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In L. japonicus, hairy root transformation is the very useful technique to generate transformed root systems in a short term. This protocol was previously described (Kumagai and Kouchi, 2003) with some modifications. After the infection of Agrobacterium rhizogenes, L. japonicus develops not only transformed but also untransformed roots. Thus, transgenic roots need to be identified by certain indications. In this protocol, we use the GFP florescent signals as such indication.

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Hairy Root Transformation in Lotus japonicus
百脉根(L. japonicus)的根毛转基因

植物科学 > 植物转化 > 农杆菌介导的转化方法
作者: Satoru Okamoto
Satoru OkamotoAffiliation: Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Japan
Bio-protocol author page: a630
Emiko Yoro
Emiko YoroAffiliation: Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Japan
Bio-protocol author page: a633
Takuya Suzaki
Takuya SuzakiAffiliation: Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Japan
For correspondence: tsuzaki@nibb.ac.jp
Bio-protocol author page: a370
 and Masayoshi Kawaguchi
Masayoshi KawaguchiAffiliation: Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Japan
Bio-protocol author page: a632
Vol 3, Iss 12, 6/20/2013, 6350 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.795

[Abstract] In L. japonicus, hairy root transformation is the very useful technique to generate transformed root systems in a short term. This protocol was previously described (Kumagai and Kouchi, 2003) with some modifications. After the infection of Agrobacterium rhizogenes, L. japonicus develops not only transformed but also untransformed roots. Thus, transgenic roots need to be identified by certain indications. In this protocol, we use the GFP florescent signals as such indication.

[Abstract]

Materials and Reagents

  1. Germination plate (1% agar in sterilized water)
  2. B5 salt
  3. Agar
  4. Sucrose
  5. Gamborg’s vitamin solution (Sigma-Aldrich, catalog number: G1019 )
  6. Meropen (Dainippon Sumitomo Pharma)
  7. LB medium
  8. Sterilized water
  9. Co-cultivation medium (see Recipes)
  10. Hairy root elongation medium (see Recipes)

Equipment

  1. Clean bench
  2. L. japonicus growth facility
  3. Surgical knife
  4. Sterilized dish (9 cm in diameter)
  5. Sterilized filter paper (7-8 cm in diameter)
  6. Sterilized square dish (10 x 14 cm)

Procedure

A.   Plant growth

  1. Sandpaper the surface of L. japonicus Gifu or MG-20 seeds, and then incubate them in 2% sodium hypochlorite solution for 5 min. Wash the seeds several times with sterilized water and incubate the seeds overnight in the sterilized water.
  2. Surface-sterilized seeds are germinated and grown in the germination plate.
  3. Place the plate vertically in a growth cabinet (For Gifu, 23 °C 24 h dark for first 3 days and 23 °C 16 h light/8 h dark for next 2 days; For MG-20, 23 °C 24 h dark for first 2 days and 23 °C 16 h light/8 h dark for next 2 days).

B.  Culture of Agrobacterium

Streak A. rhizogenes harboring the desired construct on LB plate with appropriate antibodies for 2 days at 28 °C, then spread bacteria all around sterilized dish (9 cm in diameter) and incubate for 1 day.


C.  Infection of A. rhizogenes with L. japonicus

  1. Collect the bacteria with bacteria spreader from LB plate and suspend 6 ml sterilized water.
  2. Set a sterilized filter paper in a new dish, and saturate it with bacterial suspension by pipetting.
  3. Place the juvenile plants on the saturated filter paper, and cut at the middle of the hypocotyl with surgical knife (Figure 1).
  4. Transfer the seedlings of shoot side onto co-cultivation media (cut end is need to be about 1 mm in depth from agar surface), and place the plate horizontally in a growth cabinet (23 °C 24 h dark) for 1 day.
  5. Place the plate vertically and incubate at 23 °C (16 h light/8 h dark) for 5 days.

D. Induction of hairy roots

  1. Transfer the plants onto hairy root elongation media and incubate vertically in a growth cabinet (23 °C 16 h light/8 h dark).
  2. After 10-14 days, the hairy roots should be approximately 2-5 cm in length. Pick up plants with transgenic roots expressing florescent proteins for further analysis (Figure 1).



    Figure 1. Hairy root transformation in L. japonicus

Recipes

  1. Co-cultivation medium
    1/2x B5 salt
    1/2x Gamborg’s vitamin solution
    1% agar
    Maintain pH with KOH at pH 5.5, pour into a square dish
    Mix all components except for Gamborg’s vitamin solution, and autoclave the mixture, and then add Gamborg’s vitamin solution.
  2. Hairy root elongation medium
    1/2x B5 salt
    1/2x Gamborg’s vitamin solution
    12.5 μg/ml meropen
    1% sucrose
    1% agar
    Maintain pH with KOH at pH 5.5, pour into a square dish
    Mix all components except for Gamborg’s vitamin solution and meropen, and autoclave the mixture, and then add Gamborg’s vitamin solution and meropen.

Acknowledgments

This work was supported by MEXT/JSPS KAKENHI, Japan (22870035, 23012038, 25114519 to Takuya Suzaki).

References

  1. Kumagai, H. and Kouchi, H. (2003). Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16(8): 663-668.
  2. Okamoto, S., Ohnishi, E., Sato, S., Takahashi, H., Nakazono, M., Tabata, S. and Kawaguchi, M. (2009). Nod factor/nitrate-induced CLE genes that drive HAR1-mediated systemic regulation of nodulation. Plant Cell Physiol 50(1): 67-77.
  3. Suzaki, T., Yano, K., Ito, M., Umehara, Y., Suganuma, N. and Kawaguchi, M. (2012). Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response. Development 139(21): 3997-4006.

材料和试剂

  1. 发芽板(灭菌水中1%琼脂)
  2. B5盐
  3. 琼脂
  4. 蔗糖
  5. Gamborg维生素溶液(Sigma-Aldrich,目录号:G1019)
  6. Meropen(Dainippon Sumitomo Pharma)
  7. LB培养基
  8. 灭菌水
  9. 共培养培养基(见配方)
  10. 毛根伸长培养基(参见配方)

设备

  1. 清洁长椅
  2. L。 japonicus 生长设施
  3. 手术刀
  4. 灭菌盘(直径9cm)
  5. 灭菌滤纸(直径7-8厘米)
  6. 灭菌方形皿(10×14厘米)

程序

A.   植物生长

  1. 砂纸表面的。 japonicus 岐阜或MG-20种子,然后将其在2%次氯酸钠溶液中孵育5分钟。 用灭菌水洗涤种子几次,并在灭菌水中孵育种子过夜。
  2. 表面灭菌的种子在发芽平板中萌发并生长。
  3. 将板垂直放置在生长箱中(对于Gifu,23℃24小时黑暗持续前3天,23℃16小时光照/8小时黑暗持续接下来的2天;对于MG-20,23℃24小时黑暗 前2天和23℃16小时光照/8小时黑暗,接下来的2天)

B.  培养土壤杆菌

条纹A。 在适当的抗体的LB平板上在28℃下培养所需的构建体2天,然后将细菌扩散到灭菌的培养皿(直径9cm)周围并孵育1天。


C.  感染。 rhizogenes 。 japonicus

  1. 用LB板上的细菌撒布器收集细菌,并悬浮6ml无菌水
  2. 在一个新的盘子里设置一个灭菌的滤纸,并用细菌悬浮液通过移液饱和。
  3. 将幼体植物放在饱和的滤纸上,并用手术刀切下下胚轴的中部(图1)。
  4. 将苗侧的幼苗转移到共培养培养基上(切割端需要距离琼脂表面深度约1mm),并将板水平放置在生长箱(23℃,24小时黑暗)中1天。 br />
  5. 将板垂直放置,并在23℃(16小时光照/8小时黑暗)孵育5天

D.诱导毛根

  1. 将植物转移到毛状根伸长培养基上,并在生长箱(23℃16小时光照/8小时黑暗)中垂直孵育。
  2. 10-14天后,发根长约2-5厘米。 挑取具有表达荧光蛋白的转基因根的植物用于进一步分析(图1)


    图1.在 L中的毛状根变换。 japonicus

食谱

  1. 共培养培养基
    1/2x B5盐
    1/2x Gamborg的维生素溶液
    1%琼脂
    用KOH在pH 5.5保持pH,倒入方形培养皿
    混合所有组分,除了Gamborg的维生素溶液,高压灭菌混合物,然后加入Gamborg的维生素溶液。
  2. 毛根伸长培养基
    1/2x B5盐
    1/2x Gamborg的维生素溶液
    12.5μg/ml meropen
    1%蔗糖 1%琼脂
    用KOH在pH 5.5保持pH,倒入方形培养皿
    混合所有组分,除了Gamborg的维生素溶液和meropen,并高压灭菌,然后添加Gamborg的维生素溶液和meropen。

致谢

这项工作得到MEXT/JSPS KAKENHI,日本(22870035,23012038,25114519 to Takuya Suzaki)的支持。

参考文献

  1. Kumagai,H。和Kouchi,H。(2003)。 通过在莲花日本根和根瘤中表达发夹RNA进行的基因沉默。 Mol Plant Microbe Interact 16(8):663-668。
  2. Okamoto,S.,Ohnishi,E.,Sato,S.,Takahashi,H.,Nakazono,M.,Tabata,S.and Kawaguchi,M。(2009)。 点头因子/硝酸盐诱导的CLE 基因,其驱动HAR1介导的系统结节调节。 植物细胞生理学 50(1):67-77。
  3. Suzaki,T.,Yano,K.,Ito,M.,Umehara,Y.,Suganuma,N.and Kawaguchi,M。(2012)。 Lotus japonicus中根瘤结核发育期间的皮层细胞分裂的正面和负面调节伴随着生长素反应。 开发 139(21):3997-4006。
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How to cite this protocol: Okamoto, S., Yoro, E., Suzaki, T. and Kawaguchi, M. (2013). Hairy Root Transformation in Lotus japonicus. Bio-protocol 3(12): e795. DOI: 10.21769/BioProtoc.795; Full Text



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12/6/2016 3:26:46 PM  

Bin Pang
South China Normal University


I don't know how much time do u need to put the cuting seedling on the filter paper and how to
remove the A. rhizogenes after co-cultivation

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11/24/2013 3:41:10 AM  

raju rajender arolla
ou

IS THERE ANY POSSIBILITY TO TRANSFER GENES INTO EXISTED HAIRY ROOTS

11/24/2013 11:31:48 PM  

Takuya Suzaki (Author)
Division of Symbiotic Systems, National Institute for Basic Biology, Japan

I do not have such experience. But if mobile gene or peptide were expressed in transformed hairy root, it may move to untransformed ones.

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