Transformation of sweet potato and expression of the transferred genes

为了探索获得甘薯转基因植株的有效方法，利用农杆菌介导法对甘薯愈伤组织和叶盘进行了转化和再生。选用了6个甘薯品种和1个野生二倍体甘薯(I.leucantha)和1个根癌农杆菌(A.tuefaciens)菌株EHA101。该菌株含有抗卡那霉素(NPT-II)和潮霉素抗性(HPT)基因的GUS表达载体。切除芽尖、叶柄和茎，与根癌农杆菌共培养20min。他们被转移到含有抗生素的愈伤组织诱导培养基中。1个月或2个月后，诱导出了表达GUS基因的转化愈伤组织，但这些愈伤组织都没有再生植株。在接下来的实验中，从含有2，4-D的茎尖诱导出胚性愈伤组织。从该愈伤组织中观察到高频率的植株再生。将愈伤组织与根癌农杆菌共培养3d，获得了抗性愈伤组织，并在部分愈伤组织中形成了不定胚。在两个甘薯品种Jewel和台农10号上，建立了无愈伤组织诱导的无叶盘植株再生体系。用NAA直接从叶盘上获得再生植株，或将叶盘形成的不定根转移到不含激素的培养基上获得再生植株。与根癌农杆菌共培养5d后，在添加抗生素的培养基上再生转化根。然而，这些根的生长在1~2 cm长时停止生长，没有再生植株，还需要进一步研究提高转化细胞和组织的再生频率，以获得转基因甘薯植株。

To develop an efficient method in obtaining transgenic plants of sweet potato, Agrobacterium-mediated transformation and regeneration from calli and leaf disks were carried out. Six sweet potato cultivars and a wild diploid species (I.leucantha), and an A.tumefaciens strain, EHA101 were used. This bacterial strain has a GUS expression vector which contains a kanamycin resistant (npt-II) and a hygromycin resistant (hpt) genes.Shoot apexes, petioles and stems were excised and coincubated with A.tumefaciens for 20 min. They were transferred to a callus induction medium containing antibiotics. After one or two months, transformed calli expressig a GUS gene were induced, but no plantlet was regenerated from these calli. In the following experiment, an embryogenic callus was induced from a shoot apex with 2,4-D. A high frequency of plant regeneration was observed from this callus. By three days coincubation of the calli with A.tumefaciens, antibiotic resistant calli were obtained and adventitious embryos were formed in some of the calli. However, no plant regeneration was observed from the adventitious embryos.Plant regeneration system without callus induction was newly developed with leaf disks in two sweet potato cultivars, Jewel and Tainou No.10. Regenerated plants were obtained directly from leaf disks with NAA or obtained by transferring adventitious roots formed from leaf disks onto the medium without hormones. After a coculture with A.tumefaciens for 5 days, transformed roots were regenerated on the medium with antibiotics. However, the growth of these roots stopped in 1 to 2 cm long and no plant was regenerated from these roots.Further research is needed on the improvement of regeneration frequency from transformed cells and tissues to obtain transgenic plants of sweet potato.