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Establishment of New Split-root System by Grafting
通过嫁接建立新的分根系统

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Abstract

A new split-root system was used to simulate non-uniform salt, drought or nutrient deficiency stress in the root zone, in which the root system was divided into two or more equal portions. Here, we established a split-root system by grafting of cotton seedlings. In contrast to the conventional split-root, the main roots of the new system remained intact, which provided a better system for studying cotton response to unequal treatment in the root zone. The new system was suitable for plant growth in nutrient solution and the two root systems can fully be immerged in the nutrient solution.

Keywords: Cotton(棉花), Split-root system(分根系统), Graft(嫁接), Unequal treatment(差异处理), Scion(幼枝), Stock(砧木)

Background

The split-root system has been used to study plant responses to heterogeneous soil conditions such as partial root drying, unequal salt distribution, and heterogeneous nutrient distribution. The conventional split-root system in cotton and other plants are established by dividing the lateral roots into two equal parts after cutting of the main root of a seedling (Bazihizina et al., 2009; Dong et al., 2010). The new system was suitable for plant growth in nutrient solution and for a girdling experiment because there was sufficient distance between the root and position of the graft (Kong et al., 2012 and 2016).

Materials and Reagents

  1. Plastic boxes (60 x 45 x 15 cm) (Linhui, catalog number: LH-600 )
  2. Sterilized wet sand
  3. Blade (Pao Shen Enterprises, KW-trio®, catalog number: 0 3541 )
  4. Parafilm (Bemis, catalog number: PM996 )
  5. Disposable cup (Jiaxing, catalog number: hot paper cup-HC02W )
  6. Plastic bags (Yiwu, catalog number: 10*15 )
  7. Cotton seeds (SCRC41, a commercial Bt [Bacillus thuringiensis] transgenic cotton [Gossypium hirsutum L.] which developed by the Cotton Research Center, Shandong Academy of Agricultural Sciences, Jinan)
  8. Sulfuric acid, H2SO4 (Sinopharm Chemical Reagent, catalog number: 7664-93-9 )
  9. Calcium nitrate, Ca(NO3)2 (Sinopharm Chemical Reagent, catalog number: 10124-37-5 )
  10. Potassium nitrate, KNO3 (Sinopharm Chemical Reagent, catalog number: 7757-79-1 )
  11. Magnesium sulfate, MgSO4 (Sinopharm Chemical Reagent, catalog number: 7487-88-9 )
  12. Ammonium dihydrogen phosphate, NH4H2PO4 (Sinopharm Chemical Reagent, catalog number: 7722-76-1 )
  13. EDTA·FeNa (Sinopharm Chemical Reagent, catalog number: 15708-41-5 )
  14. Orthoboric acid, H3BO3 (Sinopharm Chemical Reagent, catalog number: 10043-35-3 )
  15. Zinc sulfate, ZnSO4 (Sinopharm Chemical Reagent, catalog number: 7446-20-0 )
  16. Copper sulfate, CuSO4 (Sinopharm Chemical Reagent, catalog number: 7758-99-8 )
  17. Manganese sulfate, MnSO4 (Sinopharm Chemical Reagent, catalog number: 15244-36-7 )
  18. (NH4)6Mo7O24 (Sinopharm Chemical Reagent, catalog number: 12027-67-7 )
  19. Potassium hydroxide, KOH (Sinopharm Chemical Reagent, catalog number: 1310-58-3 )
  20. Nutrient solution (see Recipes)

Equipment

  1. Growth chamber (Percival Scientific, model: AR-41L2 )
  2. Aeration instrument (JeanPole, catalog number: B00WDWUS8W )

Procedure

  1. Seed germination
    1. Treat cotton seeds with concentrated sulfuric acid for 2-3 min and then wash the seeds with flowing tap water for 6 times.
    2. After that, the wet seeds are dried in a hot air stream (50 °C) for 8 h.
    3. Sow the acid-delinted seeds at approximately 3 cm depth in plastic boxes (60 x 45 x 15 cm) containing sterilized wet sand.
    4. Place the boxes in a growth chamber with light/dark regimes of 16 h/8 h, light intensity of 400 µmol m-2 sec-1 PAR, and temperature of 28-32 °C and relative humidity of 60-70%.

  2. Seedling growth
    1. Thin the seedlings to 100 plants per box at full emergence at 10 days after planting.
    2. When most seedlings reach the 2-true leaf stage (Figure 1-1) at 15 days after planting, carefully pull the uniform seedlings (the seedlings which germinate at the same day and have same plant height, leaf number and leaf area) out from the sand and wash the seedlings with water to remove all the sand.
      Note: Don’t irrigating the seedlings for about 3 days before grafting in order to remove the seedlings easily and induce root growth.

  3. Grafting
    1. Establish split-root systems through grafting with seedlings (Figure 1). Briefly, make a ‘/’ shaped incision with a blade on the hypocotyl 2 cm below the two cotyledons, leaving about 1/3 of the hypocotyl tissues intact. The angle between the ‘/’ shaped incision and the hypocotyl is about 60°.
    2. Cut the top of the rootstock to form a deep ‘ʌ’ at the same position of the hypocotyl from another seedling. The ‘ʌ’ section is then inserted into the ‘/’ incision of the plant and closely wrap the seedlings with Parafilm.


      Figure 1. Schematic diagram of the sequential steps of the graft split-root system. 1. Scion; 2. Stock; 3 and 4. Grafted cotton.

  4. Grafted seedling management
    1. Transfer grafted seedlings into disposable cups containing aerated nutrient solution, spray the seedlings with water and immediately cover them with plastic bags to prevent wilting (Figure 2). The air is introduced into the nutrient solution by the aeration instrument to maintaining oxygen concentration in the nutrient solution.
    2. We top the nutrient solution with deionized water every day instead of changing the whole nutrient solution, as the grafted seedling uptake a little water and nutrient each day.
    3. When a new leaf emerged from the grafted seedling at one week after grafting, remove the plastic bags and Parafilm (Figure 3).
    4. Transfer the grafted seedlings with two uniform split-root systems into a growth chamber under 28-32 °C and 60-70% relative humidity for 20 days.
    5. Renew the nutrient solutions daily during the period of growth.


      Figure 2. The grafted plant after being transplanted into disposable cup


      Figure 3. New leaf emerged from the grafted seedling at one week after grafting

Data analysis

The survival rate of grafted seedlings was more than 95% and most of them had two uniform root systems (kong et al., 2012) (Figure 4). A successfully grafted cotton seedling has two uniform root systems. At this stage, the seedling which was used as scion becomes indistinguishable from the stock.


Figure 4. Grafted cotton. The xylem (1) and phloem (2) of the grafted site.

Notes

Uniformly germinated seeds and seedling were selected to make sure the grated seedlings had two uniform split-root systems.

Recipes

  1. Nutrient solution
    1.25 mM Ca(NO3)2
    1.25 mM KNO3
    0.5 mM MgSO4
    0.25 mM NH4H2PO4
    0.05 mM EDTA·FeNa
    10 µM H3BO3
    0.5 µM ZnSO4
    0.1 µM CuSO4
    0.5 µM MnSO4
    0.0025 µM (NH4)6Mo7O24
    Adjusted pH to 6 with KOH

Note: The nutrient solutions used in this experiment needn’t be sterilized. It can be made in advance and stored in 4 °C for one month.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant Nos. 31371573 and 31501249), the special fund for Taishan Scholars (Nos. Tspd20150213 and tshw20110218), the Natural Science Foundation of Shandong Province (ZR2015QZ03), the Seed Project of Shandong Province (2014-cotton), the Agricultural Scientific and Technological Innovation Project (CXGC2016C04) and Youth Scientific Research Foundation (Grant No. 2014QNZ01) of Shandong Academy of Agricultural Sciences.

References

  1. Kong, X., Luo, Z., Dong, H., Eneji, A. E. and Li, W. (2012). Effects of non-uniform root zone salinity on water use, Na+ recirculation, and Na+ and H+ flux in cotton. J Exp Bot 63(5): 2105-2116.
  2. Kong, X., Luo, Z., Dong, H., Eneji, A. E. and Li, W. (2016). H2O2 and ABA signaling are responsible for the increased Na+ efflux and water uptake in Gossypium hirsutum L. roots in the non-saline side under non-uniform root zone salinity. J Exp Bot 67(8): 2247-2261.
  3. Bazihizina, N., Colmer, T. D. and Barrett-Lennard, E. G. (2009). Response to non-uniform salinity in the root zone of the halophyte Atriplex nummularia: growth, photosynthesis, water relations and tissue ion concentrations. Ann Bot 104:737-745.
  4. Dong, H. Z., Kong, X. Q., Luo, Z., Li, W. J. and Xin, C. S. (2010). Unequal salt distribution in the root zone increases growth and yield of cotton. Eur J Agron 33: 285-292.

简介

采用新的分裂系统模拟根系中不均匀的盐,干旱或营养缺乏胁迫,根系分为两个或更多等份。在这里,我们通过嫁接棉花幼苗建立了根系。与传统的根系相反,新系统的主要根源保持不变,为根区不平等处理棉花的响应提供了更好的系统。新系统适用于营养液中的植物生长,两种根系可以完全浸入营养液中。

背景 分裂根系被用于研究植物对不均匀土壤条件的响应,如部分根部干燥,不均匀盐分布和异质营养分布。棉花和其他植物中常规的根系系统是通过在切割幼苗的主根之后将侧根分成两等份(Bazihizina等人,2009; Dong& et al。,2010)。新系统适合于营养液和环绕实验中的植物生长,因为移植物的根部和位置之间有足够的距离(Kong& et al。,2012和2016)。

关键字:棉花, 分根系统, 嫁接, 差异处理, 幼枝, 砧木

材料和试剂

  1. 塑料盒(60 x 45 x 15厘米)(临惠,目录号:LH-600)
  2. 灭菌湿沙子
  3. 刀片(宝神企业,KW-trio ®,目录号:03541)
  4. 石蜡膜(Bemis,目录号:PM996)
  5. 一次性杯(嘉兴,目录号:热纸杯-HC02W)
  6. 塑料袋(义乌,目录号:10 * 15)
  7. 山东省农业科学院棉花研究中心济南市开发的棉花种子(SCRC41,商业Bt [苏云金芽孢杆菌]转基因棉[陆地棉]
  8. 硫酸H 2 SO 4(国药化学试剂,目录号:7664-93-9)
  9. 硝酸钙,Ca(NO 3 3)2(国药化学试剂,目录号:10124-37-5)
  10. 硝酸钾,KNO 3(国药化学试剂,目录号:7757-79-1)
  11. 硫酸镁,MgSO 4(国药化学试剂,目录号:7487-88-9)
  12. 磷酸二氢铵,NH 4 H 2 PO 4(国药化学试剂,目录号:7722-76-1)
  13. EDTA·FeNa(国药化学试剂,目录号:15708-41-5)
  14. 正硼酸,H 3 BO 3(国药化学试剂,目录号:10043-35-3)
  15. 硫酸锌,ZnSO 4(国药化学试剂,目录号:7446-20-0)
  16. 硫酸铜,CuSO 4(国药化学试剂,目录号:7758-99-8)
  17. 硫酸锰,MnSO 4(国药化学试剂,目录号:15244-36-7)
  18. (NH 4)6族Mo 7 O 24(国药化学试剂,目录号:12027-67-7 )
  19. 氢氧化钾,KOH(国药化学试剂,目录号:1310-58-3)
  20. 营养液(参见食谱)

设备

  1. 生长室(Percival Scientific,型号:AR-41L2)
  2. 曝气仪(JeanPole,目录号:B00WDWUS8W)

程序

  1. 种子发芽
    1. 用浓硫酸处理棉花种子2-3分钟,然后用流动的自来水冲洗种子6次
    2. 之后,将湿种子在热空气流(50℃)中干燥8小时。
    3. 在含有灭菌湿沙的塑料盒(60 x 45 x 15厘米)内,将约3厘米深的酸变甜的种子播种。
    4. 将盒子放在具有16h/8h,光强度为400μmol/m 2 /秒以上的亮/暗方案的生长室中,PAR的温度为28℃ -32℃,相对湿度60-70%。

  2. 幼苗生长
    1. 在种植后10天,将幼苗稀释至100个植物,完全出苗。
    2. 当大多数幼苗在种植后15天达到2叶真叶期(图1-1)时,小心地将均匀的幼苗(同一天发芽的幼苗,具有相同的植物高度,叶数和叶面积)从沙子用水冲洗幼苗以除去所有的沙子。
      注意:嫁接前不要在苗下灌溉大约3天,以便容易地除去幼苗并引起根生长。

  3. 嫁接
    1. 通过与幼苗嫁接建立分裂系统(图1)。简单地说,在两个子叶下方2厘米的下胚轴上用刀片做一个"/"形切口,约1/3的下胚轴组织完好无损。 '/'形切口与下胚轴之间的角度约为60°。
    2. 将砧木顶部切成与下一根幼苗下胚轴相同位置的深层"ʌ"。然后将"ʌ"部分插入植物的"/"切口,并用Parafilm紧密包裹幼苗。


      图1.移植物分裂根系统的顺序步骤示意图。 1. Scion;库存; 3和4.嫁接棉。

  4. 嫁接幼苗管理
    1. 将移植的幼苗转移到含有充气营养液的一次性杯中,用水喷洒幼苗,并立即用塑料袋覆盖,以防止枯萎(图2)。通过曝气仪将空气引入营养液中以维持营养液中的氧浓度。
    2. 我们每天用去离子水顶部营养液,而不是改变整个营养液,因为嫁接苗每天摄取一点水和营养。
    3. 移植后一周新移植的秧苗出现一周后,取出塑料袋和Parafilm(图3)。
    4. 将具有两个均匀分裂根系的嫁接幼苗转移到28-32℃和60-70%相对湿度下的生长室中20天。
    5. 在生长期间每天更新营养液。


      图2.移植到一次性杯中的嫁接植物


      图3.嫁接后一周从嫁接幼苗出现的新叶子

数据分析

嫁接苗的存活率超过95%,其中大部分存在两个均匀的根系(kong et al。,2012)(图4)。成功接种的棉花苗具有两个均匀的根系。在这个阶段,用作接穗的幼苗与库存无法区分。


图4.接枝棉。嫁接部位的木质部(1)和韧皮部(2)。

笔记

选择均匀发芽的种子和幼苗,以确保磨碎的幼苗具有两个均匀的分裂根系。

食谱

  1. 营养液
    1.25mM Ca(NO 3 3)2
    1.25mM KNO 3
    0.5mM MgSO 4
    0.25mM NH 4 H 2 PO 4
    0.05 mM EDTA·FeNa
    10μMH 3 3
    0.5μMZnSO 4
    0.1μMCuSO 4
    0.5μMMnSO 4
    0.0025微米(NH 4)6以下的第7个O 2 O 2/2><>           用KOH调节pH至6

注意:本实验中使用的营养液不需要消毒。可以提前制作,并在4°C下储存一个月。

致谢

这项工作得到了中国国家自然科学基金(授权号31371573和31501249),泰山学者专项资金(Tspd20150213和tshw20110218),山东省自然科学基金(ZR2015QZ03),种子项目山东省(2014-棉花),山东省农业科学院农业科技创新项目(CXGC2016C04)和青年科学研究基金(2011QNZ01)。

参考文献

  1. Kong,X.,Luo,Z.,Dong,H.,Eneji,AE and Li,W(2012)。  不均匀根区盐度对水分的影响,Na + 再循环,Na + 和H + 在棉花中的通量。 J Exp Bot 63(5):2105-2116。
  2. Kong,X.,Luo,Z.,Dong,H.,Eneji,AE and Li,W.(2016)。  H 2 2 ,ABA信号负责增加的Na + 在不均匀的根区盐度下,非生理盐水中的陆地陆地棉根部的外排和吸水量为67(8) :2247-2261。
  3. Bazihizina,N.,Colmer,TD和Barrett-Lennard,EG(2009)。对盐藻根区的不均匀盐度的响应Atriplex nummularia :生长,光合作用,水分关系和组织离子浓度。 Ann Bot 104:737-745。
  4. Dong,HZ,Kong,XQ,Luo,Z.,Li,WJ and Xin,CS(2010)。  根区的不均匀盐分布增加了棉花的增长和产量。 Eur J Agron 33:285-292。
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引用:Kong, X., Luo, Z. and Dong, H. (2017). Establishment of New Split-root System by Grafting. Bio-protocol 7(4): e2136. DOI: 10.21769/BioProtoc.2136.
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