烟草脱外壁花粉人工萌发与离体授粉实验系统的建立

通过花蕾低温处理、花药漂浮培养与花粉短时酶解程序可脱去花粉外壁,分离出烟草(Nicotianatabacum L.)的脱外壁花粉。研究了分离过程中的酶液渗透压、培养基中聚乙二醇(PEG)与蔗糖以及添加水解乳蛋白等因素对脱外壁花粉人工萌发的影响。在含30％PEG-6000与0.1％水解乳蛋白的D_2培养基中,萌发率最高达57.8％;花粉管生长正常,培养24h后一半以上的花粉管中生殖细胞分裂成精子。用微滴和贴滤纸小片的方法将30～40粒脱外壁花粉授予柱头上,近一半能萌发花粉管并在花柱中生长。采取授粉后子房培养方法,获得了种子与幼苗。从而建立了脱外壁花粉离体授粉实验系统。讨论了脱外壁花粉人工萌发与离体授粉实验系统的建立对于研究外壁在花粉萌发中的生物学功能以及开拓新的转基因技术等方面的意义。

ESTABLISHMENT OF AN EXPERIMENTAL SYSTEM FOR ARTIFICIAL GERMINATION AND IN VITRO POLLINATION WITH DE-EXINED POLLEN IN NICOTIANA TABACUM

Tobacco flower buds at mid-binucleate pollen stage were cold-treated and their anthers were then float-cultured to release the pollen, which were subsequently macerated in enzyme solution for a short time. And de-exined pollen grains were eventually isolated. The main factors affecting artificial germination of the de-exined pollen were investigated, including osmotic pressure of the enzyme solution during the isolation process, polyethylene glycol (PEG) or sucrose in the culture medium as well as supplementation of lactoalbumin hydrolysate (LH) . Finally, a medium containing D2 macroelements, 30% PEG-6000 and 0.1% LH was established, which supported the de-exined pollen to germinate well with a frequency up to 57 .8 % . After 24 h of culture, the generative cell in more than half of the pollen tubes, already divided into two sperms. Using a method of micro-suspension droplets with the aid of a small piece of filter paper, 30 to 40 de-exined pollen grains were pollinated onto the stigma, resulting in nearly half of the pollen tubes growing in the style and approximately a yield of one seed out of four de-exined pollen grains after subsequent ovary culture. The seeds were germinated into seedlings. The artificial germination of de-exined pollen can be fur- ther used as a tool for understanding the role of exine in pollen germination. The in vitro pollination with such exine-free pollen might become a new means for introducing foreign genes into the seeds and offsprings.