Study on Genetic Stability of Wild Rubus sachalinensis Leveille Based on Tissue Rapid Propagation Techniques and ISSR-PCR Reaction System

Abstract

Abstract Background： By establishing the tissue rapid propagation system, the rapid propagation and large yield were not affected by external factors, and the scarcity of wild resources was effectively solved. Analysis of test-tube plantlets and wild plantlets by ISSR molecular marker technology to detect the stability of genetic characteristics of test-tube plantlets is of great significance to the study of germplasm resources and medicinal value of Leptospiraea japonica. Result： The regeneration system of Rubus sachalinensis Leveille was established in this paper by using axillary buds as explants in vitro culture. The results showed that axillary buds of Rubus sachalinensis Leveille were sterilized in 75% alcohol for 40 s and then in 0.1% HgCl2 for 8 min had the best effect, the pollution and the browning rate of the explants reached a rather low level, with the survival rate of 78.34%. The optimum compositions for proliferation culture, strong plant culture and root inducing were MS + 6-BA 2.0 mgL-1 + KT 0.5 mgL-1 + NAA 0.1 mgL-1 + IBA 0.05 mgL-1, MS + 6-BA 2.0 mgL-1 + KT 0.5 mgL-1 + NAA 0.1 mgL-1 + IBA 0.1 mgL-1 + GA3 0.5 mgL-1 and 1/2 MS + IBA 1.5 mgL-1. The proliferation coefficient and rooting rate on these media were up to 7.71, 6.60, and 97.04% respectively. The most suitable transplanting medium for test-tube plantlets was a mixed medium of humus, vermiculite and perlite ( 2 : 1 : 1 v / v ), and the survival rate of transplanting was 100 %. This study was conducted to acquire a large quantity of wide Rubus sachalinensis Leveille plantlets and provide a basis for large-scale production.The tissue culture seedlings were amplified by PCR with simple repeat interval. Conclusion ：The results showed that the tissue culture seedlings had high genetic stability and low mutation rate compared with wild seedlings.This study not only opens up a new way for the tissue rapid propagation technology of L. japonica, but also lays a foundation for the maintenance of excellent traits and resource protection of L. japonica.