Attempt to Establish Direct Gene Transformation System to Seeds of Sweet Potato (Ipomoea batatas) Using Electroporation Method

Abstract

Aims: The purpose of this study is to get transformants by using the seeds of sweet potato and the instrument of NEPA21, an electroporation method. Study Design: As the first step, we used grafting method to get seeds from the combinations of Ipomoea crassicaulis cv. [Kidachiasagao] as stock and I. batatas (L.) Lam varieties as graft. Then, we used an electroporation method with instrument of NEPA21 to get the GUS transformants by using direct gene introduction into the seeds. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between September 2014 and December 2016. Methodology: The materials for grafting between Ipomoea crassicaulis var. [Kidachiasagao] as stock and I. batatas (L.) varieties as graft to get seeds, were provided by Kyushu-Okinawa Agricultural Research Center (KOARC), and they were cultivated in growth chambers at Minami Kyusyu U. For the judgment of crossing incompatibility (CI) among the varieties of sweet potato, the standard of KOARC was adopted. For crossing experiment of sweet potato, the flowers of any two varieties were used for pollination, and the performance was stopped before 10 am. The seeds were harvested when they matured. For the transformation of GUS into sweet potato, the seeds obtained from [Koganesengan] were used in this study. As the seeds were wrapped with hard skin, they were damaged with knife to absorb water easily. Then, the seeds were placed onto wetted filter papers laid in the dishes for 3~4 days. When the seeds germinated, they were used for transformation according to the procedures of NEPA21. After the process was finished, the container was placed in dark condition for 2 days at 25℃, and then, dyed with GUS dyeing solution. Results: 1) According to the standard of selection for varieties having crossing ability based on the result of CI conducted by KOARC, we selected 3 varieties of [Koganesengan], [Narutokintoki] and [Beniazuma] out from 6 ones held; 2) According to the standard of selection suitable to be used as either for seed or pollen we have  obtained mature  seeds  successfully  from  the  3  varieties; 3) Using the sweet potato seeds collected from [Koganesengan] and electroporation have made GUS transformant of [Koganesengan] with GUS gene expression, for the first time, detected by GUS dyeing with blue color which appeared on the surfaces of the seeds and young buds of the transformants. Conclusion: The results obtained in this study, provide a practical sweet potato transformation system by two steps: 1) Using the grafting between [Kidachiasagao] and sweet potato can consistently obtain mature seeds of sweet potato according to the judgmental standard for CI provided by KOARC, and 2) Using the combination of seeds of sweet potato and electroporation instrument of NEPA21, can get GUS transformants of sweet potato by using the direct gene transformation system. The establishment of GUS direct gene transformation system in seeds of sweet potato can provide a powerful tool for the ASG-1 gene transformation into sweet potato, which is considered as a crop, difficult to be cultured for plant regeneration. Furthermore, the system can be expected to open the way for all of the seed-set crops with a simplified and practical method to produce transformants.