紫菜苔精细胞的大量分离和生活力保存

MASS ISOLATION AND PRESERVATION OF VIABLE SPERM CELLS IN BRASSICA CAMPESTRIS VAR.PURPUREA

本工作改良了原有的渗透压冲击法和研磨法,从紫菜苔(Brassica campestris var.purpurea)花粉粒中分离出大量生活精细胞。根据荧光素二醋酸酯测定,上述二法生活精细胞的分离率分别为34%和86%。应用改良的研磨法分离的精细胞在4℃低温下最长可存活一周。对影响精细胞分离率和生活力的若干因素进行了比较。细胞学观察表明,从一粒花粉刚分离出来的一对姊妹精细胞成对相连,其中一个具长尾状延伸物,尾部也呈FDA荧光反应。对精细胞分离后的形态变化进行了观察。

The two-step osmotic shock and grinding methods reported by Yang and Zhou (1989) were modified for isolation of viable sperm cells in large quantities from pollen grains of Brassica campestris var.purpurea.Factors affecting the yield and survival of isolated sperm cells have been investigated.These included physiological status of donor flowers,sucrose concentration used for pollen hydration,basic media,protectants and osmotica supplemented in the medium etc.As a result,two procedures have been developed.For osmotic shock method, pollen grains at the day of anthesis were hydrated in 25% sucrose solution for 30 min and,after centrifugation and removal of the supernatant,the pellet was shocked by a medium containing 12.5% sucrose,0.1 g/L KNO_3,0.36 g/L CaCl_2,2H_2O,0.3% potassium dextran sulphate (PDS),0.6% bovine serum albumin (BSA),and 0.3% polyvinylpyrrolidone (PVP).The viable sperm yield was 34%.After removal of pollen wall debris by filtration and centrifug- ation,the sperm cell-rich pellets were resuspended in a medium containing 20% sucrose,5% sorbitol,0.1 g/L KNO_3,0.36g/L CaCl_2-2H_2O,0.6% BSA and 0.3% PDS,and preserved at 4℃ for two days.For grinding method,the pollen grains hydrated in 30% sucrose solution for 30 min.were resuspended in a medium containing 20% sucrose,5% sorbitol,0.1g/L QNO_s, 0.36g/L CaCl_2·2H_2O,0.3% PDS,0.6% BSA,0.3% PVP and 20 μg/ml fluorescein diacetate, then ground with a glass homogenizer to release the sperm cells.The viable sperm yield was up to 86%.Following filtration and centrifugation for removal of pollen wall debris,the sperm cells were stored at 4℃ in the same medium but without supplementation of PVP.Tested by fluorochromatic reaction,the sperm cells could survive up to one week with a gradual decline of viability.Cytological observations revealed that pairs of ellipsoidal sperm cells just released were linked together;one of the pair had a long tail-like extension which also show fluorochromasia. Soon after,the sperm cells separated and turned to be spherical.The present results open a prospect to use isolated viable sperm cells for further experimental manipulations.