甘蓝型油菜和紫菜苔花粉原生质体的大量分离

RELEASE OF POLLEN PROTOPLASTS IN LARGE QUANTITIES IN BRASSICA NAPUS AND B.CAMPESTRIS VAR.PURPUREA

第一次由两种芸苔属作物甘蓝型油菜(Brassica napus L.)和紫菜苔(B.campestris var-purpurea)的成熟花粉中成功地分离出大量原生质体。分离率最高分别达66.7%与70.4%。绝大多数花粉原生质体是生活的。在分离技术上,改进了原来在几种单子叶植物中所用的水合与酶解同时进行的“一步法”,建立了适于油菜和紫菜苔的“二步法”,即:首先将花粉经长时间水合吸胀,脱去外壁获得仅有内壁的花粉;然后通过酶解除去内壁获得花粉原生质体。研究了影响水合与酶解两个过程的各种因素。

Protoplasts were released in large quantities from mature pollen of Brassica napus L.and B.campestris var.purpurea for the first time,with a yield up to 66.7% and 70.4% respecti- vely.Most of the pollen protoplasts were viable as tested by fluorochromatic reaction with fluo- rescein diacetate.The success of isolation of pollen protoplasts in these two Brassica species re- lied on a technique modified from the previous method developed for several monocotyledo- nous flowers.The procedure included two steps:First,the pollen was hydrated in 1 mol/L of sucrose solution at 28—30℃ for ca.9h.During this process,the exine of most pollen dehis- ced and was detached from the pollen grains which were then covered by intine alone.Second, the hydrated pollen was transferred into an enzyme solution containing 1% cellulase,1% pec- tinase,1 mol/L mannitol,0.5% potassium dextran sulphate and K_3 medium salts.After 4—6 of enzymatic maceration,the intine was degradated resulting in the release of protoplasts.Fac- tors affecting the two steps have been investigated.