Anther Culture And Microspore Culture Of Asparagus Officinalis L

Garden asparagus (Asparagus officinalis L.) is the most important vegetable in the world because of its high value and good nutrition. In this study,40different varieties of A. officinalis L.from the Third International Asparagus Cultivar Trial were used as materials to study the asparagus microspore culture and anther culture technologies. One of the objectives is to determine the growth development relationship of asparagus microspore and buds or anther size and shape. Discussions were made on the factors effecting induction rate of microspore male nuclear development, including Varieties, pretreatment, hormone, and cultivation conditions. Moreover, the formation technology of regenerated seedlings from embryoids directly through anther culture was studied. Identification of the ploidy was conducted on the regenerated plants through microscope and metheds were developed for seedlings transplanting. The results were as follows:1. Asparagus microspore development period closely related with the external morphology of flower bud and anther color.The average length of flower buds’ longi-tudinal diameter, horizontal diameter, and anthers was2.099～2.284mm,1.749～1.891mm, and1.149～1.225mm respectively when microspores were in uninucleate ecce-ntric stage for the five tested asparagus varieties. It’s possible to identify the microsp-ore development period based on the bud morphological characteristics, which is hel-pful for selection of the best flower buds for microspore and anther cultures.2. The efficient cultivation system of forming asparagus embryoids induced by microspore was constructed.Bud pretreatment for4d at4℃, can significantly improve the microspore regeneration frequency. For harvesting pure and clean microspores, it was a high efficient way to extrud anther and centrifuge once with low speed (300r/min). Viability of free microspores were different among different asparagus varieties. The culture efficiency was also affected by varieties of A. officinalis L., B23was best in all tested Varieties. However, the microspore viability was also changeable in different growth period even the same variety. The B23, which had best embryoid formation rate, had highest microspore viability only in anther culture. Moreover, our results indicated that it was helpful for keeping microspore vitality in4℃, and dedifferentiation at32℃for2-3d condition. The efficiency was0.6embryoid/bud in the optimized culture system with normal embryoid development as well as high survival rate of seedlings.3. The media for anther culture had significant effects on embryoid formation and seedling survival rate for different Asparagus varieties.Variety was the key actors for embryoids induction and seedlings formation rate in anther culture. Hormone was another important factor. The embryoid formation rate and seedlings survival rate of B23were16.7%and20.0%respectively which were the highest in five different asparagus varieties (B9, B10, B23, B33, and B14). The optimal induced concentrations of hormone were different among different varieties. It was important to figure out types and concentration of the hormone combination for anther culture. The appropriate hormone combination was0.1mg/L NAA+0.5mg/L2,4-D+0.5mg/L6-BA for B9, B10, and B23. While,0.1mg/L NAA+0.5mg/L2,4-D+0.1mg/L6-BA was suitable to B14, and0.5mg/L NAA+0.1mg/L2,4-D+0.5mg/L6-BA for B33.4. There was diversity in ploidy of regeneration seedlings for microspore and anther culture, such as haploid, diploid and tetraploid. The haploid differentiation rate was different between microspore and anther culture. It was also different between embryoid and callus culture. The differentiation rate of embryoid (42.2%) was far higher than and callus tissue regeneration plant (12.5%). Total91haploid plants were identified from all regeneration plants of8asparagus varieties in this study.5. Established the seedlings adaptation procedures for A. officinalis L. anther culture and microspore regeneration seedlings.The procedures for seedlings adaptation were established:transfer the seedlings in the bottles, then out-of-bottle adaptation and followed by adaptation in greenhouse conditions. It was necessary for seedlings to go through out-of-bottle adaptation for1-2days before transplanting. The established ration of matrix components for transplanting was as follows:garden soil:peatmoss:river sands:perlite was3:1:1:1. In this condition, the survival rate of regenerated seedlings was more than95%, and the seedlings grew well with root system developed.