Koike Imari, Shimomura Koichiro, Umehara Mikihisa
Graduate School of Life Sciences, Toyo University.
Graduate School of Life Sciences, Toyo University;
J Vis Exp. 2018 Mar 15(133):56902. doi: 10.3791/56902.
Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 °C under 15 µmol m s of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.
不定芽的形成是经济作物繁殖和转基因植物再生的一项重要技术。大多数物种诱导不定芽都需要进行植物激素处理。不定芽能否被诱导取决于生长素和细胞分裂素（CK）水平之间的平衡。人们花费了大量精力来确定用作外植体的每个组织以及每个植物物种中植物激素的最佳浓度和组合。然而，在吐根中，无需植物激素处理，在培养基中的节间切段上就能诱导出不定芽。这使得我们能够评估吐根细胞分化的内在可塑性。为了在吐根中诱导不定芽，我们将节间切段在24℃、光照强度为15 μmol m⁻² s⁻¹、14小时光照/10小时黑暗周期的条件下，培养在添加0.2%结冷胶固化的无植物激素B5培养基上5周。为了研究不定芽形成过程中的植物激素动态变化，我们通过液相色谱 - 串联质谱法（LC-MS/MS）测定了切段中的内源吲哚 - 3 - 乙酸和细胞分裂素。这种方法能够以简单的方式分析内源吲哚 - 3 - 乙酸和细胞分裂素的水平。它可用于研究其他植物物种器官发生过程中内源生长素和细胞分裂素的动态变化。
