Technology Of Asparagus Officinalis L. Anther Culture And Sex Identification Of Plant Regeneration By Molecular Markers

Asparagus as an internationally recognized anti-cancer food, was called“King of Vegetable”in the world because of its nutritional value, the efficacy of anti-cancer, prevention and treatment of high blood pressure and heart disease. Although China was the largest cultivation area of asparagus and the most exports in the world, asparagus seeds in China mainly relied on imports. Due to lack of improved varieties with independent intellectual property rights, it restricted the development of the asparagus industry in China. Asparagus was a dioecious plant, and gender of which was controlled by a pair of alleles. The genotypes of male and female were Mm and mm, respectively. The male types were considered to be the super-male plants. Because of vigorous growth, good quality, high output, long life and strong disease resistance, all-male varieties would be the main direction of the asparagus breeding. To gain asparagus homozygous plants was the key of nurturing all-male varieties. And anther culture was a simple and important way to obtain asparagus homozygous plants. The factors in this study, the season, pretreatment, NAA, and 6-BA, which affected the formation of callusogenesis from asparagus anther was analysed. And an anther culture technology system of‘Apollo’,‘Champion’and‘NJ987’was established. Molecular marker technology was used to identify the sex of regeneration.The main results were as follows:1. Induction of callus from asparagus anther culture:（1） Seasons had effects on the induction of asparagus anther callus. Callus could be induced by anther which was collected in May. But it was failed in July or September.（2） The highest induction frequency period of callus was in the time of buds of 1-2 mm. When the buds were 2-3 mm and 3-4 mm, callus frequency was low. It was failed to induce callus when buds were longer than 4 mm.（3） Different varieties required different low temperature pretreatment. The highest callus formed frequency condition was 4℃, 5 days for‘Apollo’,‘Champion’and‘NJ987’, while‘Grand’was 4℃, 3 days.（4） There were some differences of genotypes in asparagus callus induction conditions. To obtain the highest inductivity and formation of buds naturally, the optimal callus induction mediums of‘Apollo’,‘Champion’and‘NJ987’were MS+2.0 mg/L 6-BA+1.0 mg/L NAA. The best medium of‘Grand’induced callus was MS+2.0 mg/L 6-BA+1.25 mg/L NAA, but no bud was induced. It could not get plant regeneration by callus dedifferentiation further.2. The best subculture medium for‘Apollo’,‘Champion’and‘NJ987’was MS+0.5 mg/L 6-BA+0.2 mg/L NAA.3. The regenerated plants varied in ploidy. 142‘Apollo’and 341‘NJ987’were ploidy identified. The chromosome ploidy of‘Apollo’was 4.93% haploid, 30.99% diploid, 24.65% triploid 15.49% tetraploid and 23.94% aneuploid; and‘NJ987’was 2.64% haploid, 78.89% diploid, 7.04% triploid 4.99% tetraploid and 6.45% aneuploid.4. Gender identification of asparagus regeneration plant with molecular markers:（1） The STS marker system which could identify the asparagus gender was optimized in this study, the best PCR system was 20μL: DNA extraction 3μL, 10×PCR Buffer （MgCl₂）₂μL, dNTP （2.5mM） 0.8μL, Taq DNA enzyme （5U/μL） 0.24μL, Asp1-T7-Pc （10U/μL） 0.2μL, Asp1-T7-Ph （10U/μL） 0.2μL, MgCl₂ （25mM） 0.4μL, ddH₂O 13.16μL. Response procedures were as follows: 94℃for 30 s, followed by 32 cycles of 94℃for 30 s, 47℃for 30 s, 72℃for 40 s; 72℃for 10 min, and preservation at 4℃.（2） In this study, 370 regeneration individuals of asparagus were detected by Asp1-T7. The result was that 251 individuals could amplify the 440 bp fragments which could be identified as male （MM, Mm and M）. The male regeneration plants number of‘Apollo’and‘NJ987’were 148 and 103, respectively.