Studies On Anther Culture Of Asparagus Officinalis L.

China is the top producer and exporter of asparagus in the world and demands a large number of seeds every year. However the fine seeds of asparagus for planting still mainly rely on importing. The"all-male"breeding method is usually adopted in Asparagus breeding. To obtain the supermale plants, it will take more than 10 years by the method of cultivating monoecious male plants, but in this study only 2 years by cultivating anthers, which greatly accelerates the asparagus breeding process.In this study, the experiments were conducted on the anther of Asparagus officinalis L. to study how some different factors affected on callus inducement and green shoot. First of all, five fine asparagus varieties were collected as experimental materials, which showed wide adaptability, high yield and disease resistance, and a large number of regenerated plants were obtained. Then regenerated plants'ploidy was detected by the method of shoot-tip chromosome number. For getting double haploid plants, the haploid shoot-tips were treated with colchicine in two different treatments. Also, to study the rootage of regenerated plants, experiments were conducted on four plant growth regulators (IBA, NAA, IAA and S-3307). In addition, the BTB identification method was used to identify the male-asparagus and female-asparagus plants. Finally, this study established a simple and efficient cultivate system of super-male asparagus plants. The major findings are as follows:1. Anther callus inductivityThe experiment analyzed the physiological state, genotype, centrifugae, high-temperature culturing, culture medium complement, illumination density affecting the anther callus inducement. The results showed that different genotypes of asparagus anther callus showed different growth characteristics in the green shoot differentiation medium. Except for the centrifugal treatment could not raise the callus inducement rate, all the other factors were in favour of forming anther callus. 2. Regeneration of anther callus plantThe experiment analyzed the regeneration affected by different kinds of callus, age in days, varieties and the culture modes. The results showed that the callus of inequable age had different differentiation ability. Among different callus age, the 30~40 days showed the highest green shoots regeneration rate. The calluses of different age had different differentiation rate. Different varieties of anther callus had different differentiation rate. Secondary culture could promote the green shoot differentiation and growth.3. Haploid identificationIt's feasible to identify the chromosome ploidy by counting the shoot-tip chromosomes of asparagus anther. The results showed that the chromosomal ploidy changed in the scope of large. There're haploid, diploid, tetraploid and aneuploid in the asparagus anther culture regeneration groups, while the diploid groups took up the majority.The haploid differentiation rate of 28.4 % from dense callus was much higher than that of 5.7 % from loose callus. And 44 haploid lines in four varieties were obtained from five test varieties of regenerated plants.4. Haploid doublingThe shoot-tips of haploid cultures were treated with colchicine in different ways to induce double haploid. Satisfactory results were achieved when shoot-tips cultured on a medium supplemented with 0.05 mg.L-1 colchicine for 15 days or on a medium supplemented with 0.10 mg.L-1 colchicine for 10 days. Their successful inductivity rates were 53.04 % and 52.98 % respectively. The stable diploid plants could be obtained after four generations culture. Ten lines of haploids (DH) in four varieties were obtained in this study.5. Test-tube seeding rootingExperiments were conducted on four plant growth regulators (IBA,NAA,IAA and S-3307) to study the rooting of Asparagus officinalis L. regenerated plants from anther. The results indicated that all the doubled buds were lack of roots and must be cultivated in the rooting culture. The results indicated that adding the admixture of IBA, NAA and IAA the culture could raise the rooting availability. Adding suitable consistency of plant growth retardant S-3307 at 0.1 mg.L-1 could increase the availability rooting rate. The optimum culture medium for rooting was 1/2MS+KT 0.1 mg.L-1 +NAA 0.1 mg.L-1 +IAA 0.5 mg.L-1 +IBA 2.0 mg.L-1+S-3307 0.1 mg.L-1。6. Sex identificationBTB Identification was used to identify the sex of asparagus seedling plants in the early growth period. The color of extraction from female plants of asparagus seedling changed significantly faster than that from male plants. And it took about 6 h for the former. After 6 hours past ,the absorption spectra of extraction from female plants was flat without peak at 620 nm, while that from male plants still remained a peak. From this result it could be concluded that 6 hours after extraction could be choosed as the suitable time for sex identification of seedling plants. By the method of BTB identification, 6 lines to 3 varieties of super-male asparagus plants were obtained from the regeneration green shoots.