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Results and discussion
In vitro screening and organogenesis of haploid calli:
For in vitro screening of haploid calli, the calli were
transferred on threshold level (T medium i.e. MS1
supplemented with 10 mg/l of culture filtrate) and growth was
recorded (Table 1). The haploid calli of
the cross (UP2338 x HD2285) F1 x maize were transferred to
MS1, and T media and it was observed that callus growth
was -16.1 per cent in the former and 1.4 per cent in later medium.
However, for (WH533 x PBW343) F1 x maize cross the callus
growth was 8.8 per cent in MS1 and 1.0 per cent in T
medium. The per cent callus growth was decreased in both the crosses
m culture filtrate treatment. It was observed that there are chances
of obtaining resistant DH plants from these screened calli. Whereas,
other two crosses namely (UP2338 x PBW343) F1 x maize and
(WH533 x HD2285) F1 x maize failed to show any callus
development and hence plantlet regeneration. The possible reason can
be the timing of embryo rescue, for these two crosses may be somewhat
different or some modifiers may be affecting embryo recovery.
Haploid callus survival at threshold level was Observed 56.5 per cent
for (UP2338 x HD2285) F1 x maize and 57.1 per cent for
(WH533 x PBW343) F1 x maize cross (Table
1). In both the crosses after callus screening the frequency of
embryoids per callus was less than one. Whereas, per cent plant
obtained after converting to DH using colchicine was 30.8 and 16.7
for the crosses (UP2338 x HD2285) F1 x maize and (WH533 x
PBW343) F1 x maize, respectively. However, number of
regenerated DH shoots from plated calli were 4 and 2 for the
respective crosses.
This reduction in obtaining regenerants could he due to colchicine
treatment given in between. Barnabas et al. (1991) and Mentewab and
Sarrafi (1997) reported similar findings. They observed that
colchicine treatment applied in induction medium slightly decreased
the frequency of embryoids and hence per cent regeneration.
Converting haploid to doubled haploids: The efficiency of colchicine
in converting haploids to doubled haploids at embryoid stage and
seedling stage is presented in Table 2. The
efficiency of converting haploid seedlings was less than the
efficiency of converting haploid calli having embryoids into doubled
haploids. When embryoids and seedlings were treated with colchicine,
in cross (UP2338 x HD2285) F1 x maize 75.0 and 57.9 per
cent DH plants were obtained, respectively. Similarly, in second
cross i.e. (WH533 x PBW343) F1 x maize 100 per cent
doubled plants were obtained by treating the embryoids in induction
medium, whereas, it was 53.9 per cent when seedlings were treated
with colchicine solution. Results showed that for converting haploids
into doubled haploids it was observed that embryoids plated on
colchicine mediated medium give higher frequency of DH plants as
compared to the whole seedlings treated with colchicine. These
results are in accordance with that of Mentwab and Sarrafi
(1997).
This investigation mainly embarked on validating in vitro
methodology for development of DHs possibly tolerant to Karnal bunt
using Indian wheat x maize crosses. Wheat x maize cross scheme as
adopted in present investigation would be quite helpful in reducing
the time of breeding programs aimed at developing disease resistant
genotypes (DH) combining favorable gene constellations in homozygous
state. Our studies indicated that culture filtrate of the fungi
Neovossia indica can be used successfully under controlled
environmental tissue culture conditions to pick up tolerant calli
which can be regenerated to whole plant.
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