多种被子植物花粉生殖细胞大量分离技术的比较研究

从11种植物的成熟花粉粒中分离出大量生活的生殖细胞。比较了四种分离方法(一步渗透压冲击法、二步渗透压冲击法、低酶法和花粉原生质体释放法)在不同植物中的效果。归纳出三类植物适于采用三种分离方法。研究了影响分离效果的若干重要因素。对5种植物的分离生殖细胞进行了纯化。经鉴定,纯化的细胞群体中80%以上的细胞是生活的。

A COMPARATIVE STUDY ON METHODS FOR ISOLATION OF GENERATIVE CELLS IN VARIOUS ANGIOSPERM SPECIES

Four methods for isolating generative cells from pollen grains, named one-step osmotic shock, two-step osmotic shock, weak .enzyme treatment and isolation via pollen protoplast preparation, were compared among 11 angiosperm species belonging to 6 families. The result revealed that each method was effective only for a group of species. The method of one-step osmotic shock is adaptable to species as Gossypium hirsutum whose pollen grains are easy to burst in a low osmotic sucrose solution. The two-step osmotic shock method is suitable for species as Nicotiana tabacum, Zephyranthes candida, Z. grandijlora, Lycoris radiata and Impatiens balsamina whose pollen grains are characterized by easy germination in sucrose solution. A sudden osmotic shock by addition of water just at the beginning of germination causes the pollen to burst. In Iris tectorium, Gladiolus gandavensis, Hemerocallis minor, Allium tuberosum and Hippeastrum vittatum pollen grains neither burst nor germinate well in simple sucrose solution, but weak enzyme treatment promotes bursting of pollen grains in large quantity. Pased on this result, it is assumed that the responce of pollen grains in sucrose solution could be used as a criterion for choosing suitable method for isolation of generative cells in a given plant species. Besides, the fourth method of isolating viapollen protoplast preparation was examined. Although it is adaptable to certain species, the isolation efficiency is not high enough. Factors affecting on the isolation result, such as pollen developmental stage, medium components, sucrose and cnzyme concentration, duration of pollen incubation, quantity of water added to were studied as well. Viability test by fluorescence microscopy showed over 30% cells were viable after purification procedure.