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Arabidopsis Leaf Explant Culture
拟南芥叶外植体培养   

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参见作者原研究论文

本实验方案简略版
The Plant Cell
Mar 2015

Abstract

In this protocol, Arabidopsis leaf explant culture is described using an adaptation of a previous method (Hu et al., 2000). Cells from the cut edges of leaf explant are able to proliferate and subsequently form calli on the callus induction medium, in which is supplemented with 2,4-D and 6-benzyl aminopurine [6-BA]. 2,4-D, one of the artificial auxin, is able to promote cell mitosis at low concentration. 6-BA, the first generation of synthetic cytokinin, plays an important role in plant cell division. 2,4-D in combination with 6-BA can effectively induce callus formation (Rashmi and Trivedi, 2014). The aim of this protocol is to analyze cell division competence of Arabidopsis plants with different genotypes. This protocol can be modified and applied to culture explants from other types of plant tissues, such as root and stem.

Keywords: Leaf explant (叶片外植体), Tissue culture (组织文化), Callus (老茧)

Materials and Reagents

  1. Plastic Petri dish
  2. Parafilm
  3. Millex-GP Filter Unit 0.22 μm (Merck Millipore Corporation, model: R4AA41572 )
  4. 2-3 weeks old sterile Arabidopsis plants (Murashige & Skoog solid medium grown)
  5. Murashige & Skoog medium (Duchefa Biochemie, catalog number: P11293.01 )
  6. Phytagel (Sigma-Aldrich, catalog number: MFCD00131909 )
  7. NaOH
  8. Sucrose
  9. 2, 4-dichlorophenoxyacetic acid (2, 4-D) (Sigma-Aldrich, catalog number: D7299 ) (see Recipes)
  10. 6-benzylaminopurine (6-BA) (Sigma-Aldrich, catalog number: 83488 ) (see Recipes)
  11. Murashige & Skoog solid medium (see Recipes)
  12. Callus induction medium (see Recipes)

Equipment

  1. Arabidopsis growth chamber
  2. Magnetic stirrer and stirring bar
  3. pH meter
  4. Scissors
  5. Tweezers
  6. Flow cabinet

Procedure

  1. Preparation of sterile Arabidopsis plants
    1. Prepare the Murashige & Skoog (MS) solid medium.
    2. Autoclave the Murashige & Skoog (MS) solid medium and pour it into Petri dishes when the temperature of the bottle reaches 50 °C.
    3. Put the Arabidopsis seeds in the 30% 84 bleach for 10 min for sterilization. Discard the bleach and wash the seeds with sterilized water for three times.
    4. Place 10~15 sterilized Arabidopsis seeds onto each Petri dish with the MS solid medium, and seal the Petri dishes with parafilm to keep sterile and moist.
    5. Store the Petri dishes in darkness at 4 °C for 2 to 4 d for vernalization.
    6. Transfer the Petri dishes to Arabidopsis growth chamber at 22 °C under a long-day condition (16 h light/8 h dark).
    7. Culture the Arabidopsis plants for about 2 to 3 weeks until the width of leaves reaches 3-5 mm (Figure 1).


      Figure 1. Arabidopsis plants culturing on the MS solid medium for 3 weeks. Bar = 1 cm.

  2. Preparation of callus induction medium
    1. Prepare and autoclave the MS solid medium.
    2. Add 100 µl of 1 mg/L 2,4-D and 1 mg/L 6-BA into the MS solid medium, named callus induction medium, when the temperature of the bottle reaches 50 °C. Ensure that the performance is done in a flow cabinet.
    3. Pour the callus induction medium into Petri dishes. Ensure that the performance is done in a flow cabinet.

  3. Leaf explant culture
    1. Ensure that all the performances from steps C11-13 are done in the flow cabinet.
    2. Cut across the leaf and midvein with sterilized scissors to yield small strips, which are about 5 mm in length and 2 mm in width, including the midvein going across the width. Ensure that similar mature rosette leaves are selected for the experiments.
    3. Select leaf strips in similar size and transfer them to the same callus induction medium with sterilized tweezers (Figure 2).


      Figure 2. Arabidopsis leaf strips. Bar = 3 mm.

    4. Seal the Petri dishes with parafilm to keep sterile and moist.
    5. Store the Petri dishes at 22 °C in darkness for 1 to 2 weeks.
    6. Observe the calli and take photographs (Figure 3).


      Figure 3. Callus growth in leaf explants (Wang and Guo, 2015). Callus growth in leaf explants of Col-0, 35S-miR159847 transgenic (lines 8), iaa28-ko mutant, and 35S-IAA28-1 and PCsVMV-IAA28m transgenic plants. The explants were photographed on day 7 without changing the medium. Bar = 2 mm.

Recipes

  1. 1 mg/ml 2,4-D
    Dissolve 20 mg 2,4-D power in 5 ml ethanol, and while stirring, gently add water to 20 ml
    Subsequently, sterilize it by filtration through a Millex-GP Filter Unit (0.22 µm), in the flow cabinet
    Stored at -20 °C
  2. 1 mg/ml 6-BA
    Dissolve 20 mg 6-BA power in 5 ml 0.1 M NaOH, and while stirring, gently add water to 20 ml
    Subsequently, sterilize it by filtration through a Millex-GP Filter Unit (0.22 µm) in the flow cabinet
    Stored at -20 °C
  3. Murashige & Skoog solid medium (1 L)
    4.4 g Murashige & Skoog medium
    30 g sucrose
    3.5 g phytagel
    Adjust pH to 5.9, using 1 M NaOH
  4. Callus induction medium (1 L)
    Murashige & Skoog solid medium
    100 µl 1 mg/L 2,4-D
    100 µl 1 mg/L 6-BA

Acknowledgments

This protocol was developed from the following published paper: Hu et al. (2000). This research was supported by the Ministry of Science and Technology (Grant2014CB138402).

References

  1. Hu, Y., Bao, F. and Li, J. (2000). Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J 24(5): 693-701.
  2. Rashmi, R. and Trivedi, M. P. (2014). Effect of various growth hormone concentration and combination on callus induction, nature of callus and callogenic response of Nerium odorum. Appl Biochem Biotechnol 172(5): 2562-2570.
  3. Wang, J. J. and Guo, H. S. (2015). Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in Arabidopsis. Plant Cell 27(3): 574-590.

简介

在该方案中,使用先前方法的适应来描述拟南芥叶外植体培养物(Hu等人,2000)。 来自叶外植体的切缘的细胞能够增殖并随后在愈伤组织诱导培养基上形成愈伤组织,其中补充有2,4-D和6-苄基氨基嘌呤[6-BA]。 2,4-D是人工生长素之一,能够在低浓度下促进细胞有丝分裂。 6-BA,第一代合成细胞分裂素,在植物细胞分裂中发挥重要作用。 2,4-D与6-BA的组合可以有效诱导愈伤组织形成(Rashmi和Trivedi,2014)。 该方案的目的是分析具有不同基因型的拟南芥植物的细胞分裂能力。 该方案可以修改并应用于来自其他类型植物组织(例如根和茎)的培养外植体。

关键字:叶片外植体, 组织文化, 老茧

材料和试剂

  1. 塑料培养皿
  2. parafilm
  3. Millex-GP过滤器单元0.22μm(Merck Millipore Corporation,型号:R4AA41572)
  4. 2-3周龄无菌拟南芥植物(生长的Murashige& Skoog固体培养基)
  5. Murashige& Skoog培养基(Duchefa Biochemie,目录号:P11293.01)
  6. Phytagel(Sigma-Aldrich,目录号:MFCD00131909)
  7. NaOH
  8. 蔗糖
  9. (2,4-二氯苯氧基乙酸(2,4-D)(Sigma-Aldrich,目录号:D7299)(参见配方)
  10. 6-苄氨基嘌呤(6-BA)(Sigma-Aldrich,目录号:83488)(参见配方)
  11. Murashige& Skoog固体培养基(见配方)
  12. 愈伤组织诱导培养基(参见配方)

设备

  1. 拟南芥生长室
  2. 磁力搅拌器和搅拌棒
  3. pH计
  4. 剪刀
  5. 镊子
  6. 流动缸

程序

  1. 制备无菌拟南芥植物
    1. 准备Murashige& Skoog(MS)固体培养基
    2. 高压灭菌 Murashige& Skoog(MS)固体培养基并倒入培养皿中 当瓶子的温度达到50℃时,进入菜肴
    3. 将拟南芥种子置于30%84漂白剂中10分钟以进行灭菌。 丢弃漂白剂,用无菌水冲洗种子三次 次。
    4. 将10?15个灭菌的拟南芥种子置于每个培养皿上 皿与MS固体培养基,并用石蜡膜密封陪替氏培养皿 保持无菌和潮湿。
    5. 将培养皿在4℃的黑暗中保存2-4天以进行春化处理
    6. 在长日照条件下(16小时光照/8小时黑暗)将培养皿转移到22℃的拟南芥生长室中。
    7. 将拟南芥植物培养约2至3周,直到叶片宽度达到3-5mm(图1)。


      图1. 拟南芥 在MS固体培养基上培养3周的植物。 酒吧= 1厘米

  2. 制备愈伤组织诱导培养基
    1. 准备并高压灭菌MS固体培养基。
    2. 加入100μl的1 mg/L 2,4-D和1mg/L 6-BA进入MS固体培养基,命名为愈伤组织诱导 介质,当瓶子的温度达到50℃时。确保这件事 性能在流动柜中完成。
    3. 将愈伤组织诱导培养基倒入培养皿中。确保性能在流动柜中完成。

  3. 叶外植体培养物
    1. 确保步骤C11-13的所有演奏在流动柜中完成。
    2. 用无菌剪刀切开叶和中脉,得到 小条,其长度为约5mm,宽度为2mm, 包括穿过宽度的中脉。确保类似成熟 ?玫瑰花叶被选择用于实验
    3. 选择叶子 条带相似的大小,并将其转移到相同的愈伤组织诱导 介质用灭菌镊子(图2)

      图 拟南芥 叶条。条= 3毫米

    4. 用石蜡膜密封培养皿以保持无菌和潮湿。
    5. 将培养皿在22℃下在黑暗中保存1-2周。
    6. 观察愈伤组织并拍摄照片(图3)。


      图3.叶外植体中的愈伤组织生长(Wang和Guo,2015)。 Callus 在转基因(第8行)的Col-0,35S-miR <159> > iaa28-ko 突变体,以及35S-IAA28-1 和PCsVMV-IAA28m 转基因植物。 在第7天拍摄外植体而不更换培养基。 条= 2mm

食谱

  1. 1mg/ml 2,4-D
    将溶于20毫升2,4-D粉末的5毫升乙醇溶解,并在搅拌下,轻轻加水至20ml 随后,通过Millex-GP过滤单元(0.22μm)过滤灭菌,在流动室
    中 储存于-20°C
  2. 1 mg/ml 6-BA
    将20mg 6-BA溶于5ml 0.1M NaOH溶液中,搅拌下,轻轻加水至20ml。
    随后,通过在流动室
    中的Millex-GP过滤器单元(0.22μm)过滤灭菌 储存于-20°C
  3. Murashige& Skoog固体培养基(1L)
    4.4g Murashige& Skoog培养基
    30克蔗糖 3.5克植物凝集素 使用1M NaOH将pH调节至5.9
  4. 愈伤组织诱导培养基(1L)
    Murashige& Skoog固体介质
    100μl1mg/L 2,4-D
    100μl1mg/L 6-BA

致谢

该协议由以下发表的论文开发:Hu等人(2000)。这项研究得到了科技部的支持(Grant2014CB138402)。

参考文献

  1. Hu,Y.,Bao,F。和Li,J。(2000)。 油菜素类固醇对细胞分裂的促进作用在拟南芥中涉及不同的CycD3诱导途径, Plant J 24(5):693-701。
  2. Rashmi,R。和Trivedi,M.P。(2014)。 各种生长激素浓度和组合对愈伤组织诱导,愈伤组织的性质和肠炎反应的影响> Nerium odorum 。 Appl Biochem Biotechnol 172(5):2562-2570。
  3. Wang,J.J.and Guo,H.S。(2015)。 由microRNA847切割INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA上调生长素信号,调节细胞增殖和侧向拟南芥中的器官生长。 植物细胞27(3):574-590。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, J., Zhang, L. and Guo, H. (2015). Arabidopsis Leaf Explant Culture. Bio-protocol 5(22): e1654. DOI: 10.21769/BioProtoc.1654.
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