活性碳和花药密度对大麦花培效率的影响及外源基因导入初探

PRELIMINARY STUDIES ON EFFECTS OF ACTIVATED CHARCOAL(AC) AND ANTHER DENSITY ON EFFICIENCY OF BARLEY ANTHER CULTURE AND GENE TRANSFER-BY PARTICLE BOMBARDMENT IN BARLEY(Hordeum vulgare L. )

在过滤灭菌的液体培养体系中研究了甘露醇预培养和诱导培养过程中活性碳和花药密度对大麦花培效率的影响。结果表明，活性碳会降低花粉胚和愈伤组织产量和质量，影响植株分化率，尤以诱导培养基中添加活性碳的影响较大。甘露醇预培养过程中的花药密度对花培效率没有明显影响，但在诱导培养过程中，较高的花药密度（６０～９０枚／ｍｌ）不利于花粉胚和愈伤组织的生长发育；而在较低的花药密度（１５～３０枚／ｍｌ）条件下，花粉胚和愈伤组织生长发育较好，绿苗分化率较高。作者尝试了利用基因枪技术对大麦花粉胚和愈伤组织进行外源基因（ＧＵＳ）导入，获得丰富的转化事件，并讨论了活性碳和花药密度对大麦花培效率产生负效应的原因及利用大麦花粉胚和愈伤组织作为基因转化受体系统的可能性。

Effects of AC and anther density on pollen embryo and callus production and green plant regeneration were studied in a filter-sterilized, liquid-medium culture system. AC could reduce much more embryo and callus production when applied to induction culture than to mannitol pre-culture, and obvious deterioration of pollen embryos and calli was observed, which resulted in a drop in green plant regeneration,while difference in anther density in mannitol pre-culture didn' t show any effects on anther culture, difference in anther density during induction culture did have an influence.That is, higher anther density (60 ￣ 90 anthers/ml) gave an adverse effect on the growth of pollen embryos and calli, causing a decrease both in pollen embryo and callus production and green plant regeneration ; lower anther density(15￣30 anthers/ml ) generated a favorable effect on the growth of pollen embryos and calli, resulting in an increase both in pollen embryo and callus production and green plant regeneration. Gene transfer into pollen embryos and calli was performed using particle bombardment.Abundant transient expression of reporter gene(GUS) occured on the bombarded pollen embryos and calli. However, we failed to obtain transgenic barley plants because the totipotency of transformed cells was probably lost during subsequent selection subculture.Some possible causes of negative effects of AC and anther density on anther culture in the present work were discussed, and the application of isolated microspores rather than microspore-derived embryos and calli to gene transfer experiments was suggested by in barley the authors.