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Pak. J. Bot., 42(5): 3613-3631, 2010.

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IMPROVING AGROBACTERIUM MEDIATED TRANSFORMATION PROTOCOL FOR INTEGRATION OF XA21 GENE IN WHEAT (TRITICUM AESTIVUM L.)

NAVEED IQBAL RAJA^{1, 2}, ASGHARI BANO², HAMID RASHID^3*, ZUBEDA CHAUDHRY⁴ AND NOSHIN ILYAS¹.

Abstract: During the present study, embryo derived calli of 3 wheat varieties viz., Chakwal-97, Inqulab-91 and Tartara-2000 were infected with Agrobacterium tumefaciens strain EHA101 carrying genes Xa21 for bacterial blight resistance. Hygromycin @ 50mg/l was lethal dose for the selection of transformed calli. Up to 90% GUS expression was observed in selected calli on the selection media containing 50 mg/l hygromycin.  The highest number (90%) of GUS expression was observed from 21 days old calli. Callus differentiation was not observed at 0-50µM application, hygromycin resistant calli were obtained from the calli treated with acetosyringone (200-400µM). The application of 400 µM acetosyringone promoted the production of resistant calli and GUS expression was found to be higher in Inqulab-91 (90%). A combined treatment of cefotaxime (500mg/l) and carbenicillin (250mg/l) are better than cefotaxime (500mg/l) or carbenicillin (500mg/l) alone for the control of bacteria.  Transient GUS expression was observed in calli of all varieties. Stable GUS expression was observed in transformed leaves and roots of all varieties especially Tartara-2000 and Inqulab-91.  The transformation was also confirmed by the presence of 1.4kbp band in PCR and Real time quantitative PCR in two successive generations of wheat.

¹Department of Botany, PMAS Arid Agriculture University, Rawalpindi. Pakistan

 ² Department of Plant Sciences, Quaid-i-Azam, University, Islamabad.

³Mohammad Ali Jinnah University, Islamabad.

⁴Deportment of Botany,Hazara University, Mansehra.