STUDY OF SOMACLONAL VARIATION IN WHEAT FOR THE INDUCTION OF SALINITY TOLERANCE

By

Saleem Akhtar

Roll No. 12

Thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR of PHILOSOPHY

In

BOTANY

DEPARTMENT OF BOTANY

GC UNIVERSITY, FAISALABAD

March 2011

Declaration The work reported in this thesis was carried out by me under

the supervision of Dr. Mubashir Niaz, Department of Botany, GC

University, Faisalabad, Pakistan.

I hereby declare that the “Study of somaclonal variation in

wheat for the induction of salinity tolerance” and the contents of

thesis are the product of my own research and no part has been

copied from any published source (except the references, standard

mathematical or genetic models / equations/ formulas / protocols

etc.). I further declare this work has not been submitted for award of

any other degree / diploma. The University may take action if the

information provided is found inaccurate at any stage.

Signature of the student Saleem Akhtar

Registration No.: 2005-GCUF-10-113

CERTIFICATE BY THE RESEARCH SUPERVISOR I certify that the contents and form of thesis submitted by

Mr. Saleem Akhtar Roll No.12 has been found satisfactory and

according to the prescribed format. I recommend it be processed for

evaluation by the external examiner for the award of degree.

Signature Name : Dr. Mubashir Niaz Stamp

Chairperson

Dean, Faculty of Science and Technology, GC University, Faisalabad

SUPERVISORY COMMITTEE

Chairman ____________________________ (Dr. Mubashir Niaz) Member ____________________________ (Dr. Muhammad Iqbal) Member ____________________________ (Dr. Naeem Iqbal)

SCRUTINIZING COMMITTEE

1. Dr. Naeem Iqbal _______________________ (Convenar)

2. Dr. Muhammad Iqbal _______________________ (Member)

3. Dr. Syed Hammad Raza _______________________ (Member)

CONTENTS

CHAPTER TITLE PAGE

SUMMARY i

ACKNOWLEDGEMENTS v

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 9

3 MATERIALS AND METHODS 52

4 RESULTS 59

5 DISCUSSION 106

REFERENCES 113

APPENDICES

LIST OF TABLES

Table No TITLE PAGE

1. CALLUS INDUCTION FREQUENCY OF 50 WHEAT VARIETIES, USING MATURE EMBRYO AS EX PLANT SOURCE UNDER DIFFERENT 2,4-D, LEVELS

61

2. RESPONSE OF WHEAT GENOTYPES TO CALLOGENESIS UNDER SALT STRESS

65

3. SALINITY EFFECT ON RFGR OF WHEAT CALLUS 71

4. NECROTIC PERCENTAGE IN CALLUS IN WHEAT GENOTYPES UNDER SALT STRESS.

72

5. REGENERATION POTENTIAL OF 20 WHEAT VARIETIES UNDER SALT STRESS (NO. OF PLANTS)

74

6. FRESH SHOOT WEIGHT OF WHEAT SOMACLONES AS AFFECTED BY SALINITY

78

7. SALINITY EFFECT ON DRY SHOOT WEIGHT OF WHEAT SOMACLONES

79

8. SALINITY EFFECT ON FRESH ROOT WEIGHT OF WHEAT SOMACLONES

81

9. SALINITY EFFECT ON DRY ROOT WEIGHT OF WHEAT SOMACLONES

82

10. EFFECT OF SALINITY ON SODIUM CONCENTRATION (mol m-3) OF 10 WHEAT VARIETIES

84

11. EFFECT OF SALINITY ON K+ CONCENTRATION (mol m-3) IN LEAF SAP OF 10 WHEAT VARIETIES AT DIFFERENT SALT LEVELS

86

12. K+/Na+ OF WHEAT SOMACLONAL AS AFFECTED BY SALINITY

87

13. EFFECT OF SALINITY ON NET PHOTO SYNTHESIS(PN) IN 10 WHEAT SOMACLONES

89

14. STOMATAL CONDUCTANCE (GS) IN10 WHEAT SOMACLONES AS AFFECTED BY SALINITY

90

15. EFFECT OF SALINITY ON PLANT HEIGHT IN 10 WHEAT SOMACLONES ALONGWITH PARENTS

94

16. SALINITY EFFECT ON NO. OF TILLERS IN WHEAT SOMACLONAL

96

17. EFFECT OF SALINITY ON NO. OF SPIKES IN 10 WHEAT SOMACLONES ALONGWITH PARENTS

98

18. SALINITY EFFECT ON NO. OF SPIKELETS IN 10 WHEAT SOMACLONES ALONGWITH PARENTS

100

19. EFFECT OF SALINTY ON 100 GRAIN WEIGHT (G) OF 10 WHEAT SOMACLONES ALONGWITH PARENTS

102

20. YIELD/PLANT (G) OF 10 WHEAT SOMACLONES ALONGWITH PARENTS AS AFFECTED BY SALINITY

104

i

SUMMARY

Selection of favorable somaclonal variants from callus culture are helping

tools to common breeding for the development of stress tolerant plants (Larkin

and Scowcroft, 1981; Dix, 1993; Ashraf, 1994). The inclusion of a variety in

selection program depends on its response to in vitro culture, especially to callus

formation and embryogenic callus production. In fact, for numerous genotypes,

results showed that plant genotypes differed in in vitro culture response (Abe

and Futsuhara, 1984; Mikami and Mirodjagh, 1999).

Salinity is a main factor hampering the crop production in arid and semi-

arid lands in world (Ashraf, 1994). Plant tissue culture methods provide a

promising approach to develop salt resistant plants. In vitro selection of salt

tolerant cultivars and regenerated plants has been reported in different crops

such as rice, barley, wheat and sunflower (Lutts et al; 1999; Sibi and Fakiri,

2000, Barakat and Abdel-Latif, 1996, and Alvarez et al., 2003). This concludes

that tissue culture techniques can be helpful to improve salt tolerance of different

plants.

Initially fifty wheat varieties were screened for their callus induction

potential under four levels of 2,4-D, i.e., 1, 2, 3 and 4 mgL-1. Uqab-2002 and AS-

2000 proved better with 90% callus induction frequency while ufaq-2002 and V-

4189 produced 85 and 80% callus induction frequency respectively. As far as the

dose of 2,4-D is concerned, maximum callus was obtained at the level of 3 mgL-1

2,4-D. Twenty genotypes i.e. (Bhakhar-2002, V-03079 and V-04189 LU-26S,

ii

Pothowar, Punjab-76, Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86

Pasban-90, Inqulab-91, Punjab-96, Uqab-2000, Chenab-70, Iqbal-2000, AS-

2000, Ufaq-2002, SA-42 and Parwaz-94,) having callus induction frequency of

70% or more were further tested for callus formation under four salinity levels,

i.e., 0,50,100 and 150mM NaCl + 3 mgL-1 2,4-D. Salinity adversely affected the

callus weight and Relative Fresh Weight Growth of callus. As the salinity

increased callus masses, Relative Fresh Weight Growth (RFWG) and necrotic

percentage in callus was decreased. Almost all the twenty genotypes produced

the callus of varying degree with and without salt. Among these genotypes V-

04189 produced good callus at 50mM salinity levels, LU-26S at 100mM level

while Uqab-2000 produced maximum calls at 150mM NaCl level. Ten wheat

genotypes, i.e., Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-

2002, Ufaq-2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90 produce granular

and morphogenic callus under salt stress condition, were evaluated for RFWG of

callus under same salinity level. Salinity again reduced the relative fresh weight

growth. The maximum reduction was noted in genotype AS-2002 while Ufaq-

2002 showed minimum reduction in RFWG.

The 20 wheat genotypes which gave good callus formation under salinity

levels were evaluated for their regeneration potential under the same salinity

levels. Once again the effect of salinity was quite dominant on the production of

plantlets. Maximum (48) shoots were developed by LU-26S followed by Pasban-

90(40) while Ufaq-2002 and AS-2000 produced 31 shoots each .These

iii

genotypes developed roots on rooting medium and turned into complete plants.

Then these plants were grown to maturity in sand culture to obtain R0 seed.

Various ions, when present in supraoptimal levels in the medium show

differential effects on the overall plant or cell growth. It is therefore, imperative to

separate toxicity effects of various ions for improvement programs. Among all the

ions, Na+ and Cl- are more damaging to growth of wheat, the seed of wheat

somaclones were sown in plastic buckets having in net house of Agricultural

Biotechnology Research Institute Faisalabad and different parameters at

seedling stage were studied. The growing plants were irrigated at regular interval

of one week with full Hoagland’s nutrient solutions. The plants were subjected to

0,50,100,150 mM levels of salinity using NaCl. The data for various

morphological, physiological and biochemical attributes were recorded

Growths attributes such as root / shoot fresh and dry weight, root / shoot

length, photosynthesis rate and stomatal conductance revealed a significant

decrease under saline conditions in all the somaclones of wheat genotypes.

Salinity also decreased the total yield. Adverse impact of salt stress on growth of

wheat was associated due to increased osmotic potential which in turn was

associated with more concentration of Na+ and less concentration of K+. High

NaCl concentration proved to be toxic for the growth of all the wheat variants. In

the parameters studies, response was similar except minor variation among

genotypes.

The so developed somaclones of various genotypes along with their

mother plants were sown in pots having artificially developed four levels of

iv

salinity, i.e., 0, 4, 8 and 12 dSm-1using NaCl salt to study and compare

agronomic traits among themselves. Here again salinity decreased plant height,

No. of tillers, spikes, spikelets, 100 grain weight and yield per plant. Maximum

100grain weight was observed in Bhakhar-2000(P) followed by AS-2000(P) as

compared to other somaclones/genotypes of wheat. Bhakhar-2000(S) was also

at par with its parent Bhakhar-2002(P). At EC level of 4dSm-1, Inqulab-91(P),

Inqulab-91(S) and Pasban-90(P) were salt tolerant. At EC 8dSm-1, LU-26(P) was

noted as the best tolerant while at EC12dSm-1 Pasban-90(P) was superior to all

other genotypes. As for as yield per plant is concerned somaclones of Ufaq-

2002, AS-2000, Bhakhar-2002 at 4, Punjab-96, Punjab-76, AS-2000, Bhakhar-

2000 at 8 and Punjab-96, Punjab-76, Chenab-70 were found to be salt tolerant

at 12dSm-1 salinity level in yield per plant.

In conclusion, salinity adversely effects the plant growth, ion accumulation,

photosynthetic activities and total yield. NaCl salinity increases Na+ concentration

and decreases K+ concentration. However, somaclones developed having salt

tolerance at different salinity levels can be sown in marginal lands where no other

crop is grown.

v

ACKNOWLEDGEMENTS

I am extremely grateful to Almighty Allah, who enabled me to complete this study.

I express my gratitude to my supervisor, Dr. Mubashir Niaz Chairman,

Department of Botany for his guidance, kindness and helpful suggestions during

the research work. His concerned supervision and generous response to my

difficulties during the research work will never be forgotten.

I offer my thanks to Mr. Makhdoom Hussain, Director Wheat Research

Institute Faisalabad and Dr. Muhammad Zaffar Iqbal, Director Agricultural

Biotechnology Research Institute AARI, Faisalabad for their affectionate

supervision, valuable advices and help in planning and execution of the present

studies and providing all the facilities during the course of my research pursuits.

Without their sincere inspiration and keen interest, the completion of this work

would have been difficult.

I would like to express my deep sense of indebtedness to:

Dr. Iftikhar Ali (late), Dr. Muhammad Iqbal and Dr. Naeem Iqbal for their

cooperation, valuable suggestions during research and for reviewing the

thesis.

Dr. Sajid-ur-Rahman, Economic Botanist and Mr. Muhammad Asif,

Assistant Research Officer for their help extended in the compilation of the

thesis.

Finally I wish to thank my family member for their prayers for my success.

(SALEEM AKHTAR)

1

CHAPTER 1

INTRODUCTION

Salinity in agricultural land is a major problem in many countries of the world.

The extent, distribution and nature of the salt-affected lands of the world is very

imperfectly known for most countries due to lack of standardization of

characterization criteria. About over 800 million hectares of soil in the world are

suffered from both salinity and sodicity (Munns, 2005). Abiotic stresses such as

salinity, water logging, temperature etc. have largely affected crop production at

global level (Taiz and Zeiger, 1998). Among these abiotic stresses salinity is major

factor that effect the plant growth (Russ et al., 2000). The salt effected soils in

Pakistan are 6.3 million hectares (Anonymous, 2003) that include about 2.4 million

hectares in Punjab, 2.1 million hectares in Sindh, 0.5 million hectares in K.P. and

about 1.3 million hectares in the Baluchistan. Out of which 3369.7 thousand

hectares has become totally unproductive due to soil salinity and the productive

lands are still continuously going out of cultivation due to salinity problem (Chaudary,

2001). The presence of high salt concentration and water logging of soils are serious

problems affecting the agricultural production in Pakistan reducing yield by about

25% (Sandhu and Qureshi, 1986). Since many of these soils are beyond the reach

of conventional reclamation techniques, either for economic reasons or for lack of

2

fresh water. A major scientific thrust has been aimed at developing suitable salt, and

water logging tolerant crops to bring these lands into agricultural productivity

( Hollaender, 1979 ). In this respect, an understanding of the plant responses to

various stresses and the mechanisms that make some species or genotypes more

tolerant than others seems essential. The accumulation of toxic ions in the

rhizosphere initially causes injury to plant roots and their presence in aerial parts

cause damage to plant metabolism by reducing growth and yield ( Francois and

Maas, 1999 ). Salt tolerance is a complex phenomenon which varies not only among

species but also depends upon the environmental conditions perpetual irrigation

without proper drainage have caused large scale soils salinity. Salinity may arise as

a result of poor drainage, use of poor quality water, redistribution of salts in soil

profiles, shallow water table and lack of proper soil and water management practices

( Ansari et al., 1998 ). In sodic soils as a rule not only the high salt concentration but

also pH is the stress factor, particularly in cases where a higher contents of sodium

carbonate in the solid and liquid phase is present. The high pH hinders the life

function of crops and limits their productivity. Sodic soils have pH value and highly

exchangeable Na+ and/or often barren sodic soils having poor physical conditions

that badly affect the water and air movement in soils. Sodicity causes impaired

plant growth ( Pessarakli and Szaboles, 1999 ). Salinity can increase concentration

of certain ions that have an inhibitory effect on plant metabolism. It can adversely

effect soil structure such as soil permeability and soil aeration, or diminish physico-

chemical effect ( Evangelous and McDonald,1999).

Man is dependent on field crops for food. In Asia only about 8% of diet is from

3

animal protein and more than 75% comes from cereals, wheat (Triticum aestivum L.)

as it is known today evolved through inter crossing of its related wild ancestors.

Unlike many cereals, wheat is mainly food rather than a food crop ( Stephen and

Carter, 1980 ). The effect of NaCl salinity is very harmful for wheat as well as for

other crops. Salinity suppresses plant growth and suppressions increases as the salt

concentration increases (Sudyova et al., 2002).

Wheat is one of the major staple foods in world. In Pakistan it is grown on an

area of 8.41 million hectares with an annual production of 21.74 million tons

(Anonymous, 2008-9) which is very low compared to world production. It’s

production potential in our country is limited due to various environmental stresses,

particularly salt stress. High population growth rate demands higher yield of wheat in

this country. At present deficit in production/demand ratio is compensated with the

import which is an additional burden to an already meager economy. Generally in

Pakistan, wheat crop is sown from the month of October to end of December, but

the suitable time for sowing is the month of November. Harvesting is done in April

and May. According to Martin & Leonard (1963 ) there are many species of Triticum.

There are three main groups are diploids, tetraploids and hexapliods having 14, 28,

and 42 chromosomes respectively. Wheat plant is exposed to many diseases, insect

pests attack and many abiotic stresses including salt stress which may cause

reduction in yield. The production profile of wheat from salt effected areas is

extremely poor. The yield in these areas can be increased considerably through the

development of salt tolerant wheat varieties. However, consistent efforts are

required to boost in crop production in these lands.

4

It is very difficult, time consuming and tedious to breed salinity tolerant

varieties through conventional means. Where as exploitation of somaclonal

variation in plants for the development of salt tolerant cultivars through tissue

culturing is advantageous over conventional methods. Exploitation of somaclonal

variation is an efficient and potent method for producing novel and useful varieties

(Larkin and Scowcroft, 1981).

Characteristic symptoms of salinity are damage to vegetative growth in

herbaceous and woody plants including a slowing of leaf and shoot extension, low

rate of dry matter accumulation by aerial tissues and conspicuous yellowing of older

(lower)leaves suggesting premature senescence and wilting. Further leaf opening

and leaf shedding are typical responses of some species to salinity stress ( Drew,

1981 ). Sodium chloride significantly reduces root and shoot fresh and dry masses,

root length and shoot length (Zoppo et al., 1999). Growing leaf cells maintain their

turgor pressure in response to the salt stress by accumulating the osmotically active

solutes ( Humayun et al., 1992 ).

There are significant difference in salinity tolerance of plants and the way in

which they partition excess of salts in various parts to sustain growth. The

depressing effect of salinity on root growth is generally less severe than effect on

growth of aerial components (Stem, leaves, and fruits). Root growth is almost always

less effected than shoot growth by increasing soil salinity, so root shoot ratio

generally increases ( Munns and Termaat,1986). Soil and water salinity directly

affects the wheat production. The reduction in growth of many crops plants by

salinity may be due to its effect on dry matter production, ionic relations, water

5

potential, physiological disorders, metabolic reactions or a combination of all these

factors. ( Ashraf et al.,1991; Ashraf and Khan 1993; Ashraf and Khan, 1994 ).

Two approaches are being used to tackle soil stress problem. The first is to

amend the soil conditions to favour crop plants. A variety of approaches can be used

to manage these salt affected soils through well established techniques such as

provision of adequate drainage and use of amendments are available for this

purpose, yet due to limitation of poor quality irrigation water, high cost of

amendments and low soil permeability. The second one is to use the genetic ability

of plants for their adaptability to adverse soil conditions. The first approach is a long

term process and a few success has been seen in our country even after spending

Rs.21 billion up to 1988 ( Aslam et al., 1993B ). However the later is short term

strategy and includes the crop cultivation on the salt effected fields. To employ this

approach an understanding of the plant responses to salinity and its mechanism

that, make some species/genotypes more tolerant than others are essential. Various

varieties of wheat do not equally respond to soil salinity. Some varieties have been

observed very sensitive to soil salinity while others have shown high salt tolerance (

Ashraf and Khanum, 1997 ). Thus there is a need to screen out or evolve wheat

cultivars suitable for cultivation in saline areas. During the last few years, “in-vitro”

selection for salt tolerance has been adopted as a technological solution to salinity

problem. ( Blumwald and Poole, 1987, Khan and Qureshi,1992 ). Cell lines of the

major crops having desirable characters, such as salt resistance, can provide the

base for the regeneration of plants and provide an insight into the mechanism of

tolerance at cellular level.

6

In vitro somatic cell and tissue culture technologies have been developed to

assist plant breeding. During the last few years tissue culture methods have been

emerged that can be under taken to improve the field crops. The cell, tissue and

anther culture as tools to use in the betterment of crop plants has now been

recognized (Green, 1977; Vasil, 1987 ). The regeneration of a complete plant is

possible today from major cereal crops; such as bread wheat, maize, rice, and

barley ( Redway et al., 1990; Vasil et al., 1990; Duncan et al., 1985; Yamada et al.,

1986; and Luhrs & Lorz, 1987 ).

The first successful tissue culture in cereal from endosperm was done by La

Rhu in 1949.. Gamborg & Eleveigh (1968) produced solution cultures of wheat using

a medium comprising of mineral salts supplemented with sucrose, vitamin B and

2,4-D. Shimada et al. (1969) established callus and single cell cultures in wheat.

Since the first report on immature embryo culture of maize (Green and Phillips,

1975) extensive documented reports in a whole range of species ( Larkin and

Scowcroft, 1981) have shown undesirable genetic and cytogenetic variation between

regenerated plants and the starting material. However, some work gave the details

on the production of agriculturally useful in vitro selected traits of agronomic values

(Sibi and Fakiri, 2000 and Alveraz et al.,2003). Regenerated plants varied for a wide

range of agro-morphological characters. The variability associated with cell and

tissue culturing has been somaclonal variation (Larkin and Scowcroft, 1981). They

further told that it might be due to the reflection of heterogeneity between the cells

and explants tissue, or a simple representation of spontaneous mutation, and are

due to the activation by cultural environmental transposition of genetic materials.

7

Clonal variation had been demonstrated many times in cereals and forage

crop for both physiological/biochemical and structural/morphological characteristics.

These changes are typically undesirable, but the occasional appearance of traits not

found in existing population more than compensates for the large populations of

unusable regenerants. Much of the literature simply reports variation following

cloning in tissue culture with little attempt to determine the importance of the

variation or its heritability. Limited research has been conducted to compare

mutation breeding with chemical mutagens or irradiation to generation of variation

through tissue culture. Clonal variation is most useful for breeding programmes

where the desired trait are not known naturally form available germplasm or is not

readily available. Examples are salt tolerance, tolerance to mineral toxicities,

tolerance to various chemicals including herbicides, tolerance to cold or heat,

tolerance to certain pests and pathogens, and variation in useful physiological

characteristics. Much of the useful of clonal variation is that the variations are

generated under in vitro conditions, which allows for in vitro selection among millions

of cells for specific characters. Large scale field screening of plants and progeny is

usually limited by available space and labour. Clonal variation in general also has

less deleterious effects on mutated plants than chemical mutation or irradiation.

Clonal variation also can be combined with the use of chemical mutagens in culture

or irradiation of explant materials before placing in tissue culture. The amount of

colonal variation among culture varieties and lines within a crop varies greatly, with

some germ plasm highly variable and some tissue culture stable. High variability in

tissue culture appears to be a heritable trait (Ahloowalia, 1982).The amount of clonal

8

variation in a crop should be greatly increased by including highly variable germ

plasm in crosses, by culturing explants from F1 or F2 plants from these crosses, and

by including chemical mutagens in the tissue culture medium or by irradiating

explants before plating.

This study is mainly is related to understand the possible useful mechanisms

which may be found in both wheat and wheat-somaclones that may be exploited to

facilitate the process of breeding or salinity tolerance. The major objectives of the

study were:

1. Exploitation of somaclonal variation for creation of genetic variability and

induction of salt tolerance.

2. Determination of salt tolerance mechanism (s) in newly developed

somaclones.

3. Development of multiple salt tolerant variants for making suitable cultivars in

all kinds of salt affected areas.

The thesis being presented is written in accordance with the rules of the G.C.

University. Faisalabad and divided / subdivided in various sections which sometimes

appear repetitive. This has done with the hope that it makes the thesis better

understandable for the readers.

9

CHAPTER 2

REVIEW OF LITERATURE

The response of plants to soil salinity is of great concern to the

Agriculturists. To meet the food requirements of the human beings, problem of

salinity become more severe every year as the non-saline soils and non-saline

waters are being exploited more intensively. Further, extension of agriculture is

imperative through the cultivation on saline soils using irrigation water with

relatively higher soluble salts and sodium absorption ratio (SAR).

Salts may affect plant growth directly through disturbing the water and

nutrient balance of soils and plants, causing specific ion effects and indirectly

through detereorating the soils for plant growth.

Two techniques have been adopted to tackle soil salinity problem. The

first one is to amend the soil conditions to favour the plant growth. The second

one is to use the genetic potential of plants to adopt adverse soil conditions. The

former is a lengthy process and a very few results have been obtained due to

either for economic reasons i.e. high cost of amendments or for lack of the

availability of fresh water. However the latter is short term strategy to exploit the

genetic potential through soma clonal variation in plants for the development of

salt tolerant cultivars through tissue culture (Gandonou et al., 2005).

10

2.1 Effect of salinity on growth parameters and yield components

Salinity is the most important environmental factor that leads to severe

crop loss, especially in the arid and semi-arid lands. The inherent roots of

adaptability to salinity tolerance lie deep in the evolution of the flora of salt

affected areas. Even then the majority of the plants are sensitive to salts,

particularly those which cannot tolerate the saline conditions permanently.

Salt tolerance is a complex phenomenon which varies not only among

species but also depends upon the environmental conditions, for example,

temperature, relative humidity, air pollution, fertility, salinity and water availability

( Maas, 1986 ).

Pessarakli et al., (1991) reported that dry matter yield and number of tillers

decreased with increasing salinity in both wheat and barley. Wheat was more

severely affected than barley. Grieve et al. (1992) reported that number of tillers

per plant , spikelets per panicle and number of seeds per penicle decreased in

wheat as the salinity increased.

Khan et al. (1992) studied the salt tolerance of eight wheat varieties under

sodic soil field conditions. It was observed that number of spikelets per spike,

1000 grain weight, straw and grain yield decreased significantly with increasing

salinity/ sodicity levels.

Begum et al. (1992) found that NaCI stress consistently decreased

the rate of germination in wheat. Due to increase in salinity accumulation of Na+

and Cl- was increased and K+ accumulation was decreased in germinating seeds.

11

Parveen and Qureshi (1992) imposed incremental NaCI stress to seven

days old seedlings of wheat in order to determine the toxicity levels of Na + and

Cl- in the leaf sap of two wheat varieties Pb-85 (sensitive) and LU-26S (tolerant).

Concentrations of Na+ and Cl- present in the leaf giving 50% reduction in fresh

weight of shoot and root were tentatively taken as toxic levels for respective ions.

Accordingly the toxic level of Na+ in both varieties were around 250 m mol kg-1,

whereas corresponding value for Cl- was about 300 m mol kg-l indicating that Na+

was probably more toxic than Cl- in wheat.

Liu and Liu (1993) studied the effect of NaCl stress in barley and found

that increasing NaCl concentration resulted in increased root dry weight but

shoot dry weight decreased.

Deleon et al. (1995) introduced a rapid in vitro screening of bread wheat

with various levels of salts. After eight days seedlings were evaluated for height,

root length and root number. The test showed tolerance to NaCl at concentration

of 50mm and 100mM.

Ashraf and Leary (1996) also reported decreased in all growth parameters

.i.e. height of stem, number of grain, length of ear, biomass weight / plant in

wheat as the salinity levels increased.

Hamada (1996) demonstrated that wheat growth in pots in 2: 1 clay: sand

mixture subjected to different salinity levels (0-160 mM NaCl) and moisture

contents (20-80%). Growth was decreased by increasing salinity and decreasing

soil water. Root and shoot water contents were mostly unaffected. NaCI up to 80

12

mM increased the net photosynthetic rate (PN) while it was decreased by the

highest salinity level and the most severe drought. The content of Na+ in the

shoots and roots of wheat increased as the salinity increased.

Grattan and Grieve (1999) suggested that crop performance may be

suffered due to nutritional disorders induced by salinity. These disorders occurs

by the effect of salinity on minerals availability, competitive ionic uptake, transport

/ partitioning within the plant cell. Salinity can affects the nutrient uptake such as

Na +, reducing K+ uptake or by Cl- reducing N03- uptake. Salt stress can also

caused a combination of complex interactions that affected plant metabolism,

susceptibility to injury or internal nutrient requirement.

Qing and Cheng (1999) investigated the differences in Na+, K+

accumulation between the mutant and the wild wheat (Triticum aestivum L ). The

wild type accumulated more in the root and leaf than the mutant under NaCl

stress. There in Na+ deposited in leaf was more significant than the root. The

mutant had a less accumulation rate of Na + than the wild type during the stress.

K+ contents in the leaves and roots of both cultivars reduced severely when

subjected to NaCl stress, but the content in the leaf and root of the wild type were

lesser than the mutant one.. The Na+ distribution in the seedlings of the mutant

and the wild type differed significantly.. When subjected to salt stress for 96 h,

the quantity of the accumulated Na+ in root was 44.3% of the total Na+ per

seedling of the mutant, whereas it was 24.3% in the wild type, which likely

resulted from the reduction of Na+ transfer from roots to shoots in the mutant.

13

Schachtman and Liu (1999) concluded that potassium uptake is essential

for plant nurishment but under the problem soils sodium has a competition with

potassium for uptake across the plasma membrane of plant cells. This happens

in high Na+: K+ ratios and the plant growth is reduced and become toxic.

Akhtar et al. (2000) conducted a pot experiment to determine the effect of

salinity and waterlogging on five wheat varieties. The experiment was comprised

of four treatments,.i.e.,NaCI altered nutrient uptake, due to an accumulation of

Na + and Cl- contents both in shoots and roots. In particular, Adamello showed

higher decrease in the Ca2+ of roots and the K+ of shoots and a higher increase

in the Na + of shoots. Even though both cultivars reacted to the stress, cv.

Adamello was more sensitive to NaCI.

Naseem et al. (2000) conducted a solution culture experiment in a green

house to screen wheat genotypes against salinity. The experiment was

conducted by growing forty wheat genotypes. There were three treatments viz.

control (non saline), 100 and 200 mol m-3 NaCI. Salinity was imposed gradually

and plants were harvested after forty days stress. An increase in salinity reduced

the vegetative growth significantly. Genotypic BWN-75 proved to be tolerant at

both the stress levels due to exclusion of Na + and Cl-, as it maintained lower Na

+ and Cl- in its leaves. The genotypes PARC-N1, PARC-N2 and Bakhtawar were

also classified as tolerant and attributed to better management of these ions.

Iqbal et al., (2001) observed that the contents of sodium and chloride in

leaf sap increased while that potassium ions decreased under salinity as

compared to control. Among the genotypes, Pb-25, Pb-28, SARC-6, KLR-I-4 and

14

Bakhtawar stopped the uptake of Na+ and favoured K+ and thus maintained high

K+: Na+ ratio.

Alem et al. (2002) cultivated seven varieties of both bread and durum

wheat each in three different places in the area of Errachidia (southeastern

Morocco). These were differed by the degree of salinity in irrigation water.

Results obtained showed that the reduction in leaf area was major mechanism

that made it possible to tolerate the effects of the reduction in the availability of

water under saline conditions in Bread wheat, that in turn, limited the reduction

in leaf area.

James et al. (2002) grew two tetraploid wheat genotypes (Wollaroi and

Line 455) that differed in the degree of salt induced leaf injury in 150 mM NaC1

for 4 weeks, to determine the characters that affected tolerance to higher internal

salt concentration. Shoot biomass of both genotypes was substantially reduced

by the increase in salinity, but genotypic differences were seen only after 3

weeks, when durum cultivar Wollaroi exhibited greater leaf injury and a larger

decrease in biomass than line 455. Salinity caused a greater reduction in

stomatal conductance of both varieties. The most responsive indicator of salinity

stress was g(s), followed by CO2 assimilation.

Rivelli et al. (2002) conducted an experiment by using four wheat lines

with different values Na + exclusion to find out that whether less Na + uptake had

an ill effect on water relations or growth rates under saline conditions. Plants

were grown hydroponically with and without150 molm-3 NaCl, and samples were

collected for the measurements of water relations, biomass, stomatal

15

conductance, and ion accumulation. After 4 weeks salt, decreased the biomass

in all varieties up to 50%, while the effect of salinity on relative growth rate

confined largely to the first week. Little difference was noticed between

genotypes in the effect of salinity on water relations, as indicated by their relative

water content and estimated turgor. This concluded that selecting lines with less

Na + accumulation for the purpose to improve salt tolerance is unlikely to

introduce limitations for osmotic adjustment.

Rajpar and Sial (2002) reported that ten hexaploid wheat cultivars, namely

Tonic, LU-26S, Q-19, 'KRLl-4, Kharchia-65, SARC-l, PAK-81, KTDH-19,

Cadenza and NIAB-20, were grown up to flag leaf stage in NaCl solution at

120mol/m3. Q-19 was significantly shorter, without tillers and having significantly

less shoot dry weight than the other cultivars. Kharchia-65, KTDH-19 and

Cadenza were taller, with higher shoot dry weight and had more tillers and main

stem leaves than most of the cultivars. Compared to other cultivars, poor

performance of Q-19 was associated with higher Na + concentration and less K+/

Na + ratio. Better performance of Kharchia was associated with less Na + and Cl-

concentration and high K+ /Na+ ratio t in the flag leaf sap, however, this trend was

not observed in KTDH-I9 and Cadenza.

Akram et al. (2002) studied the effects of various levels of salinity (10,15,

or 20 dSm-l) on the yield and yield components of salt-tolerant, medium tolerance

and susceptible wheat varieties in a pot experiment. Salinity reduced the spike

length, spikelets , grains per spikelet, grain weight, and yield per plant. The

susceptible variety was the most adversely affected.

16

Munns et al. (2003) conducted salt tolerance studies in the genus Triticum

and found that it is related with less contents of Na+ in leaves. Durum and other

tetraploid wheats generally have greater accumulation of Na+ as compare to

bread wheat, and are salt sensitive, but a durum wheat landrance line 146 was

found to havig exceptionally lesser leaf Na+contents. Generations were

developed by crossing 149 with high Na+ accumulation variety Tamaroi, as well

as between 149 and a durum landrace with greater Na + accumulation line 141.

The third leaf of parent varieties, F 1, F2, and low and high-selected F2 and F3

progenies were taken to determine Na+ uptake when grown in 150 mM NaCl.

Sodium contents were significantly less in the lower Na+ uptake Line 149

compared with high Na+ uptake Tamaroi (5-fold higher Na+ accumulation) and

Line 141 (7 -fold greater Na + accumulation). There was no inherent effect on Na

+ accumulation.

Hussain et al. (2003) studied six durum wheat lines with different sodium

concentrations in leaves to determine the effects of sodium exclusion on leaf

length and biomass production under saline soil. Leaf chlorophyll content, plan

height, ion concentration and dry matter were studied at 3 salinity levels (1, 75,

and 150 mM NaCl, . Yield and yield components were recorded on 2 different

groups of genotypes. Low Na + content lines showed much higher chlorophyll

retention than those of higher Na+ lines, the initiation of leaf senescence being

extended for a week or more under the low Na+ genotypes. The was greater

difference was recorted at 75 mM NaCl. At ear emergence, the salinity effects on

biomass were less in the low Na + than in the higher Na+ genotypes at 75 mM

17

NaCI, but no difference was observed between groups at 150 mM NaCI. At the

mature stage, salinity showed a similar trend on biomass of both genotypes at

both 75 and 150 mM NaCI. Grain yield at 150 mM NaCI was also reduced

equally in both the genotypes, being only 12% of controls. But , at 75 mM NaCI

level, there was a significant yield difference between these genotypes; yield

having high Na + genotype was only 70% of the control, whereas yield in

genotype having less Na+ was 88% of controls. The higher yield of in low Na +

genotype was because of more number of grains and grain weight in the tiller .

Iqbal (2003) carried out a pot study to see the effect of two salt species,

i.e., NaCI and Na2 SO4 on growth and physiology of spring wheat. Na+

concentration levels in both the salt species were 0, 50, 100 and 200 mM. A

significant difference was noticed in case of osmotic pressure , which is

indicates that NaCI had a greater effect on osmotic pressure. There was no

significant effect of two salt species on K+, Na + contents and K+ /Na + ratio. A

significant increase in osmotic pressure and Na+ content in the sap was

observed with the increase in Na+ concentrations whereas leaf area, K+ contents

and K+ /Na+ ratio decreased with as the Na + concentration increased.

Munns and James (2003) followed rapid and effective greenhouse

screening techniques that CaC identify genetic variation in salinity tolerance. A

set of previously unexplored tetraploid wheat genotypes, from five subspecies of

Triticum turgidum, were used in a case study for developing and validating

glasshouse screening techniques for selecting for physiologically based traits

that confer salinity tolerance. Short-term experiments (l week) measuring either

18

biomass or leaf elongation rates revealed large decreases in growth rate due to

the osmotic effect of the salt, but little genotypic differences, although there were

genotypic differences in long-term experiments. Specific traits were assessed.

Na + exclusion correlated well with salinity tolerance in the durum subspecies,

and K+ 1 Na + discrimination correlated to a lesser degree.

Saqib et al. (2004) conducted a pot trial using sandy clay loam soil during

2001-2002 to examine the interactive effects of soil compaction; salinity and

waterlogging on grain yield and yield parameters in two wheat (Triticum

aestivum) genotypes (Aqaab and l\1H-97). Compaction was done at 10%

moisture level by dropping 5 kg weight, controlled by a tripod stand for 20 times

from 0.6m height on a wooden block placed inside the pots filled with. Soil bulk

density of non-compact and compact treatments was acheived as 1.21 and 1.65

Mg m3, respectively. The required salinity level (15dS m-I) was developed

artificially by mixing the required amount of NaCl in soil before filling in the pots.

Mean reduction in grain yield was 44% under non-compact saline conditions

against 76% in the compact saline conditions. Similarly, the reduction about 20%

more for 100 grain weight and shoot length, 30% more for number of spikelets

per spike, 37% more for number of tillers per plant, and 32% more for straw

weight in compact saline treatment than in non-compact saline treatment was

noticed. Reduction of 36% in grain yield was caused by compaction alone.

Poustini and Siosemardeh (2004) used 30 genotypes of wheat (Triticum

aestivum L.) to check the leaf and grain K+/Na+ ratios and their ion selectivity

responses under NaCI salinity in a pot experiment. Significant correlations was

19

found between grain dry matter and some ionic traits which indicated that, in

spite of the adverse effects of the higher leaf Na+ contents on grain dry matter,

the mechanisms involved in the rate and/or duration of grain filling may be more

effetivet in terms of salt tolerance where grain yield is concerned. The earlier 11

cultivars could therefore be considered as the most completely salt resistant

cultivars. In genotype Kharchia-65, salinity did not affect the grain K+/Na+ ratio

and ion selectivity, but the grain filling period and harvest index of it were

reduced under saline conditions.

Ali et al. (2005) tested the wheat varieties of different origin including

commercial varieties of different provinces of Pakistan i.e., Punjab, KP and Sindh

under different salinity levels in laboratory as well as in saline field conditions in

diverse ecological zones. firstly, 16 genotypes were tested for germination at 6

different salinity levels ranging from 0-25 dS m-I (2, 5, 10, 15, 20, 25) EC = dS

m-1. Then, out of 16 genotypes, 11 were studied for the relative growth rate

under different levels of salinity and after their study in the laboratory, 9

genotypes were selected to test in the naturally saline areas of Punjab province.

in germination test, the varieties viz. Pasban-90, Sarsabz, Bakhtawar, 93032 and

933118 were less affected than other cultivars. On overall yield performance

basia, the Sarsabz cultivar produced the greater seed yield (4.37 t/ha). By These

findings, it is indicated that the genotypes viz. Sarsabz, Bakhtawar and Pasban-

90 are better resistant to saline environment as compared to other cultivars.

Katerji et at. (2005) studied that salt resistant variety Cham-I, developed

by ICARDA, produced a greater grain yield than the less salt tolerant cultivar

20

Haurani, in a lysimeter experiment but the main parameters responsible for the

pasta quality were declined considerably. Three irrigation water of different

qualities were used .i.e; fresh water as a control with EC of 0.9 dSm-1 and two

saline waters with the EC of 4 and 8 dSm-l, developed by mixing equivalent

amounts of NaCI and CaCl2 to fresh water. Salinity had a little positive effect on

the grain quality of variety Cham-l , but Haurani variety showed no salinity effect

on the grain quality.

EI-Hendawy et al. (2005) conducted a pot experiment by using thirteen

wheat genotypes grown in soil under different four salinity levels (control, 5, 10

and 15 dS m-I). At vegetative stage number of tillers, number of leaf and leaf area

per plant were recorded. Dry weight per plant at vegetative, reproductive and

maturity stages were calculated while yield components of main spike and total

grain yield at maturity stage were determined. The results revealed that number

of tiller was affected greater by salinity than the leaf number and leaf area at the

vegetative stage. Salinity decreased the dry matter per plant significantly at all

the growth stages. Number of spikelet on the main stem were decreased much

more with the increase in salinity than spike length, number of grain and 1000

grain weight at maturity stage.

Houshmand et al. (2005) conducted experiment by using eight durum

wheat cultivars including three salt-resistant and one salt-sensitive variety. The

study was done under both the saline and non-saline field conditions as well as

under glasshouse condition with saline solution culture of 0 (control), 75 and 150

mol m-3 NaCl concentrations. Data on days to heading, days to maturity, plant

21

height, number of grains per spike, grain weight per spike, 1000 grain weight,

number of spikes , grain yield and harvest index were recorded in the field

conditions. While plant dry weight, Na+, K+ and Ca+2 accumulated in the

hydroponically grown seedlings were measured after 20 days of salinity

application. In the field conditions, salinity significantly reduced (P < 0.0 I)

means of all the traits averaged on eight tested wheat genotypes.

Basal et al. (2006) reported that increasing soil salinity is becoming a

serious problem in the agriculture areas of over the world wide. The object of this

research was to find out salt-tolerant CRS accession (s) that would serve as

parental material for further salt-tolerant (M-9044-0031, M-9044-0061, M-9044-

0140 and M-9044-0150) and three putative salt-sensitive CRS accessions (M-

9044-0060, M-8744-0091 and M-8744-0175) plus Acala 1517-77, Deltapine 50,

TAM 94 /L-25 Stonville-453 and Nazilli-84 were exposed to two different salt

concentrations (125 and 250 mol m-3 NaC1)and control in completely

randomized design with ten replications. The significant differences were found

among the cotton genotypes for reduction in shoot dry weight (SDW), total dry

weight (TDW), leaf area (LA) and plant water content (PWC), SDW, TDW and

PWC were correlated positively regardless of treatment, indicating that either trait

could be used as a selection criterion. TAM94L-25, M-9044-0060, M-8744-0091

and M-9044-0140 may provide parental material for salt tolerance in upland

cotton breeding.

Hu et al. (2006) Examined the short term effects of salinity and drought

on nutrient imbalance in wheat seedlings. Wheat was sown in the greenhouse in

22

soil under saline and drought conditions for 26 days after the sowing. After

harvesting, it was observed that the reduction in plant height, accumulative

evapo transpiration, and shoot biomass under drought was quite similar to under

salinity as compared to control plants.

Eker et al. (2006) tested salt tolerance of 19 hybrid maize (Zea mays L.)

cultivars in nutrient solution culture at early growth stage under controlled

conditions. For the salinity stress treatment, NaCl was applied to the nutrient

solution at a concentration of 250 mM for 6 days before the harvesting. The

harvesting of plants wase done after the 17 days of growth and assesed for the

shoot and root dry matter production, severe leaf damage .i.e; necrotic patches

on older leaves, and the potassium (K+), sodium (Na+) and calcium (Ca+2)

contents in the roots and shoots. There were great differences among the

varieties in response to the NaCI treatment. The development time and severity

of leaf symptoms caused by 250 mM NaCI were different in different varieties.

On the base severity in leaf symptoms, the genotypes Maverik and C. 7993 were

found as the most resistant and sensitive varieties, respectively. Decreases in

the shoot dry matter production as a due to the NaCI treatment were gtreater

than the reduction in root growth. There was also a profound genotypic variation

in the concentrations of K\ Ca+2 and Na + in roots and particularly in shoots as

well. The higher salt tolerance in maize varieties based on the severity of leaf

symptoms was due to with significantly lower Na+ concentrations in shoots. The

K+/ Na+ and Ca2+/ Na+ ratios were also significantly greater in most of the

resistant varieties. The results show the extent of a large varietal variation in

23

tolerance to NaCI stress in maize that should be utlized in breeding programs

with the aim to develop maize cultivars with high NaCl tolerance during the early

stage of growth. Among the ions determined, shoot Na+ contents were a reliable

screening parameter in sorting varieties for their tolerance to salinity stress.

Ruan et al. (2007) conducted an experiment to study the differential

effects of less salinity on the growth and ion concentrations in the mainstem and

the subtillers of wheat crop; two spring wheat (Triticum aestivum L.) cultivars

(sakha 8 and Thasos) were sown in a greenhouse in soil with and without

salinity. The results told that dry weight and leaf area in the subtillers (T1 and T2)

at day 55th after sowing were greatly reduced by increase in salinity. Salinity

affected the subtiller growth more while the main stem was less affected during

the vegetative stages of growth. . Both the wheat cultivars were more sensitive to

salinity stress at vegetative stages than to the reproductive stages. The salt

resistant cultivar sakha 8 is characterized by the exclusion of sodium ions in the

leaves. Thus, under moderately saline conditions the more reduction in subtillers

may be resulted from an ionic imbalance in the salt-tolerant variety Sakha 8 and

from sodium toxicity in the salt-sensitive cultivar.

Thalj and Shalaldeh (2007) conducted an experiment to screen wheat and

barley genotypes for salinity resistant, 10 bread wheat, 12 durum wheat and II

barley genotypes were planted under saline conditions. Salinity was ranged

(20.6-21.9 and 4.5-5.5 dS m-1) for both soil and water, respectively. In wheat

genotypes potassium (K+) content, (K+/Na+) and Sodium (Na+) have showed a

strong positive correlation with seed yield in wheat genotypes, respectively

24

whereas, chloride (Cl) showed a strong negative correlation with the seed yield.

In barley genotypes, phosphorus and potassium have a strong negative

correlation with the biological yield respectively and with the straw yield,

respectively at the three leaf stage.

Adiloglu et al. (2007) determined the effects of different levels (0, 15, 30

and 60 mM) of NaCl and KCI salt on seedling growth and some biological

indexes of wheat, shoot height, stem diameter, leaves number of plant, fresh

weight of shoot and dry matter weight index were investigated. The results

showed that biological index of wheat decreased with increasing salt in

comparison to control. The adverse affect of NaCl on wheat plant was obtained

higher than that of KCI. This should carefully be considered if wheat is grown

under saline soil condition

Caterina and Giuliani (2007) conducted an experiment to study the effects

of saline irrigations on grain yield and quality of sunflower. A pot experiment was

conducted for over two crop seasons on two hybrids, a standard one (Carlos)

and a high one in oil contents (Tenor) exposed to five salinity levels of irrigation

water (0.6, 3, 6, 9 and 12 dS m-I). Soil salinity was maintained for the entire crop

cycle, and leaf ion content was monitored after maturity. Tenor cultivar

accumulated higher Na+ and Mg+2 content with lower K+ values. There were no

difference between the two hybrids for CI- content. A progressive increase in leaf

Na+, K+ and Na+ /K+ ratio with increasing salinity level was noticed. Seed weight

per head, 1000 achene's weight, and number of seeds per plant and oil yield

significantly decreased as the salt stress increased in both hybrids. The

25

percentage in seed yield decrease was greater per unit increase in EC irrigation

water. As the oil fatty acid composition is concerned, the main significant

differences as a result of salinity stress were a progressive increase in oleic acid

content, from 82.2% to 86.7% fin Tenor and from 21.8% to 27.3% for Carlos

genotype, which was associated with a decrease in linoleic acid content, .i.e;

from 5.9% to 3% for Tenor and from 66% to 61.3% for Carlos line. These results

confirmed the possible suppression of oleate desaturase under salt affect.

Turhan et al. (2008) conducted a trial to study the effects of salinity on

physiological features of sunflower (Helianthus annuus L.). Three NaCl

concentrations were used .i.e; 0.5, 1 and 1.5% NaCl. The data revealed that

plant growth was decreased with the increase in levels of NaCl. Chlorophyll

concentration and Normalized Difference Vegetation Index (NDVI) were

determined in the plants by a using m spectrorediometer. A significant (r2 =

0.76) correlation between chlorophyll and NDVI values was found.

Saqib et al. (2008) Studied the interaction between the leaf ionic

composition and grain yield and yield parameters of wheat under salinity x

sodicity and salinity alone. Experiment was carried out in soil culture in pots filled

with soil. Three treatments including control (ECe 2.6 dS m" I and SAR 4.53),

salinity (ECe 15 dS m" 1 and SAR 9.56), and salinity x sodicity (ECe 15 dS m" 1

and SAR 35) were applied. They showed that in sensitive line salinity alone

decreased less grain yield as compare to the combine effect of salinity and

sodicity.

Zheng et al. (2008) performed an experiment to investigate relieving

26

senescence potential in the new genotypes. They compared two cultivars for gas

exchanges and related physiological parameters of winter wheat, DK961 (salt-

tolerant) and IN 17 (salt-sensitive) under the different salinity levels of NaCI

concentrations. During the whole reproductive period, decrease in the leaf area

index, leaf area duration, leaf relative water content, net photosynthetic rate,

stomatal conductance, pigment contents, ions contents, and dry matter

accumulation of spikes was noted in both genotypes with the increase in salt

concentrations. However, the salt-resistant lines exhibited no significant

reductions in these parameters as compared to the control (0.3% and 0.5% salt

concentrations).

It is learnt from the literature that plant height and other agronomic traits

like growth and yield parameters are greatly decreased as the root zone salinity

level increased this study was conducted to create genetic diversity through

tissue culture for salt tolerance and other growth and yield characteristics so that

it may be used for breeding more productive varieties for salt-affected soils.

2.1.1. Photosynthesis and stomatal conductance

The rate of photosynthesis, CO2 assimilation is normally reduced due to

salinity. Perhaps this reduction is due to reduction in stomatal conductance (

Sharma, 1996 ) and consequent non availability of CO2 for carboxylaton. Nosn-

stomatal restriction in photosynthesis, caused due to the direct effects of NaCl on

photosynthetic equipment independent of stomatal closure, for several plant

species has also been observed in both halophyte and non-halophyte plants

(Brugnoli and Lauteri, 1991 ).

27

Brugnoli and Lauteri (1991) reported a decline in CO2 assimilation rate

when bean and cotton plants were sown under salinity and it is direct effect of

stress on net photosynthesis. The inhibition in photosynthetic capacity caused by

salts is apparently happens due to occurrence of patchy photosynthesis.

Seemann and Critchley (1985) indicated effective compartmentation in

that, r Phaseolus Vulgaris, broke down in plants grown under higher salt

oncentration and the Cl- was essentially needed in the chloroplast-cytoplasm and

for the compartmentation in vacuole which might have reduced the efficiency if

RuBP carboxylase in vivo.

Sultana et al. (2000) evaluated the affect of salinity on rice and found that

reduction in photosynthetic rate in the salinized plants which not depended only

in the reduction of available CO2 by stomatal closing but also depended on

combine effects of leaf water and osmotic potential, transpiration rate, stomatal

conductance and other chemical constituents like photosynthetic pigments,

soluble proteins and carbohydrates.

Ashraf and Parveen (2002) examined the response of two spring wheat

cultivars, salt tolerant SARC-1 and salt sensitive Potohar to different levels of

NaCl. SARC-1 had higher stomatal conductance (gS), net photosynthetic

rate(PN) and transpiration rate than cv. Potohar at the vegetative stage, but the

cultivars did not differ significantly in water-use efficiemcy (PN/E). at the grain

development stage, SARC-1 had significantly higher Pn and gs in the flag leaf

than cv. Potohar under salinity.

28

Ismail (2003) studied the effect of different concentrations of salinity (NaCl

up to 250 mM) on three wheat varieties for germination, dry matter production

and some relevant metabolic parameters and found increasing salinity resulted in

a significant decrease in water content and higher levels inhibited photosynthetic

activity. K+/Na+ ratio decreased significantly with the rise in salinity.

Iqbal (2003) conducted a hydroponic experiment to find out the effect of

salinity in wheat crop using four salinity levels 0, 50, 100 and 150 mM NaCl. They

found that net photosynthesis (Pn) was not significantly affected by salinity during

the morning. During noon and afternoon, Pn was significantly greater at 150 mM

NaCl level than the 50 mM NaCllevel whereas the difference between other

treatments was not significant. Pn was lower at 50 mM NaCl than in other

treatments at all sampling times. Stomatal conductance (gs) was more during the

morning than the noon and afternoon as well..

Sharma et al. (2005) studied the effect of different salinity levels on two

wheat genotypes k-65 and HD-2329 in a pot experiment and found that, stomatal

conductance (g S), transpiration rate (E) and the net photosynthesis rate (PN)

were reduced with the addition of salt. The reduction was more in HD-2329 than

that of K-65. The sodium and potassium concentrations were greater in the roots

and leaves of K-65 as compare to HD-2329. Thus at cellular level K-65 has

proved salt tolerance by adjusting PN, E, gS, and K deposition in leaves along the

excessive production of antioxidative enzyme activities (SOD and POX).

Zhang and Xing (2008) determined the damage to photosynthesis caused

by salt in Arabidopsis seedlings and reported that delayed fluorescence (DF)

29

intensity and net photosynthesis rate (Pn) were decreased in with the same way

as increasing NaCl or sorbitol concentration. Seedlings Incubated under 200 mM

NaCl gained a quike and reversible decline and subsequent lower and

irreversible loss in both the Pn and DF intensity and it is ion specific.

Li et al. (2008) found out the differences in stomatal photosynthetic

characteristics of five diploidy wheat species and pointed out that the net

photosynthesis rate showed significant correlation with stomatal conductivity, and

the changes in intercellular CO2 content showed the opposite trend to the

changes in stomatal limitation, indicating that the stomatal conductivity is the

main factor limiting the photosynthetic rate.

It is quite clear from above review that salinity affects the stomatal

conductance and net photosynthesis. It is observed the both factors are less

affected in the morning time. In some cases net photosynthesis was greater at

higher salinity levels than normal soils.

2.2. Somaclonal variation for genetic variability.

Wheat breeding by traditional means has been in practice for

centuries, but no success has been found for a breakthrough to cope with the

world’s demand. The varieties thus evolved do not last longer. Long term

objectives can not be achieved unless more genetic variability is generated. In

vitro technology complements the conventional means of wheat breeding in

generating diversity when both are combined with each other. During last

decades, achievements made for the betterment and utilization of in vitro

techniques in wheat development has been enormous especially for the

30

induction of genetic variability, production of haploid and wide hybridization in

incompatible crosses. Problem of the depletion of genetic resources combined

with losses of native germplasm has lead to disappearance of genetic diversity.

So biotechnological approaches are being evaluated to induce genetic variability

and conservation of germplasm. In vitro culture exhibit somaclonal variation and

generated plants show heritable variation (Ahloowalia, 1982).

It has been concluded by several scientists that tissue culture is a novel

source to generate heritable genetic variability (Bhaskaran and Smith, 1990 and

Larkin and Scowcroft, 1981). Several changes are involved in this system, i.e.,

callus induction and proliferation, organogenesis, plant regeneration and field

testing. Substantial genotypic differences have been reported at each step

(Ahloowalia, 1982).

The exploiting of in vitro selection and transformation are all depended on

the regeneration of wheat plants from callus. Wheat embryo culture technique is

being used widely for plant regeneration (Bhaskaran and Smith, 1990).

Regeneration in plant from embryo culture calli in wheat crop is a step oriented

method comprising of callus formation, callus multiplication, organogenesis and

plant development (Mohammad,1993). Low concentration of auxin results in

better organogenesis and plant regeneration (Sharma et al., 1981).

2.2.1. Use of somaclonal variation for development of salt tolerant plants.

31

In vitro somatic cell and tissue culture techniques have been developed to

assist plant breeding. Exploitation of somaclonal variation in plants for the

development of salt tolerant cultivars through tissue culture is advantageous over

conventional techniques which is an efficient and potent method for producing

novel and useful varieties ( Larkin and Scoweroft, 1981; Gondonou et al., 2005 ).

La Rue (1949) conducted successfully tissue culture for the first time in cereal

crops by using endosperm. Gamborg and Eveleigh (1968) successfully produced

suspension cultures of wheat and Shimada et al. (1969) reported callus culture

and cell culture in the wheat crop. The literature on use of somaclonal variation

for salinity tolerance in wheat and other crops is reviewed as under.

Kishore (1985) investigated the effect of salt on callus culture of Oryza

sativa L. and found that free praline, Na+, ,Cl- and Ca+2 were significantly

increased with the increase in NaCl level. While Yang et. al., (1990) told that in

Sorghum NaCl stress reduced the relative growth of callus and the Na+ /K+ ratio

was lower in salt tolerant callus while the accumulation of Na+ and K+ was more

in salt tolerant than in less tolerant callus.

Subbashini nd Reddy (1990) studied the rice in relation to salt stress.

They found that there is a significant effect of salt stress on rice at callus level. It

significantly reduced callus growth and K+ contents, and increased Na+ , Cl- and

paraline contents.

Trividi et al. (1991) maintained cultured of different wheat varieties on

media containing NaCl and found that under stress, the callus derived from

sensitive lines showed smaller reduction in growth rate and high intracellular K+

32

than from sensitive one. Under low level of salinity the growth of resistant

cultivars was unaffected as compare to sensitive one.

Jia et al. (1993) selected a stable NaCl tolerant variant callus line of

Setaria italica. Which produced numerous regenerated plants from tolerant calli

and found that these plants grew well in medium containing 0.6-0.8 % NaCl.

Gulati and Jaiwal (1994) exposed the salt resistant callus of Vigna radiata on

medium having stress treatment, i-e., 100 mM NaCl and noted that resistant

callus retained higher level of K+ than the sensitive one. They suggested that

higher internal K+ contents impart resistance to salt stress.

Lu and Jia (1994) treated the embryogenesis callus of pearl millet initiated

from young inflorescence segments with NaCl and found that tolerant lines

showed good resistance to stress caused by NaCl. Poustini and Baker (1994)

wheat cv. Sholeh (tolerant to salinity) and Inia-66 (susceptible) were grown at 0,

2.5 or 5 g NaCl/litre. CO2 uptake by Sholeh was decreased only at the highest

salinity level. Decreases in CO2 uptake were paralleled by reductions in

transpiration and stomatal conductance in both cultivars, and it is suggested that

the change in stomatal resistance in saline conditions may be responsible for

reduced photosynthesis.

He and Yu (1995) found that with increasing level of NaCl , the salt

tolerant callus contained more soluble sugars, reduced water content and had

higher Na+ and K+ contents.

Diaz-de-Leon et al. (1995) screened excised mature embryos of 14

cultivars of Triticum aestivum for salt tolerance on MS medium fortified with 30 g /

33

litres of sucrose and 50, 100 and 150 mM NaCl. After 8 days, the developed

seedlings were evaluated for height, root length and root number. Cultivars were

classified as tolerant (0-35% inhibition compared with control values), moderately

tolerant (36-68% inhibition) or sensitive (69-100% inhibition). In relation to height

and root length, the cultivars Kharchia and Shorawaki were tolerant at 150 mM

NaCl. NaCl did not affect root number significantly. Anions (in the presence of

Na+) and cations (in the presence of Cl-) distinctly affected some parameters.

Poustini (1995) performed a pot experiments, 2 wheat cultivars (one salt-

resistant) were watered with solutions containing 0, 2.5 or 5 g NaCl/litre from 4

weeks after sowing. Salinity decreased the number of tillers bearing spikes and

relative growth rate but did not affect net assimilation rate. Flag leaf water

potential was decreased and Na+ and K+ contents were increased by salinity. It is

concluded that the effects of salts on growth and yield parameters of wheat are

due to its effect on plant ionic status and not on the rate of carbon uptake.

Barakat and Latif (1995) exposed the embryogenic calli isolated from

immature embryos of 4 wheat cultivars to in vitro selection techniques for salt

tolerance. Investigatios were done to see the effect of NaCl on selected and

unselected cell lines. The relative growth weight of callus decreased while dry

weight increased as the salinity increased in both cell lines. The selected cell

lines gave significantly higher relative growth responses compared with the

unselected cell lines across all medium protocols and in all cultivars. Na+ and Cl-

content of both cell lines increased with increasing salinity, while K+ content

decreased. The selected cell lines of each cultivar produced significantly higher

34

amounts of Na+, K+ and Cl- than the unselected callus lines at most salinity

levels.

Barakat and latif (1996) isolated embryogenesis calli from immature

embryo of four wheat varieties and subjected them to three in vitro techniques for

salt tolerance and investigated the impact NaCl on the selected and unselected

cell lines which indicated that the relative growth rate of callus was decreased as

the level of NaCl increased in both callus lines. The sodium and chloride content

of both lines were increased with increasing salinity level while K+ content was

decreased.

Lutts et al., (1996) exposed the embryo derived callus of rice cultivars

with different in salt tolerance ability to various concentration of NaCl and

reported that higher level of NaCl induced inhibition in growth, Na+ and Cl-

accumulation as well as internal osmotic potential.

Moraru et al. (1996) studied the response of Calluses derived from

immature embryos of 3 genotypes cultured in vitro in the presence of 4 g and 6 g

NaCl/litre. Mean callus weight was determined before and after treatment. Turda

81 showed the least reduction in callus weight due to NaCl treatment (33% at 4 g

and 18.57% at 6g). The other 2 genotypes, AF93-2 & Fundulea 29, showed

reductions of 52-84 %.

Ozgen et al. (1996) used mature and immature embryos of seven wheat

cultivars of durum wheat on MS medium fortified with 2, 4-D. They observed low

frequency of callus formation in mature embryos with higher regeneration

capacity as compare to immature embryos. Regeneration through embryo culture

35

was influenced by culturing medium and varietal genetic factors which can be

controlled and environmental factors which can not be controlled

Poustini and Salmasi (1997) sown two wheat cultivars, Sholeh ( known to

be salt tolerant ) and Inia 66, in the greenhouse, with 3 salinity levels (0, 2.5 and

5 g NaCl /litre) being applied 4 weeks after sowing. Both cultivars showed

significant reductions in total DM production and significant increases in DM

remobilization under saline conditions. Sholeh performed better in terms of

vegetative growth than Inia 66, whereas the reverse was true for reproductive

growth and grain production. Inia 66 showed higher grain dry weight and harvest

index than Sholeh under salinity, due to a reduction in the grain filling period.

Barakat and Haris (1998) coducted a pot experiment in sandy soil to

evaluate 7, 11 and 9 somaclonal progeny of the respective wheat cultivars Giza

157, Yecora Rojo and West Bred 911 grown under 4 levels of salt stress (1.5, 4,

6 and 8 dS m-1). Grain yield, number of spikes, 100-grain weight, plant height

and number of days to heading were significantly influenced by both irrigation

water salinity and somaclonal genotype. Additionally, spike length in Yecora Rojo

and West Bred 911 and grain weight per spike in West Bred 911 somaclones

were significantly affected by both factors. The interaction between the 2 factors

was found significant for 100 grain weight, spike length, crop height and number

of days to tillering for all 3 cultivars. Moreover, significant interaction was found

for number of grains per spike and the grain yield only in West Bred 911

somaclones, and for number of spikes only in Yecora Rojo and West Bred 911

somaclones. The reductions noticed in grain yield were 58.2, 62.6, and 64.7% for

36

Giza 157, Yecora Rojo and West Bred 911, respectively, corresponding to an

increase in water salinity from 1.5 to 8 dS m-1. The correlations between yield

and the other yield parameters and among yield components were highly

significant and clearly observed in Yecora Rojo and West Bred 911 lines.

Correlations were of a lower magnitude in Giza 157 lines. Under salinity stress,

the somaclones GS1, GS2 and GS7 of Giza 157, YS1, YS4, YS5 and YS6 of

Yecora Rojo and WS3, WS6, WS7 and WS8 of West Bred 911 gave better grain

yields and were superior for most of the other agronomic traits compared to their

parents. These results show that it is possible to produce salt tolerant

somaclones by regenerating plants from calli cultured under salt stress

conditions.

Kishor (1998) studied the effect of salt on the callus culture of Oryza sativa

L. and found that free praline, Na+ , K+ and Ca++ were significantly increased

with the increasing level of NaCl. Ochatt et. al. (1998) exposed the salt resistant

potato callus on various levels of NaCl (60-450 m M ) and recorded a higher

callus fresh weight at 120 / or 150 mM concentration of NaCl.

Taghvaii et al. (1998) studied embryogenic callus formation of different

explants (inflorescence, immature embryo, mature embryo and primary roots) in

six spring and winter wheat cultivars and effect of differet doses of 2,4-D on

callus initiation in vitro. Significant differences were found between rate of callus

initiation of immature embryos and concentrations of 2,4-D among different

cultivars, but differences between callus initiation of inflorescence and mature

embryos were not significant. The highest amount of callus formed and the

37

amount of embryogenic callus in all cultivars were produced from immature

embryos and inflorescences. However the highest regeneration rate was

achieved from 2,4-D-free medium, and increase of NaCl concentrations in

medium, caused in reduction of callus growth.

Kopertekh and Butenko (1998) selected wheat Lines resistant to salinity .

All the lines exceeded the parental controls in terms of biomass increase under

salt stress conditions.

Arzani and Mirodjagh (1999) evaluated 28 cultivars for callus production

under saline stress using immature embryo culture. Morphogenic calli were

exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and

2.1% w/v) added to the culture medium during two subsequent subcultures (4

weeks each). Comparison of cultivars for callus induction from immature

embryos was based on callus induction frequency and fresh weight growth of

callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG)

and necrosis percent in callus were used. There were significant differences

among cultivars for potential of regeneration from immature embryos, and

Shahivandi, a native durum wheat cultivar originating from western Iran, was

superior among the cultivars tested. FWG distinguished cultivars better than

callus induction frequency did for callus induction evaluation. Hence, a range of

FWG from 1.23 to 14.65 g was observed in Mexical-75 and Omrabi-5 cultivars,

respectively. Growing calli derived from cultivars PI40100' and Dipper 6 showed

superiority for tolerating salinity under in vitro conditions.

38

Mirodjaghe et al. (1999) investigated salt tolerance of 28 cultivars of

durum wheat using in vitro technique. Calli of immature were exposed 8 NaCl

levels including 0, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8% (w/v). Assessment of calli was

conducted after 0, 8 and 16 days of subculture in the NaCl-containing medium.

Callus growth rate, relative fresh weight growth rate of callus and necrosis

percentages of callus were measured. Relative fresh weight growth rate of callus

was most reliable. PI40100 and Dipper-6 were had the best in vitro salt

tolerance.

El-Bahr et al. (2000) developed an in vitro procedure for the selection and

characterization of salt tolerant Egyptian wheat cultivars.Callus cultures

established from mature embryos of cultivars Giza-164 and Sakha-8 were

cultured using Linsmaier and Skoog's media fortified with 2 mg 2,4-D/litre were

subcultured on the same medium with NaCl (0, 2000, 4000, 6000, 8000 and

10000 ppm) for a total of 12 weeks. Callus growth (measured as fresh weight)

and the level of salt tolerance decreased with the increase in salinity. In general,

higher growth rates, but lower salt tolerance, were recorded for the calluses of

Giza-164 than those of Sakha-8. The proline content of Sakha-8 callus tissues

increased at 6000 ppm NaCl. In Giza-164, the corresponding increase was

observed at 8000 ppm. This was attributed to the higher proline level in the

calluses of Sakha-8 when cultured in saline treatments. The increase in salinity

raised the sodium level, but lowered the potassium and calcium contents, in the

calluses of both cultivars. Plantlets were obtained from Giza-164 and Sakha-8

callus tissues tolerant to 2000 and 6000 ppm NaCl, respectively. In general,

39

calluses of Sakha-8 regenerated into plantlets more efficiently than those of

Giza-164.

Ozalp et al. (2000) investigated the in vitro photosystem II (PS-II) activity

measurements for screening hexaploid wheat (Triticum aestivum) cultivars for

NaCl tolerance by studying their responses under stress and control (without

NaCl) conditions. One from the four lines under study was 'Kharchia' known as

high salt tolerant. Wheat seedlings were sown hydroponically in environmental

controlled chambers and subjected to differennt range of NaCl concentrations

(0.034, 0.17, 0.68 or 3.42 M) for a 1, 3 or 5-day period. The doses of NaCl were

applied at the proper time so that all plants were 10 days old at harvesting stage

Cell membrane stability (CMS) as recorded by a conductivity method. The PS-II

activity values were affected badly by NaCl levels and duration of treatment. By

these both methods clear the difference between salt-sensitive and salt-tolerant

cultivars. Statistical analysis explainedthat PS-II activity and CMS measurements

were well correlated (r=0.7589) It was suggested that PS-II activity can be used

as an extra screening toal besides CMS to determine salt tolerance of wheat.

Almansouri et al. (2000) studied the behavior of embryo derived calli from

three durum wheat varieties in the presence of NaCl (0-300 mM ) . the observed

that in all treatments callus growth (fresh and dry weight of callus) was not

affected in stress sensitive calli. Salt stress increased the K+ concentrations at

the callus level in all the cultivars.

Rus et al. (2000) studied the changes in the salt responses created

through long term callus cultures of cultivated tomato species ( Lycopersoin

40

esculent Mill. ). They found that callus relative growth rate and cell volumewas

tented to decreas with subculturing under both control and saline media (100

mMol/L NaCl ), although reduction in growth was much higher under stressful

medium.

Abdel-Hady et al. (2001) produced salt tolerant callus culture of three

barley (Hordeum vulgare) cutivars from immature embryo on MS medium

supplemented with different NaCL levels (4000, 8000 or 12000 ppm). They found

that growth rate of callus decreased as the salinity levels increased in the culture

medium. Callus growth rate was significantly different among tested cultivars

.

Almansouri et al. (2001) studied the deleterious effects of NaCl and PEG

on isolated wheat embryos and whole seeds germinated onto an in vitro

Linsmaier and Skoog (LS) medium and concluded that stress inhibition of

germination could not be attributed to an inhibition of mobilization of reserves and

that the major effect of PEG occurred through inhibition of water uptake. The

detrimental effects of NaCl can be linked to long term effects of accumulated

toxic ions. The behavior of the three cultivars during germination did not fully

reflect their mean level of putative stress resistance in field conditions, and

germination is, therefore, not recommended as a reliable selection criterion for

breeding purposes.

Bezerra et al. (2001) investigated the impact of different NaCl

concentrations on embryogenic calli of maiz. However, at the highest

concentration tested (150-200 mM/L), lower growth as well as lower praline

41

accumulation was observed. They reported reduction in growth at high

concentration of NaCl in the medium.

Poustini and Ciocemardeh (2001) conducted a pot experiment to

determine the impact of salt stress on the ion distribution patterns in three wheat

(Triticum aestivum) cultivars. Sodium chloride treatments (0.0, 2.5 and 5.0 g

NaCl/litre water) were applied to cultivars Inia-66, Tabasi and Sholeh. Na+

content of the plants increased under salt-stressed conditions, especially during

the grain filling period, while that of the K+ decreased under the same conditions.

The selective system of K+ over Na+ from roots to stems showed almost no

resistance to salt stress, but those from stems to the leaves and from stems to

the grains showed significantly higher efficiencies, resulting in higher K+/Na+

ratios in the grains and leaves under salt-stressed conditions. In the susceptible

Inia-66 cultivar, a higher K+/Na+ ratios were observed from roots to stems, but at

the starting point of the leaves and grains, the two salt-resistant cultivars showed

higher efficiencies for K+/Na+ selective system. It is concluded that the selective

system in ion transport between different plant parts may be considered as a salt

resistance index, which is more effective between stems and grains.

EL-Bahr et al. (2001) developed in vitro procedure for the selection and

characterization of salt tolerant Egyptian wheat cultivars and found that callus

growth (measured as fresh weight) decreased with the increase in salinity. In

general, higher growth rates, but lower salt tolerance, were recorded for calli

production. Plantlets were obtained from callus tissues tolerant to 2000 and 6000

ppm NaCl, respectively. A less genetic depended in vitro regeneration method

42

able to produce multiple shoot clusters and whole plants in four different wheat

cultivars were reported by Ahmad et al. (2002). All four genotypes were positively

responsive to shoot multiplication depending upon media composition.

Sudyova et al. (2002) tested embryogenic calluses derived from immature

embryos of three wheat (Triticum aestivum) cultivars and two triticale

(Triticosecale) genotypes for salt resistance in vitro. The 5 genotypes were: Ilona,

Hana, 8167, Ra-Tc-15 and Lasko. Tested callus culture medium was fortified

with 3, 6, and 9 g L-1 of NaCl. To weigh whole calluses in aseptic conditions as a

non destructive method was used to calculate a weight increase of callus. Ilona

showed the relative fresh weight gain of the control and tested calluses (0 vs. 9 g

L-1 NaCl) at nearly same level (25.30% vs. 20.30%). The growing calluses

obtained from wheat Ilona were found to have the highest salt resistant. After sub

cultivation on regenerating rooting medium, there were produced 9 plants, which

were cultivated to maturity. The highest salt-sensitivity was observed for in vitro

Lasko and R-Tc-15 triticale calluses. The relative fresh weight gain of callus and

regeneration potential decreased as the concentration of NaCl increased in both

triticale genotypes. The regeneration of tested Ra-Tc-15 triticale calluses was not

successful among the highest concentration of NaCl in medium (9 g l-1).

Callusing on the medium containing 12.5 mmol NaCl grew slower than grown on

medium with out added NaCl ( Tanvir et al., 2002 ).

Javed (2002A) determined the effect of salinity stress on the growth and

ion deposition in the One-month-old calluses of wheat cultivars LU-26S and

Potohar in MS liquid medium were subjected to different salt concentrations (100

43

or 200 mol m-3) to assess the effects of salinity stress on the growth and ion

accumulation in the calluses. The growth rate of calluses decreased with

increasing salt concentrations, with Potahar recording a higher growth rate

reduction than LU-26S. Callus Na+ and Cl- increased, whereas K+ increased with

increasing salt concentrations, with LU-26S recording the highest Na+ and Cl-

increment and Potohar recording the highest K+ reduction. Callus K+/Na+ ratio in

both cultivars decreased with increasing salt concentrations.

One-month-old calluses of wheat cultivars LU-26S and Potohar in MS

liquid medium were also subjected to different salt concentrations (100 or 200

mol m-3 ) by Javed (2002B) to assess the effects of salinity on the accumulation

of organic solutes in the calluses. Callus dry weight increased with increasing salt

concentrations, with LU-26S recording higher increments in dry weight compared

to Potohar. The total soluble proteins and carbohydrates, and free amino acids in

the calluses of both cultivars increased with increasing salt concentrations, with

LU-26S recording higher increments in the parameters measured compared to

Potohar.

Javed (2002C) studied one-month-old calluses of wheat cultivars LU-26S

and Potohar in MS liquid medium subjected to different salt concentrations (100

or 200 mol m-3) to assess the effects of salinity on the callus water relations. The

relative water content (RWC) of both calluses decreased with increasing salt

concentrations, with Potohar recording higher RWC reductions than LU-26S. On

the other hand, water (WP) and osmotic potential (OP), as well as turgor

potential (TP) of both calluses increased with increasing salt concentrations, with

44

Potohar recording higher WP and LU-26S recording higher OP and TP

increments.

Oudija and Ismaili (2002) tested two durum wheat genotypes (Karim and

Sebou) and two soft wheat (Sais and Merchouch) cultivars, in vitro for salt

tolerance. Salt treatments were applied during the callus initiating stage. With the

increase in salt concentration up to 10 g litre-1 resulted in a gradual decrease of

zygote germination (P_0.0001), callus embryogenesis (P_0.0001) and

callogenesis rates (P_0.0001). However, in culture media with high salt

concentration (15 and 20 g/L-1), the callus embryogenesis rate remained high.

Salt affected regeneration even when, at this stage, the culture medium was

deprived of salt. Indeed, calluses which had been initiated in 20 salt g L-1, had

the lowest regeneration rate. This concentration resulted in worthless albino

seedlings.

Muranakae et al. (2002) found out the impact of NaCl treatment on the

photosynthetic machanism in wheat (Triticum aestivum L.) cultivars differed in

salt tolerance by comparison with iso-osmotic PEG treatment. Both cultivars

similarly reduced the photosystem 2 (PS2) energy conversion efficiency

(PHIPS2) rapidly when plants were imposed to a 100 mM NaCl solution, though

no decline was detected under the iso-osmotic PEG treatment. There was no

correlation between the reduction of the leaf relative water content (RWC) and

the PHIPS2 in the two iso-osmotic stress treatments. In contrast, a decline of

PHIPS2 along with the increasing of the leaf sodium content over 4% dry matter

was detected under the NaCl treatment, while no such correlation was observed

45

with other cations. The recovery of PHIPS2 after photoinhibitory irradiation was

repressed by the NaCl treatment as the increase of the duration of the treatment.

Norin 61 subjected to the 100 mM NaCl treatment for 10 d showed a decline of

the PHIPS2 after 1 h moderate irradiation of 400 µmol m-2 s-1 PPFD. Thus the

concentrated Na+ within a leaf under salinity treatments the stability of PS2

functions may decrease and lead to photochemical inactivation.

Poustini (2002) conducted a greenhouse study to determine the impact of

salinity stress in 30 wheat cultivars (including salt-tolerant cultivars Kharchia-66,

Mahooti, Alvand, Roshan, Sorkh-tokhm, Sholeh, Tabasi, Kavir and Mahdavi).

Tap water (EC=0.6 dSm-1) was used as the control, and increasing

concentrations of NaCl in water up to an EC of 16 dS/m were given as salt

treatments. Based on the grain and shoot dry weights, seventeen cultivars

exhibited absolute salt tolerance. The significant correlations between the grain

and shoot dry weights, and grain filling period under saline conditions indicated

that extending the grain filling period, which resulted to a higher grain dry weight,

highly and positively affected the salt tolerance. The relative salt tolerance was

highest in Tabasi and Sholeh, and lowest in cultivars Atrak and Ghods. There

were some other cultivars, although not known as salt-tolerant, that showed

relative salt tolerance based on either one or both the grain and shoot growth

rates (e.g. cultivar Bessotoon). Leaf burn, particularly at 98 days after sowing,

can be taken as a rapid visual indicator of salt tolerance in wheat cultivars.

Ashraf and Shabaz (2003) screened twenty five cultivars of early CIMMYT

hexaploid wheat were evaluated for salt tolerance in a green house experiment

46

using photosynthetic capacity and water relation parameters as selection criteria.

Under salt stress (150 mM NaCl) the cultivars Frontana, Norin-10, Mayo-54,

Noreste-66, and Yaktana-54 excelled all other genotypes in shoot dry mass,

while Na(20)TPP, Inia-66, Penjamo-62, Frontana, Siete Cerros, and Jaral-66 in

grain yield per plant in both absolute and relative (percent of control) terms.

Although net photosynthetic rate (PN) declined in all cultivars due to salt stress, it

was not helpful in discriminating among cultivars according to salt tolerance.

Similarly, no positive relationships of salt tolerance of the cultivars with stomatal

conductance, leaf water potential, or turgor pressure were found. Every cultivar

used its own specific mechanism to tolerate salt stress. However, a large amount

of variation in salt tolerance observed in 25 early CIMMYT wheat cultivars can be

of considerable practical value for improving salt tolerance in the existing

commercial hexaploid wheats.

Tomar and Punia (2003) cultured mature embryos and seeds of wheat

cultivars C 306, HD 2329, UP 2338, PBW 343 and WH 533 on MS medium

supplemented with 0, 0.5, 1, 1.5 or 2% NaCl to determine a suitable medium for

in vitro salt-tolerance screening in wheat. Callus growth in WH 533 and HD 2329

was observed up to 1% NaCl concentration, whereas callus growth in UP 2338

was observed only up to 0.5% NaCl concentration. Callus growth in C 306 and

PBW 343 cultured in MS medium supplemented with NaCl was not observed.

Seed germination of PBW 343 and UP 2338 was recorded only up to 1.5% NaCl

concentration, whereas those of cultivars C 306, HD 2329 and WH 533 were

recorded up to 2% NaCl concentration.

47

Javaid-Akhtar et al. (2003) coducted a hydroponics study to determine

the salt tolerance of 20 wheat genotypes under three salinity levels .i; control,

100 and 200 mM. Seven-day-old seedlings were transplanted in thermopore

sheets with holes floating on 1/2 strength Hoagland's solution (200 litres) and

assessed after 20 days. Classification criteria was formulated according to the

salt tolerance of the genotypes based on biomass production . Salt tolerant

cultivars included 8244, 8659, 8730, B2-57 and B2-5711. Less salt tolerant

genotypes were 8290,8284, 8602-1, B4-5711, 8706-1, 8784, 8670, and 8638.

The sensitive genotypes were 8699, 5038, 8750, 8757, B4-92, B2-5713, and B2-

5734.

Zair et al.(2003) quantified somatic embryogenesis through callus culture

under salt stress conditions for 8 wheat genotypes currently cultivated in

Morocco. The genotypes were classed according to the mean number of somatic

embryos formed per immature embryo and regenerated plants per 100 explants

under saline conditions. Regenerated plants from control callus (R0-0) and callus

initiated on 10 g L-1 NaCl (R0-10) did not show significant differences concerning

plant height, spike length and grain number per ear but, the R0 plants remained

less developed than parent plants. When irrigated with a solution containing

more than 20 g L-1 NaCl, the seeds of line to Te derived from R0-10 regenerated

plants showed the best elongation of roots and coleoptiles. Moreover, a

chlorophyll fluorescence test exhibited a clear improvement in salt tolerance of

R1-10 plants at four to five-leaf stage, as compared to R1-0 plants. It is

48

concluded that plant regeneration from callus masses on high NaCl levels may

be a valid method of selection for salt tolerance.

Dang and Lang (2003) used induced mutation to exploit genetic variability

in rice cell culture to obtain salt resistant plants through in vitro selection and

found that optimum medium was well responsive to callus production in Doc Do

and Pokkali than Trang Thai Lan and KDM105 with MS+2,4-D(2mgL-1) to

embryogenic callus formation ability. Parts of the calli were transferred to NaCl

supplemented medium in an attempt to produce physiological variants from

somaclones and assesed some physiological aspects of cellular adoption in

response to salinity among cultivars selected in Soc nau, Trang Thai Lan,

AS996, these increased the %age of calli showing regeneration by 90.79%,

75.00%, 73.08%, and68.75%, respectively.

Shariatpanahi and Dabirashrafi (2003) used somaclonal variation to

produce high-yielding salt tolerant lines in barley. Embryo derived calluses were

cultured on e MS medium fortified with various doses of 2,4-D and then shifted

to the medium containing NaCl for plantlet regeneration. The %age of callus,

root and shoot were analysed and found that the genotypic effect on callus

induction was significant while the mature embryo produced the lowest callus

induction.

Khan et al. (2004) studied somaclones of sugarcane cv. CP-43/33 in the

field on saline sodic soil, and salt tolerance in these plants were evaluated based

on growth and quality parameters. The somaclones performed better than the

source plant in term of tillers per plant, stem height, number of nodes per stem.

49

Yadav et al. (2004) performed a experiment to study callus growth, to

select salt-tolerant cell lines and to regenerate whole plant from selected/non-

selected cell lines in wheat in seven genotype using NaCl @ 4,12,20,28,36& 44

dSm-1 and found that callus growth on salt-containing media was slow compared

to untreated medium. There was a continuous decrease in relative callus growth

under NaCl salinity. Evaluation of regenerated plants showed somaclonal

variation for spike length, spikelet number and grain number.

Ghannaddha et al. (2005) studies the relationship between different traits

and salt tolerance in bread wheat through tissue culture and observed that with

the increase of salt the callus size was decreased and Na+ contents of the callus

increased. Genotypes and genotypes x salt effects were significant for k+ and

K+/Na+. Similar findings were observed by Patade et. al., (2008).

Gandonou et al. (2005) studied three sugarcane varieties for in vitro salt

screening to callus induction and embryogenic callus production. The culivars

responded well to callus induction with a %age of about 82, 84 and 100% for

CP70-321, NCo310 and CP65-357, respectively. The high percentages of

embryogenic callus obtained for three varieties indicated that these lines have a

high ability for embryogenic callus production. Relative fresh weight growth of

callus was about 1.07, 1.282,and 0.925 for CP70-321, NCo310 and CP65-357

respectively. NaCl impact resulted in calli necrosis and a reduction in their

growth. However, growing calli obtained from varieties CP70-321 and NCo310

exhibited less necrosis percentage and less relative fresh weight growth

50

reduction under salt stress. They proved to be more salt tolerant in vitro than

CP65-357.

Lu, et al. (2007) established a protocol for in vitro induction of salinity

tolerant somaclonal variation, calli of tripod bermudagrass cv. TifEagle were

cultured in suspension medium. To create somaclonal variation the, calli were

subcultured for 18 months and were then subjected to three-round selection for

salt-tolerant calli by culturing on medium containing 0.3 M NaCl for ten days

followed by a recovery for two weeks. The so survived calli were regenerated on

regeneration medium containing 0.1M NaCl. Three somaclonal variant lines (2,

71, and 77) were collected and analyzed. The selected somaclonal variants

showed higher relative growth and less injury than Tifeagle under salinity stress,

indicating that they enhanced salt tolerance. In addition, they have relatively

more water content and low electrolyte leakage than Tifeagle by withholding the

irrigation, indicated that they also increased drought tolerance. The line 71 had a

higher K+/Na+ ratio under salt stress conditions, indicated that different

mechanisms for salinity resistance might exist in these three variants.

Keeping in view above information and conclusions, this study was

planned to create genetic variability and develop new cultivars of wheat by using

tissue culture techniques through callus culture for cultivation in the marginal

lands where no other crop is sown successfully. As wheat breeding by traditional

means has been in practice for centuries, but nosuccess has been made for a

breakthrough to cope with world’s demand. The varieties thus developed do not

last long. Long term objectives can be achieved unless more genetic variability is

51

generated. In vitro technology complements the conventional means of whet

breeding in generating diversity when both are combined with each other. So it is

concluded that tissue culture is a novel source to generate heritable genetic

variability to develop salt tolerant plants of wheat genotypes.

52

CHAPTER 3

MATERIALS AND METHODS

The Research pursuits on “Study of Somaclonal variation in wheat for the

induction of salinity tolerance” reported in this thesis were conducted at the

Department of Botany, Government College University, Faisalabad and partially

at the Agricultural Biotechnology Research Institute AARI, Faisalabad, Pakistan

during the year 2005-2009. This study was initiated to exploit somaclonal

variation and to benefit from its potential for wheat improvement under salt

affected soils.

GROWTH CONDITIONS AND EXPERIMENTAL TECHNIQUES

Growth conditions and techniques used in these studies in most of the

experiments are described in this chapter; other more specific to individual

experiments will be presented in the relevant chapters.

Seed source

Fifty wheat genotypes including those known salt tolerant varieties i.e. LU-

26S and Pasban-90 were obtained from Wheat research Institute, Agric.

Biotechnology Research Institute, ARRI, Faisalabad and National Agric.

Research Centre, Islamabad (Annexure-1).

53

Callus initiation

Mature seeds /mature embryo of 50 wheat genotypes ( Triticum aestivum

L) were sterilized with ethanol (100 %) followed by 0.01 % HgCl2 with

subsequent shaking for 30 seconds. Then applied three rinsing of autoclaved

distilled water under aseptic conditions. Afterwards seeds were cultured in

18x150 mm test tubes containing solidified MS medium (Murashige and Skoog,

1962) (Annexure-2) supplemented with 4 levels of 2,4-D (1, 2 ,3, and 4 mgL-1) to

induce callus & at least 10 seeds per genotype (one seed per test tube).

Prior to autoclaving, pH of the medium was maintained to 5.8 poured in

test tubes of 18x150 mm and then autoclaved at 15 psi pressure at 121o C for 30

minutes. Then the material was placed in incubation room at 28+-2oC with 12

hours photoperiod illuminated with florescent light to produced 2500 lux. Callus

induction frequency at different levels of 2, 4-D was recorded at an interval of 15

and 45 days. The responsive varieties (having 50 or > 50% callus frequency)

were subjected to different levels of salinity for further studies (Annexture-3).

The following laboratory parameters were recorded:

Callus induction

Fresh weight growth of callus(FWG)

Relative fresh weight growth(RFWG)

Frequency of necrotic calli

Regeneration

54

Plantlet production

The callus induction/ fresh weight growth of callus and frequency of

necrotic calli was calculated on percentage basis.

Relative fresh weight growth Rate

Fresh weight of the calli after harvesting from the saline medium was

determined with the help of electronic sartorious research balance (Model R-160)

and their fresh relative growth rate were calculated with the following formula:

In (FW)-In (IW).

Salinity treatment

The responsive wheat genotypes were cultured on MS medium

supplemented with 2,4-D @ 3 mgL-1 and four salinity levels to induce salt

tolerance. There were four treatments including control, and each treatment was

replicated ten times. 100 mg calli were used in each treatment. The following

treatments were studied during the course of this research work.

So Control S1 50 mM NaCl S2 100 mM NaCl S3 150 mM NaCl

55

Regeneration of plantlets

Surviving calli under salt stress were shifted to regeneration medium for

the development of plantlets. MS basal medium with out any addition of 2,4-D

was used for the regeneration of plants under same salinity levels ( Hussain et

al., 2001). The plantlets developed by this method were shifted to the pots filled

with sand and gravel under the same salinity levels under controlled conditions

up to maturity to produced Ro seeds. Then R1 plants along with parents were

treated with salt to assess their extent of salinity tolerance after following Arzani

and Mirodjagh, 1999.

The following parameters were studied at seedling stage.

Shoot fresh weight

Shoot dry weight

Root fresh weight

Root dry weight

Na+ concentration in plants

K+ concentration in plants

K+/Na+ ratio in plants

To obtain shoot dry weight, the shoots were stored in paper bags. First

these were air dried and then were oven dried at 65 +- 5oC till the weight

was constant.

56

Phenotypic measurements

On the maturity of crop, data regarding vegetative growth and yield

components were also recorded.

Plant height

Tiller/plant

Number of spike/plant

100-grain weight

Number of spikelets

Yield/plant

To study the yield components somaclones of different wheat genotypes

were grown along with their parent on artificially salinized soils in pots. The salt

levels were developed in the following way:

Original EC of the soil = A

EC to be deliverd = B

Difference of EC = B-A = C

T.S.S. = C x 10 = D meq/l

The meq/L was converted in to mg/100 g by using following relationships.

D x Eq.wt./1000 x S.P = mg/100g.

57

Plant Analysis

Plant sample collection

After uprooting the plants, soil was removed carefully from the roots. Roots

and shoots were separated and the latter were dried with tissue paper. Fresh

weight of shoots and roots was recorded immediately. The youngest fully

expanded leaf was detached, washed with distilled water, blotted and stored in

1.5 cm3 polypropylene micro centrifuge tube at freezing temperature. The leaf

was used later for tissue sap extraction. Fresh weight of shoot was again

recorded after removing the leaf and the sample was preserved in the paper bag.

To obtain dry weight, the plant shoot was dried at 70oC till the weight was

constant. Calculations were made to get the dry weight of shoot including the leaf

separated for tissue sap extraction.

Extraction of tissue sap

For the extraction of sap the plant material was frozen in 1.5 cm3 micro

centrifuge tubes, thawed and crushed by using a metal rod with a tapered end.

Small holes were made in the cap and in the base of the tube and the sap were

centrifuged out into a second centrifuged tube at about 6000xg. After further

centrifugation of second tube was done at 9000xg for 3 minutes, the sap was

diluted for the estimation for inorganic ions by following Gorham et al. (1984).

58

Determination of sodium and potassium

The tissue sap was diluted as required by adding distilled water and

sodium and potassium estimation was done by using Flame photometer with the

help of standard solutions of NaCl and KCl prepared from reagent grade salts

(Method 10a and 11a of USDA Hand book 60: Annonymous, 1954).

Physiological parameters

The following physiological data were also recorded:

Stomatal conductance

Stomatal conductance of the fully expanded leaf was measured with IRGA

(Infera red gas analyzer) from 10:30 a.m. to 1:0 p.m.

Net Photosynthesis rate

The net photosynthesis rate was also measured by IRGA (Infra red gas

analyzer).Analytical set of conditions was molar air flow per unit leaf was 335

mMm-1 S-1, atmos pressure 99.61 (Pa) PAR on leaf surface 1250 µmol m-2 S-1,

CO2 concentration 357 umol m-1 and ambient temperature for control plant was

28oC.

Statistical Analysis

Data of the experiment was subjected to statistical analysis using

completely randomized design (C.R.D.) in factorial arrangement (Steel and

Torrie, 1980) using M STAT computer soft ware. The significance among mean

and interaction values was determined by using LSD test.

59

CHAPTER 4

RESULTS

Preliminary screening of wheat genotypes for callogenesis.

Plant tissue culture has provided plant breeders with certain techniques for

creating genetic variability without involving sexual crossing. Callus cells under go

genetic changes during callogenesis and which after regeneration give rise to plantlets

genetically variable (Larkin and Scoworoft, 1981). Mature seed / embryo of fifty wheat

genotypes were cultured on solidified MS medium fortified with 4 levels of 2,4-D, i.e.,

1,2,3, and 4 mgL-1 in 18 x 150 mm test tubes (1 seed / tube). Ten replication were

followed in each genotypes for each 2,4-D levels. The rest of the procedure is

mentioned in the chapter Material and Methods. The callus initiation was recorded after

30 days of culture. After 45 days of culture callus frequency / fresh growth was recorded

(Table-1)

Responses of genotypes to callogenesis

The data reveals that all the genotypes were different from each other in

response to callogenesis. Callus induction frequency ranged from 25% to 90%. Among

the varieties maximum callus (90%) was produce by variety Uqab-2000 and AS-2002

followed by Ufaq-2002 and V-4189 with callus score of 85% and 80% respectively.

60

Minimum callus (25%) was noticed in Barani-70 and WL-711 with the callus induction

potential of 30%. All other varieties responded with ranges between these varieties.

All the wheat genotypes produced the callus at all the 2, 4-D levels. Good callus

production was recorded at the level of 3mg and 2mgL-1 2, 4-D and it was less in 1mg

and 4mg per litre. The callus produced at 3mgL-1 was more granular and morphogenic

in nature.

Interaction between varieties x 2, 4-D showed different % age of callus at

different 2,4-D levels. It ranges from 20-100 %. Ufaq-2002, SA-42, Barani-83, Uqab-

2000 and AS-2000 showed higher callus induction frequency. Less callus (20%) was

produced by Barani-70, WL-711 and V-04112. Ufaq-2002 produce maximum callus at

3mgL-12,4-D while AS-2000 at 2mgL-1. SA-42, Barni-83 and Uqab-2000 gave maximum

response at 4mgl-1 2, 4-D levels.

The objective of this study was to obtain preliminary information about callus

formation potential of 50 wheat genotypes which was done successfully. Genotypes

showing better callus response were studied further for regeneration and somaclonal

variation under 4 salinity levels. 20 genotypes that produced 70% or more callus

induction frequency i.e., Ufaq-2002, SA-42, Parwaz-94, LU-26S, Pothowar, Punjab-76,

Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86, Pasban-90, Inqulab-91, Punjab-

96, Uqab-2000, Chenab-70, Iqbal-2000, AS-2000, Bhakhar-2002, V-03079 and V-

04189 were tested for somaclonal variation study under salt stress conditions.

61

Table.1: Callus Induction frequency of 50 wheat varieties, using mature embryo as ex plant source under different 2,4-D, levels Name of Variety 2,4-D, levels Mean

1mg L-1 2mgL-1 3mgL-1 4mgL-1 Ufaq-2002 60 90 100 90 85 Mexi Pak 30 30 40 40 35 Chenab-70 40 40 50 50 45 Barani-70 20 30 30 20 25 SA-42 60 60 90 100 77.5 Blue Silver 60 60 60 60 60 Lyall Pur-73 50 40 50 50 47.5 Parwaz-94 70 70 70 70 70 PARI-73 60 60 60 60 60 Sandal 30 50 50 50 45 SA-75 50 60 50 60 55 LU-26 S 70 70 80 80 75 WL-711 30 30 40 20 30 Punjab-81 60 60 70 60 62.5 Pak-81 60 60 70 70 65 Pothowar 50 90 80 80 75 Punjab-76 60 70 90 80 75 Barani-83 60 60 80 100 75 Kohinoor-83 80 80 70 70 75 Fsd-85 70 70 80 70 72.5 Chakwal-86 60 80 80 70 72.5 Shalimar-86 50 70 70 60 62.5 Rohtas-90 60 60 70 60 62.5 Pasban-90 60 60 90 90 75 Inqlab-91 70 80 80 80 77.5 Shahkar-95 70 70 70 60 67.5 Punjab-96 60 60 90 80 72.5 MH-97 50 60 60 60 57.5 Kohistan-97 60 60 60 60 60 Uqab-2000 80 90 90 100 90 Chenab-70 80 80 80 70 77.5 Iqbal-2000 70 80 80 80 77.5 SH-2000 60 70 60 60 62.5 AS-2000 80 100 90 90 90 Sehar-2006 70 70 70 60 67.5 Shafaq-2006 60 70 60 60 62.5 Fareed-2000 50 60 60 60 57.5 Bhakar-2002 60 80 80 90 77.5 GA-2002 60 70 60 50 60 V-03094 40 50 50 50 47.5 V-03079 70 80 80 80 77.5 V-04188 30 70 40 40 45 V-04189 70 80 90 80 80 V-03138 50 60 50 50 52.5 V-04040 50 50 50 50 50 V-04067 30 60 60 50 50 V-04112 20 60 50 50 45 V-04171 30 50 60 60 50 Fsd-83 50 70 70 70 65 Punjab-85 60 60 70 60 62.5 Mean 55.6 64.8 67.6 65.22

62

Effect of salinity on callogenesis and organogenesis in 20 wheat varieties It is very difficult and time consuming to develop salinity tolerant varieties

through traditional breeding techniques. Whereas exploitation of somaclonal

variation in plants to develop salt tolerant cultivars is an efficient and potent

methods for producing novel and useful varieties (Gandonou et. al; 2005). The first

study in Pakistan on callogenesis and organogenesis in wheat under salt stress was

carried out by Mahmood and Quraishi in 1983 .Moreover, the study was restricted to

callogenesis. The present piece of study has, infact, broadened the scope of

somaclonal variation and investigations have been made on the impact of various

doses of NaCl salinity in callus formation and regeneration in 20 wheat varieties.

The mature embryo / seed of 20 wheat varieties (Ufaq-2002, SA-42, Parwaz-94,

LU-26S, Pothowar, Punjab-76, Barani-83, Kohinoor-83, Faisalabad-85, Chakwal-86,

Pasban-90, Inqulab-91, Punjab-96, Uqab-2000, Chenab-70, Iqbal-2000, AS-2000,

Bhakhar-2002, V-03079 and V-04189) which gave good response to callogenesis in

the previous studies were cultured on MS medium + 3mgL-1 2,4-D supplemented

with 4 doses of NaCl salt i.e., 0,50,100 and 150mM NaCl according to the methods

described in chapter-2. 20 test tube of each varieties having one seed per tube

were cultured on each salt level.

Callogenesis under salt stress

Differences among genotypes for callus induction potential under salt stress

were quite evident from the results presented in Table-2. Almost all the genotypes

produced callus of varying degree with and with out salt. Comparison of the cultivars

indicated earlier, the callus initiation was high in case of control (0 NaCl) and callus

63

weights ranged from 0.05169 g (Kohinoor-83) to 0.10 g (Uqab-2000), at 50 mM. The

range was from 0.039g (Pothowar) to 0.090 (LU-26S), at 100mM from 0.0280

(Faisalabad-85) to 0.0794g (LU-26S) at 150mM NaCl. The data led to the conclusion

that increase of salt in culture medium directly affect the production of callus in all

the genotypes. The callus growth at higher salt level was very low as compared to

control. Mean values showed that genotypes LU-26S proved to be more salt tolerant

followed by Uqab-2000, Inqulab-91 and V-04179 and Kohinoor-83 was the least

responsive to callus initiation. Computation of mean callus values of wheat cultivars

under cumulative salt stress given in Fig-III. Analysis of variance showed that the

differences among treatment means, genotypes mean and genotypes x treatment

induction was non significant. 10 genotypes, i.e., Ufaq-2002, Parwaz-94, LU-26S,

Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-

2002, produced granular, compact, Shiny and morphogenetic calli under salt stress

and were further valuated for relative fresh weight growth on the basis of callus

characteristics and shape. The rest ten varieties, i.e., SA-42, Pothowar, Barani-83,

Kohinoor-83, Faisalabad-85, Chakwal-86, Punjab-96, Iqbal-2000, V-03079 and V-

04189 produce necrotic calli (Table.7). The callus produced by these varieties was

loose, watery in nature and in dry form. The 10 wheat varieties (Ufaq-2002, Parwaz-

94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000,

and Bhakhar-2002) that produced good granular calli under salt stress were

evaluated for relative fresh growth rate of callus under the same salinity levels.

64

Relative fresh growth rate of callus

The relative fresh weight growth of calli (Table.3) in 10 wheat varieties Ufaq-

2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-

70, AS-2000, and Bhakhar-2002, were calculated as per formula given in chapter-3

(page-54).

Analysis of variance showed that treatment means, genotypes means and

interaction between treatment and genotype to relative fresh weight growth of callus

were non significant. Although interaction was not significant, genotype Punjab-76

showed more reduction than other varieties at all the three salinity levels, i.e., 0,

50,100 and 150 mM NaCl while variety AS-2000 reduced callus weight at 150 mM

NaCl. A trend of reduction in relative fresh growth rate with increase in salinity was

observed. Maximum callus reduction was recorded at salt level 150mM (0.1684)

followed by 100 (0.1947), 50 (0.2065) and 0 mM NaCl (0.2016)(Table-3).

Mean differences in varieties described that maximum reduction was

observed in AS-2002 (0.1831) followed by Punjab-76 (0.1884) and Chenab-70

(0.1894) respectively while minimum reduction in relative fresh weight growth was

recorded in Ufaq-2002 (0.2085). The other varieties reduced relative fresh weight

growth in between this range.

All the 10 varieties were evaluated for their regeneration potential under the

same salinity levels.

65

Table.2: Response of wheat genotypes to callogenesis under salt stress Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 0.093375 0.08926 0.079783 0.072508 0.0837315 SA-42 0.099794 0.089914 0.08417 0.074737 0.08715375Parwaz-94 0.08423 0.08049 0.07268 0.05818 0.073895 LU-26S 0.07787 0.09993 0.09037 0.0794 0.0868925 Pothowar 0.06097 0.05776 0.03905 0.003862 0.0404105 Punjab-76 0.09277 0.06374 0.07456 0.06535 0.074105 Barani-83 0.080697 0.07781 0.06727 0.05983 0.07140175Kohinoor-83 0.053406 0.05169 0.04368 0.03588 0.046164 Faisalabad-85 0.07109 0.06683 0.04842 0.02832 0.053665 Chakwal-86 0.05755 0.05655 0.05345 0.04386 0.0528525 Pasban-90 0.09962 0.09587 0.08306 0.06871 0.086815 Inqulab-91 0.1058 0.09415 0.08546 0.07123 0.08916 Punjab-96 0.05286 0.0525 0.04661 0.04257 0.048635 Uqab-2000 0.11169 0.10137 0.08889 0.07987 0.095455 Chenab-70 0.08561 0.08558 0.07824 0.07288 0.0805775 Iqbal-2000 0.0857 0.08551 0.07553 0.0713 0.07951 AS-2000 0.1007 0.09648 0.07912 0.07568 0.087995 Bhakhar-2000 0.10075 0.09425 0.08206 0.07784 0.088725 V-03079 0.10695 0.09598 0.08092 0.0781 0.0904875 V-04189 0.1014 0.09628 0.07989 0.07798 0.0888875 Mean 0.0861416 0.0815972 0.07166065 0.06190435

Analysis of variance of wheat genotypes to callogenesis under salt stress SOV DOF SS MS F Replication 9 0.008 0.001 6.0231 Salinity 3 0.124 0.041 280.4685N.S Varieties 19 0.474 0.025 169.0280N.S Salinity x Variety 57 0.214 0.004 25.4007N.S Error 711 0.105 0.000 Total 799

66

Callogenesis, Morphogenesis and Plantlet development in Wheat

67

Callogenesis in different Wheat varieties

68

Morphogenesis in Wheat

69

Development of Plantlets

70

Somaclones of different wheat varieties in pots

71

Table.3: Salinity effect RFGR of wheat Callus(mg) Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 0.2304 0.2206 0.2058 0.1774 0.20855 Parwaz-94 0.2280 0.2144 0.2042 0.175 0.2054 LU-26S 0.2248 0.2104 0.2028 0.1754 0.20335 Punjab-76 0.2068 0.1938 0.1840 0.1690 0.1884 Pasban-90 0.2244 0.2142 0.1946 0.1798 0.20325 Inqulab-91 0.2310 0.2058 0.1964 0.1742 0.20185 Uqab-2000 0.2234 0.1950 0.1886 0.1532 0.19005 Chenab-70 0.2122 0.1972 0.1934 0.1550 0.18945 AS-2000 0.2078 0.1978 0.1770 0.1500 0.18315 Bhakhar-2002 0.2272 0.2162 0.2002 0.1752 0.2047 Mean 0.2216 0.20654 0.1947 0.16842 , : Analysis of variance of RFGR of wheat callus under salt stress SOV DOF SS MS F Replication 4 0.000 0.00 2.1509 Salinity 3 0.076 0.025 500.6676N.S Varieties 9 0.015 0.002 32.3505N.S Salinity x Variety

27 0.003 0.000 2.3596N.S

Error 156 0.008 0.00 Total 199 0.102 0.00 Necrotic percentage

Data regarding necrotic calli produced under salt stress is presented in

Table.4. Salinity increased the necrotic % age in calli of wheat genotypes. As the

salinity increased, necrotic % age also increased. Among the genotypes, it is clear

that maximum necrotic % age was observed in Pothowar (35.5) followed by Punjab-

76 and Chakwal-86 with the % age of 34.5 and 34.0, respectively. While it was low

in Ufaq-2002 (4.5%). Maximum % age of necrosis (27.55) was recorded at the

salinity level of 150mM NaCl. The minimum value (15.6%) was noticed where no salt

was applied.

72

Interaction between S x V showed that all the genotypes produced necrotic in

callus masses at higher salinity levels, while it was low under control conditions.

Table.4: Necrotic percentage in callus in wheat genotypes under salt stress. Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 2 3 3 10 4.5 SA-42 20 25 26 40 27.75 Parwaz-94 5 7 7 12 7.75 LU-26S 5 5 8 10 7.0 Pothowar 30 36 36 40 35.50 Punjab-76 8 10 10 19 11.75 Barani-83 25 38 36 38 34.5 Kohinoor-83 38 30 30 42 33.75 Faisalabad-85 30 30 32 40 33.0 Chakwal-86 30 33 33 40 34.0 Pasban-90 6 7 8 12 8.2 Inqulab-91 4 9 9 16 9.5 Punjab-96 16 28 30 36 27.5 Uqab-2000 6 6 7 21 10.0 Chenab-70 7 7 7 20 10.25 Iqbal-2000 20 22 20 33 23.75 AS-2000 8 10 10 20 12.0 Bhakhar-2000 7 7 8 20 10.5 V-03079 30 30 32 40 33.0 V-04189 30 30 33 42 33.7 Mean 15.6 18.65 19.25 27.55 - Regeneration Studies in 20 Wheat Varieties under Salt Stress

This experiments were aimed at production of callus derived plants of

varieties previously screened for callogenesis under salt stress. Calli developed after

4 week of seed culture were proliferation by sub culturing 4-5 times on the same

salinity levels + 3mg L-1 2,4-D. After proliferation, calli of 20 wheat varieties (Ufaq-

2002, SA-42, Parwaz-94, LU-26S, Pothowar, Punjab-76, Barani-83, Kohinoor-83,

Faisalabad-85, Chakwal-86, Pasban-90, Inqulab-91, Punjab-96, Uqab-2000,

73

Chenab-70, Iqbal-2000, AS-2000, Bhakhar-2002, V-03079 and V-04189) were

subjected to regeneration under the same salinity levels, i.e., 0,50,100 and 150mM

NaCl. The regeneration media was comprised of MS media with out having any

auxin (2,4-D) 10 test tubes of each varieties were maintain in each somaclone

under the control environmental conditions.

The response of genotypes to plantlet development under 4 levels of NaCl is

presented in Table-5. Range of plantlets developed varies from 1 to 48 plants

among all the varieties. Maximum shoots (48) were developed by LU-26S followed

by Pasban-90(40), Ufaq-2002(31), Inqulab-91(29) and Uqab-2000(26). Minimum

shoot developed in SA-42(1).Total number of plants ranged 1 to 48. shoot

development was suppressed with increase in salinity that indicated the deleterious

effect of NaCl on morphogenetic process in wheat.

74

Table.5.Regeneration Potential of 20 wheat varieties under salt stress (No. of plants) Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Total

Ufaq-2002 15 10 4 2 31 SA-42 1 0 0 0 1 Parwaz-94 7 6 5 2 20 LU-26S 21 10 10 7 48 Pothowar 4 1 1 0 6 Punjab-76 9 3 2 1 15 Barani-83 2 0 0 0 2 Kohinoor-83 1 1 0 0 2 Faisalabad-85 4 2 0 0 6 Chakwal-86 7 1 0 0 8 Pasban-90 17 10 8 5 40 Inqulab-91 12 8 6 3 29 Punjab-96 4 1 1 0 6 Uqab-2000 13 7 5 1 26 Chenab-70 6 4 2 1 13 Iqbal-2000 6 2 2 0 10 AS-2000 12 9 7 3 31 Bhakhar-2000 10 6 6 2 24 V-03079 2 1 0 0 3 V-04189 1 1 1 0 3 Total 154 83 60 27 324

Maximum shoots developed(154) were observed where no salt was applied,

i.e., 0 mM NaCl followed by 83 and 60 at salinity levels 50 and 100mM NaCl. While

minimum plants (27) were produced at the higher salinity level of 150mM NaCl.

At control level LU-26S was at the top with maximum plant (21) while SA-42,

Kohinoor-83 and V-04189 were at the bottom with 1 plant each. At 50mM NaCl level

varieties LU-26S was also at the top at par with Pasban-90 having 10 plants each.

Minimum response to regeneration was exhibited by Pothowar at par with Kohinoor-

83, Chakwal-86, Punjab-96, V-03079 and V-04189 having 1 plant each. At 100 mM

NaCl LU-26-S once again showed best performance while SA-42, Barani-83,

75

Kohinoor-83, Faisalabad-83, Chakwal-86, and V-03079 failed to developed any

plant. At 150mM level of salinity 10 varieties SA-42, Pothawar, Barani-83, Kohinoor-

83, Faisalabad-85, Chakwal-86, Punjab-96, Iqbal-2000, V-03179 and V-04189 did

not produced any plant. While LU-26S also remained on the top at this salt level as

well.

Thus these developed plantlets were transferred on rooting medium salinized

with the same salinity levels (MS+1mgl-1 IAA). Plants of 10 wheat varieties (Ufaq-

2002, Parwaz-94, LU-26S, Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-

70, AS-2000, and Bhakhar-2002) developed roots and turned in to complete plants.

Then these complete plants having both shoots and roots were shifted to sand

culture in pots to obtain regenerator seed (R0). The R0 seeds of these varieties were

further evaluated for chemical analysis and yield components.

Plant analysis

There is no single selection criterion for salinity tolerance (Blum, 1988) that

has been tested through the process of selection with positive end results. Selection

for improvement of crop salt tolerance may be made at a number of stages in the life

cycle of a crop, although selection at seedling stage would clearly as easier and

more economical way. On the other hand, seedling or plant dry matter as an

expression of total growth is an important criterion in terms of over all yield under

stress conditions. It integrates various possible effect of response to salinity into one

measure of resistance. As root growth often expresses the relative resistance of a

76

plant to salt stress, it was also considered as a possible criterion of resistance to

salinity (Qureshi et el., 1990).

R0 seeds of 10 wheat somaclones i.e. (Ufaq-2002, Parwaz-94, LU-26S,

Punjab-76, Pasban-90, Inqulab-91, Uqab-2000, Chenab-70, AS-2000, and Bhakhar-

2002) were germinated and grown in plastic buckets containing vermiculite and

gravel (1:1 by volume) in a wire house to avoid the birds. 4 levels of salinity, non-

saline and saline (50, 100, and 150mM NaCl) were used. The control and saline

buckets were filled and drained daily. Full strength Hoagland nutrient solution were

used as the nutrient solution. The buckets were recycled with the same solution

throughout the week. Salt were mixed to the nutrient solution to develop the needed

concentration to final desired salinity levels.

10 seeds of each somalones were grown in each treatment and were split in

to 5 replication each of 2 plants. Plants were harvested 7 weeks after sowing. Fresh

weight of shoot and root were recorded while the youngest expanded leaves were

preserved in the tubes at freezing temperature for the extraction of sap. K+ and Na+

were determined from the leaf sap while shoot and root dry weight were obtained

after drying at 70oC in an oven.

Shoot fresh weight

Data given in Table.6. indicate that salinity significantly decreased the shoot fresh

weight and at all salt levels comparison of means showed significant differences

among treatment, variety and salinity x varieties.

Maximum (9.94g) shoot fresh weight was observed in control followed by 50,

100 and 150mM NaCl and it differs significantly from all other treatments. Minimum

77

fresh shoot weight (5.06g) was observed under highest level of salt. Differences in

mean in varieties showed that maximum shoot fresh weight (10.20g) was recorded

in somaclones of Ufaq-2002 at par with Inqulab-91 followed by Parwaz-94 (8.90g)

minimum shoot fresh weight (5.75g) produced by somaclone of Punjab-76 the other

varieties responded in between these range.

Interaction between S x V was also significant. Somaclones of Ufaq-2002

gave maximum shoot fresh weight (14.26 and 13.4g) at control and 50 mM NaCl

while somaclone of Punjab-76 give minimum shoot fresh weight (6.52g) at these salt

levels. In case of 100 mM NaCl variety Inqulab-91 produced maximum shoot fresh

growth weight (8.98g) and LU-26S yielded low fresh weight of shoot i.e. 5.04 g. At

higher salinity level (150mMNaCl) Inqulab-91yielded maximum fresh shoot weight of

7.84 g and Parwaz-94 was at the lowest (3.46g). It is clear from presentation given

in Fig-VIII that Ufaq-2002 was more tolerant at salinity levels 0f 0 and 50mM salt

levels while Pasban-90, LU-26S, AS-2000 and Bakhar-2002 produced more shoot

fresh weight at 50mM NaCl than control (Wyn Jones,1985). It was probably because

of the fact that low amount of salts might have rather stimulating effect on biological

activities with in the soil , especially the micro-organisms. Another reason could be

that salts might have served as nutrients at low concentrations.

Shoot dry weight

A look at the data ( Table.7 ) revealed average shoot dry weight decreased

from 1.0906 in plants from control ( 0 NaCl ) to 0.4034 g. at highest salinity level 150

mM NaCl. Differences among varietal means were also varies significantly. The

highest dry shoot weight ( 0.9785 ) was obtained in LU-26S , followed by Ufaq-2002

78

(0.9445) , Parwaz-94 ( 0.814 ) and the least being in somaclone from Uqab-2000

(0.4395).

Salinity and varietals interaction also produced significant differences.

Evaluation of somaclones in salinity were negatively related with each other.

Minimum dry shoot weight (0.204 g) was found in Bhakhar-2002 at 150mM salt level

and maximum (1.402g) was in Ufaq-2002 at where the salts were not added.

Table.6: Fresh Shoot weight of wheat somaclones (g) as affected by salinity

Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 13.40b 14.26a 7.20m 5.92p 10.20A Parwaz-94 12.94c 13.90a 5.32qr 3.46v 8.905B LU-26S 9.04h 8.84hi 5.04r 3.98tu 6.725F Punjab-76 7.42lm 6.52o 5.22qr 3.84u 5.750G Pasban-90 9.62f 9.42fg 7.36lm 5.46q 7.965C Inqulab-91 11.32e 12.12d 8.98hi 7.84jk 10.06A Uqab-2000 8.14j 8.00jk 7.66kl 6.2op 7.500 CDE Chenab-70 9.18gh 8.64i 7.40lm 4.26t 7.370DE AS-2000 8.94hi 8.90f 7.84jk 4.98rs 7.665CD Bhakhar-2002 9.40fg 8.16j 6.924n 4.66s 7.285E Mean 9.940A 9.876A 6.902B 5.060C Mean sharing the same letter do not differ significantly at P = 5% LSD V = 0.55 S= 0.35 S x V = 0.35 ,

Analysis of variance of fresh shoot weight of wheat somaclones SOV DOF SS MS F Replication 4 88.012 22.007 27.9064 Salinity 3 870.889 290.296 368.1816** Varieties 9 355.953 39.550 50.1615** Salinity x Variety

27 278.930 10.331 13.1024*

Error 156 123.000 0.788 Total 199 1716.784

79

Table.7: Salinity effect on Dry Shoot weight of wheat somaclones(g)

Name of Varity 0 mMNaCl 50 mMNaCl

100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 1.402 1.016 0.86 0.50 0.9445 Parwaz-94 1.09 0.876 0.786 0.504 0.814 LU-26S 1.47 0.932 0.798 0.714 0.9785 Punjab-76 0.844 0.83 0.652 0.498 0.706 Pasban-90 1.178 0.80 0.762 0.32 0.765 Inqulab-91 1.364 0.826 0.604 0.386 0.795 Uqab-2000 0.654 0.532 0.322 0.25 0.4395 Chenab-70 1.062 0.768 0.908 0.35 0.772 AS-2000 1.114 0.864 0.568 0.308 0.7135 Bhakhar-2002 0.728 0.624 0.292 0.204 0.462 Mean 1.0906A 0.8068B 0.6552C 0.4034D

Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.27

Analysis of variance of dry shoot weight of wheat somaclones

SOV DOF SS MS F Replication 4 3.764 0.941 19.4823 Salinity 3 11.283 3.761 77.8675** Varieties 9 4.914 0.546 11.3055** Salinity x Variety 27 1.802 0.067 1.3819* Error 156 7.535 0.048 Total 199 29.298 Root fresh weight

The data ( Table.8 ) show that the maximum root fresh weight (9.770g ) was

obtained at S1 ( 0 mM NaCl ) which decreased statistically as the salinity increased

and was minimum (5.332g ) from treatment S4 i-e. 150 mM NaCl. The relation

between salinity and fresh root weight is shown in the data.

Comparison of means among genotypes under controlled conditions

indicated that Ufaq-2002, Pasban-90, Parwaz-94 and Uqab-2000 had significantly

80

the highest root fresh weight as compare to other wheat somaclones while Punjab-

76 has the lowest value.

At salinity level 100 and 150 mM NaCl. Ufaq-2002 remained at the top while

Bhakkar-2002 and Punjab-76 were at the lowest respectively. Comparison of

genotypes on over all basis indicated that Ufaq-2002 had more root fresh weight

followed by Parwaz-94 and Inqulab-91 respectively while Punjab-76 had lowest

fresh root weight.

Root Dry Weight

A similar trend for root dry weight was observed. The data of root dry

weight presented is Table.9. Comparison of treatment means indicated that addition

of salt significantly decreased the root dry weight of wheat somaclones under all the

salinity levels. Highest dry root weight (0.7242g) at par with 0.7208g was found at 0

and 50mM NaCl respectively while it was 0.4602g at 100mM and 0.4058 at 150mM

salt level. The differences between the treatments were significant. Comparison of

different wheat somaclones revealed the highest root dry weight (1.133g) was

noticed in Ufaq-2002, while Punjab-76 yielded low dry root weight ( 0.3735 g ). At

50mM NaCl a slight increase in dry root weight was noted in Ufaq-2002 as

compared to the control (0 Mm NaCl) and Punjab-76 showed poor response(o.522g)

Ufaq -2002 also produced better root dry weight at salinity level of 100 and 150 mM

NaCl but it was less than the control. Somaclones of Punjab-76 were at the lowest in

dry root weight at both the higher levels of salt.

81

The interaction between treatments x somaclones also produced significant

Differences. Maximum root dry weight (1.266g) was recorded in Ufaq-2002

somaclones at 50 mM salt level while (0.228g) root dry weight was found in Chenab-

70 somaclones at the salinity level of 150 mM salt level.

Table.8: Salinity effect on Fresh Root Weight of wheat somaclones(g) Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 12.14a 10.46c 8.92hi 8.06k 9.785A Parwaz-94 11.3b 9.4fg 6.76m 6.2no 8.415B LU-26S 8.02k 7.28l 5.54p 4.84rs 6.420E Punjab-76 6.14o 8.08k 3.14w 4.02uv 5.345F Pasban-90 11.38b 9.18gh 5.28pq 4.18qr 7.745CD Inqulab-91 9.76de 9.72ef 7.12l 6.5mn 8.275BC Uqab-2000 11.3b 9.42efg 4.06uv 4.36tu 7.285D Chenab-70 9.24gh 8.9hi 4.48st 4.12uv 6.695E AS-2000 9.9d 9.72ef 5.3pq 5.52pq 7.660D Bhakhar-2002 8.8ij 8.52j 3.8v 4.52st 6.410E Mean 9.770A 9.068B 5.44k4C 5.332C Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 0.54 S= 0.34 S x V = 0.34 , Analysis of variance of fresh root weight of wheat somaclones SOV DOF SS MS F Replication 4 52.454 13.114 17.4630 Salinity 3 825.082 275.027 366.2469** Varieties 9 286.887 31.876 42.4489** Salinity x Variety 27 103.579 3.836 5.1086* Error 156 117.146 0.751 Total 199 1385.148

82

Table.9: Salinity effect on dry root weight of wheat somaclones Name of Varity 0 mM NaCl 50mM

NaCl 100 mM NaCl

150 mM NaCl

Mean

Ufaq-2002 1.266a 1.326a 0.96b 0.94b 1.133A Parwaz-94 0.916b 0.792b 0.48b 0.424b 0.6560B LU-26S 0.67b 0.81b 0.45b 0.354b 0.5710B Punjab-76 0.424b 0.522b 0.26b 0.276b 0.3735B Pasban-90 0.766b 0.654b 0.434b 0.302b 0.5390B Inqulab-91 0.608b 0.682b 0.578b 0.468b 0.5850B Uqab-2000 0.746b 0.554b 0.326b 0.308b 0.5085B Chenab-70 0.604b 0.614b 0.362b 0.228b 0.452B AS-2000 0.66b 0.672b 0.418b 0.374b 0.5310B Bhakhar-2002 0.582b 0.5825b 0.30b 0.28b 0.4385B Mean 0.7242A 0.7208A 0.4602B 0.4058B Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 3.05 S= 1.93 S x V = 1.93 , LSD = 3.059 LSD = 1.935 Analysis of variance of dry root weight of wheat somaclones SOV DOF SS MS F Replication 4 96.065 24.016 1.0268 Salinity 3 94.974 31.658 1.3535* Varieties 9 202.436 22.493 0.9616* Salinity x Variety 27 630.019 23.334 0.9976* Error 156 3698.837 23.390 Total 199 4672.330

83

Ionic concentration

Na+concentration in the leaf sap.

The data relating Na+ concentration in the sap of fully expanded leaf on

different wheat somaclones is presented in Table-10. On overall basis, youngest

expanded leaf had manyfold lower Na+ concentration in controlled treatment than the

salinity applications. Comparison of treatment means indicated non significant

differences in Na+ concentration under controlled conditions, but the addition of salt

significantly increase Na+ concentration under these conditions. Maximum Na+

(52.08mM )was recorded at higher salinity level .i.e. 150 mM NaCl followed by 20.81

and 7.52 at the level of 100 and 150bmM NaCl. The minimum Na+ concentration

(2.26) was observed at controlled level where no salt was applied.

Comparison of means revealed that there had a significant difference in Na+

ion concentration within genotypes. Somaclone AS- 2002 proved more salt sensitive

with the value of 27.87 mM Na+ concentration in the leaf followed by Parwaz-94 and

LU-26S with the value of 23.91 and 23.65 respectively less affected somaclones

was of variety Uqab-2000 with the mean values of 20.20 mM Na+ concentration

Interaction between the salinity and somaclones was also found statistically

significant. There was positive correlation between genotypes and salinity levels as

the salinity increased Na+ concentration also increased. Maximum Na+

concentration (70.04) was found for wheat somaclone AS- 2002 at 150 mM NaCl

level while minimum for LU- 26S at controlled level having no salt (Fig.XII)

84

Table.10: Effect of Salinity on Na+concentration (mol m-3) of 10 wheat varieties Name of Varity 0 mMNaCl 50

mMNaCl

100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 1.64z 8.02u 19.88n 46.08g 18.91F

Parwaz-94 2.22yz 10.06qr 27.66j 55.68c 23.91B

LU-26S 1.44z 8.56st 24.76l 59.82b 23.65BC

Punjab-76 2.20yz 5.14v 14.88o 44.8h 16.77G

Pasban-90 2.30y 5.18v 14.34p 46.66f 17.14G

Inqulab-91 1.84z 3.44wx 10.35q 39.7i 13.83H

Uqab-2000 2.40y 7.82u 22.32m 48.6e8 20.20E

Chenab-70 2.96x 9.06s 25.94k 55.6d 22.94C

AS-2000 3.72w 9.82r 27.86j 70.04ac 27.86A

Bhakhar-2002 1.82z 8.10tu 20.08n 55.88 21.47D

Mean 2.262D 7.522C 20.81B 52.08A

Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 0.79 S= 0.50 S x V = 0.50 ,

Analysis of variance of Na+ concentration of wheat somaclones SOV DOF SS MS F

Replication 4 18.445 4.614 2.8704

Salinity 3 74910.306 24970.102 15534.8038**

Varieties 9 3091.746 343.527 213.7208**

Salinity x Variety 27 2281.048 84.483 52.5681*

Error 156 250.749 1.607

Total 199 80552.304

85

K+ concentration in younger fully expanded leaf The effect of somaclones and salinity levels on K+ concentration in wheat

crop has been shown in Table.11. Analysis of variance indicates the somaclones,

salinity and there interaction decreased the K+ concentration in wheat plants

significantly. K+ concentration decreased with increasing soil salinity. K+ decreased

from 231.6 at controlled salt level to 133.7 mM at highest salinity level of 150 mM

NaCl. The observed decreased in K+ concentration with increasing salinity may be

due to competition among sodium and potassium because sodium antagnosis

potassium uptake due to which sodium partially can substitute potassium in certain

metabolic reactions at moderate salinity level.

Maximum K+ concentration (204 mM ) was found in Punjab-76 and minimum (183.9mM ) in the somaclone of Bhakhar-2002. The other genotypes responded in

between these two range. The resultant reduction in potassium may be the result of

growth dilution effect.

Interaction between genotypes and salinity was also observed statistically

significant. As salinity increase, K+ concentration decreased in a consistent pattern.

Maximum K+ concentration (238.0 mM) was found in Parwaz-94 at controlled salinity

level and minimum (115.4) for Ufaq-2002 at higher salinity level that is 150mM NaCl

the behaviour between genotype and salinity is presented in the data.

86

Table.11: EFFECT OF SALINITY ON K+ CONCENTRATION (Mol M-3) IN LEAF SAP OF 10 WHEAT VARIETIES AT DIFFERENT SALT LEVELS

Name of Varity 0 mMNaCl 50 mMNaCl

100 mMNaCl

150 mMNaCl

Mean

Ufaq-2002 237.2a 228.6d 208.2l 115.4v 196.900CDEParwaz-94 238.6a 217.8i 202.0m 142.6q 200.3B LU-26S 224.0fg 223.4g 211.4k 119.0t 194.4EF Punjab-76 235.6b 228.4d 214.40j 137.6r 204.0A Pasban-90 233.2c 210.4i 199.2m 137.0s 197.7BCD Inqulab-91 229.4d 215.4k 201.6n 141.6rs 193.9F Uqab-2000 226.0e 225.6j 210.2m 146.8q 196.1DEF Chenab-70 232.4c 220.2ef 194.8k 144.2p 203.6A AS-2000 237.8a 201.18h 195.4o 117.4p 199.6BC Bhakhar-2002 222.4h 218.6m 203.0o 135.4ub 183.9G Mean 231.6A 219.0B 204.0C 133.7D

Mean sharing the same letter do not differ significantly at P = 5% LSD V= 2.98 S= 1.89 SxV= 1.42

Analysis of variance of K+ concentration of wheat somaclones

SOV DOF SS MS F Replication 4 528.180 132.045 5.7337 Salinity 3 287074.475 95691.658 4155.1566* Varieties 9 6000.605 666.734 28.9512* Salinity x Variety 27 7747.775 286.953 12.4602* Error 156 3592.620 23.030 Total 199 304944.155 K+

: Na+ ratio in wheat somaclones.

K+: Na+ ratio (Table.12) assessed from leaves of wheat somaclones was quite

high under saline conditions it decreased drastically as the salinity level enhanced.

Comparison of treatments means indicated a significant decreased K+: Na+ ratio as

the salinity increased. Maximum K+: Na+ ratio 110.7 was under controlled (0mM

NaCl) while lowest (2.623) K+: Na+ ratio was observed at salinity level 150mM NaCl

and 100mM NaCl level, it was 32.21 and 10.85 respectively.

87

Comparison of means among the genotypes was also significant. Genotype

inqlab-91 had the higher K+: Na+ ratio in comparison with other genotypes. It was

followed by Ufaq- 2002 and LU- 26S at par with almost having the same K+: Na+

ratio while AS-2000 was at lowest with K+: Na+ ratio 23.92.

Interaction between salinity and genotypes was also found significantly.Ufaq-

2002 had maximum k+: Na+ ratio under control (having no salt). Genotype Inqulab-

91 showed maximum K+: Na+ ratio at all the rest salt levels with the value of 62.194,

19.696 and 3.486 respectively.

Table.12: K+/Na+ ratio of wheat somaclonal as affected by salinity Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 151.618b 28.5l 10.47r 2.5u 48.27B Punjab-94 82.778e 21.6o84 7.302t 2.55u4 34.58D LU-26S 156.038a 26.13mn6 8.542st 1.986u 48.19B Punjab-76 107.344e 43.21k 14.412q 3.06u 42.01C Pasban-90 99.97f 42.214k 14.308q 2.898u 39.95C Inqulab-91 124.9c 62.194j 19.696p 3.486u 5217H Uqab-2000 94.924g 27.558lm 9.024rs 2.93u 3361D Chenab-70 78.622h 24.872n 8.098st 2.636u 28.56E AS-2000 63.012i 22.438o 6.99t 2.078u 23.92F Bhakhar-2002 122.38d 24.894n 9.63rs 2.096u 39.75C Mean 110.7A 32.21B 10.85C 2.623D Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 2.62 V= 1.66 S x V = 1.66 , Analysis of variance of K+/Na+ ratio of wheat somaclones SOV DOF SS MS F Replication 4 66.065 16.516 0.9326 Salinity 3 365249.909 121749.970 6874.361*7 Varieties 9 14782.469 1642.497 92.7402* Salinity x Variety 27 31241.579 1157.096 65.3330* Error 156 2762.874 17.711 Total 199 414162.897

88

Physiological parameters

The present research work was undertaken to understand the mechanism of

salinity resistance in wheat somaclones and to rank them on the basis of their

salinity tolerance level. The following physiological parameters were studied.

Net photosynthesis rate (Pn)

The data presented in Table.13 shows that different levels of applied salinity

significantly reduced the net photosynthesis rate of all the wheat somaclones. The

maximum reduction was observed at higher salinity level, i.e., 150 mm NaCl. The

cultivars did differ significantly from each other. The maximum net photosynthesis

rate was observed in somaclones of Ufaq-2002 and Perwaz-94 and less

photosynthesis rate was shown by Chenab-70 somaclones. A significant interaction

( v x t ) was also observed under stress conditions.

Stomatal conductance

Data shows that different levels of imposed salinity reduced the stomatal

conductance of all the wheat cultivars but difference among them was non-

significant. The maximum reduction was observed at highest level of salinity. The

somaclones differed non-significantly from each other in stomatal conductance.

However, higher value of stomatal conductance was observed in cv.Inqulab-91. The

interaction ( v x t ) was also non-significant. The maximum stomatal conductance

was observed in somaclones of Uqab-2000 at 0, 50 and 100 mM NaCl salinity level

and it was high in Inqulab-91( somaclones) at higher salinity level ( Table.14).

89

Table.13: Effect of Salinity on Net Photo Synthesis(Pn) in 10 wheat somaclones Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 10.54c 9.72ef 8.2k 6.04q 8.625A Parwaz-94 10.68c 9.98d 8.3jk 5.26s 8.605A LU-26S 10.16d 8.66i 6.62p 4.76t 7.550C Punjab-76 9.6f 7.6lm 5.1s 3.42w 6.430E Pasban-90 9.86ef 7.42mn 5.74r 5.16s 7.015D Inqulab-91 11.42a 9.8e 8.4j 4.72t 8.585A Uqab-2000 11.04b 9.22g 7.3n 4.6t 8.085B Chenab-70 10.52c 8.88h 6.94o 3.96v 7.575C AS-2000 8.38j 6.58p 4.68t 3.56w 5.785F Bhakhar-2002 9.22g 7.72l 6.1q 4.16u 6.805D Mean 10.13A 8.586B 6.73C 4.566D Mean sharing the same letter do not differ significantly at P = 5% LSD V = 0.29 S = 0.18 SxV= 0.18 Analysis of variance of Net Photosynthesis of wheat somaclones SOV DOF SS MS F Replication 4 29.823 7.456 33.1920 Salinity 3 865.310 288.437 1284.0923** Varieties 9 176.365 19.596 87.2399** Salinity x Variety 27 37.714 1.397 6.2184* Error 156 35.041 0.225 Total 199

90

Table.14: Stomatal Conductance (gs) in10 wheat somaclones as affected by salinity Name of Varity 0 mMNaCl 50

mMNaCl 100 mMNaCl 150 mMNaCl Mean

Ufaq-2002 0.312 0.232 0.196 0.132 0.218 Parwaz-94 0.328 0.25 0.20 0.104 0.2205 LU-26S 0.328 0.278 0.204 0.14 0.2375 Punjab-76 0.234 0.182 0.122 0.086 0.156 Pasban-90 0.322 0.23 0.182 0.16 0.2235 Inqulab-91 0.372 0.322 0.27 0.204 0.292 Uqab-2000 0.388 0.292 0.234 0.142 0.264 Chenab-70 0.318 0.266 0.208 0.146 0.2346 AS-2000 0.184 0.136 0.098 0.076 0.1235 Bhakhar-2002 0.234 0.194 0.154 0.10 0.7505 Mean 0.302 0.2382 0.1868 0.129 Analysis of variance of stomatal conductance of wheat somaclones SOV DOF SS MS F Replication 4 0.0320 0.008 31.2191 Salinity 3 0.839 0.280 1102.2707NS Varieties 9 0.412 0.046 180.3509NS Salinity x Variety 27 0.053 0.002 7.7493NS Error 156 0.40 0.000 Total 199 1.375

91

Response of somaclones derived from wheat cultivars to salinity

Wheat breeding by tradition means has been in practice for centuries, but no

success has been made for a breakthrough to cope with worlds demand. The varieties

thus developed do not last long, long term objectives can not be achieved unless more

genetic variability is generated. In vitro technology complements the conventional

means of wheat breeding in generating diversity when both are combined with each

other. So biotechnological approaches are being evaluated to induce genetic variability.

In vitro cultures exhibit somaclonal variation and regenerated plants show heritable

variation (Ahloowalia,1982).the production profile of wheat from salt effected areas is

extremely poor. The yield in these areas can be increased through development of salt

tolerant wheat.

R0 regenerated plants of 10 wheat somaclones along with their parents were

tested in pots having artificially developed four salinity levels with NaCl salts .i.e. 0, 4. 8,

and 12dSm-1 ECe. Salt level were prepared as per method given in Hand Book No. 60,

United States Department of Agriculture(Anonymous,1958). A normal non- saline, non-

sodic sandy loamy soil was taken from the upper 15 cm layer of a field located in the

research area of Ayub Agricultural Research Institute, Faisalabad. The soil was first air

dried, ground and passed through 2 mm sieve and was mixed thoroughly, a

representative sample from the soil was taken for physical and chemical analyses. With

out any provision of leaching, 10kg of soil were filled in each of the 400 glazed pots the

soil was artificially salinized to different salinity level using NaCl. Calculated amounts of

92

salt) was mixed thoroughly with the soil of each pot separately, followed by

equilibrization for 15 days by giving alternate wetting and drying cycles.

10 healthy R0 seed each of both somaclones and their parents were dry-sown in

each part on 15th November 2008 and crop was harvested on 25th April,2009. During

the studies data regarding plant height, number of tillers, number of spikes, number of

spikelets , 100 grain weight and yield / plant were also recorded.

The major effects of somaclones to its parent , salinity and their interaction was

significant in almost all the parameters. It indicated that both somaclones and parents

were differential in their responses to salinity. Therefore, in discussing this results,

emphasis was given to the relative performance of various genotypes and wheat

somaclones under different stress conditions.

Plant Height:

The data presented in Table.15. represents the plant height of wheat genotypes

and somaclones at different salinity levels. The data reflects that plants attained more

height at lower salinity levels as compare to higher ones and the differences at different

salinity levels were significant. The mean plant height was 89.48, 75.82, 59.38 and

55.76 cm for 0, 4, 8 and 12 dSm-1 ECe levels. It was also noted that height of

somaclones was almost less affected than their parents. The findings are in line with the

results of different researchers working on wheat and barley (Poustini and Salmasi,

1997; Barkat and Haris, 1998; Lutts et al., 1999 and Ahmad et al., 2002.).

93

According to the data regarding plant height of individual genotypes at all the

salinity levels the maximum plant height (80cm) was attained by Ufaq.2002(S) and

minimum (64.95cm) by Chenab-70(P). The plant height of all the 20 genotypes was in

order as under:

Ufaq.2002(S) > Ufaq-2002(P) >Punjab-96(S) ) > Punjab-76(S) >LU-26S(S) > Inqulab-

91(S) >Bhakhar-2002(S) >AS-2002(S) > Chenab-70(S) > Pasban-90(S) > Uqab-

2000(S) > Punjab-76(P) > Punjab-96(P) > AS-2002(P) >LU-26S(P) > Uqab-2000(P) >

Inqulab-91(P) > Bhakhar-2000(P) > Pasban-90(P). > Chenab-70(P).

94

Table.15. Effect of Salinity on Plant Height in 10 wheat somaclones alongwith parents Name of Varity

0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean

Ufaq-2002 S 113.00a 80.80hi 70.40nopqr 55.80tuvwx 80.00A Ufaq-2002 P 96.20c 85.80fg 66.40r 56.20tuvwx 76.15B Punjab-96 S 101.80b 83.80fgh 60.40stu 55.00wx 75.25BC Punjab-96 P 83.40fgh 75.60jklm 57.20 stuvwx 56.00tuvwx 74.95BC LU-26S S 95.60c 81.80ghi 60.00stuv 56.40stuvwx 73.40CD LU-26S P 80.20hij 74.60klmn 59.00stuvwx 55.80tuvwx 71.35DE Punjab-76 S 101.80b 80.40hij 60.60st 57.00stuvwx 71.10DE Punjab-76 P 84.80fgh 77.40ijk 55.80tuvwx 55.00wx 69.60EF Pasban-90 S 81.00ghi 77.40ijk 59.60stuvw 55.80tuvwx 69.40EFG Pasban-90 P 79.60hij 73.00klmnopq 59.80stuvw 55.00wx 68.45FGH Inqulab-91S 91.80Cde 77.00ijkl 61.20s 55.40vwx 68.20FGH Inqulab-91P 84.40fgh 68.20qr 59.60stuvw 55.60uvwx 68.15FGH Uqab-2000 S 88.20def 74.00klmno 55.60uvwx 55.00wx 68.05FGH Uqab-2000 P 84.40fgh 71.80mnopq 58.00stuvwx 55.40vwx 67.70FGH Chenab-70 S 92.60cd 72.40lmnopq 57.60stuvwx 55.00wx 67.40FGHI Chenab-70 P 81.60ghi 68.60pqr 54.60x 55.00wx 67.40FGHI AS-2002 S 94.20c 69.60opqr 59.00stuvwx 55.60uvwx 67.40FGHI AS-2002 P 87.40ef 73.40klmnop 55.00wx 55.00wx 66.90HI Bhakhar-2000 S

87.20efF 80.20hij 59.00stuvwx 58.00stuvwx 66.85HI

Bhakhar-2000 P

80.80hi 70.80mnopqr 58.80stuvwx 57.20stuvwx 64.95I

Mean 89.48A 75.82B 59.38C 55.76D Mean sharing the same letter do not differ significantly at P = 5% LSD= V = 2.491 S = 1.114 SxV= 4.983 Analysis of variance of plant height of wheat somaclones SOV DOF SS MS F Replication 4 679.585 169.896 105959 Salinity 3 72885.640 24295.213 1515.2097* Varieties 19 5815.460 306.077 19.0890* Salinity x Variety 57 5457.660 95.748 5.9715* Error 316 5066.815 16.034 Total 399

95

The interaction among the varieties showed that maximum plant height

(113cm) was recorded in genotypes Ufaq-2002(S) followed by Punjab-96(S) with

101.80cm height at 0 dSm-1. The accessions Ufaq-2002(P) and Punjab-96(S)

showed best performance at 4 dSm-1 with the values of 85.80 and 83.80cm

respectively. Ufaq-2002(S) was again at the top with 70.40cm height at salinity level

8 dSm-1 while Ufaq-2002(P) maximum height of 56.20cm at salinity level 12 dSm-1 .

The minimum values were shown by Pasban-90(S), Inqulab-91(P), Chenab-70(P) at

0, 4, and 8 dSm-1 salinity levels and Punjab-96(S) along with five varieties were at

the bottom at 12 dSm-1 salt level . The remaining accessions fell in the these

extremes. The Ufaq-2002(S) and Ufaq-2002(P) stood almost at the top at all the

salinity levels which cab be used as salt tolerant wheat lines for salt affected soils.

No. of Tillers Per Plant

Data Table.16. depicts that all the genotypes and somaclones performed well

and produced more tillers per plant at lowest salinity level. Mean values recorded for

this parameter were 8.10, 4.88, 4.13 and 3.18 at 0, 4, 8 and 12dSm-1 respectively.

The differences in no. of tillers at different salinity level were statistically significant.

These results are in line with the finding of Poustini (1995), Barakat and Haris

(1998), khan et al.(2004) and Yadav et al. (2004).

Maximum tillers were produced by genotype Punjab-96(P) reflecting

maximum tolerance followed by Inqlab-91(P) whereas maximum sensitivity was

96

shown by Uqab-2000(S). When this somaclones were compared with their parent,

they were in the following order in producing the No. of tillers per plant.

Punjab-96(P) > Inqlab-91(P) > Pasban-90(P) > Inqlab-91(S) > Punjab-70(P) >

Bhakhar-2000(P) > Pasban-90(S) > Bhakhar-2000(S) > Ufaq-2002(P) > LU-26S (P)

> Punjab-96(S) >AS-2002(P) > LU26-S(S) > Uqab-2002(S) > Chenab-70(p) > Uqab-

2000(P) > Punjab-76(S) > AS-2000(S) ) > Ufaq-2002(S) > Chenab-70(S).

Interaction between the genotypes (P & S) and salt levels was found

significantly variable among themselves.

Table.16. Salinity effect on No. of Tillers in Wheat Somaclones

Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 8.6bcd 5.0klmn 3.2tuvw 2.6wxyz 4.850DE Ufaq-2002 P 8.8bc 5.4kl 3.6rstu 2.8vwxy 5.150CD Punjab-96 S 8.2cde 4.4nopq 4.2opqr 3.8qrst 5.150CD Punjab-96 P 8.8bc 7.2ghi 5.4kl 3.8qrst 6.3A LU-26S S 7.4fgh 4.6mnop 4.6mnop 3.6rstu 5.050DE LU-26S P 7.8efg 4.2opqr 4.6mnop 4.0pqrs 5.150CD Punjab-76 S 6.6ij 5.0lkmno 3.6rstu 2.2yz 4.350F Punjab-76 P 8.6bcd 5.6k 4.4nopq 3.2tuvw 5.45BC Pasban-90 S 8.2cde 5klmn 4.6mnop 4.0pqrs 5.450BC Pasban-90 P 8.8bc 5.4kl 4.8lmno 3.8qrst 5.700B Inqulab-91S 9.2b 5.2klm 4.2opqr 3.8qrst 5.600B Inqulab-91P 11a 4.8lmno 5.2klm 3.8qrst 6.200A Uqab-2000 S 6.8hij 3.4stuv 3.0uvwx 2.4xyz 3.900G Uqab-2000 P 8.6bcd 4.2opqr 3.4stuv 2.8vwxy 4.750E Chenab-70 S 6.8hij 3.2tuvw 3.2tuvw 2.0z 3.800G Chenab-70 P 8.2cde 4.2opqr 3.6rstu 3.0uvwx 4.750E AS-2002 S 6.4j 4.4nopq 3.6rstu 2.0z 4.1000FG AS-2002 P 8.2cde 5.6k 4.0pqrs 2.6wxyz 5.100CDE Bhakhar-2000 S 7.0hij 5.6k 4.6mnop 3.6rstu 5.200CD Bhakhar-2000 P 8.0def 5.2klm 4.8lmno 3.8qrst 5.450BC Mean 8.100A 4.880B 4.130C 3.180D

Mean sharing the same letter do not differ significantly at P = 5% LSD V=0.36 S=0.16 SxV= 0.72

97

Analysis of variance of No. of tillers of wheat somaclones SOV DOF SS MS F Replication 4 2.285 0.571 1.7076 Salinity 3 1367.268 455.756 1362.3312* Varieties 19 172.647 9.087 27.1617* Salinity x Variety

57 104.983 1.842 5.5054*

Error 316 105.715 0.335 Total 399

No. of Spikes

Data given in Table.17. reveal that all the mean genotypes and their

somaclones showed better performance and produced more no. of spikes at lower

salinity levels as compared to higher ones. No. of spikes were decreased as the

salinity increased. These were 8.20, 4.80, 4.08 and 3.17 at EC 0, 4, 8 and 12 dSm-1

respectively. These differences among the No. of spikes at different salinity levels

were statistically significant. The result indorsed the result of Grieve and Francoise,

1992, Khan et. al., 1992 and Poustini, 1995. Maximum No. of spikes among the

genotypes were produced by Punjab-96(P) and was at par with the Inqlab-91(P)

followed by Bhakhar-2002(S) and Pasban-90(P) whereas minimum no. of spikes

were produced by Chenab-70 (S) and Uqab-2000(S). Other genotypes were in

between these two peaks.

The interaction between salinity and genotypes/somaclones showed

that at EC 4d Sm-1 genotype Punjab-96(P) and Bhakhar-2000(S) were most tolerant

and Chenab-70(S) and Uqab-2000(S) were more salt sensitive. At EC 8 dSm-1 the

effect was similar but at EC12 dSm-1 , variety Punjab-96(P) along with LU-26S(P),

Pasban-90(P&S), Inqulab-91(P&S) and Bhakhar-2000(P&S) produced more no. of

LSD = 0.22

98

spikes which showed their improved tolerance at this level of salinity. The interaction

among the salinity x genotypes/somaclones were statasticaly significant (Fig.XIX).

Table.17. Effect of Salinity on No. of Spikes in 10 Wheat Somaclones alongwith parents

Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 8.2cde 4.8lmno 3.2tuvw 2.6wxyz 4.70H Ufaq-2002 P 8.8bc 5.4jkl 3.6rstu 2.8vwxy 5.15F Punjab-96 S 8.0def 4.4nopq 4.0pqrs 3.6rstu 5.00FGH Punjab-96 P 8.6bcd 7.2gh 5.2klm 3.8qrst 6.20A LU-26S S 7.4fg 4.6mnop 4.6mnop 3.6rstu 5.05FG LU-26S P 7.8efg 4.2opqr 4.6mnop 3.8qrst 5.10F Punjab-76 S 6.0ij 5.0klmn 3.6rstu 2.2yz 4.20I Punjab-76 P 8.2cde 5.2klm 4.4nopq 3.2tuvw 5.25EF Pasban-90 S 7.8efg 5.0klmn 4.6mnop 3.8qrst 5.30DEF Pasban-90 P 8.6bcd 5.2klm 4.8lmno 3.8qrst 5.60CDBCD Inqulab-91S 9.0b 5.2klm 4.2opqr 3.8qrst 5.55CDE Inqulab-91P 10.4a 4.8lmno 5.0klmn 3.8qrst 6.00AB Uqab-2000 S 6.6hi 3.4stuv 3.0uvwx 2.4xyz 3.85J Uqab-2000 P 8.6bcd 4.0pqrs 3.4stuv 2.8vwxy 4.70H Chenab-70 S 6.6hi 3.2tuvw 3.0uvwx 2.0z 3.70J Chenab-70 P 8.2cde 4.2opqr 3.4stuv 3.2tuvw 4.75GH AS-2002 S 9.0b 4.2opqr 3.6rstu 2.0z 4.70H AS-2002 P 8.8bc 5.2klm 4.0pqrs 2.6wxyz 5.15F Bhakhar-2000 S 8.8b 5.6jk 4.6mnop 3.8qrst 5.70BC Bhakhar-2000 P 8.6bcd 5.2klm 4.8lmno 3.8qrst 5.60CD Mean 8.20A 4.80B 4.08C 3.17D

Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.322 S= 0.144 SxV= 0.644 Analysis of variance of No. of spikes of wheat somaclones

SOV DOF SS MS F Replication 4 3.200 0.800 2.9811 Salinity 3 1445.968 481.989 1796.0917** Varieties 19 161.388 8.494 31.6525* Salinity x Variety

57 92.082 1.615 6.0200*

Error 316 84.800 0.268 Total 399

99

No. of Spikelets

Data on No. of spikelets are presented in Table.18. It narrates that all the

genotypes/somaclones produced more spikelets per ear at controlled level where no

salt was added as compared to spikelets at higher salinity level. i.e. 12 dSm-1. the

average No. of spikelets per ear was13.17, 12.31, 10.15 and 6.05 in case of 0, 4, 8

and 12dSm-1 respectively. These results are in agreement with the results of Grieve

et al.(1994); Khan et al.(1992); Barakat and Haris (1998) and Yadav et al. (2004) .

Whereas the performance of individual accessions is concerned maximum number

of spikelets per ear (11.95) was produced by Pasban-90(P) followed by Bhakhar-

2000(P) and Inqlab-91(P) with the value of 11.85 spikelets/ear by each genotype

and Inqulab-91(S) with value of 11.80 spikelets per ear. The minimum values were

observed in Chenab-70(S) and Uqab-2000(S) with the value of 9.30 and 10.05

spikelets per ear. All the remaining values fell in between these lower and higher

performance levels.

The interaction of accession with salinity levels showed that at EC 4dSm-1

Bhakhar-2000(P) and Inqulab-91(S) and LU-26S (P) were more tolerant than others

while Uqab 2000(S) and Chenab-70 (S) were salt sensitive. At EC 8dSm-1 Bhakhar-

2002(S) and Bhakhar-2000(P) showed best performance. At higher salinity level of

EC 12 dSm-1 , the accessions LU-26S (P) and Pasban-90(P) were at the top while

Punjab-96(S) was at lower side in producing the No. of spikelets per ear.

100

Table.18. Salinity effect on No. of Spikelets in 10 wheat somaclones alongwith parents

Name of Varity 0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean Ufaq-2002 S 14.00bcde 12.6ghijkl 9.0yz 8.8z 11.10CD Ufaq-2002 P 14.00bcde 13.4defgh 10.6qrstuv 9.0z 11.75A Punjab-96 S 13.00efghij 12.8fghijk 10.4rstuvw 8.4z 11.15BCD Punjab-96 P 14.00bcde 13.6cdefg 9.4wxyz 9.0yz 11.50ABC LU-26S S 13.2defghi 12.4hijklm 11.2nopqrs 10.0tuvwxy 11.70AB LU-26S P 12.8fghijk 13.8bcdef 10.6qrstuv 9.0yz 11.55ABC Punjab-76 S 11.6lmnopq 11.6lmnopq 8.8z 8.8yz 10.20EF Punjab-76 P 13.0efghij 13.2defghi 9.4wxyz 9.0yz 11.15BCD Pasban-90 S 13.4defgh 12.0jklmno 10.2stuvwx 8.6yz 11.05CD Pasban-90 P 14.2bcd 12.8fghijk 10.8pqrstu 10.0tuvwxy 11.95A Inqulab-91S 14.6abc 13.8bcdef 9.6vwxyz 9.2y 11.80A Inqulab-91P 14.6abc 13.0efghij 11.0opqrst 8.8z 11.85A Uqab-2000 S 12.6ghijkl 9.8uvwxyz 9.0yz 8.8z 10.05F Uqab-2000 P 11.2nopqrs 12.4hijklm 10.4rstuvw 8.8z 10.70DE Chenab-70 S 9.2xyz 9.8uvwxyz 9.2xyz 9.0yz 9.30G Chenab-70 P 13.4defgh 11.2nopqrs 9.6vwxyz 8.8z 10.75DE AS-2002 S 11.4mnopqr 11.0opqrst 10.4rstuvw 9.6vwxyz 10.60DEF AS-2002 P 15.4a 11.8klmnop 10.2stuvwx 9.0yz 11.60ABC Bhakhar-2000 S 13.0efghij 13.0efghij 11.6lmnopq 9.6vwxyz 11.80A Bhakhar-2000 P 14.8ab 12.2ijklmn 11.6lmnopq 8.8z 11.85A Mean 13.17A 12.31B 10.15C 6.05D

Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.57 S= 0.25 SxV= 1.14 Analysis of variance of No. of spikelets per spike of wheat somaclones

SOV DOF SS MS F

Replication 4 94.115 23.529 27.8797 Salinity 3 1083.440 361.147 427.9294** Varieties 19 196.340 10.334 12.2446* Salinity x Variety 57 225.860 3.962 4.6952* Error 316 266.685 0.844 Total 399

101

100 Grain weight.

Data given in Table.19. indicate that salinity significantly decreased the grain

yield in most of the wheat genotypes and in somaclones as well. All the accessions

performed well and produced more weight per 100 grains at lower salinity levels as

compare to higher ones. The weight per 100 grains was 3.375, 3.099, 2.841 and

2.790 grams at 0 , 4 , 8 , and 12 dSm-1 respectively. These differences in weight per

100 grains at different salinity levels were statistically significant. The results confirmed

the findings of Poustini, 1995 and Ozgen et al. 1996.

Regardless of salinity levels Bhakhar-2000 (P) with 3.301 grams proved itself the

best followed by AS-2002 (P) and Bhakhar-2000 (S) with weight of 3.253 and 3.210

grams per 100 grains. The minimum weight per 100 grains was observed in Chenab-

70(S) i.e. 2.861 grams followed by Punjab-76(S) 2.869. The rest genotypes /

somaclones performed in between these limits. Inqulab-91 (P) was most tolerant at EC

4dSm-1 followed by Inqulab-91 (S) and Bhakhar-2000 (S) while Chenab-70 (S) was

least tolerant. At EC 8dSm-1 LU-26S(P) and Bhakhar-2000 (S) noted as the best

tolerant and Chenab-76 (S) improved its tolerance while performance of Uqab-2002(S)

was most sensitive at this level of salinity. At EC 12 dSm-1 Inqulab-91 (P) showed more

tolerance while Punjab-76 (P) yielded the least weight of 100 grams).

102

Table.19. Effect of Salinty on 100 Grain weight (g) of 10 Wheat Somaclones alongwith parents

Name of Varity

0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean

Ufaq-2002 S 3.168ghijklmn 3.114ijklmnop 2.798vwx 2.772x 2.963GHI Ufaq-2002 P 3.320defgh 3.110jklmnop 2.930pqrstuvwx 2.770x 3.033FG Punjab-96 S 2.810vwx 3.030nopqrs 2.922pqrstuvwx 2.788vwx 2.888IJ Punjab-96 P 2.780vwx 3.094lmnopq 2.824uvwx 2.776wx 2.868IJ LU-26S S 3.426cde 3.086lmnopqr 2.814vwx 2.782vwx 3.027FGH LU-26S P 3.288efghijk 3.228fghijklm 2.956opqrstuvwx 2.796vwx 3.067DEF Punjab-76 S 2.906qrstuvwx 2.968opqrstuvw 2.816vwx 2.788vwx 2.869IJ Punjab-76 P 3.498cd 3.100klmnop 2.784vwx 2.770x 3.038EFG Pasban-90 S 3.040mnopqrs 3.060mnopqr 2.862stuvwx 2.772x 2.934HIJ Pasban-90 P 3.332defg 3.258efghijkl 2.898rstuvwx 2.824uvwx 3.078DEF Inqulab-91S 3.554c 3.400cdef 2.788vwx 2.786vwx 3.132CDE Inqulab-91P 3.300efghij 3.430cde 2.930pqrstuvwx 2.972opqrstuv 3.158BCD Uqab-2000 S 3.398cdef 2.850stuvwx 2.766x 2.772x 2.947GHIJUqab-2000 P 3.446cde 3.132hijklmno 2.770x 2.774x 3.030FG Chenab-70 S 3.100klmnop 2.778wx 2.776wx 2.788vwx 2.861J Chenab-70 P 3.128hijklmno 2.772x 2.810vwx 2.772x 2.871IJ AS-2002 S 3.370cdef 3.018nopqrst 2.822vwx 2.778wx 2.997FGH AS-2002 P 4.450a 3.016nopqrstu 2.776wx 2.772x 3.253AB Bhakhar-2000 S

3.810b 3.306defghi 2.952opqrstuvwx

2.770x 3.210ABC

Bhakhar-2000 P

4.374a 3.226fghijklm 2.832tuvwx 2.772x 3.301A

Mean 3.375A 3.099B 2.841C 2.790D Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.96 S= 0.04 SxV= 0.19 Annexure-22: Analysis of variance of 100 grain weight of wheat somaclones

SOV DOF SS MS F Replication 4 1.451 0.363 14.8082 Salinity 3 21.698 7.233 295.3331** Varieties 19 6.681 0.352 14.3578* Salinity x Variety 57 14.982 0.263 10.7327* Error 316 7.739 0.024 Total 399 52.551

103

Yield per plant

Data given in Table.20. told that all the genotypes / somaclones responded

differently at all the salinity levels. Increase in salinity level , significantly decreased the

yield per plant. All the accessions exhibited more yield per plant at control level i.e. 0

dSm-1 compared to salinity levels of 4, 8, and 12 dSm-1. Salinity levels reduced the

yield/ plant significantly. The yield per plant was 7.138, 3.832, 2.857 and 2.791 grams

per plant at EC 0, 4, 8 and 12 dSm-1 respectively. Similar results were reported by

Barkat and Haris, 1998. As a whole cultivar AS-2002(P) produced better yield

(5.139g/plant ) followed by Bhakhar-2000(P) , Inqulab-91 (P) and Inqulab-91 (S) i.e.

5.107, 5.068 and 5.002 g/plant.

Interaction salinity x genotype showed that Punjab-96 (P) was tolerant at EC 4d

Sm-1 followed by Inqulab-91(S) , Bhakhar-2000 (S) and Ufaq-2002(P) while Chenab-70

(S) and Chenab-70 (P) was less tolerant followed by Uqab-2000 (S). At the salinity

level of 8 dSm-1 Inqulab-91(P) and Bhakhar-2000 (S) performed better with the yield of

3.206 and 3.154 grams per plant while minimum yield (2.524g/plant) was obtained in

Ufaq-2002(S) at the same salinity level. At EC 12 dSm-1 Uqab-2002(P) responded well

later by Punjab-76 (P) and Punjab-96 (P) with the values of 2.912, 2.852 and

2.822g/plant while minimum yield per plant was recorded in LU-26S(S) Inqulab-91(P)

with the average yield of 2.748and 2.762 grams per plant.

104

Table.20. Yield/Plant (g) of 10 Wheat Somaclones alongwith parents as affected by salinity

Name of Varity

0 dSm-1 4 dSm-1 8 dSm-1 12 dSm-1 Mean

Ufaq-2002 S 7.38def 3.764opqrst 2.524w 2.768vw 4.109EFGHUfaq-2002 P 8.154d 4.500mno 2.786vw 2.788vw 4.557CDE Punjab-96 S 5.498jkl 3.426qrstuvw 2.764vw 2.804uvw 3.623IJKL Punjab-96 P 6.638efgh 6.054ghij 2.762vw 2.822uvw 4.569CDE LU-26S S 7.122ef 3.474qrstuv 2.914tuvw 2.748vw 4.064FGHI LU-26S P 5.802hijk 3.642opqrstuv 2.862tuvw 2.794uvw 3.775HIJK Punjab-76 S 4.038nopqrs 3.436qrstuvw 2.772vw 2.764vw 3.253LM Punjab-76 P 7.448de 4.230mnopq 2.836uvw 2.852tuvw 4.341DEFGPasban-90 S 6.502fghi 3.660opqrstuv 2.772vw 2.772vw 3.927GHIJ Pasban-90 P 8.218d 4.432mnop 3.038tuvw 2.778vw 4.616BCD Inqulab-91S 9.386c 4.902klmn 2.952tuvw 2.768vw 5.002ABC Inqulab-91P 10.05bc 4.256mnopq 3.206rstuvw 2.762vw 5.068AB Uqab-2000 S 5.630ijkl 2.776vw 2.768vw 2.804uvw 3.494JKL Uqab-2000 P 6.786efg 3.120stuvw 2.780vw 2.912tuvw 3.900GHIJ Chenab-70 S 3.530pqrstuv 2.762vw 2.766vw 2.780vw 2.960M Chenab-70 P 6.892efg 2.762vw 2.804uvw 2.764vw 3.806HIJ AS-2002 S 4.988klm 2.808uvw 2.762vw 2.776vw 3.333KLM AS-2002 P 11.300a 3.710opqrstu 2.774vw 2.770vw 5.139A Bhakhar-2000 S

7.024ef 4.830lmn 3.154stuvw 2.788vw 4.449DEF

Bhakhar-2000 P

10.370b 4.100mnopqr 3.144stuvw 2.808uvw 5.107A

Mean 7.138A 3.832B 2.857C 2.791D Mean sharing the same letter do not differ significantly at P = 5% LSD V= 0.46 S= 0.50 SxV= 0.9

Analysis of variance of yield per plant of wheat somaclones

SOV DOF SS MS F Replication 4 28.071 7.018 12.7714 Salinity 3 1254.752 418.251 761.1618** Varieties 19 162.480 8.552 15.5628* Salinity x Variety 57 301.918 5.297 9.6395* Error 310 173.639 0.549 Total 399 1920.859

105

CHAPTER 5

DISCUSSION

During the last few years tissue culture methods have been emerged that can

be used for the improvements of crop plants. The potential value of cell, tissue and

anther culture technique to use in the improvement of field crops has been described

(green,1977; vasil 1987). The regeneration of whole plant is possible today from

cereal species; such as bread wheat (Redway et al., 1990; Vasil et al.,1990,) maiz

(Duncan et al., 1985) rice (Vamada et al., 1986) and barley (Luhrs & Lorz, 1987)

In the present studies all the wheat genotypes responded differently to callus

formation. Genotypes Uqab-2002, V-o4189, Ufaq-2002 and Inqulab-91 produced

excellent callus induction frequency having the range from 80-90% under 3.0mgL-1

2,4-D. Different concentration of 2,4-D were used to find out the callogenic response

of seed as explant of wheat. The effect of 2,4-D (2-4dichlorophynoxy acetic acid) as

auxin for callus formation and growth has been adopted for wheat, (Abdrabou and

Moustafa,1993). The excellent callus formation was noticed at 3mgL-1 2,4-D after the

culture duration of 5-6 weeks. These results are in accordance with Mohmand

(1993) who found good callus from mature seed embryos of spring and winter

genotypes of wheat and In contrast to Barabanova et al; (1988) they achieved callus

induction at 1.5mgL-1 2,4-D while Bartok and sagi (1990) found callus formation on

6.0-8.0mgL-1 2,4-D which are quite opposite to the present result. Shah et al. (2003)

106

also supported the finding of present studies who achieved best callus at 3,5 mgL-1

2,4-D. Twenty wheat genotypes which produced callus induction frequency in the

range of 70-90 were further evaluated for callus induction potential under different

levels of salt.

Differences among genotype for callus induction potential under salt stress

were quite evident from the results. Almost all the twenty genotypes produce callus

of varying degree with and without salt. Wheat varieties Kohinoor-83 produce more

callus at 50mM NaCl. While LU-26S produce more callus at all four salinity levels i.e.

50,100 and 150mM NaCl . The data indicate that the callus formation was

dependent on genotype which is according to the finding of Van Sint Jan et al. 1990

in rice, Bomineni and Jauhar,1996; Ozgen et al.1996; Arzani and Mirodjagh,1999 )

in durum wheat and Hess and Carman,1998, in Bread wheat where a high impact of

genotypes on callus induction was found.

Salinity affected the growth rate and relative fresh weight growth as the

salinity increase callus weight and RFWG was also decreased. The relative fresh

weight growth rate after four weeks of culture were 0.1894, 0.1884 and 0.1831 for

Chenab-70, Punjab- 76 and AS-2002 respectively while minimum reduction in

RFWG was recorded in Ufaq-2002. These results are according to the data reported

by Karadimova and Bambova (1993) in durum wheat. They found that higher

concentration of NaCl caused brownish colour and appearent necrosis and reduced

callus growth. Similar results were reported for other genotypes of durum wheat by

Arzani and Mirodjagh in 1999

107

Calli of 10 responsive wheat genotypes under salt stress were subjected to

regeneration and plantlet development on MS medium without having any auxin

(2,4-D). This is also in accordance with those of Varshney et al. (1991), Chen et al.

(1992) and Hussain et. al, (2001) who used immature embryo of Trticum aestivum

as ex-plant and observed plant regeneration on hormone free MS media.

The effect of salts was quite dominant on the development of plant which

decreased as the salinity level increased. Maximum shoots (48) were developed by

LU-26S followed by Pasban-90(40) and Ufaq-2002 with 31 plants. Minimum shoot

development was observed in SA-42 (1). Ozgen et al. (1996) and Poustini and

Salmasi (1997) supported these results who observed that plant regeneration was

apparently influenced by culture medium and found significant reduction in total dry

production

The finding of superior genotypes for salt tolerance together with their high

embryogenic callus induction potential guide us to recommend these varieties as a

good model to observe physiological mechanism involved in in vitro salt tolerance

and in vitro selection for salt tolerance in wheat.

Keeping in view the importance of wheat crop and increasing problem of

salinity, the present studies were under taken to develop wheat somaclones with a

better potential to grow in saline areas where wheat is either grown inefficiently or

not at all. As a finding of these studies , a sufficient callus and plantlets have been

obtained which were used for the salinity evaluation for further parameters. These

research pursuits were carried out in the wire house in pots having artificially

developed saline soils.

108

In saline soils where salts are present in great concentration The growth of

plant is affected adversely through various ways such as osmotic effects, specific ion

effect and nutritional imbalance; all occurring probably simultaneously (Flowers et

al.,1991 ). Initial retarted growth in saline soils is induced by the decreased water

potential of medium due to higher salt presence (Munns et al., 1995). A major effect

of high concentration of Na+ and Cl-1 in the root medium is the suppression of uptake

of essential nutrients such as k+, Ca++ ,Na+ etc. ( Gohrum and wyn jones,1993). In

the present study the slowly increase in NaCl salinity in rooting media reduced the

shoot and root yield, shoot and root length and potassium where as sodium

accumulation was increased. Akhter et al. 2001 and Pervaiz et al. 2002, have also

observed similar adverse effect of salts. The reduction in shoot and root yield of all

the genotypes with the addition of salt may be due to the decrease of physiology

availability of water with increasing in solute suction from saline media or presence

of toxic ions in plants. This data is according to those of Ehret et al. 1990 and

Pervaiz et al. 2002. less reduction in shoot length is because of the toxic effect of the

salts added.

Among gas exchange attributes by applying salinity treatments, there is a

decrease in sub-stomatal conductance. However the significant reduction was there

in the photosynthetic rate and the stomatal conductance (gs). This indicated that an

adverse effect of salinity on the stomatal opening was possible there which tended

to reduce the photosynthetic rate and stomatal conductance in leaves. These results

are in line to those of Sharma (1996) who found that different gas exchange

characteristics declined in leaves under salinity. Similarly Yamamoto et al. (1994)

109

resulted that there was a significant change in gas exchange characteristics

response to salinity and observed a significant reduction in net photosynthesis.

In the present study the photosynthetic rate was significantly decreased due

to salinity stress with increasing concentration of NaCl which is in agreement with

some earlier studies with different crops such as in wheat (Ashraf et al.,2003).

Brassica ( Nazir et al.,2001 ) and sunflower (Ashraf and Rehman,1999). As it has

been earlier studied that this reduction in photosynthetic rate is associated with

stomatal or non- stomatal factors (Debey,2005). In the present study stomatal

conductance was reduced due to salt stress with an accompanied decrease in

photosynthetic rate (A) thus indicating that stomatal limitation was the only factor in

reducing photosynthesis under salt stress.

As for as the mineral contents are concerned, salinity enhanced the sodium

and reduced the potassium contents gradually, different amount of sodium and

potassium concentrations in their root and shoot at all the salinity levels which was

probably due to difference in dry matter yield as well as their individual genetic

behavior.

This tells the suppression effect of sodium on potassium uptake. Epstein

(1977) observed that the basic reason of potassium deficiency in case if salinity was

a competitive inter relationship among sodium and potassium. There is an apparent

relationship in sodium and potassium contents. Although there is proof that cultivar

with high potassium levels can tolerate the presence of high sodium (Caterina and

Guiliani, 2001). The maximum K+ concentration was found in somaclones of Punjab-

76. The relative shoot dry yield produced at high salinity level has been considered

110

tentively to consider as the standard (most rational) critical of relative salt tolerance

of wheat genotypes while the result due to other growth parameters were evaluated

against it. A consistent pattern among the growth parameters and salt tolerance of

genotypes tested was found. Thus the rating of tolerance to salinity based on growth

parameters can not be considered worthwhile. In assessing the plant to salinity

tolerance, both the shoot and root yield at higher salinity level has been used by

different workers. It is found that the considering the relative growth value is

necessary when comparing genotypes that are differed widely in growth habit or, are

grown under various environment conditions (Mass and Hoffman, 1977) more over,

research is required to develop such correlation among genotypes to exploit their

potentials in salt tolerance. In present study an addition of sodium chloride to the

rooting medium caused higher accumulation of sodium ions in leaf and roots which

is commonly seen in salt stress studies (Ashraf and Naz, 1994). However increase

in accumulation of Na+ ions in leaves under saline environments was in all wheat

somaclones, thus indicating the same toxic effect of increase accumulation of Na+

under saline conditions, plant growth is stunted through osmotic effects as well as

specific ion toxicity and nutritional disturbance, it is clear that only accumulation of

Na+ may have a role in increasing the osmotic potential of wheat somaclone..

Estimates of grain yield is another complexity to the salinity response, not just due to

the crops must be grown in uncontrolled environments for long periods of time, but

the conversion of shoot biomass is complex. Highly significant correlations between

the inorganic ions to salt resistance, there were significant negative correlations

between Na+ - contents and yield components of plants. These results are according

111

to the findings of Thalji and Shalaldeh, (2007) and Salam et al. (1999). They also

found the negative correlations between Na+ and yield component in contrast to

Islam et al. (1997) who failed to find such correlations between Na+ and growth

characteristics in barely. There was a positive correlation between K+/Na+ ration and

yield and its components. Positive correlations were also found between K+ and

yield component. These correlations were also obtained for the shoot and root fresh

weight. This supports the conclusions of Thalji and Shalaldeh, (2007) and Salam et

al. (1999) in wheat genotypes that salinity resistance is made up of two factors.

Physiological tolerance or a small relative decrease in growth, and complete

tolerance a high intrinsic growth rate both with and without salinity. The yield

factors, number of grain per plant, 100 grain weight and grain yield per plant were

affected by salts more badly than were plant height. The findings are in good

agreement with the results of Saqib et al. (2004) they found significant decrease in

yield per plant with increasing salinity during experiment of commercial wheat

varieties. Similarly, in hydroponics experiment Salam et al. (1999) also recorded a

decrease in these three yield components when wheat plant were sown under salt

stress. At salinity level 4dSm-1 somaclones of LU-26S, Inqulab-91 and Bhakhar-

2000 performed in plant yield/plant as compare to their parent genotypes and all

other cultivars. At salinity level 8 dSm-1, somaclones of Pasban-90 and LU-26S

were at par with parent genotypes in yield/plant. At 12dSm-1 somaclones of Pasban-

90 and Inqulab-91 were also at par with their parent plants. The rest of the

somaclones produced less yield as compare to their parent plants. These findings

are in accordance with the results of Khan et al. (2004) who observed that

112

somaclones performed better than the source plant. Yadav et al. (2004) also

observed that regenerated plants showed somaclonal variation for spike length,

spikelet number and grain number. Zair et al. (2003) find out that regeneration of

plant from callus initiation on high NaCl levels is a valid method of selection for salt

tolerance.

As the conventional method of variety development takes excessively long

time for the release of a variety due to non availability of desirable variability. This is

found that tissue culture technique can be an important tool in observing the

economy of time and fast selection of desirable variants. Many new traits have

already been identified by this technique. If any character is found to be superior to

mother plant, the somaclones after testing can be released as a new variety.

113

REFERENCES

ABDEL-HADY, M.S., WAHEED, M.S. AND HUSSAIN, M.M. (2001). Salt tolerance in barley using tissue culture. Annu. Agric. Sci. 46:103-115.Cairo, Egypt.

ABDRABOU, R.T. and MOUSTAFA. R.A.K.. (1993). Effect of 2,4-D concentration and two levels of sucrose on callus induction and plantlet formation in two wheat cultivars. Annu. Agric. Sci. Cairo, Egypt. No.1 (special issue), 41-46.

ABE, T.Y and FUTSHARA, Y. (1984). Varietal difference of plant regeneration from root callus tissue in rice. Japan J. Breed. 34: 147- 155.

ADILOGLU, S., ADILOGLU, A. AND OZKIL, M. (2007). Effect of different levels of NaC1 and KCI on growth and some biological indexes of wheat plant. Pak. J. Biol. Sci., 10:1941-1943.

AHLOOWALIA, B. S. (1982). Plant regeneration from callus culture in wheat. Crop Sci. 22: 405-410.

AHMAD, A., ZONG,H. WANG W. and. STICKLEN.M.B.2002. Shoot apical meristem. In-vitro regeneration and morphogenesis in wheat (Triticum aestivum). In-vitro cell development Biol. Plant, 38: 163-167.

AKHTAR, J., HAQ, M. A., AHMAD, K., SAQIB M. AND SAEED, M.A. (2005). Perfomance of cotton genotypes under saline condition. Caderno de pesquisa Ser. Bio. 17(1):29-36.

AKHTAR, J., HAQ, T., SHAHZAD, A. HAQ, A., IBRAHIM.M. AND ASHRAF.N.. (2003). Classification of different wheat genotypes in salt tolerance categories on the basis of biomass production. Int. J. Agri. Biol. 5 (3): 322-325.

AKHTAR, J., NASEEM, MAHMOOD, A. K. NAWAZ, S. QURESHI R.H. AND ASLAM. M. (2001). Response of some seleacted wheat (Triticum aestivum L.) genotypes to salinity: Growth and ionic relations. Pak. J. Soil. Sci., 19:1-7.

AKHTAR, J., SAQIB,M., QURESHI, R.H. AND ASLAM, M. (2002). Effect of salinity and sodicity on grain yield, different yield components and ionic relations of different wheat genotypes. P.46. In: Abstract of the paper presented in the 9th International Congress of Soil Science. Marc 18-20, 2002, NIAB, Faisalabad, Pakistan.

AKHTAR, J., SHAHZAD, A. QURESHI R.H. AND ASLAM, M. (1998). Performance of selected wheat genotypes grown under saline and hypoxic environment. Pak. J. Soil Sci., 15: 146-153.

114

AKHTAR, J., SHAHZAD, A., QURESHI R. H., NASEEM, A. and Muhammad, K. (2000). Testing of wheat (Triticum aestivum L.) genotypes against Salinity and Waterlogging. Pak. J. Boil. Sci., 3(7): 1134-1137.

AKHTAR, S., WAHID A. AND RASUL. E. (2003). Emergence, growth and nutrients composition of sugar cane sprouts under NaCI salinity. Biol. Plant, 46(1):113-116.

AKRAM, M., . HUSSAIN, M., AKHTAR S. AND RASUL, E. (2002). Impact of NaC1 salinity on yield components of some wheat accessions / varieties. Int. J. Agri. Bio. 4(1): 156-158.

ALAM, S. M., KHAN, M. A., MUJTABA S. M. AND SHEREEN, A. (2002). Influence of aqueous leaf extract of common lambsquarters and NaC1 salinity on the germination, growth, and nutrient content of wheat. Acta physiologiae Plantarum, 24 (4): 359-364.

AL-ANSARI F. M, (2003). Salinity tolerance during germination in two arid-land varieties of wheat (Triticum asetivum L.). Seed Science & Technology. 31(3): 597-603.

ALBASSAM, B.A. (2001). Effect of nitrate nutrition on growth and nitrogen assimilation of pearl millet exposed to sodium chloride stress. Journal of Plant Nutrition, 24(9): 1325-1330.

ALEM, C., LABHILILI, M., BRAHMI, K. JLIBENE M., BASRALLAH, N. AND FILALU-MALTOUF, A. (2002). Hydrus and photosynthetic adaptations of common and durum wheat to saline stress. Comptes Rendus Biologies. 325 (11): 1097-1109.

ALI, Y., ASLAM, Z. SARWAR G. AND HUSSAIN, F. (2005). Genotypes and environmental interaction in advanced lines of wheat under salt-affected soils environment of Punjab. Int. J. Envi. Sci. Tech., 2(3) 223-228.

ALMANSOURI, M., KINET, J.M. AND LTTS., S. (2000). Physiological analysis of salinity resistance in Triticum turidum var. durum Desf. Callus versus whole plant responses Options Mediterraneennes. Serie A, Seminaires Mediterraneennes. 40: 263-265.

ALMANSOURI, M., KINET, J.M. AND LUTTS, S. (2001). Effect of salt and osmotic tresses on germination in durum wheat (Triticum durum). Plant Soil. 231: 243-254.

AL-MUTAWA, M.M. AND EI-KATONY, T.M. (2001). Salt tolerance of two wheat genotypes in response to the form of nitrogen. Agronomie 21: 259-266.

ALVERAZl, L., TOMARO. M. and BENAVIDES, P.M. (2003). Changes in polymines, praline and ethylene in sunflower calluses treated with NaCl, Plant cell, Tissue and Organ culture. 00:1-9.

115

ANDRIA, R., LAVINI, A. LOVENZI, F. MATORELLA, CALLENDERELLI,A. TEDESCHI, D. P. AND HAMDY, A. (1997). Growth analysis of field-grown sunflower (Helianthus annuus L.) under different salt concentration of irrigation water. 381-394. In: paper presented in Anonymous, 2005. Statistics of Pakistan. Ministry of Food, Agriculture and Livestock wing, Economic Division, Islamabad.

ANONYMOUS. (1999). Agricultural Statistics of Pakistan. Govt. of Pakistan. Ministry of Food, agriculture and Livestock Division, Economic Wing, Islamabad.

ANONYMOUS. (2003). Agricultural statistics of Pakistan, Ministry for Food, Agriculture and Livestock, Islamabad.

ANONYMOUS. (2008-09). Agricultural Statistics of Pakistan, Ministry for Food, Agriculture and Livestock, Islamabad.

ANONYMOUS. (200I). Agricultural Statistics of Pakistan. (2000-2001). Ministry of Food, Agricultural and Livsestock Division, Economic Wing, Islamabad.

ANSARI, R., NAQVI, S.S.M., KMAREAR, N.E. ALUM, S.M. KHAN ,M.A. AND SAHIRAZI, M.U. (1998). Soil salinity as an important environmental problem and its management. In: Proceeding of International symposium. Agro environmental issue and future strategies towards 21st century. (eds. Sial J.K., M.Y. Bhatti and S.M. Azeemi).Univ. Agri., Faisalabad.

ARZANI. And MIRDOJAGH, S.S. (1999). Response of durum wheat cultivars to plant cell, Tissue and Organ culture. 58:67-72.

ASHARAF, M.Y. AND BHATTI. A.S. (2000). Effect of salinity on growth and chlorophyll content in rice. Pak. J. Sci. Ind. Res., 43:130-131.

ASHRAF M. (2001). Relationship between growth and gas exchange chrematistics in some salt-tolerant amphidiploid Brassica species in relation to their to their diploid parents. Environmental and Experimental Botany. 45:155-163.

ASHRAF, M AND SHAHBAZ. M. (2003). Assessment of genotypic variation in salt tolerance of early CIMMYT hexaploid wheat germplasm using photosynthetic capacity and water relations as selection criteria. Photosynthetica. 41: 273-280.

ASHRAF, M. (2004). Some important physiological selection criteria for salt tolerance in plants. Flora. 199:361-376.

ASHRAF, M. AND AHMAD, S. (2000). Influence of sodium and chloride characteristics in salt sensitive lines of cotton. Field Crops Res., 66:115-127.

116

ASHRAF, M. AND KHANUM, A.( 1997). Relationship between ion accumulation and growth in two spring wheat lines differing in salt tolerance at different growth stages. J. Agron. And Crop Sci. 178: 39-51.

ASHRAF, M. AND LEARY, J.W.O.( 1996). Response of some newly developed salt tolerant genotypes of spring wheat to salt stress, yield components and ion distribution. J. Agron. And Sci., 17(20): 91-100.

ASHRAF, M. AND MCNEILLY, T. (1988). Variability in salt tolerance of nine spring wheat cultivars. Crop Sci. 160:14-21.

ASHRAF, M. AND MCNEILY. T. (2004). Salinity tolerance in Brassica oilseeds. J. Plant, Sci. 23: 157-174.

ASHRAF, M. AND NAZ, F.( 1994). Response of some arid zone grasses to potassium deficiency. Aceta Physiol. Planta. 16:769-801.

ASHRAF, M. AND PARVEEN, N. (2002). Photosynthetic parameters at the vegetative stage and during grain development of two hexaploid wheat cultivars differing in salt tolerance. Biologia Plantarum. 45(3): 401-407.

ASHRAF, M. AND REHMAN, H. (1999). Mineral nutrition status of corn in relation to nitrate and long term water logging. Plant. Nutr. 22:1253-1268.

ASHRAF, M.,. KHAN, A.H. AND NAQVI, S.S.M. (1991). Effect of salinity on seedling growth and solute accumulation in two wheat genotypes. Reehis 10 (1): 30-31.

ASHRAF, M.Y. AND KHAN, A.H. (1993). Effect of NaCl on nitrogen status of Sorghum. In: current development of salinity and drought tolerance in plants. (eds. S.S.M. Naqvi., M.R. Ansari., T.J. Flower AND A.R. Azmi ). 84-88.

ASHRAF, M.Y. AND KHAN, A.H. (1994). Solute accumulation and growth of sorghum under NaCl and Na2 SO4 salinity strees. Sci. Int.6 (4): 337-379.

ASHRAF, M.Y. AND NAQVI, M.H. (1996). Germination seedling growth and mineral nutrients of Mungbean (Vigna radiate L.) grow under NaCl and Na2SO salinity. Pak.J. Agri. engg. Vet. Sci., 12 (1-2).

ASHRAF. M., ABIDA K., AND ASHRAF, M. Y. (2003). Alleviation of salt stress in pearl millet (Pennisetum glaucum ( L.) R. Br.) through seed treatments. Agronomic. 23:227-234.

ASLAM, M. AND MUHAMMAD, R. (1972). Distribution of cations in leaves of salt sensitive genotypes of sunflower under saline condition. J. PI. Nutrition.18:2379-2388.

117

ASLAM, M., ASHRAF, M. AND AHMAD, S. (2000). Influence of sodium chloride on ion accumulation, yield components and fiber characteristics in salt-tolerant and salt-sensitive lines of cotton (Gossypium hirsutum L). Field Crops Res. 66: 115-117.

ASLAM, M., QURESHI, R. H. AND AHMAD, N. (1993). A rapid screening technique for salt tolerance in rice. Plant and Soil. 150: 99-107.

ATIS, E, (2004). Economic impacts on cotton production due to land degradation in the Gediz Delta, Turkey. Land use policy. 23(2): 181-186.

BADR-UZ-ZAMAN, ALI, A., SALEEM, M. AND HUSSAIN, K. (2002). Growth of wheat as affected by sodium chloride and sodium sulphate salinity. Pak. J.Biol. Sci. 5(12)1313-1315.

BAJAJ, Y.P.S. (1985). Somaclonal variation and cryopreservation of germplasm in wheat Amer. J. Bot. 72: 874.

BARABANOVA, E.A., BANNIKOVA,V.P. AND GIRKO,V.S. 1988. Plant regeneration from cultured embryos of winter wheat. Biologiya Kul tiviruemykh Kletok Biotechnologiya. 1:110,Novsibirsk,USSR.

BARAKAT, M.N. AND ABDEL-LATIF, T.H. (1995). In vitro selection for salt-tolerant lines in wheat.II.In vitro characterization of cell lines and plant regeneration. Alex. J.Agri.Res.40 (3): 139-165.

BARAKAT,M.N. AND ABDEL-LATIF T.H. (1995). In vitro selection for salt tolerant lines in wheat. Effect of salt on the embryogenic cultures. J.Agri.Res.40 (1):77-95.

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: 27-140.

BARAKAT. M.N. AND EL-HARIS, M.K. (1998). In vitro selection for salt-tolerant lines in wheat III. Agronomic evaluation of the progeny under salt stress conditions. Alex. J. Agric. Res. 43: 21-32.

BARTOK,T. AND SAGI,F.(1990). A new endosperm supported callus induction method for wheat (Triticum aestivum L.). Cereal Research Institute, Hungary. Plant cell, Tissue and Organ culture. 69: 545-64.

BASAL, H., EDEMIRAL, M.A. AND CANAVAR, O. (2006). Shoot biomass production of converted race stocks of upland cotton (Gossypium hirsutum) exposed to salt stress. Asian J. plant Sci. 5(2): 238-242.

BASU, S., GANGOPADHYAY,G. AND MUKHERJEE, B.B. (2002). Salt tolerance in rice. In vitro: implication of accumulation of Na+, K+ and praline. Plant cell, tissue and organ culture. 69:55-64.

118

BEGUM, F., KARMWER, J. L., FATTA, Q.A. AND MANIRUZZAMAN, A.F. (1992). The effect of salinity on germination and its correlation with K+, Na+ and C1- accumulation in germinating seed of Tritcum aestivum L. (cv. Akbar). Plant and Cell physiol. 33(7): 1009-1014.

BERNARDO, M.A., DIEGUEZ, E.T., JONES, H.G., CHAIREZ, F.A., OJANGUREN, C.L.T. AND A.L. CORTES, (2000). Screening and classification of cowpea genotypes for salt tolerance during germination. Int. J. Exp. Bot. 67:71-84.

BEY, R.S. (2005). Photosynthesis in plants under stressful conditions. In: Hand Book of photosynthesis.Z't' (eds. M. Pcssarakli). 717-718. C.R.C. Press, New York, USA..

BEZERRA, J. S., WILLADINO, L. AND CAMARA,T.R. (2001). Embryogenesis callus growth of maize submitted to salt stress. Seientia Agricola. 58: 259- 263.

BHASKARN, S. AND SMITH, R.H. (1990). Regenration in the cereals tissue culture. A review. Crop Sci. 30: 1328-1336.

BIDINGER F. R. AND HASH,C. T. (2003). Pearl millet. In: Physiology and biotechnology integration in plant breeding. (eds. Nguyen, HT., Blum, A). Marce Dekker, New York. 225-270.

BLUM, A. (1988). Plant breeding for stress environment. CRC Press, Inc. Boca Raton Florida.

BLUMWALD, E., AND POOLE, R.J. (1987). Salt tolerance in suspension culture of sugar beet induction of Na+ / H+ antiport activity at the tonoplast by growth in salt. Plant Physiol. 83: 884-887.

BOMMINENE, V.R. AND. JAUHAR, P.P (1996). Regeneration of plantlets through isolated scutellum culture of durum wheat. Plant Sci. 116:197-203.

BONGI, G. AND LORETO, F. (1989). Gas-exchange properties of salt stressed olive (Olea europea L.) leaves. Plant Physiol. 90: 1408-1446.

BRUGNOLI, E. AND LAUTERI,M. (1991). Effects of salinity on stomatal conductance photosynthetic capacity and carbon Isotope discrimination of salt tolerant (Gossypium hirsutum L.) and salt sensitive (Phaseolus vulgaris L.) C3 non-halophytes. Plant Physiol. 95: 628-635.

CAMER, G.R., Schmidt, C.L. and Bidart,C. (2001). Analysius of cell wall hardening and cell wall enzymes of salt-stressed maize (Zea mays ) leaves. Aust.J. Plant Physiol. 28: 101-109.

119

CANNELL, R.Q. AND JACKON, M.B. (1981). Alleviating aeration stress. In: Modifying the root environment to reduce crop stress. ( eds. G.F. Arkin and H.M. Taylor). 141-192. Am. Soc. Agric. Engg., St. Joseph, Missouri.

CATERINA, R. AND GIULIANI, M.M. (2007). Influence of salt stress on seed yield and oil quality of two sunflower hybrids. Annals of applied Biology. 151 (2): 145-154.

CHAUDARY, M.R., (2001). Gypsum efficiency in the amelioration of saline-sodic/soils. Int. J. Agri. Biol. (3): 276-280.

CHEMMAN, J. M., (1988). Mechanisms of salinity tolerance in plants (Reviews). Plant Physiol. 87: 547550.

CHERI,S. AND REDDY, M.P. (2003). Evolution of NaCI tolerance in the callus structure 01' Suaeda mudiflora Moq. Physio!. Planta. 46(2): 193-198.

CHHIPA, B.R. AND LAL, P. (1995). Na/K ratios as basis of salt tolerance in wheat. Aus. J. Agr. Rse. 46(3): 533-539.

CICEK, N. AND CAKIRLAR, H. (2002). The effect of salinity on some physiological parameters in two maize culiivars. Bulg. J. P!. Physio!., 28 (1-2): 66-74.

CURTIS, P.S.AND LAUCHLI, A. (1987). The effect of moderate salt on leaf anatomy in Hibiscus Cannabinus (Kenaf) and its relation to leaf area. Amer. J. Bot., 74(4): 538-542.

DANG, M.N. AND N.T. LANG. (2003). In vitro selection for salt tolerance in rice. Omonrice. 11: 68-73.

DELEON, I.L.D., CARRILLOLAGUNA, M., RATARAM, S. AND MUJEEB KAZI, A. (1985). Rapid in vitro screening of some salt tolerant bread wheat. Cereal Res. Communications. 23(4): 383-389.

DEY,R.S.(2005). Photosynthesis in plants under stressful conditions. In: Hand book of photosynthesis. (eds. M. Pessarkali). 717-718. C.R.C. Press, New York, USA.

DREW, M.C. (1981). Plants response to anaerobic conditions in soil and solution cultures. In: Commentraries in Plant Science. (eds. H. Smith). 2:209-223.

DUDITS, D., NEMET, G. AND HAYDU, Z. (1975). Study of callus growth and organ formation in wheat tissue culture. Can. J. Bot. 53: 957-963.

EBRAHIMZADCH, I-I., MCIGHANY, F. AND REHIMINAN, H. (2000). Role of mineral ions in salt tolerance of two wheat (Triticum astivum L.) cullivars. Pak. J. Bot., 32 (2): 265- 271.

120

EHRET, D.L., REDMAN, R.E., HARVEY,B.L. AND CIPYWNYK, A.(1990). Salinity induced calcium deficieccies in wheat and barley. Plant and Soil.128:143-151.

EKER, S. G. COMERTPAY, O. KONUFIKAN, A.C., ULGER, L., OZTURK AND CAKMAK, S. (2006). Effect of salinity stress on Dry Matter production and ion Accumulation in Hybrid Maize Varieties. Turk. J. Agric., 30: 365-373.

EL-BAHR, M.K., AL-NAGGAR, A.M.M., ABUSTEIT, E.O., RADY, M.R. AND EID S.A.M. (2001). In vitro selection and characterization of salt tolerance in Egyptian wheat. Egyptian J. Agron., 22: 65-84.

EL-HENDAWY, S. E., HU, Y., YAKOUT, G.M., AWAD, A.M., HAFIZ, S.E. AND SCHMIDHALTER, U. (2005). Evaluating salt tolerance of wheat genotypes using multiple parameters. Europe . J. Agr. 22(3): 243-253.

EPSTEIN, K. 1977. Genetic potential for solving problems soil mineral stress. Adaptation of crops to salinity. 73-82.

EVANGELOUS AND MC DONALD. (1999). Inflence of sodium on soils of humid regions. In: Hand book of plant and Crop Stress. 240. ( eds. M. Pessarakli). Marcel Dekker Inc., New York.

FLOWERS , T. J., A. GARCIA, M. KOYAMA, A.R. YEO, (1997). Breeding for salt tolerance in crop plants the role of molecular biology. Acta. Physiol. Planta. 19:427-433.

FLOWERS , T. J., HAJIBAGHCRI, M.A. AND YEO, A.R. (1991). Ion accumulation in the European Society of Agronomy, Colamar, France. ISBN 2-95.5:124-0-2.

FLOWERS T. J. AND YEO, A.R. (1995). Breeding for salinity resistance in crop plants: Aust. J. Plant Physiol. 22: 875-884.

FRANCOIS, L.E. AND MAAS. (1999). Crop response and management of salt affected soil. In: Handbook of Plant and Crop Stress. 169-201. (ed. M. Pessarakli ). Marcel kar Inc., New York.

GABALLAH, M. S. AND MOURSY, M. (2004). Reflectants Application for Increasing wheat plant Tolerance against Salt Stress. Pak. J. Bio. Sci. 7(6): 956-62.

GAMBORG, O.L. AND EVELEIGH, D.E. (1968). Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem. 46:417-421.

GAMBRELL, R.P., DELAUNE, R.D. AND PATRICK, W.H.J.R. (1991). Redox processes in soils following oxygen depletion. In: Plant life under oxygen deprivation. 3-21. (eds. M.B. Jackson, D.D. Davies and H. Lambers). UPB Academic Publ. The Hague, The Netherland.

121

GANDONOU, C., ABRINI, J. IDAOMAR, M. AND KALISENHAJI, N. (2005). Response of sugarcane (Saccharum sp.) varieties to embryogenic callus induction and in-vitro salt stress. African J. Biotech. 4(4): 350-354.

GARG, B.K., KATHJU, S., VYAS AND LAHIRI, A.N. (1997). Alleviation of sodium chloride induced inhibition of growth and nitrogen metabolism of clustcrbcan high calcium. J. Biologia Plantarum. 39 (3): 395-401.

GHANNADHA, M.R., OMIDI, M., SHSHI, R.A. AND POUSTINI, K. (2005). A study of salt tolerance in genotypes of bread wheat using tissue culture and germination test. Iranian J. Agri. Sci. 36(1):75-85.

GIORIO, P., SORRENTINO, G.S., CASERTA, P. AND TEDESCHI, P. (1996). Leaf area development of field grown sunflower (Helianthus annuus L.) plants irrigated with saline water. Helia.19: 17-28.

GOOD A.G. AND CROSBY, W.L. (1989). Anaerobic induction of alnine aminotransferase in barley root tissue. Plant Physiol. 90:1305-1309.

GORHAM, J. (1993). Mechanisms of salt tolerant in halophytes. In: Advance course on halophyte utilization in agriculture. 3-34., 12-26 Sept. Agadir, Morocco.

GORHAM, J. (1994). Salt tolerance in the Triticeae. K/Na discrimination in some perennial wheatgrasses and their amphiploids with wheat. J. Exp. Bot. 45:441-447.

GORHAM, J. AND WYN JONES, R. G. (1993). Utilization of Triticeae for improving salt tolerance in wheat. In: Towards the rational use of high salinity tolerants plants. 27-33. (eds. Leith, H. and A. A. Massoum). Kluwcr Acad. Pub. The Netherlands.

GORHAM, J., BRISTOL, A., YOUNG, E.M., WYN JONES, R.G. AND KASHOUR,G. (1990). Salt tolerance in the Triticeae: K/Na discrimination in barley. J. Exp. Bot. 41:1095-1101.

GORHAM, J., BRISTOL, A., YOUNG, E.M.,WYN JONES, R.G. AND KASHOUR,G.(1991). The presence of the enhanced K/Na discrimination trait in diploid Triticum species. Theor. Appl. Genet. 82:729-736.

GORHAM, J., FOSTER, B.P., BUDERWICZ, E., WYN JONES, R.G. MILLER, T.E. AND LAW, C.N. (1986). Salt tolerance in the Triticeae: Solute accumulation and distribution in an amphidiploid derived from Triticum aestivum cv. Chinese Spring and Thinopyrum bessarabicum. J. Exp. Bot. 37:1435-1449.

GORHAM, J., MCDONNELL, E. AND WYN JONES, R.G.(1984). Salt tolerance in the Triticeae: I. Leymus sabulosus. J. Exp. Bot. 35:1200-1209.

122

GORHAM, J., WYN JONES, R.G. AND MCDONNELL, E. (1985). Some mechanisms of salt tolerance in crop plants. Plant Soil. 89:15-40.

GORHUM, J. (1984). Ion contents of wheat grown under Nac1 and Na2 SO4 salinity. PaK. J. Biol, Sci., 6(17): 1512-1514.

GOTO, S. AND PATRICK, W.H.JR. (1974). Transformation of iron in a waterlogged soil as influenced by redox potential & Ph . Soil Sci. Soc. Amer. Proc. 38:66-71.

GRATTANA, S.R. AND GRIEVE, C.M. (1999). Salinity-mineral nutrient relations in horticultural crops. Scientia Horticulturae 78: 127-157.

GREEN, C.E.1977. Prospects for crop improvement in the field of cell culture. Hort. Science; 12: 131-134.

GRIEVE C.M., FRANCOIS AND MASS, E.V. (1993). Salinity affects the timing of phasic development in spring wheat. Crop Sci., 34: 1544-1549.

GRIEVE, C.M., LESCH, S.M., FRANCOIS, E.E. AND MASA, E.V. (1992). Analysis of main spike yield components in salt-stressed wheat. Crop Sci. 32(3): 697-703.

HAGEMEYER, J. AND WAISEL Y. (1989). Influence of NaCl, cd(NO3)2 and air humidity on transpiration of tamarix aphylla. Physiol. Plant. 75:280-284.

HAJOR, A.S., ZIDAN, M.A. AND AL-ZAHRUNI, H.S. (1996). Effect of salinity stress on the germination, growth, and some physiological activities of black cumini Nigella sativa L.). Arab Gulf J.Sci. Res. 14: 445-454.

HAMADA, A. M., (1996). Effected of NaC1, water stress or both on gas exchange and growth of wheat. Biologia Plantarum. 38(3): 405-412.

HANSEN, J.I. AND ANDERSON, F.O.(1981). Effects of phragmites australis roots and redox potentials, nitrification and bacterial numbers in the sediment. In: A. 9th Nordic symposium on Sediments. 72-88. (eds, Broberg and T. Tiren ).

HANSON, A.D., JACOBSEN, J.V. AND ZWAR, J.A. (1984). Regulated expression of three alcohal dehydrogenase genes in barley aleurone layers. Physiol. 75:573-81.

HASEGAWA, P.M., BRCSSAN, R.A., ZHU, I.K. AND BOHNERT, H.J.(2000). Plant cellular and molecular response to high salinity. - Annu. Rev.Plant Physiol. Plant mol. Biol. 51: 463-499.

HASHMI, A.A., SHAFIULLAH, (2003). NASSD background paper. IN: Agriculture and security. 136. IUCN PAKistan, Northern Areas Programme, Gilgit.

123

HE, D.Y. AND YU, S.W. (1995). In vitro selection of a high praline producing variant from rice callus and studies on its salt tolerance. Aeta Phyto Physiological Siniea. 21: 65-72.

HEIKAL, M.M.D., BERRY, W.L. AND WALLACE, A. (1989). Interactions in plant growth response between the osmotic effect of sodium chloride and high concentration of the trace element nickel. Soil Sci. 147(6):422-425.

HESS, J.R., AND CARMAN, J.G. (1998). Embryogenic competence of immature wheat embryos: genotype, donor plant envinronment, and endogenous hormone level. Crop Sci. 38: 249-253.

HESTER ,M.W., MCNDCISSOHN, A., MCKCE, K.K., PCZCSHKI, S.R., ERNST, W.H.O. AND CHABBO, A. (2001). Species and population variation to salinity stress in Panicum hemitomcn.spartina patens and Spar/ina altcrniflora morphological and pbY);oglf.::al constrains. Environ.Exp. Bot., 46: 277- 297

HESTER, M.W., MENDEISSOHN, I.A., MEKOE, K.L., PEZESHKI, S.R. ERNS, W.H.O. AND CHABBO, A. (2001). Species and population variation to salinity stress in Panicum hemitomen spartina patens and spartina alterniflora, morphological and physiological constraints., Environ. Exp.Bot., 40:277-297.

HOAGLAND, D.R., AND ARNON, OJ. (1950). The water-culture method for growing plants without soil. Californii Agric. Exp. Stn. Circular 347.

HOFFMAN, N.E., BENT, A.F. AND HANSON, A.D. (1986). Induction of Lactate Dehydrogenase Isozymes by oxygen deficit in barley root tissue. Plant Physiol. 82:658-663.

HOLLAENDER, A. (1979). The Biosaline Concept: An Approach to the Utilization of Underexploited Resources. Plenum Press, New york.

HOLLINGTON, P. A., ROYO, A., ARAGUES, R., AND WYN JONES, R.G. (1990). The effect of variable salinity on the yield and yield components of wheat cultivars and an amphiploid between wheat and Thinopynun bcssarabicum, In: Proceedings of 1st Congress.European Society of Agronomy. 231-254. (ed. A Scr-ifc). Paris, 5-7 December 1990.

HOSSEINI, G. AND THENGANE, R.J.(2007). Salinity Tolerance in Cotton (Gossypium hirsutum L.) Genotypes. Int. J. Bot. 3(1): 48-55.

HOUSMAND, S., ARZANI, A., MAIBODY, S.A.M. AND FEIZI, M. (2005). Evaluation of salt tolerant genotypes of durum wheat derived from in vitro and field experiments. Field crops Research. 91: 345-354.

HU, Y., AND SCHMIDHALTER, U. (2008). Spatial and temporal quantitative analysis of cell division and elongation rate in geowing wheat leaves under saline condition. Inte. J. Pla. Biol., 50:76-83.

124

HU, Y., AND SCHMIDHALTER, U. (2007). Effect of salinity on the composition, number and size of epidermal cells along the mature blade of wheat leaves. J. Int. Pla. Bio. 49(7): 1016-1023.

HU, Y., FROMM, J. AND CHMIDHALTER, U. (2005). Effect of salinity on tissue architecture in expanding wheat leaves. Planta., 220:838-848.

HU, Y., BURUCS, Z. AND SCHMIDHLTER, U. (2006). Short- term effect of drought and salinity on growth andmineral elements in wheat seedlings. J. Plant Nutri. 29(12): 2227-2243.

HUANG, B., JOHNSON, J.W. NESMITH, S. AND BRIDGES, D.C. (1994). Growth, physiological and anatomical responses of two wheat genotypes to water logging and nutrient supply. J. Exp. Bot. 45:193-202.

HUMAYUN, A., TOMAS, D. AHMAD, N. (1992). Control of wheat leaf growth under saline conditions. II water relation parameters. Pak. J. Agri. Sci.29(4): 391-396.

HUSSAIN S., MUNNS, R. AND CONDON, A.G. (2003). Effect of sodium exclusion trait on chlorophyII retention and growth of durum wheat in saline soil. Aust. J. Agric. Res. 54: 589-597.

HUSSAIN, M., KHAN, G.S., SHAHEEN, M.S., AND AHMAD, A. (2001). Somaclonal variation in regenerated plants of ten wheat genotypes. J. Agric. Res., 39(1): 1-7.

HUSSAIN, M.K. AND REHMAN, O.U. (1995). Breeding sunflower for salt tolerance.

HWANG, S.Y. AND VAN TOAI, T.T. (1991). Abscisic acid induces anaerobiosis tolerance in corn. Plant Physiol. 97:593-597.

IFTIKHAR, A., IQBAL, N., IQBAL, M., ASLAM, Z. AND RASUL, E. (2000). Germination and sccdl: Ig growth of rice (Oryza sativc L j under saline condition. Pakistan Journal of Biological Sciences, Vol. 3 (2):350-351.

IIMI. (1995). Salinity and sodicity research in Pakistan. Workshop (one day) Feb. 6, 1995, Lahore, Pakistan. Int. Irri. Management Inst. Lahore, Pakistan. IQBAL, R.M. 2003. Diurnal variations in net photosynthesis and other gas exchange parameters of spring wheat under saline conditions. J. Appl. Sci. 3(10): 650-658.

IQBAL, M.S., NASEEM, A., MAHMOOD, K. AND AKHTAR, A. (2001). Comparative performance of wheat (Tritcum aestivum L.) Genotypes under Salinity Stress II: Ionic composition. Online J. Bio. Sci., 1(2): 43-45.

IQBAL, R. M. (2003). Leaf area and ion contents of wheat 2'!OWI1 under NaCI and Na2S04. Pakistan Journal of Biological Sciences. 8(17): 1512-1514.

125

IQBAL, R.M. (2003). Leaf Area and Ion Contents of Wheat Grown under NaC1 and Na2 SO4 Salinity. Pak. J. Biol, Sci., 6(17): 1512-1514.

IQBAL, R.M. (2005). Effect of different salinity levels on partitioning of leaf area and dry matter in wheat. Asian Journal of Plant Sciences. 4(3): 244-248.

ISLA, R., ROYO, A. AND ARAGUES, R. (1997). Field screening of barley cultivars to soil salinity using a sprinkler and a drip irrigation system. Plant Soil. 197: 105-117.

ISMAIL, A.M. (2003). Effect of salinity on the physiological responses of selected lines/variety in wheat. Aceta Agronmica Hungarica. 51(1): 1-9.

JACOBY, B. (1965). Sodium retention in excised bean stem. Physiol. Plant. 18:730-739.

JAFRI, A.Z. AND AHMAD, R. (1994). Effect of salinity on leaf development, stomatal size and its distribution in cotton (Gossypium hirsutim L.). Pak. J. Bot., 27:297-303.

JAMES, R.A., RIVELLI, A.R., MUNNS, R., AND CAEMMERER, S. (2002). Factors CO2 assimilation, leaf injury and growth in salt-stressed durum wheat. Funct. Plant Biol. 29: 1393-1403.

JAMIL M., LEE, C.C., REHMAN, S.U., LEE, B.D., ASHRAF, M. AND RHA, E.S. (2005). Salinity tolerance of Brassica species at germination and early seedling growth. Electronic Journal of Environmental, Agricultural and food Chemistry ,4 (4 ):970-976.

JAVAID, A; TANVEER-UL-HAQ; ARMGHAN, S., ANWAR-UL-HAQ, M., IBRAHIM, M. AND ASHRAF, N. (2003). Classification of different wheat genotypes in salt tolerance categories on the basis of biomass production. Int .J. Agri. and Biol. 5(3):322-325.

JAVED, F. A. (2002A). In vitro salt tolerance in wheat. II.Organic solute accumulation in callus. Int. J. Agri. and Biol. 4(4): 462-464.

JAVED, F. (2002B). In vitro salt tolerance in wheat. III. Water relations in callus. Intl. J. Agric. Biol. 4(4): 465-467.

JAVED, F. (2002C). In vitro salt tolerance in wheat. I. Growth and ions accumulation. Intl. J. Agric. Biol. 4: (4) 459-461.

JESCHKE, W.D. (1980). Involvement of proton fluxes in K+-Na+ swlectivity at the plasma lemma: K+ dependent net extrusion of sodium in the barley roots and the effect of anion and pH on sodium fluxes. Z. Pflanzenphusiol. 98:155.

126

JESCHKE, W.D. (1984). K+-Na+ exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In: Salinity tolerance in plants. Strategies for Crop Improvement. (eds. R.C. Staples and G.H. Toenniessen. ). 37-66. John Wiley and Sons, New York.

JESCHKE, W.D. AND NASSERY, H. (1981). K+-Na+ selectivity in roots of Triticum, Halianthus and Alium. Physiol. Plant. 52:217-224.

JESCHKE, W.D. AND DREW., M.C. (1984). Effects of flooding on growth and metabolism of herbaceous plants. P. 47-128. In: T.T. Kozlowski (ed.) Flooding and plant growth. Academic Press, Inc. London.

JESCHKE, W.D. AND WOLF, O. (1988). Effect of NaC1 salinity on growth,development, ion distribution and ion translocation in castor bean ( Ricinus communis L.). J. plant Physiol. 132:45-53.

JESCHKE, W.D., STELTER, W., REISING, B. AND BEHL, R. (1983). Vacuolar Na/K exchange, its occurrence in the root cells of Hordeum, Atriplex and Zea and its significance for K/Na discrimination in roots. J. Exp. Bot. 34:964-979.

JIANG, L., DUAN, L., TIAN, X., WANG, B., ZAHANG, H. ZHANG, M. AND LI, Z. (2006). Nac1 salinity stress decreased Bacillus thuringiensis (Bt) protein content of transgenic Bt cotton (Gossypium hirsutum L.) seedlings. J. Envir. Exper Bot., 55(3): 315-320.

JOENS, M.P. AND STENHOUSE, J.W. (1983). Salt tolerance of mangrove swamp rice varieties. Int. Rice Res. News-Letter. 8:8-9.

JOHANSON, J.G. AND CHEESEMAN, J.M.(1983). Uptake and distribution of sodium and potassium by corn seedlings. II. Role of the mesocotyle in sodium exclusion. Plant Physiol. 73:153-158.

KADER, A.A., MOHAMEDIN, A.A.M. AND AHMAD, K.K.A. (2006). Growth and yield of sunflower as affected by different salt affected soils. Int. J. Agri. Bio., 583-587.

KAHLOWN, M.A. AND AZAM, M. (2000). Effect of saline drainage effluent on soil health and crop yield. Agric. Water Manage. 62(2): 127-138.

KAHLOWN, M.A. AND ZAM, M. (2002). Individual and combined effect of water Logging and salinity in the Indus basin. Irrig. And Drain. 51: 329-338.

KALAJI, M.H. AND PIETKIEWICZ, S. (1993). Salinity effects on plant growth and other physiological process. Acta Physiol. Plant. 15:89-124.

KANWAL, H. (1999). Response sunflower (Helianthus annuus L.) to different levels of NaC1 salinity under two N fertilizers levels. M.Sc. Thesis. Dept. Bot., Univ. Agri. Faisalabad.

127

KAOW.T., TSAI, T.T. AND SHIH, C.N. (2003). Phtosynthetic gas exchange a fluorescence of three wild soybean species in response to NaCl treatments. Photosynthetica. 41:415-419.

KARADIMOVA, M. AND BAMBOVA, G.1993. Increased NaCl-tolerance in selection. In vitro cell. Dev. Boil. 9:180-82.

KATERJI, N., HOORN, J. W.V., FARES, C., HAMDY, A., MASTROFRILLI,M AND OWEIS, T. (2005). Salinity effect on grain quality of two durum wheat varieties differing in salt tolerance. Agricultural Water Management. 75(2):85-91.

KHAN, G.S. (1993). Characterization and genesis of saline-sodic soils in Indus Plains of Pakistan. Ph.D. Thesis, Dep. Soil Sci. Univ. Agri. Faisalabad, Pakistan.

KHAN, M., ABDUL RAUF, MAKHDOOM, M.I. AND AHMAD, A. (1992). Salt tolerance of eight wheats (Triticum aestivum L.). under saline-sodic field conditions. Sarhad J. Agric., 8(5): 593-599

KHAN, M.A. AND ABDULLAH, Z. (2003). Reproductive physiology of two wheat cultivars differing in salinity tolerance under dense saline sodic soil. Food, Agriculture and Environment. 1(3&4): 185-189.

KHAN, N.I., HHSSAIN, M., MIRZA, M.S. AND KHAN, G.S.(1992). Premlinary screening of wheat germ plasm for callogenesis. J. Agric. Res., 30(1):1-4.

KHAN, S.J., KHAN, M.A., AHMAD, H.K., KHAN, R.D. AND ZAFAR,Y. (2004). Somaclonal variation in sugarcane through tissue culture and subsequent screening for salt tolerance. Asian J. Plant Sci. 3 (3): 330-334.

KHATHAR, D. AND KUHAD, M.S. (1999). Stage sensitivity of wheat cultivars to short term salinity stress. Ind. J. Plant Physiol., 5: 26-31.

KHATUN, S. AND FLOWERS, TJ. (1995). Effect of salinity on seed set in rice. Plant Cell Environ., 18:6l-70.

KISHOR, P.B.K. (1998). Effect of salt stress on callus cultures of Oryza sativa L. J. Exp Bot., 39: 235-240.

KOPERTEKH, L.G. AND BUTENKO, R.G. (1998). Testing of regenerant wheat plants for salt resistance. Russ. Agri. Sci., (1): 19-22;

LA RUE, C.D. (1949). Cultures on the endosperm of maize. Am. J. Botany, 34: 585-586.

128

LAGUDAH, D.P. SCHACHTMAN AND HARE, R.A. (2002). Avenues for increasing salt tolerance of crops, and the role of physiologically based selection traits. Plant Soil. 247: 93-105.

LARKIN, P.J. AND SCOWCROFT, W.R. (1981). Somaclonal variation: A novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet., 60: 197-214.

LAUCHLI, A. (1990). Calcium, salinity and the plasma membrane. In: Current topics in plant physiology. ( eds. T. Leonard and P.K. Helper ) 1:26-33. An American society of plant physiologist, Maryland, USA.

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. (ed. K.K. Tanji ).113-37. Amer. Soc. Civil Eng., New York.

LEILA, B.A., GABALLAH, M.S., EL-ZEINY, H.A. AND KHALI, H. (2007). Effect of Antitransparent application on yield and fatty acid of sesame cultivars grown under saline condition. J. Appl. Sci. Res., 3(9): 879-885.

LI, C., SONG, M., JIQING, C., YONGGANG, XIUFEN, W. AND YONGFENG, W. (2008). Differences in stomatal and photosynthetic characteristics of five diploidy wheat species. Aceta Eclogica Sinica. 28(7): 3277-3283.

LIN C.C. AND KAO, C.R.I. (1995). Nael stress ill rice seedlings. The influence of calcium on root growth .Bot, Bull. Acad. Sin. 36:41-45.

LIU, W. AND LIU, Y.L. (1993). NaCl-stress and tolerable Cl- contents in barley seedlings. J. Nanjing Agric. Univ. 16(1): 16-20.

LU, S., XINXIANG, P., ZHENFEL, G., GENGYUN, Z., ZHONGCHENG, W., CONGYING, W.,CHAOSHU, P., ZHEN, F. AND JIHUA, W. (2007). In vitro selection of salinity tolerant variants from trioloid bermudagrass(Cynodon transvaalensis x C. dactylon) and their physiological responses salt and drought stress. Plant Cell. Reports.26 (6):1413-1429.

LU, W. AND JIA, J.F. (1994). Selection of a salt tolerance cell line from embryogenesis callus of millet and studies on its physiological and biochemical characteristics. Aeta Agronomica siniea., 29;241-247.

LUHRS, R. AND LORZ, H. (1987). Initiation of morphenogenic cell suspension and protoplast culture of barley. Planta, 175: 71-85.

LUTTS,S., KINET, J.M. AND BAUHARMONT, J. (1999). Effect of various salts and of mannitol on ion and praline accumulation in relation to osmotic adjustment in rice callus culture. J. Plant Physiol.149:186-195.

129

LYNCH, J. AND LAUCHLI, A. (1988). Salinity effects intracellular calcium in corn root protoplasts. Plant Physiol. 87:351-356.

LYNCH, J., CRAMER, G.R. AND LAUCHLI, A. (1987). Salinity reduces membrane associated calcium in corn root protoplasts. Plant Physiol. 83:390-394.

LYNCH, J., POLITO, V.S. AND LAUCHLI, A. (1989). Salinity stress increases cytoplasmic Ca+ activity in maize root protoplasts. Plant Physiol. 90:1271-1274.

MACKINNON, C. (1986). Morphogenic responses of wheat calli derived from develop-mentally mature embryo explants. Diss. Abstr. Int. Bot. 46: 3297.

MACKINNON, C., UNDERSON, G. AND NABORS, M.W. (1986). Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum. Plant cell Rep.5: 349-351.

MADDOCK, S.E. (1985). Cell culture, somatic embryogenesis and plant regeneration in cereals. In Bright, Tissue and Cell Culture. S.W.J. jones MGK (eds.) Dordrecht Martunus Nijhoff Publ. p. 131-174.

MAHAJAN, T.S. AND SONAR, K.R. (1980). Effect of NaCl and Na2SO4 on dry matter accumulation and uptake of N,P and K by wheat. J. Maharashtra Agric. Univ. 96(3): 622-638.

MAHMOOD, N. AND QURAISHI, A. (1983). Effect of salinity on callogenesis and organogenesis in four wheat varieties. Plant Tissue Cultutre, PARC, Islamabad.

MAHMOOD, T., YOUNAS, M., MEHDI, S.M. AND NIAZI, M.H.K. (1991). Comparative physiological studies in rice (Oryza sative L.) under normal and saline conditions, Pak. J Soil Sci., G, 1 1-17.

MAKHDOOM, M.U., MEMON, M.H. AND DERO, S.R. (1985). Germination and growth of wheat crops as affected by different salinity levels. Sindh. J. Agric. Res., 5(1): 93-101.

MALMSTROM, B.G. (1985). Cytochrome c oxide as a proton pump. A transition-state mechanism. Biochem. Biophys. Acta. 811:1-12.

MALTBY, E. (1991). Wetlands-their status and role in the biosphere. In: plant life under oxygen deprivation. (eds. M.B. Jackson,D.D. Davis and H. lambers ) 3-21. SPB Academic Publishing bv., The Hague, The Netherlands.

MARAIM, K.B. (1990). Physiological parameters of salinity tolerance in C4 turf grasses. Dissertation Abst. International B. Sci. and Eng. 51:484p.

130

MARCAR, N. E. AND TERMAAT. (1990). Effect of root zone solutes on Eucalyptus camaldulensis and Eucalyaptus bicostata seedlings; responses to Na+, Mg+ and Cl-, Plant Soil 125:245-254.

MARCAR, N.E. (1993). Waterlogging modifies growth, water use and ions concentrations in seedlings of salt-treated Eucalyptus camaldulensis, E. robusta and E. globules. Aust. J. Plant Physiol. 20:1-13.

MARSCHNER, C.M., (1995). Salinity affects the timing of phasic development in spring wheat. Crop Sci. 34: 1544-1549.

MARSCHNER, H. (1995). Mineral nutrition of higher plants. 2nd Ed. - Academic Press. In: Mineral nutrition in higher plants. (eds. Marschner, H.). 77-542. Academic Press, San Diego. London.

MARSCHNER,H. (1971). Why can sodium replace potassium in plans? Potassium Biochem. Physiol. Colloq. In Potash Inst. 8:50-63.

MARTIN, J.H. AND LEONARD, W.H. (1963). Cereal Crops. The Mcmillan Company, New York. 275-287.

MARTIN, P.K., TAEB, M. AND KOEBNER, R.M.D. (1993). The effect of photoperiod insensitivity on the salt tolerance of amphiploids between bread wheat (Triticum aestivum) and Sand couch grass (Thynopyrum bessarabicum). Plant Breeding. 111:283-289.

MASS, E.V., J.A. POSS, AND G.J. HOFFIMAN, (1986). Salinity sensitivity on sorghum at three growth stages. Irrig. Sci. 7:1-11.

MASS,E.V. (1986). Salt tolerance of plants. Appl. Agri. Res.1:12-26.

MASS,E.V. AND HOFFMAN, G.J. (1977). Crop salt tolerance-Current assessment .J. Irrg. Drainage Div. Amer. Soc. Civil Engg. 103:115-134.

MAAS, E.V. (1993). Plant growth response to salt stress. In: Towards the rational use of the growth and mineral nutrition of pepper cultivars. Plant high salinity tolerant plants. ( eds. Lieth and A.AI Masoom ) Vol.1:279-291. Kluwer Academic Publishers. Nutr. 20: 1085-1094.

MAAS, E.V. AND NIEMAN, R.H. (1978). Physiology of plant tolerance to salinity.. In: Crop tolerance to suboptimal land conditions.(ed. G. A. Gung ). 77-299. Soil Sci. Soc. Amer. Spec. Pub., Madison, U.S.A.

MATHIAS, R.J., FUKUI, K. AND LAW, C.N. (1983). Cytoplasmic effect on tissue culture response of wheat callus. Theor. Appl. Genet. 72:70-74.

MATSUDA, K. AND RIAZI, A. (1981). Stress-induced osmotic adjustment in growing regions of barley leaves. Plant Physiol. 68:571-6.

131

MAXWELL, K, AND JOHNSON, G.N. (2000). ChlorophyII fluorescence a practical guide. J. Exp. Bot. 51(345): 659-668.

MCCUNE, D,C. AND SILBERMAN, D.H. (1991). Responses of plants to simulated saline drift as affected by species and conditions ofexposure. Environ. Pollution. 70:57-69.

MCGUIRE, P.E. AND DVORAK, J. (1981). High salt-tolerance potential in wheat grasses. Crop Sci. 21:702-705.

MCMANMON, M. AND CRAWFORD, R.M.M. (1971). A metabolic theory of flooding tolerance. The significance of enzymem distribution and behavior. New Phytol. 70:299-306.

MEDHI, S.M.,. RANJHA, A.M., SARFRAZ, M. AND HASSAN, G. (2001). Response of wheat to potassium Application in Six Soil Series of Pakistan. On line J.Bi0I. Sci., 1(6): 429-431.

MEHDI, S.S., JAVED, K. AND ZAFAR, S.S. (2000). Relationship of sunflower (Helianthus annuus L.) cultivars for seedling traits across NaC1 treatments. Sci. Int. 12:99-102.

MELLONI, Y., HARIPYRIYA, C.V., PATIL, S.G., HEBBARA, M. AND SASTRY, J.A. (2003). Effect of salinity on germination and early growth in sunflower (Helianthus annuus L.). Helia. 21:95-101.

MENEGUZZO, S., NAVARI-IZZO, F. AND IZZO, R. (2000). NaC1 effects on water relations and accumulation of mineral nutrients in shoots, roots and cell sap of wheat seedlings. J. Pla. Physiol., 156(5/6): 711-716.

MIAN, M.A. (1988). Land resources of Pakistan. In: Manging soil resources. 10-16. Published by Soil Sci. Soc. Pakistan.

MIKAMI,T. AND KINOSHITA,T. (1988). Genottypic effects on the callus formation from different explants of rice, Oryza sativa L. Plant Cell, Tissue and Organ Culture. 12(30:311-314.

MIRODJAGH, S.S., ARZANI, A. MIRODJAGH, S.S. AND ARZANI, A. (1999). Assessment of durum wheat (Triticum turgidum var. durum) cultivars for in vitro salt tolerance. J. Sci.Technol. Agri. Nat. Resour., 3: 21-34.

MOHMAND, A.S. (1993). Tissue culture variability in wheat germ- plasm: callus initiation and long-term plant regeneration and maintenance. Pakistan journal of scientific society and industrial Research, 36(8):306-309.

MORARU-I; RADUCANU, F. AND PETCU,E. (1996). In vitro reaction of three winter wheat genotypes to NaCl treatment. Probleme-de-Genetica-Teoretica-si-Aplicata.28:2,93-98.

132

MUHAMMED. S., NEUE, H.U. AND MENDOSA, B.S. (1986). Effect of gypsum on the growth and mineral nutrition of some K-efficient rices in costal saline-sodic soils in Philippines. J. Crop Sci. 11:213-220.

MUNK, D.S. AND ROBBERTS, B. (1995). Growth and development of Pima and Acala cotton on saline soils. Proceedings, Beltwide Cotton Cong. San Antonio, TX, USA. 1995:90-92.

MUNNS, R. (1985). Na+, K+ and Cl- in xylum sap flowing to shoots of NaCl treated barley. J. Exp. Bot. 36:1032-42.

MUNNS, R. (1993). Physiological processes limiting plant growth in saline soils. Some dogmas and hypotheses. Plant Cell Environ. 16: 15-24.

MUNNS, R. (2002). Comparative physiology of salt and water stress. Plant. Cell Environ. 25:239-250.

MUNNS, R., AMI R.A. JAMES. (2003). Screening methods for salinity tolerance: a case study with tetraploid wheat. Plant Soil. 253:201-218.

MUNNS, R. AND TERMAAT, A. (1986). Whole plant-responses to salinity. Aust. J. Plant Physiol. 13:143-160.

MUNNS, R., (2002). Effect of saline conditions on the emergence and establishment of various cultivars of wheat. Sarhad. J. Agri. 10:1-19.

MUNNS, R., (2005). Genes and salt tolerance: bringing them together. New phytol. 167: 645-663.

MUNNS, R., SCHACHTMAN, D.P. AND CONDON, A.G. (1995). The significance of a two-phase growth response to salinity in wheat and barley. Australian Journal of Plant Physiology 22,561-569.

MUNNS, R., GREENWAY, H. AND KIRST, G.O. (1983). Halotolerant eukaryotes. In: Encyclopedia of Plant Physiol. (eds. O.L. Lange, P.S. Nobel, C.B. Osmand and H. Ziegler ), New Series. Vol. 12:59-135, Springer-Verlag, Berlin.

MURANAKA, S., SHIMIZU, K. AND KATO, M. (2002). A salt-tolerant cultivar of wheat maintains photosynthetic activity by suppressing sodium uptake. Photosynthetica. 40:509-515.

MURASHIGE, T. AND SKOOG, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15:473-479.

NAIDOO, G. AND RUGHUNANAN, R. (1990). Salt tolerant in the succulent, coastal halophytes, Sarcocomia natalensis. J. Exp. Bot. 41:497-502.

133

NAKAMURA, S., SHIMIZU, K. AND KATO, M. (2002). Ionic and osmotic effect of salinity on single leaf photosynthesis in two wheat cultivars with different drought tolerance. Photosynthetica. 4(20): 201-207.

NASEEM, A., M.S. IQBAL, K. MAHMOOD AND J. AKHTAR, (2001). Comparative of wheat (Triticum aestivum L.) Genotypes under Salinity Stress. 1:Growth and Yield parameters. On line J. Bio. Sci. 1(1): 33-35.

NASEEM, A., IQBAL, M.S. AND QURESHI, R.H. (2000). Response of wheat (Triticum aestivum L.) to varying levels of salinity. Sci. Int. 11:92-109.

NATH, J., RAJ, M., SINGH, A., DEV, S. AND SINGH, R. (1982). Performance of sorghum, sunflower and wheat as affected by salinity of irrigation water. Transaction of Indian Society of Desert Technology and University centres of Desert studies., 7(2): 59-61.

NAVARI-IZZO, F., UARTAEEI, M.P.Q. AND IZZO, R. (1990). Water stress induced changes in protein and free amino acids in field grown maize and sunflower. Plant Physiol. Biochem. 28:531-537.

NAWAZ, S., QURESHI, R.H., ASLAM, M. AKHTAR, J. AND PARVEEN, S. (1998). Comparative performance of different wheat varieties under salinity and water logging. II. Lonic relations. Pak. J. Bio. Sci., 1: 357-359.

NAZIR, N., ASHRAF, M. AND EJAZ, R. (2001). Genomic relationships in oil seed Brassica with respect to salt tolerance, photosynthetic capacity and ion relations. Pak. J. Bot., 33: 483-50 I.

NETONDO, G.W., ONYANGO, J.C. AND BECK, E. (2004). sorghum and salinity: II.Gas exchange and chloroflurescence of sorghum under salt stress. J. Crop Sci. 44 : 806- 811.

NEUMANN, P., (1997). Salinity resistance and plant revisited. Plant, cell and Environment, 20: 1193-1198.

NONAMI, H. AND. BOYERS, J.S. (1989). Turgor and growth at low water potentials. Plant Physiol. 69;789-804.

NURITDINOV, N. AND VARTAPETIAN, B.B. (1980). Translocation of 12C-sucrose in cotton plant under condition of root anaerobiosis. Fiziol. Rast. (Moscow) 27:814-820.

OCHATT, S.J., MORCONI, P.L., RADIEE, S., ARNOZIS, P.A. AND CASO, O.H. (1998). In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants. Plant Cell, Tissue Organ Cult.,55:1-8.

134

OHARA, J.F. AND STREET, H.F. (1978). Wheat callus culture the initiation, growth and organogenesis of callus derived from various explant sources. Ann. Bot. 43: 1029-1038.

OPIK, H. (1973). Effect of anaerobiosis on respiratory rate, cytochrome oxidase activity and mitochondrial structure in coleoptiles of rice (Oryza sativa L). J. Cell Sci. 12:725-739.

OSMOND, C.B. AND GREENWAY, H. (1972). Salt response of carboxylation enzymes from species differing in salt tolerance. Plant Physiol. 49:260-263.

OUDIJA, F. AND ISMAILI, M. (2002). Effect of the concentration of NaCl on somatic embryogenesis and on the regeneration capacities of wheat. African Crop Sci. J.10: 211-219.

OVESNA, J, AND LHOTOVA, M. (1987). Study of callus growth and plant regeneration in wheat tissue culture. Sci. Agri. Bohemo Slovaca. 19: 243-253.

OZALP,V.C; OKTEM, H.A., NAQVI, S.M.S. AND YUCEL, Y. (2000). Photosystem II and cellular membrane stability evaluation in hexaploid wheat seedlings under salt stress conditions. J.PlantNutri.23: 2,275-28.

OZGEN, M., TURET, M., OZEAN, S. AND SANEAK, C. (1996). Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes. Plant Breeding, 115(6): 455-458.

PALTA, J.P. (1990). Stress interaction at the celluler and memrane level. Hort. Sci. 25:1377-1381.

PARAIDA, A. K., DAGAONKAR, V.S. PHALAK, M.S. UMALKAR, G.V. AND AURANGABADKAR, L.P. (2007). Alterations in photosynthetic pigments, protein and osmotic components in cotton genotypes subjected to short term drought stress followed by recovery. J. plant Biotech. Reports. 1(1): 37-48.

PARVEEN, S. AND QURESHI, R.H. (1992). Toxicity levels of Na+ and C1- in wheat leaves cell sap. Pak. J. Agri. Sci., 29(3):148-152.

PATADE. V.Y., SUPRASANNA, P. AND BAPAT, V.A. (2008). Effect of salt stress in relation to osmotic adjustment on sugarcane (Saccharum officinarum L.) callus cultures. Pl. Growth Regulation. 1573-1587(Online).

PEARSON, J. AND HAVILL, D.C. (1988). The effect of hypoxia and sulphide on culture grown wetland and non-non wetland plants.II. metabolic physiological changes. J. Exp. Bot. 39:431-439.

PERVAIZ, Z., AFZAL, M., XIAOE, Y. AND ANCHCNG, L. (2002). Physiological Parameters of Salt Tolerance in Wheat. Asian Journal of Plant Sciences. (1)4: 478-481.

135

PESSARAKLI, M. AND SZABOLES. (1999). Soil salinity sodicity as stress factor. In: Hand book of Plant and Crop stress. (ed. M. Pessarakali.) , Marcel Dekker Inc. New York.

PESSARAKLI, M., TUCKER, T.C. AND NAKABAYASHI, K. (1991). Growth response of barley and wheat to salt stress. J. Plant Nutri., 14(4): 331-340.

PICCHIONI, G.A., MIYAMOTO, S. AND STOREY, J.B. (1991). Rapid testing of salinity effects on Pistachio seedling rootstock. J. Amer. Soc. Hort. Sci. 11;555-559.

PITMAN, M.G. (1965). Transpiration and the selective uptake of potassium by barley seedlings (Hordeum vulgare cv. Bolivia). Aust. J. Biol. Sci. 18:987-999

PITMAN, M.G. (1975). Whole plants. In: D.A. Baker and J.L. Hall (ed.) Ion transport in Cells and Tissues. North Holland, Amsterdam.

PITMAN, M.G. (1976). Ion uptake by plant roots. In: Transport in plants II, Part B, Tissues and Organs. Encyclopedia of plant Physiology, (eds. U.Luttge and M.G. Pitman ) New Series, Vol. 2.5-128. Springer-Verlag, Berlin.

PITMAN, M.G. AND SADDLER, H.D.W. (1967). Active sodium and potassium transport in cells of barley roots. Proc. Nati. Acad. Sci. U.S.A. 57: 44-49.

PITMAN, M.G.., LAUCHLI, A. AND STELTZER, R. (1981). Ion distribution in barley seedlings measured by electron probe x-ray microanalysis. Plant physiol. 68: 673-679.

POLJAKOFF-MAYBER, A. (1975). Morphological and anatomical changes in plants in response to salinity stress. In: Plants in saline environments. ( A. Poljakoff-mayber and J. Gale ). 97-117.Springer-Verlang, Berlin Heidelberg, New York

POLJAKOFF-MAYBER, A. AND GREENWAY, H. (1974). Effect of high sodium chloride concentration in the growth medium on activity of glucose-6- phosphate dehydrogenasefrom pea roots. Aust. J. Plant Physiol. 1:483-489.

POLLARD, A. AND WYN JONES, R.G. (1979). Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta. 144:291-298.

PONNAMPERUMA, F.N., (1984). Salinity tolerance in plants with different strategies for crop improvement. Wiley, New York, 255-271.

PORATH, E. AND POLJAKOFF-MAYBER, A. (1968). The effect of salinity in the growth medium on carbohydrate metabolism in pea root tips. Plant Cell Physiol. 44:1031-1034

POUSTINI, K. AND A. SIOSEMARDEH, (2004). Ion distribution in wheat cultivars in response to salinity stress. Field crops Research. 85: 125-133.

136

POUSTINI, K. AND SIOSEMARDEH, A. (2004). Ion distribution in wheat cultivars in response to salinity stress. Field-Crop-Res., 85: 125-133.

POUSTINI.K. (1995). Physiological responses of two wheat cultivars to salinity stress. Iranian-Journal-of-Agricultural-Sciences. 26:2,57-64.

POUSTINI.K. (2002). An evaluation of 30 wheat stress. Iran.J.Agri.Sci. 33:1, 57-64.

POUSTINI.K. AND CIOCEMARDEH, A. (2001). K+/Na+ ratio and ion selectivity in response to salt stress in wheat. Iran.J.Agri.Sci. 32:3,525-532.

POUSTINI.K. AND BAKER, A.D. (1994). Photosynthetic responses of two wheat cultivars to salinity. Iranian-Journal-of-Agricultural-Sciences.25(1): 61-69.

POUSTINI.K. AND SALMASI, S.Z. (1997). Effect of salinity on dry matter production and remobilization in two wheat cultivars. Iranian-Journal-of-Agricultural-Sciences. 28: 4, 11-17.

PUA, E.C. AND THROPE, T.A. (1986). Differential response of non selected and Na2SO4 selected callus cultures of Beta vulgaris L. to salt stress. J. Plant Physiol. 123: 241-248.

PURVIS, A.C. AND WILLIAMSON, R.E. (1972). Effect of flooding and gaseous composition of the environment on growth of corn. Agron. J. 64:674-678.

PUSHPALATHA, T. AND PADMANABHAN, C. (1998). Studies of in vitro testing for salt tolerance in callus cultures of rice (Oryza sativa L.) Current Agric. 22 (1-2): 109-113.

QADIR, M. AND SHAMS, M. (1997). Some agronomic and pgysiological aspects of salt tolerance in cotton (G. hirsutum L. ). J. Agron. Crop Sci., 179(2): 101-106.

QASIM M., ASHRAF, M., ASHRAF, M.Y., REHMAN, S.U. AND RHA, E.S. (2003). Salt-induced changes in two canola cultivars differing in salt tolerance. Biological Plantarurn (4): 629-632.

QING, G.F. AND CHENG, T.Z. (1999). Difference in Na+, K+ accumulation in the salt-tolerant mutant and the wild type of wheat during exposure to NaC1 stress. Acta Botanica Sinica. 41(5):515-518.

QURESHI, F.A. AND SPANNER, D.C. (1973). The effect of nitrogen on movement of tracers down to the stolon of Saxifraga tormentosa, with some observations on the influence of light. Planta. 110: 131-141.

QURESHI, R.H. (1986). Extent, characteristics and constraints of sodic soils in Asia. In: Project Designed Workshop for developing Collaborative Res. Program for the Improvement of rice Yields in Problem Soils. Int. Rice Res. Inst. Los Danos, laguna, Phillipines, 24-28 Nov. 1986.

137

QURESHI, R.H., RASHID, A. AND AHMAD, N. (1990). A procedure for quick screening of wheat cultivars for salt tolerance.p. In: Genetics aspects of plant mineral nutrition. (eds. N. ElBassam, M. Damborth and B.C. Laughman ).215-324. Kluwer Acad. Pub. The Netherland.

QURESHI, R.H., BARRETT-LENNARD, (1998). Saline Agriculture for irrigated Land in Pakistan, Kluwer Academic Publisher, Netherlands, 19-28.

QURESHI, R.H., NAWAZ, N. AND MAHMOOd, T. (1993). Performance of selected tree species under saline-sodic field conditions in Pakistan. In; Towards the rational use of high salinity tolerant plants, (eds. H.Lieth and A. Al Masoom ) Vol. 2:259-269. Kluwer Acad. Pub., Neitherlands.

QURESHI, T.M . ASHRAR, M.Y., BANO, M. AND HUSSAIN, F. (2000). Physiological responses or Eucalyptus under saline environment. Ionic composition in selected salt tolerant and salt sensitive provinces of the Eucalyptus camul-dulcnsisI'ev: J. BioI. Sci., 3 :2112-2115.

RAFIQ, M. (1975). Saline, Saline-alkali and waterlogged of the Indus Plain-their characteristics, cause of formation and measures needed for reclamation. 308-321. In; Proc. Int. Conference on Waterlogging and salinity, 13-17 October 1975, Univ. Engg. Tech. Lahore, Pakistan.

RAFIQ, M. (1990). Soil resources andsoil related problems in Pakistan. In: Soil Physics-application under stress environments.(eds. M. Ahmad, M.E. Akhtar and M.I. Nizami ). 16-23. BARD, PARC. Islamabad, Pakistan.

RAHMAN, M.S . MIYAKE, T.I. AND TAKEOKA, Y. (2001). Effect of sodium chloride salinity on seed germination and early seedling growth of rice (Oryza sativa) Pak. J. Biol. Sci., 4:351-355.

RAHMAN, S., AHMAD, B., SHAFI, M. AND BAKHAT, J. (2000). Effect of different salinity levels on the yield and yield components of wheat cultivars. Pak. J. Biol. Sci 3:1161-1163.

RAINS, D.W. (1972). Salt transport by plants in relation to salinity. Ann. Rev. Plant Physiol. 23:367-388.

RAINS, D.W. (1981). Salt tolerance-new developments. In: Advances in Food Producing systems for Arid and Semi Arids. Academic Press. New York.

RAINS, D.W. AND EPSTEIN, E. (1967a). Sodium absorption by barley roots: Its mediation by mechanism 2 df alkali cat ion transport. Plant Physiol. 42:319-323.

RAINS, D.W. AND EPSTEIN, E. (1967b). Prefrential absorption of poyassium by leaf tissue of the mangrove A vicennia marina. An aspect of halophytic competence in coping with salt. Aust. J. Giol. SCi. 20:847-857.

138

RAJPUR AND SIAL, N.B. (2002). Effected of hydroponic salinity (120 molm-3 NaC1) on the ion uptake and growth of different wheat varieties. Pak. J. Appl. Sci., 2(5): 541-543.

RANGAN, T.S. AND VASIL, I.K. (1982). Sodium chloride tolerant embryogcnie cell lines of Pennisetum americnum (L.). Annals of Botany. 2(1):59-64.

RASHID, A. (1986). Mechanisms of salt tolerance in wheat (Triticum aestivum L.). Ph. D. Thesis, Univ. Agri. Faisalabad, Pakistan.

RATHERT, G., DOERING, H.W AND WITT, J. (1981). Influence of extreme K:Na ratios high substrate salinity on plant metabolism of crops differing in salt tolerance. III. K/Na effects on the carbohydrate pattern bush bean and sugarbeat plants in response to the salt tolerance of the species. J. Plant Nutr. 4:131-141.

RAWSON, H.M. AND MUNNS, R. (1984). Leaf expansion in sunflower as influenced by salinity and short-term changes in carbon fixation. Plant, cell Environ. 7:207-213.

RAWSON, H.M.,. RICHARDS, R.A. AND MUNNS, R. (1988). An examination of selection criteria for salt tolerance in wheat, barley and triticale. Australian Journal of Agricultural Research 39,759-772.

RAYMOND, J.M. AND SIMPSON, E.S. (1986). The interaction of genotype and culture medium on the tissue culture response of wheat callus. Plant Cell, Tissue Organ Cult. 7:31-37.

REDDY, K.R. AND PATRICK, W.H.JR. (1975). Effect of alternate aerobic and anaerobic conditions redox potential, organic matter decomposition, and nitrogen loss in a flooded soil. Soil Biol. Biochem. 7;87-94.

REDWAY, F.A., VASIL, V., LU, D. AND VASIL, I.K. (1990). Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Ttriticum aestivum L.). Theoretical and Applied Genetics, Gainesville., FL, 32611, USA. 79(5):609-617.

REHMAN, O.U. AND HUSSAIN, M.K. (1998). Effect of salinity on growth and development of cultivated sunflower (Helianthus annuus L.). Pak. J. Sci., 50:45-53.

REINERT, J. AND BAJAJ, Y.P.S. (1976). Applied and Fundamental aspects of plant cell, tissue and organ culture. 144-147.

RENGEL, Z. (1992). The role of calcium in salt toxicity. Plant Cell Environ. 7:207-213.

139

RICHARDS, L.A. (1954). Diagonosis and improvement of saline and Alkali soils. U.S.D.A. Hand book. No.60. Washington D.C. USA.

RIVELLI, A.R., JAMES, R.A., MUNNS, R. AND CONDON, A.G. (2002). Effect of salinity on water relations and growth of wheat genotypes with contrasting sodiukm uptake. Funct Plant Biol. 29: 1065-1074.

RIVELLI-A.R., LOVCLLI, S. NARDIELLO, I., PEMIOLA, M AND GHERBIN, P. (2002). Growth and yield response of paper sorghum to irrigation with saline water. Agronomia. 36:4,333-338.

ROBINSON, S.P. AND JONES,G.P. (1986). Accumulation of glycinebetaine in chloroplast provides osmotic adjustment during salt stress. Aust. J. Plant Physiol. 13:639-668.

RUAN, Y., EL-HENDAWY, S.E. AND SCHMIDHALTER, U.Y. (2007). Differential effect of moderate salinity on growth and ion contents in the mainstem and subtillers of two wheat genotypes. J. S. Sci. Pla. Nutr., 53: ((6) 782-791.

RUS, A.M., RIOS, S., OLMOS, E., SANTA-CRUZ, A. AND BOLARIN, M.C. (2000). Long term culture modifies the salt responses of callus lines of salt tolerant and salt sensitive tomato species. J. Plant Physiol. 157: 413-420.

RUSH, D.W. AND EPSTEIN, E. (1981). Comparative study on the sodium, potassium and chloride relations of a wild halophytic and a domestic salt-sensitive tomato species. Plant Physiol.68:1308-13.

RYAN, S.A., BRETTEL, R.I.S. AND CROF, W.R.S. (1984). Heritable somaclonal variation in wheat. Ibid. 67: 443-455.

SABBAH,S. AND Tal, M. (1990). development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress. Plant cell Tissue and Organ culture 21:119-128.

SACALA, E., BIEGUN, A.,DEMCZUK, A. AND GRZYS,E. (2005). Effect of NaCI and supplemental calcium on growth parameters and nitrate reductase activity in maize. Acta Societatis Botanicorum Poloniae. 74(2): 119-123.

SAIRAM, R.K., RAO, AND SRIVASTAVA, G.C. (2002). Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, antioxidant activity and osmolyte concerntration. Plant Science. 163(5): 1037-1046.

SALAM, A., HOLLINGTON, P.A., GORHAM, J., WYN JONES, R.J., AND GLIDDON, C. (1999). Physiological Genetics of Salt Tolerance in wheat (TriticlIlIl acstivum L.) Perfon11ance of Wheat Varieties\ Inbred Lines and Reciprocal f\ Ilybrids under Saline: Conditions. J. Agronomy & Crop Science .183:145-156.

140

SALIM, M. AND PITMAN, M.G. (1983). Effect of salinity on ion uptake and growth of mung bean plants (Vigna Rodiata L.). Aust. J. Plant Physiol. 10:393-407

SALIM,M. (1989). Effects of salinity and relative humidity on growth and ionic relations of plants. New Phytol. 113;12-20.

SALMA, S., TRIVEDI, S., BUSHEVA, M., ARAFA, A. A., GARAB, G. AND ERDEI, L, (1994). Effects of NaC1 salinity on growth, cation accumulation, chloroplast structure and function in wheat cultivars differing in salt tolerance. J. Pla. Physiol. 144(2): 241-247.

SANCOKA, H., SHIOTA, K., KURBAN, H., CHAUDHRY, M.I., RCMACHANDRA, G.S. AND FUJITA, K. (1999). Effect of salinity on growth and solute accumulation in two lines. differing in salt tolerance. Soil Sci. Plant Nutr., 45(4):873-880.

SANDHU, G.G. AND QURESHI, R.H. (1986). Salt affected soil of Pakistan and their utilization. Reclaim. Reveg. Res., 5: 105-113.

SANTOS, C.L., DOS, V. AND CALDCRRA, G. (1999). Comparative responses of llclianthus annuus plants and calli exposed to NaCI : growth rate and osmotic regulation in intact plant and calli. J. Plant Physio!., ISS :769-777.

SAQIB, M., AKHTAR, J AND QURESHI, R.H. (2004). Pot study on wheat growth in saline and warerlogged compacted soil I. Grain yield and yield components. Soil and Tillage Research. 77: 169-177.

SAQIB, M. AKHTAR, J., PERVAIZ, S., QURESHI, R.H. AND ASLAM, M. (2002). Comparative growth performance of five cotton genotypes (G. hirsutum) against different levels of salinity. Pak. J. Soil Sci. 39(2): 69-75.

SAQIB, M., AKHTAR, J. AND QURESHI, R.H. (2008). Sodicity intensifies the effect of salinity on grain yield and yield components of wheat. J. Plant Nutr. 31(4): 689-701).

SAYED, O.H. (2003). Chlorophyll fluorescence as a tool in cereal crop research. Photosynthetica 41: 321-330.

SCHACHTMAN, D. AND LIU, W. (1999). Molecular pieces to the puzzle of the interaction between potassium and sodium uptake in plants. Trends in plant Science. 4(7): 281-287.

SCHACHTMAN, D.D. AND MUNNS, R. (1992). Sodium accumulation in leaves of Triticum species that differ in salt tolerance. Aust.J. Plant Physiol. 19;331-40.

SCHACHTMAN, D.P., BLOOM, A.J. AND DVORAK, J. (1989). Salt tolerant Triticum x Lophopyrum derivatives limit the accumulation of sodium and chloride ion salt stress. Plant cell and environment, 121(1): 47-55.

141

SCHUBERT, S. AND LAUCHLI. A. (1990). Sodium exclusion mechanisms at the root surface of two maize cultivars. Plant Soil .123:205-209.

SEAR, R.G. AND DECKARD, E.L. (1982). Tissue culture variability in wheat callus induction and plant regeneration. Crop Sci. 22:546-550.

SEEMANN, J. R. AND SHARKEY, T.D. (1986). Salinity and nitrogen effects on photosynthesis, ribulose-1-5-bisphosphate carboxylase and metabolite pool size in phaseolus vulgaris L. Plant Physiol. 82:555-560.

SEEMANN, J.R. AND CRITCHLEY, C. (1985). Effect of salt stress on the growth, ion content, stomatal behavior and photosynthetic capacity of a salt-sensitive species, phaseolus vulgaris L. Plant Soil .123:205-209.

SHAFAQAT, M.N., MUSTAFA, G., MIAN, S.M. AND QURESHI, R.H. (1998). Evaluation of physiological aspects of stress tolerance in wheat. J. Soil Sci., 14:85-89.

SHANNON, M.C. (1978). Testing salt tolerance variability among tall wheat grass lines. Agron, J. 70:719-722.

SHANNON, M.C. (1984). Breeding, selection and the genetics of salt tolerance.p. In: Salinity tolerance in plants, strategies for crop improvement. (eds. R.C. Staples and G.H. Toenniessen. ). 231-235. John Wiley and saons, New York.

SHANNON, M.C. AND FRANCOIS, L.E. (1978). Salt tolerance of three cantaloupe cultivars (Cucumis melo L.). J. Amer. Soc. Hort. Sci. 103:127-130.

SHANNON, M.C., DALTON, F.N. AND EL-SAYED, S.F. (1993). Phsiological response of crops to sea water: minimizing constraints that limit yield. In: Towards the rational use of high salinity tolerance in plants. (eds. H. Lieth and A. Al Masoom ). vol 2:3-12. Kluver Academic publishers, Netherlands.

SHARIATPANAHI, M.E. AND DABIRAShraft, O. (2003). Production of salt-tolerant barley (Hordeum vulgare L.) lines using somaclonal variation. Iranian J. Agri. Sci. 34(2):367-377.

SHARMA, G.C., BELLO, L.L., SARPA, V.T. AND PETERSON, C.M. (1981). Callus initiation and plant regeneration from triticale embryos. Ibid. 21: 113-118.

SHARMA, N., GUPTA, N., GUPTA, S. AND HASEGAWA, H. (2005). Effect of NaCl salinity on photosynthetic rate, transpiration rate, oxidative stress tolerance in contrasting wheat genotypes. Photosynthetica. 43(4): 609-613.

142

SHARMA, S.K. (1989). Effect of salinity on growth, ionic and water relations of three wheat genotypes differing in salt tolerance. Ind. J. Plant Physiol., xxxii (3): 200-205.

SHARMA, S.K. (1996). Soil salinity affect on transpiration and net photosynthesis rate, stomatal conductance and Na+ and Cl- contents in durum wheat. Biol. Plant., 38:519-523.

SHIMADA, T., SASAKUMA, T. AND TSN-UNEWAKI, K. (1969). In vitro culture of wheat tissues. Callus formation, plant organ differentiation and single cell culture. Can. J. genet. Cytol. 11: 294-304.

SHOMER-LLAM, A. AND WAISAL, Y. (1973). The influence of Na on the balance between the C3 and C4 carbon fixation pathways. Physiol. Plant. 29;190

SIAL, N.B. AND KHANUAM, N.V. (1998). wheat response to different salinity levels. Pak. J. Agric., 14:77-80.

SIBI, ML, Fakiri, M (2000). Androgenese et gynogenese, sources de vitrovariation et de tolerance a la salinite chez l’orge Hordeum vulgare Secheresse. 11(2): 125-132.

SILBCRBUSH, M. AND BEN-ASHER, J. (2001). Simulation study of nutrient uptake by plants from soil less culture as affected by salinity build lip and transpiration. Plant Soil, 233(I): 65-69.

SINGH, K. N. AND RANA, R.S. (1985). Genetic variability and character association in wheat varieties grown on sodic soils. Ind. J. Agric. Sci. 44: 612-615.

SINGH, K.N. AND CHATRATH, R. (2001). Salinity Tolerance. In: Application of physiology in wheat Breading. (eds. Reynold, M.P.,J.I.Ortiz. Monasterio and A.McNab).101-110. Mexico, D.F.CIMMYT.

SINGH, R.U., MINHAS, P.S., CHAUHAN, C.P.S. AND GUPTA, R.K. (1992). Effect of high salinity and SAR waters on salinization, sodication and yields of pearlmillet and wheat Agricultural Water Management. 21(1-2):93-105.

SLATYER, R.O. (1961). Effects of several osmotic substrates on water relations of tomato. Aust. J. Biol. Sci. 14:519-540.

SMILLIE, R.M. AND NOTT, R. (1982). Salt tolerance in crop plants monitored by chlorophyll fluorescence in vitro. Plant Physiol. 70:1049-1054.

SONG, J., FAN, H., ZHAO, Y., JIA, Y. AND DU, X. (2008). Effect of salinity on germination, seedling emergence, seedling growth and ion accumulation of a euhalophyte suaeda salsa in an intertidal zone and on saline inlan. Aquatic Botany., 88(4): 331-337.

143

SRIVASTAVA, J.P. AND JANA, S. (1984). Screening wheat and barley germplasm for salt tolerance. In: Salinity tolerance in plants, stratigies for crop improvement. (eds. R.C. Staples and G.H. Toenniessen ). 381-387. John Wiley and Sons, New York.

STARK, Z. AND CZAJKOWSKA, E. (1981). Fuction of roots in NaCl sressed bean plants. In: structure and fuction of plan roots.(eds. R. Brouwer et al., ).381-387. Junk Publishers. The Hauge/boston/London.

STEEL, R. G. D. AND TORRIE, J.H. (1980). Principles and procedures of statistics a biological approach. 2 nd Ed., McGraw Hill Inc., NY. USA.

STEFHEN, R. AND CARTER, L.P. 1980. Crop Production. In: Principles and Practices. Montana state University.

STEPFUHN, H., AND WALL, K.G. (1997). Grain yield from spring-sown Canadian wheat grown in saline rooting media. Can. J. Plant Sci., 77: 63-68.

STEVCNINCK, VAN. R. F.M., MURALITHARAN, M.S., AND CHANDLER, S.F. (199-2). Effects of Na2S04, K2S04 and KCI on growth and ion uptake of callus culture of Vaccinum corymbosum L. cv. Blue Crop Ann. Bot., 69:459-462.

STOREY, R. AND WYN JONES, R.G.. (1978). Salt stress and comparative physiology in the Gramineae. I. Ion relation of two salt and water stressed barley cultivars. California Mariout and Arimer. Aust. J. Plant Physiol. 5:801-816.

STROGONOV, B.P. (1962). Physiological basis of salt tolerance of plants. Akademia, Nauk U.S.S.R., Moskwa, U.S.S.R.

SUBBASHNI, K. AND REDDY, G.M. (1990). Effect of salt stress on enzyme activities In callus culture of tolerant and susceptible rice cultivars. Ind. J. Exp. Biol. 28; 277-279.

SUDHIR.P. AND. MURTHY, S.D.S. (2004). Effects of salt stress on basic processes of Photosynthesis . Photosynthetica. 42 (4): 481-486.

SUDYOVA, SLIKOVA, V, S. AND OALOVA, z. (2002). Testing wheat (Triticum aestivum L.) and triticale (Triticosecale Witt.) callus to salt tolerance. Acta Fytotech. Zootech., 5: 67-71.

SULTANA, N., IKEDA, T. AND ITOH, R. (2000). Effect of NaCL salinity on photosynthesis and dry matter accumulation in developing rice grains. Environ. and Expt. Botany.42 (2): 181-183.

SYVERTSEN, J.P., BOMAN, B. AND TUCKER, D.P.H. (1989). Salinity in Florida citrus production. Proc. Florida State Hort. Soc. 102:61-64.

144

SZABOLES, I. (1991). Desertification and salinization. In: Plant Salinity Research Proc. Int. Conf. Agric. Management of Salt-affected Areas.(eds. R. Chouk-allah ).3-18.,26April-3May 1991, Agadir Institu. Agronomique et veterinarie Hassan II- C.H.A. Agadir, Morocco.

SZABOLICS, M.C. (1989). Salt Affected Soils. The Chemical Rubber Company Press,

TAGHVAII, M., MAJIDI-HERAVAN, E., SARMADNIA, G., MAZAHERI, D. AND SHAKIB, M.A. (1998). Salt tolerance of calluses of six wheat cultivars obtained from tissue culture on different media. Seed and Plant. 13: 60-68.

TAIZ, L. AND E. ZEIGER. (1998). Plant Physiology (2nded.). Sinauer Associates, Inc.,Sunderland, Massachusetts. 103-122.

TAIZ, L. AND ZEIGER, E. (2002). Plant physiology. 3rd edition, Sinauver Associates Innc. Publishers, Sunderland.

TARIQ, R.M. (1982). Screening of different wheat varieties for salt tolerance. M.Sc. Thesis, Deptt. Soil Sci. Univ. Agri., Faisalabad.

THALJI, T, AND SHALALDEH, G. (2007). Screening wheat and barley genotypes for salinity resistance. J. Agr, 6(1); 75-80.

TOMAR, P.C. AND PUNIA, M.S. (2003). In vitro screening for salt tolerance of cultivars of wheat [Triticum aestivum (L.) em. Thell]. Am. J. Agri. Biol. Res. 8: 91-96.

TOMOS, A.D. (1998). Cellular water relations of plants. In: Water Science Reviews 3. (eds. F. Franks ).186-277. Cambridge University Press.

TORRES, C.B. AND BINGHAM, F.T. (1973). Salt tolerance of Maxican wheat. I. Effect of NO3 and NaCl on mineral nutrition, growth and grain production of four wheats. Soil Sci. Soc. Amer. Proc. 37:711-715.

TURHAN, H., GENC, L., SMITH, S.E., BOSTANCI, Y.B. AND TURKMEN, O.S. (2008). Assessment of effect of salinity on the early growth stage of sunflower using spectral discrimination techniques. Afri. J. Bio. 7(6): 750-756.

UPPAL, S., BEHL, R.K., MIXWAGNER, G. AND ELBASSAM, N. (1996). Callus induction and plant regeneration from embryos in bread wheat (Triticum aestivum L.). Landbauforsehung Volkenrode, 46(4): 157-165.

VAN DER MOEZEL, WATSON, L.E., PEARCE-PINTO, G.V.N. AND BELL, D.T. (1988). The response of six Eucalyptus species and Casuarina obesa to the combined effect of salinity and waterlogging. Aust. J. Plant Physiol. 15:465-474.

145

VAN DER MOEZEL, P.G., PEARCE-PINTO, G.V.N. AND BELL, D.T. (1991). Screening for salt and waterlogging tolerance in Eucalyptus and Melaleuca species. Forest Ecology and Management. 40:27-37.

VAN SINT JAN V, SAKLI-SENHAJI N, BOUHARMONT J. (1990). Comparison different varieties de riz (oryza sativa L.) pour leur aptitude a la culture in vitro.Belg. J. Bot. 123(1/2) : 36-44

VARSHNY, A., KENT, T., SHARMA, V.K. AND KOTHARI, S.L. (1996). High frequency plant regeneration from immature embryo culture of Triticum aestivum and T. durum. Cereal research Communications,24(4):409-416.

VASIL, I.K. (1987). Biotechnology and food security for the 21st century: real world perspective. Nature Biotechnolo.,16:399-400.

VASIL, I.K. (1998). Biotechnology and food security for the 21st century: Real. World Perspective ; Nature Biotechno., 16: 399-400.

VASIL, V., REDWAY, F AND VASIL, I.K. (1990). Regeneration of plants from embryogenic suspension culture protoplasts of wheat ( Triticum aestivum L. ). Biotech., 8(5): 429-434.

WAHEED, R.A., NAQVI, M.H., TAHIR, G.R., NAQVI, S.H.M., KIRDA, C. AND MOUTONNET, P. (1999). Some studies on pre-planned soil moisture irrigation scheduling of field crops. Crop yield response to deficit irrigation. 180-195.

WAINWRIGHT, S.J. (1980). Plants in relation to salinity. Adv. Bot. Res. 8:221-261.

WAISEL, Y. (1972). Biology of halophytes. Acad. Press, London.

WEIMBERG, R. (1967). Effect of sodium chloride on the activity of a soluble malate dehydrogenase from pea seeds. J. Biol. Chem. 242:3000-3006.

WEIMBERG, R. (1970). Enzyme levels in pea seedlings grown on highly salinized media. Plant Physiol. 46:466-470.

WEST, D.W. AND TAYLOR, J.A. (1980). The Response of Phaesolus vulgaris L. to root zone anaerobiosis, waterlogging and high sodium chloride. Ann. Bot. 46:51-60.

WYN JONES, R.G. (1981). Salt tolerance. In: Physiological processes limiting plant productivity.(eds. C.B. JJonton ).271-292. Butterworth, London.

WYN JONES, R.G. (1985). Salt tolerance. Chemistry in Britian, 454-459.

146

WYN JONES, R.G. AND STOREY, R. (1978). Salt stress and comparative physiology in the Gramineae. IV. Comparision of salt stress in Spartina x townsendii and three barley cultivars. Aust. J. Plant Physiol. 5;839-850.

WYN JONES, R.G., BRADY, C.J AND SPEIRS, J. (1979). Ionin and osmotic relations in plant cells. In: Recent advances in the biochemistry of cereals.(eds. D.L. Laidman and R.G. Wyn jones ).63-104. Academic Press, London.

WYN JONES, R.G., GORHAM, J. AND MCDONNELL, E. (1984). Organic and inorganic solute contents as selection criteria for salt tolerance in Triticeae.p. In: Salinity tolerance in plants, Strategies for crop improvement.(eds. R.C. Staples and G.H. Toenniessen ) 189. Jhon Wiley and Sons. New York.

YADAV, N., SANJOGTA, U., SEHRAWAT, A.R. AND SINGH, K.P. (2004). In vitro callus growth, selection of NaCl tolerant cell lines and plant regeneration in wheat. National J. Plant-Improvement. 6(2):130-131.

YAMADA, Y., AND TANG, D.T. (1986). Plant regeneration from protoplast derived callus of rice ( Oryza sativa L. ). Plant Cell Rep., 5: 85-88.

YAMAMOTO, Y., MATSUMOTO, O. AND TANABE, K. (1994). Dry matter production and translocation of CIJ assimilates in Taro (Colocasia euculcnta Scho I) plants grown from standard corns and those propagated by in vitro culture. J. Jap. Soc. Hort. Sci., 63:575-580.

YANG, Y.W., NEWTON, R.J AND MILLER, F.R. (1990). Salinity tolerance in sorghum-I I I. Cell Culture response to sodium chloride in S. bicolor and S. Halepense. Crop Sci., 30:781-785.

YEO, A.R. AND FLOWERS T.J. (1982). Accumulation and localization of sodium ions with in the shoots of rice (Oryza sativa L.) varities differing in salinity tolerance. Physiol.Plant. 56:343-348.

YEO, A.R., KRAMER, D., LAUCHLI, A AND GULLASCH, J. (1977). Ion distribution in salt-stessed mature Zea mays roots in relation to ulrastucture and reyention of sodium. J. Exp. Bot. 28: 17-29.

YEO, A.R., LEE, K.S., IZARD, P., BOURSIER, P.J AND FLOWERS, T.J. (1991). Short- and long-term effects of salinity on leaf growth in rice. (Oryza sativa L.). J. Exp. Bot. 42: 881-889.

ZAIR, I., CHLYAH, A., SABOUNJI, K., TITTAHSEN, M. AND CHLYAH, H. (2003). Salt tolerance improvement in some wheat cultivars after application of in vitroselection pressure. Plant Cell Tiss-Org. Cult. 73 (3): 237-244.

147

ZHANG, L. AND XING, D. (2008). Rapid determination of the damage to photosynthesis caused by salt and osmotic stresses using delayed fluorescence of chloroplasts. Photochem. Photobiol. Sci. 7: 352-360.

ZHENG L., SHANNON, C.M., GRIEVE, C.M. (2002). Evaluation of salt tolerance in rice genotypes by multiple agronomic parameters. Euphytica. 127: 235-245.

ZHENG, Y., WANG, Z. SUN, X. AND JIANG, G. (2008). Higher salinity tolerance cultivars of winter wheat relieved senescence at reproductive stage. Envi. & Expe. Bot., 62(2): 129-138.

ZHU, J.K. (2001). Plant salt tolorence. Trends Plant Sci., 6: 66-72.

ZIDAN, I., AZAIZEH, H. AND NEUMANN, P.M. (1990). Does salinity reduce growth in maiz root epidermal cells by inhibiting their capacity for cell wall acidification. Plant Physiol. 93: 7-11.

ZOPPO, M., DEL, L., GALLESH, A., .OMIIS, H., PARDOSSI AND SAVIOZZI, F. (1999). Effect of salinity on water relations, Sodium accumulation, Chlorophyll content and proteolytic enzymes in a wild wheat. Boil. Plant., 42 (1): 97-104.

APPENDICES Annexure-1: List of Wheat Germplasm used to exploit the somaclonal variation

Sr.No. Genotype Sr.No. Genotype

1 Ufaq-2002 26 Pasban-90 2 Mexi Pak 27 Inqalab-91 3 Chenab-70 28 Shahkar-95 4 Barani-70 29 Punjab-96 5 SA-42 30 MH-97 6 Blue Silver 31 Kohistan-97 7 Lyallpur-73 32 Uqab-2000 8 Parwaz-94 33 Chenab-70 9 PARI-73 34 Iqbal-2000

10 Sandal 35 SH-2000 11 SA-75 36 AS-2000 12 LU-26S 37 Sehar-06 13 WL-711 38 Shafaq-06 14 Punjab-81 39 Fareed-2000 15 Pak-81 40 Bhakhar-2000 16 Pothowar 41 GA-2002 17 Punjab-76 42 V-03094 18 Barani-83 43 V-03079 19 Kohinor-83 44 V-04188 20 Fsd-83 45 V-04189 21 Fsd-85 46 V-03138 22 Punjab-85 47 V-04040 23 Chakwal-86 48 V-04067 24 Shalimar-86 49 V-04112 25 Rohtas-90 50 V-04171

Annexure-2: Screening of Wheat Genotypes for Salt Tolerance

Sr.No. Genotype Sr.No. Genotype 1 Ufaq-2002 11 Pasban-90 2 SA-42 12 Inqalab-91 3 Parwaz-94 13 Punjab-96 4 Lu-26S 14 Uqab-2000 5 Pothohar 15 Chenab-2000 6 Punjab-76 16 Iqbal-2000 7 Barani-83 17 AS-2000 8 Kohinoor-83 18 Bhakkar-2000 9 Fsd-83 19 V-03079

10 Chakwal-86 20 V-04189

Annexure-3: Composition of Murashige-Skoog Media for plant tissue and cell culture Macronutrients mg/l mM NH4NO3 1650 20.6 KNO3 1900 18.8 CaCl2

. 2H2O 440 3.0 MgSO4

. 7H2O 370 1.5 KH2PO4 170 1.2 Micronutrients mg/l uM KI 0.83 5 H3BO3 6.2 100 MnSO4

. 4H2O 22.3 100 ZnSO4

. 7H2O 8.6 30 Na2Mo04

. 2H2O 0.25 1.0 CuSo4

.5H2O 0.025 0.1 CoCL2

. 6H2O 0.025 0.1 Fe-Versenate (EDTA) 43.0 100 Vitamins & Hormones mg/l mg/l Inositol 100 100 Nicotinic acid 0.5 1.0 Pyridoxine. HCl 0.5 1.0 Thiamine. HCl 0.1 10.0 IAA 1-30 -- Kinetin 0.04-10 0.1 Sucrose 3000 pH 5.7 --

Annexure-4: Physical and chemical properties of the soil used.

Determination unit value Mechanical analysis Sand % 66.9 Silt % 16.5 Clay % 16.6 Textural class - sandy clay loam Electrical conductivity of dS/m 2.7 Saturation extract (ECe ) - Saturation percentage 32.7 pHs - 7.7 Soluble anions Co3 me/l absent Hco3 “ 6.03 Cl “ 5.0 So4 (By difference) “ 18.0 Soluble cations Ca + Mg “ 13.6 Na “ 13.7 K “ 0.5 .

Annexure-5: Designated, achieved and after crop harvest levels of ECe (dSm-1) ______________________________________________________________________ Soil Designed Achieved After Crop Harvest ECe ECe ECe ______________________________________________________________________ S1 2.7 2.7 2.82 S2 4.0 4.15 4.76 S3 8.0 8.50 8.60 S4 12.0 13.0 12.64 Anneure-6: Salt (mg/100g soil) to develop designed levels of ECe (dS/m) Salt ECe______________________________ ___________________________________________________________________ 0 4 8 12 NaCl - 28.87 101.38 177.90 _____________________________________________________