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Agrobacterium rhizogenes-Based Transformation of Soybean Roots to Form Composite Plants
发根农杆菌转化大豆根部形成复合植物   

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Abstract

Transgenic soybean roots of composite plants are a powerful tool to rapidly test the function of genes and activity of gene promoters. No tissue culture is needed, thus avoiding loss of valuable material due to contamination. This is a simple technique that requires less training and care than tissue culture techniques. Furthermore, it takes less time to produce transgenic roots than techniques using sterile tissue culture. If the transgenic roots are to be challenged with a pathogen, there is no need to produce axenic pathogens with this technique, because sterile tissue culture medium is not used. Therefore, there is no agar medium on which contaminants may grow resulting in obscured results or diseased roots. Here, we describe the production of transgenic soybean roots on 7-day-old soybean seedlings using Agrobacterium rhizogenes. These composite plants may be grown in the greenhouse for further experimentation, such as to determine the effect of gene expression on nematode development.

Materials and Reagents

  1. 17 x 100 mm, 14 ml culture tubes designed for bacteria
  2. Sodium hypochlorite (Commercial Bleach-Clorox)
  3. Soybean seeds (William 82) (Glycine max)
  4. Agrobacterium rhizogenes strain K599 (Homemade culture)
  5. PRAP15, pRAP17 or other plasmid
  6. Tetracycline hydrochloride, min. 95% (Sigma-Aldrich)
  7. Murashige & Skoog Medium- including vitamins (Duchefa Biochemie, catalog number: P08805.02 )
  8. Ethyl alcohol (The Warner Graham Company, Gas, catalog number: 64-17-5 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 64-17-5 ”.
  9. Anti-bacterial soap (Clean & smooth)
  10. MS salts
  11. 3% sucrose
  12. Casein digest peptone
  13. Yeast extract
  14. Dipotassium phosphate (K2HPO4)
  15. Potassium phosphate (K3PO4)
  16. Glycerol
  17. KOH
  18. MS media (see Recipes)
  19. Terrific Broth, Modified (Research products International Corp., catalog number: 31237 ) (see Recipes)
  20. Fertilizer-Peters Excel 13-2-13 150 ppm nitrogen (pH 6.1) (see Recipes)

Equipment

  1. 162 pots flat trays and 32 pot flats (Myers Lawn&Garden)
  2. Promix Flexible Purpose (Pro-Mix, model: Flex Loose 2.8 cuft )
  3. Heated growing mat (Home depot)
  4. 1 L culture flasks (Pyrex)
  5. Spectrophotometer UV-120-02 (Shimadzu Corporation, catalog number: 204-00010-08 )
  6. Centrifuge with a GSA rotor (model: Sorvall RC-5B Refrigerated Super speed Centrifuge)
  7. Plastic pasteur pipette (Corning, Falcon®, catalog number: 357575 )
  8. A desiccator attached to a vacuum pump
  9. G10 Gyrotory shaker (New Brunswick Scientific)
  10. Gyrotory water bath Shaker (LabX, New Brunswick Scientific, model: G76 )
  11. Buckets (Home depot)
  12. Growth chamber
  13. Stainless steel scissors (Roboz Surgical Instrument Co., catalog number: RS-5912 )
  14. Dark Reader Spot Lamp-SL85 (Clare Chemical Research)
  15. Orange filter glasses (Clare Chemical Research)
  16. Patio/Paver Sand. Multi-purpose (Pavestone) 
  17. 50 ml disposable plastic beakers
  18. Clear plastic tub
  19. Rotating platform
  20. Thermo Fisher Scientific Nalgene Spherical-Bottom Centrifuge Bottle, Polycarbonate (PC)-Jade Scientific Inc./ NalgeneTM Spherical-Bottom Polycarbonate Centrifuge Bottle (Thermo Fisher Scientific, catalog number: 3123-0250 )
  21. Vacuum 380 mm Hg (Torr)

Procedure

  1. Preparing the soybean seedlings
    1. Count soybean seeds as needed, approximately 162 seeds per DNA construct. Sort the seeds to remove seeds that are diseased, small, or damaged. Remove weed seeds and other extraneous material. The approximate number of soybean cv. Williams 82 seed can be determined by weight using 22.5 grams equals 100 seeds. Seeds from other soybean cultivars may have a different weight per 100 seeds. Sterilize soybean seeds by soaking in 10% bleach for 10 min. 300 ml of 10% bleach in a 1 L plastic beaker works well for up to about 300 grams of seed. Add the seed to the bleach solution, swirl briefly to sink the seeds, and allow the seeds to rest quietly for the remainder of the exposure. Pour off the bleach solution. The seeds were rinsed about 10 times in quick succession with RO [reverse osmosis] water to remove the bleach. Alternatively, run RO water into the beaker until it is full and the seeds are suspended. Continue to run the RO water into the beaker for about 20 sec. Drain the water from the seeds. Repeat 4 times. Drain the seeds well, but gently. Handle the seeds gently, as they are now fragile.
    2. Plant the soybean seeds 7 to 9 days before the transformation date. The seeds germinate and the seedlings grow faster in the summer and slower in the winter. In a 9 x 18 cell flat, plant 2 seeds per cell containing hydrated Promix potting media. Thus, one 8 x 18 cell flat provides enough plants (324) for two constructs. Hydrate the Promix by mixing it with water in a 5-gallon bucket.
      Place the 162 cell flat on a clean surface, and fill the individual pots with the hydrated Promix, packing each pot with firm pressure, using one finger per pot. Fill with more ProMix to make it level with the top of the flat. Form a hole to plant the seed by dibbling the Promix with an extra large sharpie pen of something similar to a depth of 2 cm. Plant 2 seeds per cell; lightly cover the seeds with ProMix; and add more ProMix to cover as needed. The covering ProMix is placed lightly and not pressed. Water the seeded flats until the Promix is moderately saturated. Place the 162 pot flat directly on the growing bench or heated growing mat, without any open flats underneath. Do not water the flat again until the top layer of Promix has begun to dry to a light brown color. Water lightly, as needed, until the plants emerge (Figure 1).
      Note: In the summer, in Maryland, USA, the seedlings are ready on the 7th or 8th day after planting. At this time the first cotyledonary leaves are partially open. During the winter and cooler seasons, the seedlings may not be at the correct stage until the 9th or 10th day after planting, you can use a heated germination mat in the winter season.


      Figure 1. Soybean (William 82) seedlings 7 days after planting

  2. Prepare the Agrobacterium culture
    1. Prepare the small Agrobacterium rhizogenes cultures the week before the transformation week.
      Inoculate 5 ml of the appropriate culture medium with A. rhizogenes. For cultures of A. rhizogenes strain K599 carrying pRAP15 constructs (Figure 2), the inserts were cloned into the gene expression vector pRAP15 between the attR1 and attR2 sites designed for directional cloning. In this vector, the Figwort Mosaic Virus (FMV) promoter drives expression of the inserted gene. To enable visualization of transformed roots, the vector contains the gene encoding enhanced green fluorescent protein controlled by the rolD promoter that gives strong root expression. A gene encoding tetracycline resistance (TetR) provides antibiotic selection. Inoculate into TB [Terrific Broth] media with 5 μg/ml Tetracycline. Grow two culture tubes of each clone, 5 ml per tube, in the temperature range of 73 F to 74 F [22.8 to 23.3 °C]. The incubation temperature must not rise significantly above 77 F [25 °C] or K599 may expel or modify the plasmids. Shake the tubes at about 200 rpm. Use round bottom 17 x 100 mm 14 ml culture tubes designed for bacteria to avoid clumping of the bacteria. The incubation time will be about 48 to 50 h.
    2. Prepare large cultures of A. rhizogenes.
      One or two days before transformation, use the 5 ml cultures to inoculate 300 ml of Terrific Broth (TB) media-5 μg/ml tetracycline for each clone for an overnight incubation at around 73 F to 74 F [22.8 to 23.3 °C]. Inoculate the large culture with one or two of the small cultures depending on culture concentration. The incubation temperature must not rise significantly above 77 F [25 °C] or K599 may expel or modify the plasmid. The 1 L culture flasks are shaken at 160 rpm and 2 L flasks are shaken at 140 rpm for 12 h. Check OD600; they should be in the 0.5 to 0.6 range. The cultures can be stored at 4 °C for 1-2 h until needed.
      Note: On the day before transformation, if the lab temperature is expected to be above 75 F [24 °C], turn on the air conditioning to cool both the room and the MS media. The MS media is kept at room temperature or 75 F [24 °C]. Inspect the plantlets to be transformed for disease. Remove plantlets having cotyledons with yellow areas, very tall thin stems, or any other attribute that would make them weak. The plantlets should be dark green with thick, blemish-free cotyledons and thick stems. If the plantlets are not strong enough for the rigors of the transformation, grow the plants again with fresh seed and prepare new bacterial cultures.
    3. Pour the cultures into capped tubes designed for a GSA rotor and balance them. Centrifuge at 5,000 rpm [4,068 x g] at 4 °C for 30 min to collect the A. rhizogenes. (After this point the bacterial cultures do not have to be sterile anymore.)
    4. Resuspend the pellets in 20 ml MS media. Use a disposable plastic Pasteur pipette to gently resuspend the culture. Transfer about 15ml of the resuspended culture to 190 ml MS. Mix by swirling. Read the OD600. Adjust the culture concentration to about OD600 = 0.5 to 0.6 with MS media.

  3. Transformation of the base of the soybean seedling with Agrobacterium
    1. Add 30 ml MS - bacterial solution to 50 ml plastic disposable beakers that will hold the cut plants easily (6 plastic beakers with bacterial solution are needed per construct). With a sharp safety razor blade, excise the seedlings at soil level and immediately place them in beakers containing A. rhizogenes culture. Ensure that the base of the seedling is submerged in the culture. Add about 28 cut plants per beaker. Place the beakers of excised seedlings into a vacuum chamber.
    2. Apply vacuum 380 mm Hg (Torr) for 30 min to vacuum infiltrate the co-cultivation solution into the plants. Release the vacuum slowly to avoid potential damage to the plants.


      Figure 2. Gateway overexpression vector containing FMV promoter, eGFP and Tet gene pRAP 15 digested with EcoRI(E), HindIII(H), EcoRI/HindIII(E/H) and HindIII/SpeI(H/S) restriction enzymes

    3. Co-cultivate the plants overnight for about 15 h. The plants are placed inside of a clear plastic tub, (Figure 3) which is placed on a rotating platform, moving at 65 rpm. The temperature is held at around 73 F to 74 F [22.8 to 23.3 °C]. The co-cultivation temperature must not rise significantly above 77 F [25 °C] or K599 may expel or modify the plasmids needed for effective transformation.


      Figure 3. The plantlets are placed inside of a clear plastic tub for co cultivation

    4. Wash the plants with RO water. For each gene, fill two 1 L beakers with RO water up to about 1 cm from the top edge. Remove the plants from one co-cultivation beaker and dip the stem ends about 8x into the first wash beaker and then do the same in the second wash beaker. Place the plants into a clean beaker containing reverse osmosis water. Place the beakers of washed plants in a plastic tub, and store them in the uncovered tub overnight. For each gene, use new, fresh RO water and beakers to avoid cross-contamination. The plants may be stored in the small beakers for up to 4 days after the post co-cultivation wash. All of the wash water should be autoclaved before discarding (Figure 4).


      Figure 4. The plantlets are washed with RO water to remove the Agrobacterium

      Note: Do not use regular tap water or any water that may contain chlorine, fluorides, or any other water purifying reagent. Fill enough 1 L beakers the night before the post co-cultivation wash to wash all of the plants. This allows RO water to equilibrate to room temperature. Air condition the lab if necessary.

    5. Incubate the plants at about 73 F to 74 F [22.8 to 23.3 °C] under growth lights with a light cycle of about 15 h for about 42 h [two overnights after the post co-cultivation wash] in a growth chamber. The growth lights are about 15 cm from the plants. If the distance is too far, the plants will elongate excessively and possibly jeopardize their health and durability during their recovery in ProMix.

  4. Planting the soybean seedlings in the greenhouse
    1. Before planting the soybean plantlets, pre-wet enough ProMix in a bucket with greenhouse tap water to fill three 32 cell flats fitted within a standard perforated flat per DNA construct. The ProMix should be quite wet; even sodden. Pack the ProMix into the pots with firm hand pressure. Just before planting the plants, sprinkle the ProMix with water from a watering can. Use a marker pen as a dibble to create 2 to 3 cm holes in the ProMix for plant insertion. Insert the plants into the ProMix to about 2 to 3 cm, or close to the cotyledons, and lightly compress the ProMix down with four fingers encircling the stem. After planting the plants, thoroughly sprinkle the ProMix with water from a watering can. The planting day is day 0.
      Note: Do not use ProMix with Biofungicide. It may interfere with assays of roots involving nematodes and fungi.
    2. Water the plants gently with a watering can for the first 4 days after planting in ProMix. Switch to watering with a wand on day 5. Water with plain water for the first 9 days after planting in ProMix. On day 10, begin feeding the plants 3 days with a Spot Sprayer or watering can and 1x Peters Excel 13-2-13 150 ppm nitrogen (pH 6.1).
    3. If restricted shoot growth is desired, the top of the plants above the 2nd trifoliate (on about day 15) may be cut off. Use stainless steel scissors that have been cleaned with alcohol or anti-bacterial soap. This time period will vary with the season and growing conditions. During the summer months, two toppings may be necessary. Plants were grown in a greenhouse under natural light supplemented daily with a 12 h:12 h light: dark cycle of artificial light. Greenhouse temperatures were maintained at 25 °C (days) and 14 °C (nights) with 20-50% humidity.

  5. Evaluate the transformation by checking the soybean roots and trimming the untransformed roots
    1. On about day 38, (Figure 5) gently remove the soil from all plants in each clone group by placing them in a 4 gallon autoclavable bucket. Gently massage the root mass to remove Promix. Rinse the roots with room temperature water. One bucket can be used to wash about 50 plants. Store the de-soiled plants in a 2 gallon bucket containing 0.5x Peters Excel 13-2-13 75 ppm nitrogen (pH 6.1).


      Figure 5. Soybean plants are in the greenhouse and growing well 38 days after transformation

    2. In a dark room, use the Dark Reader Spot Lamp-SL85 and orange filter glasses to visualize the GFP roots (Figure 6). Discard plants with no GFP roots. Identify 20 or more of the best composite plants with good GFP roots for the experiment. The transformation efficiency ranges from 65 to 90%.
    3. With scissors, cut off the non-GFP roots on the composite plants. Maintain the composite plants in the greenhouse with their roots submerged in 0.5x Peters Excel 13-2-13 at about 75 ppm nitrogen (pH 6.1) for two nights in order to minimize wilting after they are replanted in sand.


      Figure 6. Soybean roots exposed to Dark Light® to visualize GFP-expressing roots

    4. Plant the composite plants in flooded, sterile sand to help them recover from the removal of non-GFP roots. Place 2 cm of sand in each cell. Place the root of the plant in the cell, with the base of the seedling 1 cm below the top of the cell. Gently sprinkle wet sand around and on top of the roots. Build a compact cone of sand 2 to 3 cm tall around the base of the seedling to hold it upright. Place the cell in a flat. Flood the flat with 0.5x Peters Excel 13-2-13 75 ppm nitrogen. Leave the plants in flooded sand for 2 days.
    5. Transfer the plants to perforated flats containing wet Promix about 2.5 cm deep. Allow the plants to drain overnight.
    6. The next day, inoculate with nematodes.
      Note: If the plantlets appear wilted after the non-GFP roots are excised, put them in the shade to protect them from direct sunlight until they have recovered.

Recipes

  1. MS media
    Preparation
    1 L
    MS salts
    4.4 g
    3% sucrose
    30 g
    Adjust the pH to 5.7
    If the MS media is to be used within a day, there is no need to autoclave.
  2. Terrific Broth, Modified
    RPI Premixed Terrific Broth
    Contents of the Terrific Broth premix
    1 L
    Casein digest peptone
    12 g/L
    Yeast extract
    24 g/L
    Dipotassium phosphate (K2HPO4)
    9.4 g/L
    Potassium phosphate (K3PO4)
    2.2 g/L
    Preparation
    1 L
    Add TB premix to 1 L water
    47.6 g
    Glycerol
    4 ml
    No need to pH the media
    Autoclave 20 min
  3. Scotts Peters Excel 13-2-13 16x Fertilizer
    Prepare 1 gallon of water with 13 pellets KOH
    Mix each solution well
    Prepare 1 gallon of water with 10 tablespoons [10T] Peters Excel13-2-13, Scotts Peters Excel 13-2-13 32x
    Mix Combine the two solutions and mix well separately
    pH 6.2

Acknowledgments

This protocol is adapted from our previous work, including (Matthews et al., 2013; 2014 and Youssef et al., 2013). We thank Hunter Beard for technical support. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable. The authors have no conflict of interest.

References

  1. Matthews, B. F., Beard, H., Brewer, E., Kabir, S., MacDonald, M. H. and Youssef, R. M. (2014). Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots. BMC Plant Biol 14: 96.
  2. Matthews, B. F., Beard, H., MacDonald, M. H., Kabir, S., Youssef, R. M., Hosseini, P. and Brewer, E. (2013). Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode. Planta 237(5): 1337-1357.
  3. Youssef, R. M., MacDonald, M. H., Brewer, E. P., Bauchan, G. R., Kim, K. H. and Matthews, B. F. (2013). Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes. BMC Plant Biol 13: 67.

简介

复合植物的转基因大豆根是快速测试基因功能和基因启动子活性的有力工具。不需要组织培养,因此避免了由于污染导致的有价值材料的损失。这是一种简单的技术,比组织培养技术需要更少的培训和护理。此外,产生转基因根的时间比使用无菌组织培养的技术要少。如果转基因根将用病原体攻击,则不需要用该技术产生无性病原体,因为不使用无菌组织培养基。因此,不存在其上可能生长污染物的琼脂培养基,导致​​模糊的结果或患病的根。在这里,我们描述使用发根土壤杆菌(Agrobacterium rhizogenes)在7日龄大豆幼苗上生产转基因大豆根。这些复合植物可以在温室中生长用于进一步实验,例如确定基因表达对线虫发育的影响。

材料和试剂

  1. 17×100mm,14ml用于细菌的培养管
  2. 次氯酸钠(商业Bleach-Clorox)
  3. 大豆种子(William 82)(大豆)
  4. 发根土壤杆菌菌株K599(自制培养物)
  5. PRAP15,pRAP17或其他质粒
  6. 盐酸四环素, 95%(Sigma-Aidrich)
  7. Murashige& Skoog Medium - 包括维生素(Duchefa Biochemie,目录号:P08805.02)
  8. 乙醇(The Warner Graham Company,Gas,目录号:64-17-5)
    注意:目前,它是"Sigma-Aldrich,目录号:64-17-5"。
  9. 抗菌皂(清洁&光滑)
  10. MS盐
  11. 3%蔗糖
  12. 酪蛋白消化蛋白胨
  13. 酵母提取物
  14. 磷酸二钾(K 2 HPO 4)
  15. 磷酸钾(K 3 PO 4)
  16. 甘油
  17. KOH
  18. MS介质(参见配方)
  19. Terrific Broth,Modified(Research products International Corp.,目录号:31237)(参见配方)
  20. Fertilizer-Peters Excel 13-2-13 150ppm氮(pH 6.1)(参见配方)

设备

  1. 162盆平托盘和32个盆栽平台(迈尔斯草坪花园)
  2. Promix灵活用途(Pro-Mix,型号:Flex松散2.8立方英尺)
  3. 加热生长垫(家庭仓库)
  4. 1L培养瓶(Pyrex)
  5. 分光光度计UV-120-02(Shimadzu Coopration,目录号:204-00010-08)
  6. 用GSA转子离心机(型号:Sorvall RC-5B制冷超速离心机)
  7. 塑料巴斯德移液管(Corning,Falcon ,目录号:357575)
  8. 连接真空泵的干燥器
  9. G10 Gyrotory振动器(New Brunswick Scientific)
  10. Gyrotory水浴摇床(LabX,New Brunswick Scientific,型号:G76)
  11. 铲斗(家得宝)
  12. 生长室
  13. 不锈钢剪刀(Roboz Surgical Instrument Co.,目录号:RS-5912)
  14. Dark Reader Spot Lamp-SL85(Clare Chemical Research)
  15. 橙色滤光镜(Clare Chemical Research)
  16. 天井/摊铺机砂。多用途(Pavestone)
  17. 分光光度计(Shimadzu Coopration,型号:UV-120-02)
  18. 50 ml一次性塑料杯
  19. 清除塑料桶
  20. 旋转平台
  21. Thermo Fisher Scientific Nalgene球形底部离心瓶,聚碳酸酯(PC)-Jade Scientific Inc./Nalgene TM球形底部聚碳酸酯离心瓶(Thermo Fisher Scientific,目录号:3123-0250)
  22. 真空380 mm Hg(Torr)

程序

  1. 准备大豆苗
    1. 根据需要计数大豆种子,每个DNA构建体约162个种子。 对种子进行排序以去除患病,小或损坏的种子。 除去杂草种子和其他外来物质。大概数量 的大豆cv。 Williams 82种子可以使用22.5的重量来测定 克等于100粒种子。来自其他大豆品种的种子可以具有a 不同重量每100粒种子。浸泡大豆种子消毒 10%漂白10分钟。 300ml 10%漂白剂在1L塑料烧杯中 适用于高达约300克种子。将种子添加到漂白剂中 ?溶液,短暂地旋转以沉下种子,并允许种子休息 静静地为剩下的曝光。倒掉漂白溶液。 ?将种子用RO快速连续冲洗约10次 [反渗透]水除去漂白剂。或者,运行RO 水倒入烧杯中直到其充满并且种子悬浮。 继续运行RO水进入烧杯约20秒。排出 ?水从种子。重复4次。排出种子好,但轻轻。 ?轻轻处理种子,因为它们现在是脆弱的
    2. 植物 大豆种子转化前7?9天。种子 发芽和幼苗在夏天生长更快,而在慢 冬季。在9×18细胞平,植物每个细胞含2个种子 水合Promix灌封介质。因此,一个8×18单元平面提供 用于两个构建体的足够的植物(324)。通过混合水合Promix 用水在5加仑桶。
      将162电池平放在干净 ?表面,并用水合Promix填充各个盆,包装 ?每罐用坚固的压力,每罐使用一个手指。填写更多 ProMix使它与平顶的水平。形成一个洞来种植 种子通过用一个特大的锐利的笔钻出Promix 类似于2厘米的深度。植物每个细胞2粒种子;轻轻 用ProMix覆盖种子;并根据需要添加更多ProMix来覆盖。的 ?覆盖ProMix轻轻放置,不按压。浇水播种 直到Promix中度饱和。将162锅平放 直接在生长台或加热生长垫上,没有任何开放 下面的单位。不要再把水弄平,直到顶层 Promix已开始变干为浅棕色。轻轻地,水 需要,直到植物出现(图1) 注意:在夏天,在 马里兰州,美国,幼苗在7天或8天天准备好了 种植。此时,第一子叶叶部分打开。 ?在冬季和寒冷季节,幼苗可能不在 正确的阶段,直到种植后第9天或第10天天,您可以使用 在冬季加热发芽垫。


      图1.种植后7天的大豆(William 82)幼苗

  2. 准备农杆菌文化
    1. 在转化周前一周准备小的发根土壤杆菌培养物。
      用A接种5ml合适的培养基。毛根纲。 对于A的培养物。含有pRAP15构建体的毛霉属菌株K599 (图2),将插入片段克隆到基因表达载体中 pRAP15之间的attR1和attR2位点设计用于定向 克隆。在这个载体中,玄参花叶病毒(FMV)启动子驱动 插入基因的表达。使变换的可视化 根,载体含有编码增强绿色荧光的基因 蛋白质由产生强根的rol D启动子控制 表达。编码四环素抗性(TetR)的基因提供 抗生素选择。接种到TB [Terrific Broth]培养基中 μg/ml四环素。生长每个克隆的两个培养管,每管5ml, ?在73°F?74°F [22.8?23.3℃]的温度范围内。的 培养温度不得上升至明显高于77°F [25°C]或 ?K599可以驱逐或修饰质粒。在约200摇动管 rpm。使用圆底17 x 100毫米14毫升培养管设计 细菌以避免细菌结块。孵化时间为 ?约48至50小时
    2. 准备大型文化的A。毛根纲。
      转化前一天或两天,使用5ml培养物 接种300ml Terrific Broth(TB)培养基-5μg/ml四环素 每个克隆在约73°F至74°F下过夜温育[22.8℃ 23.3℃]。接种大文化与一个或两个小 培养取决于培养浓度。孵育温度 ?不得显着超过77°F [25°C]或K599可能会排出或 修饰质粒。将1L培养瓶在160rpm和2L下振荡 ?将烧瓶在140rpm振荡12小时。检查OD 600 ;他们应该在 0.5?0.6的范围。培养物可以在4℃保存1-2小时,直到 ?需要。
      注意:在转换前一天,如果实验室 温度预计高于75°F [24°C],打开空气 空调冷却房间和MS媒体。 MS媒体是 保持在室温或75°F [24℃]。检查小植株 转化为疾病。删除具有黄色子叶的小植株 区域,非常高的细茎,或任何其他属性,将使他们 ?弱。小植株应该是深绿色,厚,无瑕疵 子叶和厚茎。如果小植物的强度不够 严格的转化,用新鲜种子再次种植植物 并准备新的细菌培养物。
    3. 倒入培养物上盖 ?设计用于GSA转子并平衡它们。离心机5000 rpms [4,068×g ]在4℃下30分钟收集 A。毛根纲。 (后 ?这一点细菌培养不再是无菌的。)
    4. 将沉淀重悬在20ml MS培养基中。使用一次性塑料 巴斯德吸管轻轻地重悬培养。转移约15ml 再悬浮培养至190ml MS。通过旋转混合。读取OD 600 。 用MS将培养物浓度调节至大约OD 600 = 0.5至0.6 媒体。

  3. 用土壤杆菌转化大豆幼苗的基础
    1. 添加30毫升MS-细菌溶液到50毫升塑料一次性烧杯 这将容易切割植物(6个塑料烧杯细菌 溶液)。用锋利的安全剃刀刀片, 在土壤水平上切除幼苗并立即将其放入烧杯中 ?包含。发根培养物。确保幼苗的基部 被淹没在文化中。每个烧杯中添加约28个切割植物。地点 将切下的幼苗的烧杯放入真空室中。
    2. 应用 真空度为380 mm Hg(Torr),真空渗透30 min 共培养溶液进入植物。缓慢地释放真空 避免对植物的潜在损害。


      图2.网关 包含FMV启动子,eGFP和Tet基因pRAP15的过表达载体 ?用EcoRI(E),HindIII(H),EcoRI/HindIII(E/H) HindIII/SpeI(H/S)限制酶

    3. 共同培养 植物过夜约15小时。植物被放置在a 透明塑料桶(图3),其放置在旋转平台上, 以65rpm移动。温度保持在约73°F至74°F [22.8℃] 23.3℃]。共培养温度不能显着上升 高于77°F [25°C]或K599可以驱逐或修改所需的质粒 有效转换。


      图3.将小植物放置在用于共培养的透明塑料盆中

    4. 用RO水洗涤植物。对于每个基因,填充两个1L烧杯 其中RO水从顶部边缘至多约1cm。从中除去植物 ?一个共培养烧杯并将茎末端约8x浸入第一个 ?洗涤烧杯,然后在第二个洗涤烧杯中做同样的。放置 植物放入含有反渗透水的干净烧杯中。放置 烧杯在一个塑料桶洗涤的植物,并且存储他们 露天浴缸在夜间。对于每个基因,使用新的,新鲜的RO水和 烧杯以避免交叉污染。植物可以储存在 小烧杯最多4天后共培养洗涤。所有 的洗涤水应在丢弃前进行高压灭菌(图4)

      图4.将小植株用RO水洗涤以除去土壤杆菌

      注意:不要使用普通自来水或任何可能含有的水 氯,氟化物或任何其它水净化试剂。填充足够1 ?L烧杯前后夜间共培养洗洗全部 植物。这允许RO水平衡到室温。空气 ?如有必要,请对实验室进行条件。

    5. 孵化植物 约73°F至74°F [22.8至23.3℃] 周期约15小时约42小时[两个晚上过后 共培养洗涤]。生长灯约15 ?cm。如果距离太远,植物会 细长过度,可能危及他们的健康和耐久性 ?在他们的恢复期间在ProMix。

  4. 在温室中种植大豆苗
    1. 在种植大豆苗之前,在桶中预先湿润ProMix ?用温室自来水填充三个32个单元格单元内 穿孔的标准平板/DNA构建体。 ProMix应该是相当 湿;甚至sodden。用稳定的手压力将ProMix装入锅中。 ?就在种植植物之前,用水喷洒ProMix 喷壶。使用记号笔作为钻头,创建2到3厘米的孔 ProMix用于植物插入。将植物插入ProMix中 约2至3厘米,或靠近子叶,轻轻压缩 ProMix下来用四个手指环绕茎。种植后 植物,用喷壶彻底洒上ProMix。 种植日为天0.
      注意:不要将ProMix与Biofungicide一起使用。它可能干扰涉及线虫和真菌的根的测定。
    2. 浇水植物轻轻地用喷壶前4天 后种植在ProMix。在第5天用魔杖切换到浇水 ?用纯水在ProMix种植后的前9天。在天 10,用Spot Sprayer或喷壶开始给植物3天 和1x Peters Excel 13-2-13 150ppm氮(pH 6.1)。
    3. 如果 需要限制性枝条生长,植物的顶部高于第二 三叶(约在第15天)可能被切断。使用不锈钢 已用酒精或抗菌皂清洁的剪刀。 这个时间段将随季节和生长条件而变化。 在夏季,可能需要两次浇头。植物 在温室中在自然光下生长,每天补充12 h:12h光:人造光的暗循环。温室气温 保持在25℃(天)和14℃(夜),20-50%湿度。

  5. 通过检查大豆根和修剪未转化的根来评估转化
    1. 在第38天,(图5)轻轻地从所有植物中去除土壤 每个克隆组通过将它们置于4加仑可高压灭菌的桶中。 轻轻按摩根块,以删除Promix。用房间冲洗根 ?温度水。一个桶可以用来洗约50棵植物。 将去污的植物储存在含有0.5x Peters的2加仑桶中 Excel 13-2-13 75ppm氮(pH6.1)

      图。 5.大豆植物在温室中,并在转化后
      38天生长良好
    2. 在黑暗的房间,使用Dark Reader Spot Lamp-SL85和橙色过滤器 ?眼镜以可视化GFP根(图6)。丢弃没有植物 GFP根。识别20个或更多最好的复合植物与好 GFP根进行实验。转化效率范围从 65至90%。
    3. 用剪刀,剪掉非GFP根上 复合材料厂。在温室中保持复合植物 它们的根在约75ppm处浸没在0.5x Peters Excel 13-2-13中 氮(pH6.1)两天,以便在它们之后最小化萎。 ?在沙子中重新种植

      图6.暴露于暗光的大豆根以显现表达GFP的根

    4. 植物复合植物在淹没,无菌砂中帮助他们 从去除非GFP根中恢复。每个2厘米的砂 细胞。将植物的根部放在细胞中,与其基部 幼苗低于细胞顶部1cm。轻轻地洒湿沙 ?并在根的顶部。建立一个紧凑的2至3厘米高的砂锥 围绕幼苗的基部以保持其直立。将单元格放在 平面。用0.5x Peters Excel 13-2-13 75ppm氮气冲洗平板。 离开植物在洪水沙子2天。
    5. 将植物转移到含有约2.5厘米深的湿Promix的穿孔平面。让植物过夜。
    6. 第二天,接种线虫 注意:如果小植株在非GFP根后出现枯萎 切除,将它们遮蔽,以保护他们免受阳光直射 他们已经恢复。

食谱

  1. MS媒体
    准备
    1升
    MS盐
    4.4克
    3%蔗糖 30克
    将pH调节至5.7
    如果要在一天内使用MS培养基,则无需高压灭菌。
  2. 可怕的肉汤,修改
    RPI预混巧克力肉汤
    Terrific Broth预混料的内容
    1升
    酪蛋白消化蛋白胨
    12 g/L
    酵母提取物
    24克/升
    磷酸二钾(K 2 HPO 4)
    9.4克/升
    磷酸钾(K 3 PO 4)
    2.2 g/L
    准备
    1升
    将TB预混物加入1L水中
    47.6克
    甘油
    4 ml
    无需pH介质
    高压灭菌器20分钟
  3. Scotts Peters Excel 13-2-13 16x肥料
    用13粒KOH制备1加仑水
    将每种溶液很好地混合
    用10汤匙准备1加仑的水[10T] Peters Excel13-2-13,Scotts Peters Excel 13-2-13 32x
    Mix合并两种溶液并分别混匀
    pH 6.2

致谢

该协议改编自我们以前的工作,包括(Matthews等人,2013; 2014和Youssef等人,2013)。我们感谢猎人胡子的技术支持。提及商业名称,专利产品或供应商不构成美国农业部对产品的担保或保证,也不意味着批准其他可能适用的其他产品或供应商。作者没有利益冲突。

参考文献

  1. Matthews,B.F.,Beard,H.,Brewer,E.,Kabir,S.,MacDonald,M.H.and Youssef,R.M。(2014)。 拟南芥基因,AtNPR1,AtTGA2和AtPR-5,赋予部分抗性当在转基因大豆根中过表达时,大豆胞囊线虫( Heterodera glycines )。
  2. Matthews,B.F.,Beard,H.,MacDonald,M.H.,Kabir,S.,Youssef,R.M.,Hosseini,P.and Brewer,E。 通过对大豆中100种基因对其主要病原体,大豆胞囊的功能代谢研究的工程化抗性和过敏性线虫。 237(5):1337-1357。
  3. Youssef,R.M.,MacDonald,M.H.,Brewer,E.P.,Bauchan,G.R.,Kim,K.H。和Matthews,B.F。(2013)。 AtPAD4的异位表达拓宽了大豆对大豆胞囊和根结线虫的抗性。 BMC Plant Biol 13:67.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Matthews, B. F. and Youssef, R. M. (2016). Agrobacterium rhizogenes-Based Transformation of Soybean Roots to Form Composite Plants. Bio-protocol 6(2): e1708. DOI: 10.21769/BioProtoc.1708.
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