhatullah et al. (2002) also
reported that NaCl damaged cells and restricted the growth activities in potato at higher salt
levels. During the selection of salt-tolerant cell lines in potato, Queiros et al. (2007) had also
reported decrease in fresh weight. This reduction of callus growth at high salinity stress was
also linked to lesser absorption of water, ionic imbalance and induction of oxidative stress
(Hasegawa et al., 2000; Errabii et al., 2007). In the present investigation, the reason of better
tolerance of Cardinal as compared to Desiree might be due to its inherent tolerance level.
This is apparently achieved by several mechanisms that may include regulation of K+, Na+
and Cl- uptake across the plasma membrane and/or compartmentalization of Na+ and Cl- in
the vacuole (Greenway and Munns, 1980; Jeschke, 1984; Binzel et al., 1985; Parida and Das,
2005).
The concept of in vitro selection is to exploit the genetic variation known to occur in
plants by screening cell cultures for resistance to disease, insects, herbicide or any abiotic
stress. The procedure of in vitro selection typically involves subjecting cells in cultures to a
suitable selection pressure and recovering any variant cell line/s that is/are resistant to that
particular stress. These variant lines are then used to regenerate whole plants. During this
investigation, a direct recurrent selection procedure was employed to select salt-tolerant cell
lines in potato cvs. Cardinal and Desiree. Results have shown more than 50% reduction in
relative fresh weight in both the cultivars above 100 mM NaCl. Callus morphology
correspondingly changed from off-white to blackish-brown above 100 mM to acutely-
necrotic at 140 mM NaCl. This decrease in growth of callus cultures at higher salt
concentration in potato is considered as a common phenomenon (Benavides et al., 2000;
104
Sotiropoulos et al., 2006; Queiros et al., 2007). Perhaps not surprising that such type of
growth reduction was also observed in other plant species, e.g., Cicer arietinum (Pandey and
Ganapathy, 1984), sugarcane (Gandonou et al., 2005), Chrysanthemum morifolium Ramat
(Hossain et al., 2007) and Jatropha curcas (Kumar et al., 2008). Thus under stress
conditions, one of the strategies that higher plants in general have probably adopted is to
slow down their growth and metabolism (Zhu, 2001). One other possibility is to better utilize
and manage the available resources under nutritional imbalance, osmotic and metabolic
disturbances. This reduction in growth not only helps the plants to save the energy for
defense purpose but also limits the risk of heritable damage (May et al., 1998). Change in
callus morphology (brownish to black) at higher salt concentrations may directly be linked to
cell death at higher salt concentrations.
In this study, regeneration of plants from salt-stressed callus cultures of both the
potato cultivars was highest on salt-free (control) medium. On the other hand, regeneration
potential was completely inhibited at concentrations higher than 60 in Desiree and 80 mM
NaCl in Cardinal. It has been observed previously by many workers that the presence of salt
in the medium generally reduced or even completely inhibited the plant regeneration
(Vajrabhaya et al., 1989; Subhashini and Reddy, 1989; Lutts et al., 1999). These results are
also corroborating previous studies of El-Enany (1997) and Hassanein, (2004) on
regeneration of tomato under salt stress in which they obtained relatively lesser regeneration
response in salt-treated calluses. This phenomenon is justified partly due to the loss of
regeneration potential during the long periods required for selection (Nabors, 1990) or the
presence of high concentrations of NaCl in the regeneration medium (Shankhdhar et al.,
2000). In the present study, it has been observed that regeneration potential of recurrently-
105
selected callus cultures (100 mM NaCl-treated) on salt-free medium was not much different
as compared to the control ones. Regeneration of selected salt-tolerant callus cultures on salt-
free regeneration medium is well documented in literature (Li and Heszky, 1986; Ben-
Hayyim and Goffer, 1989; Jaiswal and Singh, 2001). In contrast to these results several
workers have obtained regeneration of selected salt-tolerant calluses on salt-containing media
(Heszky et al., 1986; Reddy and Vaidyanath, 1986; Beloualy and Bouharrmont, 1992; Ochatt
et al., 1999). Regeneration response was more pronounced in Desiree as compared to
Cardinal. The overall vigor (number of shoot and nodes) of regenerated plants from salt-
treated callus cultures was relatively less in comparison with the control.
When well-acclimatized recurrently-selected plants were treated with 100 mM NaCl
and compared with control plants without any salt treatment to check their acquired salinity
tolerance, it was observed that recurrently-selected plants showed higher fresh/dry weight
and number of tubers as compared to control ones in both the cultivars. Similar growth
behavior in selected salt-tolerant lines of potato was observed by Van-Swaaij et al. (1986),
Ochatt et al. (1999) and Queiros et al. (2007).
Proteins have been suggested as an important molecular marker for the improvement
of salt tolerance using genetic engineering techniques (Pareek et al., 1997). The influence of
different NaCl concentrations on protein contents in plants of two potato cultivars was also
estimated in the present study. A slight decrease in protein contents of in vitro Cardinal
cultures was observed as the concentration of NaCl gradually increased in the media.
However, there was an increase in protein contents in Desiree plants when subjected to
increasing salt concentrations. In case of in vitro recurrently-selected plants, protein contents
were higher as compared to control (non-selected ones) in both the cultivars. These results
106
were in agreement with the reports of other workers. Cano et al. (1998) studied the growth
and physiological responses to salinity of two inter-specific hybrids between the cultivated
tomato (Lycopersicon esculentum Mill.) and its wild salt-tolerant species (Lycopersicon
pennellii) and compared to those of their parents. They concluded that protein contents
increased with salinity in all the genotypes. The findings for cv. Desiree were hence nearly in
line with those of Cano et al. (1998). These salt-inductive proteins were also reported in
potato plants by Rahnama and Ebrahimzadeh (2004). More recently, Queiros et al. (2007)
also observed this increasing trend of soluble and insoluble proteins in potato cultures during
the selection of salt-tolerant cell lines. These higher protein contents might be attributed to
synthesis of stress-induced proteins (Kumar et al., 2008) that may be helpful for maintaining
the osmotic imbalance. Salt-responsive proteins were also suggested to be quite valuable for
further analysis of general cellular adaptive mechanism to abiotic stress. Salt has two
antagonistic effects on protein; firstly they tend to break electrostatic bonds and secondly
increase hydrophobic interactions (Melander and Horvath, 1977; Ashraf and Harris, 2004).
During the germination of peas for instance, salinity lowered the protein and peptide contents
stimulating protein hydrolysis and this hydrolysis was considered a primary effect of the salt
(Uprety and Sarin, 1975). This decrease in protein contents under salinity stress was also
observed in several other studies (Ashraf and Waheed, 1993; Streb and Feierabend, 1996;
Niknam et al., 2006). Interestingly, both possibilities are given in the literature (although
seemingly species-specific) and hence partially justify the variable response of the two tested
cultivars in this study.
It is quite evident from the literature that many plants up-regulate several antioxidant
enzymes (Peroxidase, Catalase, Superoxide dismutase etc) to scavenge reactive oxygen
107
species (ROS) produced in response to salt stress (Mittova et al., 2000; Rahnama et al.,
2003; Ashraf and Harris 2004; Batkova et al., 2008). Peroxidase is an important antioxidant
enzyme, implicated in several metabolic functions such as cell wall formation, cell
elongation and detoxifies ROS in plants under stress (Lagrimini et al., 1990; Biggs and Fry,
1997; Rahnama et al., 2003). The peroxidase activity as observed in this study exhibited a
slightly decreasing trend in Cardinal though an increasing one in Desiree with an increasing
NaCl level in the medium. This contrasting behavior of POD activity may be corroborated
with the better tolerance of Cardinal to salt as compared to Desiree. In the present
investigation, recurrently-selected plants had higher POD, CAT and SOD activities as
compared to the control ones in both the cultivars. The above-mentioned antioxidant
enzymes play a necessary role in detoxification of ROS produced under stressful conditions
(Hernandez et al., 2000; Rahnama et al., 2003). Quite recently, Kumar et al. (2008) reported
that SOD activity increased in salt-treated callus cultures of Jatropha curcas as compared to
non-treated controls. Similarly, an increase in SOD activity was also reported by
Sreenivasulu et al. (2000) and Cherian and Reddy (2003). SOD normally converts more
toxic O2●- radicals to less toxic H2O2 (Scandalios, 1993) and to neutralize H2O2 other
enzymes such as peroxidase and catalase are produced (Dionisiosese and Tobita, 1998). So
the increase in peroxidase and catalase activity in this study seems to be in agreement with
such previous reports on these enzyme behavior. It is, therefore perhaps safer to infer from
the results of this investigation that the increase in growth and biochemical parameters were
corresponding to a shifting behavior of plants from being sensitive-to-relatively-more-
tolerant-ones.
108
In conclusion, higher levels of NaCl in this investigation severely suppressed the
growth of both the in vitro plants as well as callus cultures of the two tested cultivars of
potato. The results from this study also highlighted a strong possibility for the selection of
salt-tolerant cell lines of potato followed by efficient plant regeneration. The results from this
work in the light of contemporary literature indicated a probable genetic modification at
cellular level resulting in an acquisition of salt tolerance that was also evident in enhanced
biochemical activity of proteins and antioxidant enzymes. Although a potential NaCl-tolerant
cell line was selected and maintained during the present work but apparently lot of work
regarding biochemical and physiological aspects of salinity tolerance still remains elusive
and deserves further experimentation not only under in vitro but also in greenhouse and/or
field conditions to draw meaningful conclusions.
109
Chapter 6A
Role of Ascorbic Acid in Amelioration of Salt Tolerance in Potato (cvs.
Cardinal and Desiree)
RESULTS
6.1: Effect of Ascorbic Acid Pretreatment to In vitro Salinized Plants and
Callus Cultures of two Cultivars of Solanum tuberosum L. cvs. Cardinal
and Desiree
Work regarding this aspect is attached as Annexure 9D (Sajid and Aftab, 2009).
6.2: Regeneration Potential of Ascorbic Acid-pretreated and Non-
pretreated Callus Cultures at Different Concentration of NaCl
To determine regeneration potential, ascorbic acid-pretreated and non-pretreated
callus cultures after two subcultures on MS medium supplemented with various salt
concentrations after 60 days were shifted to the regeneration medium (MS basal medium
supplemented with 2.64 µM NAA and 1.0 µM TDZ). It was observed that in both the
cultivars of potato, ascorbic acid-pretreated callus cultures had better regeneration potential
as compared to non-pretreated ones. The regeneration potential, however, decreased
correspondingly with an increase in NaCl concentration. At higher than 60 in cv. Desiree and
80 mM NaCl in cv. Cardinal, plant regeneration ceased completely in the non-pretreated
callus cultures. On the other hand, pretreatment with ascorbic acid significantly enhanced the
regeneration potential of calluses of both the cultivars even at higher NaCl concentration of
80 and 100 mM, respectively in Desiree and Cardinal (Fig. 6.1).
110
Fig. 6.1: Regeneration potential of ascorbic acid-pretreated and non-pretreated callus
cultures of Solanum tuberosum cvs. Cardinal and Desiree at day 60 on salt-free
regeneration medium
Data are the means from 30 replicate cultures at each salt treatment.
111
6.3: Amelioration of Salinity Tolerance by Foliar Application of Ascorbic
Acid in Potato cv. Cardinal
Looking at a positive correlation between exogenous applications of ascorbic acid in
in vitro cultures, experiments on whole plant level under normal greenhouse conditions using
foliar sprays were also performed. Foliar spray of 0.5 mM ascorbic acid to salt-treated (120
mM) plants considerably enhanced their growth parameters (fresh/dry weight of tubers,
number of shoots/ length) as compared to salt-treated plants without ascorbic acid
pretreatment (Table 6.1). The mean fresh/dry weight of tubers (17.59 and 3.10 g) from salt-
treated plants did show an increase to 21.84 and 4.44 g respectively when treated with
ascorbic acid. However, control plants had relatively higher biomass accumulation (fresh/dry
weight of tubers) as compared to salt-treated plants by ascorbic acid application. Shoot
length was also significantly increased from 12.32 to 15.03 cm in control and 12.86 to 14.59
cm in 120 mM NaCl in ascorbic acid-treated plants. Number of shoots was increased in
ascorbic acid-treated plants from 1.5 to 2.4 in control and 2.9 to 3.2 in salinized ones.
Biochemical parameters (protein contents and antioxidant enzymes activity) were also
significantly increased by foliar spray of ascorbic acid showing a positive effect of ascorbic
acid under stress conditions (Table 6.1, Fig. 6.2A-D).
112
Table 6.1: Effect of foliar application of ascorbic acid on growth and biochemical
parameters of potato plants (cv. Cardinal) with or without supplemental NaCl
treatment of potting mix†
Salinity treatment
Ascorbic acid
treatment (0.5 mM)*
Fresh wt. of
Tubers (g)
Dry wt. of
Tubers (g)
Shoot length (cm)
Number of
shoots
Protein contents(mg/g)
POD activity
(units/ml enzyme)
CAT activity
(units/ml enzyme)
SOD activity
(units/mg protein)
Control (without
NaCl)
T 31.54 ± 2.37
5.99 ± 2.30
15.03 ± 4.61
2.40 ± 1.87
4.73 ± 0.91
2.05 ± 2.19
10.18 ± 0.45
22.70 ± 2.85
NT 24.6 ± 2.41
4.69 ± 2.19
12.32 ± 4.34
1.50 ± 2.01
4.17 ± 2.02
1.50 ± 2.11
9.58 ± 2.15
20.50 ± 4.73
120 mM NaCl
T 21.84 ± 1.17
4.44 ± 2.60
14.59 ± 6.78
3.20 ± 1.35
4.70 ± 0.98
1.46 ± 0.98
8.10 ± 1.35
28.09 ± 0.94
NT 17.59 ± 5.80
3.10 ± 1.24
12.86 ± 3.71
2.90 ± 2.50
3.78 ± 0.95
1.12 ± 1.28
6.37 ± 1.23
22.62 ± 5.44
Effect of medium S S NS S NS S S S
Effect of ascorbic acid NS S NS NS NS S S S
Effect of medium and ascorbic acid NS NS NS NS NS NS NS NS
†Clay-loam soil constituted the potting mix.
*With (T) or without (NT) foliar treatment.
Significant (S) or non significant (NS) at P < 0.01 according to F-test.
Data were the means ± S.E. from 15 replicate per treatment.
113
Fig. 6.2 (A-D): A Comparison of growth of ascorbic acid-treated or non-treated salt-
stressed Cardinal plants
Fig. 6.2 B: Control plants without salt and with 60 days foliar treatment of ascorbic acid (0.5 mM).
Fig. 6.2 C: Salt treated-plants without ascorbic acid treatment. Four such plants (one in each pot) are shown.
Fig. 6.2 D: Cardinal plants at day 60 after salt (120 mM) and ascorbic acid (0.5 mM) treatment.
A B
C D
Fig. 6.2 A: Control plants of cv. Cardinal without salt and ascorbic acid treatment.
114
DISCUSSION
Ascorbic acid is a powerful reducing agent found usually in millimolar
concentrations in plants, and is proposed to play an important role in scavenging reactive
oxygen species (O2, H2O2, OH- etc) generated during stress conditions in plants and animals
(Foyer, 1993; Smirnoff, 1995, 2005). It also plays multiple roles in plant growth, such as cell
division, cell enlargement, acting as co-factor for many enzymes and stomatal regulation
(Asada, 1999; Lee and Kader, 2000; Conklin 2001; Barth et al., 2006). Moreover, ascorbic
acid is also considered as an important molecule that regulates the peroxidase activity in
actively dividing cells (Stasolla and Yeung, 2007). It is generally accepted that over-
production of peroxidase under stress conditions leads to the deposition of phenolic
compounds. This higher concentration of phenols affects the architecture of cell wall and
ultimately reduction in cell elongation or cell division (Fry, 1986). Higher concentrations of
ascorbic acid thus might be helpful in enhancing regeneration by regulating the peroxidase
activity under NaCl stress. Looking at these facts, effect of exogenously-applied ascorbic
acid was studied on regeneration potential under various NaCl concentrations in both the
cultivars of potato.
In the present investigation, regeneration potential correspondingly decreased with an
increase in salt level in MS medium. Plant regeneration was completely inhibited above 60
mM NaCl concentration in Desiree and 80 mM in Cardinal. The reduction in regeneration
potential might have been due to the fact that salinity is reported to decrease cell division,
cell elongation and meristematic activity (Rehman et al., 2000; Munns, 2002). On the other
hand, ascorbic acid pretreated salinized-callus cultures showed a better regeneration potential
as compared to non-pretreated ones at all the tested salt levels in both the cultivars. This high
115
regeneration potential in ascorbic acid-pretreated callus cultures may also be associated with
the utilization of ascorbic acid in cellular metabolism (Loewus and Helsper, 1982) that
probably enhanced cell division and differentiation (Liso et al., 1984; Conklin, 2001) or
resulted in reactivation of the apical meristem (Stasolla and Yeung, 2007). Another possible
reason could have been the inhibition of several reactive oxygen species (Padh, 1990) by the
accumulation of antioxidant enzymes as indeed observed during the present investigation.
Similar observations regarding positive effect of ascorbic acid application on plant growth
under salt stress have been observed by several research groups (Shaddad et al., 1990;
Khodary, 2004; Khan et al., 2006; Arafa et al., 2009). The general view-point mentioned by
these workers was that ascorbic acid not only seemed to counteract adverse effects of salinity
on plant growth and development but also probably influenced certain metabolic processes in
plants.
Endogenous ascorbic acid can be increased by exogenous application of ascorbic acid
by foliar spray, as it is readily available to the plants through stomata (Mozafar and Oertli,
1993; Gadallah, 2000; Chen and Gallie, 2004). In view of the results obtained from this
study, the response of potato plants to high level of salinity was reflected by decrease in
tuber fresh/dry weight, shoot length and shoot numbers/plant. However, foliar application of
ascorbic acid promoted these growth parameters in both the control as well as in salt-treated
plants. Consistent findings reported on the beneficial effects of the exogenous application of
ascorbic acid in ameliorating the adverse effects of salt stress can be seen in the
contemporary literature in diverse plant species (Arrigoni et al., 1997; Shalata and Neumann,
2001; Beltagi, 2008). The results from the present study showed an increasing trend in
protein contents though non-significant either in 120 mM NaCl and ascorbic acid-treated
116
plants or the plants treated with ascorbic acid only. An increase in protein profile in response
to the ascorbic acid treatment was also observed by Beltagi (2008). Enhanced protein
contents could be partially linked to an increased activity of antioxidant enzymes against
salinity stress environment. Likewise by foliar application of ascorbic acid, antioxidant
enzyme activities increased significantly in both control as well as in salt-treated potato
plants. Up-regulation in the activity of peroxidase, catalase and superoxide dismutase
indicates that these enzymes are somehow involved in the neutralization process of reactive
oxygen species in potato as well. The results from the present investigation strongly support
Bor et al. (2003), Athar et al. (2008), and Sajid and Aftab (2009) where an efficient
antioxidant system was shown to correlate with salinity tolerance in sugar beet, wheat and
potato. These results are also in agreement with those of Dolatabadian and Jouneghani,
(2009) who reported that major enzymes (peroxidase, catalase and superoxide dismutase)
involved in scavenging reactive oxygen species increase significantly by the application of
ascorbic acid to salt stress bean plants.
In conclusion, this study indicates that salinity is a serious constraint to potato growth
as it alters several morphological and biochemical characteristics. Exogenous application of
ascorbic acid (pretreatment to nodal segments and callus cultures before inoculation/added in
medium) or foliar spray to pot-grown plants has shown to have reduced its effect
substantially in both the cultivars (Cardinal and Desiree). Ascorbic acid perhaps minimized
the oxidative damage by increasing the amount of antioxidant enzymes that in turn was
reflected in better growth parameters in the two tested potato cultivars. The information
gathered from this study necessitates further work both under in vitro as well as green-house
and field conditions to evaluate and harness the potential benefits it holds.
117
Chapter 6B
Role of Salicylic Acid in Amelioration of Salt Tolerance in Potato (cvs.
Cardinal and Desiree)
RESULTS
6.4: Salinity Tolerance and Effect of Salicylic Acid
6.4.1: Effect of Salicylic acid on Growth Characteristics of Salinized Potato
Plants (cvs. Cardinal and Desiree)
In this phase of the present investigation, MS (Murashige and Skoog, 1962) medium
containing 60 mM NaCl was supplemented with different concentrations (0, 0.125, 0.25,
0.50 and 0.75 mM) of salicylic acid (SA) to observe effect on potato growth (cvs. Cardinal
and Desiree) under in vitro conditions. In all the tested salicylic acid treatments, maximum
shoot length (2.39 cm) was observed in medium M3 (MS + 60 mM NaCl + 0.125 mM SA)
followed by M5 (medium containing 0.50 mM SA) where the shoot length was 2.26 cm in
Cardinal. In case of Desiree, maximum shoot length (2.36 cm) was observed in M4 medium
(medium containing 0.25 mM SA). Shoot length was increased at all salicylic acid levels in
comparison with plants containing only NaCl in the medium (M2). The results were
somewhat different in Cardinal, where shoot length was decreased to 1.66 cm and 1.69 cm
(observed in medium M4 and M6, respectively) in comparison with salt-stressed plants
(Table 6.2).
There were significant differences in rooting behavior between the salicylic acid-
treated or non-treated plants of Desiree (Table 6.3). Roots were generally absent in salt
stressed Desiree plants. In salicylic acid-supplemented medium, maximum growth of roots
was observed in M4 (0.25 mM SA) medium. However, this type of behavior was
118
diametrically different in Cardinal where the root length was observed to decrease in
salicylic acid-treated plants as compared to salinized-plants without SA treatment.
Salicylic acid treatment to salinized plants improved the rooting behavior, as an
increase in root number (from 0.90 to 1.20) was observed using M4 medium (0.25 mM SA)
in Cardinal (Table 6.2) and (0 to 1.30) in Desiree (Table 6.3). Salinzied plants showed
bunchy appearance due to the formation of more shoots with shorter internodal distances
(Fig. 6.3 a, b). An application of salicylic acid in the medium resulted in decreased number
of shoots in both the cultivars. The situation was different in case of number of nodes, where
the treatment with SA increased the number of nodes. Maximum number of shoots (2.80 and
2.40) was observed on M2 medium (MS + 60 mM NaCl) in cultivar Cardinal and Desiree,
respectively. Maximum number of nodes (7.10 and 7.50) was observed on M3 and M4
medium respectively in cvs. Cardinal and Desiree. Statistically a non-significant difference
was observed in case of number of shoots (Cardinal) and number of nodes (Desiree) at
different salicylic acid treatments.
Maximum fresh and dry weight was observed on MS medium without NaCl and SA
(M1) as in case of all the other growth parameters. When salicylic acid-treated salinized
plants were compared with the plants treated with salt only, increase in fresh and dry weight
(0.13 and 0.02g, respectively) was observed on M3 medium in Cardinal while the same trend
was observed on M4 and M5 medium in Desiree (Table 6.2 and 6.3). Mean values were
significantly different for fresh weights in both the cultivars and so was the case with the dry
weights in the cultivar Cardinal. The situation was rather different in case of dry weights in
cv. Desiree where the results were non-significant in statistical terms.
119
Table 6.2: Effect of salicylic acid on different growth parameters in Solanum tuberosum
L. cv. Cardinal
*M1 to M6 media designated for MS supplemented with NaCl and salicylic acid given
against each.
Values are mean ± S.E from 30 replicate cultures.
Means followed by the same letter(s) are not significantly different at P ≤ 0.05.
Values are significant (S) or non-significant (NS) at P ≤ 0.05.
Medium* NaCl + SA (mM)
Shoot length (cm)
Number of shoots
Root length (cm)
Number of roots
Number of nodes
Fresh wt. (g)
Dry wt. (g)
M1 0 + 0 9.09 ± 2.09a
1.40 ± 0.45a
6.61 ± 1.61a
6.40 ± 1.77a
14.90 ± 3.31a
0.45 ± 0.12a
0.04 ± 0.010a
M2 60 + 0 1.86 ± 0.70b
2.80 ± 0.87a
3.27 ± 1.71b
0.90 ± 0.43b
5.20 ± 1.60b
0.11 ± 0.03b
0.01 ± 0.003b
M3 60 + 0.125 2.39 ± 0.43b
2.80 ± 0.53a
0.52 ± 0.34b
0.70 ± 0.40b
7.10 ± 0.98b
0.13 ± 0.02b
0.02 ± 0.002b
M4 60 + 0.250 1.66 ± 0.28b
2.70 ± 0.63a
0.92 ± 0.45b
1.20 ± 0.55b
6.50 ± 0.82b
0.10 ± 0.02b
0.01 ± 0.002b
M5 60 + 0.500 2.26 ± 0.36b
2.20 ± 0.36a
1.01 ± 0.43b
0.80 ± 0.39b
6.80 ± 0.93b
0.10 ± 0.02b
0.01 ± 0.002b
M6 60 + 0.750 1.69 ± 0.14b
2.70 ± 0.61a
0.24 ± 0.18b
0.30 ± 0.21b
6.30 ± 0.52b
0.07 ± 0.01b
0.01 ± 0.001b
Significance (P ≤ 0.05) S NS S S S S S
120
Table 6.3: Effect of salicylic acid on different growth parameters in Solanum tuberosum
L. cv. Desiree
*M1 to M6 media designated for MS supplemented with NaCl and salicylic acid given
against each.
Values are mean ± S.E from 30 replicate cultures.
Means followed by the same letter(s) are not significantly different at P ≤ 0.05.
Values are significant (S) or non-significant (NS) at P ≤ 0.05.
Medium*
NaCl + SA (mM)
Shoot length (cm)
Number of shoots
Root length (cm)
Number of roots
Number of nodes
Fresh wt. (g)
Dry wt. (g)
M1 0 + 0 6.03 ± 2.03a
0.90 ± 0.35b
4.97 ± 1.72a
3.60 ± 1.38a
6.60 ± 2.34a
0.40 ± 0.14a
0.03 ± 0.01a
M2 60 + 0 1.26 ± 0.28b
2.40 ± 0.63a
0.00 ± 0.00b
0.00 ± 0.00b
4.50 ± 1.10a
0.10 ± 0.02b
0.01 ± 0.00b
M3 60 + 0.125 1.49 ± 0.35b
0.90 ± 0.28b
0.66 ± 0.45b
0.20 ± 0.13b
3.80 ± 0.88a
0.05 ± 0.01b
0.01 ± 0.00b
M4 60 + 0.250 2.36 ± 0.28b
2.20 ± 0.29a
0.71 ± 0.28b
1.30 ± 0.47b
7.50 ± 0.64a
0.11 ± 0.01b
0.01 ± 0.00b
M5 60 + 0.500 1.68 ± 0.31b
1.60 ± 0.50ab
0.25 ± 0.18b
0.20 ± 0.13b
4.50 ± 0.78a
0.09 ± 0.02b
0.02 ± 0.01ab
M6 60 + 0.750 1.82 ± 0.29b
1.30 ± 0.26ab
0.61 ± 0.56b
0.30 ± 0.15b
5.60 ± 0.82a
0.08 ± 0.01b
0.01 ± 0.00b
Significance (P ≤ 0.05) S S S S NS S NS
121
Fig. 6.3: Comparison of potato shoots (cv. Desiree) at various salicylic acid levels.
Culture vessel at left is CONTROL (0 mM NaCl + 0 mM SA), whereas the rest of the
five culture vessels from left to right are showing a comparison of shoot length at 0,
0.125, 0.25, 0.50 and 0.75 mM SA respectively in MS medium containing 60 mM NaCl
6.3 (a) 6.3 (b)
Fig. 6.3 (a): Bunchy appearance of shoots (arrows) in potato plants
(Desiree) exposed to salt stress (60 mM NaCl, 1.6x).
Fig. 6.3 (b): Bunchy appearance of shoots (arrows) in potato plants
(Cardinal) exposed to salt stress (60 mM NaCl, 2.0x).
122
6.4.2: Effect of Salicylic acid on Protein Contents of the Salinized Cardinal and
Desiree Plants
Protein contents showed generally an increasing trend in salicylic acid-treated
salinized plants as compared to only salt-treated potato plants in both the cultivars.
Maximum protein accumulation (1.17 and 0.88 mg/g) was recorded at 0.75 and 0.50 mM
salicylic acid treatment in cv. Cardinal and Desiree, respectively. Protein contents did not
change at 0.125 and 0.50 mM concentration of SA in cv. Cardinal. In cv. Desiree, protein
contents at 0.50 were maximum and then decreased sharply with further rise in SA
concentration. Mean values were significantly different from each other for protein contents
in both the cultivars (Fig. 6.4 A&B). For protein content, there was a statistically significant
difference between salicylic acid-treated and non-treated potato plants of both the cultivars.
123
Fig. 6.4: Effect of different SA concentrations on protein contents of in vitro-grown
potato plants (cvs. Cardinal and Desiree)
Values are mean (± S.E) from 30 replicate cultures.
Cultivars: Cardinal (A) and Desiree (B).
124
DISCUSSION
The present investigation reports the effect of salicylic acid on different growth and
biochemical features of salt stressed potato plants (cvs. Cardinal and Desiree). Salinity was
found to strongly inhibit the plant growth since high concentrations of NaCl cause ion
imbalance and osmotic stress in many plants (Maggio et al., 2000). These effects may lead to
the development of other types of stresses such as oxidative damage to plants that may be
responsible for reduced plant growth (Zhu, 2001). Similar results were noted in the present
study where application of high concentration of NaCl (60 mM) to in vitro-grown potato
plants adversely affected several of their growth (shoot/root length/number, number of
nodes, fresh and dry weight) as well as biochemical (protein contents) parameters. This
general response to salt stress is also reported for other potato cultivars (Benavides et al.,
2000), as well as for other plant species (Rodriguez et al., 1997; Hernandez et al., 1999;
Rashid et al., 1999).
In the present study, treatment of salt-stressed Cardinal and Desiree plants, with
different concentrations (0.125, 0.25, 0.50 or 0.75 mM) of salicylic acid resulted in increased
growth of both the tested potato cultivars. These results support the previous studies in which
increase in salt tolerance in maize plants was observed by the application of salicylic acid. It
enhanced the growth parameters (fresh, dry weight and length of shoots and roots) in plants
as compared to only salt stressed-plants (Khodary, 2004). Same results have been reported
earlier in case of salt stressed cucumber plants where SA application resulted in higher
values for above-mentioned growth parameters (Yildirim et al., 2008). Increase in shoot and
root growth was observed by El-Tayeb et al. (2006) in case of copper-stressed plants of
Helianthus annus L. which were treated with salicylic acid. These ameliorative effects of
125
salicylic acid on growth of stressed-plants may be due to the fact that SA potentiates the
generation of reactive oxygen species and increases the production of H2O2 in plants that in
turn reduce the oxidative damage under saline stress, as described, for example, in case of
wheat (Wahid et al., 2007).
In this study, it was observed that as the concentration of salicylic acid was increased
in the medium from 0.25 mM, it decreased the growth of both the cultivars. This might be
due to the toxic effects of salicylic acid at higher concentrations. Previously, adverse effects
of high SA concentrations (above 1.0 mM) were observed on bean and tomato plants when
grown in high and low temperature stresses (Senaratna et al., 2000).
During this study, it was observed that the two tested cultivars of potato in terms of
biochemical and growth parameters responded differently to different salicylic acid
treatments. As in case of Cardinal, where applications of salicylic acid (0.125-0.175 mM)
resulted in reduction of root length as compared to salt stressed plants without salicylic acid
treatment. However in Desiree, SA application showed a positive effect on root length
(highest at 0.250 mM). This behavior of rooting in Cardinal seems to be due to the fact that
different cultivars of the same species behave differently to different chemicals and mode of
their application in in vitro conditions. Horvath et al. (2007b) has previously reported that
salicylic acid pre-treatment decreased the drought tolerance of one wheat cultivar (Chinese
spring) while increased in another (Cheyenne).
The literature reveals that SA induces the abiotic stress tolerance in plants by
regulating the expression of certain receptor protein kinases (RPKs). These protein kinases
have been found to initiate response to specific stress-signals, as described, for example, after
wounding in Brassica oleracea (Pastuglia et al., 1997) or in peaches (Bassett et al., 2005). In
126
the present investigation, protein contents showed an increasing trend in both potato cultivars
as compared to plants given only salinity stress. This increase in protein contents was more
in cv. Cardinal as compared to cv. Desiree. The accumulation of protein in SA-treated plants
is rather well documented in literature (Mc-Cue et al., 2000; Kang et al., 2003; El-Tayeb et
al., 2006). This increase in protein contents by salicylic acid application was also previously
reported in heat-stressed plants (Cronje and Bornman, 1999).
Overall, exogenously-applied SA enhanced the growth of both the cultivars of potato.
This improvement in growth behavior might be due to the ameliorating effect of salicylic
acid since it promotes seed germination, enhances the uptake of water and also acts as
signaling molecule under salt stress. It can be interpreted from the results that SA application
with high concentrations did not confer much tolerance to NaCl stress in potato cultivars in
comparison to moderate SA concentrations, especially 0.125 mM and 0.25 mM proved very
effective in enhancing growth in Cardinal and Desiree, respectively. These results hint at a
possibility that moderate concentrations of salicylic acid may, in future, be helpful in
improving yield of plants under saline conditions.
127
Chapter 7
LITERATURE CITED
Abd-Elaleem, K.G., Moawi, R.S., Khalafalla, M.M. (2009). Effect of plant growth regulators
on callus induction and plant regeneration in tuber segment culture of potato
(Solanum tuberosum L.) cultivars. Diamant. African Journal of Biotechnology 8:
2529-2534.
Aburkhes, M., Fahmi, N., Benhemida, A., Nafali, M. and Zeiglem, A. (1984). Virus-free
potatoes by tissue culture in Libya. Acta Horticulturae 289: 77-79.
Aftab, F. and Iqbal, J. (1999). Plant regeneration from protoplasts derived from cell
suspension of adventive somatic embryos in sugarcane (Saccharum sp. hybrid cv.
CoL-54 and cv. CP-43/33). Plant Cell, Tissue and Organ Culture 56: 155-162.
Aftab, F., Mansouri, K. and Preece, J.E. (2005). The influence of environment, media, and
zerotol on forcing and in vitro establishment of softwood shoots from large stem
segments of Acer saccharinum L. and Fraxinus pennsylvanica marsh. Propagation of
Ornamental Plants 3: 111-116.
Aftab, F., Zafar, Y., Malik, K.A. and Iqbal, J. (1996). Plant regeneration from embryogenic
cell suspensions and protoplasts in sugarcane (Saccharum spp. hybrid cv. CoL-54).
Plant Cell, Tissue and Organ Culture 44: 71-78.
Afzal, I., Basra, S.M.A., Ahmad, N. and Farooq, M. (2005). Optimization of hormonal
priming techniques for alleviation of salinity stress in wheat (Triticum aestivum L.).
Caderno de Pesquisa serie Biologia 17: 95-109.
Agarwal, B., Singh, U. and Maitreyi, B. (1992). In vitro clonal propagation of tea (Camellia
sisnensis L.). Plant Cell, Tissue and Organ Culture 30: 1-5.
Agarwal, S. and Pandey, V. (2004). Antioxidant enzyme responses to NaCl stress in Cassia
angustifolia. Biologia Plantarum 48: 555-560.
128
Agastian, P., Kingsley, S.J. and Vivekanandan, M. (2000). Effect of salinity on
photosynthesis and biochemical characteristics in mulberry genotypes.
Photosynthesis 38: 287-290.
Aghaei, K., Ehsanpour, A.K., Balali, G. and Mostajeran A. (2008). In vitro screening of
potato (Solanum tuberosum L.) cultivars for salt tolerance using physiological
parameters and RAPD analysis. American-Eurasian Journal of Agriculture and
Environmental Sciences 3: 159-164.
Ahloowalia, B.S. (1982). Plant regeneration from callus culture in potato. Euphytica 31: 755-
759.
Alamgir, A.N.M. and Ali, M.Y. (1999). Effect of salinity on leaf pigments, sugar and protein
concentrations and chloroplast ATPase activity of rice (Oryza sativa L.). Bangladesh
Journal of Botany 28: 145-149.
Al-Hakimi, A.M.A. and Hamada, A.M. (2001). Counteraction of salinity stress on wheat
plants by grain soaking in ascorbic acid, thiamine or sodium salicylate. Biologia
Plantarum 44: 253-261.
Allakhverdiev, S.I., Sakamoto, A., Nishiyama, Y., Inaba, M. and Murata, N. (2000).
Inactivation of photosystem I and II in response to osmotic stress in synechcoccus,
contribution of water channels. Plant Physiology 122: 1201-1208.
Alscher, R.G., Donahue, J.L. and Cramer, L.L. (1997). Reactive oxygen species and
antioxidants relationships in green cells. Physiologia Plantarum 100: 224-233.
Alvarez, I., Tomaro, L.M. and Benavides, P.M. (2003). Changes in polyamine, proline and
ethylene in sunflower calluses treated with NaCl. Plant Cell, Tissue and Organ
Culture 74: 51-59.
Al-Wareh, H., Trolinder, N.L. and Goodin. J.R. (1989). Callus initiation, shoot regeneration
and micropropagation of three potato cultivars. Horticultural Science 24: 680-682.
129
Anjum, M.A. and Ali, H. (2004). Effect of culture medium on shoot initiation from calluses
of different origin in potato (Solanum tuberosum L.). Biotechnology 3: 194-199.
Arafa, A.A., Khafagy, M.A. and El-Banna, M.F. (2009). The effect of glycine or ascorbic
acid on grain germination and leaf structure of sorghum plants grown under salinity
stress. Australian Journal of Crop Sciences 3: 294-304.
Arfan, M., Athar, H.U.R. and Ashraf, M. (2007). Does exogenous application of salicylic
acid through the rooting medium modulate growth and photosynthetic capacity in two
differently adapted spring wheat cultivars under salt stress? Journal of Plant
Physiology 6: 685-694.
Arrigoni, O. (1994). Ascorbate system in plant development. Journal of Bioenergetics and
Biomembranes 26: 407-419.
Arrigoni, O., Calabrese, G., De-Gara, L., Bitonti, M.B. and Liso, R. (1997). Correlation
between changes in ascorbate and growth of Lupinus albus seedlings. Journal of Plant
Physiology 150: 302-338.
Asada, K. (1999). The water-water cycle in chloroplast: scavenging of active oxygen and
dissipation of excess photons. Annual Review Plant Physiology and Plant Molecular
Biology 50: 601-639.
Ashraf, M and Foolad, M.R. (2007). Roles of glycine betain and proline in improving plant
abiotic stress resistance. Environmental and Experimental Botany 59: 206-216.
Ashraf, M. (1994). Breeding for salinity tolerance in plants. Critical Review in Plant
Sciences 13: 17-42.
Ashraf, M. (2009). Biotechnological approach of improving plant salt tolerance using
antioxidants as markers. Biotechnological Advances 27: 84-97.
Ashraf, M. and Ali, Q. (2008). Relative membrane permeability and activities of some
antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica
napus L.). Environmental and Experimental Botany 63: 266-273.
130
Ashraf, M. and Harris, P.J.C. (2004). Potential biochemical indicators of salinity tolerance in
plants. Plant Science 166: 3-16.
Ashraf, M. and Waheed, A. (1993). Responses of some genetically diverse lines of chickpea
(Cicer arietinum L.) to salt. Plant and Soil 154: 257-266.
Ashraf, M., Athar, H.U.R., Harris, P.J.C. and Kwon, T.R. (2008). Some prospective
strategies for improving crop salt tolerance. Advances in Agronomy 97: 45-110.
Athar, H.U.R., Khan, A. and Ashraf, M. (2007). Exogenously applied ascorbic acid alleviates
salt-induced oxidative stress in wheat. Environmental and Experimental Botany 63:
224-234.
Athar, H-U-R., Khan, A. Ashraf, M. (2008). Inducing salt tolerance in wheat by
exogenously-applied ascorbic acid through different modes. Journal of Plant
Nutrition 32: 1799-1817.
Badawi, M.A., El-Agroudy, M.A. and Attia, A.N. (1995). Effect of planting date and N, P, K
fertilization on growth and yield of sugar beet (Beta vulgaris L.). Journal of
Agricultural Sciences 20: 2683-2689.
Badoni, A. and Chauhan, J.S. (2009). Single node callus culture: improvement for
micropropagation of Solanum tuberosum (cv. Kufri Himalini). Nature and Science 7:
99-103.
Bajaj, Y.P.S. (1987). Biotechnology in Agriculture and Forestry Vol. 3: Potato. Springer-
Verlag, Berlin.
Bajji, M., Kinet, J.M. and Lutts, S. (1998). Salt stress on roots and leaves of Atriplex halmus
L. and their corresponding callus culture. Plant Science 137: 131-142.
Bannister, J.V., Bannister, W.H. and Rottilio, G. (1987). Aspects of the structure, function
and application of superoxide dismutase. Critical Review of Biochemistry 22: 111-
180.
131
Barakat, M.N. and Abdel-Latif, T.H. (1996). In vitro selection of wheat callus tolerant to
high levels of salt and plant regeneration. Euphytica 91: 127-140.
Barkosky, R.R. and Einhellig, F.A. (1993). Effects of salicylic acid on plant water
relationship. Journal of Chemical Ecology 19: 237-347.
Barth, C., De-Tullio, M. and Conklin, P.L. (2006). The role of ascorbic acid in the control of
flowering time and the onset of senescence. Journal of Experimental Botany 57:
1657-1665.
Bartoli, C.G., Simontacchi, M., Tambussi, E., Beltrano, J., Montalde, E. and Puntarulo, S.
(1999). Drought and watering dependent oxidative stress: effect on antioxidant
content in Triticum aestivum L. leaves. Journal of Experimental Botany 50: 375-383.
Bassett, C.L., Nickerson, M.L., Farrell, R.E., Artlip, T.S., El-Ghaouth, A., Wilson, C.L. and
Wisniewski, M.E. (2005). Characterization of a Slocus receptor protein kinase-like
gene from peach. Tree Physiology 25: 403-411.
Basu, S., Gangopadhyay, G. and Mukherjee, B.B. (2002). Salt-tolerance in rice in vitro:
implication of accumulation of Na+, K+ and proline. Plant Cell, Tissue and Organ
Culture 69: 55-64.
Basu, S., Gangopadhyay, G., Mukherjee, B.B. and Gupta, S. (1997). Plant regeneration of
salt adapted callus of indica rice (var. Basmati 370) in saline conditions. Plant Cell,
Tissue and Organ Culture 50: 153-159.
Batkova, P., Paspisilova, J. and Synkova, H. (2008). Production of reactive oxygen species
and development of antioxidative system during in vitro growth and ex vitro transfer.
Biologia Plantarum 52: 413-422.
Beers, R.F. and Sizer, I.W. (1952). A spectrophotometric method for measuring the
breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 195:
133-140.
132
Bekheet, S.H., Taha, S.H., Sawires, E.S. and Elbahr, M.K. (2000). Salt stress in tissue culture
of Asparagus officinalis. Egyptian Journal of Horticulture 27: 275-287.
Beloualy, N. and Bouharmont, J. (1992). NaCl tolerant plants of Poncirus trifoliata
regenerated from tolerant cell lines. Theoretical and Applied Genetics 83: 509-514.
Beltagi, M.S. (2008). Exogenous ascorbic acid (vitamin C) induced anabolic changes for salt
tolerance in chick pea (Cicer arietinum L.) plants. African Journal of Plant Sciences
2: 118-123.
Benavides, M.P., Marconi, P.L., Gallego, S.M., Comba, M.E. and Tomaro, M.L. (2000).
Relationship between antioxidant defense systems and salt tolerance in Solanum
tuberosum. Australian Journal of Plant Physiology 27: 273-278.
Ben-Hayyim, G. and Goffer, Y. (1989). Plantlet regeneration from NaCl-selected salt
tolerant callus culture of Shamouti orange (Citrus sinensis L.). Plant Cell Reports 7:
680-683.
Benkema, H.P., Vanderzaag, D.E. (1990). Introduction to potato production. Centre of
Agricultural Publishing and Documentation, Wangeningen, pp 13.
Beversdorf, W.D. (1990). Micropropagation in crop species. In: Progress in Plant Cellular
and Molecular Biology. Nijkamp, H.J.J., Van-Der Plas, L.H.W. and Van Aartrijk, J
(Eds), Kluwer Academic Publishers, Dordrecht, pp 3-12.
Bhaskaran, S., Smith, R.H. and Schertz, K. (1983). Sodium chloride-tolerant callus of
Sorghum bicolor L. Moench. Journal of Plant Physiology 112: 459-463.
Bhojwani, S.S. and Razdan, M.K. (2004). Cell culture. In: Plant tissue culture theory and
practices. Elsevier Publishers Netherlands, pp. 63-64.
Biggs, K.J. and Fry, S.C. (1997). Phenolic cross-linking at the cell wall. In: physiology of
cell expansion during plant growth. Cosgrove, D.J. and Knievel. (Eds), American
Society of Plant Physiologist. Rockville, M.D, pp 46-57.
133
Binzel, M.L., Hasegawa, P.M., Handa, A.K. and Bressan, R.A. (1985). Adaptation of
Tobacco cells to NaCl. Plant Physiology 79: 118-125.
Blokhina, O., Virolainen, E. and Fagerstedt, K.V. (2003). Antioxidants, oxidative damage
and oxygen deprivation stress: A review. Annals of Botany 91: 179-94.
Bohnert, H.J. and Jensen, R.G. (1996). Strategies for engineering water stress tolerance in
plants. Trends in Biotechnology 14: 89-97.
Bolar, J.P., Norelli, J.L., Aldwinckle, H.S. and Hanke, V. (1998). An efficient method for
rooting and acclimation of micropropagated apple cultivars. Hort-Science 37: 1251-
1252.
Bor, M., Ozdemir, F. and Turkan, I. (2003). The effect of salt stress on lipid peroxidation and
antioxidants in leaves of sugar beet (Beta vulgaris L.) and wild beet (Beta maritima
L.). Plant Science 16: 77-84.
Borsani, O., Valpuesta, V. and Botella, M.A. (2001). Evidence for a role of salicylic acid in
the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings.
Plant Physiology 126: 1024-1030.
Bowler, C., Van-Montagu, M. and Inze, D. (1992). Superoxide dismutase and stress-
tolerance. Annual Review of Plant Physiology and Plant Molecular Biology 43: 83-
116.
Bowman, W.D. (1987). Physiological differentiation to salt stress in the C4 non-halophytic
Andropogan glomeratus. 14th International Botany Congress. Berlin (West), pp 74.
Boyer, J.S. (1982). Plant productivity and environment. Science 218: 443-448.
Bruns, S. and Caesar, K. (1990). Shoot development and tuber yield of several potato
cultivars under high salt concentrations at different stages of development. Potato
Research 33: 23-32.
134
Burgutin, A.B., Butenko, R.G., Kaurov, B.A. and Iddagoda, N. (1996). In vitro selection of
potato for tolerance to sodium chloride. Russian Journal of Plant Physiology 43: 524-
531.
Caldiz, D.O. (1994). Genetic improvement and associated physiological changes in the
potato. In: Genetic Improvement of Field Crops. Slafer, G.A. (Eds), Marcel Dekker,
New York, pp. 361-441.
Cano, E.A., Perez-Alfocae, F., Moreno, V., Caro, M. and Bolarin, M.C. (1998). Evaluation
of salt-tolerance in cultivated and wild tomato species through in vitro shoot apex
culture. Plant Cell, Tissue and Organ Culture 53: 19-26.
Capelle, S.C., Mok, D.W.S., Kirchner, S.C. and Mok, M.C. (1983). Effects of thidiazuron on
cytokinins autonomy and metabolism of N6- (2- isopentyl) [8-14C] adenosine in
callus tissue of Phaseolus lunatus L. Plant Physiology 73: 796- 802.
Casanova, E., Zuker, A., Trillas, M.I., Moysset, L. and Vainstein, A. (2003). The rolC gene
in carnation exhibits cytokinin and auxin-like activities. Scientia Horticulturae 97:
321-331.
Chalupa, V. (1987). Effect of benzylaminopurine and thidiazuron on in vitro shoot
proliferation of Tilia cordata Mill, Sorbus aucuparia L. and Robinia pseudoacacia L.
Biologia Plantarum 29: 425-429.
Chalupa, V. (1988). Large-scale micropropagation of Quercus robur L. using adenine type
cytokinin and thidiazuron to stimulate shoot proliferation. Biologia Plantarum 30:
414-421.
Chandler, S.F. and Vasil, I.K. (1984). Selection and characterization of NaCl-tolerant cells
from embryonic cultures of Penisetum purpureum schum (Napier grass). Plant
Science Letter 37: 157-164.
Chauhan, V., Prathapasenan, G. and Chauhan, V. (2000). Growth characteristics and ion
contents of rice callus under the influence of NaCl and hydroxyproline. Acta
Physiologia Plantarum 22: 39-44.
135
Chee, P.P. (1990). High frequency of somatic embryogenesis and recovery of fertile
cucumber plants. Hort-Science 25: 792-793.
Chen, Z. and Gallie, D.R. (2004). The ascorbic acid redox state controls guard cell signaling
and stomatal movement. Plant Cell 16: 1143-1162.
Cherian, S. and Reddy, M.P. (2003). Evaluation of NaCl tolerance in the callus cultures of
Suaeda nudiflora Moq. Biologia Plantarum 46: 193-198.
Choukr-Allah, R. (1995). The potential halophytes in the development and rehabilitation of
arid and semi-arid zones. In: Halophytes and biosaline agriculture. Choukr-Allah, R.,
Malcolm, C.V. and Hamdy, A. (Eds), Marcel Dekker, Inc., New York, pp. 1-13.
Chowdhury, B. and Mandal, A.B. (2001). Microspore embryogenesis and fertile plantlet
regeneration in a salt-susceptible and salt-tolerant rice hybrid. Plant Cell, Tissue and
Organ Culture 65: 141-147.
Conklin, P.L. (2001). Recent advances in the role and biosynthesis of ascorbic acid. Plants
Cell and Environment 24: 383-394.
Cronje, M.J. and Bornman, L. (1999). Salicylic acid influences Hsp70/Hsc70 expression in
Lycopersicon esculentum: dose and time dependent induction or potentiation.
Biochemical and Biophysical Research Communication 265: 422-427.
Cutt, J.R. and Klessing, D.F. (1992). Salicylic acid in plants: a changing perspective.
Pharmaceutical Technology 16: 25-34.
Davenport, S.P. (2003). Behavior of antioxidant defense system in the adaptive response to
salt stress in Helianthus annus L. cells. Plant Growth Regulation 40: 81-88.
Debergh, P.C. and Read, P.E. (1991). Micropropagation. In: Micropropagation, Debergh,
P.C. and Zimmerman, R.H. (Eds), Kluwer Acad. Publisher Dordrecht. The
Netherlands, pp 1-13.
136
De-Gracia, E. and Martinez, S. (1995). Somatic embryogenesis in Solanum tuberosum L. cv.
Desiree from stem nodal sections. Journal of Plant Physiology 145: 526-530.
Delauney, A.J. and Verma, D.P.S. (1993). Proline biosynthesis and osmoregulation in plants.
Plant Journal 4: 215-223.
Dhindsa, R.S. (1991). Drought stress, enzymes of glutathione metabolism, oxidative injury,
and protein synthesis in Tortula ruralis. Plant Physiology 95: 648-651.
Dionisiosese, M. and Tobita, S. (1998). Antioxidant response of rice seedlings to salinity
stress. Plant Science 135: 1-9.
Dix, P.J. and Street, H.E. (1975). Sodium chloride-resistant cultured cell lines from
Nicotiana sylvestris and Capsicum annum. Plant Science Letters 5: 231-237.
Dolatabadian, A. and Jouneghani, R.S. (2009). Impact of exogenous ascorbic acid on
antioxidant activity and some physiological traits of common bean subjected to
salinity stress. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 37: 165-172.
Edriss, M.H., Badawy, M.A., Fathi, S. and Barkouki, T.M. (2003). Propagation of potato
using tissue culture technique. Acta Horticulturae (ISHS) 434.
Ehsanpour, A.A., Madani, S. and Moseini, M. (2007). Detection of somaclonal variation in
potato callus induced by UV-C radiation using RAPD-PCR. Genetic Application and
Plant Physiology 33: 3-11.
Elavumoottil, O.C., Martin, J.P. and Moreno, M.L. (2003). Changes in sugars, sucrose
synthase activity and proteins in salinity tolerant callus and cell suspension cultures
of Brassica oleracea L. Biologia Plantarum 46: 7-12.
El-Enany, A.E. (1997). Shoot regeneration and protein synthesis in tomato tissue cultures.
Biologia Plantarum 39: 303-308.
137
Elkahoui, S., Hernandez, J.A., Abdelly, C., Ghrir, R. and Limam, F. (2005). Effects of salt
on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus
suspension cells. Plant Science 168: 607-613.
Elstner, E.F. (1986). Metabolism of activated oxygen species. In: The Biochemistry of
Plants: Biochemistry of Metabolism. Davies, D.D (Eds), Academic Press, San Diego.
Volume 11, pp. 253-315.
El-Tayeb, M.A. (2005). Responses of barley grains to the interactive effect of salinity and
salicylic acid. Plant Growth Regulation 45: 215-224.
El-Tayeb, M.A., El-Enany, A.E and Ahmed, N.L. (2006). Salicylic acid induced adaptive
response to copper stress in sunflower (Helianthus annus L.). Plant Growth
Regulation 50: 191-199.
Errabii, T., Bernard, C., Gandonou, C.B., Essalmani, H., Abrini, J., Idamar, M. and Senhaji,
N.S. (2007). Effect of NaCl and manitol induced stress on sugarcane (Sacchrum sp.)
callus cultures. Acta Physiologia Plantarum 29: 95-102.
Esna-Ashari, M. and Villierst, A. (1998). Plant regeneration from tuber discs of potato
(Solanum tuberosum L.) using 6-benzylaminopurine (BAP). Potato Research 41: 371-
382.
Evans, D.E., Coleman, J.O.D. and Kearns, A. (2003). Role of callus in embryogenesis,
organogenesis, and cell culture. In: Plant Cell Culture. Oxford Brookes University,
Oxford, UK Bios Scientific Publishers, pp. 64-65.
Evens, D.A., Sharp, W.R. and Flick, C.E. (1981). Growth and behavior of cell culture:
embryogenesis and orgenogenesis. In: Plant tissue culture: methods and applications
in agriculture. Thorpe T.A. (Eds), Academic press. New York, pp 45-113.
F.A.O. (2005). Global network on integrated soil management for sustainable use of salt-
affected Soils. Rome, Italy: FAO Land and Plant Nutrition Management Services.
138
F.A.O. (2008). Hidden treasure. International year of the potato. Food and agriculture
organization of the United Nations. http://www.potato2008.org/en/index.html.
Fallon, K.M. and Phillips, R. (1989). Responses to water stress in adapted and un-adapted
carrot cell suspension cultures. Journal of Experimental Botany 40: 681-687.
Farhatullah., Abbas, Z. and Abbas, S.J. (2009). In vitro effect of gibberellic acid on
morphogenesis of potato explant. International Journal of Agriculture and Biology 9:
181-182.
Farhatullah., Rashid, M. and Raziuddin. (2002). In vitro effect of salt on the vigor of potato
(Solanum tuberosum L) plantlets. Biotechnology 1: 73-77.
Fengyen, L. and Han, L. (2002). Effect of exogenous hormones on micropropagation of in
vitro virus free potato plantlets. Chinese Potato Journal 16: 214-216.
Fidalgo, F., Santos, A., Santos, I. and Salema, R. (2004). Effects of long-term salt stress on
antioxidant defense system, leaf water relations and chloroplast ultra-structure of
potato plant. Annals of Applied Biology 145: 185-192.
Fila, G, Franz, W.B, Sylvie, M., Zoran, C. and Jaleh, G. (2006). Relationships between leaf
conductance to CO2 diffusion and photosynthesis in micropropagated grapevine
plants, before and after ex-vitro acclimatization. Journal of Experimental Botany 57:
2687-2695.
Flowers, T.J., Yeo, A.R. (1995). Breeding for salinity resistance in crop plants-where next?
Australian Journal of Plant Physiology 22: 875-884.
Foyer, C.H. (1993). Ascorbic acid. In: Antioxidant in higher plants. Alscher R.G. and Hess,
J.L. (Eds), Boca Raton, FL: CRC Press, pp 51-58.
Fratini, R. and Ruiz, M.L. (2002). Comparative study of different cytokinins in the induction
of marphogenesis in lental (Lens culinaris Medik). In vitro Cellular and
Developmental Biology-Plant 38: 46-51.
139
Fridovich, I. (1988). The biology of oxygen radicals: general concepts. In: Oxygen radicals
and tissue injury. Halliwell, B. (Eds). Proceedings of an Upjohn Symposium. The
Upjohn, Maryland, pp. 1-5.
Fridovich, I. (1991). Molecular oxygen; Friend and foe. In: Active oxygen/oxidative stress
and plant metabolism. Pell, E.J. and Steffen, K.L. (Eds), American Society of Plant
Physiologists, Rockville, pp 1-5.
Fry, S.C. (1986). Cross-linking of matrix polymers in the growing cell wall of angiosperms.
Annual Review of Plant Physiology 37: 165-186.
Gadallah, M.A.A. (2000). Effects of acid mist and ascorbic acid treatment on the growth,
stability of leaf membranes, chlorophyll content and some mineral elements of
Carthamus tinctorius, the safflower. Water, Air and Soil Pollution 118: 311-327.
Gandonou, C.B., Abrini, J., Idaomar, M. and Senhaji, S.N. (2005). Response of sugarcane
(Saccharum sp.) varieties to embryogenic callus induction and in vitro salt stress.
African Journal of Biotechnology 4: 350-354.
Gandonou, C.B., Errabii, T., Abrini, J., Idaomar, M. and Senhaji, S.N. (2006). Selection of
callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to
salt stress. Plant Cell, Tissue and Organ Culture 87: 9-16.
Garratt, L.C., Janagoudar, B.S., Lowe, K.C., Anthony, P., Power, J.B. and Davey, M.R.
(2002). Salinity tolerance and antioxidant status in cotton cultures. Free Radical
Biology and Medicine 33: 502-511.
Goodwin, P.B. Kim, Y.C. and Adisarwanto, T. (1980). Propagation of potato by shoot-tip
culture. Potato Research 23: 9-18.
Gopal, J., Iwama, K. and Jitsuyama, Y. (2008). Effect of water stress mediated through agar
on in vitro growth of potato. In vitro Cellular and Developmental Biology-Plant 44:
221-228.
140
Gopal, J., Minocha, J. L. and Dhaliwal, H.S. (1980). Microtuberization in potato (Solanum
tuberosum L.). Plant Cell Reports 17: 794-798.
Gossett, D.R., Millhollon, E.P., Lucas, M.C., Banks, S.W. and Marney, M.M. (1994). The
effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and
salt-sensitive cultivars of cotton. Plant Cell Reports 13: 498-503.
Greenland, D.J. (1984). Exploiting plants: rice. Biologist 31: 291-295.
Greenway, H. and Munns, R. (1980). Mechanism of salt tolerance in non-halophytes. Annual
Review of Plant Physiology 31: 149-190.
Gu, R., Liu, Q., Pei, D. and Jiang, X. (2004). Understanding saline and osmotic tolerance of
Populus euphratica suspended cells. Plant Cell, Tissue and Organ Culture 78: 261-
265.
Gueta-Dahan, Y., Yaniv, Z., Zilinkas, B.A. and Ben-Hayyim, G. (1997). Salt and oxidative
stress: similar and specific responses and their relation to salt-tolerance in citrus.
Planta 203: 460-469.
Gunes, A., Inal, A., Alpaslan, M., Eraslan, F., Bagci, E.G. and Cicek, N. (2007). Salicylic
acid induced changes on some physiological parameters symptomatic for oxidative
stress and mineral nutrition in maize (Zea mays) grown under salinity. Journal of
Plant Physiology 164: 728-736.
Halliwell, B. and Gutteridge, J.M.C. (1985). Free radicals in biology and medicine.
Clarendon Press. Oxford, pp. 1-51.
Haque, A.U., Samad, M.A. and Shapla, T.L. (2009). In vitro callus induction and
regeneration in potato. Bangladesh Journal of Agricultural Research 34: 449-456.
Hare, P.D. and Van-Staden, J. (1994). Inhibitory effect of TDZ on the activity of cytokinin
oxidase isolated from soybean callus. Plant Cell Physiology 35: 1121-1125.
141
Harinasut, P., Poonsopa, D., Roengmongkol, K. and Charoensataporn, R. (2003). Salinity
effects on antioxidant enzymes in mulberry cultivar. Science Asia 29: 109-113.
Harms, C.T. and Oertli, J.J. (1985). The use of osmotically adapted cell cultures to study salt
tolerance in vitro. Journal of Plant Physiology 120: 29-38.
Hasegawa, P., Bressan, R.A., Zhu, J.K. and Bohnert, H.J. (2000). Plant cellular and
molecular responses to high salinity. Annual Review of Plant Physiology and Plant
Molecular Biology 51: 463-499.
Hassan, N.S. and Wilkins, D.A. (1988). In vitro selection for salt tolerant lines in
Lycopersicon peruvianum. Plant Cell Reports 7: 463-465.
Hassan, N.S., Shaaban, L.D., Hashem, E.A. and Saleem, E.E. (2004). In vitro selection of
water stress tolerant callus line of Helianthus annus L. cv. Myak. International
Journal of Agriculture and Biology 6: 13-18.
Hassanein, A.M. (2004). Effect of relatively high concentrations of mannitol and sodium
chloride on regeneration and gene expression of stress-tolerant (Alhagi graecorum)
and stress sensitive (Lycopersicon esculentum L.) plant species. Bulgarian Journal of
Plant Physiology 30: 19-36.
Hawkins, H.J. and Lips, S.H. (1997). Cell suspension cultures of Solanum tuberosum L. as a
model system for N and salinity response. Effect of salinity and NO3− uptake and
PM-ATPase activity. Journal of Plant Physiology 150: 103-109.
Hazarika, B.N. (2003). Acclimatization of tissue cultured plants. Current Science 85: 1704-
1712.
Hernandez, J.A., Campillo, A., Jimenez, A., Alarcon, J.J. and Sevilla, F. (1999). Response of
antioxidant systems and leaf water relations to NaCl stress in pea plants. New
Phytologist 141: 241- 251.
142
Hernandez, J.A., Jimenez, A., Mullineaux, P. and Sevilla, F. (2000). Tolerance of pea (Pisum
sativum L.) to long-term salt stress is associated with induction of antioxidant
defenses. Plant Cell and Environment 23: 853-862.
Hernandez, J.A., Olmos, E., Corpas, F.J., Sevilla, F. and Del-Rio, L.A. (1995). Salt induced
oxidative stress in chloroplast of pea plants. Plant Science 105: 151-167.
Heszky, L.E., Li, S.N. and Horvath, Z.S. (1986). Rice tissue culture and its application to
breeding II. Factors affecting the plant regeneration during subculture of diploid and
haploid callus. Cereals Research Communications 14: 289-296.
Heuer, B. and Nadler, A. (1995). Growth and development of potato under salinity and water
deficit. Australian Journal of Agricultural Research 46: 1477-1486.
Ho, W.J. and Vasil, I.K. (1983). Somatic embryogenesis in sugarcane (Saccharum
officinarum L.): Growth and plant regeneration from embryogenic cell suspension
culture. Annals of Botany 51: 719-726.
Hoagland, D.R. and Arnon, D.I. (1950). The water culture method for growing plants
without soil. California Agricultural and Experimental Statistics Circulation number
347.
Horton, D. and Sawyer, R.L. (1985). The Potato as a world food crop, with special references
to developing areas. In: Potato Physiology. Li, P.H. (Eds), Academic Press Inc.,
USA, pp. 2-32.
Horvath, E., Pal, M., Szalai, G., Paldi, E. and Janda, T. (2007b). Exogenous 4-
hydroxybenzoic acid and salicylic acid modulate the effect of short-term drought and
freezing stress on wheat plants. Biologia Plantarum 51: 480-487.
Horvath, E., Szalai, G. and Janda, T. (2007a). Induction of abiotic stress tolerance by
salicylic acid signaling. Plant Growth Regulation 26: 290-300.
143
Hossain, Z., Mandal, A.K.A., Data, S.K. and Biswas, A.K. (2007). Development of NaCl-
tolerant line in Chrysanthemum morifolium Ramat. Through shoot organogenesis of
selected callus line. Journal of Biotechnology 129: 658-667.
Hulme, J., Higgins, E.S. and Sheild, R. (1992). An efficient genotype-dependent method for
regeneration of potato plants from leaf tissue. Plant Cell, Tissue and Organ Culture
31: 161-167.
Hussain, I., Muhammad, A., Rashid, H. Chaudhary, Z., Naqvi, S.M.S. and Asghar. (2005).
Morphogenic potential of three potato (Solanum tuberosum L.) cultivars from diverse
explants a prerequisite in genetic manipulation. Pakistan Journal of Botany 37: 889-
898.
Hussey, G. and Stacey, N. J. (1981). In vitro propagation of potato (Solanum tuberosum L.).
Annals of Botany 48: 787-796.
Hutchinson, M.I. and Saxena, P.K. (1996). Role of purine metabolism in TDZ induced
somatic embryogenesis of geranium (Pelargonium horturum L. Bailey). Journal of
Plant Physiology 149: 573-579.
Hutchinson, M.I., Krishma-Raj, S. and Saxena, P.K. (1996). Morphological and
physiological changes during thidiazuron induced somatic embryogenesis in
geranium (Pelargonium hortorum L.) Hypocotyl culture. International Journal of
Plant Sciences 157: 110-117.
Iba, K. (2002). Acclimative response to temperature stress in higher plants: approaches of
gene engineering for temperature-tolerance. Annual Review Plant Biology 53: 225-
245.
Iqbal, J. and Schraudolf, H. (1977). Changes in protein biosynthesis during gibberellic acid
induced induction and formation of antheridium in the Fern (Anenmia phyllitiolis).
Development, Growth and Differentiation 19: 85-92.
144
Irfan, A. (1992). Proceedings of the workshop on Seed Potato Health. Seed Potato Health
Laboratory Project, Sahiwal. Federal Seed Certification Department. Ministry of
Food and Agriculture Cooperation, pp. 1-2.
Jain, M., Chengalrayan, K., Abouzid, A. and Gallo, M. (2007). Prospecting the utility of a
PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum
spp. hybrid) plants. Plant Cell Reports 26: 581-590.
Jain, S.M. (2001). Tissue culture-derived variation in crop improvement. Euphytica 118:
153-166.
Jaiswal, R. and Singh, N.P. (2001). Plant regeneration from NaCl tolerant callus/cell lines of
chickpea. International Chickpea and Pigeon pea Newsletter 8: 21-23.
Jaiswal, V.S. and Naryan, P. (1985). Regeneration of plantlet from callus of stem segment of
adult plants of Ficus religiosa L. Plant Cell Reports 4: 256-258.
Jaleel, C.A., Gopi, R., Manivannan, P. and Panneerselvam, R. (2007). Antioxidative
potentials as a protective mechanism in Catharanthus roseus (L.), (Eds), G. Don.
Plants under salinity stress. Turkish Journal of Botany 31: 1-7.
Jarret, R.L., Hasegawa, P.M. and Erickson, H.T. (1980). Effects of medium components on
shoot formation from cultured tuber discs of potato. Journal of the American Society
for Horticultural Science 105: 238-242.
Jason, S., Senaratna, T. and Sivasithamparam, K. (2006). Salicylic acid induces salinity
tolerance in tomato (Lycopersicon esculentum cv. Rome): associated changes in gas
exchange, water relations and membrane stabilization. Plant Growth Regulation 49:
77-83.
Jaya-Sree, T., Pavan, U., Ramesh, M., Rao, A.V., Jagan. M., Reddy, K. and Sadanandam, A.
(2001). Somatic embryogenesis from leaf cultures of potato. Plant Cell, Tissue and
Organ Culture 64: 13-17.
145
Jefferies, R.A. (1996). Evaluation of seedling selection for salinity tolerance in potato
(Solanum tuberosum L.). Euphytica 88: 207-213.
Jeschke, W.D. (1984). K+, Na+ exchange in cellular membranes, intra-cellular
compartmentation of cations and tolerance. In: Salinity tolerance in plants: Strategies
for crop improvement. Staples, R.C. and Teoenniesson, G.H. (Eds), John Wiley and
Sons, New York, pp 37-66.
Jones, R.L. (1994). Gibberellins: their physiological role. Agriculture Review of Plant
Physiology 24: 571-598.
Kang, G., Wang, C., Sun, G. and Wang, Z. (2003). Salicylic acid changes activities of H2O2
metabolizing enzymes and increases the chilling tolerance of banana seedlings.
Environmental and Experimental Botany 50: 9-15.
Karlidag, H., Yildirim, E. and Turan, M. (2009). Salicylic acid ameliorates the adverse effect
of salt stress on strawberry. Science and Agriculture 66: 180-187.
Katerji, N., Van-Hoorn, J.W. Hamdy, A. and Mastrorilli, M. (2003). Salinity effect on crop
development and yield, analysis of salt-tolerance according to several classification
methods. Agricultural Water Management 62: 37-66.
Kerns, H.R. and Meyer, M.M. (1987). Diligence finds chemical key to micropropagation a
new maple. American Nurseryman 165: 104-110.
Khan, M.A., Ahmad, M.Z. and Hameed, A. (2006). Effect of sea salt and L-ascorbic acid on
the seed germination of halophytes. Journal of Arid Environment 67: 535-540.
Khan, M.H., Panda, S.K. (2002). Induction of oxidative stress in roots of Oryza sativa L. in
response to salt stress. Biology Plant 45: 625-627.
146
Khan, W., Prithiviraj, B., Smith, D.L. (2003). Photosynthetic responses of corn and soybean
to foliar application of salicylates. Journal of Plant Physiology 160: 485-492.
Khatun, N., Bari, M.A., Islam, R., Huda, S., Siddique, N.A., Rahman, M.H. and Mollah,
M.U. (2003). Callus induction and regeneration from nodal segment of potato cultivar
Diamant. Journal of Biological Sciences 3: 1101-1106.
Khedr, H.A., Abbas, M.A., Abdel-Wahid, A.A., Quick, W.P. and Abo-Gadallah, G.M.
(2003). Proline induces the expression of salt-stress responsive proteins and improve
the adaptation of Pancratium maritimum L. to salt-stress. Journal of Experimental
Botany 54: 2253-2262.
Khodary, S.E.A. (2004). Effect of salicylic acid on growth, photosynthesis and carbohydrate
metabolism in salt stressed maize plants. International Journal of Agricultural
Biology 6: 5-8.
Khrais, T., Leclerc, Y. and Donnelly, D.J. (1998). Relative salinity tolerance of potato
cultivars assessed by in vitro screening. American Journal of Potato Research 75:
207-210.
Koca, H., Ozdemir, F. and Turkan, I. (2006). Effect of salt-stress on lipid peroxidation and
superoxide dismutase and peroxidase activities of Lycopersicon esculentum and L.
pennellii. Biologia Plantarum 50: 745-748.
Kogan, M.J., Kristoff, G., Benavides, M.P. and Tomaro, M.L. (2000). Effect of pre-treatment
with ethanolamine on the response of Helianthus annus L. to salt stress. Plant Growth
Regulation 3: 87-94.
Kuldybear, M.M., Amerkhanova, M.B. and Dzhakibaeva, G.T. (1995). Effect of
phytohormones (kinetin/2, 4.D) on induction of callus tissue growth in tissue culture
of potato cultivar Nevskii. Izu. Akad Nauk. Resp. Kaz. Ser. Biol 2: 84-87.
Kumar, N., Pamidimarri S, D.V.N., Kaur, M., Boricha, G. Reddy, M.P. (2008). Effect of
NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes
activity in callus cultures of Jatropha curcas. Biologia 63: 378-382.
147
Kusvuran, S., Ellialtioglu, S., Yasar, F. and Abak, K. (2007). Effects of salt stress on ion
accumulation and activity of some antioxidant enzymes in melon (Cucumis melo L.)
Journal of Food, Agriculture and Environment 5: 351-355.
Lagrimini, L.M., Bradford, S. and Rothstein. (1990). Peroxidase-induced wilting in
transgenic tobacco plants. The Plant Cell 2: 7-18.
Landberg, T. and Greger, M. (2002). Differences in oxidative stress, heavy metal resistant
and sensitive clones of Salix viminalis. Journal of Plant Physiology 159: 65-75.
Lapichino, G., Lee, S.P., Chen, T.H.H. and Fuchigami, L.H. (1991). In vitro regeneration in
Solanum commersonii. Journal of Plant Physiology 137: 734-738.
Lauchli, A. and Epstein, E. (1990). Plant responses to saline and sodic conditions. In:
Agricultural salinity assessment and management. ASCE manuals and reports on
engineering practice No. 71. Tanji, K.K. (Eds). ASCE New York, pp 113-137.
Lechno, S.E., Zamsk. and Tel-Or, E. (1997). Salt stress induced responses in cucumber
plants. Plant Cell Physiology 150: 206-211.
Lee, S.K. and Kader, A.A. (2000). Pre-harvest and post-harvest factors influencing vitamin C
contents of horticultural crops. Post-harvest Biology and Technology 20: 207-220.
Leone, A., Costa, A., Tucci, M. and Grillo. (1994). Adaptation versus shock response to
polyethylene glycol-induced low water potential in cultured potato cells. Physiologia
Plantarum 92: 21-30.
Levy, D. (1992). The response of potatoes (Solanum tuberosum) to salinity: plant growth and
tuber yields in the arid deserts of Israel. Annals of Applied Biology 120: 547-555.
Li, S.N. and Heszky, L.E. (1986). Testing of salt (NaCl) tolerance and regeneration in callus
culture (n, 2n) of rice. In: Genetic Manipulation in Plant Breeding. Horn, W. Jensen,
J.C. Odenbach, W. and Schieder. O. (Eds), Walter de Gruyter and Corporation.,
Berlin, New York, pp. 617-619.
148
Liang, L.F., Keng, C.L. and Lim, B.P. (2006). Selection of cell lines for the production of
rosmarinic acid from cell suspension cultures of Orthosiphon stamineus Benth. In
vitro Cellular and Developmental Biology-Plant 42: 538-542.
Lindeque, J.M., Vander-Mescht, A., Slabbert, M.M. and Henn, G. (1991). Variation in
phenotype and proteins in plants regenerated from cell suspensions of potato cv. BPI.
Euphytica 54: 41-44.
Liso, R, Calabrese, G., Bitonti, M.B and Arrigoni, O. (1984). Relationship between ascorbic
acid and cell division. Experimental Cell Research 150: 314-320.
Liu, S.H., Fu, B.Y., Xu, H.X., Zhu, L.H., Zhai, H.O. and Li, Z.K. (2007). Cell death in
response to osmotic and salt stresses in two rice (Oryza sativa L.) ecotypes. Plant
Science 172: 897-902.
Liu, T. and Van-Staden, J. (1999). Selection and characterization of sodium chloride-
tolerant callus of Glycine max (L.) Merrcb. Acme. Plant Growth Regulation 31: 195-
207.
Loewus, F.A. and Helsper, J.P.F.G. (1982). Metabolism of L-Ascorbic acid in plants. In:
Ascorbic acid, chemistry and uses. Seib, A. and Tolbert, A. (Eds), American
Chemical Society, Washington, pp 249-261.
Loewus, M.A. (1999). Biosynthesis and metabolism of ascorbic acid in plants and analogs of
ascorbic acid in fungi. Phytochemistry 52: 193-210.
Luck, H. (1974). Methods in enzymatic analysis 2nd edition. Bergmeyer Academic New
York, pp 885.
Lutts, S., Almansouri, M. and Kinet, J.M. (2004). Salinity and water stress have contrasting
effects on the relationship between growth and cell viability during and after stress
exposure in durum wheat callus. Plant Science 167: 9-18.
Lutts, S., Kinet, J.M. and Bouharmont, J. (1999). Improvement of rice callus regeneration in
the presence of NaCl. Plant Cell, Tissue and Organ Culture 57: 3-11.
149
Maggio, A., Reddy, M.P. and Joly, R.J. (2000). Leaf gas exchange and solute accumulation
in the halophyte Salvadora persica grown at moderate salinity. Environmental and
Experimental Botany 44: 31-38.
Magioli, C., Rocha, A.P.M., De-Oliveira, D.E. and Mansur, E. (1998). Efficient shoot
orgenogensis of eggplant (Solanum melongena L.) induced by thidiazuron. Plant Cell
Reports 17: 661-663.
Mahajan, S. and Tuteja, N. (2005). Cold, salinity, and drought stresses: an overview.
Archives of Biochemistry and Biophysics 444: 139-158.
Malik, N.J. (1995). Potato in Pakistan. World mate, Islamabad, Pakistan.
Mansour, M.M.F. (2000). Nitrogen containing compounds and adaptation of plants to
salinity stress. Biologia Plantarum 43: 491-500.
Marconi, P.L., Benavides, M.P. and Caso, O.H. (2001). Growth and physiological
characterization of regenerated potato (Solanum tuberosum) plants affected by NaCl
stress. New Zealand Journal of Crop and Horticultural Science 29: 45-50.
Marschner, H. (1995). Mineral nutrition of higher plants. Academic Press: London.
Martinez, C.A., Maestri, M. and Lani, E.G. (1996). In vitro salt tolerance and proline
accumulation in Andean Potato (Solanum spp.) differing in frost resistance. Plant
Science 116: 177-184.
Mass, E.V. (1985). Crop tolerance to salinity sprinkling water. Plant and Soil 89: 273-284.
May, M.J., Vernoux, T. Leaver, C. Montagu, M.V. and Inze, D. (1998). Glutathione
homeostasis in plants: implications for environmental sensing and plant development.
Journal of Experimental Botany 49: 649-667.
McCoy, T.J. (1987). Characterisation of alfalfa (Medicago sativa L.) plants, regenerated
from selected NaCl-tolerant cell lines. Plant Cell Reports 6: 417-422.
150
Mc-Cue, P., Zheng Z., Pinkham, J.L. and Shetty, K. (2000). A model for enhanced pea
seedling vigour following low pH and salicylic acid treatments. Process in
Biochemistry 35: 603-613.
Mc-Cue, R.F. and Hanson, A.D. (1990). Drought and salt-tolerance toward understanding
and application. Trends in Biotechnology 8: 358-362.
Mc-Hughen, A. and Swartz, M. (1984). A tissue culture derived salt-tolerant line of flax
(Linum usitatissimum). Journal of Plant Physiology 117: 109-118.
Mc-Kersie, B.D., Bowley, S.R., Harjanto, E. and Leprince, O. (1996). Water-deficit
tolerance and field performance of transgenic alfalfa over expressing superoxide
dismutase. Plant Physiology 111: 1177-1181.
Melander, W. and Horvath, C. (1977). Salt affected on hydroponic interaction in
precipitation and chromatography of proteins: an interpretation of the lycotropic
series. Archives of Biochemistry and Biophysics 183: 200-215.
Meloni, D.A., Oliva, M.A., Martinez, C.A. and Cambraia, J. (2003). Photosynthesis and
activity of superoxide dismutase, peroxidase and glutathione reductase in cotton
under salt stress. Environmental and Experimental Botany 49: 69-76.
Menconi, M., Sgherri, C.L.M., Pinzino, C. and Navari-Izzo, F. (1995). Activated oxygen
production and detoxification in wheat plants subjected to a water deficit program.
Journal of Experimental Botany 46: 1123-1130.
Merja D. and Stasa. (1997). In vitro regeneration and propagation of potato and its genetic
homogeneity determination by means of protein polymorphism of tubers. ISHS Acta
Horticulturae 462: I Balken Symposium on vegetables and potato, pp.153.
Michalczuk, L., Cooke, T.J. and Cohen, J.D. (1992). Auxin levels at different stages of carrot
somatic embryogenesis. Phytochemistry 31: 1097-1103.
Miki, Y., Hshiba, M. and Hisajima, S. (2001). Establishment of salt stress-tolerant rice plants
through step up NaCl treatment in vitro. Biologia Plantarum 3: 391-395.
151
Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant
Science 7: 405-410.
Mittova, V., Tal, M., Volokita, M. and Guy, M. (2003). Up-regulation of the leaf
mitochondrial and peroxisomal antioxidative system in response to salt-induced
oxidative stress in the wild salt tolerant tomato species Lycopersicon pennellii. Plant
Cell and Environment 26: 845-856.
Mittova, V., Volokita, M., Guy, M. and Tal, M. (2000). Activities of SOD and the ascorbate
glutathione cycle enzyme in subcellular compartments in leaves and roots of the
cultivated tomato and its salt-tolerant relative Lycopersicon pennellii. Physiologia
Plantarum 110: 42-51.
Mondal, T.K., Bhattacharya, A., Sood, A. and Ahuja, P.S. (1998). Micropropagation of tea
(Camellia sinensis L.) O. kuntze using thidiazuron. Plant Growth Regulation 26: 57-
61.
Mozafar, A. and Oertli, J.J. (1993). Vitamin-C (ascorbic-acid): uptake and metabolism by
soybean. Journal of Plant Physiology 141: 316-321.
Muller, A.J. and Grafe, R. (1978). Isolation and characterization of cell lines of Nicotiana
tabacum lacking nitrate reductase. Molecular and General Genetics 161: 67-76.
Muller-Moule, P., Conklin, P.L. and Niyogi, K.K. (2002). Zeaxanthin deficiency enhances
the high light sensitivity of an ascorbate deficient mutant of Arabidodpsis. Plant
Physiology 133: 1-13.
Munns, R. (2002). Comparative physiology of salt and water stress. Plant, Cell and
Environment 25: 239-250.
Munns, R. and James, R.A. (2003). Screening methods for salt tolerance: a case study with
tetraploid wheat. Plant and Soil 253: 201-218.
Murashige, T. (1974). Plant propagation through tissue culture. Annual Review of Plant
Physiology 25: 135-166.
152
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiologia Plantatrum 15: 473-497.
Murthy, B.N.S., Murch, S.J. and Saxena, P.K. (1995). Thidiazuron induced somatic
embryogenesis in intact seedling of peanut (Arahis hypogaea L.) Endogenous growth
regulator level and significance of cotyledons. Physiolgia Plantarum 94: 268-276.
Muscolo, A., Sidari, M., Panuccio, M.R. (2003). Tolerance of kikuyu grass to long-term salt
stress is associated with induction of antioxidant defences. Plant Growth regulation
41: 57-62.
Muthukumarasamy, M., Dutta-Gupta, S. and Panneerselvam, R. (2000). Enhancement of
peroxidase, polyphenol oxidase and superoxide dismutase activities by triadimefon in
NaCl stressed Raphanus sativus L. Biologia Plantarum 43: 317-320.
Mutlu, S., Atici, O. and Nalbantoglu, B. (2009). Effect of salicylic acid and salinity on
apoplastic antioxidant enzymes in two wheat cultivars differing in salt tolerance.
Biologia Plantarum 53: 334-338.
Nabors, M.W. (1990). Environmental stress Resistance. In: Plant cell lines selection,
procedures and applications. Dix, P.J. (Eds), Weinheim, New York, Basel
Cambridge, pp. 168-185.
Nabors, M.W. and Dykes, T.A. (1985). Tissue culture of cereal cultivars with increase of
salt, drought and acid tolerance. In: Biotechnology, International Rice Research
Institute Manila, Philippines, pp.121-138.
Nabors, M.W., Gibbs, G.E., Bernstein, C.S. and Meis, M.E. (1980). NaCl-tolerant tobacco
plants from cultured cells. Z. Pflanzenphysiol 97: 13-17.
Nabors, M.W., Kroskey, C.S and McHugh, D.M. (1982). Green spots are predictors of high
callus growth rates and shoot formation in normal and in salt-stressed tissue cultures
of oat (Avena sativa L.). Z. Pflanzenphysiol 105: 341-349.
153
Nagib, A., Hossain, S.A., Alam, M.F., Hossain, M.M., Islam, R. and Sultana, R.S. (2003).
Virus free potato tuber seed production through meristem culture in tropical Asia.
Asian Journal of Plant Sciences 2: 616-622.
Nakano, Y. and Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate-specific
peroxidase in spinach chloroplast. Plant Cell Physiology 22: 867-880.
Nasir-ud-Din, S. (2006). In vitro propagation of elite indigenous potato (Solanum tuberosum
L.) Var. indurkani. Bangladesh Journal of Plant Sciences 1: 212-216.
Nasrin, S., Hossain, M.M., Khatun, M.F., Alam and Mondal, M.R.K. (2003). Induction and
evaluation of somaclonal variation in potato (Solanum tuberosum L.). On line Journal
of Biological Sciences 3: 183-190.
Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D. and Hancock, J.T. (2002). Hydrogen
peroxide and nitric oxide as signalling molecules in plants. Journal of Experimental
Botany 53: 1237-1247.
Niknam, V., Bagherzadeh, M., Ebrahimzadeh, H. and Sokhansanj, A. (2004). Effect of NaCl
on biomass and contents of sugars, proline and proteins in seedlings and leaf explants
of Nicotiana tabacum grown in vitro. Biologia Plantarum 48: 613-615.
Niknam, V., Razavi, N., Ebrahimzadeh, H. and Sharifizadeh, B. (2006). Effect of NaCl on
biomass, protein and proline contents, and antioxidant enzymes in seedlings and calli
of two Trigonella species. Biologia Plantarum 50: 591-596.
Noctor, G. and Foyer, C.H. (1998). Ascorbate and glutathione: Keeping active oxygen under
control. Annual Review of Plant Physiology and Plant Molecular Biology 49: 249-
279.
Ochatt, S. J., Marconi, P.L., Radice, S., Arnozis, P.A. and Caso, O.H. (1999). In vitro
recurrent selection of potato: production and characterization of salt-tolerant cell lines
and plants. Plant Cell, Tissue and Organ Culture 55: 1-8.
154
Ochatt, S.J., Power, J.B. (1989). Selection of salt and drought-tolerance in protoplast-and
explants-derived tissue cultures of colt cherry (Prunus avium × pseudocerasus). Tree
Physiology 5: 259-266.
Omidi, M. and Shahpiri, A. (2003). Callus induction and plant regeneration in vitro in potato.
Acta Horticulture (ISHS) 619: 315-322.
Ostroshy, M., Nazarian, F. and Struik, P.C. (2009). Effect of temperature fluctuation during
in vitro phase on in vitro micro-tuber production in different cultivars of potato
(Solanum tuberosum L.). Plant Cell, Tissue and Organ Culture 98: 213-218.
Ozkaynak, E. and Samanci, B. (2005). Yield and yield componants of greenhouse, field and
seed bed grown potato (Solanum tuberosum L.) plantlets. Akdeniz Universitesi Ziraat
Fakultesi Dergisi 18: 125-129.
Padh, H. (1990). Cellular functions of ascorbic acid. Biochemistry and cell Biology 68:
1166-1173.
Paek, K.Y., Chandler, S.F. and Thorpe, T.A. (1988). Physiological effects of Na2SO4 and
NaCl on callus cultures of Brassica campestris (Chinese cabbage). Physiologia
Plantarum 72: 160-166.
Palma, F., Lluch, C., Iribarne, C., Garrido-Garcia, J.M. and Tejera-Garcia, N.A. (2009).
Combined effect of salicylic acid and salinity on some antioxidant activities,
oxidative stress and metabolic accumulation in Phaseolus vulgaris. Plant Growth
Regulation 58: 307-316.
Panda, S.K. and Upadhyay, R.K. (2004). Salt stress injury induces oxidative alteration and
antioxidative defense in the roots of Lemna minor. Biologia Plantarum 48: 249-253.
Pandey, R. and Ganapathy, P.S. (1984). Isolation of sodium chloride-tolerant callus line of
Cicer arietinum L. Plant Cell Reports 3: 45-47.
Pareek, A., Singla, S.L. and Grover, A. (1997). Salt responsive proteins/genes in crop plants.
In: Strategies for improving salt tolerance in higher plants. Jaiwal, P.K. Singh, R.P.
155
and Gulati, A. (Eds), Oxford and IBH, Publication Corporation. New Delhi, pp. 365-
391.
Parida, A., Das, A.B. and Das, P. (2002). NaCl stress causes changes in photosynthetic
pigments, proteins and other metabolic components in the leaves of a true mangrove,
Bruguiera parviflora, in hydroponic cultures. Journal of Plant Biology 45: 28-36.
Parida, A.K., Das, A.B., Mittra, B. and Mohanty, P. (2004). Salt-stress induced alterations in
proteins profile and protease activity in the mangrove. Bruguiera parviflora
Zeitschrift für Naturforschung, Z. Naturforsch 59: 408-414.
Parida, K.A. and Das, A.B. (2005). Salt tolerance and salinity effects on plants. Ecotoxicol
and Environment Safety 60: 324-349.
Pastuglia, M., Roby, D., Dumas, C. and Cock, J.M. (1997). Rapid induction by wounding
and bacterial infection of an S gene family receptor like kinase gene in Brassica
oleracea. Plant Cell 9: 49-60.
Patnaik, J. and Debata, B.K. (1997). In vitro selection of NaCl-tolerant callus lines of
Cymbopogon martini (Roxb.) wats. Plant Science 124: 203-210.
Pereira, J.E.S. and Fortes, G.R. (2003). Protocol for potato propagative material production
in liquid medium. Pesquisa Agropecuaria Brasileira 38: 1035-1043.
Perl-Treves, R. and Galun, E. (1991). The tomato Cu, Zn superoxide dismutase genes are
developmentally regulated and respond to light and stress. Plant Molecular Biology
17: 745-760.
Petrova, A. and Dedicova, B. (1992). Somatic embryogenesis in Solanum tuberosum L. cv.
Desiree from unripe zygotic embryos. Journal of Plant Physiology 139: 539-542.
Pijut, P.M., Michler, C.H. and Voclker, T.M. (1991). Effect of embryo explants orientation,
thidiazuron, and agar on eastern white pine (Pinus strobus L.) adventitious shoot
initiation. Proceeding of international Symposium on application of biotechnology to
tree culture, protection and utilization. Columbus OH, pp. 126.
156
Piqueras, A., Hernandez, J., Olmos, E., Hellin, E. and Sevilla, F. (1996). Changes in
antioxidant enzymes and organic solutes associated with adaptation of citrus cells to
salt stress. Plant Cell, Tissue and Organ Culture 45: 53-60.
Pospisilova, J., Synkova, H., Haisel, D., Catsky, J., Wilhelmova, N. and Sramek, F. (1999).
Effect of elevated CO2 concentration on acclimation of tobacco plantlets to ex vitro
conditions. Journal of Experimental Botany 50: 119-126.
Potluri S, D.P. and Devi-Prasad, P.V. (1993). Influence of salinity on axillary bud cultures of
six lowland varieties of potato (Solanum tuberosum L.). Plant Cell, Tissue and Organ
Culture 32: 185-191.
Potluri S, D.P. and Devi-Prasad, P.V. (1994). Salinity effects on in vitro performance of
some cultivars of potato (Solanum tuberosum L.). Revista Brasileira Fisiologia
Vegetal 6: 1-6.
Preece, J.E. and Sutter, E.G. (1991). Acclimatization of micropropagated plants to the
greenhouse and field. In: Micropropagation. Technology and Application. Debergh,
P.C.and Zimmerman, R.H. (Eds), Kluwer Academic Publishers, Dordrecht-Boston-
London, pp. 71-93.
Queiros, F., Fidalgo, F., Santos, I. and Salema, R. (2007). In vitro selection of salt-tolerant
cell lines in Solanum tuberosum L. Biologia Plantarum 51: 728-734.
Qureshi, A. and John, I. (1985). Organogenetic callus formation from various potato
explants. Pakistan Journal of Agriculture Research 6: 271-273.
Racusen, D. and Johnstone, D.B. (1961). Estimation of protein in cellular material. Nature
191: 292-493.
Rahnama, H. and Ebrahimzadeh, H. (2004). The effect of NaCl on proline accumulation in
potato seedlings and calli. Acta Physioloia Plantarum 26: 263-270.
157
Rahnama, H., Ebrahimzadeh, E. and Ghareyazie, B. (2003). Antioxidant enzymes responses
to NaCl stress in calli of four potato cultivars. Pakistan Journal of Botany 35: 579-
586.
Rai, V.K., Sharma, S.S. and Sharma, S. (1986). Reversal of ABA-induced stomatal closure
by phenolic compounds. Journal of Experimental Botany 37: 129-134.
Ranalli, P. (1997). Innovative propagation methods in seed tuber multiplication programmes.
Potato Research 40: 439-453.
Rashid, A., Qureshi, R.H., Hollington, P.A. and Wyn-Jones, R.G. (1999). Comparative
responses of wheat (Triticum aestivum L.) cultivars to salinity at the seedling stage.
Journal of Agronomy and Crop Science 182: 199-207.
Rashid, V.A. (2002). Induction of multiple shoots by thidiazuron from caropsis cultures of
minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of
embryogenic callus cultures. Plant Cell Reports 21: 9-13.
Raskin, I. (1992). Role of salicylic acid in plants. Annual Review of Plant Physiology and
Plant Molecular Biology 43: 439-463.
Reddy, P.J. and Vaidyanath, K. (1986). In vitro characterization of salt-stress effects and the
selection of salt-tolerant plants in rice (Oryza sativa L.) Theoretical and Applied
Genetics 71: 757-760.
Rehman, M.S., Matsumuro, T., Miyake, H. and Takeoka, Y. (2000). Salinity-induced
ultrstructural alterations in leaf cells of rice (Oryza sativa L.). Plant Production
Science 3: 422-429.
Roca, W., Roca, M.R. and Bryam, J.E. (1978). A tissue culture method for the rapid
propagation of potatoes. American Potato Journal 55: 691-701.
Rodriguez, P., Dell-Amico, J., Morales, D., Sanchez-Blanco, M.J. and Alarcon, J.J. (1997).
Effects of salinity on growth shoot water relations and root hydraulic conductivity in
tomato plants. Journal of Agricultural Science 128: 439-444.
158
Rosell, G., De-Bertoldi, F.G. and Tizio, R. (1987). In vitro mass tuberization as a
contribution to potato micropropagation. Potato Research 30: 111-116.
Sabbah, S. and Tal, M. (1990). Development of callus and suspension cultures of potato
resistant to NaCl and mannitol and their responses to stress. Plant Cell, Tissue and
Organ Culture 21: 119-128.
Sairam, R.K. and Srivastava, G.C. (2002). Changes in antioxidant activity in sub-cellular
fractions of tolerant and susceptible wheat genotypes in response to long-term salt
stress. Plant Science 162: 897-904.
Sairam, R.K., Rao, K.V. and Srivastava, G.C. (2002). Differential response of wheat
genotypes to long term salinity stress in relation to oxidative stress, antioxidant
activity and osmolyte concentration. Plant Science 163: 1037-1046.
Sairam, R.K., Srivastava, G.C., Agarwal, S. and Meena, R.C. (2005). Difference in
antioxidant activity in response to salinity stress in tolerant and susceptible wheat
genotypes. Biologia Plantarum 49: 85-91.
Sajid, Z.A., Aftab, F. (2009). Amelioration of salinity tolerance in Solanum tuberosum L. by
exogenous application of ascorbic acid. In vitro Cellular and Developmental Biology-
Plant 45: 540-549.
Sawada, H., Shim, I.S. and Usui, K. (2006). Induction of benzoic acid 2-hydroxylase and
salicylic acid biosynthesis-modulation by salt stress in rice seedlings. Plant Science
171: 263-270.
Scandalios, J.G. (1993). Oxygen stresses and superoxide dismutases. Plant Physiology 101:
7-12.
Seabrook, J.E.A. and Douglass, L.K. (2001). Somatic embryogenesis on various potato
tissues from range of genotype and ploidy levels. Plant Cell Reports 20: 175-182.
159
Seabrook, J.E.A., Douglass, L.K. and Tai-George, C.C. (2001). Segregation for somatic
embryogenesis on stem-internode explants from potato seedlings. Plant Cell, Tissue
and Organ Culture 65: 69-73.
Seckin, B., Sekmen, A.H. and Turkan, İ. (2009). An enhancing effect of exogenous mannitol
on the antioxidant enzyme activities in roots of wheat under salt stress. Plant Growth
Regulation 28: 12-20.
Senaratna, T., Touchell, D., Bunn, E. and Dixon, K. (2000). Acetyl salicylic acid (Asprin)
and salicylic acid induce multiple stress tolerance in bean and tomato plants. Plant
Growth Regulation 30: 157-161.
Shaddad, M.A. (1990). The effect of proline application on the physiology of Raphanus
sativus plants grown under salinity stress. Biologia Plantarum 32: 104-112.
Shah, J. (2003). The salicylic acid loop in plant defense. Current Opinion in Plant Biology 6:
365-371.
Shah, K., Russinova, E., Gadella, T.W.J., Willemse, J. and De-Vries, S. (2002). The
Arabidopsis kinase-associated protein phosphatase controls internalization of the
somatic embryogenesis receptor kinase 1. Genes Development 16: 1707-1720.
Shakirova, F.M., Sakhabutdinova, A.R., Bezukova, M.V., Fatkhutdinova, R.A. and
Fatkhutdinova, D.R. (2003). Changes in the hormonal status of wheat seedlings
induced by salicylic acid and salinity. Plant Science 164: 317-322.
Shalata, A. and Neumann, P.M. (2001). Exogenous ascorbic acid (vitamin C) increases
resistance to salt and reduce lipid peroxidation. Journal of Experimental Botany 52:
2207-2211.
Shalata, A. and Tal, M. (1998). The effect of salt-stress on lipid peroxidation and
antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative
Lycopersicon pennellii. Physiologia Plantarum 104: 169-174.
160
Shankhdhar, D., Shankhdhar, S.C., Mani, S.C. and Pant, R.C. (2000). In vitro selection for
salt-tolerance in rice. Biologia Plantarum 43: 477-48.
Sharma, S.K., Bryan, G.J., Winfield, M.O. and Millam, S. (2007). Stability of potato
(Solanum tuberosum L.) plant regenerated via somatic embryos axillary bud
proliferated shoots, microtubers and true potato seeds: a comparative phenotypic,
cytogenetic and molecular assessment. Planta 226: 1449-1458.
Shaterian, J., Waterer, D., De-Jong, H. and Tanino, K.K. (2005). Differential stress response
to NaCl salt application in early and late maturing diploid potato (Solanum sp.)
clones. Environmental and Experimental Botany 54: 202-212.
Shepard, J.F. and Totten, R.E. (1977). Mesophyll cell protoplasts of potato-isolation,
proliferation and plant regeneration. Plant Physiology 60: 313-316.
Shibli, R.A., Abu-Ein, A.M and Ajlouni, M.M. (2001). In vitro and in vivo multiplication of
virus-free ‘Spunta’ potato. Pakistan Journal of Botany 33: 35-41.
Shigeoka, S., Takahiro, I., Masahiro, T., Yoshiko, M., Toru, T., Yukinori, Y. and Kazuya, Y.
(2002). Regulation and function of ascorbate peroxidase isoenzymes. Journal of
Experimental Botany 53: 1305-1319.
Shirin, F., Hossain, M., Kabir, M.F., Ray, M. and Sarker, S.R. (2007). Callus induction and
plant regeneration from internodal and leaf explants of four potato (Solanum
tuberosum L.) cultivars. World Journal of Agricultural Sciences 3: 1-6.
Shonjani, S. (2002). Salt sensitivity of rice, maize, sugar beet, and cotton during germination
and early vegetative growth. PhD. Thesis. Institute of Plant Nutrition, Justus Liebig
University, Giessen.
Sibi, M.L. and Fakiri, M. (2000). Androgens et gynogenes, sources de vitrovariation et de
tolerance a la salinite chez I’orge Hordeum vulgare? Secheresse 11: 125-132.
Smirnoff, N. (1993). The role of active oxygen in the response of plants to water deficit and
desiccation. New Phytologist 125: 27-58.
161
Smirnoff, N. (1995). Metabolic flexibility in relation to the environment. In: Environment
and plant metabolism. Smirnoff, N. (Eds) Oxford, Bios Scientific Publishers, pp 1-16.
Smirnoff, N. (1996). The function and metabolism of ascorbic acid in plants. Annals of
Botany 78: 661-669.
Smirnoff, N. (2005). Ascorbate, tocopherol and cartenoids: metabolism, pathway engineering
and functions. In: Antioxidants and reactive oxygen species in plants. Smirnoff, N.
(Eds). Blackwell publishing Ltd., Oxford, UK, pp 53-86.
Smirnoff, N. and Wheeler, G.L. (2000). Ascorbic acid in plants: biosynthesis and function.
Critical Reviews in Plant Sciences 19: 267-290.
Sopory, S., Jacobsen, E., and Nienzel, G. (1978). Production of mono-haploid embryoids of
plantlets in cultures anthers of Solanum tuberosum. Plant Science Letter 12: 47-54.
Sotiropoulos, T.E., Dimassi, K.N., Tsirakoglou, V. and Therios, I.N. (2006). Responses of
two Prunus rootstocks to KCl induced salinity in vitro. Biologia Plantarum 50: 477-
480.
Spooner, D.M. and Salas, A. (2006). Structure, biosystematics, and genetic resources. In:
Handbook of potato production, improvement, and post-harvest management. Gopal,
J. and Paul Khurana, S.M. (Eds). Haworth's Press, Inc., Binghampton, New York, pp.
1-39.
Sreenivasulu, N., Grimm, B., Wobus, U. and Weschke, W. (2000). Differential response of
antioxidant compounds to salinity stress in salt-tolerant and salt-sensitive seedlings of
fox-tail millet (Setaria italica). Physiologia Plantarum 109: 435-442.
Stasolla, C. and Yeung, E. (2007). Cellular ascorbic acid regulates the activity of major
peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic
embryos. Plant Physiology and Biochemistry 45: 188-198.
Stevens, J., Senaratna, T. and Sivasithamparam, K. (2006). Salicylic acid induces salinity
tolerance in tomato (Lyycopersicon esculentum cv. Roma): associated changes in gas
162
exchange, water relations and membrane stabilization. Plant Growth Regulation 49:
77-83.
Streb, P. and Feierabend, J. (1996). Oxidative stress responses accompanying photo-
inactivation of catalase in NaCl-treated rye leaves. Botanica Acta 109: 125-132.
Struik, P.C. and Wiersema, S.G. (1999). Seed potato technology. Wageningen, The
Netherlands: Wageningen Press, pp 383.
Subhashini, K. and Reddy, G.M. (1989). In vitro selection for salinity and regeneration of
plants in rice. Current Science 58: 584-586.
Sutter, E.G. (1988). Stomatal and cuticular water loss from apple, cherry, and sweet gum
plants after removal from in vitro culture. Journal of American Society of
Horticultural Science 133: 234-238.
Svetek, J., Borut, K., Barbara, V. and Milan, S. (1999). Lateral domain diversity in
membranes of callus and root cells of potato as revealed by EPR spectroscopy.
Physiologia Plantarum. 105: 499-505.
Swapna, T.S. (2003). Salt stress induced changes on enzyme activities during different
developmental stages of rice (Oryza sativa L.). Indian Journal of Biotechnology 138:
251-258.
Szabolcs, I. (1994). Soil resilience and sustainable land use. CABI, Wallingford, Oxon, UK.
Tal, M. (1996). Somaclonal variation for salt tolerance in tomato and potato. In:
Biotechnology in agriculture and forestry. Bajaj Y.P.S (Eds) vol. 3: Somaclonal
variation in crop improvement II. Springer-Verlag, Berlin, pp. 132-145.
Tari, I., Csiszar, J., Szalai, G., Horvath, F., Pecsvaradi, A. and Kiss, G. (2002). Acclimation
of tomato plants to salinity after a salicylic acid pre-treatment. Acta Biologia
Szegediensis. 46: 55-60.
163
Tasgin, E., Atici, O. and Nalbantoglu, B. (2003). Effect of salicylic acid and cold on freezing
tolerance in winter wheat leaves. Plant Growth Regulation 41: 231-236.
Tasgin, E., Atici, O., Nalbantoglou, B. and Popova, L.P. (2006). Effects of salicylic acid and
cold treatments on protein levels and on the activities of antioxidants enzymes in the
apoplast of winter wheat leaves. Phytochemistry 67: 710-715.
Teixeira, J. and Pereira, S. (2007). High salinity and drought act on an organ-dependent
manner on potato glutamine synthetase expression and accumulation. Environmental
and Experimental Botany 60: 121-126.
Tester, M. and Davenport, R. (2003). Na+ tolerance and Na+ transport in higher plants.
Annals of Botany 91: 503-527.
Thomas, T.D. (2003). Thidiazuron induced multiple shoot induction and plant regeneration
from cotyledonary explants of mulberry. Biologia Plantarum 46: 529-533.
Thomas, T.D. and Puthur, J.T. (2004). Thidiazuron induced high frequency shoot
organogenesis in callus from Kigelia pinnata L. Botanical Bulletin of Academia
Sinica 45: 307-313.
Torabi, F., Majad, A. and Ehsanpour, A.A. (2008). Plant regeneration from cell suspension
culture of potato (Solanum tuberosum L.). Pakistan Journal of Biological Sciences
11: 778-782.
Tsugane, K., Koboyashi, K., Niwa, Y., Ohba, Y., Wada, K. and Koboyashi, H. (1999). A
recessive Arabidopsis mutant that grows photo-autotrophically under salt stress
shows enhanced active oxygen detoxification. The Plant Cell 11: 1195-1206.
Turhan, H. (2004). Callus induction and growth in transgenic potato genotypes. African
Journal of Biotechnology 3: 375-378.
Uprety, D.C. and Sarin, M.N. (1975). Physiological study of salt-tolerance in Pisum sativum
L. II. Mechanism of salt action during germination. Acta Agronomy. Academy of
Sciences Hungary 24: 188-191.
164
Vajrabhaya, M., Thanapaisal, T. and Vajrabhaya, T. (1989). Development of salt-tolerant
lines of KDML and LPT rice cultivars through tissue culture. Plant Cell Reports 8:
411-414.
Van-Nieuwkerk, J.P., Zimmerman, R.H. and Fordham, I. (1986). Thidiazuron stimulation of
apple shoots proliferation in vitro. Hort-Science 21: 516-518.
Van-Swaaij, A.C., Jacobsen, E., Kiel, J.A.K.W. and Feenstra, W.J. (1986). Selection,
characterization and regeneration of hydroxyl proline resistant cell lines of Solanum
tuberosum: Tolerance to NaCl and freezing stress. Physiologia Plantarum 68: 359-
366.
Vargas, T.E., De-Garcia, E. and Oropeza, M. (2005). Somatic embryogenesis in Solanum
tuberosum from cell suspension cultures: histological analysis and extracellular
protein patterns. Journal of Plant Physiology 162: 449-456.
Veljovic-Jovanovic, S.D., Pignocchi, C., Noctor, G. and Foyer, C.H. (2001). Low ascorbic
acid in the vtc1 mutant of Arabidopsis is associated with decrease growth and
intracellular redistribution of the antioxidant system. Plant Physiology 127: 426-435.
Vinocur, B. and Altman, A. (2005). Recent advances in engineering plant tolerance to abiotic
stress: achievements and limitations. Current Opinion in Biotechnology 16: 1-10.
Vinterhalter, D., Vinterhalter, B., Calovia, M. (1996). The relationship between sucrose and
cytokinin in the regulation of growth and branching in potato cv. Desiree shoot
cultures. Acta Horticulturae 319-323.
Visser, C., Qureshi, J.A., Gill, R., Malik, K.A. and Saxena, P.K. (1992). Morpho-regulatory
role of thidiazuron: Substitution of auxin and cytokinins requirement for the
induction of somatic embryogenesis in geranium hypocotyls culture. Plant
Physiology 99: 1704-1707.
Vreugdenhil, D., Boogaard, Y., Visser, R.G.F. and De-Bruijin, S.M. (1998). Comparison of
tuber and shoot formation from in vitro cultured potato explants. Plant Cell, Tissue
and Organ Culture 53: 197-204.
165
Wahid, A. and Close, T.J. (2007). Expression of dehydrins under heat stress and their
relationship with water relations of sugarcane leaves. Biologia Plantarum 51: 104-
109.
Wahid, A., Perveen, M., Gelani, S. and Basra, S.M.A. (2007). Pretreatment of seed with
H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage
and expression of stress-proteins. Journal of Plant Physiology 164: 283-294.
Wang, D. and Zhang, N. (2002). Studies on the single cell culture and plant regeneration of
potato. In: High technology and potato industry. Chen, Y and Qu, D. (Eds), pp. 1-9.
Wang, Y. and Nil, N. (2000). Changes in chlorophyll, ribolose bisphasphate carboxylase-
oxygenase, glycine betaine contents, photosynthesis and transpiration in Amaranthus
tricolor leaves during salt stress. Journal of Horticulture Sciences and Biotechnology
75: 623-627.
Wattimena, G., McCown, B. and Weis, G. (1983). Comparative field performance of
potatoes from microculture. American Potato Journal 60: 27-33.
Yasmin, S., Nasiruddin, K.M., Begum, R. and Talukder. S.K. (2003). Regeneration and
establishment of potato plantlet through callus formation with BAP and NAA. Asian
Journal of Plant Sciences 2: 936-940.
Yildirim, E., Turan, M. and Guvene, I. (2008). Effect of foliar salicylic acid applications on
growth, chlorophyll, and mineral content of cucumber grown under salt stress.
Journal of Plant Nutrient 31: 593-612.
Yoshida, S., Ogawa, M., Suenaga, K. and Ye, H.C. (1983). Induction and selection of salt
tolerant mutant rice by tissue culture: Recent progress at IRRI. In: Cell and Tissue
Culture Techniques for Cereal Crop Improvement. Science Press, Beijing, pp. 237-
254.
Yu, B.P. (1994). Cellular defenses against damage from reactive oxygen species. Physiology
Review 74: 139-162.
166
Yusnita, S., Geneve, R.L. and Kester S.T. (1990). Micropropagation of white flowering
eastern redbud (Cercis Canadensis var. alba L.). Journal of Environment and
Horticulture 8: 177-179.
Zel, J., Mlakar, M.M., Vilhar, B., Grill, D. and Guttenberger, H. (1999). The efficient
regeneration of the potato (Solanum tuberosum L.) cv. Igor in vitro. Special issue: 2nd
Slovenian symposium on plant physiology with international participation, Gozd
Martuljek, Slovenia, September 30-October 2. 1998. Phyton (Horn) 39: 277-282.
Zhang, J. and Kirkham, M.B. (1994). Drought stress induced changes in activities of
superoxide dismutase, catalase and peroxidase in wheat species. Plant Cell
Physiology 35: 785-791.
Zhang, N. and Dai, Z.X. (2000). Studies on the establishment of high quality cell suspension
culture in potato. Chinese Potato Journal 14: 195-197.
Zhu, D. and Scandalios, J.G. (1994). Differential accumulation of manganese-superoxide
dismutase transcripts in maize in response to abscisic acid and high osmoticum. Plant
Physiology 106: 173-178.
Zhu, J.K. (2001). Plant salt tolerance. Trends in Plant Science 6: 66-71.
Zimmerman, T.W., Williams, K., Joseph, L., Wiltshire, J. and Kowalski, J.A. (2007).
Rooting and acclimatization of cassava (Manihot esculenta) ex vitro. Acta
Horticultureae (ISHS) 738: 735-740.
167
Annexure: 1
Formulation of MS Medium (Murashige and Skoog, 1962) for the Preparation of Stock
Solutions
A) Macronutrients (20x)
Components Stocks Final concentration in MS
Medium (NH4) NO3 20 × 1650 = 33000 mg/L 1650 mg/L
KNO3 38000 1900 MgSO4.7H2O 7400 370
KH2PO4 3400 170 CaCl2.2H2O 8800 440
B) Micronutrients (100x)
MnSO4.4H2O 22.3 × 100 = 2230 mg/L 22.3 mg/L ZnSO4.7H20 860 8.6 H3BO3.7H2O 620 6.2
KI 83 0.83 Na2MoO4.2H2O 25 0.25
CuSO2.5H2O 2.50 0.025 CoCl2.6H2O 2.50 0.025
C) Vitamins (200x)
Glycine 2 × 200 = 400 mg/L 2.0 mg/L Nicotinic acid 100 0.5
Pyridoxine HCl 100 0.5 Thiamine HCl 20 0.1
D) Iron (200x)
Na2EDTA.2H2O 33.6 × 200 = 6720 36.2 FeSO4.7H2O 5560 27.8
E) Myo-inositol (100x) Myo-inositol 100 × 100 = 10000 100
168
Annexure: 2
Preparation of Stock Solutions for MS (Murashige and Skoog, 1962) Medium
A) Macronutrients
Macronutrients stock for MS medium was prepared at the final concentration of 20x
(Annexure 1, section A). All the salts were weighed individually and dissolved separately in
distilled water. Then they were mixed together in a conical flask already containing an
appropriate amount of distilled water so as to avoid precipitation. Calcium chloride was
added at the last otherwise it always forms precipitation. The solution was then transferred to
a 1000 ml capacity volumetric flask to make up the final volume.
B) Micronutrients
Stock solution of micronutrients was prepared 100 times more concentrated than the
final volume (100x). All the salts of micronutrients as given in Annexure 1, under section
“B” were weighed and dissolved separately and made up to the final volume as described
above in section A.
C) Fe-EDTA
Iron EDTA stock solution was prepared at a concentration of 200x. The salts for this
stock solution are given in Annexure 1, section C. The prepared 200x stock was poured in an
amber-colored bottle and stored in refrigerator. For the preparation of 1liter of MS medium,
5 ml of this stock solution was used.
D) Vitamins
Vitamins of MS medium were prepared as 200x. Separately dissolved vitamins (as
given in Annexure 1, section D) were transferred to a 500 ml volumetric flask and final
169
volume was made with distilled water. For the preparation of 1 liter medium, 5 ml of vitamin
stock was used.
E) Myo-inositol
Stock solution of myo-inositol was prepared separately as 100x. It was prepared by
dissolving 10 g of myo-inositol in 1000 ml of distilled water and 10 ml of this stock was
taken for 1 L MS medium.
Annexure: 3
Preparation of Stock Solutions of Growth Regulators
Auxins (2, 4-D, NAA, IBA etc.) were dissolved initially in a little quantity of 0.1 N
NaOH while the initial solvent for cytokinins (BAP, TDZ etc.) was 0.1 N HCl. Once
dissolved, the final volume was made up with distilled water in an appropriate volumetric
flask and stored at 4°C in refrigerator till use.
Annexure: 4
Preparation of 1 liter MS Medium
One liter MS medium for callus induction and proliferation was prepared
in a manner given below.
Medium Components Volume of Stock solution
1) Macronutrients 50 ml/L
2) Micronutrients 10
3) Vitamins 05
4) Myo-inositol 10
5) Iron-EDTA 05
6) Sugar 30 g/L
7) Agar (Oxoid, Hampshire, England) 7 g/L
8) pH 5.8
170
Annexure: 5
Preparation of Reagents for the Estimation of Peroxidase, Catalase and Superoxide
dismutase
Reagents for Peroxidase Estimation
a) Guaiacol (20 mM)
It was prepared by dissolving 0.240 ml in a small amount of water and then volume
raised up to 100 ml.
b) H2O2 (12.3 mM)
Prepared by dissolving 0.14 ml of 30% H2O2 in water and raise volume up to 100 ml.
These solutions were always prepared fresh.
Reagents for Catalase Estimation
a) Reagent A (50 mM Potassium Phosphate Buffer, pH 7.0 at 25°C)
Prepared 200 ml in deionized water using Potassium Phosphate. Adjusted to pH 7.0
at 25°C using 1 M KOH.
b) Reagent B [Substrate Solution: 0.036 % (w/w) Hydrogen Peroxide (H2O2) Solution]
Prepared in Reagent A using Hydrogen Peroxide, 35% (w/w). Determined the A240nm
of this solution using Reagent A as a blank. The A240nm should be between 0.550 and 0.520
absorbance units. Added hydrogen peroxide to increase the absorbance and Reagent A to
decrease the absorbance.
c) Reagent C (Catalase Solution)
Immediately before use, prepared a solution containing 50-100 units per ml in cold
Reagent A.
171
Reagents for Superoxide dismutase Estimation
a) Phosphate buffer (pH 7.8)
Dissolved 6.9 g NaH2PO4.H2O in 900 ml distilled water and adjusted to pH 7.8 by
10% NaOH. Final volume was made up to 1 liter with distilled water.
b) Riboflavin solution
Dissolved 7.5 mg of riboflavin in 100 ml distilled water. It was always prepared fresh
and kept in darkness.
c) Sodium cyanide
Dissolved 13 g sodium cyanide in 1 liter distilled water.
d) Nitroblue tetrazolium (NBT): (Prepared fresh and kept in darkness)
Dissolved 137 mg NBT in 10 ml distilled water.
e) Methionine: (Prepared fresh and kept in darkness)
Dissolved 14.9 mg methionine in 10 ml phosphate buffer.
f) EDTA
Dissolved 245 mg of di-sodium salt of EDTA in 10 ml buffer solution.
Preparation of Reaction Mixture
The reaction mixture was prepared as follows.
1. 1 ml NaCN
2. 10 ml methionine
3. 10 ml EDTA
4. 1 ml NBT
5. 1 ml Riboflavin
The final volume was made up to 100 ml with buffer solution. This mixture was prepared
away from a direct light source and kept in a dark bottle.
172
Annexure: 6
Culture Media Used for the Establishment of Cell
Suspension Culture in Solanum tuberosum cvs. Cardinal
and Desiree
Components Quantity
Salts Murashige and Skoog Basal salts
Myo-inositol 100 mg/l
Thiamine 0.1
Pyridoxine 0.5
Nicotinic acid 0.5
Glycine 2.0
Yeast extract 1000
Sucrose 25000
Citric acid 50
Ascorbic acid 50
Kinetin 0.5
2, 4-D 0.5
pH 5.7-5.8
173
Annexure: 7
Composition of AA (Muller and Grafe, 1978) Medium
Components Stock Final concentration in the medium
A. Macronutrient (100x)
CaCl2. 2H2O 44 g/L 440 mg/L
KH2PO4 17 g/L 170 mg/L
MgSO4.7H2O 37 g/L 370 mg/L
B. Micronutrient (100x)
MnSO4. H2O 1.69 g/L 16.9 mg/L
ZnSO4. 7H2O 860 mg/L 8.6 mg/L
H3BO3 620 mg/L 6.2 mg/L
CuSO4.5H2O 2.5 mg/L 25µg/L
Na2MoO.2H2O 25 mg/L 250 µg/L
CoCl2. 6H2O 2.5 mg/L 25 µg/L
KI 83 mg/L 830 µg/L
C. Iron Stock (100x)
FeSO4.7H2O 2.8 g/L 28 mg/L
Na2 EDTA 3.7g/L 37 mg/L
D. Vitamins
Nicotinic acid - 50 mg/L
Thiamine HCl - 50 mg/L
Pyridoxine HCl - 10 mg/L
Myo-inositol - 10 mg/L
E. Amino acid (20x)
Glutamine 17.7 g/L 877 mg/L
Aspartic acid 5.32 g/L 266 mg/L
174
Arginine 4.56 g/L 288 mg/L
Glycine 1.5 g/L 75 mg/L
KCl was added separately as dry powder: 2940 mg/L
Sucrose: 30 g/L
2, 4 dichlorophenoxyacetic acid: 13.5 µM
Annexure: 8
Composition of Hoagland Solution (Hoagland and Arnon, 1950)
Components Stock (g/L) ml stock solution for 1 litter Hoagland
solution A. Macronutrients
KH2PO4 136 1.0
KNO3 101 5.0
Ca (NO3)2. 4H2O 236 5.0
MgSO4.7H2O 246 2.0
B. Micronutrients
H3BO3 2.86 1.0
MnCl2. 4H2O 1.81 1.0
ZnSO4. 7H2O 0.22 1.0
CuSO4.5H2O 0.80 1.0
H2MoO4.H2O 0.02 1.0
Fe-EDTA 37.33 1.0
![S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase …...S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis1[OPEN] Yanjun Zhang,a,2 Li Zhao,a,2](https://img.pdfslide.us/doc/110x75/5fbb495347cd1d50e62c72c8/s5hdmr6-encodes-a-salicylic-acid-5-hydroxylase-s5hdmr6-encodes-a-salicylic.jpg)
