AGROBACTERIUM MWDIKT^X) GENETIC TRANSEORMATION AND REGENERATION OF PUTATIVE TRANSGENICS IN GROUNDNUT {Arachis hypogaea L.) WITH THE MANNITOL 1-PHOSPHATE DEHYDROGENASE GENE FOR STRESS TOLERANCE
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Date
2009-04
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JAU,JUNAGADH
Abstract
Groundnut is a major oil seed crop of the world. The biotic and
abiotic stress happens to be the major constraints in groundnut
production. The plant mtlD genes, can be engineered into the desired
crop plants by genetic transformation and regeneration there of resulting
in the development of transgenic plants. Putative transgenics of
groundnut {Arachis hypogaea L.) with mtlD gene were produced efficiently
by Agrobacterium mediated genetic trcmsformation. The De-embiyonated
cotyledons from cultivars GG-20 were co-cultured with the Agrobacterium
strain LBA4404 harbouring a binary vector pCAMVmtlD containing mtlD
(mannitol 1-phosphate dehydrogenase gene) and nptll (neomycin
phosphotransferase genes) as selectable marker. A high percentage of
(77.10%) de-embryonated cotyledon explants regenerated to form
multiple shoot buds which were subsequently sub-cultured on MS
medium containing 30 mg/L kanamycin for employing high selection
pressure for screening transformants. From the total regenerated shoots
34% were completely resistant to kanamycin whereas, all the control
plants bleached in selection medium. The putative transgenic plants with
well-developed roots were first established in Hoagland medium and
finally transferred to earthen pots for hardening. Hundred percent
rooting was observed in rooting medium and the hardening of the plants
from de-embryonated cotyledons.
The integration of the transgene was assessed by the PGR
amplification of the genomic DNA from the transformants with nptll gene
specific primers. In the PGR analysis, out of the total number of
established kanamycin resistant plants the DNA was isolated from 43
plants selected. In the PGR using specific primers out of 43 putative
transgenic plants, 37 plants (86.04%) were found positive for the nptll
gene and mtlD gene producing the expected size of band. Fifteen
independently transformed putative transgenic plants were maintained
and forwarded for further analysis and confirmation. Fertile plants were
obtained which flowered normally and in all the positive plants
maintained in pots normal pod formation was observed. The pods were
collected, dried properly cmd stored for progression of the generation and
further analysis.
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AGRICULTURAL BOTANY