Development of an efficient regeneration and Agrobacteriummediated.pdfVIP

In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-013-9544-6 BIOTECHNOLOGY Development of an efficient regeneration and Agrobacterium -mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants Hongyan Dai Wenran Li Wenjuan Mao Lei Zhang Guofen Han Kai Zhao Yuexue Liu Zhihong Zhang Received: 20 March 2013 /Accepted: 2 July 2013 / Editor: J. Forster # The Society for In Vitro Biology 2013 Abstract Apple has become a model species for Rosaceae Keywords Apple . Regeneration .Agrobacterium -mediated genetic and genomic research, but it is difficult to obtain trans- transformation . Cotyledon . Cocultivation time genic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transforma- Introduction tion system for crab apple (Malus micromalus) using cotyle- dons as explants. The proximal cotyledons of M. micromalus, Malus , the apple genus, includes over 30 primary species excised from seedlings that emerged from mature embryos belonging to the Rosaceae family (Korban 1986). Apples cultured for 10– 14 d in vitro, were suitable as explants for were certainly one of the earliest fruits to be gathered. regeneration and Agrobacterium -mediated transformation. Ancient civilizations were growing apples at least 2,500 yr Cotyledon explants were cocultivated for 3 d with ago, and superior seedlings were actively selected for, as was Agrobacterium tumefaciens strain EHA105 harboring the bina- propagation through budding and grafting around 2,000 yr ry vector pCAMBIA2301 on regeneration medium. ago (Roach 1985). The domestic apple (Malus × domestica Kanamycin-resistant buds were produced on coty