Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture
Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture
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DOI：
10.1007/s00425-007-0565-4
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发表时间：
2007-10-01
期刊：
影响因子：
4.3
通讯作者：
Howell, Stephen H.
中科院分区：
文献类型：
--
作者：
Che, Ping;Lall, Sonia;Howell, Stephen H.
在组织培养中，拟南芥的根外植体通过两个步骤获得再生芽，这两个过程需要在富含生长素的愈伤组织诱导培养基(CIM)上预培养，然后在富含细胞分裂素的芽诱导培养基(SIM)上培养。在CIM预培养过程中，根外植体获得了对芽诱导信号做出反应的能力。在CIM预培养过程中，根外植体中的中柱细胞经历细胞分裂和去分化，失去中柱细胞特异性标记的表达。这些细胞在CIM预培养1d后才能形成绿色愈伤组织，在CIM预培养2~3d后才能形成芽。可逆的DNA合成抑制剂干扰了形成新梢的能力的获得。通过芯片分析鉴定了需要CIM预孵育才能上调SIM的基因，包括反应调节因子15(ARR15)、多聚半乳糖酶抑制蛋白2(PGIP2)和WUSCHEL(WUS)。这些基因是获得愈伤组织能力的发育标记，因为对ARR15和PGIP2上调的CIM预培养要求与形成绿色愈伤组织的能力的获得很好地相关，而对WUS上调的CIM预培养要求与对芽形成的CIM预培养要求相匹配。与ARR15不同的是，另一种细胞分裂素诱导的A型ARR基因ARR5在SIM上上调，但这种诱导不需要CIM预先孵育。这些发现表明，在CIM预培养过程中，与芽再生相关的各种事件的能力是逐渐获得的，并且一组通常在SIM上上调的基因被CIM预培养所缓解的过程所抑制。
Arabidopsis shoots regenerate from root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium (CIM) followed by incubation on a cytokinin-rich shoot induction medium (SIM). During CIM preincubation, root explants acquire competence to respond to shoot induction signals. During CIM preincubation, pericycle cells in root explants undergo cell divisions and dedifferentiate, losing the expression of a pericycle cell-specific marker. These cells acquire competence to form green callus only after one day CIM preincubation and to form shoots after 2-3 days CIM preincubation. Reversible DNA synthesis inhibitors interfered with the acquisition of competence to form shoots. Genes requiring CIM preincubation for upregulation on SIM were identified by microarray analysis and included RESPONSE REGULATOR 15 (ARR15), POLYGALACTURONASE INHIBITING PROTEIN 2 (PGIP2) and WUSCHEL (WUS). These genes served as developmental markers for the acquisition of competence because the CIM preincubation requirements for ARR15 and PGIP2 upregulation correlated well with the acquisition of competence to form green callus, and the CIM preincubation requirements for WUS upregulation matched those for shoot formation. Unlike ARR15, another cytokinin inducible, A-type ARR gene, ARR5, was upregulated on SIM, but the induction did not require CIM preincubation. These findings indicate that competencies for various events associated with shoot regeneration are acquired progressively during CIM preincubation, and that a set of genes, normally upregulated on SIM, are repressed by a process that can be relieved by CIM preincubation.