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Grafting of Potato Plants
马铃薯嫁接实验   

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Abstract

Grafting is a very useful technique for testing the transmission of long-distance signals in plants and is used in agriculture and gardening for different purposes. This protocol, based on a previously published one (Jackson et al., 1998), describes a grafting method for potato plants, which has successfully been used to test the transmission of tuber-inducing signals (Martin et al., 2009; González-Schain et al., 2012). We describe first the procedures for plant growth and then the grafting procedure. Although this method has been used for grafting plants that were initially grown in vitro, it should work as well with plants grown from tubers in soil. This protocol probably works well for other plant species, with small modifications.

Keywords: Grafting(嫁接), Potato(马铃薯), Solanum tuberosum(马铃薯)

Materials and Reagents

  1. Murashige & Skoog medium including vitamins (Duchefa Biochemie, catalog number: M0222 )
  2. Sucrose
  3. MES (Sigma-Aldrich, catalog number: M8250 )
  4. ddH2O
  5. KOH
  6. GelriteTM (Duchefa Biochemie, catalog number: G1101.5000 )
  7. Soil composed of blocking compost (Plantaflor® Presstopfsubstrat) and sand in a 3:1 proportion
  8. KNO3
  9. NH4NO3
  10. K2HPO4
  11. KH2PO4
  12. Ca(NO3)2.4H2O
  13. MgSO42.7H2O
  14. FeSO42.7H2O
  15. Kelamix
  16. Fertilization solution: modified Hoagland’s solution (Johnson et al., 1957) diluted 1/60 (see Recipes)
  17. 2MS medium (see Recipes)

Equipment

  1. Plant growth chamber
  2. Laminar flow hood
  3. Sterile forceps
  4. Glass jars, glass tubes or plastic containers suitable for in vitro growth
    Note: Equipments 1-4 is not required if plants are grown from tubers in soil.
  5. Sterile scalpels
  6. Pots
  7. Stakes
  8. Plant twist ties (e.g. Garden Odyssey, catalog number: T001 )
  9. Beaker
  10. Paper surgical tape (e.g. 3 M Micropore medical tape, catalog number: 1530-0 )
  11. Transparent plastic bags
  12. Adhesive tape

Procedure

  1. Micropropagate in vitro grown plants under sterile conditions in a flow hood, according to steps 2-4.
  2. Carefully hold the plant stem with sterile forceps and use a sterile scalpel to cut a single-node stem piece.
    Note: The forceps and scalpel can be surface sterilized by dipping them in ethanol and passing them through a flame.
  3. Put the cutting in fresh 2MS medium.
  4. Repeat steps 2 and 3 as many times as required.
    Note: It is advisable to propagate 10% more cuttings than the number of plants required for grafting. This number will depend on the purpose of the grafting. For our tuberization experiments, in order to have at least 10 successful grafted plants per type, we made between 12 and 20 grafts. For example, to test the transmission of signals between 2 genotypes A and B, the following grafts will be required: A/B, B/A, A/A (as control) and B/B (as control), as well as intact A and B plants. To make 15 grafts of each type and leave 15 intact controls, you will need 45 A and 45 B plants. Then, propagate 50 cuttings per genotype.
  5. Grow plants for approximately two weeks at 23 °C under long day conditions (LD: 16 hours light and 8 h darkness), until the roots are well developed.
  6. Pull the plants out of the medium, taking care not to damage the stem or the roots.
  7. Wash the roots by dipping into water and carefully remove the remaining medium.
  8. Transfer the plants to soil, previously watered with fertilization solution.
  9. Cover the plants with a plant propagator cover, an inverted transparent plastic bag or cling film to maintain the humidity high.
  10. Grow the plants under appropriate conditions, watering them as needed with fertilization solution during the whole procedure.
    Note: We usually grow the plants at 23 °C under LD conditions in the greenhouse. Plants grown under LDs are more vigorous than plants grown under short days, at least for Solanum tuberosum ssp. andigena.
  11. After 3 days, allow the humidity to slowly decrease for 3-4 more days to gradually acclimate the plants from high humidity (in vitro) to low humidity (greenhouse). If you are using a propagator, open first one vent, one day later open another vent and 2-3 days later remove the cover. If you are using a plastic bag or cling film, make small holes (approximately 0.5 cm diameter, 40 holes/m2), gradually increase the size of the holes during the next 2-3 days and finally remove the cover.
  12. Tie the plants to stakes as needed. Upright plants are grafted more easily than sprawling or bending plants.
  13. Grow the plants for at least 3 weeks.
    Note: We have successfully grafted 3-5 week old plants, but plants of other ages could also be grafted. Grafting younger plants is more difficult as the stem is weaker and is more easily damaged during the grafting procedure.
  14. Start with the plant that will be used as scion. Use a scalpel to cut the stem in a V shape below the third or fourth leaf from the apex.
  15. Put the scion in water while the plant that will be used as stock is cut.
  16. Use a scalpel to cut the stem of the stock plant in a V shape below the third or fourth leaf from the apex.
    Note: Grafting works best when the diameters of the scion and stock are similar.
  17. Add a drop of water to the top of the cut stock.
    Note: Water prevents the stock and scion from drying out.
  18. Slightly trim the end of the scion stem, maintaining the V-shape, immediately before inserting the scion into the stock.


  19. Remove the water from the cut stock with a tissue paper, but do not dry it completely. Leave just enough water to keep the slit of the stock moist.
  20. Immediately insert the scion into the top of the stock. If the scion and the stock do not fit well, slightly trim one of them to improve the fitting.
  21. Wrap the graft junction with paper surgical tape to keep the scion in place.
  22. Cover the scion with an inverted transparent plastic bag slightly bigger than the scion and use adhesive tape to close the bag around the stem, without tightening, below the graft junction. This maintains a high humidity, which helps the graft to succeed. Alternatively, use a big plastic bag to cover all the plants or groups of plants.
    Note: Try to keep the bag loose enough to prevent it from clinging to the plant leaves. Holding a side of the bag to the stake using adhesive tape can help to keep the bag in place. Avoid moving the grafted plants to prevent the scion from detaching from the stock.


  23. Grow the plants for 2 days in the LD greenhouse or under the same conditions used from step 10. Avoid direct sunlight. During this time - and until the graft heals - check the plants daily. If axillary branches grow in the stock, remove them.
  24. Cut off a corner of the plastic bag to allow the humidity to slowly decrease.
  25. One day later, cut off a second corner of the bag.
  26. Two days later, remove the plastic bag.
  27. Wait a few more days until the graft heals.
    Note: It can take up to 7-10 days for the graft to heal. When the grafting is successful, the scion looks healthy and vigorous and resumes growth. Otherwise, scions wilt quickly.
  28. Your grafted plants are ready.
    Note: Grafting is easy and works very well in potato plants, with efficiencies as high as 90-100%. However, it is a good idea to practice before starting to graft your valuable plants. Experienced grafters usually obtain higher efficiencies than inexperienced ones.

Recipes

  1. 2MS medium (1 L)
    4.40 g Murashige & Skoog medium including vitamins
    20 g Sucrose
    0.5 g MES
    Add ddH2O to approximately 800 ml
    Adjust pH to 5.8 with 1 N KOH
    Add ddH2O to final volume
    Add 2 g GelriteTM
    Autoclave
    Pour into appropriate sterile containers before the medium gelifies.
  2. Fertilization solution (1 L)
    0.849 g KNO3
    0.096 g NH4NO3
    0.209 g K2HPO4
    0.490 g KH2PO4
    0.590 g Ca(NO3)22.4H2O
    0.173 g MgSO42.7H2O
    0.167 g SO4Fe2.7H2O
    0.035 g Kelamix
    0.400 g microelements Extra AZ (Intraplex Agrichem)
    Dissolve in 800 ml dH2O
    Adjust volume to 1 L

Acknowledgments

This protocol was adapted from Jackson et al. (1998) and Martin et al. (2009) and was optimized in our laboratory by Nahuel D. González-Schain. This work was funded by the Spanish Ministry of Education and Science (grants BIO2002-00933 and BIO2005-00717, co-financed by the European Regional Development Fund) and the Xarxa de Referència en Biotecnologia of the Generalitat de Catalunya. P.S.-L. was supported by the Ramón y Cajal and I3 programs of the Spanish Ministry of Education and Science.

References

  1. Gonzalez-Schain, N. D., Diaz-Mendoza, M., Zurczak, M. and Suarez-Lopez, P. (2012). Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner. Plant J 70(4): 678-690.
  2. Jackson, S. D., James, P., Prat, S. and Thomas, B. (1998). Phytochrome B affects the levels of a graft-transmissible signal involved in tuberization. Plant Physiol 117(1): 29-32.
  3. Johnson, C. M., Stout, P. R., Broyer, T. C., Carlton, A. B. (1957). Comparative chlorine requirements of different plant species. Plant Soil 8(4): 337-353.
  4. Martin, A., Adam, H., Diaz-Mendoza, M., Zurczak, M., Gonzalez-Schain, N. D. and Suarez-Lopez, P. (2009). Graft-transmissible induction of potato tuberization by the microRNA miR172. Development 136(17): 2873-2881.

简介

接枝是一种非常有用的技术,用于测试植物中长距离信号的传输,并用于农业和园艺用于不同的目的。 该方案基于先前公开的方案(Jackson等人,1998)描述了马铃薯植物的接枝方法,其已经成功地用于测试块茎诱导信号的传播(Martin等人, et al。,2009;González-Schain et al。,2012)。 我们首先描述植物生长的程序,然后描述嫁接程序。 虽然这种方法已经用于移植最初在体外生长的植物,但是它应当与从土壤中的块茎生长的植物一起使用。 这个协议可能适用于其他植物物种,有小的修改。

关键字:嫁接, 马铃薯, 马铃薯

材料和试剂

  1. Murashige& 包括维生素(Duchefa Biochemie,目录号:M0222)的Skoog培养基
  2. 蔗糖
  3. MES(Sigma-Aldrich,目录号:M8250)
  4. ddH sub 2 O
  5. KOH
  6. Gelrite (Duchefa Biochemie,目录号:G1101.5000)
  7. 由阻挡堆肥(Plantaflor ® Presstopfsubstrat)和砂以3:1比例组成的土壤
  8. KNO 3
  9. NH 4 3
  10. K 2 HPO 4
  11. KH 2 PO 4
  12. Ca(NO 3)2 sub 2 4H 2 O
  13. MgSO 4 4 2 7H O
  14. FeSO 4 2 7H <2> O
  15. Kelamix
  16. 施肥溶液:以1/60稀释的修改的Hoagland's溶液(Johnson等人,1957)(参见Recipes)
  17. 2MS介质(参见配方)

设备

  1. 植物生长室
  2. 层流罩
  3. 无菌钳
  4. 适用于体外生长的玻璃瓶,玻璃管或塑料容器
    注意:如果植物是从土壤中的块茎生长,则不需要设备1-4。
  5. 无菌手术刀

  6. 风险
  7. 植物系带(例如 Garden Odyssey,目录号:T001)
  8. 烧杯
  9. 纸胶带(例如 3 M Micropore医用胶带,目录号:1530-0)
  10. 透明塑料袋
  11. 胶带

程序

  1. 微量繁殖体外植物在无菌条件下在流动罩中根据步骤2-4生长植物。
  2. 用无菌镊子小心握住植物茎,并使用无菌手术刀切割单节茎片 注意:镊子和手术刀可以浸泡在乙醇中并通过火焰进行表面灭菌。
  3. 将切割放入新鲜的2MS培养基中
  4. 根据需要重复步骤2和3多次。
    注意:建议传播比移植所需植物数量多10%的插条。这个数量将取决于接枝的目的。对于我们的结核实验,为了每种类型具有至少10个成功接枝的植物,我们制备了12-20个移植物。例如,为了测试2种基因型A和B之间的信号传递,将需要以下移植物:A/B,B/A,A/A(作为对照)和B/B完整A和B植物。为了使每个类型15移植并留下15完整的控件,你将需要45 A和45 B植物。然后,每个基因型传播50个扦插。
  5. 在长日照条件下(LD:16小时光照和8小时黑暗),在23℃下生长大约两周,直到根发育良好。
  6. 将植物从培养基中取出,小心不要损坏茎或根。
  7. 通过浸入水中清洗根,小心地除去剩余的介质
  8. 将植物转移到土壤,先前用施肥溶液浇灌
  9. 用植物繁殖器盖子,倒置的透明塑料袋或保鲜膜覆盖植物,以保持湿度高
  10. 在适当的条件下种植植物,在整个过程中根据需要用肥料溶液浇灌它们 注意:我们通常在温室中在LD条件下在23℃下生长植物。在LD下生长的植物比在短日内生长的植物更有活力,至少对于马铃薯(Solanum tuberosum ssp。 andigena。
  11. 3天后,使湿度缓慢降低3-4天,以逐渐使植物从高湿度(体外)适应到低湿度(温室)。如果你使用传播者,打开第一个通风口,一天后打开另一个通风口,2-3天后取下盖。如果您使用塑料袋或保鲜膜,制作小孔(直径约0.5厘米,40孔/米<2>),在接下来的2-3天内逐渐增加孔的尺寸,最后取下盖子。
  12. 根据需要将植物绑在树桩上。直立植物比蔓延或弯曲植物更容易移植
  13. 生长植物至少3周。
    注意:我们已经成功地移植了3-5周龄的植物,但是其他年龄的植物也可以移植。移植较年轻的植物更加困难,因为茎杆较弱,并且在移植过程中更容易损坏。
  14. 从将用作接穗的植物开始。使用手术刀从顶点切割第三叶或第四叶下方的V形茎
  15. 把接穗放在水中,而用作原料的植物被切断
  16. 使用手术刀在顶点的第三叶或第四叶下方切割V型植物茎。 注意:当接穗和股票的直径相似时,移植效果最好。
  17. 将一滴水添加到切割纸的顶部。
    注意:水可防止原料和接穗干燥。
  18. 在将接穗插入鱼苗之前,稍微修剪接穗茎的末端,保持V形。


  19. 用薄纸从切割的纸上除去水,但不要完全干燥。 留下足够的水,以保持原料的缝隙湿润
  20. 立即将接穗插入库存的顶部。如果接穗和股票不适合,稍微修剪其中一个,以改善拟合
  21. 用纸胶带包住移植连接处,以保持接榫就位。
  22. 用一个比接穗略大的倒置的透明塑料袋盖住接穗,并使用胶带封闭袋周围的茎,而不收紧,在移植接头下面。这保持高湿度,这有助于移植成功。或者,使用一个大塑料袋来覆盖所有的植物或植物群 注意:尽量保持袋子足够宽,以防止它粘在植物叶子上。使用胶带将袋的一侧保持在桩上可以帮助保持袋在适当位置。避免移动嫁接的植物,以防止接穗从原料上脱落。


  23. 在LD温室中或在与步骤10相同的条件下使植物生长2天。避免阳光直射。在这段时间 - 直到移植物愈合 - 每天检查植物。如果腋毛枝在种群中生长,则将其除去
  24. 切掉塑料袋的一角,使湿度缓慢下降。
  25. 一天后,切断了包的第二个角落。
  26. 两天后,取出塑料袋。
  27. 再等几天,直到移植物愈合。
    注意:移植物可能需要7-10天才能愈合。 当嫁接成功时,接穗看起来健康和活力,恢复生长。 否则,接穗会快速消失。
  28. 你的嫁接植物已经准备好了。
    注意:嫁接很容易,在马铃薯植物中效果很好,效率高达90-100%。 然而,在开始嫁接您的有价值的植物之前练习是一个好主意。 经验丰富的嫁接者通常比没有经验的嫁接者效率更高。

食谱

  1. 2MS培养基(1L)
    4.40g Murashige& 包括维生素的Skoog培养基 20克蔗糖
    0.5 g MES
    将ddH <2> O加到约800ml
    用1N KOH将pH调节至5.8 将ddH 2 O添加到最终量
    加入2g GelriteTM
    高压灭菌器
    在培养基凝胶化之前,倒入适当的无菌容器中
  2. 施肥溶液(1 L)
    0.849克KNO 3
    0.096g NH 4 NO 3 sub。 0.209g K 2 HPO 4
    0.490g KH 2 PO 4 sub/
    0.590g Ca(NO 3)2 Sub 2 O 2 br 2 O 2 br(NO 3) /> 0.173g MgSO 4·7H 2 O O
    0.035克Kelamix
    0.400克微量元素Extra AZ(Intraplex Agrichem)
    溶于800ml dH 2 O中 将音量调整为1 L

致谢

该协议改编自Jackson等人(1998)和Martin等人(2009),并且在我们的实验室中由NahuelD.González-Schain优化。这项工作由西班牙教育和科学部资助(授予BIO2002-00933和BIO2005-00717,由欧洲区域发展基金共同资助)和加泰罗尼亚通用的Xarxa deReferènciaen Biotecnologia。 P.S.-L.得到西班牙教育和科学部的Ramóny Cajal和I3计划的支持。

参考文献

  1. Gonzalez-Schain,N.D.,Diaz-Mendoza,M.,Zurczak,M。和Suarez-Lopez,P。(2012)。 马铃薯CONSTANS以可植入物传播的方式参与光周期性块茎化。 Plant J 70(4):678-690。
  2. Jackson,S.D.,James,P.,Prat,S.and Thomas,B。(1998)。 植物色素B影响涉及块茎化的可移植传播信号的水平。 Plant Physiol 117(1):29-32。
  3. Johnson,C.M.,Stout,P.R.,Broyer,T.C.,Carlton,A.B。(1957)。 不同植物物种的氯比较要求植物土壤 8(4) :337-353。
  4. Martin,A.,Adam,H.,Diaz-Mendoza,M.,Zurczak,M.,Gonzalez-Schain,N.D.and Suarez-Lopez,P.(2009)。 马铃薯的移植物传播诱导 通过微小RNA miR172进行的小块化。 136(17):2873-2881。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Suárez-López, P. (2012). Grafting of Potato Plants. Bio-protocol 2(23): e303. DOI: 10.21769/BioProtoc.303.
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very, very good
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