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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。
The exine pollen of tobacco (Nicotiana tabacum L.) was isolated by cryogenic treatment of bud, anther floating culture and pollen short enzymolysis procedure. The effects of enzyme osmotic pressure, polyethylene glycol (PEG) and sucrose in the medium, and the addition of hydrolyzed milk protein on the germination of detached pollen were studied. In D 2 medium containing 30% PEG-6000 and 0.1% hydrolyzed milk protein, the germination rate was up to 57.8%. The pollen tube grew normally, and more than half of germ cells in pollen tube were split into sperm after culturing for 24h. Thirty to forty de-exserted pollen was applied to the stigma with microdroplets and filter paper pieces, with nearly half able to germinate and grow in the style. After pollination, ovary culture method was used to obtain seeds and seedlings. Thus, an ex-pollen experiment system of ex-pollen wall pollen was established. The establishment of an experimental system for artificial germination and pollination in vitro pollination was discussed in order to study the biological functions of exine in pollen germination and explore the new transgenic technology.