Olsen F L
Department of Physiology, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Hereditas. 1991;115(3):255-66. doi: 10.1111/j.1601-5223.1992.tb00568.x.
Two different techniques for mechanical isolation of microspores from the barley cultivar 'Igri' have been evaluated. The anthers were subjected to mannitol pretreatment prior to microspore isolation, which was performed either by maceration with a pestle or by blending of the excised anthers. The microspores were purified by centrifugation and washing and cultured in liquid medium on a membrane support. In the following four weeks the microspores developed into embryoids, which were subsequently regenerated to plants on solid medium. Microblending of the anthers was found to be more reproducible than pestle maceration, and the yield of large microspores was 100% higher using this method. With the microblending technique a mean of 9.4 green plants and 0.4 albino plants were regenerated per plated anther while a mean of only 2.8 green and 0.17 albino plants per anther were regenerated from microspores isolated after pestle maceration of the anthers. Microspores isolated from mass cultures were also cultured as single cells in microdroplets, and it was shown that microspores isolated from 3-5 days old mass cultures could develop into plants although at a low frequency (0.3%). Finally, the potential of using microinjection for transforming embryogenic microspores has been evaluated.
对从大麦品种“Igri”中机械分离小孢子的两种不同技术进行了评估。在小孢子分离之前，花药要进行甘露醇预处理，小孢子分离可通过用研杵研磨或对切下的花药进行匀浆来完成。小孢子通过离心和洗涤进行纯化，并在膜支持物上的液体培养基中培养。在接下来的四周里，小孢子发育成胚状体，随后在固体培养基上再生为植株。发现花药的微量匀浆比研杵研磨更具可重复性，使用这种方法大孢子的产量高出100%。采用微量匀浆技术，每个接种的花药平均再生出9.4株绿色植株和0.4株白化植株，而通过花药研杵研磨后分离的小孢子每个花药平均仅再生出2.8株绿色植株和0.17株白化植株。从大量培养物中分离的小孢子也以单细胞形式在微滴中培养，结果表明，从3 - 5天大的大量培养物中分离的小孢子虽然频率较低(0.3%)，但仍能发育成植株。最后，评估了使用显微注射转化胚性小孢子的潜力。
