Department of Horticulture, University of Georgia, Athens, GA, USA.
Department of Health and Natural Sciences, Middle Georgia State University, Macon, GA, USA.
Methods Mol Biol. 2021;2290:271-284. doi: 10.1007/978-1-0716-1323-8_18.
Various steps of micropropagation include selection of suitable explant, establishment of adventitious shoot induction cultures, proliferation, rooting, and acclimatization of the resulting plantlets. A systematic protocol is provided for the micropropagation and Agrobacterium tumefaciens-mediated genetic transformation of a fast growing, multipurpose tree, Paulownia elongata. Our studies show that optimum shoot induction is on half leaf with petiole explant on MS medium supplemented with 25 μM thidiazuron and 10 μM indole-3 acetic acid. Micropropagation protocols provided here are applicable to explants collected from the primed in vitro raised seedlings on MS medium containing 2.5 μM 6-benzylaminopurine (BAP) or actively growing shoots collected from greenhouse or field growing plants. We also discuss a possible role of "Python" script guided protocol optimization for higher and consistent multiplication of shoots that can be very helpful for scaled up production in commercial settings. To facilitate future plant improvement and gene editing possibilities, an A. tumefaciens based genetic transformation protocol and molecular identification of transgenic plants using Polymerase Chain Reaction (PCR) and Reverse Transcriptase-PCR (RT-PCR) techniques have also been optimized.
微繁殖的各个步骤包括选择合适的外植体、建立不定芽诱导培养、增殖、生根和培养的幼苗的适应环境。为快速生长、多用途的泡桐属植物的微繁殖和根癌农杆菌介导的遗传转化提供了一个系统的方案。我们的研究表明，在补充有 25μM 噻苯隆和 10μM 吲哚乙酸的 MS 培养基上，半叶带叶柄的外植体最适合诱导出芽。这里提供的微繁殖方案适用于从含有 2.5μM 6-苄基氨基嘌呤（BAP）的 MS 培养基上预先培养的体外幼苗或从温室或田间生长的植物中收集的活跃生长的芽。我们还讨论了“Python”脚本指导的协议优化对于提高和稳定芽的繁殖的可能作用，这对于在商业环境中进行规模化生产非常有帮助。为了促进未来的植物改良和基因编辑的可能性，还优化了基于根癌农杆菌的遗传转化方案和使用聚合酶链反应（PCR）和逆转录-聚合酶链反应（RT-PCR）技术对转基因植物的分子鉴定。
