Maruyama Tsuyoshi E, Tsuruta Momi, Ueno Saneyoshi, Kawakami Kiyohisa, Bamba Yukiko, Moriguchi Yoshinari
Department of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research Institute, Tsukuba, Japan.
Verde Co., Ltd., Toyohashi, Japan.
Front Plant Sci. 2022 Feb 8;13:825340. doi: 10.3389/fpls.2022.825340. eCollection 2022.
Sugi (Japanese cedar, ) is the most important forestry tree species in Japan, covering 44% of the total artificial forest area. Large amounts of pollen released from these forests each spring cause allergic reactions in approximately 40% of the population, which are a serious social and public health problem in Japan. As a countermeasure, there is an urgent need to reforest using male-sterile plants (MSPs; pollen-free plants); however, the production of MSPs conventional methods is inefficient, time consuming, and requires considerable resources in terms of labor and space. In the present paper, we described an improved and simplified methodology for the efficient propagation of pollen-free Japanese cedar, combining the use of genetic markers (marker-assisted selection or marker-aided selection) for the early selection of male-sterile genotypes and the use of somatic embryogenesis (SE) for the clonal mass propagation of seedlings. We describe all the stages involved in the production process of somatic seedlings. Our results demonstrated that this methodology easily and efficiently produces MSPs with a discrimination rate of 100% in a short period of time. Production of 243.6 ± 163.6 cotyledonary embryos per plate, somatic embryo germination, and plantlet conversion frequencies of 87.1 ± 11.9% and 84.8 ± 12.6%, respectively, and a 77.6 ± 12.1% survival rate after acclimatization was achieved. Moreover, we also describe an easy method for the collection of somatic embryos prior to germination, as well as an efficient and practical method for their storage at 5°C. Finally, a representative schedule for the propagation of pollen-free sugi somatic seedlings is presented as a reference for practical uses. This methodology will definitively help to accelerate the production of MSPs across Japan.
柳杉（日本柳杉）是日本最重要的林业树种，占人工林总面积的44%。每年春天，这些森林释放出大量花粉，导致约40%的人口出现过敏反应，这在日本是一个严重的社会和公共卫生问题。作为一种应对措施，迫切需要使用雄性不育植株（MSP；无花粉植株）进行重新造林；然而，用传统方法生产MSP效率低下、耗时，并且在劳动力和空间方面需要大量资源。在本文中，我们描述了一种改进和简化的方法，用于高效繁殖无花粉日本柳杉，该方法结合了使用遗传标记（标记辅助选择）来早期选择雄性不育基因型，以及使用体细胞胚胎发生（SE）来进行幼苗的克隆大量繁殖。我们描述了体细胞幼苗生产过程中涉及的所有阶段。我们的结果表明，这种方法能够在短时间内轻松、高效地生产出鉴别率为100%的MSP。每平板产生243.6±163.6个胚性子叶，体细胞胚胎萌发率和植株转化率分别为87.1±11.9%和84.8±12.6%，驯化后成活率为77.6±12.1%。此外，我们还描述了一种在萌发前收集体细胞胚胎的简便方法，以及一种在5°C下储存它们的高效实用方法。最后，给出了一个无花粉柳杉体细胞幼苗繁殖的代表性时间表，作为实际应用的参考。这种方法肯定有助于加速全日本MSP的生产。
