Studies On Isolated Microspore Culture In Non-Heading Chinese Cabbage And Broccoli

The isolated microspore culture technology is useful for crop breeding, which can speed up the breeding processes, shorten breeding periods and get a lot of haploid or homozygous diploid, especially for cross-pollination crops such as non-heading Chinese cabbage and broccoli. It also plays an important role in the research of crop genetics and breeding, genetic analysis, construction of genetic map and molecular markers. Here, we studied the isolated microspore culture in non-heading Chinese cabbage and broccoli to find out the effects of the main factors on the microspore embryogenesis and plant regeneration, to optimize isolated microspore culture system. It is of great significance to speed up the breeding efficiency and promote the research of genetic analysis on Brassica vegetables.The results are as follows,Total of 20 genotypes of non-heading Chinese cabbage and 10 genotypes of broccoli were used to determine the buds length and the correspond developmental stage of microspore and to research the effect of genotypes on non-heading Chinese cabbage and broccoli microspore embryogenesis. It was found that the development of microspore nuclear is asynchronous in a bud, while there is a corresponding relation between the microspore developmental stage and bud length. In general,2～3 mm non-heading Chinese cabbage buds and 3-4 mm broccoli buds were chosen for isolated microspore culture; 13 non-heading Chinese cabbage genotypes and 4 broccoli genotypes were induced to embryos, respectively accounting for 65% and 40%, the highest frequency of microspore-derived embryo were 12.17 embryos per bud and 4.30 embryos per bud repectively. Therefore, the embryogenesis ability of different Brassica genotypes varies significantly.The genotypes of ’LBC-80’,’LBC-82’ and ’LBC-83’ were used to study the effect of density of microspores, activated charcoal and temperature stress on microspore embryogenesis. The density was 1-2 buds/mL and activated charcoal was 0.50mg/mL, which was beneficial for the microspore embryogenesis. Heat shock is essential for embryogenesis, while cold pretreatment is not. Microspore embryogenesis was significantly enhanced using the combination of cold pretreatment and heat shock stress treatment.With the higher rate of microspore-derived embryo genotypes’LBC-82’and’A47-4’ as the materials, the microspore were stained with DAPI (4’,6-diamidino-2-phenylindole) every other day, using fluorescent microscope examination, observed the developmental process of microspore embryogenesis. According to the mode of the first division of microspore embryogenesis, there were A and B developmental pathway in the process of non-heading Chinese cabbage and broccoli microspore embryogenesis. After heat shocking for Id, the volume of most survival microspore increased, a few cells began to occur symmetrical and unsymmetrical division, and the number of division cell increased at the 4th day; with the further development of microspore embryogenesis, the structure of four cell proembryo, multicellular regiment, ball embryo, heart embryo, torpedo embryo and cotyledon embryo would appear one after another; From the 18 day, embryos were visible in the petri dishes.The genotypes of ’LBC-549%’ A36-5’,’A47-4’ and ’A48-3’ were used to study the effect of embryo age, concentration of agar and coconut juice on microspore plant regeneration. The results were shown as follows, embryo age were 20～29 days in liquid NLN medium transferred into B5 solid medium, the plant regeneration rate was higher; the 1% concentration of agar was better than the 0.78% concentration agar, because it is more advantageous to the further development of embryo and plant regeneration; adding 10% coconut juice to the MS solid medium could promote the growth of the regeneration plants from the non-heading Chinese cabbage and broccoli isolated microspore culture, make them strong and root developed, and can effectively avoid the phenomenon of vitrification and defoliation.The study also applied the flow cytometry to identify the chromosome ploidy of regeneration plants. The ploidy analysis results showed that the natural doubling rate of non-heading Chinese cabbage was 100%, the natural doubling of broccoli genotypes ’A36-5’ and ’A47-4’ were also higher, both over 90%, but the genotype ’A48-3’ was very low, only 9.09%.