Agrobacterium tumefaciens-MEDIATED GENETIC TRANSFORMATION USING rd29A::DREB1A GENE TO DEVELOP MOISTURE STRESS TOLERANCE IN BRINJAL (
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Date
2011
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Abstract
Moisture stress is one of the major abiotic constraints affecting crop
productivity in brinjal (Solanum melongena L.). Water use efficiency under
drought conditions is thought to be one of the most promising traits to improve
and stabilize crop yields under intermittent water deficit. In the present
investigation, genetic improvement for this trait of moisture stress tolerance
was carried out through transgenic research in brinjal cv. Utakal Anushree.
An efficient and reproducible in vitro regeneration protocol which is a basic
need for this research was developed. High frequency indirect somatic
embryogenesis was achieved with cotyledonary explants in basal MS salts
supplemented with 2,4-D (2.0 mg/l) for callus induction and BAP (2.0mg/l) for
shoot induction. Multiple shoot regeneration (20.0 shoot buds/explants) in
vitro was also achieved with shoot-tip explants through direct regeneration in
MS medium fortified with BAP (2.0mg/l). Direct somatic embryogenesis which
has better applicability in the improvement of crop since plant regeneration
from callus cultures is often associated with genetic and cytological variations
was pursued for genetic transformation. Kanamycin sensitivity to the normal
tissues of brinjal plants in vitro was optimized at 100mg/l for selecting putative
transformants. A transcription factor DREB1A from Arabidopsis thaliana,
which specifically interacts with the dehydration response element (DRE), a
cis–acting promoter element and induces expression of stress tolerance
genes, driven by the stress inducible promoter from the rd29A gene, was
introduced in to the drought-sensitive brinjal cultivar Utkal Anushree
by Agrobacterium tumefaciens- strain GV3107 with the binary vector
pCAMBIA2300. A transformation frequency of 6.4% was reported after
molecular analysis with PCR amplification using gene-specific primers.The
stress inducible expression of DREB1A in these transgenic plants did not
result in growth retardation or visible phenotypic alterations. Gene expression
studies on the basis of physio-biochemical analysis like membrane stability
index, proline content,relative water content suggest that the transgenic
brinjal plant can be advanced for further generation and toxicology studies for
clearance of biosafety issues for the release of transgenic brinjal.