Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen
Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen
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DOI：
10.1007/s00299-008-0647-1
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发表时间：
2009-03-01
影响因子：
6.2
通讯作者：
Geitmann, Anja
中科院分区：
文献类型：
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作者：
Daher, Firas Bou;Chebli, Youssef;Geitmann, Anja
模式植物拟南芥的一个罕见的弱点是与其雄配子体的萌发和花粉管的体外产生有关的技术问题。拟南芥花粉是三细胞的，与具有双细胞花粉的物种相比，其体外萌发率低得出了名。这一缺陷严重影响了基于该细胞系统的实验的重复性。再加上从该物种采集花粉的繁琐，这些都阻碍了拟南芥花粉的标准使用，因为这些实验需要大量的花粉管，而且萌发率需要高度重复性。在大量采集花粉后冷冻储存花粉的可能性是解决这些问题的潜在方法，但需要确保持续存活和可再生的萌发能力的方法。本研究的目的是对冷冻保存的拟南芥花粉萌发条件进行优化。我们优化了常规用于花粉体外萌发的各种培养基组分的浓度。我们发现，一般情况下，4 mM的钙、1.62 mM的硼酸、1 mM的钾、1 mM的镁、18%的蔗糖和22.5摄氏度的温度是花粉萌发的最佳条件。然而，不同的实验装置可能会偏离这一通用协议的要求。我们建议如何将这些优化的方法最佳地用于不同的实际实验，从光学和电子显微镜对花粉管的形态观察到它们大量用于分子和生化分析，或者用于特定介质硬度至关重要的实验装置。
One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5A degrees C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical.