Study On Somaclonal Variation Of Callus Regeneration System In Betula Platyphalla

In this paper, the leaves of 5 clones of Betula platyphalla were cultured on IS media supplemented with different concentrations of BA. A rapid regeneration system was developed via the establishment of callus. The ploidy of regenerated plantlets from callus was surveyed; and the AFLP polymorphism of genome DNA of the regenerated plantlets from callus was performed. The conclusions were as follows:1.The medium IS+ (3~5mg/L) BA+ 0.5mg/L KT +2%sucrose was the most suitable medium for the induction of callus of Betula platyphalla; The induction rate exceeds 80%. Adventitious buds could form from callus cultured on IS+BA 0.4mg/L+2%sucrose medium; the differentiation rate exceeds 90%.2. Leaf explants of Betula platyphalla directly formed adventitious shoots from wound regions within 1 month; when cultured on IS+ (10.0~15.0mg/L) BA+0.5mg/L KT +2%sucrose for 20 days and then transferred to IS+0.4mg/L BA+2%sucrose medium for 10 days.3. The percentage of diploid cells in regenerated plants decreased rapidly with the increase of BA concentration and culture time; the variation of chromosome number of regenerated plants which induced by 5.0mg/L BA was smaller than others of this experiment; the variation rate was only 40.75%.4. The percentage of polymorphism fragments of genomic DNA increased obviously form 38.73% to 61.33% with the increase of BA concentration; the percentages of polymorphism fragments of regenerated plants which subcultured for 1~5 times were 61.78%, 74.91 %, 76.13%, 72.51% and 78.81% respectively; This indicated the variation of genom was exacerbated with the crease of subculture time.5. The main reasons Betula platyphalla somaclonal variation occurred were high concentration BA and subculture time by the genetic variation analysis on cytology and DNA level.6. 13 pairs of primer were screened for AFLP of Betula platyphalla genomic DNA. AFLP technique was improved to make it suitable for polymorphism analysis of Betula platyphalla genomic DNA.7. Medium IS supplemented with 5.0 mg/L BA +0.5mg/L KT+2%sucrose was the best medium for rapid propagation of Betula platyphalla with small variation in tissue culture.8. The results indicated that the callus, which were cultured for one week was the best material for observed chromosome. It was easier to obtain ideal division cells by the method that callus were treated with 0.2% colchicine for 1.5~2h. The optimum hydrolyze condition was that the callus was soaked in 1N HCl for 10~ 15min or in 2% pectinase and cellulase for 30min at room temperature.9. The results indicated that various concentrations EMS and inducing time had important effect on the survival rate of callus. High concentration EMS (treated for 1 hr. or 2 hr) or low concentration EMS (treated for 5 hr or 6 hr) can lead to half death of callus. Variations in number of chromosomes in callus by mutation treatment were observed. It was found that the ratio of diploid ,aneuploid and polyploid of induced callus were higher than the control. The percentages of abnormal cells increased from 60% to 89.93%, EMS exacerbated the variation in the callus cells.