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Transformation of sweet potato and expression of the transferred genes

甘薯的转化及转入基因的表达

基本信息

批准号：
03660004
负责人：
KAKEDA Katsuyuki
金额：
$ 1.6万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03660004/
关键词：
Sweet potato Ipomoea Transgenic plant Gene transfer Molecular genetics Ipomcea

项目摘要

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.

为探索获得甘薯转基因植株的有效方法，以甘薯愈伤组织和叶盘为外植体，进行了农杆菌介导的转化和植株再生研究。使用了六个甘薯栽培品种和一个野生二倍体物种（I.leucantha），以及一个根癌农杆菌菌株EHA 101。该菌株具有含卡那霉素抗性基因（npt-Ⅱ）和潮霉素抗性基因（hpt）的GUS表达载体。一两个月后，诱导出表达GUS基因的转化愈伤组织，但这些愈伤组织没有再生出植株。在随后的实验中，用2，4-D从茎尖诱导胚性愈伤组织。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3天，获得了抗生素抗性愈伤组织，并在部分愈伤组织中形成了不定胚。本研究以宝石和台欧10号两个甘薯品种的叶片为外植体，建立了不经愈伤组织诱导的植株再生体系。再生植株可直接从含有NAA的叶盘上获得，也可将叶盘形成的不定根转移到不含激素的培养基上获得。在与根癌农杆菌共培养5天后，转化根在含有抗生素的培养基上再生。但这些根在1 ~ 2cm长时就停止生长，没有再生植株，提高转化细胞和组织的再生频率以获得转基因甘薯植株还需进一步研究。