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.

为了开发获得甘薯转基因植物的有效方法，进行了农杆菌介导的愈伤组织和叶盘的转化和再生。使用了六个甘薯品种和一个野生二倍体物种（I.leucantha），以及一个农杆菌菌株 EHA101。该细菌菌株具有 GUS 表达载体，其中含有卡那霉素抗性 (npt-II) 和潮霉素抗性 (hpt) 基因。切下芽尖、叶柄和茎，并与根癌农杆菌共孵育 20 分钟。将它们转移至含有抗生素的愈伤组织诱导培养基中。一两个月后，诱导表达GUS基因的转化愈伤组织，但没有从这些愈伤组织再生出小植株。在以下实验中，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物再生。通过将愈伤组织与根癌农杆菌共孵育三天，获得了抗生素抗性愈伤组织，并且在一些愈伤组织中形成了不定胚。然而，没有观察到不定胚的植株再生。在两个甘薯品种Jewel和Tainou 10中，新开发了带有叶盘的无愈伤组织诱导的植株再生系统。再生植物直接从含有NAA的叶盘获得或通过将叶盘形成的不定根转移到不含激素的培养基上获得。与根癌农杆菌共培养5天后，转化的根在含有抗生素的培养基上再生。然而，这些根的生长在1至2厘米长时就停止了，并且没有从这些根再生出植物。如何提高转化细胞和组织的再生频率以获得甘薯转基因植物还需要进一步的研究。