In-planta genetic transformation with DREB1A for enhancing cold tolerance in black gram (Vigna mungo L.)
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Date
2014
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Abstract
Crop improvement through transgenic technology is one of the most impeding and viable option
for the advance methods of crop improvement programme. Recent developments in plant
biotechnology made researchers to produce superior cultivars of crops by transferring key genes
into the plant genome by using plant transformation techniques. There are two important prerequisites
to develop transgenic plants traditionally being followed is the susceptibility to
Agrobacterium and the ability of the transformed cells to regenerate to whole plant. However,
there are many recalcitrant plant species which are not amenable to in vitro regeneration, thus
making it very difficult to develop transgenic in those crop species. Therefore, the alternate
strategy of genetic transformation like in-planta genetic transformation has been followed here
in this investigation. One of the promising targets for in-planta transformation has been shoot
apical meristem. The apical meristem targeted strategy involves in-planta inoculation of embryo
axes of germinating seeds with Agrobacterium and allowing them to grow into seedlings ex
vitro. Since differentiating cells are targeted, the plants developed in the T
generation are
chimeric and stable transformants are obtained in the T
generation. In the present study, an
attempt was made to establish efficient in-planta transformation method for black gram cv.
PU40, a recalcitrant pulse crop using cotyledonary nodes of the full grown seedlings as source
tissues for genetic transformation. In this investigation, transgenic black gram plants have been
produced by a tissue-culture independent Agrobacterium tumefaciens – mediated transformation
procedure. Agrobacterium strain GV3107 harboring the binary vector pCAMBIA2300 that
carries the genes for transcription activator with a stress inducible promoter (rd29A:DREB1A)
and neomycin phosphotransferase (npt II) was used for transformation of black gram cv. PU40.
Apical meristem of the differentiated embryo of the germinating seedling is infected with
Agrobacterium. Since the transgene is integrated into the cells of already differentiated tissues,
the T
0
1
plants are chimeric and stable integration could be seen only in the T
transformants were identified by PCR analysis with gene- specific primer, positive control and
negative control. Percentage survival was maximum in variety Prasad and least in cv. PU40,
calculated as cv. Prasad-71.42%, cv. PU40-66.67%. There were 15 plants in T
generation. 7
plant (T
) of cv. PU40 was analysed by PCR amplification with gene specific primer and none
of the 7 plants ( T
1
) showed the integration of transgene, which probably requires more number
of such plants need to be screened. The in planta protocol need to be further optimized by
increasing the no of plants for agrobacterium infection in T
1
0
0
1
generation. T
0
to get sufficient no of seeds for T
analysis.