Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong, 793022, Meghalaya, India.
Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong, 793022, Meghalaya, India.
Cryobiology. 2019 Apr;87:60-67. doi: 10.1016/j.cryobiol.2019.02.003. Epub 2019 Feb 15.
Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ± 0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ± 0.5%) with the incorporation of 30 μM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%-63%, providing a suitable technique for storage of this threatened orchid.
兰花及其可持续性是需要全球保护努力的重要问题。硬叶兜兰（一种濒危兰花）是兰花中过度开发的物种之一，被列入 IUCN 红色名录和 CITES 附录 I。随着改进的冷冻保存技术的发展，该物种的保护和商业化前景将得到加强。本研究报告了使用玻璃化（Vit）和包埋-玻璃化（E-Vit）技术成功冷冻保存硬叶兜兰原球茎的情况。该研究比较了在预处理和恢复阶段添加四种抗氧化剂、三种生长培养基、搅拌和半固体培养基对初始恢复的影响。Vit 的冷冻保存恢复率为 27%，E-Vit 为 37%。在这两种情况下，搅拌培养可使恢复率提高约 10%，但 E-Vit 的恢复率明显更好。控制组的最佳恢复率（51.2±0.9%）记录在使用 0.5 M 蔗糖预培养的包埋原球茎中，用 PVS2 处理 45 min，在 1/2 MS（L/S）液体培养基中在搅拌下恢复 10 天，然后转移到半固体培养基中。通过在液体预培养和液体和半固体再生长培养基中加入 30 μM 谷胱甘肽，进一步提高了恢复率（62.7±0.5%）。该新方案将 E-Vit 冷冻保存的恢复率从最初的 37%-63%提高到了 63%-63%，为这种濒危兰花的贮藏提供了一种合适的技术。
