Studies on regeneration and transformation for improving salinity tolerance in chickpea (Cicer arietinum L.)
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Date
2007
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Publisher
CCSHAU
Abstract
Experiments were conducted to develop efficient regeneration and
transformation protocol in HC-5 and HK-2 cultivars of chickpea. Three
explants (shoot tips, cotyledonary nodes and embryo axis, attached with
one cotyledon) in case of HC-5 cultivar and two explants (shoot tips and
embryo axis attached with one cotyledon) in case of HK-2 cultivar were
used for in vitro regeneration studies. Maximum shoot regeneration
frequency was observed from shoot tips, cotyledonary nodes, embryo axis
attached with single mature cotyledon when placed on S2 (0.1 mg/l), S1
(BAP 5.0 mg/l) and S2 (0.1 mg/l) media respectively, in cutlivar HC-5
(98.8% in all three cases) as compared to the cultivar HK-2 which
showed maximum response of 95% and 96.6% respectively in shoot tips
and embryo axis when placed on S10 medium (TDZ 1.0 mg/l). Embryo
axis explant proved to be the best for in vitro shoot regeneration both in
HC-5 and HK-2 cultivars of chickpea. The explants from HC-5 cultivar
gave better shoot regeneration frequency as compared to HK-2 cultivar.
The regenerated shoots both the cultivars gave best root regeneration in
R2 medium (IBA 1.0 mg/l and NAA 1.0 mg/l).
When transplanted to soil, in vitro regenerated plantlets of HC-5
cultivar survived for a maximum of 18 days and gave a survival
frequency of 13.3% whereas plantlets of HC-5 cultivar survived for a
maximum of 8 days and gave a survival rate of 10%. Inoculation of
transplanted in vitro regenerated plantlets of HC-5 with PGPR cultivars
greatly increased the number of survival days.
Agrobacterium strain EHA 105 (pBI 121) harboring salinity
tolerance rice Hal-2 like gene driven by promoter was introduced in the
two chickpea cultivars.
In transformation experiments, embryo axis attached with the
single mature cotyledon was used as explant and cocultivated with EHA
105 strain (pBI 121). Selection of transformed shots was carried out on
the medium containing Kanamycin (100 mg/l), afotaxime (250 mg/l) and
NaCl (200 mM). In HC-5, out of 190 explants, 9 putative transgenic
shoots were recovered giving transformation frequency of 2.3%. In
contrast to this, in HK-2, 7 kanamycin resistant transgenic shoots were
obtained from 190 explants, giving transformation frequency of 3.3%.
Genotypically, chickpea cultivar HC-5 responded better than HK-2 for
both multiple shoot formation and Agrobacterium mediated
transformation studies.
Description
Keywords
Chickpea, Regeneration, Transformation, Salinity toleranceand Rice hal 2 like gene