Studies on the Introduction of Alien Chloroplast DNA into Protoplast

本研究将玉米叶绿体导入番茄原生质体，不仅是为了选育高光合作用植株，也是为了明确C_4叶绿体导入C_3植株的可能性。研究结果如下：(1)在蔗糖溶液的30% ~ 45%界面，离心后可获得充足的玉米叶绿体。(2)作为受体，白化或黄化原生质体或无活性的原生质体是可取的。以杂色番茄愈伤组织或幼苗为材料，在添加百草枯或阿特拉津的培养基上或在黑暗条件下生长。当酶、果胶酶Y-23、纤维素酶“Onozuka”RS和Driselase组合使用时，原生质体数量最多。(3)利用电脉冲电压将玉米叶绿体导入番茄原生质体。然而，玉米的叶绿体对于番茄原生质体来说看起来太大了，由于它们的浮力，它们在溶液中被分离出来。因此，引种率不到1%。(4)对番茄原生质体进行了3年的反复培养，未观察到原生质体的再生。为了检验原生质体培养技术，还对生菜进行了原生质体培养试验，获得了许多植株。(5)体外培养条件对番茄驯化率有影响。(6)为检查外源叶绿体的引入情况，研究了ctDNA分析技术。通过ctdna的差异，可以区分栽培番茄和野生番茄。虽然本实验没有获得番茄植株，但认为每个步骤都是可行的。在未来，将出现引进外来c4叶绿体的个体。

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.