Sorghum is the major grain crop in semi arid tropics. Until recently the genetic improvement of sorghum for abiotic stresses has been carried out thorugh traditional plant breeding methods. Non-availability of efficient transformation techniques is one of the limitations 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 medicated gene transfer.
The cDNA of annexin gene isolated from Brassica juncea, which is 954 bp length, was cloned into pTZ57R vector by T/A cloning for creating restriction sites, followed by cloning in pRT100 vector to mobilize CaMV35S promoter and terminator. The expression cassette 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 wich pCAMBIA 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 dya old calli were bombarded with annexin gene construct. The putative transgenics wee 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 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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