Agrobacterium-mediated transformation of Cry1Ab gene into Tectona grandis L. (teak)
2017
Onwimol, P. | Chanprame, S. | Chanprame, S.
The binary vector pCAMBIA1302-Ab, containing Cry1Ab and hygromycin phosphotransferase (hpt) genes, was used for transformation of in vitro teak nodes and multiple shoots clumps. The explants were submerged in a suspension culture of Agrobacterium tumefaciens strain EHA105 (OD sub 600 = 1.2), sonicated for 3 min, and then vacuum-infiltrated for 5 min. Co-cultivation was performed for 3 days on MS medium containing 100 uM acetosyringone. Two months after selection on medium containing 50 mg L sup-1 hygromycin, calli formed. Total RNA was extracted from these calli and subjected to first-standed RT-PCR analysis of the Cry1Ab gene. The presence of Cry1Ab transcript was confirmed in all selected calli. A single shoot developed from a multiple shoot clump cultured on selected medium. The 4-weeks-old putative transformed shoot was also positive for Cry1Ab and hpt genes through PCR. However, chimera tissue was observed in this shoot. The presence of Cry1Ab protein in transgenic tissue was also determined using a Bt-Cry1Ab/1 Ac ELISA Kit. Expressed Cry1Ab protein in the putative transformed shoot was confirmed in leaves containing Cry1Ab and hpt genes.
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