State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), Harbin, China.
State Key Laboratory of Tree Genetics and Breeding (Chinese Academy of Forestry), Beijing, China.
PLoS One. 2021 Oct 14;16(10):e0258654. doi: 10.1371/journal.pone.0258654. eCollection 2021.
To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.
迄今为止，仅有少数关于落叶松和其他针叶树成功遗传转化的报道，主要是因为外源基因的转化和整合较为困难。本研究于 2017 年 6 月 27 日、7 月 1 日、7 月 4 日、7 月 7 日和 7 月 16 日采集杂交落叶松杂种雄球果（杂种落叶松 3 倍体×落叶松 9 倍体）。通过不同植物生长调节剂及其浓度的组合，研究了胚性愈伤组织的诱导。结果表明，7 月 1 日是最佳诱导阶段；最高诱导率为 10.83%，在 BM 培养基（Button 培养基，S1 表中列出了其配方）中，2,4-D（2,4-二氯苯氧乙酸）浓度为 1.0mg/L、KT（细胞分裂素）浓度为 0.2mg/L 时培养。在增殖培养基中培养 12 天，增殖速度最快，达到 323.08%，同时还能保持新鲜活力。体细胞胚发生的适宜预培养培养基为含 10g/L 肌醇和 60g/L 蔗糖的 1/4BM 培养基。45mg/L ABA（脱落酸）和 75g/L PEG4000（聚乙二醇 4000）的组合可促进体细胞胚的数量，达到最大值，每 1gFW 为 210 个左右。采用农杆菌介导的转化方法，以培养 12 天的胚性愈伤组织为转化受体。结果表明，感染液（农杆菌悬浮液）的最佳 OD600 为 0.5，共培养时间为 2 天，潮霉素（Hyg）筛选浓度为 4mg/L。本研究中，抗性愈伤组织的转化率为 32.1%。这为目前落叶松遗传转化效率低提供了参考。本研究可为落叶松的遗传工程创新和育种提供参考，也为落叶松及其他针叶树优良种质资源的快速繁殖和遗传工程育种奠定了一定基础。
