GENETIC TRANSFORMATION OF CHICKPEA (Cicer arietinum L.)USING A Cry2Aa GENE DRIVEN BY CaMV 35S PROMOTER
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Date
2017-01
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AAU, Jorhat
Abstract
Chickpea (Cicer arietinum L.) is a widely cultivated grain legume for nutritious protein rich seeds. In spite of its nutritional importance, its productivity has been constrained largely by a devastating insect pest Helicoverpa armigera. Conventional breeding is limited to impart resistance against Helicoverpa infestation due to lack of gene within the genepool. Genetic transformation has significantly contributed to develop insect resistant lines for better sustainability of this important food crop. Transgenic chickpea lines generated using a cry2Aa gene regulated by Arabidopsis Rubisco small sub unit (AraSSU) gene promoter conferred complete protection to Helicoverpa but with a phenotypic drag. Therefore, developing more transgenic lines with optimum levels of Cry2Aa protein and complete protection against pod borer is necessary to complement or replace the existing lines. In the present investigation, a cry2Aa gene driven by CaMV35S promoter or targeting the Cry2Aa protein to the chloroplasts using transit peptide linked to the gene was selected for optimal levels of expression. The binary vectors having chimeric cry2Aa genes were used for transformation of a model plant, tobacco to test the suitability of these constructs for their efficiency. In all, 51 transgenic tobacco lines were generated using these cry2Aa gene constructs and found that the level of expression ranged from 0.100 to 205 ng/mg of fresh weight (FW). The Cry2Aa tobacco lines with AraSSU and AraSSU-CTP promoter accumulated highest (>150 ng/mg FW tissue) levels of protein in the leaves. However, a tobacco line expressing about 202 ng/mg FW of Cry2Aa protein showed reduced plant growth. Using above constructs, 34 transgenic chickpea lines were generated. Molecular analyses revealed that the primary transgenic (T0) lines accumulated low levels (0.01 – 15.0 ng/mg FW) of Cry2Aa protein. The T1 progeny of these lines showed the transmission and expression of transgene into the next generation. The spatiotemporal expression of cry2Aa gene in these lines was compared with an existing high expressing line (BS6H) by immunohistochemical assay. The study revealed the expression of Cry2Aa protein in the non-green vascular tissues (vascular tissues) of the line BS6H which showed stunted phenotype due to accumulation of high level of Cry2Aa protein. However, no variation in the level of expression was observed during various plant developmental stages. We also studied expression of stress related
genes in two different light regimes. The expression of stress related gene was not observed in these light regimes except for the gene ABC transporter. A down-regulation of ABC transporter was observed in the plant grown under bright light. During the present investigation, tobacco transformation revealed that, these binary vectors were suitable for genetic transformation. However, efforts to generate more chickpea lines using these chimeric cry2Aa genes are to be made to identify lines with optimum level of expression with complete protection to target pest.
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