高加索三叶草与白三叶杂交后代育性恢复及固氮性研究

Caucasian clover and white clover are excellent legume. They play an important role in modern animal husbandry and ecological protection, with a genetic relationship. For most parts of Inner Mongolia Autonomous Region the climate demonstrates extreme drought and cold in the winter. Only the Caucasian clover can adapt to this kinds of climates up to now, and showed some good agronomic characters, cultivation and utilization prospects. But in the first year of planting, Caucasian clover growed and developed slowly, with poor nitrogen-fixing ability for whole growing stages. The native white clover in Inner Mongolia can survive only in Daxinganling forest where it has heavy snow in the winter and spring. White clover showed some excellent characters like rapid growth, high yield and good quality, higher nitrogen-fixing ability. But it has poor ability to adapt to the dry and low temperature and dry heat. During preliminary studies, the techniques of artificial distant hybridization and embryos in vitro culture were used to obtain a large number of F1 hybrids, and planted in the greenhouse and field successfully. Because F1 hybrids are not sterile, the objective of this project is to reveal their infertility mechanism and restore their fertility by using parent two-way backcross combined with the embryos in vitro culture techniques and chromosome doubling techniques, then to select fertile BC1 plants. Further study is to inoculate a variety of clover rhizobium to fertile BC1 plants, then select effective rhizobia and determine the nitrogen fixation ability of the BC1 roots. Various experimental aspects will make full use of cell biological and molecular biological detection technology to improve research efficiency. Finally some new clover lines with a number of good traits of parents, including some new features like early accelerating growth rate, higher nitrogen-fixing ability and adaptability to Extreme climate, will be selected.

高加索三叶草和白三叶都是优良豆科牧草，在畜牧业和生态保护中有重要作用，具有亲缘关系。但内蒙古多数地区极端干旱、寒冷，目前只有前者适应性强，农艺性状优良，栽培利用前景好。但其种植早期生长发育缓慢，固氮能力差。后者自然仅存于冬春积雪较多的大兴安岭林区，生长迅速，量高质优，固氮能力强，但适应干燥低温和干燥高温的能力差。通过前期研究，利用人工远缘杂交和胚离体培养技术，已获得了大量杂交F1代植株，并在温室和田间栽植成功。由于F1代不育，本项目的目标是在揭示其不育机理的情况下，通过父、母本双向回交，结合胚离体培养技术及染色体加倍技术，恢复其育性，选择可育的BC1植株。再利用多种三叶草根瘤菌接种，筛选使BC1结瘤固氮的根瘤菌，同时测定BC1根系固氮性。各实验环节，充分利用细胞生物学和分子生物学检测技术，提高研究效率。最终选择一批兼有双亲优良性状，前期生长速度加快，具有一定固氮能力，适应性强的可育新品系。

高加索三叶草具有极强的抗寒和耐旱性，生产力高，耐牧性好，适于内蒙古等北方地区种植，但固氮性差；白三叶固氮性强，但抗寒性和抗旱性都较差。通过杂交，试图选育能够结合两种三叶草优点的后代，为北方地区开辟新的豆科牧草种质资源。前期研究通过胚拯救和组培技术获得大量高加索三叶草与白三叶杂交F1代植株。由于杂交亲本染色体倍性不同，这些后代植株不育。本项目的重点内容是恢复杂交F1代育性，提高可育后代的固氮性和选择培育优良株系。获得育性的途径有人工回交胚拯救组培技术；田间回交技术；染色体加倍技术等。研究取得了理想的结果，包括（1）获得田间回交结实杂交后代株系48份，这些株系的种子都能正常成苗，有42份生产出新的后代种子，育性完全恢复；（2）通过人工回交胚拯救技术获得了8株以白三叶为回交父本的BC1白后代，已在田间正常开花，可通过根蘖进行扩繁，育性正在恢复；（3）利用染色体加倍技术使40个杂交F1代植株恢复育性，正常结实并能通过根蘖扩繁，形成株系；（4）提纯出父本白三叶的根瘤菌。利用白三叶根瘤菌接种，使多个可育杂交后代株系实现了结瘤。但其固氮性目前正在测试中；（5）选择了一批性状优良的可育后代株系，进行了综合评价。建立了永久性材料保存评价圃，可以通过根蘖扩繁，形成稳定的株系或者品系群并建立档案，利于今后的进一步研究。产生了多叶型变异植株，成为该研究的重要发现。这些成果将大大丰富内蒙古等地三叶草的种质资源，打破内蒙古仅有1个能够完全越冬度夏三叶草品种的格局，为进一步选育兼有双亲优良性状、适应北方条件的三叶草新品系打下了坚实的物质基础。