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Results
Lethality of toxin was tested on five levels of toxin conc.
and increase in fresh weight was determined after 3 weeks of
growth (Fig. 1a). At the highest
toxin conc. in B4T medium, almost all the calli died. At the
next lower level of toxin conc. in B3T medium, 10-30% of the
calli survived and a growth rate of around 30% was observed.
At the lower levels in B1T and B2T no significant effect on
growth of calli compared to control B0T level was observed.
A control experiment with uninoculated fungal growth medium,
processed in the same way as the toxin preparation was
tested with different levels of conc. in callus growing
medium. There was no effect on growth of callus (Fig.
1b).
Selection of resistant calli was done by putting small
pieces of calli (10/petri dish) containing toxic medium and
transferring the insensitive calli after 3 weeks of growth
to the next cycle (Fig. 1c & d).
After the 1st cycle on toxic medium the two genotypes showed
24% survival of calli (Table 1).
In the 2nd cycle 6-7% of the calli still died but there
after only 2-4 % of the calli died indicating insensitivity
to toxic medium was exhibited after 2nd cycle of
selection.
After 4 selection cycles the resistant calli were grown on
maintenance medium without hormones and parts of good
looking calli were placed on regeneration medium. The
frequency of regeneration varied from 25 to 48%. As compared
to this maximum of 16% green shoot formation frequency was
observed. The observation on in vivo reaction of
leaves of regenerated plants showed 7 plants were
insensitive to the pathogen and 2 showed intermediate
reaction (Fig. 1e).
Discussion
In addition to the efforts made in resistance breeding
through conventional approaches, attempts have been made in
the last few years to produce novel resistant plants through
cell culture techniques. In the present work helminthosporal
and victoxinine toxins produced by the fungus in the liquid
culture filtrate were used for selection of resistant calli.
The earlier experiments on selections conducted by
Gengenbach and Green (1975) and Hartman et al (1984)
increased the conc. of toxin in the media slowly and others
have used low to moderate conc. of toxin (Behnke 1979;
Thanutong et al, 1983). But here a high toxin conc. from the
begining was employed to kill in the first cycle of
selection itself 70-80% of the calli.
The previous report on these genotypes has revealed that 14
to 25% regeneration potentiality is retained by old callus
cultures (Chawla and Wenzel 1987a). It revealed 16% green
shoot formation after selection in one of the genotypes.
Also, the plants regenerated from calli resistant to the
toxins expressed the resistance character. However, the
character was not expressed as a qualitative trait in all
cases. The regenerated plants also showed intermediate
reaction i.e. few symptoms and at a later stage. This
variability of the regenerated plants in the trait selected
for has been found in rapeseed for Phoma lingam
resistance (Sacristan 1982) and in barley for fusaric acid
toxin (Chawla and Wenzel 1987b). The variation for
resistance obtained in the plants indicates that probably
the nuclear genome has an influence on the resistance rather
than mitochondrial DNA as reported for Helminthosporium
maydis resistance in maize (Brettell et al 1980).
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