Abstract:

Isolated microspore embryogenesis of Chinese kale was observed by an improved DAPI fluorescence staining technology and the effects of 2,4-D on the embryogenesis of six genotypes were compared. In addition,the ploidy and seed setting of regenerated plant lines obtained were identified. The results showed that B（equal division）pathway was the main pathway of microspore embryogenesis in Chinese kale and A（unequal division）pathway also occurred. Once the microspore nucleus undergoes asymmetrical division or symmetrical division accompanied by unequal division of cytoplasm at first time,its polarity can be established,and the formation of various embryoids seems to have their own developmental routes. The effect of 2,4-D on embryo yield varied with genotype. 0.01-0.1 mg · L^-1 2,4-D had no significant effect on the yields of all type microspore embryos in three genotypes：‘Lubao’,‘Qiancui’and‘Zemei’. 0.1 mg · L^-1 2,4-D significantly reduced the cotyledon embryo yield of ‘Xiacui’,but did not affect the yield of other type embryoids. The addition of 0.05 and 0.1 mg · L^-1 2,4-D significantly increased the yields of globular and torpedo embryo of ‘Shunbao’,respectively,and therefore their total embryo yield increased significantly. For‘Shunbao 2’,which has strong ability of embryo formation,the yields of heart-shaped,globular,other type and total embryos increased significantly with the increase of 2,4-D concentration,in contrast,the yield of cotyledon embryo decreased significantly. The addition of 2,4-D had no significant effect on the microspore initiation division of‘Shunbao 2’,but could maintain or improve the re-division ability of the cells and lead to a reduction of the degradation or abortion during the process of embryogenesis,thus increasing the embryo yield. Flow Cytometry（FCM）detection of 150 regenerated plant lines revealed that 72.7% were haploid lines,22.7% were DH lines,and other lines including aneuploid and chimera only accounted for the remaining 4.6%. DH lines or haploid and diploid chimeras could set seed normally after pollination at bud stage,while other aneuploids（including chimeras）could not produce seeds or had a very low seed setting rate. In order to reduce the cost of FCM detection,haploid,DH and haploid + diploid chimera can be determined according to the presence or absence of pollen,the seed setting after bud pollination,leaf morphology and growth potential,and then the ploidy of other regenerated plants which have pollen but no seed or have poor seed can be identified by FCM.

Key words: Chinese kale, isolated microspore, embryogenesis pathway, 2,4-D