AGROBACTERIUM MEDIATED GENETIC TRANSFORMATION AND REGENERATION OF PUTATIVE TRANSGENICS IN GROUNDNUT (Arachis hypogaea L.) WITH THE NUCLEOCAPSID PROTEIN GENE OF PEANUT STEM NECROSIS VIRUS
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Date
2006-03
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JAU, JUNAGADH
Abstract
Putative transgenics of Groundnut (Arachis hypogaea L.) were produced by
Agrobacterium mediated genetic transformation. The De-embryonated
cotyledons and immature leaf explants from cultivars TMV-2 and K-134 were
co-cultured with the Agrobacterium tumefaciens strain LBA 4404 harbouring
the coat protein gene of the Peanut Stem Necrosis Virus (PSNV-Cp) in sense
orientation in pBI121 and in antisense orientation in pBinAR vector with the
neomycin-phosphotransferase (npt II) gene as selectable marker. A high
percentage of 67% explants from the de-embryonated cotyledons and 58%
explants from the immature leaves regenerated to form multiple shoot buds
which were subsequently sub-cultured on MS medium containing 250 mg/L
kanamycin for employing high selection pressure for screening transformants.
From the total regenerated shoots 38% of the shoots obtained from deembryonated
cotyledons and 33% from immature leaves were completely
resistant to kanamycin whereas, all the control plants died in selection
medium. The putative transgenic plants with well-developed roots were first
established in plastic cups 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 and immature
leaves was 85% and 60% respectively. In the GUS assay of kanamycin
resistant putative transformant obtained from de-embryonated cotyledons cocultured
with sense (S-7) construct about 71% and in case of immature leaves
above 72% tested positive for GUS activity.
The integration of the transgene was assessed by the PCR
amplification of the genomic DNA from the transformants with PSNV-Cp gene
specific primers. In the PCR analysis, out of the total number of established
kanamycin resistant plants the DNA was isolated from 93 plants selected at
random. Out of 93 putative transgenic plants, 72 plants (77.42%) were found
positive for the PSNV-Cp gene producing the expected size of band. In the
PCR analysis of 30 randomly selected GUS positive plants, 20 putative
transformants (66%) tested positive for both GUS and PCR. Around 200
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
and stored for progression of the generation and further analysis.
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Botany