Cryopreservation method of rhynchostylis retusa pollen

A technology of ultra-low temperature preservation and drilling beak orchid, which is applied in the fields of plant preservation, botanical equipment and methods, and application, can solve the problems of inability to meet breeding needs, decreased pollen vigor, low seed setting rate, etc., and achieves good antifreeze effect and enhanced Vitality, good drying effect

Active Publication Date: 2017-11-24
DONGGUAN AGRI SCI RES CENT
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Problems solved by technology

At present, there is no report on the long-term storage method of orchid orchid pollen. Using the conventional pollen preservation method of orchids, that is, the pollen pieces are dried in anhydrous calcium chloride and stored in a -20°C refrigerator. Orchid pollen can only be kept for a period of time under such storage conditions, and its vitality will plummet after about 1 month. If there is a difference of more than 1 month between the flowering

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  • Cryopreservation method of rhynchostylis retusa pollen

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Experimental program
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Effect test

Embodiment 1

[0026] S1.采粉和干燥:在天气晴朗的上午9:00~11:00,取开花当天的新鲜花粉块放入茶叶袋中,然后置于放有变色硅胶的培养皿中,在4℃条件下放置2h。

[0027] S2.冻存:将步骤1的花粉放入2ml冻存管中,加入1ml的预处理液(含有0.4M蔗糖、2M甘油的BK无机盐储备液,pH5.7),在室温下(27±2℃)静置10min,然后将预处理液吸出,加入1ml改良的玻璃化溶液MPVS2 [30% (w / v)甘油+15% (w / v)乙二醇+10% (w / v)丙二醇+5% (w / v)DMSO,用含有0.4M蔗糖的BK无机盐储备液配制,pH5.7],投入液氮保存。

[0028] S3.解冻:冻存的钻喙兰花粉在使用时先将冻存管取出,置于室温下(27±2℃)或用自来水冲洗30min,然后将冻存管中的原有的MPVS2溶液吸出一半,并加入相应体积的冲洗液(含有1.2M蔗糖的BK无机盐储备液),静置5min,如此重复3次。解冻后的花粉可按常规方法直接用于授粉。

Embodiment 2

[0030] S1.采粉和干燥:在天气晴朗的上午9:00~11:00,取开花当天的新鲜花粉块放入茶叶袋中,然后置于放有变色硅胶的培养皿中,在0℃条件下放置1h。

[0031] S2.冻存:将步骤1的花粉放入2ml冻存管中,加入1ml的预处理液(含有0.4M蔗糖、2M甘油的BK无机盐储备液,pH5.7),在室温下(27±2℃)静置10min,然后将预处理液吸出,加入1ml改良的玻璃化溶液MPVS2 [30% (w / v)甘油+15% (w / v)乙二醇+7.5% (w / v)丙二醇+7.5% (w / v)DMSO,用含有0.4M蔗糖的BK无机盐储备液配制,pH5.7],投入液氮(LN)保存。

[0032] S3.解冻:冻存的钻喙兰花粉在使用时先将冻存管取出,置于室温下(27±2℃)或用自来水冲洗30min,然后将冻存管中的原有的MPVS2溶液吸出一半,并加入相应体积的冲洗液(含有1.2M蔗糖的BK无机盐储备液),静置5min,如此重复3次。解冻后的花粉可按常规方法直接用于授粉。

Embodiment 3

[0034]1. Collecting pollen and drying: From 9:00 to 11:00 in the morning when the weather is fine, take the fresh pollen block of the flowering day and put it in a tea bag, then put it in a petri dish with color-changing silica gel, and keep it at 4°C Leave it for 3h.

[0035] 2. Cryopreservation: put the pollen in step 1 into a 2ml cryopreservation tube, add 1ml of pretreatment solution (BK inorganic salt stock solution containing 0.4M sucrose, 2M glycerol, pH5.7), and store at room temperature (27± 2°C) for 10 minutes, then suck out the pretreatment solution, add 1ml of modified vitrification solution MPVS2 [30% (w / v) glycerol + 15% (w / v) ethylene glycol + 10% (w / v) Propylene glycol + 5% (w / v) DMSO, prepared with BK inorganic salt stock solution containing 0.4M sucrose, pH5.7], put into liquid nitrogen (LN) for storage.

[0036] 3. Thawing: Take out the cryopreservation tube before using the frozen orchid orchid pollen, place it at room temperature (27±2°C) or rinse it with...

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Abstract

The invention discloses a cryopreservation method of rhynchostylis retusa pollen. The cryopreservation method comprises the following steps: collecting and drying the pollen, freezing and thawing. In the pollen drying process, the pollen is placed in a breathable bag, a drying agent is isolated from the pollen to prevent pollen pollution, the breathable bag is good in breathability, and the drying effect is very good; furthermore, the concentration of DMSO in a vitrifying solution PVS2 is reduced, propylene glycol is added on the basis of original components of the PVS2, and the propylene glycol is an anti-freezing agent with permeability and low toxicity. Therefore, by the cryopreservation method of the rhynchostylis retusa pollen, provided by the invention, the pollen vitality can be effectively guaranteed for a long time; the cryopreservation method is simple and easy to implement; the pollen can be taken out and thawed for pollination at any time, so that the cryopreservation method has an important practicing and promotional significance in breeding and production of rhynchostylis retusa.

Description

technical field [0001] 本发明属于超低温保存技术领域,具体地,涉及一种钻喙兰花粉的超低温保存方法。 Background technique [0002] 钻喙兰( Rynchostylis retusa )是兰科(Orchidaceae)树兰亚科(Epidendroideae)万代兰族(Vandeae)指甲兰亚族(Aeridinae)钻喙兰属( Rynchostylis )的一种附生型兰花,它花色淡雅且具芳香,小花密集,花序饱满而下垂,形似一条毛茸茸的狐狸尾巴,因此其商品被称为狐狸尾兰。钻喙兰主要分布于南亚、东南亚诸国以及我国的贵州和云南。 [0003] 钻喙兰具有很高的观赏价值,也是育种的好材料,但钻喙兰在育种中的利用率远不如同属下的海南钻喙兰(( Rynchostylis gigantea ). 目前在英国皇家园艺学会(RHS)上登录的以钻喙兰属植物为亲本之一的30个杂交属、326个杂交种中,只有很少的一部分是由钻喙兰与其近缘属下的种和品种之间杂交获得的。 导致这个状况的原因之一就是钻喙兰的花粉不易保存。 钻喙兰的花粉寿命很短,在离开植株体后自然条件存放的花粉其活力直线下降,12小时后花粉萌发率降至10%以下,而在杂交育种过程中,常会出现花期不遇的情况,给杂交育种工作带来很大的不便。 [0004] 兰科植物的花粉外包裹着类脂膜,因此花粉粒粘连成花粉块,而不同物种的花粉粒在大小、含水量等方面存在差异,导致不同种类的花粉在保存条件上也会有区别。目前尚没有关于钻喙兰花粉长期保存方法的报道,利用常规的兰科植物花粉保存方式,即将花粉块在无水氯化钙中干燥后置于-20℃冰箱中冷冻保存,我们发现钻喙兰的花粉在这样的保存条件下只能保持一段时间,约1个月以后其活力直线下降,如在实际操作中遇到与另一个种或品种花期相差1个月以上,常规的保存方法将无法满足育种需求。而在大多数植株组织、花粉超低温保存中使用的冷冻保护剂PVS2无法在钻喙兰花粉的超低温保存中发挥很好的效果,利用PVS2进行超低温保存后我们发现花粉活力下降较快,授粉后结实率也很低。因此,我们需要一种效果良好的长期保存技术来克服钻喙兰育种中存在的时间空间限制。 Contents of the invention [0005] 本发明的目的是为了克服现有...

Claims

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Application Information

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IPC IPC(8): A01N3/00
CPCA01N3/00
Inventor 谢佩吾马锞李汉文李早文伍爱萍江南
Owner DONGGUAN AGRI SCI RES CENT
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