Shoot Tip Cryopreservation and Elimination of SMV and CMV Virus from Infected Stocks of Pinellia ternata (Thunb.) Breit.

Abstract

Cryopreservation, an effective protocol for long-term conservation, requires minimal maintenance and space. Genetic resource collection of Pinellia ternata (Thunb.) Breit. is essential for breeding and genetic diversity. The purpose of this study is to develop a vitrification cryopreservation protocol and virus-free technology of P. ternata. Shoot tips of dormant buds recovered from cryopreservation yield a regeneration rate of 75.0% and the main steps of the optimized protocol are as following: 0.8 mm-long shoot tips excised from in vitro dormant tubers subjected to cold-acclimation for 14 days, and then treated with LS for 20 min and incubated in plant vitrification solution (PVS2) for 40 min followed by plunging directly into liquid nitrogen. After rapid thawing at 38°C for 2 min, the shoot tips were unloaded in a 1.2 M sucrose solution for 20 min and then inoculated into 1/2MS medium (half-strength salts) supplement with KT 0.5 mg·L−1, NAA 0.2 mg·L−1, 3% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. No obvious phenotypic variation was observed. Inter-simple sequence repeat (ISSR) detected only 0.063% variation bands in 44 cryo-derived plantlets of P. ternata. By means of shoot tips cryotherapy or thermotherapy in vitro plantlets at 35℃ for 4 weeks followed by shoot tips culture for regenerated plants, SMV and CMV could be efficiently eradicated. Thus, cryotherapy would hold great promise as an effective approach for validly eradication of P. ternata viruses, and can be simultaneously applied to long-term collect P. ternata germplasm and produce virus-free plants.