兰花花粉的 V 型 cryo-plate 法冷冻保存。
Cryopreservation of Orchid Pollinia Using the V Cryo-Plate Method.
机构信息
Department of Plant Science, Textile and Design, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin, Thailand.
Department of Science and Mathematics, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin, Thailand.
出版信息
Cryo Letters. 2021 Jan-Feb;42(1):25-32.
BACKGROUND
Preserving pollinia viability and fertility for pollination at specific times of the year is very important in orchid breeding. One possible method is cryopreservation using aluminium cryo-plate which is increasingly used for the long-term storage of plant genetic resources.
OBJECTIVE
To determine a V cryo-plate method for the cryopreservation of orchid pollinia and apply it to some Thai orchid species for breeding.
MATERIALS AND METHODS
Pollinia of Rhynchostylis gigantea (L.) Ridl. were collected from completely open flowers in the morning and then were placed on aluminium cryo-plates embedded in alginate gel, then immersed in loading solution containing 2 M glycerol and 0.4 M sucrose for 15 min at room temperature (29oC), and then dehydrated with PVS2 solution for 0-60 min at 29oC. The cryo-plates with pollinia were directly plunged into liquid nitrogen for 40 min, and rapidly warmed in 1 M sucrose for 15 min. The cryopreserved and non-cryopreserved pollinia were used for hand-pollinating flowers of the same species for producing hybrids.
RESULTS
The viability of non-cryopreserved and cryopreserved pollinia dehydrated with PVS2 was 100%. The highest capsule set after pollinating flowers with cryopreserved pollinia dehydrated with PVS2 for 40 min was also up to 100%. The protocol for cryopreservation of R. gigantea (L.) Ridl pollinia using V cryo-plate method was successfully applied for the cryopreservation of nine Thai orchid species. The exposure time to PVS2 affected the pollinia viability (range 40-100 %; average 93%) and capsule set (range 20-100 %; average 78%) of the nine species. The successful capsule set and seed production after pollination with cryopreserved pollinia in all orchid hybrids were observed. Seed germinated into protocorms and developed to plantlets cultured on modified Vacin and Went (1949) agar medium.
CONCLUSION
Cryopreservation of freshly collected orchid pollinia using V cryo-plate method is an efficient tool for the long-term storage of plant germplasm and for orchid breeding.
背景
在兰花育种中，将花粉活力和生育力保存到一年中的特定时间非常重要。一种可能的方法是使用铝制 cryo 板进行 cryopreservation，该方法越来越多地用于植物遗传资源的长期储存。
目的
确定一种 V 型 cryo 板方法，用于兰花花粉的 cryopreservation，并将其应用于一些泰国兰花物种的育种。
材料和方法
从完全开放的花朵中收集 Rhynchostylis gigantea (L.) Ridl. 的花粉粒，然后将其放置在嵌入藻酸盐凝胶的铝制 cryo 板上，然后在室温（29°C）下将其置于含有 2 M 甘油和 0.4 M 蔗糖的加载溶液中 15 分钟，然后用 PVS2 溶液在 29°C 下脱水 0-60 分钟。将带有花粉粒的 cryo 板直接浸入液氮中 40 分钟，然后在 1 M 蔗糖中快速升温 15 分钟。使用未经 cryopreservation 和经 PVS2 脱水的 cryopreserved 花粉粒对同种花朵进行 hand-pollinating，以产生杂种。
结果
未经 cryopreservation 和经 PVS2 脱水的 cryopreserved 花粉粒的活力均为 100%。用 PVS2 脱水 40 分钟的 cryopreserved 花粉粒授粉后，花粉囊的设置率也高达 100%。使用 V 型 cryo 板方法对 Rhynchostylis gigantea (L.) Ridl. 花粉粒进行 cryopreservation 的方案成功应用于 9 种泰国兰花物种的 cryopreservation。暴露于 PVS2 的时间影响 9 种物种花粉粒的活力（范围为 40-100%；平均值为 93%）和花粉囊的设置（范围为 20-100%；平均值为 78%）。观察到所有兰花杂种授粉后 cryopreserved 花粉粒的成功花粉囊设置和种子生产。种子在改良的 Vacin 和 Went（1949）琼脂培养基上培养的 protocorms 中发芽并发育成幼苗。
结论
使用 V 型 cryo 板方法对新鲜采集的兰花花粉粒进行 cryopreservation 是长期保存植物种质资源和兰花育种的有效工具。
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