利用离体受精技术探索水稻远缘杂交育种新途径

Distant hybridization is one of important ways in improving crop varieties. The sperm cells and egg cells are isolated from the cultivated rice and wild rice, then formed hybrid artificial zygotes using in vitro fusion method. And take advantage of the characteristics that the zygote cells easily division and in vivo embryogenesis regeneration, the artificial hybrid zygotes are cultured as hybrid plants. The in vitro fertilization technique can overcome the obstacles of stigmas, style and ovary in the fertilization process. The method can create the conditions for breeding new rice varieties that have excellent resistance. The exploration not only overcomes incompatibility problems of the distant hybridization, but also solves the ploidy problems in somatic hybridization, making the rice heterosis better utilized. In this project, based on successful isolating sperm cells and eggs, especially to explore vitro sperm and egg of rice induced vitro fusion technology and artificial zygote culture technique, and induce plant regeneration of zygotic cells, which will establish an experimental system of rice in vitro fertilization. This project not only creates a new way to distant hybridization in crop breeding, but also to the exploration of studying the mechanism of fertilization in higher plants, which has a high value and theoretical significance.

远缘杂交是是农作物品种改良的重要途径之一。将栽培稻和野生稻的精、卵细胞分离出来，在体外诱导精、卵细胞融合形成人工合子；利用合子细胞具有易分裂，且遵循体内胚胎发生途径再生出植株的特点，将人工合子培养成为杂种植株。离体受精技术排除了柱头、花柱和胚珠等体细胞组织对受精过程的障碍，为选育具优良抗性的水稻新品种创造了条件。这种探索既克服了远缘杂交种中出现的物种受精不亲和性问题，又解决了体细胞杂交中出现的倍性问题，使水稻的杂种优势更好地得到利用。本项目在我们已成功地分离出水稻精细胞和卵细胞的基础上，重点探索诱导水稻精卵细胞的体外融合技术和人工合子的培养技术，并诱导合子细胞的植株再生，建立水稻的离体受精实验体系。本项目的研究不仅在水稻等作物的育种中开创了一条远缘杂交新途径，还可对高等植物的受精机理进行探索研究，具有很高的应用价值和理论意义。

远缘杂交是是农作物品种改良的重要途径之一。将水稻的精、卵细胞分离出来，在体外诱导精、卵细胞融合形成人工合子；利用合子细胞具有易分裂，且遵循体内胚胎发生途径再生出植株的特点，将人工合子培养成为杂种植株。离体受精技术排除了柱头、花柱和胚珠等体细胞组织对受精过程的障碍，为选育具优良抗性的水稻新品种创造了条件。这种探索既克服了远缘杂交种中出现的物种受精不亲和性问题，又解决了体细胞杂交中出现的倍性问题，使水稻的杂种优势更好地得到利用。结果如下：用直接爆破法和离体—活体技术法分离收集水稻精细胞，用酶解和解剖方法分离收集水稻的卵细胞。将分离的精、卵细胞用电融合介导尝试了体外诱导融合。在合适的渗透压（11%甘露醇）和添加0.04%牛血清蛋白的溶液中，用交流电场AC为20-35 V, 10-25 s使精、卵细胞排队，直流电场DC在400-600 V，45-50 μs的脉冲穿孔条件可诱导45%的水稻精、卵细胞融合和75%以上的水稻卵细胞之间的融合。尝试了水稻人工合子的单细胞培养但未获成功。本项目的研究不仅在水稻等作物的育种中开创了一条远缘杂交新途径，还可对高等植物的受精机理进行探索研究，具有很高的应用价值和理论意义。