Studies on the Introduction of Alien Chloroplast DNA into Protoplast

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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.