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 S2
1
Department of Botany, Gulbarga University, Gulbarga 585 106, India
2
Srinidhi 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 in crop improvement programmes3-5. Toxin produced

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
____________
*Author for correspondence:
Tel: 91-8472-263291
E-mail: srinathraom@rediffmail.com
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
188 INDIAN J BIOTECHNOL, APRIL 2010

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)

Concentration of Culture period (d)

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

721±1.17b 67±0.63b 1046±1.16b 102±1.0b 1272±1.15b 122±1.20b
5
(-15.37) (-27.17) (-28.40) (-31.08) (-40.47) (-44.0)
612±1.28c 60±0.66c 882±1.11c 83±1.28c 1047±1.42c 101±0.96c
10
(-28.16) (-34.78) (-39.63) (-43.91) (-51.0) (-53.66)
542±1.59d 52±0.76d 676±1.16d 68±1.09d 896±1.60d 87±0.91d
20
(-36.38) (-43.47) (-53.73) (-54.05) (-58.07) (-60.00)
368±1.26 37±0.63e 462±1.31e 45±0.81e 564±1.3e 55±0.98e
30
(-56.80) (-59.78) (-68.37) (-69.59) (-73.60) (-74.77)
238±1.38f 20±0.80f 22.8±1.54f 21±0.44f 219±0.55f 18±0.55f
40
(-72.06) (-78.26) (-84.39) (-85.81) (-89.75) (-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
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
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
189
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.
190 INDIAN J BIOTECHNOL, APRIL 2010
Acknowledgement
The authors are thankful to Head, Department of
Botany for providing facilities.
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