CYTOKININ HYPERSENSITIVE1 (CKH1) and CKH2 affect to cytokinin responses in tissue culture

Analysis of calli derived from the wild type (Ler), the ckh1 and ckh2 mutants cultured on media in the absence of cytokinin (control), in the presence of low (25 ng/ml kinetin) or high (200 ng/ml kinetin) levels of cytokinin, or in the presence of Trichostatin A (TSA). In these conditions, a constant 2,4-dichlorophenoxyacetic acid (2,4-D) was included as an auxin. Hypocotyl segments of the wild type (Ler), ckh1-1 and ckh2-1 mutants were cultured for 3 days on media containing 100 ng/ml 2,4-D in the absence of kinetin or in the presence of low (25 ng/ml) or high (200 ng/ml) kinetin or 100ng/ml TSA, and then collected and rapidly frozen by liquid nitrogen. Total RNA was extracted by using an RNeasy plant kit (Qiagen; USA), and 10 µg of total RNA was used for target preparation by using

本研究对源自野生型（Ler）、ckh1与ckh2突变体的愈伤组织开展分析：上述愈伤组织分别培养于不含细胞分裂素（对照组）、含低浓度（25 ng/ml激动素）或高浓度（200 ng/ml激动素）细胞分裂素的培养基，或是添加曲古抑菌素A（TSA）的培养基中，所有培养体系均添加恒定浓度的2,4-二氯苯氧乙酸（2,4-D）作为生长素。将野生型（Ler）、ckh1-1与ckh2-1突变体的下胚轴切段，置于含100 ng/ml 2,4-D且不含激动素、或添加低浓度（25 ng/ml）/高浓度（200 ng/ml）激动素、或添加100 ng/ml曲古抑菌素A的培养基中培养3天，随后收集样本并通过液氮快速冷冻。采用RNeasy植物总RNA提取试剂盒（Qiagen公司，美国）提取总RNA，并取10 µg总RNA用于靶标制备，具体操作内容未完整提供。