Nontaswatsri Chalermsri, Fukai Seiichi
Department of Horticulture, Faculty of Agricultural Production, Maejo University, Chiangmai, 50290, Thailand.
Methods Mol Biol. 2006;344:311-20. doi: 10.1385/1-59745-131-2:311.
Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/Lthidiazuron (TDZ), 0.1 mg/L alpha-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 microM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants.
康乃馨是切花产业中的一种重要作物,对新的改良品种的需求正在增长。然而,由于缺乏高效的常规技术,康乃馨的遗传转化目前受到限制。在本章中,我们介绍了一种简单有效的方法,用于将基因导入康乃馨节段外植体并随后进行不定芽再生。为了实现高不定芽再生,将5至8厘米长的嫩枝上第一至第三节的节段外植体接种在含有1.0毫克/升噻苯隆(TDZ)、0.1毫克/升α-萘乙酸(NAA)、20克/升蔗糖和2克/升结冷胶的Murashige和Skoog(MS)培养基上培养10天。然后将外植体切成8个径向段,并转接至含有1.0毫克/升苄氨基嘌呤(BA)、0.1毫克/升NAA、20克/升蔗糖和2克/升结冷胶的MS培养基上。为了进行有效的遗传转化,将预培养3至5天的节段外植体浸入农杆菌悬浮液中10分钟,然后在用水和50微摩尔乙酰丁香酮(AS)浸湿的滤纸上共培养。共培养后,将外植体切成8个径向段,并转接至选择培养基上,直至从外植体上再生出转化芽。