Abstract
In the present study, a vitrification method for shoot tip cryopreservation and virus eradication in P. ternata is described as follows: After 7 days of cold acclimation of second-generation in vitro tubers in dormancy, shoot tips (0.8-1 mm in length) were excised from in vitro dormant tubers, transferred to cryovials, and treated with Loading Solution (LS) for 20 min. Subsequently, the shoot tips were incubated in Plant Vitrification Solution (PVS2) for 40 min, followed by direct immersion in liquid nitrogen (LN) along with the vitrification solution. After rapid thawing at 38 °C for 2 min, the shoot tips were immersed in Unloading Solution for 20 min, then transferred to 1/2 MS medium (half-strength salts) supplemented with Kinetin (KT) 0.5 mg·L(- 1), α-Naphthylacetic acid (NAA) 0.2 mg·L(- 1), 3% (w/v) sucrose, and 8 g L(- 1) agar (pH 5.8) for recovery. Morphologically, plantlets regenerated from cryopreserved shoot tips were identical to non-cryopreserved controls. Inter-Simple Sequence Repeat (ISSR) analysis revealed a mere 0.063% band variation among 44 cryopreserved plantlets. Attempts were also made to eliminate SMV and CMV by shoot tip culture for twice, cryotherapy, and thermotherapy combined with shoot tip culture. All methods received high survival rates and regeneration rates (over 70.0%). Frequency of virus-free plantlets produced by cryotherapy was 85.7% for SMV and 57.1% for CMV, which were higher than by shoot tip culture for twice (80% for SMV and 40% for CMV) and three treatments of thermotherapy followed by shoot tip culture, including 35 °C for 4 weeks (88% for SMV and 48% for CMV), 38 °C for 2 weeks (85.7% for SMV and 28.0% for CMV) and 35 °C for 2 weeks (85.7% for SMV and 14.3% for CMV). This technology can simultaneously support production of virus-free plants and long-term conservation of P. ternata germplasm, which is critical for agricultural production and breeding. Moreover, cryopreservation of shoot tips dissected from dormant buds proves to be a viable strategy for addressing low regeneration rates post-cryogenic treatment.