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: srinathraom@rediffmail.com

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.

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