GENETIC TRANSFORMATION FOR POD BORER RESISTANCE USING crylA (b) IN PIGEONPEA [Cajanus cajan (L) Milisp.] cv. ICPL-8863 (MARUTI)
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Date
2004-01-23
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UNIVERSITY OF AGRICULTURAL SCIENCES BANGALORE
Abstract
A study was undertaken to standardize in vitro plant regeneration
and Agrobacterium mediated transformation procedure for pigeonpea
(Cajunus cajan) cv. ICPL-8863 (Maruti). For regeneration direct
organogenesis was attempted using different explants viz., shoottip (ST),
cot}dedonaiy node (CN), half colydedon with cotyledonary node {V2 CNC)
and CNC. These were cultured on various levels of benzyl amino purine
(BAP) (1, 2, 3, 4 mg 1 -) and thidiazuron (TDZ) (0.01, 0.05, 0.1, 0.5 mg h^).
CNC found to produce average of 1.69 shoots/explant and was better
among all explants used. Among different levels of BAP and TDZ tried,
BAP 2 mg 1-1 was found to be better for multiple shoot and shootbud
induction. Shootbuds were cultured on MS with reduced levels of
cytokinins and TDZ 0.05 mg pi gave better elongation compared to otherlevels.
elongated shoots were rooted on MS with IBA (0.1-0.5 mg pi).
Among all the levels tried 0.2 mg pi IBA gave good healthy roots.
For transformation Agrobacterium strains EHA 105 harboring
pBinBtl plasmid [cryl A(b)] and GV2260 harboring pCAMBIA1301
plasmid {gus) with nptll as selectable marker, which confers kanamycin
resistance were used. Initially kanamycin sensitivity of control explants
was tested at different growth stages. Inhibitory levels at different stages
were used for selection of transformants. Precultivation of explants on MS
with 2 mg pi BAP for two days prior to cocultivation resulted in increased
survival. Explnats w^ere cocultured for two daj'S in dark and transferred to
selection medium (with kanamycin and cefotaxime). Approximately 1.4
per cent shoots obtained were cry positive.
In in planta approach plants were treated with Agrobacterium
inoculum at different growth stages. Germinating seeds were injected with
GV2260 strain and shoots were histochemically assayed and 0.9% of
shoots were gus positive. In seedling dip and flower injection methods
crylA (b) gene was transferred and confirmed through PGR analysis
(9/54, 11/26 plants were cry positive respectively). Thus efficient
regeneration and transformation protocol has been standardized for
pigeonpea cv. ICPL-8863 (Maruti).
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