Upload
s-ramgopal-rao
View
3
Download
1
Embed Size (px)
DESCRIPTION
sunflower callus
Citation preview
Indian Journal of Biotechnology
Vol 9, April 2010, pp 187-191
Effect of Alternaria helianthi culture filtrate on callus and regeneration of plantlets
from tolerant callus in sunflower (Helianthus annuus L.)
Srinath Rao1* and Ramgoapl S
2
1Department of Botany, Gulbarga University, Gulbarga 585 106, India 2Srinidhi Institute of Science and Technology, Ghatkesar 501 301, India
Received 9 January 2009; revised 19 June 2009; accepted 28 August 2009
A protocol for selecting callus line tolerant to culture filtrate of Alternaria helianthi (ACF), which causes severe blight
disease, was developed in sunflower using 40% ACF as selection pressure. In vitro plantlets were obtained from the selected
callus on Murashige and Skoog’s (MS) medium fortified with 2.0 mg/L N-6-Benzyl amino purine (BAP) and 0.5 mg/L
α-Naphthalene acetic acid (NAA). The plantlets obtained were rooted on MS medium fortified with 1.0 mg/L NAA or IBA.
NAA proved better for rooting than IBA. In the preliminary experiments, in vitro developed plants from selected callus
showed fewer symptoms of disease when spore suspensions of A. helianthi, was sprayed on the leaves.
Keywords: Alternaria helianthi, Helianthus annuus, culture filtrate, disease tolerance, in vitro regeneration
Introduction
Sunflower (Helianthus annuus L.) is the third
important major oil seed crop in the world after
soybean and groundnut. In India, the area under
cultivation of sunflower is about 15 million hectares
accounting for about 200 million tonnes of seeds
annually. The cultivation of sunflower in India is
mainly confined to southern states of Karnataka,
Tamil Nadu and Andhra Pradesh, with Karnataka
accounting for nearly 60% of total area and 40% of
total production in the country1.
Susceptibility to fungal diseases is a major limiting
factor for the increased sunflower production in India.
Of the four major diseases (Alternaria blight, rust,
downy mildew and root/colour rot) of sunflower,
Alternaria blight caused by Alternaria helianthi is a
major threat, especially in South India.
Breeding for resistance is one of the major areas for
research. However, the germplasm screening to date
have indicated lack of genetic resistance to the disease
in cultivated sunflower, even all the other wild species
are susceptible2. The advances made in the area of
plant cell and tissue culture and the eventual
regeneration of entire plants from them have
established this technology as a powerful tool for use
in crop improvement programmes3-5
. Toxin produced
by fungi or crude culture filtrate are used as selective
agents and plants have been regenerated via tissue
culture technique6-13
. The present study is aimed at
selecting sunflower callus tolerant to culture filtrate of
A. helianthi and regenerating plantlets resistant to
culture filtrate.
Materials and Methods Seeds of sunflower var. Modern were obtained
from Regional Agricultural Research Station, Raichur
(UAS, Dharwad, India). Seeds were surface sterilized
with HgCl2 for 3 min and germinated on filter paper
bridge in culture tubes containing sterilized water.
Cotyledons were used as the source of explants from
1-wk-old in vitro raised seedlings.
The explants were inoculated on MS medium14
supplemented with various concentrations of 2,4-D,
NAA alone or in combination with Kn. For shoot
induction the media was supplemented with Kn and
BAP at various concentrations.
Preparation of Culture Filtrate
Pure cultures of Alternaria helianthi were obtained
by single spore isolation method. 1.0 mL of such
spore suspension was spread uniformly on potato
dextrose agar (PDA) and incubated at 27 ± 2°C and
grown for 15 d. The spores were isolated from
infected leaves. The fungus was subcultured on PDA
____________
*Author for correspondence:
Tel: 91-8472-263291
E-mail: [email protected]
INDIAN J BIOTECHNOL, APRIL 2010
188
broth and host seed extract and allowed to grow at
27 ± 2°C for 24 d. After this, mycelial mat was
separated from the culture broth by sequential
filtrations with three layers of cheese cloth and then
using Buchner funnel under vacuum. Finally it was
filtered by using Whatman filter paper (No. 42). For
sterilization, the culture filtrate was filter sterilized
using millipore filters (0.22 µ) under aseptic
conditions1. This filter sterilized culture filtrate was
used for further studies.
Alternaria culture filtrate (ACF) was concentrated
to ¼ of its volume at 42°C on a water bath and finally
sterilized with 0.22 µ millipore filter; it was then
mixed with MS callus induction medium to achieve
concentrations of 5, 10, 20, 30 and 40% (v/v). The pH
of the medium was adjusted to 5.7. In each case, an
ACF free medium was also prepared which, served as
control.
ACF resistant callus line was isolated following the
direct selection procedure of biochemical variants15
.
For selection studies, two types of selection strategies
were employed: (1) callus exposed to stepwise
increase of ACF concentration in the medium or (2)
sudden exposure directly to 40% ACF supplemented
media containing MS+2.0 mg/L 2,4-D+0.5 mg/L Kn
and subcultured after 30 d on the same medium with
or without ACF. The calli pieces were gently
macerated for uniform exposure of the cells to ACF.
The culture tubes were incubated at 16/8 h light and
dark period provided by cool fluorescent tubes.
Results and Discussion
From the data presented in the Table 1 it is clear
that the growth of callus, measured in terms of fresh
and dry weight, decreased gradually with an increase
in the concentrations of ACF (Fig. 1). Cell survival
was almost non-existent at 40% ACF and entire callus
turned brown and cell death occurred within few days.
However, few pockets of surviving cells were
observed in about 5% of culture (Fig. 1e & Fig. 2a).
Fresh and dry weights of the callus were used as a
convenient indicator for studying the effect of
abiotic16-22
and biotic stress11,23,24-26
.
There are several reports that culture filtrates of
pathogens affect growth of callus, viz., bean callus
showed differential growth when cultured on toxin
filtrate of halo blight bacterium, Pseudomonas
phaseolicola23
. Callus from susceptible plants of
maize showed sensitivity to the toxin produced by
H. maydis24
. Inhibitory effect of Pseudomonas and
Alternaria toxin on the growth of protoplast derived
callus in tobacco has been demonstrated25
. Similarly,
culture filtrate of P. citrophthora in citrus25
and
Fusarium11,12,26
, inhibited the growth of the callus in
Table 1—Effect of ACF on growth (fresh and dry weight) of cotyledon derived sunflower callus reared on
MS medium + 2.4-D (2.0 mg/L) + Kn (0.5 mg/L)
Culture period (d) Concentration of
ACF in the medium
(%)
10
20
30
FW DW FW DW FW DW
Control (0) 852±1.49a 92±0.63a 1461±1.31a 148±0.68a 2137±1.12a 218±0.66a
5 721±1.17b
(-15.37)
67±0.63b
(-27.17)
1046±1.16b
(-28.40)
102±1.0b
(-31.08)
1272±1.15b
(-40.47)
122±1.20b
(-44.0)
10 612±1.28c
(-28.16)
60±0.66c
(-34.78)
882±1.11c
(-39.63)
83±1.28c
(-43.91)
1047±1.42c
(-51.0)
101±0.96c
(-53.66)
20 542±1.59d
(-36.38)
52±0.76d
(-43.47)
676±1.16d
(-53.73)
68±1.09d
(-54.05)
896±1.60d
(-58.07)
87±0.91d
(-60.00)
30 368±1.26
(-56.80)
37±0.63e
(-59.78)
462±1.31e
(-68.37)
45±0.81e
(-69.59)
564±1.3e
(-73.60)
55±0.98e
(-74.77)
40 238±1.38f
(-72.06)
20±0.80f
(-78.26)
22.8±1.54f
(-84.39)
21±0.44f
(-85.81)
219±0.55f
(-89.75)
18±0.55f
(-91.74)
ACF – Alternaria culture filtrate.
Mean ± Standard error.
Initial inoculums – 150 (± 10) mg.
FW=Fresh weight and DW = Dry weight expressed in terms of mg.
Values in parenthesis are percent reduction to respective control callus.
Data represents average of 3 replicates each replicate consisting of 10 culture tubes.
Means followed by same superscript in a column is not significantly different at = 0.05 level.
RAO & RAMGOAPL: REGENERATION OF SUNFLOWER FROM ALTERNARIA HELIANTHI TOLERANT CALLUS
189
tobacco28
, bengalgram11
and redgram12
. Inhibition of
callus growth by culture filtrate of Phomopsis has
been reported in sunflower26
.
Cell survival was almost non-existent at 40% ACF,
except for few pockets of cells. Therefore, this
concentration of 40% was used for selection. Pockets
of calli developed at this concentration (Fig. 1d) were
subcultured twice in the same medium for 60 d at an
interval of 30 d each, and once on AFC free medium,
followed by again subculturing on ACF supplemented
medium after two months, a callus line exhibiting
continuous growth without much discoloration was
visually identified as ACF tolerant line and
regeneration of plantlets was attempted. Use of
surviving callus line from culture filtrates of
pathogens for regeneration of plants has been reported
earlier in bengalgram11
and redgram12
.
Approximately 150 mg of selected callus was
transferred to regeneration medium containing MS
salts supplemented with 2.0 mg/L BAP+0.5 mg/L
NAA. Within few days green shoot buds appeared
on callus which gave rise to 3-4 multiple shoots
(Fig. 2b). Regeneration from callus via stepwise
increase in ACF was very less probably because of
the long culture period (270 d) before transferring to
regeneration medium. However, regeneration from
selected callus via sudden exposure to high
concentration (40%) ACF (growth period 120 d) was
better. It is reported that selection of cell lines at very
high toxin concentration with three or more cycles of
selection and transfer on non-toxin media give better
results29
.
The regenerated plantlets (4-5 cm long) were
transferred to rooting medium consisting of MS salts
supplemented with 1.0 mg/L NAA or IBA. NAA
proved better than IBA for induction of roots
(Fig. 2c). There are several reports where plants have
been regenerated from tolerant callus using culture
filtrate as selective agent and the plantlets thus
obtained showed resistance to respective pathogens,
whose culture filtrate was used as a selective agent.
Plants have been regenerated from callus, which show
resistance to culture filtrate of Phytophthora infestans
in potato7,8
, to Phoma lingum in B. napus30
, to
V. dahliae in egg plant31
, to Fusarium species in
Medicago sativa32
, strawberry33
, bengalgram11
, pea34
and redgram12
; and Alternaria spp. in geranium35
.
From the literature surveyed and from the present
studies it can be concluded that in a wide variety of
plants and particularly in sunflower, callus line
showing resistance to culture filtrate of pathogens can
be selected and plants can be regenerated from the
resistant callus, which in preliminary studies showed
resistance to Alternaria blight. Selection of tolerant
callus via direct selection from high ACF
concentration was a better technique than selection of
callus by exposing to stepwise increase in
concentration of ACF.
Fig. 1—a, Control callus; b, Callus grown on 10% ACF; c, Callus
grown on 20% ACF; d, Callus grown on 30% ACF; & e, Callus
grown on 40% ACF
Note: progressive browning and inhibition of callus growth in
c, d and e.
Fig. 2—a, Alternaria culture filtrate (ACF) resistant (Selected
AR-40) callus of sunflower reared on MS+2,4-D (2.0 mg/L)+Kn
(0.5 mg/L)+40% ACF; b, 40% ACF resistant shoot cultures of
sunflower reared on MS+BAP (2.0 mg/L) + NAA (0.5 mg/L); c,
Induction of roots on MS+NAA (1.0 mg/L); & d, Acclimatized
plant
INDIAN J BIOTECHNOL, APRIL 2010
190
Acknowledgement The authors are thankful to Head, Department of
Botany for providing facilities.
References 1 Ramgopal, S, Selection of disease resistant cell lines in
sunflower (Helianthus annuus L.) through cell and tissue
culture. Ph D Thesis, Gulbarga University, Gulbarga. 2002.
2 Seetharam A & Virupakshappa K, Present status and future
directions of sunflower breeding in India, in Sustainability in
oilseeds, edited by M V R Prasad (Indian Society of Oilseeds
Research, Hyderabad) 1994, 54-61.
3 Scowcroft W R, Somatic cell genetics and plant
improvement, Adv Agron, 29 (1977) 39-82.
4 Murashige T, The impact of plant tissue culture on
agriculture, in Frontiers of plant tissue culture, edited by T A
Thorpe (University of Calgary Printing Services, Calgary,
Alberta) 1978, 15-26.
5 Vasil I K, Scowcroft W R & Frey K J (eds), Plant
improvement and somatic cell genetics (Academic Press Inc.,
New York) 1982.
6 Carlson P S, Methionine sulfoximine resistant mutants of
tobacco, Science, 1980 (1973) 1366-1368.
7 Behnke M, Selection of potato callus for resistance to culture
filtrates of Phytophthora infestans and regeneration of
resistant plants, Theor Appl Genet, 55 (1979) 69-71.
8 Behnke M, General resistance to late blight on Solanum
tuberosum plants regenerated from callus resistant to culture
filtrates of Phytophthora infestans, Theor Appl Genet, 56
(1980) 151-152.
9 Ling D H, Vidhyasekharan P, Borromeo E S, Zapata F J &
Mew T W, In vitro screening of rice germplasm for
resistance to brown spot disease using phytotoxin, Theor
Appl Genet, 71 (1985) 133-135.
10 Venkatachalam P, Rao K S, Kishore P B K & Jayabalan N,
Regeneration of late leaf spot resistant groundnut plants from
Carcosporidium personatum culture filtrate treated callus,
Curr Sci, 74 (1998) 61-65.
11 Rao S & Padmaja M, Selection of chickpea cell lines
resistant to culture filtrate of Fusarium oxysporum sp. ciceri,
Phytomorphology, 50 (2000) 41-46.
12 Rao S, Basavaraj K & Kaviraj C P, In vitro selection of
pigeon pea cell lines and regeneration of plantlets from
tolerant callus to culture filtrate of Fusarium odum Buttler,
Plant Cell Biotechnol Mol Biol, 7 (2006) 69-72.
13 Crino P, Culture filtrate as selective agent of resistance to
phytopathogenic fungi, in Toxins in plant disease
development and evolving biotechnology, edited by K
Upadhyaya & K G Mukharjee (Oxford and IBH Publishers,
New Delhi) 1997, 183-208.
14 Murashige T & Skoog F, A revised medium for rapid growth
and bioassay with tobacco tissue culture, Physiol Plant, 48
(1962) 473-497.
15 Maliga P, Resistant mutants and their use in genetic
manipulation, in Frontiers of plant tissue culture, edited by T
A Thorpe (University of Calgary Printing Services, Calgary,
Alberta) 1978, 381-392.
16 Dix P J & Street H E, NaCl resistance cultured cell line from
Nicotiana sylvestis and Capsicum annum, Plant Sci Lett, 5
(1975) 231-237.
17 Croughan T P, Stavarek S J & Rains D W, Selection of NaCl
tolerant line of cultured alfalfa cells, Crop Sci, 18 (1978)
959-963.
18 Rains D W, Croughan T P & Stavarek S J, Selection of salt
tolerant plants using tissue culture, in Genetic engineering of
osmoregulation, edited by D W Rains, R D Valentine & A
Hollaender (Plenum Press, New York) 1980, 279-292.
19 Kavi Kishor P B, Effect of salt stress on callus cultures of
Oryza sativa L., J Exp Bot, 39 (199) (1988) 235-240.
20 Rao S & Krupanidhi K, Regeneration of plantlets from
sodium chloride tolerant cell lines in pigeon pea [Cajanus
cajan (L.) Millsp.], in Role of biotechnology in pulse crops,
edited by Jafar Nizam, A Irfan Khan & S A Farook, 1996,
149-153.
21 Sangwan V, Babber S & Varghese T M, Effect of chloride
salinity on relative growth and solute content of Cicer
arietinum L. calli, Indian J Plant Physiol, 1 (1997) 26-28.
22 Rao S & Patil P, Effect of sodium chloride on callus culture
of Cicer arietinum L, Bull Pure Appl Sci, 17 B (1998) 81-87.
23 Bajaj Y P S & Saettler A W, Effect of culture filtrates of
Pseudomonas phaseolicola on growth of excised roots and
callus tissue culture of bean, Phytopathology, 58 (1968)
1041-1042.
24 Gengenbach B G, Green G E & Denovan C M, Inheritance
of selected pathotoxin resistance in maize plants regenerated
from cell cultures, Proc Natl Acad Sci USA, 74 (1977) 5113-
5117.
25 Vardi A, Epstein E & Breiman A, Is the Phytophthora
citrophthora culture filtrate a reliable tool for the in vitro
selection of resistant citrus variants? Theor Appl Genet, 72
(1986) 569-574.
26 Dozet B & Vasic D, In vitro techniques for selection of
sunflower for resistance to Diaporthe I Phomopsis helianthi
Munt , Helia, 18 (22) (1995) 37-44.
27 Thanutong P, Furusawa I & Yamamoto M, Resistant tobacco
plants from protoplast-derived callus selected for their
resistance to Pseudomonas and Alternaria toxins, Theor Appl
Genet, 66 (1983) 209-215.
28 Bhatt D P, Selvapandian A, Mehta A R & Bhatt P N,
Inhibitory effect of Fusarium oxysporum on suspension
cultures leaf discs and on intact tobacco plants, Proc 6th Int
Congr Plant Tissue Culture, University of Minnesota,
Minnesota, 3-8 August 1986, 306.
29 Chawla H S & Wenzel G, In vitro selection for fusaric acid
resistant barley plants, Plant Breed, 99 (1981 a) 159-163.
30 Sacristan M D, Resistance responses to Phoma lingum of
plants regenerated from selected cell and embryogenic
cultures of haploid Brassica napus, Theor Appl Genet, 61
(1982) 193-200.
31 Allechio R, Antonidi C & Palenzona D, Karyotypic
variability in plants of Solanum melongena regenerated from
callus grown in presence of culture filtrate of Verticillium
dahliae, Theor Appl Genet, 67 (1984) 267-271.
32 Hartman C L, McCoy T J & Knons T R, Selection of alfalfa
(Medicago sativa) cell lines and regeneration of plants
resistant to the toxin(s) produced by Fusarium oxysporum sp.
medicaginis, Plant Sci Lett, 34 (1984) 183-194.
RAO & RAMGOAPL: REGENERATION OF SUNFLOWER FROM ALTERNARIA HELIANTHI TOLERANT CALLUS
191
33 Hideyoshi-Toyada, Horikoshi K, Yamno Y & Ouchi S,
Selection of Fusarium wilt disease resistance from
regenerants derived from leaf callus of strawberry, Plant Cell
Rep, 10 (1991) 167-170.
34 Kumar A, Srivastava D K, Gupta V K & Kohli V K, Cellular
selection of characterization of pea lines resistant to culture
filtrate of Fusarium oxysporum, in Trends in plant tissue
culture and biotechnology, edited by L K Pareek & P L
Swarnkar (Arobios Publishers, India) 2001, 162-168.
35 Shukla R S, Chauhan S S, Saxena G, Banerjee S & Kumar,
Resistant to leaf blight caused by Alternaria alternata in
geranium (Pelargonium graveolens var. Algerian) calli
clones, Int Conf Integrated Plant Disease Management for
Sustainable Agriculture, IARI, New Delhi, 1997, 447.