拟南芥幼苗的子叶创伤

Debora Gasperini; Ivan F. Acosta; Edward E. Farmer

doi:10.21769/BioProtoc.1712

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Cotyledon Wounding of Arabidopsis Seedlings

拟南芥幼苗的子叶创伤

DG Debora Gasperini email

IA Ivan F. Acosta

EF Edward E. Farmer

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1712 浏览次数: 11891

评审: Arsalan DaudiKaisa KajalaAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Genetics, featuring study using the protocol.

Jun 2015

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

一种分离拟南芥叶绿体内腔的简单超声方法

Jingfang Hao and Alizée Malnoë

2023年08月05日 1139 阅读

量化玉米胚根发育的平板生长测定

Jason T. Roberts [...] David M. Braun

2023年10月20日 1401 阅读

在莱茵衣藻中检测和定量程序性细胞死亡：以S-硝基谷胱甘肽为例

Lou Lambert and Antoine Danon

2024年08月05日 820 阅读

Abstract

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.

Keywords: Plant (植物)

Jasmonate (茉莉)

JA-Ile (JA-ILE-)

Materials and Reagents

Single wounding of seedling cotyledons
1. Sterile 6 cm x 6 cm nylon mesh, 200 μm pore size (Lanz-Anliker AG, custom made)
2. Standard 9 cm round Petri plates
3. 3M^TM Micropore^TM Tape, 1.25 cm x 9.14 m, hypoallergenic tape, standard roll (3M, catalog number: 1530-0 )
4. Two 25 G x 5/8” hypodermic needles, 0.5 mm x 16 mm (BD Bioscience, catalog number: 305760 )
5. Arabidopsis thaliana sterile seeds, e.g., MYC2p-GUS reporter line (Gasperini et al., 2015)
6. Sterile double distilled H₂O (ddH₂O)
7. Murashige and Skoog (MS) medium (Duchefa Biochemie, catalog number: M0221.0050 )
8. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
9. Agar (AppliChem GmbH, catalog number: A2111 1000 )
10. Standard 9 cm round Petri plates filled with 30 ml of solid half-strength Murashige and Skoog (0.5x MS) medium with 0.7% agar (see Recipes)
Repetitive wounding of seedling cotyledons
1. 3M^TM Micropore^TM Tape, 2.5 cm x 9.14 m, hypoallergenic tape, standard roll (3M, catalog number: 1530-1 )
2. One 25 G x 5/8” hypodermic needle, 0.5 mm x 16 mm (BD Bioscience, catalog number: 305760 )
3. One 36 gauge beveled NanoFil needle, 110 µM outer diameter (World Precision Instruments, catalog number: NF36BV )
4. Standard 12 cm x 12 cm square Petri plates
5. Racks for vertical growth (e.g., Milian, catalog number: 086680 )
6. Arabidopsis thaliana sterile seeds, e.g., CYCB1;1p-GUS reporter line (Colón-Carmona et al., 1999)
7. Sterile ddH₂O
8. Standard 12 cm x 12 cm square Petri plates filled with 70 ml 0.5x MS with 0.85% agar (see Recipes)
GUS staining
1. Staining dish [e.g., 12 well suspension culture plate (Greiner Bio-One GmbH, Cell Star^®, catalog number: 665102 )]
2. Ice bucket with ice
3. Microscopy slides (e.g., Thermo Fisher Scientific, Menzel-Glaser，catalog number: AD00000112E ) and coverslips (e.g., Thermo Fisher Scientific, Menzel-Glaser, catalog number: BB024060A1 )
4. 90% (v/v) acetone in ddH₂O
5. 50 mM sodium phosphate buffer (pH 7.0)
6. 70% (v/v) ethanol (EtOH) in ddH₂O
7. Potassium Ferrocyanide [e.g., Potassium hexacyanoferrate(II) trihydrate (Sigma-Aldrich, catalog number: 60279 )]
8. Potassium Ferricyanide [e.g., Potassium hexacyanoferrate(III) (Merck Millipore Corporation, catalog number: 104973 )]
9. 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid (X-Gluc) (e.g., Biosynth, catalog number: B-7300 )
10. Chloral hydrate (e.g., Sigma-Aldrich, catalog number: 23100 )
  Safety note: acute toxicity, avoid inhalation.
11. Glycerol (e.g., Sigma-Aldrich, catalog number: G6279 )
12. GUS staining solution (see Recipes)
13. Chloride hydrate solution (see Recipes)

Equipment

Sterile hood (laminar flow hood)
Fine forceps style 4 (e.g., Dumont, catalog number: 0508-4-PO )
Micropipettes (P20, P200, P1000)
Phytotron or plant growth chamber set with the following growth conditions: 21 °C, 100 μE m^-2 s^-1 light intensity, 14 h light/ 10 h dark photoperiod
Portable dissecting stereomicroscope
Vacuum pump
37 °C incubator
Stereomicroscope [e.g., Leica Microsystems, model: Leica MZ16A fitted with a camera (model: DFC310FX )]
Differential Interference Contrast (DIC) microscope [e.g., Leica Microsystems, model: Leica DM5500 fitted with a camera (model: DFC420 )]

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Gasperini, D., Acosta, I. F. and Farmer, E. E. (2016). Cotyledon Wounding of Arabidopsis Seedlings. Bio-protocol 6(2): e1712. DOI: 10.21769/BioProtoc.1712.