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Floral Dip Transformation in Lepidium campestre
花器官浸蘸法转化绿独行菜   

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Abstract

Floral dip is a very common technique to stably transform Arabidopsis thaliana (Clough and Bent, 1998; Martinez-Trujillo et al., 2004; Zhang et al., 2006) and has also been adapted to some other plant species (Curtis and Nam, 2001; Tague, 2001; Bartholmes et al., 2008). Here, we describe this method optimized for transformation of the Brassicaceae plant Lepidium campestre (L. campestre).

Keywords: Floral dip(蘸花), Lepidium campestre(绿独行菜), Plant transformation(植物转化), Agrobacterium tumefaciens(农杆菌介导法)

Materials and Reagents

  1. L. campestre seeds
  2. Agrobacterium strain GV3101 (carrying a binary vector for plant transformation including resistance gene for bacterial selection and a T-DNA containing a Basta-resistance gene)
  3. Seedling substrate (60% white peat; 20% frozen black peat; 20% coconut pulp) (Klasmann-Deilmann GmbH, recipe number: 080 )
  4. Osmocote mini (The Scotts Company)
  5. Triabon (COMPO)
  6. Beef extract
  7. Yeast extract
  8. Peptone
  9. Sucrose
  10. MgSO4
  11. Rifampicin (chromosomal resistance)
  12. Gentamycin (helper plasmid resistance)
  13. Further antibiotics depending on the transformation vector
  14. Silwet L-77 (Lehle Seeds, catalog number: VIS-02 )
  15. 0.01% basta solution (Bayer CropScience GmbH, catalog number: 79011725 )
  16. Soil (see Recipes)
  17. YEB Medium (see Recipes)
  18. Infiltration medium (see Recipes)

Equipment

  1. Greenhouse for plant cultivation
  2. Cold-room for plant vernalization
  3. Vermiculite (1-2 mm)
  4. 28 °C incubator with shaking
  5. Beaker
  6. Centrifuge with temperature control
  7. Spectrophotometer
  8. Magnetic stirrer
  9. Small plastic bags

Procedure

  1. Germinate L. campestre seeds on soil, keeping the seeds moist and covered until seedlings emerge.
  2. Transfer seedlings to individual pots and grow them for 6 weeks with a 16 h photoperiod at 20 °C and 8 h without illumination at 15 °C.
  3. Vernalize the plants for 8 weeks at 4 °C with 8 h of illumination.
  4. Move the plants back to greenhouse conditions. After 1-2 weeks, inflorescences should emerge.
  5. Ideally, the first dipping should take place shortly before the first flowers open.
  6. 2 days before dipping, grow a 5 ml over-night culture of GV3101 at 28 °C under constant rotation at 200 rpm in YEB medium.
  7. Use this culture to inoculate 500 ml of YEB medium and cultivate as above for 24 h.
  8. Pellet Agrobacterium cells by centrifugation at 16 °C and 5,500 x g for 15 min.
  9. Resuspend the cells in approximately 20 ml of infiltration medium by vortexing.
  10. Add infiltration medium to reach a final OD600 of 2.0 (should result in approximately 400-600 ml).
  11. Keep the cells at room temperature in the dark for approximately 2 h.
  12. Put the Agrobacterium solution in a beaker and stir it constantly using a magnetic stirrer.
  13. At least 10-15 plants should be treated per transformation construct, in order to generate several independent transgenic lines (transformation efficiency of ~0.4% results in approximately 1 transformant per plant)。
  14. Gently spread and open up the plants inflorescence using your finger in order to allow the liquid to reach as much of the surface as possible.
  15. Immerse the plant’s inflorescence for 5 sec into the Agrobacterium solution.
  16. Cover inflorescences with a plastic bag to retain humidity.
  17. Keep the plants without direct illumination for 24 h (i.e. Switch off all lights directly above your plants and close the shades. It does not need to be completely dark, but strong light it known to reduce motility and infectivity of Agrobacteria).
  18. Repeat the dipping weekly, until no new flowers are produced (3-5 runs).
  19. Cultivate the plants until all fruits are yellow and dry (approximately 3-6 weeks).
  20. Collect the seeds by manually opening the fruits.
  21. Germinate the seeds until cotyledons emerge.
  22. Spray seedlings with 0.01% Basta solution in 3-4 day intervals until no further seedlings emerge and Basta-resistant plants can be clearly distinguished from Basta-sensitive plants (approximately 2 weeks).
  23. After a few days, Basta-sensitive seedlings stop growing and die while transformed plants continue to grow.
  24. Confirm transgene integration by phenotyping PCR or Southern blotting.

Recipes

  1. Soil
    Consisting of 8:1:1 seedling substrate, Vermiculite and sand supplemented with fertilizer (1 g/L of each Osmocote mini and Triabon)
  2. YEB Medium
    5 g/L beef extract
    1 g/L yeast extract
    5 g/L peptone
    5 g/L sucrose
    0.5 g/L MgSO4
    Supplemented with rifampicin (50 µg/ml), gentamycin (25 µg/ml) and other antibiotics depending on the transformation vector.
  3. Infiltration medium (always prepare freshly)
    5% sucrose
    0.02% silwet L-77

Acknowledgments

We thank Andreas Mühlhausen and Klaus Mummenhoff (Department of Botany, University of Osnabrück, Germany) for their kind cooperation in our project on fruit dehiscence. Many thanks also to Pia Nutt for support with plant transformation, Florian Rümpler for help with statistics, and Lydia Gramzow for bioinformatics support. Thanks also go to Dominik Schmidt (Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena, Germany) for providing us with Agrobacterium strains. This protocol has been adapted based on previously published work (Clough and Bent, 1998; Curtis and Nam, 2001; Tague, 2001; Martinez-Trujillo et al., 2004; Zhang et al., 2006; Bartholmes et al., 2008). Our work was supported by a grant from the Deutsche Forschungsgemeinschaft to G.T. (TH 417/6-1).

References

  1. Bartholmes, C., Nutt, P. and Theiβen, G. (2008). Germline transformation of Shepherd's purse Capsella bursa-pastoris by the ‘floral dip’method as a tool for evolutionary and developmental biology. Gene 409(1): 11-19.
  2. Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium‐mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735-743.
  3. Curtis, I. S. and Nam, H. G. (2001). Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method–plant development and surfactant are important in optimizing transformation efficiency. Transgenic research 10(4): 363-371.
  4. Lenser, T. and Theißen, G. (2013). Conservation of fruit dehiscence pathways between Lepidium campestre and Arabidopsis thaliana sheds light on the regulation of INDEHISCENT. Plant J 76(4): 545-556.
  5. Martinez-Trujillo, M., Limones-Briones, V., Cabrera-Ponce, J. L. and Herrera-Estrella, L. (2004). Improving transformation efficiency of Arabidopsis thaliana by modifying the floral dip method. Plant Mol Biol Rep 22(1): 63-70.
  6. Tague, B. W. (2001). Germ-line transformation of Arabidopsis lasiocarpa. Transgenic research 10(3): 259-267.
  7. Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W. and Chua, N.-H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646.

简介

花苞是稳定转化拟南芥的非常常见的技术(Clough和Bent,1998; Martinez-Trujillo等人,2004; Zhang等人,,2006),并且还适应于一些其它植物物种(Curtis和Nam,2001; Tague,2001; Bartholmes等人,2008)。 在这里,我们描述该方法优化用于十字花科植物(Lepidium campestre)(L. campestre)的转化。

关键字:蘸花, 绿独行菜, 植物转化, 农杆菌介导法

材料和试剂 L。 campestre 种子 农杆菌菌株GV3101(携带用于植物转化的二元载体,包括用于细菌选择的抗性基因和含有Basta抗性基因的T-DNA) 幼苗基质(60%白泥炭; 20%冷冻黑泥炭; 20%椰子浆)(Klasmann-Deilmann GmbH,配方编号:080) Osmocote mini(The Scotts Company) Triabon(COMPO) 牛肉提取物 酵母提取物 蛋白胨 蔗糖 MgSO 4 4 / 利福平(染色体抗性) 庆大霉素(辅助质粒抗性) 其他抗生素取决于转化载体 Silwet L-77(Lehle Seeds,目录号:VIS-02) 0.01%basta溶液(Bayer CropScience GmbH,目录号:79011725) 土壤(见配方) YEB中(见配方) 渗透介质(参见配方) 设备 植物栽培温室 用于植物春化的冷室 蛭石(1-2毫米) 28°C孵育器摇动 烧杯 带温度控制的离心机 分光光度计 磁力搅拌器 小塑料袋 程序 发芽。 油菜种子在土壤上,保持种子潮湿和覆盖直到幼苗出现 转移幼苗到个别盆,并在20℃下16小时光周期生长6周,在15℃不照明下生长8小时。 在4℃下用8小时的照明春化植物8周 将植物移回温室条件。 1-2周后,应出现花序 理想情况下,第一次浸渍应在第一次开花前不久进行 在浸渍前2天,在YEB培养基中在200rpm恒定旋转下在28℃下培养5ml的GV3101的过夜培养物。 使用该培养物接种500ml YEB培养基并如上培养24小时 通过在16℃和5,500xg离心15分钟来沉淀土壤杆菌细胞。 通过涡旋将细胞重悬在约20ml的浸润培养基中 加入浸润介质以达到2.0的最终OD值600(应该导致约400-600ml)。 保持细胞在室温黑暗中约2小时。 将农杆菌溶液置于烧杯中,并使用磁力搅拌器不断搅拌 每个转化构建体应处理至少10-15个植物,以产生几个独立的转基因品系(约0.4%的转化效率导致每株植物约1个转化体)。 使用手指轻轻地展开和打开植物花序,以使液体尽可能多地到达表面。 将植物的花序浸泡在土壤杆菌溶液中5秒钟。 用塑料袋覆盖花序以保持湿度。 保持植物没有直接照明24小时(关闭所有的灯直接在你的植物和关闭树荫下,它不需要是完全黑暗,但强光,已知降低动力和感染性 of the Agrobacteria )。 每周重复浸渍,直到没有新鲜花卉产生(3-5次) 栽培植物,直到所有的水果是黄色和干燥(约3-6周) 通过手动打开水果收集种子。 发芽种子,直到子叶出现。 以3-4天的间隔用0.01%Basta溶液喷洒幼苗,直到不再出现幼苗,并且可以将Basta抗性植物与Basta敏感植物清楚地区分开(约2周)。 几天后,Basta敏感的幼苗停止生长,死亡,而转化的植物继续生长。 通过表型PCR或Southern印迹确认转基因整合 食谱 土壤 包括8:1:1幼苗基质,蛭石和补充肥料(每个Osmocote迷你和Triabon 1g/L)的沙子 YEB中等 5g/L牛肉提取物 1g/L酵母提取物 5 g/L蛋白胨 5g/L蔗糖 0.5g/L MgSO 4 4/h 补充利福平(50μg/ml),庆大霉素(25μg/ml)和其他抗生素,取决于转化载体。 渗透介质(总是准备新鲜) 5%蔗糖 0.02%silwet L-77 致谢 我们感谢AndreasMühlhausen和Klaus Mummenhoff(德国Osnabrück大学植物系)在我们的水果开裂项目中的良好合作。还非常感谢Pia Nutt支持植物转化,FlorianRümpler帮助统计学,Lydia Gramzow支持生物信息学。还要感谢Dominik Schmidt(分子生态系,马克斯普朗克化学生态研究所,德国耶拿)为我们提供农杆菌菌株。该协议已经基于以前公开的工作进行了修改(Clough和Bent,1998; Curtis和Nam,2001; Tague,2001; Martinez-Trujillo等人,2004; Zhang等人, , 2006; Bartholmes等人, 2008)。我们的工作得到了德意志研究所对G.T.的资助。 (TH 417/6-1)。 参考文献 Bartholmes,C.,Nutt,P。和Theiβen,G。(2008)。 牧羊人钱包的种系转化 Capsella bursa-pastoris 409(1):11-19。通过'花卉dip'方法作为进化和发育生物学的工具。 Clough,S.J。和Bent,A.F。(1998)。 花苞片:草酸杆菌介导的 Arabidopsis thaliana Plant J 16(6):735-743。 Curtis,I.S。和Nam,H.G。(2001)。 转基因萝卜( Raphanus sativus L. 通过花浸渍方法 - 植物发育和表面活性剂对于优化转化效率是重要的。转基因研究 10(4):363-371。 Lenser,T.和Theißen,G。(2013)。 保护水果开裂路径 植物J 76( 4):545-556。 Martinez-Trujillo,M.,Limones-Briones,V.,Cabrera-Ponce,J.L.and Herrera-Estrella,L。(2004)。 通过修改花浸法来提高拟南芥的转化效率 。 Plant Mol Biol Rep   22(1):63-70。 Tague,B.W。(2001)。 拟南芥lasiocarpa的种系转化。 转基因研究 10(3):259-267。 Zhang,X.,Henriques,R.,Lin,S.-S.,Niu,Q.-W。 和Chua,N.- (2006)。 农杆菌 - 介导的< em> Arabidopsis thaliana 使用花浸法。 Nat Protoc 1(2):641-646。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lenser, T. and Theißen, G. (2014). Floral Dip Transformation in Lepidium campestre. Bio-protocol 4(15): e1201. DOI: 10.21769/BioProtoc.1201.
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