Microtuber Induction And Genetic Transformation Of Colorful Potato (Solanum Tuberosum L.)

Potato (Solarium tuberosum L.) is an important crop and industrial raw materials worldwide, which plays an important role in people’s diet nowadays. But serious diseases have greatly hampered the development of the potato industry. The enhancement of consumer’s demand for good quality potatoes and the coming challenge after entering WTO also bring out potato researchers new objectives. The experiment was studied the effect of different conditions on the formation and growth of microtubers systematically. A regeneration system available for potato transformation was established considering by many factors, including phytohormones combinations, time of pre-culture and co-culture, Agrobacterium concentration and infection time, the concentration of bactericides and the effect of selection. In this study, GFP::UPM1gene was transferred into colorful potato by Agrobacterium-mediated method. More than4independent transformants were obtained and confirmed by PCR and confocal laser scanning microscope analysis.The main results were showed as following:1、Establishment of the regeneration system and microtuber induction system for potato. We found75%alcohol30s,0.1%HgCl2and supplemented with2%Tween solution treated for6minutes was the most convenient form of sanitizing for explants. The optimal medium for aseptic potato seedlings was half strength Murashige and Skoog-based (MS) medium supplement with0.5g/L actived carbon and200mg/L KH2PO4; the best medium for microtuber induction was MS medium supplemented with6-benzylaminopurine (6-BA)5.0mg/L, actived carbon0.5g/L and600mg/L chlormequat(CCC); and the culture condition was dark culture after weak light culture, the temperature was26℃.2、A high frequency genetic transformation system was developed, which lay the foundation of future genetic engineering breeding of potato. GFP::UPM1gene was transformed into the leaf and tube potato slices by Agrobacterium tumefaciens transfection, and in which4regeneration potato plants had confirmed by PCR detection and confocal laser scanning microscopy observation, GFP::UPM1gene has successfully integrated into the potato genome. During transformation research, we found that suitable pre-culture time, infection time and co-culture time can increase the induction rate of resistance buds. The most efficient pre-culture time is2days and the optimal concentration of Agrobacterium was OD6000.6, infected6minutes; Afert2days co-culture,250mg/L Carb was used as the most effective bactericides concentration. Different explant types need different selective concentration, HygromycinB(HmB) was used as the selective agent, the optimal concentration was20mg/L at the stage of shoot and root formation.