Cryopreservation of Pleione bulbocodioides (Franch.) Rolfe protocorm-like bodies by vitrification

In this study, a vitrification cryopreservation system for the protocorm-like bodies of monolingual orchids was successfully established and the factors involved in cryopreservation by vitrification, including preculture, loading, cryoprotection, cooling, rewarming, and recovery, were investigated. Histological observation, biochemical and genetic analyses were carried out in every step. The results revealed a high survival rate of 93.33% when the protocorm-like bodies were precultured in 0.4 M sucrose for 2 days, then loaded in 50% plant vitrification solution 2 (15% glycerin, 7.5% ethylene glycol, 7.5% dimethyl sulfoxide, 7.5% sucrose and 62.5% liquid MS) at 25 °C for 40 min, and subsequently treated with modified PVS (25% glycerin, 15% ethylene glycol, 2% polyethylene glycol, 13% dimethyl sulfoxide, 15% sucrose and 30% liquid MS) at 0 °C for 50 min, which improved the vitrification efficiency. The samples were rewarmed directly in a water bath at 60 °C for 2 min after storage in liquid nitrogen. The PLBs were then transferred to a solid medium with 1.0 mg L⁻¹ 6-benzyl adenine, 0.2 mg L⁻¹α-naphthylacetic acid, and 0.5 g L⁻¹ activated charcoal. The PLBs were maintained in the dark for 3 weeks before growth in the light in a culture room. Biochemical analyses revealed that the content of soluble protein and sugar increased during the early stage but decreased during cryopreservation. Histological analyses displayed visible nuclei, few damaged cells, and denser cytoplasm in the cryopreserved PLBs. Inter-simple sequence repeat molecular marker analysis revealed no genetic changes in vitrified PLBs, validating the use of this protocol as an efficient long-term conservation option for this species.