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Grafting Arabidopsis
拟南芥嫁接实验   

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Abstract

In Arabidopsis thaliana, hypocotyl micrografting has been used to investigate transport of flowering signals, mobile silencing signals and other peptides, proteins and secondary compounds. The effects of transported signals on target tissues require that a good vascular connection is re-established across the graft junction between the cut hypocotyls (stumps) of the root (rootstock) and shoot (scion) tissues. We outline here a method that requires only that the cut stumps be placed in close proximity, so that they touch, followed by 3-5 days of undisturbed recovery time during which the grafts are allowed to dry out somewhat. This method is quick, easy to monitor and has up to 90% success rate.

Keywords: Grafting(嫁接), Arabidopsis(拟南芥), Hypocotyl(下胚轴), Rootstock(砧木), Scion(接穗)

Materials and Reagents

  1. Young Arabidopsis seedlings (grown for 3-6 days on ½ MS containing agar plates)
  2. Sterile distilled water
  3. 96 % ethanol

Equipment

  1. Sterile 9 cm petri dishes
  2. Sterile standard Whatman No. 1 filter paper circles Grade 1:11 µm with diameter about 0.5 cm smaller than the sterile petri dish (autoclaved wrapped in foil beforehand) (8.5 cm diameter) (Whatman)
  3. Sterile cellulose nitrate filters (Whatman, catalog number: NC 45 ST)
  4. Sterile forceps
  5. Sterile 1 ml pipette tips
  6. 1 ml pipettor
  7. Micropore tape (3 M, MicroporeTM)
  8. Sapphire knife holder (World Precision Instruments, catalog number: 500317 ) or diamond knife plus blade (Electron Microscopy Sciences, Diamond Dissecting Knife, Type M-M, catalog number: 72025 )
  9. Sapphire blade (World Precision Instruments, catalog number: 504072 ) or diamond blade (Electron Microscopy Sciences, Blade Resharpening, Type M-M, catalog number: 72025-R ; Electron Microscopy Sciences, Replacement Blade, Type M-M, catalog number: 72025-L )
  10. Flow cabinet mounted dissecting microscope with a minimum of 5x magnification
    Note: Any brand we have tried worked.
  11. Parafilm
  12. Hot glass bead sterilizer (any brand) unless using 96 % ethanol for sterilization

Procedure

  1. Preparation for grafting
    Require:
    1. Sterile 9 cm petri dishes.
    2. Sterile regular filter paper with diameter about 0.5 cm smaller than the sterile petri dish (autoclaved wrapped in foil beforehand). 
    3. Sterile cellulose nitrate filters (not to be autoclaved, are wrapped in sterile packing).
    4. Sterile forceps. 
    5. Sterile distilled water.
    6. Sterile 1 ml pipette tips.
    7. 1 ml pipettor. 
    8. Micropore tape.
    9. Ethanol for flaming, unless using a hot glass bead steriliser for forceps.
    10. Sapphire or diamond knife.
    11. Dissecting microscope with attached lamp for use in laminar flow cabinet.


      Figure 1. Gluing the sapphire knife
      Note: The sapphire blade must be superglued into its holder before use. If the ethanol dip used for sterilisation loosens it, it falls out. This is not needed if using a diamond blade.

  2. Grafting Arabidopsis seedlings
    1. Set up the materials in the laminar flow cabinet as below: Rinse hands in 70% ethanol, wipe laminar flow surface and dissecting microscope with 96% ethanol.


      Figure 2. Setup of equipment in the laminar flow cabinet

    2. Add two sterile filter papers to a sterile petri dish. Wet the filter papers with 2 ml sterile water. Place two cellulose nitrate filters side-by-side on top of these and add an addtional 1 ml sterile water. The purpose of the filters is to provide a firm, yet water penetrable layer that allows both the grafting procedure and subsequent growth of the seedlings to occur. The filters must not be too wet, but must not be so dry that the plants dry out during and after grafting.
    3. Use young seedlings that have been grown vertically on ½ MS containing agar plates for 3-6 days, no more than 2-3 cm in total length, with the cotyledons sufficiently expanded to be almost horizontal-see Figure 4. The first two true leaves should not be visible or only barely visible.


      Figure 3. Putting seedlings onto the nitrocellulose plates

    4. Dip a pair of forceps in ethanol and flame, or, place forceps in hot glass bead steriliser (250 °C) for approx. 60 sec. After either procedure, press tips of forceps into the agar to cool so seedlings are not damaged by heat. Under the dissecting microscope, select 6-8 very straight seedlings for use as the scions, and place them on the top half of the wet cellulose nitrate filters. Replace the lid on the agar plate containing seedlings, and store vertically to one side of the cabinet.
    5. Now take a second plate of seedlings and use the dissecting microscope to select 6-8 very straight seedlings for use as the rootstocks of the grafts, and place them on the bottom half of the wet cellulose nitrate filter. Replace the lid on the agar plate containing seedlings, and store vertically to one side of the cabinet.
    6. Take the sapphire/diamond knife, dip into 96% ethanol and hold upright in the cabinet until dry - this knife must not be flamed or heated as it destroys the blade.
    7. Orient the seedlings under the microscope so they are ready to cut. They should all be oriented so that their cotyledons are lying horizontally on the nitrocellulose membrane as in the left-most image in Figure 4. Use the sapphire/diamond knife to remove the cotyledons from all seedlings, and move these to one side of the filter paper, or remove with forceps.
    8. Now use the knife to cut the scion seedlings cleanly, and exactly horizontally, across the hypocotyl just below the cotyledon stumps-as in the image second from right in Figure 4.


      Figure 4. Aligning seedlings for cutting

    9. Make sure the rootstock seedlings were also lying with their cotyledons flat and horizontal. Now cut the rootstock seedlings cleanly, and exactly horizontally, across the hypocotyl just below the cotyledon stumps.
    10. Very important: With the forceps, very gently grab a rootstock piece by the longest root hairs on the oldest part of the root just below the hypocotyl and place it so the cut stump touches the cut stump of a scion cutting. Repeat for all rootstocks. It is very important for the graft efficiency to minimize moving or touching the scion after it has been cut! The most successful grafts are those in which the two phloem strands match up from rootstock to scion, and this is ensured if the cotyledons are lying at the same angle before cutting. Successful grafts also have rootstock and scion well-matched for size, with the “stumps” cut very cleanly and squarely, so that the cut ends of rootstock and scion can be pressed very closely together, as in the righthand image in Figure 5. Separation between rootstock and scion, as in the central image of Figure 5, generally results in failure to connect.


      Figure 5. Scion/rootstock connection after cutting

    11. With care, the reciprocal cuttings can be placed, stump to stump, to form the reciprocal grafts.
    12. The cellulose nitrate filter paper must be kept moist, but not running wet, at all times.
      After all grafts are complete, the paper must be just damp, not wet, or the grafts will be less successful. When transferred to the growth room, there must not be too much water in the base of the vertical petri dish. It should look as in Figure 6.


       Figure 6. Amount of water that should be present in the petridish

    13. When grafting is complete, seal the petri dish with three layers of micropore tape and keep the grafts vertical at all times. Sealing with parafilm is not recommended-it is important that the grafts dry out somewhat in order to reconnect and they also need good aeration. 
    14. Place petri dishes with grafts into growth room. They should remain undisturbed for at least 3 days, and can be left for up to 5 days. They may need addition of a little sterile water on day 3 or 4, but not too much!

Notes

  1. Good sharp double-edge razor blades will probably work as well as the sapphire or diamond knives, if used only a few times and discarded as soon as there is any resistance to cutting. Similarly, when the cutting is less smooth with either sapphire or diamond scalpels, exchange the blade. Diamond scalpels work very well, are robust, retain their sharp cutting edge for several months, and can be re-sharpened, but they are very expensive! Stainless steel surgical scalpels may also work well but these have not been tested by us. The advantage of the sapphire and diamond blades is that they are transparent, so it is easy to see exactly where you are cutting, which is important when dissecting very small tissues.
  2. The scion hypocotyls are cut immediately below where the cotyledon petioles attach to the hypocotyl. This helps to prevent adventitious roots growing from the scion and causing the graft to fail, especially if it takes a little longer for vascular tissue to rejoin. In general, adventitious roots emerging from the scion indicate a less successful graft union. If the adventitious roots are removed when still small, the graft will generally survive.

Acknowledgments

Note that a similar protocol has recently been described (Marsch-Martinez et al., 2013).

References

  1. Andersen, T. G., Nour-Eldin, H. H., Fuller, V. L., Olsen, C. E., Burow, M. and Halkier, B. A. (2013). Integration of biosynthesis and long-distance transport establish organ-specific glucosinolate profiles in vegetative Arabidopsis. Plant Cell 25(8): 3133-3145.
  2. Chen, A., Komives, E. A. and Schroeder, J. I. (2006). An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis. Plant Physiol 141(1): 108-120.
  3. Flaishman, M. A., Loginovsky, K., Golobowich, S. and Lev-Yadun, S. (2008). Arabidopsis thaliana as a model system for graft union development in homografts and heterografts. J Plant Growth Regul 27(3): 231-239.
  4. Turnbull, C. G., Booker, J. P. and Leyser, H. M. (2002). Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant J 32(2): 255-262.
  5. Marsch-Martinez, N., Franken, J., Gonzalez-Aguilera, K. L., de Folter, S., Angenent, G. and Alvarez-Buylla, E. R. (2013). An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings. Plant Methods 9(1): 14.

简介

在拟南芥中,下胚轴微量移植已经用于研究开花信号,移动沉默信号和其他肽,蛋白质和次级化合物的转运。 运输信号对靶组织的影响需要在根(根茎)和芽(接穗)组织的切割的下胚轴(树桩)之间的移植物接合处重建良好的血管连接。 我们在这里概述了一种方法,其仅需要将切割的树桩放置在非常接近的位置,使得它们接触,随后是3-5天的不受干扰的恢复时间,在此期间允许移植物稍微干燥。 这种方法快速,易于监控,并具有高达90%的成功率

关键字:嫁接, 拟南芥, 下胚轴, 砧木, 接穗

材料和试剂

  1. 拟南芥幼苗(在含有琼脂平板的1/2MS上生长3-6天)
  2. 无菌蒸馏水
  3. 96%乙醇

设备

  1. 无菌9厘米培养皿
  2. 无菌标准Whatman 1号滤纸圆圈等级1:11μm,直径比无菌培养皿(预先高压灭菌包装箔)(直径8.5cm)(Whatman)小约0.5cm
  3. 无菌硝酸纤维素过滤器(Whatman,目录号:NC 45 ST)
  4. 无菌钳
  5. 无菌1ml移液器吸头
  6. 1 ml移液器
  7. 微孔胶带(3M,Micropore TM
  8. 蓝宝石刀架(World Precision Instruments,目录号:500317)或金刚石刀加刀片(Electron Microscopy Sciences,Diamond Dissecting Knife,Type M-M,目录号:72025)
  9. 蓝宝石刀片(World Precision Instruments,目录号:504072)或金刚石刀片(Electron Microscopy Sciences,Blade Resharpening,MM型,目录号:72025-R; Electron Microscopy Sciences,Replacement Blade,MM型,目录号:72025-L)
  10. 流动柜安装解剖显微镜,最小放大5倍
    注意:我们尝试过的任何品牌都有效。
  11. parafilm
  12. 热玻璃珠灭菌器(任何品牌),除非使用96%乙醇灭菌

程序

  1. 准备嫁接
    要求:
    1. 无菌9厘米培养皿。
    2. 无菌普通滤纸,直径比无菌培养皿小约0.5厘米(预先用高压灭菌包装箔)。
    3. 无菌硝酸纤维素过滤器(不进行高压灭菌,包装在无菌包装中)
    4. 无菌钳。
    5. 无菌蒸馏水。
    6. 无菌1 ml移液器吸头
    7. 1 ml移液器。
    8. Micropore磁带
    9. 乙醇燃烧,除非使用热玻璃珠灭菌器镊子
    10. 蓝宝石或金刚石刀。
    11. 用于层流柜的附带灯泡的解剖显微镜

      图1.粘贴蓝宝石刀
      注意:蓝宝石刀片必须先被固定在其支架上 使用。 如果用于灭菌的乙醇浸泡松开它,它掉出来。   如果使用金刚石刀片,则不需要。

  2. 接枝拟南芥幼苗
    1. 在层流柜中设置材料如下:用70%乙醇冲洗双手,用96%乙醇擦拭层流表面和解剖显微镜。


      图2.层流柜中的设备设置

    2. 添加两个无菌滤纸到无菌培养皿。用2ml无菌水湿滤纸。放置两个硝酸纤维素过滤器并排在这些之上,并添加1毫升无菌水。过滤器的目的是提供坚固的,但是水可渗透的层,其允许秧苗的嫁接程序和随后的生长发生。过滤器不能太湿,但不能太干,植物在移植期间和移植后会变干
    3. 使用在含有琼脂平板的1/2MS中垂直生长的幼苗3-6天,总长度不超过2-3cm,子叶充分膨胀至几乎水平 - 参见图4.前两片真叶不应该是可见的或只是几乎看不见

      图3.将籽苗放在硝酸纤维素板上

    4. 浸入一对镊子在乙醇和火焰,或,将镊子放在热玻璃珠消毒器(250°C) 60秒。在任一过程之后,将镊子的尖端压入琼脂中以冷却,从而幼苗不被热损坏。在解剖显微镜下,选择6-8非常直苗用作接穗,并将其放置在湿硝酸纤维素过滤器的上半部分。替换含有幼苗的琼脂平板上的盖子, 并垂直存储在机柜的一侧
    5. 现在取第二块幼苗,并使用解剖显微镜选择6-8非常直线的幼苗用作移植物的根茎,并将它们放置在湿硝酸纤维素过滤器的下半部分。更换含有幼苗的琼脂板上的盖子,并垂直存放在橱柜的一侧
    6. 拿蓝宝石/金刚石刀,浸入96%乙醇,直立在橱柜里,直到干燥 - 这刀不能燃烧或加热,因为它破坏了刀片。
    7. 在显微镜下使幼苗定向,以便它们准备切割。它们应该被定向为使得它们的子叶水平地位于硝酸纤维素膜上,如在图4中的最左边的图像中。使用蓝宝石/金刚石刀从所有幼苗中移除子叶,并将它们移动到过滤器的一侧纸张,或用镊子取出
    8. 现在使用刀切割接穗幼苗清洁,准确地水平,穿过下胚轴正下方子叶残端 - 如图中右侧第二个图像。


      图4.对齐切割的树苗

    9. 确保砧木幼苗也躺着与他们的子叶平和水平。现在切割根茎幼苗干净,正确水平,跨越下面的子叶树桩下胚轴。
    10. 非常重要的是:用镊子,用根尖最轻的根毛,在下根下的根的最老部分轻轻地抓住根茎,并将其放置,使切割的树桩触及接缝切割的切根。对所有砧木重复此操作。这是非常重要的移植效率,以最小化移动或接触接穗后,它已被切割!最成功的移植物是其中两个韧皮部链从砧木到接穗匹配的移植物,并且如果子叶在切割之前以相同的角度躺下,则这被确保。成功的移植物还具有良好匹配的尺寸的根茎和接穗,"树桩"非常干净和正方形地切割,使得砧木和接穗的切割端可以非常紧密地压在一起,如图5的右图所示。分离在根茎和接穗之间,如图5的中心图像,通常导致无法连接

      图5.切割后的Scion /根茎连接

    11. 小心地,可以放置相互切割,残留到残端,以形成相互移植物
    12. 硝酸纤维素滤纸必须保持湿润,但不能潮湿。
      在所有移植物完成后,纸张必须刚刚潮湿,而不是湿的,或者移植物将不太成功。当转移到生长室时,垂直培养皿的底部不能有太多的水。它应该如图6所示。


        图6.在培训中应存在的水量

    13. 当移植完成时,用三层微孔带密封陪替氏培养皿并保持移植物在任何时候垂直。不建议使用石蜡膜密封 - 重要的是,移植物有些干燥,以便重新连接,并且它们还需要良好的曝气。 
    14. 将培养皿与移植物放入生长室。他们应该保持不受干扰至少3天,并可以留下长达5天。他们可能需要在第3天或第4天添加少许无菌水,但不要太多!

笔记

  1. 良好的锋利的双刃剃刀刀片可能工作,以及蓝宝石或金刚石刀,如果使用只有几次,一旦有任何阻力切割,抛弃。类似地,当切割对于蓝宝石或金刚石解剖刀而言不太平滑时,更换刀片。钻石手术刀工作得很好,坚固,保持锋利的刀刃几个月,可以重新磨尖,但他们是非常昂贵的!不锈钢外科手术刀也可能工作良好,但这些没有经过我们的测试。蓝宝石和金刚石刀片的优点是,它们是透明的,所以很容易看到你正在切割,这是很重要的,当解剖非常小的组织。
  2. 接下来的胚轴是在子叶柄附着到下胚轴的下面切开的。这有助于防止从接穗生长的不定根,并导致移植失败,特别是如果需要一段时间更长的血管组织重新加入。一般来说,从接穗中出现的不定根表明不太成功的移植联合。如果不小根在仍然小的时候被去除,移植物通常会存活

致谢

注意,最近已经描述了类似的协议(Marsch-Martinez等人,2013)。

参考文献

  1. Andersen,T.G.,Nour-Eldin,H.H.,Fuller,V.L.,Olsen,C.E.,Burow,M。和Halkier,B.A。(2013)。 生物合成与长距离运输的整合在植物中确定器官特异性硫代葡萄糖苷配置文件拟南芥/em>。植物细胞 25(8):3133-3145。
  2. Chen,A.,Komives,E.A。和Schroeder,J.I。(2006)。 成熟拟南芥植物的改良嫁接技术展示了长距离拍摄,植物生理学141(1):108-120。植物生理学中的植物生长素根植物转运。
  3. Flaishman,M.A.,Loginovsky,K.,Golobowich,S。和Lev-Yadun,S。(2008)。 拟南芥 thaliana 作为在同种移植物和异种移植物中的移植联合发展的模型系统。 27植物生长调节 27(3):231-239。
  4. Turnbull,C.G.,Booker,J.P。和Leyser,H.M。(2002)。 在拟南芥中测试长距离信号的微观技术。 Plant J 32(2):255-262。
  5. Marsch-Martinez,N.,Franken,J.,Gonzalez-Aguilera,K.L.,de Folter,S.,Angenent,G.and Alvarez-Buylla,E.R。 拟南芥幼苗的高效平面无颈圈接枝方法 。植物方法 9(1):14.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Andersen, T. G., Liang, D., Halkier, B. A. and White, R. (2014). Grafting Arabidopsis. Bio-protocol 4(13): e1164. DOI: 10.21769/BioProtoc.1164.
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