Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas).

Aims: Sweet potato (Ipomoea batatas) cv. “Miyazakibeni” was used as material for shoot apex culture, reverse transcription-polymerase chain reaction (RT-PCR) and clonal propagation to establish an efficient and practical virus-free seedling supply system in production of vegetatively reproductive plants. Study Design: At first, efficient plant regeneration was achieved from shoot apex culture of sweet potato. Secondly, RT-PCR method was used to detect the sweet potato feathery mottle virus (SPFMV) viral infection of tuber surface of edible sweet potato using the RNAs from the plants obtained from shoot apex culture. Finally, the virus-free plants verified by RT-PCR were propagated clonally by culture of suckers cut from stems of the virus-free plants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2008 and December 2012. Methodology: The best efficiency for material sterilization was tested using different concentrations (0.1% - 1.5%) of sodium hypochlorite solution (SHS) and the treated times (5 min – 20 min). Theshoot apexes less than 0.3mm in size were cultured on Komamine and Nomura (1998) (KN) medium and Murashige and Skoog (1962) (MS) medium. The regenerated plants were used for RNA extraction and then, used for RT-PCR for detection of SPFMV. Based on the result of RT-PCR, the suckers cut from stems of virus-free plants were cultured and propagated clonally and routinely within a short period. Results: The combination of 0.3% of SHS and 10 and/or 20 min gave the best result (100%) of surviving rate for material sterilization. The culture of shoot apexes less than 0.3 mm in size gave plant regenerating rates of 82% and 65% on KN and MS medium, respectively. The results of RT-PCR of RNAs from plants obtained from shoot apex culture and plants of SPFMV infection showed that SPFMV virus was clearly removed by shoot apex culture conducted in this study. For clonal propagation, 80-100% of suckers cut from the stems of the virus-free plants detected grew into complete plants after 6 weeks of culture, indicating that the virus-free plants could be routinely propagated 5 times in number each time and repeatable by the short circle. The sweet potato produced in field showed no symptom called as russet crack-like symptom (RC-LS) even after cultivation two seasons. Conclusion: Overall, an efficient and practical virus-free seedling supply system was established in sweet potato by the three steps of 1) virus-free plant regeneration from shoot apex culture, 2) quick detection of SPFMV using RNA of the regenerated plants by RT-PCR, and 3) the verified virus-free plants were propagated clonally and routinely within a short period using culture of suckers cut from the stems of virus-free plants.

Establishment of Agrobacterium-Mediated Transformation System in Sweet Potato (Ipomoea batatas) by Culture of Leaf Segments for Functional Analysis of ASG-1, an Apomixis- Specific Gene.

Aims: In order to do the functional analysis of apomixis-specific gene (ASG-1), which was isolated from apomictic guineagrass, the sweet potato was used to establish an Agrobacterium-mediated transformation system. Study Design: At first, plant regeneration was achieved from the culture of leaf segments of sweet potato. Based on it, a binary vector pSMA35H2-NG for transformation of ASG-1 was used for establishment of a suitable procedure for plant regeneration of transformants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2009 and December 2012. Methodology: The leaf segments were used for somatic embryogenesis and plantlets regeneration. For the preliminary transformation, a GUS gene set in pSMA35H2-NG was introduced into the Agrobacterium strain GV3101/PMP9, and the Agrobacterium was used to infect the callus derived from leaf segments of sweet potato “Miyazakibeni” and the callus derived from seeds of rice “Nipponbare”. For the plasmid construction, the GUS was replaced by ASG-1, named as pSMA35H2/ASG1. The resultant plasmid was mobilized into Agrobacterium strain GV3101/PMP9 for transformation. For detection of ASG-1, DNAs of the transgenic plantlets were used for PCR, using the primers designed according to ASG-1 and hygromycin, respectively. Results: 1) When the leaf segments were sterilized with sodium hypochlorite solution of 0.3% and 0.4% for 15 min, 100% of surviving rates was achieved. And the segments cultured on Murashige and Skoog (1962) gave 100% of callus formation rates. 2) When the calli were placed onto Komamine and Nomura (1998) medium for differentiation, somatic embryogenesis was obtained with white color and grain-like tissue, and plantlets with multiple shoot-like tissues were obtained from the somatic embryo. 3) For the preliminary transformation, the calli showed GUS blue spots gradually on the surface. 4) When the pSMA35H2/ASG1 was used to the transformation of the embryogenic calli, the plantlets were developed through multiple shoots. 5) The specific bands of ASG-1 and hygromycin were observed from the PCR products of the plantlets’ DNAs, respectively. Conclusion: Overall the above results, the procedure using the binary vector pSMA35H2/ASG1 containing ASG-1 revealed, as the first case, that Agrobacteriummediated transformation system in sweet potato was established using the culture of leaf segments in this study.