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Stable Transformation in Lotus japonicus
百脉根(L. japonicus)稳定转基因方法   

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Abstract

This is a protocol to produce stable transgenic plants in Lotus japonicus, which is established based on methods previously reported (Handberg and Stougaard, 1992; Stiller et al., 1997; Thkjaer et al., 1998) with some modifications. In this protocol, hygromycin is used to select transgenic plants.

Materials and Reagents

  1. Germination plate (1% agar in sterilized water)
  2. B5 salt (Wako Pure Chemical Industries, catalog number: 399-00621 )
  3. Gamborg’s vitamin solution
  4. BAP (Wako Pure Chemical Industries, catalog number: 020-07621 )
  5. 1-Naphthaleneacetic acid (NAA)
  6. Acetosyringone
  7. MES
  8. (NH4)2SO4
  9. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  10. Meropen (Dainippon Sumitomo Pharma)
  11. Hygromycin B
  12. LB medium (nacalai tesque, catalog number: 20066-95 )
  13. Co-cultivation medium (see Recipes)
  14. YMB medium (see Recipes)
  15. Callus medium (see Recipes)
  16. Shoot elongation medium (see Recipes)
  17. Root induction medium (see Recipes)
  18. Root elongation medium (see Recipes)

Equipment

  1. Clean bench
  2. L. japonicus growth facility (we use several types of Biotrons such as LH-410S purchased from NIPPON MEDICAL & CHEMICAL INSTRUMENTS CO., LTD.)
  3. Surgical knife
  4. Sterilized dish ( 9 cm in diameter x 2 cm)
  5. Sterilized magenta box
  6. Sterilized filter paper (7 cm in diameter)
  7. Sterilized filter paper (6 x 6 cm)
  8. Vermiculite (Hakugen)

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 L. japonicus Gifu or MG-20 seeds are germinated and grown in the germination plate.
  3. Place the plate vertically in a growth cabinet (Gifu; 23 °C 24 h dark for first 3 days, 23 °C 16 h light/8 h dark for next 2 days, MG-20; 23 °C 24 h dark for first 2 days, 23 °C 16 h light/8 h dark for next 2 days). Incubation time in the darkness is depends on ecotypes (hypocotyl growth speed of Gifu is slower than MG-20).

B.  Culture of Agrobacterium

Streak A. tumefaciens 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. We use AGL1 as agrobacteria strain.


C.  Infection of A. rhizogenes with L. japonicus

  1. Put about 5 mm piles of sterilized filter papers (6 x 6 cm) in a new dish and pour 20-25 ml co-cultivation medium (Figure 1A). Pouring the medium to the bottom of a dish and then it gradually permeates to the top of pilled filter papers. It enables us to recognize the saturation of filter papers with the medium.
  2. Collect the bacteria with bacteria spreader from LB plate and suspend 5 ml YMB medium.
  3. Put a sterilized filter paper (7-8 cm in diameter) in a new dish and saturate it with bacterial suspension by pipetting.
  4. Place the juvenile plants on the filter paper and cut their hypocotyls into about 3 mm length with surgical knife.
  5. Transfer the hypocotyl pieces onto the top of pilled filter papers saturated with co-cultivation medium and place the plate in a growth cabinet (23 °C 24 h dark) for 6 days (Figure 1B).

       
    Figure 1. Infection of A. tumefaciens with L. japonicas

D. Callus induction

  1. Transfer the hypocotyl pieces from co-cultivation medium onto callus medium (about 1 cm thick) and place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for 2-5 weeks (Figure 2A). Transfer the hypocotyl pieces onto new medium every 5 days.
  2. In 2-3 weeks, calluses should start to develop from the tips of hypocotyls. When they become more than 1 mm in size, cut off them from the hypocotyls and transfer them onto new medium (Figure 2B). You can trash white hypocotyls as it is unlikely that calluses develop from them.

       
    Figure 2. Callus induction

E.  Shoot induction

Transfer the calluses onto callus medium without hygromycin B (about 1 cm thick) and place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for no longer than 7 weeks. Transfer the calluses onto new medium every 5 days (Figure 3). Shoots start to appear in about 3 weeks.


Figure 3. Shoot induction

F. Shoot elongation

When shoots start to appear, transfer the calluses onto shoot elongation medium (about 1 cm thick) and place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for no longer than 6 weeks. Transfer the calluses onto new medium every 5 days (Figure 4).


Figure 4. Shoot elongation

G. Root induction

1. When shoots become about 1 cm in length, cut off the shoots from the calluses and stick them shallowly into root induction medium (about 1 cm thick).
2. Place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for 10 days (Figure 5).


Figure 5. Root induction

H. Root elongation

Transfer and stick the shoots into root elongation medium (about 3 cm thick) and place the magenta box in a growth cabinet (23 °C 16 h light/8 h dark) until their root length become about 2-3 cm (Figure 6).


Figure 6. Root elongation

I. Naturalization

Transplant the plants into 100% vermiculite and grow them with high humidity at least first 1 week (23 °C 16 h light/8 h dark), and keep growing them for further analysis. To keep high humidity, we cover the pots of plants with Saran wrap.

Recipes

  1. Co-cultivation medium (pH 5.5)
    1/10x B5 salt
    1/10x Gamborg’s vitamin solution
    0.5 μg/ml BAP (Gifu)
    0.2 μg/ml BAP (MG-20)
    0.05 μg/ml NAA
    5 mM MES (pH 5.2)
    20 μg/ml acetosyringone
    Prepare 1/10x B5 salt solution and adjust pH, and autoclave it. Add remaining components after autoclave.
  2. YMB medium (100 ml)
    0.2 g mannitol
    0.04 g yeast extract
    0.02 g MgSO4.7H2O
    0.01 g NaCl
    Mix all components and autoclave the mixture.
    Add 1 ml 0.3 M potassium phosphate buffer (pH 6.8) before use.
  3. Callus medium (pH 5.5)
    1x B5 salt
    1x Gamborg’s vitamin solution
    2% sucrose
    0.5 μg/ml BAP (Gifu)
    0.2 μg/ml BAP (MG-20)
    0.05 μg/ml NAA
    10 mM (NH4)2SO4
    0.3% phytagel
    12.5 μg/ml meropen
    15-40 μg/ml HygromycinB (you need to find the optimal concentration of HygromycinB because its purity is different among materials)
    Mix 1x B5 salt and 2% sucrose and adjust pH, and add 0.3% phytagel. Autoclave the mixture. Add remaining components after autoclave.
  4. Shoot elongation medium (pH 5.5)
    1x B5 salt
    1x Gamborg’s vitamin solution
    2% sucrose
    0.2 μg/ml BAP
    0.3% phytagel
    12.5 μg/ml meropen
    Mix 1x B5 salt and 2% sucrose and adjust pH, and add 0.3% phytagel. Autoclave the mixture. Add remaining components after autoclave.
  5. Root induction medium (pH 5.5)
    1/2x B5 salt
    1/2x Gamborg’s vitamin solution
    1% sucrose
    0.5 μg/ml NAA
    0.4% phytagel
    12.5 μg/ml meropen
    Mix 1/2x B5 salt and 1% sucrose and adjust pH, and add 0.4% phytagel. Autoclave the mixture. Add remaining components after autoclave.
  6. Root elongation medium (pH 5.5)
    1/2x B5 salt
    1/2x Gamborg’s vitamin solution
    1% sucrose
    0.4% phytagel
    12.5 μg/ml meropen
    Mix 1/2x B5 salt and 1% sucrose and adjust pH, and add 0.4% phytagel. Autoclave the mixture. Add remaining components after autoclave. Magenta boxes are used for this root elongation medium.

Acknowledgments

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

References

  1. Handberg, K. and Stougaard, J. (1992). Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. The Plant Journal 2(4): 487-496.
  2. Stiller, J., Martirani, L., Tuppale, S., Chian, R.-J., Chiurazzi, M. and Gresshoff, P. M. (1997). High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus. J Exp Bot 48(7): 1357-1365.
  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.
  4. Thykjaer T., Schauser L., Danielsen D., Finneman J., Stougaard J. (1998). Transgenic plants: Agrobacterium-mediated transformation of the diploid legume Lotus japonicus. In Cell Biology: A laboratory handbook. Second edition vol 3. Academic Press.

简介

这是在莲花日本(Lotus japonicus)中产生稳定转基因植物的方案,其基于先前报道的方法建立(Handberg和Stougaard,1992; Stiller等人,1997; Thkjaer 。,1998),并进行了一些修改。 在该方案中,潮霉素用于选择转基因植物。

材料和试剂

  1. 发芽板(灭菌水中1%琼脂)
  2. B5盐(Wako Pure Chemical Industries,目录号:399-00621)
  3. Gamborg的维生素溶液
  4. BAP(Wako Pure Chemical Industries,目录号:020-07621)
  5. 1-萘乙酸(NAA)
  6. Acetosyringone
  7. MES
  8. (NH 4) 2 4
  9. Phytagel(Sigma-Aldrich,目录号:P8169)
  10. Meropen(Dainippon Sumitomo Pharma)
  11. 潮霉素B
  12. LB培养基(nacalai tesque,目录号:20066-95)
  13. 共培养培养基(见配方)
  14. YMB介质(参见配方)
  15. 愈伤组织培养基(参见食谱)
  16. 射击伸长介质(见配方)
  17. 根诱导培养基(见配方)
  18. 根伸长培养基(见配方)

设备

  1. 清洁长椅
  2. L。 (我们使用几种类型的生物素,例如从NIPPON MEDICAL& CHEMICAL INSTRUMENTS CO。,LTD。购买的LH-410S)。
  3. 手术刀
  4. 灭菌盘(直径9cm×2cm)
  5. 灭菌品红色盒
  6. 灭菌滤纸(直径7cm)
  7. 灭菌滤纸(6 x 6厘米)
  8. 蛭石(Hakugen)

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。用无菌水洗涤种子几次,并在灭菌水中孵育种子过夜
  2. 表面灭菌的日本G>或MG-20种子在萌发平板中萌发并生长。
  3. 将板垂直放置在生长箱中(Gifu; 23℃24小时黑暗,前3天,23℃16小时光照/8小时黑暗,接下来的2天,MG-20; 23℃24小时黑暗,天,23℃16小时光照/8小时黑暗,接下来的2天)。黑暗中的孵育时间取决于生态型(Gifu的下胚轴生长速度比MG-20慢)。

B. 培养土壤杆菌

条纹A。在适当的抗体的LB平板上在28℃下将所需的构建物包埋2天,然后将细菌扩散到灭菌的盘(直径9cm)周围并孵育1天。我们使用AGL1作为农杆菌菌株。


C.  感染。 rhizogenes 。 japonicus

  1. 将约5毫升的无菌滤纸(6×6厘米)在新的盘中,并倒入20-25毫升共培养培养基(图1A)。 将培养基倒入培养皿的底部,然后逐渐渗透到填充的滤纸的顶部。 它使我们能够用介质识别滤纸的饱和度
  2. 用LB板上的细菌扩散器收集细菌,并悬浮5ml YMB培养基
  3. 将灭菌过的滤纸(直径7-8厘米)放入新的培养皿中,用细菌悬浮液通过移液使其饱和。
  4. 将少年植物放在滤纸上,用手术刀切下下胚轴约3毫米长。
  5. 将下胚轴片段转移到用共培养培养基饱和的填充滤纸的顶部,并将板置于生长室(23℃24小时黑暗)中6天(图1B)。

       
    图1.感染 A。 tumefaciens 与 L。 japonicas

D.愈伤组织诱导

  1. 将下胚轴片从共培养培养基转移到胼us培养基(约1cm厚)上,并将板置于生长室(23℃16小时光照/8小时黑暗)2-5周(图2A)。每5天将下胚轴转移到新培养基上。
  2. 在2-3周内,愈伤组织应该从下胚轴的尖端开始发育。当它们的尺寸超过1mm时,将它们从下胚轴切下并转移到新培养基上(图2B)。你可以垃圾白 下胚轴,因为它不太可能从它们发展愈伤组织。

       
    图2.愈伤组织诱导

E.  拍摄感应

转移愈伤组织到胼us培养基上没有潮霉素B(约1厘米厚),并将板在生长室(23℃16小时光/8小时黑暗)不超过7周。 每5天将愈伤组织转移到新培养基上(图3)。 拍摄开始出现在大约3周。


图3.射击诱导

F.射击伸长

当枝条开始出现时,将愈伤组织转移到枝条延长培养基(约1cm厚)上,并将该板置于生长室(23℃16小时光照/8小时黑暗)中不超过6周。 每5天将愈伤组织转移到新培养基上(图4)。


图4.射击伸长率

G.根诱导

1.当枝条长度变为约1cm时,将枝条从愈伤组织切下,并将其轻微粘在根诱导培养基(约1cm厚)中。
2.将板置于生长箱(23℃16小时光照/8小时黑暗)中10天(图5)。


图5.根源诱导

H.根伸长

转移和粘附芽到根伸长介质(约3厘米厚),并将洋红盒放在生长箱(23℃16小时光/8小时黑暗),直到它们的根长度变成约2-3厘米(图6) 。


图6.根伸长率

I.归化

将植物移植到100%蛭石中,并且在高湿度下至少第一个星期(23℃16小时光照/8小时黑暗)使其生长,并且保持生长它们用于进一步分析。 为了保持高湿度,我们用Saran包装覆盖植物盆。

食谱

  1. 共培养培养基(pH 5.5)
    1/10x B5盐
    1/10x Gamborg的维生素溶液
    0.5μg/ml BAP(Gifu)
    0.2μg/ml BAP(MG-20) 0.05μg/ml NAA
    5mM MES(pH 5.2)
    20μg/ml乙酰丁香酮
    准备1/10x B5盐溶液并调整pH值,并高压灭菌。  高压灭菌后加入剩余的组分。
  2. YMB培养基(100ml) 0.2克甘露醇 0.04g酵母提取物
    0.02g MgSO 4 .7H 2 O 2 0.01克NaCl 混合所有组分,高压灭菌混合物 在使用前加入1ml 0.3M磷酸钾缓冲液(pH 6.8)
  3. 愈伤组织培养基(pH 5.5)
    1x B5盐
    1x Gamborg的维生素溶液
    2%蔗糖 0.5μg/ml BAP(Gifu)
    0.2μg/ml BAP(MG-20)
    0.05μg/ml NAA
    10mM(NH 4)2 SO 4 4
    0.3%植物凝集素 12.5μg/ml meropen
    15-40μg/ml潮霉素B(您需要找到最佳浓度的潮霉素B,因为它的纯度因材料而异)
    混合1×B5盐和2%蔗糖并调节pH,并加入0.3%植物凝胶。 高压灭菌混合物。 高压灭菌后加入剩余的组分
  4. 芽伸长培养基(pH 5.5)
    1x B5盐
    1x Gamborg的维生素溶液
    2%蔗糖 0.2μg/ml BAP
    0.3%植物凝集素 12.5μg/ml meropen
    混合1×B5盐和2%蔗糖并调节pH,并加入0.3%植物凝胶。 高压灭菌混合物。 高压灭菌后加入剩余的组分
  5. 根诱导培养基(pH 5.5)
    1/2x B5盐
    1/2x Gamborg的维生素溶液
    1%蔗糖 0.5μg/ml NAA
    0.4%植物凝集素 12.5μg/ml meropen
    混合1/2×B5盐和1%蔗糖并调节pH,并加入0.4%植物凝胶。 高压灭菌混合物。 高压灭菌后加入剩余的组分
  6. 根伸长培养基(pH 5.5)
    1/2x B5盐
    1/2x Gamborg的维生素溶液
    1%蔗糖 0.4%植物凝集素 12.5μg/ml meropen
    混合1/2×B5盐和1%蔗糖并调节pH,并加入0.4%植物凝胶。 高压灭菌混合物。 高压灭菌后加入剩余的组分。 洋红盒用于这种根伸长介质。

致谢

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

参考文献

  1. Handberg,K。和Stougaard,J。(1992)。 Lotus japonicus ,一个自动的,用于经典和分子遗传学的二倍体豆科植物。植物学杂志 2(4):487-496
  2. Stiller,J.,Martirani,L.,Tuppale,S.,Chian,R.-J.,Chiurazzi,M。和Gresshoff,P.M。 在模式豆科植物中的转基因植物的高频转化和再生 em> 。 J Exp Bot 48(7):1357-1365。
  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质细胞分裂的阳性和阴性调节伴有生长素反应。 139(21):3997-4006
  4. Thykjaer T.,Schauser L.,Danielsen D.,Finneman J.,Stougaard J.(1998)。 转基因植物:土壤杆菌 - 介导的二倍体豆科植物Lotus japonicus的转化。在 Cell Biology:A laboratory handbook 中。 第二版vol 3. Academic Press
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sasaki, T., Suzaki, T. and Kawaguchi, M. (2013). Stable Transformation in Lotus japonicus. Bio-protocol 3(12): e796. DOI: 10.21769/BioProtoc.796.
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mohammed dawood
university of yzngzhou
HOW TO USE PHYTAGEL IN MEDIUM BECAUSE I WANT SOLIDIFICATION MEDIUM ADJUST PH TO 4 BUT WHEN I USE AGARE MEDIUM CAN NOT BE SOLID SO I WANT SAK I ADD HPYTAGEI BEFORE AUTOCLAVING OR AFTER
9/6/2014 8:48:13 AM Reply
Takuya Suzaki
Division of Symbiotic Systems, National Institute for Basic Biology, Japan

Phytagel should be added the medium before autoclaving. The pH of the medium does not affect the solidification in this case.

9/24/2014 1:22:23 AM