三种植物花粉原生质体的大量分离与初步培养

MASS ISOLATION AND CULTURE OF POLLEN PROTOPLASTS FROM THREE PLANT SPECIES

利用花粉水合导致外壁破裂,使内壁得以酶解,分离出鸢尾(Iris tectorum Maxim)、风雨花(Zephyranthes grandiflora Lindl)和萱草(Hemerocallis fulva L.)三种植物成熟花粉的原生质体。初步分离出萱草四分体至成熟花粉(单核早期除外)各时期的原生质体。经过纯化处理,获得较纯净的有生活力的原生质体群体。萱草花粉原生质体的培养实现了细胞壁再生、管状及其它多种结构的形成以及生殖核的一、二次分裂。

The exine which contains chemically resistant sporopollenin makes a severe obstacle for obtaining pollen protoplasts.A technique was established for protoplast isolation in large number from mature pollen grains in three plant species i.e.Iris tectorum,Zephyranthes grandiflora and Hemerocallis fulva.Dehiscence of exine by the force of pollen hydration and consequentely exposure of intine to the direct action of enzymatic maceration led to suc- cessful release of protoplasts from pollen grains.The highest isolation rate up to 90% pro- toplasts was obtained from mature pollen grains of Z.grandiflora.Pollen protoplasts from tetrads to mature pollen,except early uninucleate pollen stage,were prepared in H.fulva. Purified pollen protoplasts were obtained by repeated centrifugation and density gradient centrifugation and proved to be viable by fluorochromatic reaction with FDA.When cul- tured in vitro,the pollen protoplasts of H.fulva underwent regeneration of cell wall,forma- tion of various tube-,nodule-,bead- and dumbbell-shaped structures and sometimes division of the generative nucleus into 2—4 nuclei.Exogenous hormone showed promotive effect on the growth of above mentioned structures.