Real-time Analysis of Lateral Root Organogenesis in Arabidopsis
拟南芥侧根器官形成的实时分析
发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1446 浏览次数: 9695
评审: Samik BhattacharyaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2014
Abstract
Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavý et al., 2013; Marhavý et al., 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis.
Materials and Reagents
- Arabidopsis seedlings (5-6 days old) expressing Green Fluorescent Protein (GFP) or analogous reporters (YFP, RFP, CFP, mCherry and others) (Chalfie et al., 1994; Shaner et al., 2007)
- MilliQ Water (H2O)
- Sucrose (VWR International, catalog number: 27483.294 )
- Murashige and Skoog basal salt mixture (MS salts) (Duchefa Biochemie, catalog number: M0221.0050 )
- 2- [N-morpholino] ethanesulfonic acid (MES) (Duchefa Biochemie, catalog number: M1503.0100 )
- Potassium hydroxide (KOH) (Merck KGaA, catalog number: 1.05021.1000 )
- Agar (LAB M, catalog number: MC029 )
- Ethanol (EtOH) (Sigma-Aldrich, catalog number: 32221 -2.5L)
- Seeds sterilization by ethanol (see Recipes)
- ½ MS media (see Recipes)
- Growth conditions (see Recipes)
Equipment
- Growth chamber to grow plant material
- Square plates 120 x 120 x 17 mm (Greiner Bio-One GmbH, catalog number: 688102 )
- Chambered cover glass (VWR, Kammerdeckgläser, Lab-TekTM, NuncTM - eine kammer, catalog number: 734-2056 )
- Inverted confocal microscope (Zeiss, model: LSM 700 )
Note: Fully motorized X, Y, Z scanning stage is required to perform multi-position time-lapse experiment.
- Objectives: 20x [suitable to monitor early phases of the lateral root primordia (LRP) initiation, Figure 2], 40x or 60x (suitable to monitor LRP from the stage I onwards, Figure 3)
- Fluorescence signal detection system for GFP and other fluorescent reporters (Shaner et al., 2007)
Software
- Software operating the confocal microscope
- ImageJ (Abramoff et al., 2004)
- CellseT (Pound et al., 2012)
- Microsoft Excel
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Marhavý, P. and Benková, E. (2015). Real-time Analysis of Lateral Root Organogenesis in Arabidopsis. Bio-protocol 5(8): e1446. DOI: 10.21769/BioProtoc.1446.
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分类
植物科学 > 植物细胞生物学 > 细胞结构
植物科学 > 植物发育生物学 > 形态建成
细胞生物学 > 细胞成像 > 共聚焦显微镜
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