Genetic Characters And Fertility Restoration Of The Hybrids Between Trifolium Ambiguum Bieb. And T. Repens L.

The majority species of Trifolium forage was with the characters of rich nutrition, strong ability of nitrogen fixation, good palatability and planting rapidly. In order to create new forage germplasms of parental superior characters, hexaploid Trifolium ambiguum Bieb. of strong resistance to drought and cold which from New Zealand as the famale parent was hybridized with the tetraploid Trifolium repens L. of strong ability of nitrogen fixation which from Greater Higgnan Mountains, and the hybrid F1which were completely sterile were successfully gotten. In this study, several different levels from the morphology and biology, cytological basis, anatomy and molecular markers of T. ambiguum Bieb., T. repens L. and their hybrid F1were identified by analysis, and fertility restoration of hybrids was preliminarily studied also. The main results were as follows:1. The identification results of the somatic chromosome number showed that the hybrids F1of T. ambiguum Bieb. and T. repens L. were pentaploid (2n=5x=40), the karyotype formula of them was2n=5x=40=34m+6sm. They were the real hybrid offsprings of T. ambiguum Bieb. and T. repens L.. The PMCM I chromosome pairing configuration of T. ambiguum Bieb., T. repens L. and their hybrids were0.06Ⅰ+23.97Ⅱ,0.04Ⅰ+15.98Ⅱ and9.07Ⅰ+14.33Ⅱ+0.23Ⅲ+0.03Ⅳ respectively. The meiosis of hybrids Fi was irregular. Univalents existed universally and multivalents occurred in a low frequency in metaphase I.Laggards and chromatid bridge appeared in anaphase I2. The anatomical structures of roots, stems, leaves among T. ambiguum Bieb., T. repens L. and their hybrids were quite different. Initially determined that the drought tolerance was T. ambiguum Bieb.> hybrid F1> T. repens L.3. The growth period and full-year growth day of hybrids F1was99d and201d respectively, which were between their parents. The growth rate of hybrids F1similar to T. repens L. in earl)’and later growth period but significantly more than their parents in the medium period, showed strong heterosis. Plant type of F1was similar to T. ambiuuum Bieb.. the morphological features in plant height, leaf length, leaf width, stem diameter, root length, root diameter, number of branches were all in the middle of both parents except stem length and showed significant difference.4. Root nodule were found on the hybrids F1of T. ambiguum Bieb. and T. repens L..but the ability of nitrogen fixation was still absent. 5. Photosynthetic physiological characteristics of T. ambiguum Bieb., T. repens L. and their hybridsshowed that the hybrids F1with a higher net photosynthetic rate and lower transpiration rate.Their water use efficiency was significantly higher than parents, which suggested a potential of high yield.6. MS+2,4-D (0.1mg/L)+6-BA (2mg/L) were the appropriate induction media of stems of hybrids F,. MS+NAA (0.5mg/L)+6-BA (1.0mg/L)+KT(1.0mg/L) was the suitable differentiation media for the callus from stems of F], and the1/2MS medium which had no any hormones was the suitable rooting medium, vermiculite was the suitable matrix to harden and transplant the regenerated plants of hybrids F1, with highest survival rate up to93.75%.7. Backcross have not succeeded in seeding, the chromosome doubling is the reasonable method of resuming fertility to the hybrid.8. Highest efficiency of chromosome doubling was to treat the seedlings of the hybrids F1, the suitable colchicine concentration and treatment time for them was2000mg/L and48h and the variation rate was6.67%. but with frequent chimerism. The suitable colchicine concentration and treatment time for regenerated plants of hybrids F1was1000mg/L and48h and the variation rate was5.88%, but with lower survival rate which was26.47%. The suitable colchicine concentration and treatment time for callus of hybrids F1was250mg/L and48h and with a lower variation rate which was4.08%. but easily to get the pure decaploid.9.1SSR molecular maker was used to analyze the genetic relationship among T. ambiguum Bieb.. T. repens L., hybrids F1and regenerated plants of hybrids F1. The results showed that170bands of ISSR markers were amplified with16suitable primers, the average was10.63.138bands were polymorphic, and the percentage of polymorphism bands reached81.18%. The genetic distance between Caucasian clover and white clover was larger; the hybrid F1was closer to female parents than male parents and there was just a slight variation between F1hybrids and regeneration of F1...