Wang Xiaoping, Chen Shanshan, Zhang Haonan, Luo Ping, Zhou Fangping, Zeng Bingshan, Xu Jianmin, Fan Chunjie
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, China.
Key Laboratory of State Forestry and Grassland Administration on Tropical Forestry, Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China.
Front Plant Sci. 2023 Jan 17;13:1011245. doi: 10.3389/fpls.2022.1011245. eCollection 2022.
, as an economically important species for wood and paper industries, remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. × clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of was established using × DH32-29 as material. The leaves from microshoots that were subcultured for 20-25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L α-naphthaleneacetic acid (NAA) and 0.24 mg·L forchlorfenuron [callus-inducing medium (CIM)]. After 15 days, explants were transferred to a medium containing 0.10 mg·L NAA and 0.50 mg·L 6-benzyladenine (shoot-inducing medium, SIM) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Moreover, an -mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with strain EHA105 grown to a bacterial density of 0.3 (OD). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L cefotaxime (Cef), 100 mg·L timentin, and 15 mg·L kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After inducing roots in root-inducing medium supplemented with 200 mg·L Cef and 75 mg·L Kan, completely transgenic plants were obtained. Using the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of × DH32-29 and gene function analysis in .
作为木材和造纸工业中具有重要经济价值的树种，由于缺乏高效稳定的遗传转化系统，利用转基因技术进行遗传改良仍然是一项挑战，尤其是在栽培优良无性系方面。×无性系DH32-29在中国南方种植最为广泛，但它相对较难进行不定芽再生，这阻碍了遗传转化系统的建立。在此，以×DH32-29为材料建立了高效的不定芽再生和转化系统。将继代培养20-25天的微枝上的叶片浸入添加了0.02 mg·Lα-萘乙酸（NAA）和0.24 mg·L氯吡脲的液体木本植物培养基[愈伤组织诱导培养基（CIM）]中。15天后，将外植体转移到含有0.10 mg·L NAA和0.50 mg·L 6-苄基腺嘌呤的培养基[芽诱导培养基（SIM）]中进行不定芽诱导。不定芽的最高再生效率为76.5%。此外，优化了农杆菌介导的遗传转化系统。叶片预培养7天，用生长至细菌密度为0.3（OD）的EHA105菌株感染30分钟。在黑暗中共培养72小时后，将叶片转移到添加了100 mg·L头孢噻肟（Cef）、100 mg·L替门汀和15 mg·L卡那霉素（Kan）的CIM中15天以诱导愈伤组织。然后，将外植体转移到添加相同浓度抗生素的SIM中，每15天更换新鲜培养基，直到出现抗性不定芽。在添加200 mg·L Cef和75 mg·L Kan的生根诱导培养基中诱导生根后，获得了完全转基因植株。使用上述方法，转化频率可达1.9%。这为×DH32-29的遗传改良和基因功能分析提供了有力途径。
