Fki Lotfi, Bouaziz Neila, Sahnoun Nahed, Swennen Rony, Drir Noureddine, Panis Bart
Laboratory of Plant Biotechnology, Faculty of Sciences, University of Sfax, Sfax, Tunisia.
Cryo Letters. 2011 Nov-Dec;32(6):451-62.
We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.
我们描述了一种针对枣椰树品种‘Barhee’的胚性愈伤组织（PEMs）的高效冷冻保存方案的开发。通过将幼嫩叶片小块接种在添加了每升0.3毫克2,4 - D的MS培养基上诱导产生原胚。如在植株结果后所观察到的，这些体外培养条件能产生与母本一致的植株（即“真实类型”植株）。与标准玻璃化方案相比，超快速滴液玻璃化技术被证明更具优势。蔗糖预培养显著提高了冷冻保存后的恢复率。当胚性愈伤组织在0℃下用PVS2处理30分钟时，冷冻处理后的最高再生率达到63.3%；当在25℃下用PVS2处理15分钟时，再生率为56.7%。冷冻保存的胚性愈伤组织在2至3周后出现了再生的最初迹象。冷冻保存并未影响植物材料的形态发生能力。此外，从冷冻保存的材料中可以建立高度增殖的悬浮培养物。1个月后，500毫克冷冻保存的胚性愈伤组织产生的体细胞胚总数达到1030±50个单位。对从冷冻保存材料再生的枣椰树进行的形态学研究证实了冷冻保存后克隆材料的稳定性。
