Shim Youn-Seb, Pauls K Peter, Kasha Ken J
Department of Plant Agriculture, Crop Science Building, University of Guelph, Guelph, ONN1G2W1, Canada.
Genome. 2009 Feb;52(2):166-74. doi: 10.1139/g08-112.
The objective of this study was to determine when DNA synthesis occurred during pretreatments of cultured barley (Hordeum vulgare L.) microspores and during their preparation for particle bombardment. Based on this information, an investigation of the influence of cell cycle stage on the ability to obtain homozygous transgenic plants by particle bombardment will be presented in paper II of this series. It was hypothesized that the introduction of foreign genes at the G1 cell cycle stage in cultured uninucleate microspores would produce homozygous transgenic plants. Experiments were conducted with two different commonly used pretreatments to induce microspore embryogenesis: cold (4 degrees C) for 21days and cold plus 0.3 mol/L mannitol for 4 days. After pretreatment, the microspores were placed in a higher osmotic medium for 4 h prior to and for 18 h following bombardment. It was confirmed that during the cold plus mannitol pretreatment, there was no apparent change in the cell cycle stage, with the majority of the microspores remaining at the G1 stage. While in the cold for 21 days, the microspores progressed slowly through to G2, with a few progressing further into the mitosis and binucleate stages. Hourly DNA density measurements that were taken during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment indicated that DNA synthesis began during this period at 25 degrees C, while at 4 degrees C, there was no apparent change in cell cycle stage or in DNA density. Thus, one might expect to find a higher frequency of homozygous doubled haploids by maintaining the temperature low during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment than following the 21 day cold pretreatment. However, it is also not known what effect the temperatures during the whole high-osmotic treatments will have on the rate and time of incorporation of the transgene.
本研究的目的是确定在培养的大麦（Hordeum vulgare L.）小孢子预处理期间以及在其准备进行粒子轰击的过程中，DNA合成何时发生。基于这些信息，本系列论文的第二篇将探讨细胞周期阶段对通过粒子轰击获得纯合转基因植物能力的影响。据推测，在培养的单核小孢子的G1细胞周期阶段引入外源基因将产生纯合转基因植物。实验采用了两种不同的常用预处理方法来诱导小孢子胚胎发生：4℃低温处理21天以及4℃低温加0.3 mol/L甘露醇处理4天。预处理后，小孢子在轰击前置于较高渗透压的培养基中4小时，并在轰击后置于其中18小时。已证实，在低温加甘露醇预处理期间，细胞周期阶段没有明显变化，大多数小孢子仍处于G1期。而在4℃低温处理21天的情况下，小孢子缓慢进入G2期，少数进一步进入有丝分裂和双核期。在低温加甘露醇预处理后的4小时渗透调节期内每小时进行的DNA密度测量表明，在25℃时DNA合成在此期间开始，而在4℃时，细胞周期阶段和DNA密度均无明显变化。因此，可以预期，与21天低温预处理相比，在低温加甘露醇预处理后的4小时渗透调节期内保持低温，会发现纯合双单倍体的频率更高。然而，整个高渗处理期间的温度对转基因整合的速率和时间会有何种影响也尚不清楚。
