Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient
Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient
复制标题
DOI：
10.1038/nprot.2016.107
复制
发表时间：
2016-10-01
期刊：
影响因子：
14.8
通讯作者：
Honys, David
中科院分区：
文献类型：
--
作者：
Dupl'akova, Nikoleta;Dobrev, Petre I.;Honys, David
研究雄配子体(MG)发育的动力学已被证明是植物科学界具有挑战性的。在这里，我们描述了我们分离拟南芥MG发育阶段的方案，该方案基于通过不连续的Percoll浓度梯度对花粉进行离心法。该Percoll渐变可以使用吸管形成，并且它不需要渐变生成器。分离的发育孢子纯度高达70%，大多数分离的纯度远高于80%。利用这一方法，我们可以将花粉发育的四个不同阶段--单核小孢子(UNM)、双细胞花粉(BCP)、三细胞未成熟花粉(TCP)和成熟花粉粒(MPG)分开。分离过程的持续时间(不包括花序的切割)为6h，如果在Percoll密度分离之前使用真空清洗法去除MPG，则分离过程缩短至4h。
Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.