Coelho Natacha, González-Benito María Elena, Martín Carmen, Romano Anabela
IBB/CGB, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Faro, Portugal.
Departamento de Biologia Vegetal, Universidad Politecnica de Madrid, Ciudad Universitaria, Madrid, Spain.
Cryo Letters. 2014 Mar-Apr;35(2):119-28.
Thymus lotocephalus is a rare endemic species from the Algarve, Portugal, and is legally protected by Portuguese and European legislation.
The aim is to develop a cryopreservation protocol for T. lotocephalus shoot tips, as an alternative approach for the long-term conservation of this species.
Several methods (droplet-vitrification, vitrification and encapsulation-dehydration) were tested. Conditions regarding the subculture period, cold-hardening and preculture were optimized. Cryopreserved shoot tips were also assessed for their genetic stability using RAPD markers.
Droplet-vitrification presented the best results. The best regrowth of cryopreserved shoot tips obtained eight weeks after rewarming was 67%. This was accomplished with four weeks subculture period of in vitro-donor plants at 25 degree C, preculture of excised shoot tips for one day on MS medium containing 0.3 M sucrose, treatment in PVS2 for 60 min, and MS supplemented with 0.2 mg per L zeatin as recovery medium. The assessment using RAPD markers observed variation at a low frequency and shoots regenerated from cryopreserved apices showed normal development compared to the regular in vitro-grown shoots.
Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.
多头百里香是一种来自葡萄牙阿尔加维地区的珍稀特有物种，受到葡萄牙和欧洲法律的保护。
旨在开发一种用于多头百里香叶尖的冷冻保存方案，作为该物种长期保存的替代方法。
测试了几种方法（液滴玻璃化法、玻璃化法和包埋脱水法）。优化了继代培养周期、低温驯化和预培养的条件。还使用RAPD标记评估了冷冻保存的叶尖的遗传稳定性。
液滴玻璃化法效果最佳。解冻后八周获得的冷冻保存叶尖的最佳再生长率为67%。这是通过将离体供体植物在25℃下继代培养四周、将切下的叶尖在含有0.3M蔗糖的MS培养基上预培养一天、在PVS2中处理60分钟以及使用添加了每升0.2毫克玉米素的MS作为恢复培养基来实现的。使用RAPD标记进行的评估观察到低频变异，与常规离体培养的芽相比，从冷冻保存的顶端再生的芽显示出正常发育。
因此，液滴玻璃化法是一种用于冷冻保存多头百里香叶尖的可行方法。
