Nagahara Shiori, Takeuchi Hidenori, Higashiyama Tetsuya
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Plant Reprod. 2015 Mar;28(1):33-46. doi: 10.1007/s00497-015-0256-4. Epub 2015 Feb 12.
Key message: New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research.
新的配子纯合突变体。在被子植物中，单倍体雄配子（精子细胞）在受精过程中与单倍体雌配子（卵细胞）融合，形成携带父本和母本来源染色体的合子。拟南芥中的几个受精缺陷突变体，包括生殖细胞特异性1（gcs1）/无融合生殖2突变体，其精子细胞无法与雌配子融合，由于雄配子或雌配子不育，只能作为杂合系维持。在此，我们报告通过热诱导去除GCS1转基因成功产生了gcs1纯合突变体。以gcs1纯合突变体为父本，配子融合缺陷的观察频率很高；在我们使用胚珠进行的半体内受精试验的直接观察中，培养物中100%排出的精子细胞未能显示配子融合。雌蕊中超过70%的胚珠接受了第二条花粉管以尝试恢复受精。此外，gcs1突变体的精子细胞在雌蕊中能以低频率使雌配子受精。这种产生纯合受精缺陷突变体的策略将有助于植物繁殖研究中的新方法。
