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Aug 2020
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Micrografting in Arabidopsis Using a Silicone Chip
利用硅芯片进行拟南芥微嫁接   

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Abstract

The micrografting technique in the model plant Arabidopsis has been widely used in the field of plant science. Grafting experiments have demonstrated that signal transductions are systematically regulated in many plant characteristics, including defense mechanisms and responses to surrounding environments such as soil and light conditions. Hypocotyl micrografting is a powerful tool for the analysis of signal transduction between shoots and roots; however, the requirement for a high level of skill for micrografting, during which small seedlings are microdissected and micromanipulated, has limited its use. Here, we developed a silicone-made microdevice, called a micrografting chip, to perform Arabidopsis micrografting easily and uniformly. The micrografting chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path to hold hypocotyl. All micrografting procedures are performed on the chip. This method using a micrografting chip will avoid the need for training and promote studies of systemic signaling in plants.


Graphic abstract:



A silicone chip for easy grafting


Keywords: Grafting (嫁接), Micrografting (微型嫁接), Arabidopsis (拟南芥), Seedling (秧苗), Systemic signaling (系统信号), Long-distance signaling (长途信号), Silicone (硅树脂), Microdevice (微型装置)

Background

Grafting has served as a method to assemble two different types of plants, not only in agriculture but also in the field of plant science. Although plants do not have a nervous system, many studies using grafting experiments have demonstrated that plants properly integrate information from the entire body (Wang, 2011; Goldschmidt, 2014; Notaguchi and Okamoto, 2015; Thomas and Frank, 2019). In response to changes in their surrounding environments, plants generate information signals locally at the site of reception and transport signaling molecules systemically to transmit information. Inside plants, organ-to-organ communication is achieved via vascular tissues using a wide variety of molecules such as phytohormones, small RNAs, mRNAs, and proteins.


To study systemic signaling, especially between shoots and roots, micrografting, also known as seedling grafting, has been used. Micrografting is a method for assembling a scion and a rootstock that are cut out of seedlings from different plants. Since the first micrografting technique in the model plant Arabidopsis was demonstrated by Turnbull et al. (2002), micrografting has been widely used in a great number of studies and has contributed to the identification of molecules involved in systemic signaling such as flowering, defense against biotic infection, nutrient absorption and allocation, and drought stress response (Tsutsui and Notaguchi, 2017).


However, the micrografting technique requires dexterous operation and practice to achieve a high rate of success. Several papers have developed improved methods using modified procedures and equipment (Notaguchi et al., 2009), an agar-base support (Marsch-Martínez et al., 2013), and a fine pin to align the scion and the stock (Huang and Yu, 2015); nevertheless, micrografting remains challenging for beginners. To overcome the difficulties in conventional micrografting methods and make this technique accessible to those who study systemic signaling, we recently developed a microdevice made of silicone rubber, called a micrografting chip (Tsutsui et al., 2020). The device was fabricated by Micro-Electro Mechanical Systems using poly(dimethylsiloxane) (PDMS). The micrografting chip supports the manipulation of entire micrografting processes and facilitates the generation of uniformly grafted plants. This method has been applied in studies on plant biology using Arabidopsis mutants (Tsutsui et al., 2020; Shinozaki et al., 2020; Kurotani et al., 2020) and molecular mechanisms of grafting (Notaguchi et al., 2020). Here, we show the procedure for micrografting using a micrografting chip, which is slightly modified in its structure from Tsutsui et al. (2020) (Figure 1).



Figure 1. Structure of a micrografting chip. A. Image of one unit of the micrografting chip. B. Layout of the unit of the micrografting chip. C. Images of the micrografting chip (top) and its cover (bottom). D. Layouts of the micrografting chip (top) and its cover (bottom).

Materials and Reagents

  1. Disposable gloves (Kimberly-Clark, catalog number: 52816)

  2. Square Petri dishes with grid (Simport Scientific, catalog number: D210-16)

  3. Petri dishes (ASNOL, catalog number: GD90-15)

  4. 1.5 ml tubes (Trefflab, catalog number: 96.07246.9.01)

  5. Disposable surgical scalpel No.11 (KAI, catalog number: 511-A)

  6. Disposable dropper 2 ml (Maruemu, catalog number: 0806-03)

  7. Filter paper No.1, 70 mm (ADVANTEC, catalog number: 00011070)

  8. Hybond-N+ membrane (GE Healthcare, catalog number: RPN119B)

  9. Dissecting needle

  10. Surgical tape (3M, catalog number: 1530-0)

  11. Aluminum foil (UACJ)

  12. KimWipes (CRECIA, catalog number: S-200)

  13. Seeds of Arabidopsis thaliana

    Note: Fresh seeds must be used. For successful micrografting, a scion and a stock at the same developmental stage are required. Old seeds show variation in the timing of seed germination and subsequent growth, hampering graft establishment. We used Arabidopsis thaliana ecotype Col-0 mainly, but other ecotypes can also be used.

  14. Murashige-Skoog medium (Duchefa Biochemie, catalog number: M0221)

  15. Sucrose (WAKO, catalog number: 190-00013)

  16. Agar powder (Nacalai tesque, catalog number: 01028-85)

  17. MES hydrate (Sigma-aldrich, catalog number: M2933)

  18. 76.9-81.4%(v/v) ethanol (KANEICHI, catalog number: 4987556210311)

  19. Potassium hydroxide (WAKO, catalog number: 168-21815)

  20. Bleach (KAO, catalog number: 017598)

  21. 1 N KOH (see Recipes)

  22. 1% agar plates (see Recipes)

  23. 2% agar plates (see Recipes)

  24. Hybond-N+ membrane (GE Healthcare, catalog number: RPN119B)

  25. 0.4% agar for resuspension of sterilized seeds (see Recipes)

  26. Filter paper (see Recipes)

  27. Sterilized water (see Recipes)

  28. Bleach solution (see Recipes)

  29. Sterilized seeds (see Recipes)

Equipment

  1. pH meter (HORIBA, catalog number: F-51)

  2. Pipette P20 (INMEDIAM, catalog number: FA10003P)

  3. Micrografting chip (Bio Medical Science Inc., catalog number: BGA-GRC020) (Figure 1)

    Note: We used the micrografting chips as disposable. The purchased chips are sterilized and ready to use after opening the package.

  4. Super clean zone creator (Koken, catalog number: KOACH T 500-F)

  5. Dissecting microscope (OLYMPUS, catalog number: SZX10)

  6. Precision tweezers (Inox-biology, catalog number: 11252-00)

  7. Vannas spring scissors (Fine Science Tools, catalog number: 15019-10)

  8. Plant growth chamber (Biotron, catalog number: LPH-411S)

  9. Bio-clean bench (AIRTECH, BLB-1306)

Procedure

Note: All procedures using a microscope should be performed under sterile conditions. We use a super clean zone creator KOACH T 500-F (Koken) that provides flow of filtered air (Figure 2A). A bio-clean bench can be its substitute. In all procedures, an experimenter must wear gloves and sterilize all equipment and areas by wiping with tissue paper saturated with 70% ethanol.

In the case that it is difficult to perform grafting under sterile conditions, the sucrose content of the agar media needs to be reduced to 0.2% or less. Even if it is difficult to prepare entirely sterile conditions, seed sterilization, wiping equipment with ethanol, and cleaning experimental spaces as much as possible are still preferred.


  1. Seed sterilizing

    1. Place dozens of seeds into a 1.5-ml tube, resuspend them in 1 ml 10% bleach solution (the effective chlorine concentration is 0.6%) for 5 min, and wash with 1 ml sterilized water 5 times.

    2. After sterilization, resuspend the seeds in 0.4% agar and place them in dark at 4°C for at least three days before sowing, but do not exceed 1 week.

      Note: To synchronize the timing of seed germination, incubation at 4°C for more than three days is important.


  2. Seed sowing (Day 1)

    1. Leave 1% agar plates at room temperature.

    2. Separate Hybond-N+ membrane from its mat and place the membrane on the 1% agar plate (Figure 2B).

    3. Place several micrografting chips on the membrane.

    4. Sow a seed (that has been stratified at 4°C for more than 3 days in 0.4% agar) onto a seed pocket and load 0.4% agar onto the pocket and a root path using a Pipette P20 (Figure 2C).

      Steps B4-B6 are demonstrated in Video 1.

      Note: To avoid confusion, the seeds for scions and stocks must be sown separately on different chips.


      Video 1. Seed sowing on a micrografting chip


    5. Cover the chip with the chip cover (Figure 2C).

    6. Place the chip upright on the membrane (Figure 2B).

    7. Seal the plate with surgical tape.

    8. Wrap the plate in aluminum foil.

      Note: To ensure that the seedlings get etiolated, wrap the plate doubly. Otherwise, a crack of light will prevent the seedlings from being etiolated and the cotyledons cannot pass through a micro-path in the chip during incubation.

    9. Incubate the plate at 22°C in a growth chamber for about 48-58 h.

      Note: From Steps B7 to B9, handle the plates with care; do not lay the chips down.



      Figure 2. Overview of micrografting procedures. A. A workbench for micrografting. B. Micrografting chips were placed on 1% agar media after sowing seeds. Up to 10 chips (40 seedlings) can be placed on a single plate. C. The steps of micrografting. The red arrowhead on Day 1 indicates the end of a pipette tip for seed sowing. The black arrowhead on Day 3 indicates a hook of the seedling incubated under dark conditions. Scale bars: 500 µm.


  3. Transferring to light conditions (Day 3)

    1. Take the plates from the growth chamber and unwrap the aluminum foil.

    2. Place the plate at 22°C under continuous light for about 48 h.

      Note: Since the timing of germination and the growth speed of germinated Arabidopsis seedlings depend on seed freshness, genetic strains, and experimental environmental conditions, it is important for each experimenter to find the optimal incubation period in the dark by initially observing the growth of etiolated seedlings. When the hook is passing by the second top pillars, it is the best timing to unwrap the foil for that seedling (Figure 2C, Day 3). In our case, we opened the lids of the dishes temporally and observed the plant growth using a stereomicroscope under sterile conditions. More practically, we also observed the seedling growth with the naked eye. Since the timing of seed germination and the growth pace of seedlings vary among seedlings, the foil should be removed at the time when the hooks of the “majority” of the seedlings are passing by the second top pillars as described above. In our case, we obtained around 60-90% of seedlings growing properly at the grafting step. Waiting for slow-growing seedlings is not advised, since the hypocotyls of the remaining seedlings will elongate too much. As another indicator of optimal timing to uncover the foil, we used the emergence of cotyledons from the top of the chips. When we saw a couple of etiolated seedlings in the dishes start to expand their cotyledons above the chip, it was good timing. If you see none of the seedlings appearing from the chip, cover the plate again and extend the incubation.


  4. Micrografting (Day 5)

    After four days of incubation (two days in the dark and two days in the light), a seedling passes through the chip and the cotyledons expand over the chip as shown in Figure 2C, Day 5.

    Note: The following procedures must be performed quickly to avoid desiccation; within 5 min for each chip. Desiccation, especially at the graft junction, significantly hampers graft establishment.

    1. Leave 2% agar plates at room temperature and place Hybond-N+ membrane on them.

      Notes:

      1. The 2% agar plates are used for post-grafting growth to prevent too high a humidity in the plates.

      2. The Hybond-N+ membrane is used to avoid root growth into the medium and make transplanting easy. In our tests of several kinds of filter membrane, the Hybond-N+ and cellulose nitrate filter membranes did not inhibit plant growth.

    2. Place an autoclaved filter paper onto a Petri dish and wet the paper slightly with sterilized water using a disposal dropper (Figure 3A). Before use, wash the inside of the dropper a few times with sterilized water.

      Note: Excessive amounts of water inhibit graft establishment. The recommended amount of water is around 1.1-1.2 ml per filter paper.

    3. Transfer the chip onto the wet filter paper and lay down it (Figure 3B).

    4. Remove the chip cover (Figure 3B, Video 2).

      Note: Chip covers will not be used in the following process.


      Video 2. Removal of a chip cover

      For Steps D5-D10, the procedures are shown in Video 3.


      Video 3. Micrografting procedure

    5. Cut the hypocotyl along the slit using the tip of a disposable surgical scalpel for the preparation of both stock and scion parts (Figures 3C and 3D).

      Notes:

      1. A sharp scalpel is essential for successful grafting. If the scalpel is blunt, use a new one; otherwise, a deformed cut end caused by a blunt scalpel will markedly decrease the success rate of micrografting.

      2. If the seedlings are not well secured in the chips, push the hypocotyls gently into the micro-paths with tweezers and then perform grafting.

    6. For stock preparation, remove its cut shoot (Figure 3C).

    7. Pick up a scion, a shoot, from a chip by grabbing one of the cotyledons with precision tweezers and transfer it onto the stock placed on another chip (Figures 3C and 3D).

      Note: Do not grab the scion forcefully with the tweezers. Large damage causes failure of grafting.

    8. Align the hypocotyl of the scion with that of the stock with tweezers by gently touching the scion and the stock to adjust their positions (Figures 3C and 3D).

    9. If required, remove the water around the graft junction using a twisted KimWipe.

      Note: Residual water on the surface of grafted seedlings, especially at the graft junction, inhibits graft formation and increases the frequency of adventitious root formation. Here, there is no need to cover the grafted plants with a chip cover because it keeps the graft junction humid and inhibits graft establishment.

    10. Place the chip upright.

    11. Transfer the chip carefully onto the membrane on the 2% agar plate prepared in Step D1, so that the grafted plants are not misaligned.

    12. Seal the plate with surgical tape.

    13. Place the plates in the growth chamber at 27°C under continuous light conditions for four days.

      Note: In the case that the light intensity is high in the growth chamber, cover the plates with a KimWipe to reduce the intensity and decrease photodamage to the grafted seedlings. In our case, we set the growth chamber at 100-140 μmol·m-2·s-1 continuous light conditions and cover the plates with a sheet of Kimwipe, reducing the intensity to 60-80 μmol·m-2·s-1.



      Figure 3. Micrografting. A. The workbench setting for micrografting. 1, a dissecting microscope; 2, an LED light; 3, a bottle of sterilized deionized water; 4, a dissecting needle; 5, precision tweezers; 6, an autoclaved filter paper; 7, a Petri dish; 8, a disposable surgical scalpel; 9, a disposable dropper. B. A micrografting chip before (top) and during (bottom) removal of the chip cover. C. The detailed procedures for the micrografting steps on Day 5. Scale bars: 500 µm. D. Illustration of the micrografting procedures.


  5. Removal of adventitious roots (Day 9, 4 days after grafting)

    Adventitious roots frequently emerge on the scion hypocotyls. The following steps are designed to remove them and we strongly recommend performing these because adventitious roots hamper graft formation.

    Note: The following procedures must be performed quickly to avoid desiccation; within 5 min for each chip.

    1. Take the plates from the growth chamber and sterilize them by wiping the surface with ethanol and tissue paper.

    2. Place the plates under the stereomicroscope and open the lid.

      For Steps E3 to E5, the procedures are shown in Video 4.


      Video 4. Removal of adventitious roots from scions


    3. Lay the micrografting chips down using tweezers and check whether adventitious roots are formed on the hypocotyl of the scion.

    4. If adventitious roots are growing on the scion, cut and remove them using Vannas spring scissors or smash them with the tip of the tweezers (Figure 4A).

    5. Place the micrografting chip upright.

    6. Seal the plate with surgical tape.

    7. Wrap the plates in a KimWipe to protect the grafted plants from strong light and place them in the growth chamber at 27°C under continuous light for two days.


  6. Pulling the grafted plants out of the micrografting chip (Day 11, 6 days after grafting)

    1. Leave the new 1% agar plates at room temperature.

    2. Take the plates from the growth chamber and sterilize them by wiping the surface with ethanol and tissue paper.

    3. Place the plates on the stereomicroscope and open the lid.

      For Steps F4 to F6, the procedures are shown in Video 5.

    4. Pull the grafted plants from the micrografting chip carefully using a dissecting needle or tweezers.


      Video 5. The transfer of grafted plants onto a new plate

    5. Observe the grafted plants. If you see adventitious roots in the scion, remove them using Vannas spring scissors or tweezers.

    6. Transfer the grafted plants onto the new 1% agar plate (Figure 4B).

    7. Seal the plate with surgical tape.

    8. Place the plates vertically in the growth chamber at 22°C under continuous light.


  7. Confirmation of graft establishment (Day 15, 10 days after grafting)

    If you observe healthy root growth on the rootstock and true leaf expansion on the scion without further adventitious root formation (Figure 4C), the grafting is judged to be established, and the grafted plants can be transferred onto the soil/media for subsequent experiments.



    Figure 4. The procedures performed until graft establishment. A. A grafted seedling at 4 days after grafting, before and after removal of adventitious roots. The arrowheads indicate the position of an emerged adventitious root. B. Grafted seedlings at 6 days after grafting. The successful grafts were transferred to a new plate. The asterisk indicates an unsuccessful graft. C. The grafted seedling at 10 days after grafting showing vigorous growth of true leaves and roots. Scale bars: 500 µm (A); 1 mm (B) and (C); 5 mm.

Data analysis

We described the protocol for our micrografting chip. We previously reported that the success rate of micrografting using our chip was around 48-88% (Tsutsui et al., 2020), and this success rate bears comparison with that of conventional micrografting methods in Arabidopsis (Turnbull et al., 2002; Yin et al., 2012; Marsch-Martínez et al., 2013). Using this system, we demonstrated the utility of the chip by testing the effect of grafting conditions and investigating the role of genes using the mutant lines (Tsutsui et al., 2020).

Notes

  1. During all processes until the grafted plants are pulled out of the chip (Day 11, 6 days after grafting), the plates should always be kept horizontal and handled carefully to prevent laying the micrografting chips down.

  2. To obtain seedlings growing through the micropath of the micrografting chip, use enough grown large-sized seeds. To obtain well-grown large-sized seeds, the plants for seed propagation must be grown with adequate water, serving to form a robust root system and thick stalks. Arabidopsis grows well under dry conditions rather than wet conditions. In addition, keep watering until most of the seeds are mature; otherwise, immature seeds are formed and mixed in the seed pool.

  3. When sowing seeds in seed pockets, completely fill the spaces in the pocket and the root path with 0.4% agar. Too little a supply of 0.4% agar causes incomplete seedling growth, and too much a supply of 0.4% agar induces floating of the seeds from the pocket when covering with a chip cover.

  4. Sometimes 0.4% agar turns into a solid. After 0.4% agar is autoclaved and cooled at room temperature while stirring, it will be in liquid form. If 0.4% agar still becomes solid, it can be used after stirring or reducing the agar concentration.

  5. Sometimes the chips are lifted up or fall backward as a result of the force of seedling growth. In these cases, the roots are detached from the media and the seedling growth is arrested. Moreover, the hypocotyls start to bend toward the outside of the micropath by gravitropism. These seedlings are not suitable for grafting. If the chips only lay down for a short period and there are no such problems, it is possible to continue the grafting procedures.

  6. The chip cover should be aligned with the line of the top edge of the micrografting chip. When removing the chip cover, seedlings may come off the micrografting chip by attaching themselves to the cover. To avoid this, remove the chip cover gently from one side. Even if the seedlings come off, place the hypocotyls back into the micropaths of the chips and perform grafting.

  7. To minimize damage to the seedlings, try not to touch the hypocotyls, especially around the grafting area and do not pluck the seedlings. When manipulating and adjusting the position of the seedlings, grab only one of the cotyledons, hook the seedling with the tweezers or gently push the seedlings to adjust their position using the side of the tweezers, not the tip.

  8. After grafting, the scions sometimes float due to static electricity on the Petri dishes and attach to the lids. Such scions cannot be restored. To avoid these accidents, it is effective to rinse the inside of the lids with sterile water before use.

Recipes

  1. 1 N KOH

    Dissolve 2.8 g potassium hydroxide in 50 ml deionized water.

  2. 1% agar plate

    1. Dissolve 2.3 g Murashige-Skoog medium, 5 g sucrose, and 0.5 g MES hydrate in 1 L deionized water.

    2. Adjust pH to 5.7 using 1 N KOH.

    3. Add 10 g agar powder and autoclave.

    4. Cool the medium to around 60°C and pour 33 ml into a square Petri dish under a bio-clean bench.

    5. To remove the excess moisture from the plates, dry them for 30-45 min with their lids halfway open.

    6. The plates can be stored at 4°C for up to 1 month.

  3. 2% agar plate

    1. Dissolve 2.3 g Murashige-Skoog medium, 5 g sucrose, and 0.5 g MES hydrate in 1 L deionized water.

    2. Adjust pH to 5.7 using 1 N KOH.

    3. Add 20 g agar powder and autoclave.

    4. Cool the medium to around 60°C and pour 33 ml into a square Petri dish under a bio-clean bench.

    5. To remove the excess moisture from the plates, dry them for 30-45 min with their lids halfway open.

    6. The plates can be stored at 4°C for up to 1 month.

  4. Hybond-N+ membrane

    Cut the Hybond-N+ membranes into squares of approx. 1 cm × 3 cm and place them into a glass Petri dish. Then, autoclave the membrane pieces and dry them in a 60°C drying oven overnight.

  5. 0.4% agar for resuspension of sterilized seeds

    Dissolve 0.4 g agar powder in 100 ml deionized water and autoclave it with a stir bar. Cool down the autoclaved 0.4% agar to room temperature while stirring, which prevents the medium from jelling.

  6. Filter paper

    Place the filter papers into a glass Petri dish, autoclave them, and then dry them in a 60°C drying oven.

  7. Sterilized water

    Autoclave deionized water.

  8. Bleach solution

    Mix 1 ml bleach with 9 ml sterilized water. This solution must be prepared at the time of use and cannot be stored.

Acknowledgments

The present procedures are derived from Tsutsui et al. (2020). This protocol was used in the previous study published by Notaguchi et al. (2020). This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (18KT0040, 18H03950, and 20H03273) and the Canon Foundation (R17-0070) to M.N. We greatly appreciate all the authors of Tsutsui et al. (2020) and Y. Hakamada, M. Hattori, R. Masuda, M. Ishihara, I. Yoshikawa, and A. Yagi for technical support.

Competing interests

The authors declare no conflicts of interest related to this work.

References

  1. Goldschmidt, E. E. (2014). Plant grafting: new mechanisms, evolutionary implications. Front Plant Sci 5: 727.
  2. Huang, N. C. and Yu, T. S. (2015). A pin-fasten grafting method provides a non-sterile and highly efficient method for grafting Arabidopsis at diverse developmental stages. Plant Methods 11: 38.
  3. Kurotani, K., Tabata, R., Kawakatsu, Y., Sugita, R., Okayasu, K., Tanoi, K. and Notaguchi, M. (2020). Autophagy is induced during plant grafting for wound healing. bioRxiv: 2020.02.14.949453.
  4. Marsch-Martínez, 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.
  5. Notaguchi, M., Daimon, Y., Abe, M. and Araki, T. (2009). Adaptation of a seedling micro-grafting technique to the study of long-distance signaling in flowering of Arabidopsis thaliana. J Plant Res 122(2): 201-214.
  6. Notaguchi, M., Kurotani, K. I., Sato, Y., Tabata, R., Kawakatsu, Y., Okayasu, K., Sawai, Y., Okada, R., Asahina, M., Ichihashi, Y., Shirasu, K., Suzuki, T., Niwa, M. and Higashiyama, T. (2020). Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases. Science 369(6504): 698-702.
  7. Notaguchi, M. and Okamoto, S. (2015). Dynamics of long-distance signaling via plant vascular tissues. Front Plant Sci 6: 161.
  8. Shinozaki, D., Notaguchi, M. and Yoshimoto, K. (2020). Importance of non-systemic leaf autophagy for suppression of zinc starvation induced-chlorosis. Plant Signal Behav 15(5): 1746042.
  9. Thomas, H. R. and Frank, M. H. (2019). Connecting the pieces: uncovering the molecular basis for long-distance communication through plant grafting. New Phytol 223(2): 582-589.
  10. Tsutsui, H. and Notaguchi, M. (2017). The Use of Grafting to Study Systemic Signaling in Plants. Plant Cell Physiol 58(8): 1291-1301.
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  13. Wang, Y. Q. (2011). Plant grafting and its application in biological research. Chin Sci Bull 56: 3511-3517.
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简介

[摘要] m个在icrografting技术的模式植物拟南芥已被广泛用于在植物科学领域。嫁接实验表明,信号转导在许多植物特征中受到系统调节,包括防御机制和对周围环境(如土壤和光照条件)的反应。胚轴显微移植是用于拍摄之间的信号转导的分析的有力工具小号和根S; ħ Ó wever ,所述要求为高水平的技能为显微移植, 在此期间,小籽苗米icrodissected和显微操作,公顷小号限制了其使用。在这里,我们开发了一种称为微嫁接芯片的硅胶微型装置,可以轻松、一致地进行拟南芥微嫁接。微移植芯片具有串联排列的单元,每个单元由用于种子发芽的种子袋和用于容纳下胚轴的微路径组成。所有的微移植程序都在芯片上进行。这种方法使用一个显微移植芯片将避免需要进行培训和促进全身信号研究植物。


图文摘要:



一个硅智P代表易于移植




[背景]嫁接已成为组装两种不同类型植物的一种方法,不仅在农业中,而且在植物科学领域中也是如此。虽然PL蚂蚁没有神经系统,许多研究使用嫁接实验已经证明,植物正确地集成信息从在整个身体(王,2011 ;施密特2014; Notaguchi和冈本,2015年,托马斯·弗兰克,2019)。响应于变化中及其周围的环境,植物在接收和传输信号分子的全身性给发送信息的站点本地产生的信息信号。在植物内部,器官间的交流是通过维管组织使用植物激素、小 RNA、mRNA 和蛋白质等多种分子实现的。

为了研究系统性的信号,尤其是芽和根,显微移植之间,也被称为嫁接苗,已被使用。微型嫁接是一种方法用于assembl荷兰国际集团接穗和砧木其是来自不同植物的幼苗切出。由于在第一显微移植技术的模式植物拟南芥通过特恩布尔证明等。(2002),显微移植已被广泛用于研究大量和已做出了贡献的参与全身信令如开花,德芬分子识别小号Ë针对生物感染,营养吸收和分配,和干旱应激反应(Ť sutsui和野田口,2017 年)。

然而,在显微移植技术需要灵巧的操作和实践,以实现一个成功率高。有几篇论文已经开发了改进的方法使用修饰的方法和设备(Notaguchi等人,2009),琼脂-基部支撑(进行曲-马丁内斯等人。,2013)和一个细销以对准的接穗和所述股票(黄和于,2015);ñ evertheless,显微移植遗体挑战初学者。为了克服传统方法显微移植的困难,使这项技术进入到那些谁研究的系统性的信号,我们最近开发的硅橡胶制成的微型装置,称为一个显微移植芯片(筒井等人。,2020)。该装置由微机电系统使用聚(二甲基硅氧烷)(PDMS)制造。微嫁接芯片支持整个微嫁接过程的操作,并促进均匀嫁接植物的产生。该方法已在研究中得到了应用,使用植物生物学拟南芥突变体(筒井等人,2020;筱崎等人。,2020;黑谷等人。,2020)和分子机制小号(Notaguchi接枝的等人。,2020)。这里,我们示出了程序对使用微嫁接米icrografting芯片,其被略微在其结构从筒井改性等。(2020 年)(图 1)。


图1. 微移植芯片的结构。一个。我的法师显微移植芯片的一个单位。乙。大号的显微移植芯片的单元的ayout。Ç 。微移植芯片(顶部)及其盖子(底部)的图像。d 。微移植芯片(顶部)及其盖子(底部)的布局。

关键字:嫁接, 微型嫁接, 拟南芥, 秧苗, 系统信号, 长途信号, 硅树脂, 微型装置

材料和试剂

1.一次性手套(Kimberly-Clark,目录号:52816)      

2.广场P ETRI菜ES与电网(Simport科学,目录号:D210-16)      

3.培养皿ES (ASNOL,目录号:GD90-15)      

4. 1.5 ml 管(Trefflab,目录号:96.07246.9.01)      

5.一次性手术刀No.11(KAI,目录号:511-A)      

6.一次性滴管2毫升(Maruemu,目录号:0806-03)      

7.滤纸No.1,70 mm(ADVANTEC,目录号:00011070)      

8. H ybond-N+ 膜(GE Healthcare,目录号:RPN119B)      

9.解剖针      

10.手术胶带(3M,目录号:1530-0)   

11.铝箔(UACJ)   

12. KimWipe s (CRECIA,目录号:S-200)   

13.拟南芥种子   

注意:必须使用新鲜种子。为了成功的微嫁接,需要在同一发育阶段的接穗和股票。旧种子在种子萌发和随后的生长时间上表现出变化,阻碍了嫁接的建立。我们主要使用拟南芥生态型 Col-0,但也可以使用其他生态型。

14. Murashige-Skoog 培养基(Duchefa Biochemie,目录号:M0221)   

15.蔗糖(WAKO,目录号:190-00013)   

16.琼脂粉(Nacalai tesque,目录号:01028-85)   

17. MES水合物(Sigma-aldrich,目录号:M2933)   

18. 76.9 - 81.4%(v/v)乙醇(KANEICHI,目录号:4987556210311)   

19.氢氧化钾(WAKO,目录号:168-21815)   

20.漂白剂(KAO,目录号:017598)   

21. 1 N KOH(见食谱)   

22. 1%琼脂板小号(见配方)   

23. 2%琼脂板小号(见配方)   

24. Hybond-N+ 膜(GE Healthcare,目录号:RPN119B )   

25. 0.4% 琼脂用于重新悬浮灭菌种子(见配方)   

26.滤纸(见食谱)   

27.消毒水(见食谱)   

28.漂白溶液(见配方)   

29.消毒种子(见食谱)   

设备

pH计(HORIBA,目录号:F-51)
移液器 P20(INMEDIAM,目录号:FA10003P)
中号icrografting芯片(生物医学科学公司,目录号:BGA-GRC 020 )(图1)
注意:我们使用微移植芯片作为一次性的。购买的芯片经过消毒,打开包装后即可使用。

超净区创建器(Koken,目录号:KOACH T 500-F)
解剖显微镜(OLYMPUS,目录号:SZX10)
精密镊子(Inox-biology,目录号:11252-00)
Vannas弹簧剪刀(Fine Science Tools ,目录号:15019-10)
植物生长室(Biotron,目录号:LPH-411S)
生物净化台 (AIRTECH, BLB-1306)
 

程序

注:所有的程序U唱显微镜应进行下无菌条件下小号。我们使用超级洁净区创建器 KOACH T 500-F (Koken),提供过滤空气流(图 2A)。一个生物洁净工作台可以替代它。在所有程序中,实验者必须戴上手套,并通过用 70% 乙醇饱和的薄纸擦拭对所有设备和区域进行消毒。

的,因为它是难以进行无菌条件下接枝的情况下,所述蔗糖含量的的琼脂培养基需要小号减少到0.2%或更小。即使是难以制备完全无菌条件小号,种子消毒,擦拭设备用乙醇,和清洁实验空间尽可能仍然优选的。

A.小号EED消毒      

P花边几十种子到1.5 - ml管,重悬在1ml 10%的漂白剂溶液(有效的氯浓度为0.6%)5分钟,和洗涤用1毫升无菌水5次。
灭菌后,悬浮的种子在0.4%琼脂,并放置在黑暗中,在4℃下至少三天播种前,但不超过1的一周。
注意:要同步种子发芽的时间,在 4°C 下孵育三天以上很重要。

B.       播种(第 1 天)

将 1% 琼脂平板置于室温下。
将H ybond-N+ 膜与其垫分开,并将膜放在 1% 琼脂板上(图 2B)。
将几个微移植芯片放在膜上。
将种子(在4°C 下在 0.4% 琼脂中分层超过 3 天)播种到种子袋上,然后使用移液器 P20 将 0.4% 琼脂装入袋子和根路径(图 2C)。
步骤s B 4 - B 6 a在视频1 中进行了演示。

注:为了避免混淆,在为接穗和股票的种子必须进行播种分别在不同的芯片。

 

视频1.在微移植芯片上播种

覆盖与芯片的芯片盖(图2C)。
将芯片直立放置在膜上(图 2B)。
用手术胶带密封板。
用铝箔包裹盘子。
注:为了确保在苗黄化获得,双重包裹板。否则,一个光的裂纹将防止所述被黄化苗和子叶不能通过微路径通过在阴气p期间我ncubation。

孵育在22所述板℃下在生长室中约48 - 58小时。
注意:从小号TEP小号乙7至乙9,小心处理板; 不要放下筹码。

 

图 2. 微移植程序概述。一个。用于微移植的工作台。乙。播种后将微量嫁接芯片置于 1% 琼脂培养基上。一个盘子上最多可以放置 10 个芯片(40 个幼苗)。Ç 。微移植的步骤。在第1天的红色箭头表示端移液器吸头用于种子播种。第 3 天的黑色箭头表示在黑暗条件下孵化的幼苗的钩子。比例尺:500 µm。

C.转移到光照条件下(第3天)      

从生长室采取板和解开的铝箔。
将板置于22 °C 下连续光照约 48 小时。
注:由于发芽的定时和所述的发芽拟南芥幼苗生长速度依赖于种子的新鲜度,遗传品系,与实验环境条件,它对于每个实验者来发现最佳潜伏期是重要的暗imnitially观察的黄化的生长幼苗。当钩子经过第二根顶柱时,是为幼苗打开箔纸的最佳时机(图 2C ,第3天)。在我们的例子中,我们打开了盖子的菜时间和观察到使用植物生长立体ü的nDer无菌条件。更实际地,我们也观察到与幼苗生长的裸眼。由于种子萌发和定时幼苗的生长速度变化苗中,箔片应在添去除Ë当所述的的“多数”的钩子的幼苗被第二顶支柱传递如上所述。在我们的例子中,我们获得了大约60 - 90%的幼苗长出荷兰国际集团在嫁接正确一步。等待缓慢-育苗不建议,因为剩下的幼苗下胚轴将拉长太多。作为揭开箔的最佳时机的另一个指标,我们使用了从芯片顶部出现的子叶。当我们看到一对情侣在黄化苗的菜开始以扩大其在芯片上面子叶,这是很好的时机。如果你看到没有任何的苗木出现荷兰国际集团从芯片,再覆盖板和延伸的孵化。

D.微移植(第5天)      

经过四天的孵化(两天在黑暗中,两天在光明中),幼苗穿过芯片,子叶在芯片上扩展,如图 2C,第5天所示。

注意:以下程序必须快速执行以避免干燥;每个芯片5 分钟内。干燥,特别是在移植物交界处,显著阻碍移植建立。

将 2% 琼脂平板置于室温下,并在其上放置H ybond-N+ 膜。
笔记:

的2%琼脂板用于进行后-接枝生长吨ö防止过高一个在板的湿度。
Hybond-N+ 膜用于避免根在培养基中生长并使移植变得容易。在我们的测试š几种过滤膜时,的Hybond-N +和硝酸纤维素滤膜没有抑制植物生长。
P花边高压灭菌的滤纸到P ETRI菜和微湿纸与使用处置滴管(图3A)灭菌水。使用前,清洗滴管内几次用无菌水。
注意:过量的小号水抑制移植建立。将R的水ecommended量为约1.1 -每滤纸1.2毫升。

Ť转让(BOT)的芯片到湿的滤纸上并放下它(图3B)。
ř EMOVE芯片盖(图3B,视频2)。
注意:Chip c overs 将不会在以下过程中使用。

 

视频2.去除芯片盖

˚F或步骤D5 - D10 ,该程序示于视频3。

 

V IDEO 3.微嫁接procedu重

切割沿狭缝胚轴使用的一次性外科手术刀对股票和接穗份(图的制备的尖端小号3C和3 d)。
笔记:

一把锋利的手术刀对于成功的嫁接至关重要。如果手术刀很钝,请使用新的;Ô therwise,变形的切断端引起由钝手术刀将标志着编LY减少显微移植的成功率。
如果幼苗没有很好地固定在芯片中,请用镊子将下胚轴轻轻推入微路径,然后进行嫁接。
对于股票准备,删除其切割拍摄(图 3C)。
拿起接穗,一拍,从芯片通过抓住之一的子叶瓦特第i个精密镊子,并将其转移到库存放置上另一芯片(图小号3C和3 d)。
注意:不要抢接穗强行用的镊子。大的损伤导致嫁接失败。

通过轻触接穗和库存来调整它们的位置(图对准接穗与股票的用镊子的胚轴小号3C和3 d)。
如果需要,除去水绕所述接枝结使用扭曲金W¯¯ IPE。
注意:嫁接幼苗表面的残留水分,特别是在嫁接处,会抑制嫁接形成并增加不定根形成的频率。在这里,没有必要覆盖嫁接植株带芯片盖,因为它保留的移植结潮湿,抑制移植建立。

将芯片竖直放置。
Ť转让(BOT)的芯片小心到在2%琼脂制备板的膜在小号TEP D1 ,以使得接枝的植物不对齐。
用手术胶带密封板。
在连续光照条件下,将板置于 27°C 的生长室中四天。
注意:在这种情况下的光强度是在生长室高,盖上金板W¯¯ IPE到强度和减小的光损伤降低至嫁接苗。在我们的例子中,我们将生长室设置在 100 - 140 μmol·m -2 ·s -1连续光照条件下,并用一张 Kimwipe 覆盖板,将强度降低到6 0 - 80 μmol·m -2 ·s -1 。

 

图 3. 微移植。一个。用于微移植的工作台设置。1、解剖显微镜一台;2、一个n个LED灯;3、灭菌去离子水一瓶;4、解剖针一根;5、精密镊子;6、蒸压滤纸一张;7、一个培养皿;8、一次性手术刀一把;9、一次性滴管一支。乙。在(顶部)和过程中(底部)去除芯片盖之前的微移植芯片。Ç 。详细程序用于在第一天显微移植步骤5.比例尺:500μm左右。d 。显微移植程序的插图。

E.去除不定根(第9 天,嫁接后 4 天)      

不定根经常出现在接穗下胚轴上。以下步骤旨在去除它们,我们强烈建议执行这些步骤,因为不定根会阻碍嫁接形成。

注意:以下程序必须快速执行以避免干燥;每个芯片5 分钟内。

从生长室中取出板,用乙醇和纸巾擦拭表面进行消毒。
将板放在立体显微镜下并打开盖子。
 

˚F或小号TEPS E3到E5,这些过程被示在视频4。

 

视频4.从接穗中去除不定根

大号AY的显微移植芯片向下使用是否在接穗的胚轴形成不定根镊子和检查。
我˚F不定根正在生长的接穗,切割和除去它们使用Vannas弹簧剪刀或粉碎他们与尖端的镊子(图4A)。
P蕾丝显微移植片直立。
用手术胶带密封板。
将板包裹在Kim W ipe 中,以保护嫁接植物免受强光照射,并将它们放置在 27 °C的生长室中,连续光照两天。
 

F.将嫁接的植株从微嫁接芯片中拔出(嫁接后第11天,第6天)      

离开的在室温下的新的1%琼脂平板上。
从生长室中取出板,用乙醇和纸巾擦拭表面进行消毒。
将板放在立体显微镜上并打开盖子。
 

˚F或小号TEPS F4到F 6 ,该过程在视频5中示出。

P ULL从显微移植芯片嫁接植株小心使用解剖针或镊子。
 

V记意5. Ť他吨移植植物的转让(BOT)到一个新的板

观察嫁接的植物。如果您在接穗中看到不定根,请使用Vannas弹簧剪刀或镊子将其移除。
将嫁接的植物转移到新的 1% 琼脂板上 (图 4B)。
用手术胶带密封板。
在连续光照下将板垂直放置在 22 °C的生长室中。
 

G.移植物建立的确认(移植后第15 天、10 天)      

如果您在砧木上观察到健康的根生长和接穗上的真叶扩张而没有进一步的不定根形成(图 4C),则判定嫁接已建立,嫁接的植物可以转移到土壤/培养基上进行后续实验。

 

图 4. 在移植物建立之前执行的程序。一个。嫁接后4天、去除不定根前后的嫁接苗。箭头表示出现的不定根的位置。乙。嫁接后第6天嫁接幼苗。成功的移植物被转移到一个新的板上。星号表示移植失败。Ç 。嫁接后第10天的嫁接苗显示出真叶和根的旺盛生长。比例尺:500 µm (A);1 毫米 (B) 和 (C);5 毫米。

数据分析

我们描述了我们的微移植芯片的协议。我们曾经报道过使用我们的芯片微型嫁接成功率约为48 - 88%(筒井等,2020) ,而这个成功率承担与日相比较于在传统的显微移植方法拟南芥(特恩布尔等人。2002; Yin等人,2012 年;Marsch-Martí n ez等人,2013 年)。使用该系统,我们通过测试的接枝条件的影响和投资证明芯片的效用IGAT荷兰国际集团的使用突变体品系的基因的作用(筒井等人。,2020)。

笔记

在将嫁接植物从芯片中拔出之前的所有过程中(嫁接后第 11 天,第 6 天),平板应始终保持水平并小心处理以防止将微嫁接芯片放下。
要获得通过显微移植芯片的微路径生长的幼苗,请使用足够长的大种子。为了得到良好的生长大尺寸的种子,种子繁殖植物必须与要生长充足的水,服务,以形成一个坚固的根系统和厚茎。拟南芥在干燥条件下生长良好,而不是在潮湿条件下。此外,继续浇水,直到大部分种子都是成熟; o否则,会形成未成熟的种子并在种子库中混合。
当s Ó瓦特荷兰国际集团在种子口袋种子,完全填充的空间中的口袋和用0.4%琼脂的根路径。太少一个的0.4%琼脂的原因不完全供给幼苗生长,并且t ○○多一个的0.4%琼脂诱导浮置的种子供应小号覆盖时从袋与一个芯片盖。
有时 0.4% 的琼脂会变成固体。0.4%琼脂经高压灭菌,室温搅拌冷却后,即为液态。如果0.4%琼脂仍变为固体时,可以使用后搅拌或REDUC荷兰国际集团的琼脂浓度。
有时,由于幼苗生长的力量,芯片被抬起或向后落下。在这些情况下,根部与培养基分离并且幼苗生长被阻止。此外,牛逼他下胚轴开始向micropath由向地外弯曲。这些幼苗不适合嫁接。如果芯片只放置很短的时间并且没有此类问题,则可以继续进行嫁接程序。
芯片盖应与微移植芯片上边缘的线对齐。移除芯片盖时,幼苗可能会通过将自身附着在盖上而从微移植芯片上脱落。为避免这种情况,请从一侧轻轻取下芯片盖。即使我˚F幼苗COM ë断,对花边胚轴放回芯片的micropaths并执行接枝。
为了减少损失,以苗,尽量不给碰下胚轴,特别是在移植区不拔苗。当操纵和调节苗,抢的位置的仅一个的子叶,钩与所述镊子的幼苗或轻推苗来调整它们的位置使用镊子的侧面,而不是尖。
嫁接后,接穗有时漂浮由于静电Ø ñ的P ETRI菜肴和连接到盖子。这样的接穗是无法恢复的。为了避免这些事故的发生,是有效的清洗了使用前用无菌水的盖子里面。
 

食谱

1 N 氢氧化钾
将 2.8 g 氢氧化钾溶解在 50 ml 去离子水中。

1% 琼脂平板
溶解2.3克Murashige-Skoog培养基,将5g蔗糖,在1份去离子水和0.5g MES水合物。
使用1 N KOH将pH 值调整为 5.7 。
甲DD 10克琼脂粉末和高压釜。
冷却介质到约60 ℃,并倒33毫升在一个正方形下AB培养皿IO-洁净工作台。
以除去过量的水分从所述板,干燥它们30 - 45分钟,其盖半开。
这些板可以在 4 °C下储存长达1个月。
2% 琼脂平板
溶解2.3克Murashige-Skoog培养基,将5g蔗糖,在1份去离子水和0.5g MES水合物。
使用1 N KOH将pH 值调整为 5.7 。
添加20 g 琼脂粉和高压釜。
冷却介质到约60 ℃,并倒33毫升在正方形P下AB ETRI盘IO-洁净工作台。
以除去过量的水分从所述板,干燥它们30 - 45分钟,其盖半开。
这些板可以在 4 °C下储存长达1个月。
Hybond-N+ 膜
切的Hybond-N +膜进入正方形的约 1厘米× 3厘米且p花边它们放入一个玻璃P ETRI菜。然后,对膜片进行高压灭菌,并在 60°C 的烘箱中干燥过夜。

0.4% 琼脂,用于重新悬浮灭菌种子
将 0.4 g 琼脂粉溶解在 100 ml 去离子水中,并用搅拌棒高压灭菌。冷却蒸压0.4%琼脂至室温瓦特往往微不足道搅拌下,这防止了介质从耶林。

过滤纸
P花边滤纸到玻璃P ETRI菜,高压灭菌它们,然后在烘箱中60℃干燥干燥它们。

无菌水
高压灭菌去离子水。

漂白液
将 1 毫升漂白剂与 9 毫升无菌水混合。此溶液必须在使用时配制,不能储存。

致谢

本程序源自 Tsutsui等人。(2020)。Notaguchi等人先前发表的研究中使用了该协议。(2020) 。这项工作得到了支持日本科学授予在援助科学研究(18KT0040,18H03950的推广,并20H03273)和佳能基金会(R17-0070)至MN我们非常赞赏筒井的所有作者等. (2020年)和Y.袴,M.服部,R.增田,M.石原,一吉川,和A.八木的技术支持。

利益争夺

作者宣称没有利益冲突有关日是工作。

参考

Goldschmidt, E. E. (2014)。植物嫁接:新机制,进化意义。前植物科学5:727。
Huang, NC 和 Yu, TS (2015)。针扣嫁接方法为在不同发育阶段嫁接拟南芥提供了一种非无菌和高效的方法。种植方法11: 38。
Kurotani, K., Tabata, R., Kawakatsu, Y., Sugita, R., Okayasu, K., Tanoi, K.和Notaguchi, M. (2020) 。自噬是在植物嫁接伤口愈合过程中诱导的。bioRxiv :2020.02.14.949453。
Marsch-Martínez , N., Franken, J., Gonzalez-Aguilera, KL, de Folter, S., Angenent, G. 和 Alvarez-Buylla, ER (2013)。对于一种有效的扁平的表面自由凸缘接枝方法拟南芥拟南芥幼苗。 植物方法9(1): 14。              
Notaguchi, M.、Daimon, Y.、Abe, M. 和 Araki, T. (2009)。幼苗微嫁接技术在拟南芥开花长距离信号传导研究中的应用。J Plant Res 122(2): 201-214。
Notaguchi, M., Kurotani, KI, Sato, Y., Tabata, R., Kawakatsu, Y., Okayasu, K., Sawai, Y., Okada, R., Asahina, M., Ichihashi, Y., Shirasu , K., Suzuki, T., Niwa, M. 和 Higashiyama, T. (2020)。β -1,4-葡聚糖酶促进植物嫁接中的细胞-细胞粘附。科学369(6504):698-702。              
Notaguchi, M. 和 Okamoto, S. (2015)。通过植物维管组织的长距离信号动力学。前沿植物科学6:161。              
Shinozaki, D.、Notaguchi, M. 和 Yoshimoto, K.(2020 年)。非系统性叶片自噬对抑制锌饥饿诱导的萎黄病的重要性。植物信号行为15(5): 1746042。
托马斯,人力资源和弗兰克,MH(2019 年)。连接碎片:通过植物嫁接揭示长距离通信的分子基础。新植醇223(2): 582-589。
Tsutsui, H. 和 Notaguchi, M.(2017 年)。使用嫁接研究植物系统信号。植物细胞生理学58(8): 1291-1301。
Tsutsui, H.、Yanagisawa, N.、Kawakatsu, Y.、Ikematsu, S.、Sawai, Y.、Tabata, R.、Arata, H.、Higashiyama, T. 和 Notaguchi, M.(2020)。用于测试拟南芥系统信号的微移植装置。植物学杂志103(2):918-929。              
Turnbull, CG, Booker, JP 和 Leyser, HM (2002)。用于测试拟南芥长距离信号的微移植技术。植物学杂志32(2):255-262。
王,Y. Q.(2011)。植物嫁接及其在生物学研究中的应用。中国科学公牛56:3511 - 3517。
Yin, H., Yan, B., Sun, J., Jia, P., Zhang, Z., Yan, X., Chai, J., Ren, Z., Zheng, G. 和 Liu, H. ( 2012)。嫁接结合发育:一个微妙的过程,涉及接穗和砧木之间的细胞间通讯,以促进局部生长素的积累。J Exp Bot 63(11):4219-4232。            
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tsutsui, H., Kawakatsu, Y. and Notaguchi, M. (2021). Micrografting in Arabidopsis Using a Silicone Chip. Bio-protocol 11(12): e4053. DOI: 10.21769/BioProtoc.4053.
  2. Notaguchi, M., Kurotani, K. I., Sato, Y., Tabata, R., Kawakatsu, Y., Okayasu, K., Sawai, Y., Okada, R., Asahina, M., Ichihashi, Y., Shirasu, K., Suzuki, T., Niwa, M. and Higashiyama, T. (2020). Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases. Science 369(6504): 698-702.
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Ranjana Gautam
University of Hyderabad
Hi. i am a student at CSIR-IHBT. I have to perform some signaling assays. Kindly let me know from where I can get these chips.
8/17/2021 4:15:11 AM 回复
Michitaka Notaguchi
Nagoya University Nagoya

Hi Ranjana,

You can purchase them from the following website.
https://anyonescience.com/english/2021/02/17/micrografting-chip/

Thank you,
Michi Notaguchi

8/20/2021 3:07:34 AM 回复