Studies on the Introduction of Alien Chloroplast DNA into Protoplast

外源叶绿体DNA导入原生质体的研究

基本信息

批准号：
01560044
负责人：
TANAKA Takayuki
金额：
$ 1.86万
依托单位：
Kyushu Tokai 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：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

In the present study, the introduction of corn chloroplast into tomato protoplast was conducted not only for breeding high photosynthesis plants but also for making clear the possibility of the introduction of C_4 chloroplasts into C_3 plants. The results are as follows ;(1) Adequate corn chloroplasts were avalilable from the 30 % - 45 % interface of sucrose solution after centrifuging.(2) As the recipient, albino or etiolated protoplasts or those with inactive chroloplast are desirable. As materials, callus or the seedlings from variegated tomato, grown on the media with Paraquat or Atrazine, or under dark condition were used. Maximum protoplasts in number were obtained when the combination of enzymes, pectolyase Y-23, cellulase "Onozuka" RS and Driselase were applied.(3) The introduction of corn chloroplasts into tomato protoplasts were done by electric pulse voltage. The chloroplasts of corn, however, looked like too big for the tomato protoplast to be introduced and they were isolated in the solution because of their buoyancies. Therefore, the introduction rates were achieved less than 1 %.(4) Regeneration from the tomato protoplasts was not observed, though the protoplast culture were repeatedly tried for three years. To check the technique of protoplast culture, the protoplast culture of lettuce was also tried and many plants were obtained.(5) Acclimination rates of the tomato cultures were affected by the in vitro conditions.(6) To check the introduction of alien chloroplasts, the technique of analysis of ctDNA was investigated. By the difference of their ctDNAs, cultivated (L. esculentum) and wild (L. pimpinellifolium) tomato could be identified.Though no plants of tomato were obtaind in this experiment, each step were considered to be feasible. In a future, individuals introduced alien C_4 chloroplasts will appear.

本研究将玉米叶绿体引入番茄原生质体，不仅是为了培育高光合作用植物，也是为了明确将C_4叶绿体引入C_3植物的可能性。结果如下：(1)离心后蔗糖溶液30％～45％界面处可获得足够的玉米叶绿体。(2)作为受体，应选择白化或黄化的原生质体或叶绿体无活性的原生质体。作为材料，使用来自杂色番茄的愈伤组织或幼苗，在含有百草枯或莠去津的培养基上生长，或在黑暗条件下生长。当果胶酶Y-23、纤维素酶“Onozuka”RS和Driselase组合使用时，获得了最大数量的原生质体。(3)通过电脉冲电压将玉米叶绿体引入番茄原生质体。然而，玉米的叶绿体看起来太大，无法引入番茄原生质体，并且由于其浮力而在溶液中被分离。因此，引入率低于1％。(4)尽管三年来反复尝试原生质体培养，但未观察到番茄原生质体的再生。为了检验原生质体培养技术，还尝试了生菜的原生质体培养，并获得了许多植株。（5）番茄培养物的驯化率受到体外条件的影响。（6）为了检验外来叶绿体的引入，研究了ctDNA的分析技术。通过ctDNA的差异，可以识别栽培番茄（L. esculentum）和野生番茄（L. pimpinellifolium）。虽然本实验没有获得番茄植株，但每个步骤都被认为是可行的。未来，将会出现引入外来C_4叶绿体的个体。