Studies On Genetic Variation In The Process Of Somatic Embryogenesis In Litchi"Litchi Chinensis Sonn.

The mechanisms of genetic variation of the in-vitro embryogenic cultures in the process of somatic embryogenesis were studied through cytological markers, biochemical markers and molecular markers in litchi [Litchi chinensis Sonn.], which could provide new clues for the study of the mechanism of somatic embryogenesis and somaclonal variation, and also provide scientific information for overcoming vitrified embryoids, abnormal embryoids and the low inversion rate of somatic embryos in litchi.Embryogenic and nonembryogenic calli were induced and screened from the culture of young embryos and anthers in the Litchi cultivars "Yuanhong"and "Xiafanzhi". The chromosome numbers of the nonembryogenic calli, embryonic calli, somatic embryos, and the root-tips of abnormal and normal plantlets were examined by the routine method combined with a simplified method using the alcohol-HCl (1:1) resolution for direct lysis of tissue and cell and using methylacetic acid-iron-haematoxylin for staining. The results showed that the variation of the chromosome numbers existed in all the above cells ,tissues or organs. Besides the normal chromosome number of 2n=30, some abnormal cells with the chromosome numbers of 2n= 15-29 or 2n=31-60 occurred. The results also showed that it was difficult to obtain the stable cell clones in which the chromosome numbers were stable in a number through the screening from embryogenic calli; however, some cell clones with relatively stable chromosome numbers in a narrow range were obtained.In this experiment, three cell lines were obtained. They were Cell Line I ( most cells with the normal chromosome number 2n=30), Cell Line II(most cells with the chromosome numbers 2n<30) and Cell Line Ill(cells with the chromosome numbers 2n=15 ~ 60). Different plant growth regulators, the time of subcultures, genotypes and explants had some effect on the numbers of the chromosomes.The POD and EST isoenzymatic patterns were analyzed by the discontinuous polyacrylamide gel electrophoresis (PAGE), The differencesof the numbers of the belts and strength of the belt activity existed in the tree types of the screened cell lines; moreover, there also existed some differences in POD and EST isoenzymatic patterns among the different embryoids which were induced from the same cell line.The results of randomly amplified polymorphic DNA (RAPD) showed there were differences between embryogenic and nonembryogenic calli, vitrified embryoids, white embryoids and the three screened cell lines, which proved that there existed the variations of genetic substance during the process of subcultures and differentiation of embryogenic calli in litchi.In a word, there existed wide genetic variations in the chromosome numbers, biochemical and molecular markers among the embryogenic calli and these variation also existed during the process of somatic embryogenesis from embryogenic calli to embryoids in litchi. Therefore, we considered that the key factor that influenced the regeneration of litchi somatic embryogenesis was the existence of the wide variations in the embryonic cells and the transfer characteristics of these variations in litchi.