GENETIC TRANSFORMATION OF SORGHUM (Sorghum bicolor (L.) Moench) FOR ABIOTIC STRESS TOLERANCE USING ANNEXIN GENE
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Date
2004
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Professor Jayashankar Telangana State Agricultural University
Abstract
Sorghum is the major grain crop in semi arid tropics. Until recently the genetic improvement of sorghum for abiotic stresses has been carried out through traditional plant breeding methods. Non-availability of efficient transformation techniques is one of the 8 1.1imitations for the application of biotechnology for genetic improvement of this crop. - Annexins are known to be multigene family members with diversified functions. Their role in oxidative stress tolerance was implicated in E.coli (Gidrol et al., 1996). Present investigation was aimed at developing transgenic sorghum with annexin gene through microprojectile mediated gene transfer. The cDNA of annexin gene isolated fiom Brassica juncea, which is 954 bp length, was cloned into pTZ57R vector by T/A cloning for creating restriction sites, followed by cloning in pRTlOO vector to mobilize CaMV35S promotei and terminator. The expressioncassette was cloned in pCAMBIA 1300 binary vector. Bombardment parameters like pressure of the helium gas, flight distance of the microprojectile and type of explant, which influence the efficiency of bombardment, were optimized with E CAMBIA 1305.1 vector as this vector has the GUS as reporter gene. Sorghum shoot apices isolated from 3d old seedlings were used as explant to induce calli on MS medium containing 2,4-D. Thirteen day old calli were bombarded with annexin gene construct. The putative transgenics were selected and maintained till rooting on hygromycin selection pressure. PCR verification of putative transgenics was done using nptII and hpt primers. The amplification products obtained were 1050 bp and 350 bp respectively confirming the 'presence of transgene. Annexin gene specific primers also yielded the expected 954 bp product. The amplification profiles of the putative transgenic plants confirmed the integration of the transgene in the genomic DNA. The PCR positive transgenics were transferred to green house for acclimatization. Further molecular characterization of these plants to analyze the number of copies of the inserted transgene has to be carried out using Southern blotting.
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D7083