Wheat Information Service
Number 69: 8 - 12(1989)

Resistant wheat plants against Helminthosporium sativum from embryo derived callus cultures

H.S. Chawla¹ and G. Wenzel²

1 Plant Breeding Dept., G.B. Plant Univ., Pantnagar-263145, U.P. India

2 Institute for Resistance Genetics, D-8059 -Grunbach, F.R.G.

Introduction

Wheeler and Luke (1955) first used phytotoxins in resistance breeding. Correlation of resistance to a parasite and to its toxins is a necessary prerequisite for such a use of phytotoxin. Another important factor is high regeneration ability of the genotype. Large scale screening of cell populations using in vitro procedures successfully produced resistant plants against phytotoxins (reviewed by Wenzel 1985).

In this experiment toxins from Helminthosporium sativum P.K. & B. were used for selection. This fungus produces two toxins namely, helminthosporal (Ludwig 1957) and victoxinine (Pringle 1976) which causes seedling blight, root rot, head blight and leaf spot. This paper describes the selection of immature embryo derived calli of wheat against phytotoxins of H. sativum and the testing of regenerated plants.

Materials and Methods

The callus cultures were established from immature embryos of the wheat varieties 'Atys' and 'Pitic 62' as described by Chawla and Wenzel (1987a).

Isolation of toxin

Helminthosporium sativum P.K. & B. collection strain no. 62606 (supplied by Dr. H. Nirenberg, Berlin) was used in the study. For the preparation of toxic culture filtrate, the seeding production flasks were prepared by the method of Pringle and Scheffer (1963) in modified Fries medium and then 1 ml of this medium was put in 20 ml of modified Fries medium and kept at 25C for 28 days. The culture filtrate containing the phytotoxic compounds was filtered through several layers of cheese cloth and then through Sartorius filter (0.2micro) to discard the mycelium and spores. The culture filtrate was concentrated in vacuo at 40C to 10% of the original volume. An equal volume of cold methanol was added and the solution was stored overnight at 5C. Precipitates were removed by filtration, washed with cold methanol and this methanol was removed in a vaccum freeze drier. The oily substance formed was dissolved in methanol and further dilutions were made with water. Toxicity of purified culture filtrate was tested by root bioassay method (Pringle and Braun 1957).

Testing of lethality of toxins on callus

Small pieces of callus (approx. 30 mg) were placed in a petri dish containing different units of toxin concentration and fresh weight increase of each inoculum was determined after 3 weeks.

Callus selection and regeneration procedures

After 3-4 months of callus initiation, small calli pieces of about 30 mg fresh weight were put in a petri dish with 10 ml of toxic medium. Continuous method of selection was performed in which 4 cycles of selections were done on toxic medium by transferring healthy calli after 3 weeks interval. After selection the resistant calli were grown on maintenance medium and parts of calli were put on regeneration medium containing 0.35 mg/l naphthalene acetic acid and 1 mg/l 6-benzyl-amino-purine. Regenerated plants were transferred to soil and grown under semicontrolled green house conditions.

In vivo testing of plants against pathogen

Leaves of regenerated plants from callus after selection and non-toxin treated control plants were inoculated with 5 microl spore suspension (10⁶/ml), and kept in humid chambers. In susceptible plants, dark brown spots appeared on the leaves within 3-4 days after inoculation. The plants from in vitro cultures were categorised as resistant (R), intermediate (I) and susceptible (S).

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