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Materials and methods
Crossing program and embryo rescue: Karnal bunt susceptible wheat
varieties UP 2338 and WM 533 were crossed with Karnal bunt tolerant
varieties HD2285 and PBW343. The resultant four F1's were
crossed with different maize lines (Prabhat, ViJay, Accessions; 803,
1344, 645 and 1040 used as pollinator). As soon as the primary spikes
of healthy, vigorous plant emerged from the boot, the outer two
florets were emasculated and the inner one was removed. The
pollination was done after 3 to 5 days after emasculation. Maize
pollens were collected by tapping the tassels into a petriplate. The
wheat pistils were pollinated using a fine brush and then bagged.
Immediately after pollination the solution containing 50 mg/l, 2,4-D
+ 100 mg/l GA3 was injected about a centimeter above the
upper most node through leaf sheath as suggested by Pienaar et al.
(1997). Spikelets of these pollinated spikes were again flooded with
the same solution after 24 hours using injection syringe and covered
with another brown paper bag. The treated spikes were harvested
between 13 to 16 days after pollination (Dhaliwal et al. 1995;
Riera-Lizarazu and Mujeeb-Kazi 1990; Suenaga et al. 1997) and were
taken to the laboratory in the abovementioned solution. The green
parthenocarpic caryopses (GPCs) were removed from florets by bending
them backward with a forceps. GPCs were surface sterilized and the
haploid embryos from the disinfected GPCs were excised under laminar
flow hood with scalpel and plated on callus induction medium i.e.
MS1(MS medium + 5mg/1 2,4-D).
In vitro screening at callus level: Well developed calli of
different genotypes of wheat (HD2285 as tolerant and WH157, PBW373,
KH65 and WH147 as susceptible to Karnal bunt) were transferred to
fungal filtrate mediated callus induction media (MS1) with
2 to 50 nil of fungal toxin and without it (control). The callus
cultures were incubated at 25 + or - 1C for 20 days. The final weight
of the plated calli was recorded on 21st day before their transfer
to-culture filtrate free medium. On the basis of decrease in callus
weight in comparison to control the, threshold level (T) was
determined.
For in vitro screening, haploid calli obtained from embryo
rescue, Were transferred to MS1 medium having threshold
level of culture filtrate and the callus growth was recorded. The
well-developed calli were cultured and subcultured on regeneration
media i.e. MS3(MS Medium supplemented with 0.5 mg/l of IAA
and 1.0 mg/l of BAP) to get embryoids that further developed into
plantlets.
Converting haploids into doubled haploids: The haploid calli
differentiated embryoids were transferred to MS1 medium
having 400 mg/l colchicine for three days and again the embryogenic
calli were transferred to regeneration (MS3) media to get doubled
haploid plantlets. As an alternative approach for converting haploid
plantlets obtained from haploid calli into doubled haploids the
plantlets having two to three sprouts were taken. The roots of these
plants were trimmed and washed. Colchicine solution (0.05%) was
taken. in a tube and a piece of filter paper was floated. This
solution was sterilized in autoclave, on which subsequently the plant
was placed for 24 hours at 24C. The plants were rinsed in tap water
for half an hour and then placed in a similar test tube having
distilled water and a filter paper floating on it. Then, the plants
were placed on filter paper and kept for three days at 4C. The shoots
of the treated plants were cut back to 15 cm before transferring to
the pots. Root tip analysis was conducted following acetocarmine
stain procedure to determine the ploidy level.
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