Genetic stability of wild <i>Rubus sachalinensis</i> Léveillé cultivated using tissue culture and rapid propagation techniques examined with the ISSR-PCR system

Summary Background – Rapid plant growth and high yield can be achieved via the adoption of tissue culture and rapid propagation systems. Hence, the influence of external factors can be eliminated, and the problems caused by the scarcity of wild resources can be addressed. The comparative ISSR molecular marker analysis of test-tube and wild plantlets can reveal the genetic stability of test-tube plantlets. Thus, it is of great significance for studying germplasm resources, such as those of Rubus sachalinensis Léveillé. This study was conducted to acquire a large quantity of Rubus sachalinensis Léveillé plantlets and provide a basis for its large-scale production. Result – A regeneration system for Rubus sachalinensis Léveillé was established in this study using axillary buds as explants in vitro. Tissue culture seedlings were amplified using PCR with simple repeat intervals. Axillary buds of Rubus sachalinensis Léveillé sterilized in 75% alcohol for 40 s and then in 0.1% HgCl 2 for 8 min were the most effective. Their contamination and browning rates were rather low, and they reached a survival rate of 78.34%. The optimum medium compositions for proliferation, strong plant culture, and root induction were MS + 6-BA 2.0 mg L -1 + KT 0.5 mg L -1 + NAA 0.1 mg L -1 + IBA 0.05 mg L -1 (proliferation coefficient: 7.71), MS + 6-BA 2.0 mg L -1 + KT 0.5 mg L -1 + NAA 0.1 mg L -1 + IBA 0.1 mg L -1 + GA 3 0.5 mg L -1 (proliferation coefficient: 6.60), and ½ MS + IBA 1.5 mg L -1 (rooting rate: 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 following transplantation was 100%. Conclusion – Rubi Ramulus is the dried stem of Rubus sachalinensis Léveillé. It is used to treat cold, immature fever, stroke fever, cough, “heyi” fever and other diseases. Since the twentieth century, with the rapid development of Mongolian medicine industry and the increasing amount of Mongolian medicinal materials, the wild resources are decreasing day by day. It severely restricts the healthy development of Mongolian medicine industry. In order to protect the wild resources, this study established the tissue rapid propagation system for the first time. Using adventitious buds as explants, it can cultivate high quality seedlings in a short period of time. It overcomes the scarcity of wild Rubus sachalinensis Léveillé and protects the ecological environment. At the same time, the tissue culture seedlings with good reproducibility, low mutation rate and high genetic stability were obtained by PCR amplification with simple repeat interval. It not only opened up a new way for the rapid propagation of raspberry tissue, but also laid a foundation for the preservation of fine characters and the protection of resources.