Genetic Transformation Via Biolistic Gene Gun Method and Regeneration of Common Bean

Abstract

The objective of the present research was to utilize biotechnology to improve agronomical traits such as drought stress tolerance in common bean (Phaseolus vulgaris L.) as important recalcitrant crop species via a particle gun "Genebooster TM " for the bombardment of plant tissues with DNA. The process involves the high velocity acceleration of microprojectiles carrying foreign DNA, penetration of the cell wall and membrane by microprojectiles, and delivery of the DNA into plant cells. The initial phase of the two common bean varieties "Fönix" and "Maxidor" focused on the development of a highly regenerable tissue culture procedure amenable to the particle gun physical method. The results showed that MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA were the optimal for shoot induction from meristematic ring of cotyledonary node explants in the both bean varieties. The author has developed a tissue culture protocol that enables the "meristematic ring" that develops below axillary shoots on nodal bean explants to produce shoot buds, and developed an electric-discharge particle acceleration procedure to produce transgenic plants in the two common bean varieties. The plasmid pFF19K, harboring the kanamycin resistance gene nptII as a selectable marker and pFF19-mtlD harboring the mtlD gene which confers mannitol (drought stress) tolerance were used for adapting transformation in the two common bean varieties. The results indicated that the successful delivery of plasmids DNA and efficient gene transfer into common bean meristematic tissues included 20 bar nitrogen pressure, 135 mm shooting distance, 1.1 µm diameter size of microprojectiles, and twice bombarding of the target tissues. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 7.6% and 8.4% in "Fönix" and "Maxidor" bean varieties, respectively. Transgenic plants were recovered using both kanamycin (100 mg/l) and mannitol (1.2 M/l) screening to introduce the neomycin phosphotransferase II (nptII) and the mannitol-1-phosphate dehydrogenase (mtlD) genes into the two common bean varieties. The introduced nptII and mtlD genes have been shown to confer strong resistance and tolerance in transgenic beans to kanamycin and mannitol stress applications. The transgenic bean plants were healthy and the method has proven to be applicable to common bean crop. To my knowledge, this is the first report on the production of common bean transgenic plants with mtlD gene using biolistic gun-mediated gene transfer system.