GENETIC TRANSFORMATION AND REGENERATION OF COM- MON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM

Abstract

Agrobacterium-mediated transformation and regeneration systems has been achieved for two varieties “Fönix” and “Maxidor” of common beans. Transformation ability of cotyledonary nodes explant were tested by A. tumefaciens EHA101 (pEHA101::pTd33) with the nptII and gusA genes, or A281 (pRGG bar H1) with the bar and gusA genes. Transgenic for both bean varieties with nptII gene, or bar gene were produced through this approach. Among different selection methods, 100 mg/l kanamycin, or 4 mg/l herbicide phosphinothricin turned to be optimal, resulting in the highest transformation efficiency. Transgenic common bean plants demonstrated enhanced growth ability under antibiotic kanamycin, or herbicide phosphinothricin stress conditions. The increased resistance was also reflected by delayed development of damage symptoms caused by kanamycin, or phosphinothricin stress. Cotyledonary nodes were found to be effective in formation of multiple shoots cultured on selection MS medium supplemented with 1 mg/l BA, 0.1 mg/l NAA and plus 100 mg/l kanamycin, or 4 mg/l phosphinothricin. Stable expression of the gusA was observed in various parts of the transformed tissues. Optimal transformation conditions were obtained for both bean varieties by co-cultivating cotyledonary node explants with Agrobacterium in MS regeneration medium plus 100 µM acetosyringone for 3 days. The nodal region was punctured, wounded and infected with Agrobacterium strain by overnight immersion in bacterial suspension plus 100 µM acetosyringone. This wounding pattern permits Agrobacterium to penetrate deeper and more completely throughout the tissue, and increasing the probability of infecting plant cells. After inoculation for 3 hours and co-cultivation for 3 days, the explant nodes were cultured on MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA, pH 5.7. The explant nodes were transferred three times at three day intervals to fresh MS medium plus 500 mg/l carbenicillin and 800 mg/l cefotaxime to eliminate Agrobacterium overgrowth of the target region. After cocultivation for 24 hours on MS medium, the explants nodes were transferred to a selective medium containing 100 mg/l kanamycin, or 4 mg/l phosphinothricin. Gene expression of kanamycin resistance (nptII), or herbicide phosphinothricin resistance (bar) and gusA in transformed buds and shoots for both bean varieties was demonstrated by selection test and gusA histochemical analysis. Shoots and buds of transformed explants continued on selective media. Putative transformed green shoots were transferred onto kanamycin, or phosphinothricin containing medium to verify their ability to root. Expression of the β-glucuronidase reporter gene was verified by histochemical staining.