Poormassalehgoo Andisheh, Kaniecka Elżbieta, Mirzaei Mohamadreza, Goto-Yamada Shino
Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
Doctoral School of Exact and Natural Sciences, Jagiellonian University, Krakow, Poland.
Sci Rep. 2025 Jul 23;15(1):26836. doi: 10.1038/s41598-025-11801-5.
Genetic transformation of plants is pivotal for advancing biotechnology, with success depending largely on effective selection methods. Marchantia polymorpha has emerged as a model plant due to its evolutionary importance, ease of manipulation, and simple genetic structure. However, inconsistent antibiotic performance and limited studies on optimal selection agent concentrations have posed challenges. This study aimed to optimize selection agent use in M. polymorpha genetic transformation. We assessed the effects of five antibiotics (hygromycin, kanamycin, G418, neomycin, and gentamicin) and the herbicide chlorsulfuron on M. polymorpha gemmae growth by evaluating survival, morphological properties, area, and fresh weight of the thallus on these selection media. For each agent, we identified the minimum lethal concentration for nontransgenic plants and safe thresholds for transgenics, balancing false-positive prevention with reduced toxicity. This study effectively determined optimal concentrations of selective agents for M. polymorpha gemmae transformation. Hygromycin, G418, and chlorsulfuron have broad selective concentration ranges of 5-100, 2-100 µg/ml, and 20-400 ng/ml, respectively, enabling efficient transformant selection. On the other hand, kanamycin, gentamicin, and neomycin had narrow selection concentration ranges, making the separation of transformed and nontransformed gemmae less effective. Notably, we observed cross-activity of the gentamicin resistance enzyme with G418, a phenomenon also confirmed in tobacco. This unexpected cross-activity underscores the need for careful marker selection while also highlighting its potential for strategic antibiotic use. Specifically, this cross-activity could improve the usability of the aacC1 selection marker, making it a more versatile tool for molecular biological analysis. Our findings enhance transformation protocols for M. polymorpha and possibly other plant species.
植物的遗传转化对于推动生物技术发展至关重要，其成功很大程度上取决于有效的选择方法。由于其在进化上的重要性、易于操作以及简单的遗传结构，多歧藻已成为一种模式植物。然而，抗生素性能不一致以及对最佳选择剂浓度的研究有限带来了挑战。本研究旨在优化多歧藻遗传转化中选择剂的使用。我们通过评估这些选择培养基上叶状体的存活率、形态特性、面积和鲜重，来评估五种抗生素（潮霉素、卡那霉素、G418、新霉素和庆大霉素）以及除草剂氯磺隆对多歧藻芽孢生长的影响。对于每种试剂，我们确定了非转基因植物的最低致死浓度和转基因植物的安全阈值，在防止假阳性和降低毒性之间取得平衡。本研究有效地确定了多歧藻芽孢转化的选择性试剂的最佳浓度。潮霉素、G418和氯磺隆分别具有5 - 100、2 - 100μg/ml和20 - 400 ng/ml的广泛选择浓度范围，能够有效地选择转化体。另一方面，卡那霉素、庆大霉素和新霉素的选择浓度范围较窄，使得转化和未转化的芽孢分离效果较差。值得注意的是，我们观察到庆大霉素抗性酶与G418有交叉活性，这种现象在烟草中也得到了证实。这种意外的交叉活性强调了在选择标记时需要谨慎，同时也突出了其在战略性抗生素使用方面的潜力。具体而言，这种交叉活性可以提高aacC1选择标记的可用性，使其成为分子生物学分析中更通用的工具。我们的研究结果改进了多歧藻以及可能其他植物物种的转化方案。
