【作者】 张健；

【导师】 吕柳新；

【作者基本信息】 福建农林大学 ， 果树学， 2006， 博士

【摘要】 多胚现象是柑桔研究及育种实践中的一个重要问题，也是柑桔杂交育种最大的障碍，多胚性柑桔品种在杂交育种时，由于珠心胚的干扰抑制，致使有性胚在早期败育死亡，导致育种过程中很难获得真正的杂种苗，使多胚性柑桔杂交育种工作受到很大的限制。因此，探索高效的合子胚分离技术，在合子胚尚未败育之前将其分离出来进行培养，克服珠心胚的干扰，挽救早期败育的合子胚，从而获得杂种苗，提高杂种率，是多胚柑桔杂种实生苗培育的有效途径，具有重要的实践意义。本研究初步建立了多胚性柑桔早期合子胚的显微分离、离体培养及合子苗的鉴定体系，成功显微分离了柑桔早期合子胚并通过离体培养再生植株，具体结果如下：1、多胚性柑桔早期合子胚显微分离的最适时期为授粉后50—55d。利用显微操作系统，采用自制钢针(针尖大小控制在50μm左右)代替传统的微细玻璃针，可提高多胚性柑桔早期合子胚的分离效率。2、MS、White和MT三种基本培养基都适合柑桔早期合子胚的离体培养，柑桔早期合子胚成熟培养的最佳植物生长调节剂浓度配比为IAA1mg／L+BA20mg／L，适当提高培养基中蔗糖浓度(最适浓度为10％)、添加适量水解乳蛋白(最适浓度为400mg／L)有助于柑桔早期合子胚的生长发育。3、固体培养与悬浮培养方式均适合柑桔早期合子胚的成熟培养，但悬浮培养方式更便于观察调控。4、培养成熟的柑桔合子胚转入分化培养时，不需改变成熟培养时的植物生长调节剂配方，只需将蔗糖浓度降至2％，由暗培养改为光照培养即可自然再生植株。在此基础上若添加少量ZT(0.2mg／L)，能促进合子胚再生植株的速度。由成熟培养转入分化培养，再生植株的最佳时期为：a、固体培养的合子胚及其次生胚发育至子叶状形态出现时，即可认为是发育成熟，可以转接至分化培养基中再生植株。b、液体培养的合子胚子叶长度大约在1—1.5cm(培养时间约8星期)时，即可认为是发育成熟，可以转接至分化培养基中再生植株。5、经反复实验，确立了适合柑桔基因组DNA的快速提取方法和PCR反应的优化体系，筛选出6条扩增多态性较高的引物，对再生小苗进行了RAPD鉴定分析，从73株再生小苗中，鉴定出了61株合子苗，合子苗的比例达到83.6％。综合本研究所有研究结果表明，利用显微操作系统，显微分离多胚性柑桔早期合子胚，并进一步培养再生植株，可获得高比例的合子苗，是排除多胚性柑桔珠心胚的干扰抑制，促进多胚性柑桔有性杂交育种的有效手段。更多还原

【Abstract】 Polyembryony is a serious handicap in Citrus breeding. It results in the abortion of the zygotic embryo, due to the competition with the nucellar embryos, which are often more vigorous. Within this context, microseparation and in vitro culture of early zygotic embryos from polyembryony Citrus is very important because it makes possible the use of immature hybrid embryos and accelerates the sexual hybridization breeding in Citrus.The systems of microseparation and in vitro culture of zygotic embryos from polyembryony Citrus and identification of Citrus hybrids were preliminary established in this research. Some early zygotic embryos of Citrus were microseparated and regenerated into seedlings successfully by this way. The results are as follows:1、The best stage to separate zygotic embryos was 50-55 days after pollination. It can boost the efficiency of zygotic embryos separation to use steel needles with 50μm large diameter needlepoint under the micromanipulator.2、MS, White and MT basic medium are all suitable for the culture of zygotic embryos. The addition of 1mg/Lof IAA and 20mg/L of BAto the medium provides best conditions for the mature culture of zygotic embryos of Citrus. It is favourable for the development of early zygotic embryos to increase the osmotic pressures of media and supplement lactalbumin hydrolysate (LH). The suitable concentration of sucrose was 10% and LH was 400mg/L.3、Cultured in solid or liquid medium are all suitable for the development of early zygotic embryos of Citrus. But zygotic embryos cultured in solid medium can be more ease to be observed and controlled.4、It doesn’t need to change the combination of IAA and BA when developed zygotic embryos change to differentiation culture. Seedlings can be regenerated from mature zygotic embryos when the concentration of sucrose decreased to 2% and cultured in illumination, and it can also accelerate the speed of regeneration with addition of 0．2mg/L of ZT in this way. The best stage to change to differentiation culture is the stage when cotyledons came out from the zygotic embryos cultured in solid medium or the cotyledons of the zygotic embryos cultured in liquid medium are 1-1．5 cm long.5、The method of isolation of genomic DNA from Citrus and the optimal amplification system and program for RAPD-PCR were established after iterative experiments. 61 zygotic seedlings were identificated from 73 regenerated seedlings by random amplified polymorphic DNA(RAPD) with 6 primers of 10 bases. The proportion of zygotic seedlings is 83．6%.All results above showed that it is a efficiency method to get rid of the inhibition from nucellar embryos and accelerate the sexual hybridization breeding in polyembryony Citrus to microseparate the zygotic embryos of Citrus by micromanipulator and culture them in vitro into regenerated seedlings.更多还原