Study On Cross Breeding Technology Of Stylo And Related Reproductive Biology

There are many ways to cultivate new varieties of forage, but to Stylosanthes, a kind of cleistogamy tropical forage, hybridization is still the effective one. However, until now, there was no report of hybridization method of stylo. To establish a plant form of stylo variety cultivating with high-yield, high-quality and strong resistance traits, the specific time and method of hybridization was determined by measuring the pollen vitality、stigma receptivity, and the phenotypes of F1 of hybrids were observed with whole stain-clearing technique.The results were as following:(1) Germination in vitro is the best method to indentify pollen viability of stylo. The medium for germination was:10% Sucrose,10% PEG-6000、0.01% H3BO3、0.03% CaCl2·2H2O、 0.01% KNO3、pH 6.5. Cultured in 30℃ for 10 min, it showed the pollen with a high viability over 80% from 10:00 to 11:00. The pollen collected at 10:00 can be conserved 84 h in 4℃, but only 48 h in room temperature. The air temperature had an impact on pollen viability. The viability is just 34% in a gloomy day with a low temperature of 21℃ at 10:00, but can reach 84% in a sunny day at 28℃.(2) Benzidine-hydrogen peroxide was used to measure the stigma viability. The vibrant period was from pre-flowering 20 h to after-flowering 9 h, and at the times flower in full bloom at 10:00～12:00 stigma showed the highest viability.(3) Based on the results above, September to December were determined as the best months for fertilization. Since the day-time highest temperature of November in Hainan can reach 29 ℃, the plants need a short stay indoor after fertilization. The best castration time is 18:00～20:00 in one day before flower bloom, as the bud swells and pollen is not loose at that time.(4) Blooming process and time of stylo907 is following:under the condition of average daily temperature 28℃, the flower firstly open at 7:00, but standard and wing neither open at that time. Until 8:00, the standard opened, and then followed by wing at 10:00. A number of flowers exposed stigma at 13:00, and all the opened-flowers were withered before 16:30. The statistics of flower-opened number and seed number showed 6-15 flowers opened and 3-8 seeds were got in each inflorescence. The spike of Stylosanthes is complex, and each small flower was coated with a bract. The outmost bract coating flower was defined as the first flower, inside turn as second flower, third flower and so on. Under the natural conditions:the seed ratio of second flower is 76.7%,40% and 13.3% of the fifth and ninth, respectively; under the condition of artificial hybridization, the seed ratio is:60%,46.7% and 3.3% of the second, fifth and ninth, respectively. During 2011-2012,7 hybrid combinations were carried out, and then 107 seeds were obtained. Parts of seeds were conserved and the other was planted in field.(5) Microscopic observation of Stylo hybrid embryos was applied after pretreatments including Carnoy’s Fluid (95% Ethanol: Glacial acetic acid=3:1) fixing, Ehrlich’s Hematoxylin dye and Methylis salicylas. After flower bloom,, the zygote can be distinguished in 1 day, then developed into a multi-cellular pre-embryo in 3 day, a globular embryo in 5 day, a heart-shaped embryo in 7 day, and turned to a Torpedo-shaped embryo at last around 10 day. The embryo development also affected by temperature, under the condition of, it takes 10 days to develop to Horseshoe-shaped embryo in 29℃, but 15 days in 22°.(6) ITS sequence was used to identify the Stylo Anthrax pathogen isolates. Ribosomal DNA-ITS sequence comparing analysis of the pathogens isolated from Stylo.39 and Stylo.80 showed: excluding the primer region, the Ribosomal DNA-ITS sequence length of these two strains were 576bp and 574bp, respectively. The results of BLAST in GenBank (NCBI) indicated it had a 100% homology to the strain Colletotrichum gloeosporioides.(7) The flowers of hybrids from the parent plants stylo 1979(male sterility, yellow-flower) and the stylo45 (fertile, white-flower) were yellow. All 10 Fl generation from Stylo 1979 have a ratio of fertility and sterility 4:6. Inoculated 7 kinds of Stylo with spores suspension of the pathogen isolates. The morbidity of stylo 39 is 100, stylo 907 is 17.3, but the hybrids from stylo 39 and stylo 907 is 63.5.