[Isolation of sperm cells of Allium tub rosum Roxb].

Pollen grains of Allium tuberosum Roxb broke and released their content including generative cells using osmotic shock method. In a medium containing 0.05% CaCl2, 0.01% Boric acid, 0.01%KH2PO4, 15%PEG 10% sucrose (710 mOsmol/kg H2O) 86% pollen grains germinated and grew out pollen tubes, which broke after transferred into 6% mannitol solution, and released tube content including generative cell. When pollen grains were cultured in the same medium but adding 0.1% casein, a few generative cells divided into two sperm cells. Stigmas of Allium tuberosum Roxb were pollinated at second day after anthesis and the styles grow 3 h in vivo. Then the styles were cut and cultured in a medium for about 6-8 h, some pollen tubes grew out of the cut end of the style. The cut end of the style was transferred into a solution containing 6% mannitol to burst pollen tubes. Pairs of sperm cells of Allium tuberosum Roxb were released and collected using a micromanipulator.

[Isolation of egg cells from Brugmansia aurea Lagerh "Goildens Kornett"].

Viable egg cells of Brugmansia aurea Lagerh "Goildens Kornett" were isolated using mechanical dissection and enzymic digestion. The ovules were cut from middle part and pushed its micropylar position using a dissection needle. Generally the three cells of egg apparatus were released from cut end of ovule. When the ovules put an isolating solution only containing 0.04% CaCl2, 1% BSA and 12% mannitol, 7 egg apparatus could be isolated from 40 ovules within 2 h. However, it is some difficult to separate egg cell from two synergids. When ovules were first incubated in an enzymic solution containing 1% Pectinase (Serva), 1% Cellulase (Onozuka RS) and 12% mannitol for 30 min, and then transferred into the above-mentioned isolating solution to dissect, 8 egg apparatus could be isolated from 40 ovules within 2h and egg cell is easy to separate from two synergids. The isolated egg cells of Brugmansia aurea Lagerh "Goildens Kornett" could be used in vitro fertilization to explore fertilization mechanism and in egg development using molecular methods.

[Anther development and sperm isolation of Pseudostellaria heterophylla (Miq.)].

Anther wall is general and tapetum is glandular. The process of meiosis of microspore mother cells is simultaneous and the tetrads are tetrahedral. The mature pollen of Pseudostellaria heterophylla (Miq.) is tree-celled. There are 22-30 germ pores on the pollen wall. Many pollen grains could burst in 10% mannitol or 15% sucrose solution and release a pair of sperm cells which could keep alive for 25-50 min by FDA fluorescence. Using micromanipulator the released sperm cells could be collected. When pollen grains were put into a solution containing 0.03% CaCl2, 0.01% H3BO3, 0.01% KH2PO4 and 20% PEG for 2-5 min, they would germinate and the pollen tubes would reach 815 microm at 2h after cultured. A pair of sperms would enter into pollen tube when it grew to 500-600 microm. The fluorescence of both sperms would be observed clearly in pollen tube after DAPI staining. When the pollen tubes were burst in a bursting solution, a pair of sperms would be released from pollen tube.

[Cytochemical observation on the developing anthers of Allium cepa L].

Polysaccharides was detected using the periodic-acid-Schiff's (PAS) technique and lipid detected using Sudan black during the anther development of Allium cepa L. Before the meiosis of microspore mother cells there were a few lipid drops in endothecium cells and little polysaccharides in tapetal cells which did not differentiate completely in young anthers. At the stage of tetrad there were still few polysaccharides and lipid material in young anther, and only cell wall of anther wall and callose wall of tetrads displayed the feature of polysaccharids. The size of tapetal cells began to increase at this stage. During microspore development the tapetal cells reached its maximal size, and many lipid drops were accumulated in the cells. However, few lipid drops and starches appeared in microspores. At early stage of 2-celled pollen, the vegetative cell of 2-celled pollen began to accumulate starches. Tapetal cells degenerated at this stage and its lipid drops concentrated to form lipid block. Then the starches in 2-celled pollen disappeared with pollen development, and many lipid drops were accumulated in vegetative cell of nearly mature pollen.