Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km(r)) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T-1, T-2, and T-3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T-1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.