搜索

Potato Transformation
马铃薯转基因   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

This is a protocol to produce stable transgenic potato plants (Solanum tuberosum cv. Désirée) by Agrobacterium-mediated genetic transformation, which is established based on a method described by (Jung et al., 2005) with some modifications. Agrobacterium tumefaciens strain LBA4404 carrying the desired construct is used to infect internodal explants to produce stable transgenic potato plants. Plantlet screening and molecular analyses are employed to confirm the expression of transgene in generated transgenic potato lines.

Keywords: Agrobacterium-mediated transformation(农杆菌介导转化), Agrobacterium tumefaciens strain LBA4404(根癌农杆菌LBA4404菌株), Potato plantlet(马铃薯植株)

Materials and Reagents

  1. Potato (S. tuberosum cv. Désirée) tissue culture plantlets
  2. 70% ethanol
  3. Bleach (6% sodium hypochlorite)
  4. Tween 20
  5. Acetosyringone (Fisher Scientific, catalog number: AC11554 )
  6. Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
  7. Selective antibiotics
  8. Mannitol (Fisher Scientific, catalog number: M120 )
  9. Glycine
  10. Nicotinic acid (Sigma-Aldrich, catalog number: N0761 )
  11. Pyridoxine HCl (Sigma-Aldrich, catalog number: P6280 )
  12. Thiamine HCl (Sigma-Aldrich, catalog number: T1270 )
  13. Folic acid (Fisher Scientific, catalog number: BP2519 )
  14. Biotin (Fisher Scientific, catalog number: BP232 )
  15. MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
  16. Inositol (Fisher Scientific, catalog number: AC122261000 )
  17. Sucrose
  18. 6-benzylamino purine (BAP) (Sigma-Aldrich, catalog number: B3408 )
  19. 1-naphthalene-acetic acid (NAA) (Sigma-Aldrich, catalog number: N0640 )
  20. 3-indoleacetic acid (IAA) (Sigma-Aldrich, catalog number: I2886 )
  21. Trans-zeatin-riboside (Sigma-Aldrich, catalog number: Z0876 )
  22. YM medium (see Recipes)
  23. MSVI vitamins (see Recipes)
  24. JHMS vitamins (see Recipes)
  25. 3R vitamins (see Recipes)
  26. CIM medium (see Recipes)
  27. 3C5ZR plates with selective agent (see Recipes)
  28. Propagation medium (see Recipes)
  29. TPS buffer (see Recipes)

Equipment

  1. Sterile magenta boxes (sterilized by autoclaving)
  2. Sterile petri dishes (Fisher scientific, catalog number: FB0875712 )
  3. Sterile forceps and scalpel (sterilized by heat treatment using a Bunsen burner)
  4. Sterile glass tubes with caps (sterilized by autoclaving) (tubes, Fisher Scientific, catalog number: 14-961-34 ; caps, Fisher Scientific, catalog number: 14-957-91D )
  5. Sterile inoculating loop (Fisher Scientific, catalog number: 22-363-604 )
  6. Tissue grinders
  7. Glass culture tubes (sterilized by autoclaving)
  8. 3M Micropore tape (Fisher Scientific, catalog number: 1530-0 and 1530-1 )
  9. Agrobacterium glycerol stock
  10. 50 ml conical centrifuge tubes
  11. Tissue culture biosafety cabinet
  12. Incubator shaker (New Brunswick Scientific, model: C24KC )
  13. Centrifuge
  14. S. tuberosum growth chambers (we use several types of growth chambers such as I-66LLVL from Percival)
  15. Hot block or water bath
  16. PCR thermal cycler (Bio-Rad Laboratories, model: C1000 touch)

Procedure

  1. Generation and propagation of potato tissue culture plantlets (performed under sterile conditions)
    1. Excise sprouts (at least 2.5 to 4 cm long) from potato tubers or stem segments (containing at least one internode) from plants and surface sterilize these plant pieces:
      1. Wash with 70% ethanol for 1-2 min.
      2. Use a sterile forceps to transfer plant pieces to 10% bleach containing trace amount of Tween 20 (1 μl/ml) and soak for 10-12 min.
      3. Transfer plant pieces to 70% ethanol and rinse for 1-2 min.
      4. Wash with plenty of sterile distilled water to remove any trace of ethanol.
    2. Trim the sterilized plant pieces with sterile scalpel to remove damaged cell layers and culture each plant piece on propagation medium in a glass tube at 24 °C under a 16-h/8-h light/dark cycle. Plantlets originated from the sterilized plant pieces are generally established for approximately four to six weeks.
    3. Cut approximately 1 cm-long nodal stem segments from plantlets (Figure 1), and push firmly into a magenta box or glass tube containing propagation medium, leaving the top part of the stem exposed. Put six segments per box or one segment per glass tube, and seal the box cover or tube cap with micropore tape. Around twenty four plantlets are suitable for transformation of one construct.
    4. Cultivate in a growth incubator at 24 °C under a 16-h/8-h light/dark cycle for four to six weeks.


      Figure 1. Potato plantlet growing in the propagation medium. Nodal and internodal sections are indicated as shown.

  2. Agrobacterium preparation
    1. Four days prior to infection, streak out Agrobacterium tumefaciens LBA4404, carrying the desired construct (transgene), on an YM plate with appropriate antibiotics. Incubate the plate at 28 °C for three days.
    2. The day before infection, inoculate 10 ml of YM with Agrobacterium colonies and culture at 28 °C on an incubator shaker till OD00 reaches 0.7 (usually need overnight culturing).
    3. On the day of inoculation, centrifuge the Agrobacterium culture at 9,000 x g for 15 min at room temperature and re-suspend the pellet in 10 ml CIM liquid medium.
    4. Add 50 μl of 0.074 M acetosyringone to the Agrobacterium suspension (the final concentration of acetosyringone is 0.37 mM). Use within one hour, as cells will begin to aggregate.

  3. Potato transformation (performed under sterile conditions)
    1. On the day of infection, cut internodal stem sections (Figure 1) from four to six weeks plantlets into about 1 cm pieces and transfer to CIM plates.
    2. Incubate explants with 10 ml Agrobacterium inoculum for 20 min, occasionally swirling the plates. We usually use 100 stem pieces per construct.
    3. Use sterile forceps to transfer stem pieces from the bacterial culture to new CIM media plates. Label plates, seal with micropore tape, and place them back to the 24 °C incubator in the dark.
    4. After three days, transfer stem pieces to 3C5ZR medium plates with appropriate antibiotics and 500 μg/ml timentin. Timentin is used to inhibit the growth of A. tumefaciens LBA4404.
    5. Place the plates in the growth incubator (24 °C, 16-h/8-h light/dark cycle) and transfer the stem pieces to fresh 3C5ZR medium plates every seven to ten days, until plantlets emerge (Figure 2).
    6. When plantlets are at least half a centimeter long, cut them from explants and transfer to culture tubes containing 10 ml of propagation medium with the appropriate antibiotics and timentin at 100 μg/ml. Sterile inoculating loops can be used to gently push the base of the plantlet into the medium. Label and seal the tubes with micropore tape.
    7. Grow plants for 3 - 4 weeks until roots are established. Discard any plants that do not grow roots within one month, as they are not transgenic.
    8. Cut and re-propagate the transgenic plants as needed.


      Figure 2. Potato internodal sections cultivated on the 3C5ZR medium. Plantlets (indicated) are emerging from the infected nodal or internodal sections.

  4. PCR screening
    1. Cut approximately one-half to one square centimeter of leaf tissue to a 1.5 ml centrifuge tube.
    2. Add 200 μl TPS to each tube.
    3. Grind the tissue in TPS with a tissue grinder to obtain a uniform slurry.
    4. Incubate samples at 75 °C for 20 min.
    5. Centrifuge at 13,400 x g for 10 min.
    6. Transfer supernatant to a new tube and add an equal volume of isopropanol to each tube. Mix tubes by inversion.
    7. Centrifuge at 13,400 x g for 10 min. DNA pellet should be visible at this point.
    8. Remove supernatant and wash pellet with 70% ethanol. Centrifuge at 13,400 x g for 5 min.
    9. Remove supernatant, air-dry pellet and resuspend pellet in 30 μl of sterile distilled water or TE containing RNaseA (100 μg/ml).
    10. Use 1 μl of DNA as template for PCR screening of transformants.
    11. Typical PCR program:
      First step: 95 °C for 3 min
      Second step: 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 1 min x 30 cycles
      Third step: 72 °C for 5 min

Recipes

The following media are prepared and stored at room temperature. The poured medium plates containing antibiotics can be stored at 4 °C for up to one month. Vitamins can be prepared, aliquoted and stored at -20 °C.

  1. YM Medium (1 L)
    0.4 g yeast extract
    10 g mannitol*
    0.1 g NaCl
    0.2 g MgSO4.7H2O
    0.5 g KH2PO4
    10 g Agar (for plates)
    Autoclave
    *Make and autoclave separately, make 10 g/100 ml and use 100 ml/L
  2. MSVI vitamins
    2 mg/ml glycine
    0.5 mg/ml nicotinic acid
    0.5 mg/ml pyridoxine HCl
    0.4 mg/ml thiamine HCl
  3. JHMS vitamins
    0.4 mg/ml folic acid
    50 μg/ml biotin
  4. 3R vitamins
    1 mg/ml thiamine HCl
    0.5 mg/ml nicotinic acid
    0.5 mg/ml pyridoxine HCl
  5. CIM medium (1 L)
    4.3 g MS salt
    1 ml MSVI vitamins
    1 ml JHMS vitamins
    0.1 g inositol
    30 g sucrose
    1 mg 6-benzylamino purine (BAP)
    2 mg 1-naphthalene-acetic acid (NAA)
    10 g agar (for plates)
    Adjust pH to 5.6 with KOH and autoclave
  6. 3C5ZR medium (1 L)
    4.3 g MS salt
    1 ml 3R vitamins
    0.1 g inositol
    30 g sucrose
    10 g agar (for plates)
    Adjust pH to 5.9 with KOH and autoclave
    0.5 mg 3-indoleacetic acid (IAA) *
    3 mg trans-zeatin-riboside *
    500 mg Timentin*
    Selection agent **
    * Add after sterilization
    ** Use appropriate selection agent and concentration for construct
  7. Propagation medium (1 L)
    4.3 g MS salt
    0.17 g NaH2PO4.H2O
    0.1 g inositol
    0.4 mg thiamine HCl
    30 g sucrose
    2.5 g gelrite or 10 g agar
    Adjust pH to 6.0 with KOH and autoclave
  8. TPS buffer
    100 mM Tris HCl (pH 8.0)
    100 mM EDTA (pH 8.0)
    1 M KCl

Acknowledgments

This work was supported by funding from USDA-ARS.

References

  1. Cheng, Z. M., Schnurr, J. A. and Kapaun J. A. (1998). Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17: 646-649.
  2. Chronis, D., Chen, S., Lu, S., Hewezi, T., Carpenter, S. C., Loria, R., Baum, T. J. and Wang, X. (2013). A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism. Plant J 74(2): 185-196.
  3. Jung, C. S., Griffiths, H. M., De Jong, D. M., Cheng, S., Bodis, M. and De Jong, W. S. (2005). The potato P locus codes for flavonoid 3',5'-hydroxylase. Theor Appl Genet 110(2): 269-275.

简介

这是通过土壤杆菌介导的遗传转化产生稳定的转基因马铃薯植物(马铃薯(Solanum tuberosum)cv.Désirée)的方案,其基于由(Jung& em。等人,2005),其具有一些修改。 携带所需构建体的根癌农杆菌菌株LBA4404用于感染节间外植体以产生稳定的转基因马铃薯植物。 植物筛选和分子分析用于确认转基因在所产生的转基因马铃薯品系中的表达

关键字:农杆菌介导转化, 根癌农杆菌LBA4404菌株, 马铃薯植株

材料和试剂

  1. 组织培养小植株的马铃薯( S.tuberosum cv。Désirée)
  2. 70%乙醇
  3. 漂白剂(6%次氯酸钠)
  4. 吐温20
  5. Acetosyringone(Fisher Scientific,目录号:AC11554)
  6. Timentin( Phyto Technology Laboratories ®,目录号:T869)
  7. 选择性抗生素
  8. 甘露醇(Fisher Scientific,目录号:M120)
  9. 甘氨酸
  10. 烟酸(Sigma-Aldrich,目录号:N0761)
  11. 盐酸吡哆醇(Sigma-Aldrich,目录号:P6280)
  12. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  13. 叶酸(Fisher Scientific,目录号:BP2519)
  14. 生物素(Fisher Scientific,目录号:BP232)
  15. MS盐(Caisson Laboratories,目录号:MSP01-50LT)
  16. 肌醇(Fisher Scientific,目录号:AC122261000)
  17. 蔗糖
  18. 6-苄氨基嘌呤(BAP)(Sigma-Aldrich,目录号:B3408)
  19. (NAA)(Sigma-Aldrich,目录号:N0640)
  20. (IAA)(Sigma-Aldrich,目录号:I2886)
  21. 反 - 玉米素 - 核糖核苷(Sigma-Aldrich,目录号:Z0876)
  22. YM介质(参见配方)
  23. MSVI维生素(参见食谱)
  24. JHMS维生素(见配方)
  25. 3R维生素(见配方)
  26. CIM介质(参见配方)
  27. 具有选择剂的3C5ZR板(参见配方)
  28. 传播介质(参见配方)
  29. TPS缓冲区(参见配方)

设备

  1. 无菌洋红色盒(高压灭菌)
  2. 无菌培养皿(Fisher scientific,目录号:FB0875712)
  3. 无菌镊子和手术刀(使用本生灯进行热处理消毒)
  4. 具有盖的灭菌玻璃管(通过高压灭菌灭菌)(管,Fisher Scientific,目录号:14-961-34;帽,Fisher Scientific,目录号:14-957-91D)
  5. 无菌接种环(Fisher Scientific,目录号:22-363-604)
  6. 组织研磨机
  7. 玻璃培养管(高压灭菌)
  8. 3M Micropore带(Fisher Scientific,目录号:1530-0和1530-1)
  9. 农杆菌甘油原液
  10. 50ml锥形离心管
  11. 组织培养生物安全柜
  12. 孵育摇床(New Brunswick Scientific,型号:C24KC)
  13. 离心机
  14. tuberosum生长室(我们使用几种类型的生长室,如来自Percival的I-66LLVL)
  15. 热块或水浴
  16. PCR热循环仪(Bio-Rad Laboratories,型号:C1000 touch)

程序

  1. 马铃薯组织培养小植株的生成和繁殖(在无菌条件下进行)
    1. 从植物的马铃薯块茎或茎段(含至少一个节间)消化芽(至少2.5至4cm长),并对这些植物表面进行表面灭菌:
      1. 用70%乙醇洗涤1-2分钟
      2. 使用无菌镊子将植物片段转移到含有微量Tween 20(1μl/ml)的10%漂白剂中并浸泡10-12分钟。
      3. 将植物块转移到70%乙醇,冲洗1-2分钟
      4. 用大量无菌蒸馏水清洗,以除去任何痕量的乙醇
    2. 用无菌手术刀修剪灭菌的植物碎片,以去除损伤的细胞层,并在24小时/16小时/8小时光照/黑暗循环下,在玻璃管中的每个植物片段在繁殖培养基上培养。 植物 来源于灭菌的植物碎片通常建立大约四至六周。
    3. 从小植物切下约1厘米长的节段茎段(图1),并牢牢地推入含有繁殖培养基的洋红盒或玻璃管,使茎的顶部暴露。 每个盒子放置6个片段或每个玻璃管放置一个片段,并用微孔胶带密封盒盖或管盖。 大约二十四个小植物适于转化一个构建体
    4. 在24℃,16小时/8小时光照/黑暗循环下,在生长培养箱中培养4至6周。

      图1.在传播介质中生长的马铃薯苗。节点和节间部分如图所示。

  2. 土壤杆菌制剂
    1. 在感染前4天,在具有适当抗生素的YM平板上划线携带所需构建体(转基因)的根癌土壤杆菌LBA4404。将板在28℃下孵育三天。
    2. 在感染前一天,用农杆菌菌落接种10ml YM,并在恒温摇床上在28℃下培养直至OD 00达到0.7(通常需要过夜培养)。 br />
    3. 在接种当天,在室温下以9000×g离心土壤杆菌培养物15分钟,并将沉淀重悬于10ml CIM液体培养基中。
    4. 向土壤杆菌悬浮液中加入50μl的0.074M乙酰丁香酮(乙酰丁香酮的最终浓度为0.37mM)。在一小时内使用,因为单元格将开始聚合
  3. 马铃薯转化(在无菌条件下进行)
    1. 在感染的当天,将4至6周小植株的节间茎段(图1)切成约1cm的块并转移至CIM平板。
    2. 孵育外植体与10毫升农杆菌接种20分钟,偶尔涡旋板。我们通常每个结构使用100个茎片。
    3. 使用无菌镊子将细菌培养物的茎片转移到新的CIM培养基平板上。标签板,用微孔胶带密封,并将它们放回到24℃的孵化器在黑暗中
    4. 三天后,将茎片转移到具有适当抗生素和500μg/ml特美汀的3C5ZR培养基平板上。特美汀用于抑制em的生长。 tumefaciens LBA4404。
    5. 将板置于生长培养箱中(24℃,16小时/8小时光照/黑暗循环),并将茎片段每7至10天转移到新鲜的3C5ZR培养基平板上,直到小植株出现(图2)。
    6. 当小植株至少半厘米长时,将它们从外植体切下并转移到含有10ml具有100μg/ml适当抗生素和特美汀的繁殖培养基的培养管中。可以使用无菌接种环将小植株的基部轻轻推入培养基中。用微孔胶带标记并密封试管。
    7. 种植植物3 - 4周,直到根成熟。 丢弃在一个月内不生根的植物,因为它们不是转基因的
    8. 根据需要切割和再繁殖转基因植物

      图2.在3C5ZR培养基上培养的马铃薯节间切片。小植株(指示的)从感染的节或茎节中出现。

  4. PCR筛选
    1. 将约一半至一平方厘米的叶组织切成1.5ml离心管
    2. 每管加入200μlTPS
    3. 用组织研磨机研磨TPS中的组织以获得均匀的浆料
    4. 在75°C孵育样品20分钟。
    5. 以13,400×g离心10分钟。
    6. 将上清液转移到新管中,并向每个管中加入等体积的异丙醇。 通过倒置混合管。
    7. 以13,400×g离心10分钟。 DNA沉淀在这一点应该是可见的
    8. 除去上清液,用70%乙醇洗涤沉淀。 以13,400×g离心5分钟。
    9. 去除上清,风干沉淀,并重悬在30μl无菌蒸馏水或含RNaseA(100μg/ml)的TE中。
    10. 使用1μlDNA作为模板,用于转化体的PCR筛选
    11. 典型PCR程序:
      第一步:95℃3分钟
      第二步:94℃30秒,58℃30秒,72℃1分钟×30个循环
      第三步:72℃5分钟

食谱

制备以下培养基并在室温下储存。 含有抗生素的灌注的培养基平板可以在4℃下储存长达一个月。 可以制备维生素,等分并保存在-20℃。

  1. YM中(1 L)
    0.4g酵母提取物
    10克甘露醇*
    0.1克NaCl
    0.2g MgSO 4。7H 2 O 2 0.5g KH 2 PO 4 sub/
    10g琼脂(用于板)
    高压灭菌器
    *分别制作和高压釜,使10 g/100 ml,并使用100 ml/L
  2. MSVI维生素
    2mg/ml甘氨酸 0.5mg/ml烟酸
    0.5mg/ml盐酸吡哆醇
    0.4mg/ml硫胺素HCl
  3. JHMS维生素
    0.4mg/ml叶酸
    50μg/ml生物素
  4. 3R维生素
    1mg/ml硫胺素HCl
    0.5mg/ml烟酸
    0.5mg/ml盐酸吡哆醇
  5. CIM培养基(1L)
    4.3克MS盐
    1 ml MSVI维生素
    1 ml JHMS维生素
    0.1g肌醇
    30克蔗糖 1mg 6-苄基氨基嘌呤(BAP)
    2mg 1-萘 - 乙酸(NAA)
    10g琼脂(用于平板)
    用KOH和高压釜将pH调节至5.6
  6. 3C5ZR培养基(1L) 4.3克MS盐
    1 ml 3R维生素
    0.1g肌醇
    30克蔗糖 10g琼脂(用于平板)
    用KOH和高压釜
    调节pH至5.9 0.5mg 3-吲哚乙酸(IAA)*
    3mg反式玉米素 - 核糖核苷*
    500毫克特美汀*
    选择代理**
    *灭菌后添加
    **使用适当的选择剂和浓度的结构
  7. 繁殖培养基(1 L)
    4.3克MS盐
    0.17g NaH 2 PO 4 subO 2。 H O 0.1g肌醇
    0.4mg硫胺素HCl 30克蔗糖 2.5g gelrite或10g琼脂 用KOH和高压釜将pH调节至6.0
  8. TPS缓冲区
    100mM Tris HCl(pH8.0) 100mM EDTA(pH8.0) 1 M KCl

致谢

这项工作得到了美国农业部的资助。

参考文献

  1. Cheng,Z.M.,Schnurr,J.A。和Kapaun J.A。(1998)。 Timentin作为遗传转化中根癌土壤杆菌的替代抗生素 >。 Plant Cell Rep 17:646-649
  2. Chronis,D.,Chen,S.,Lu,S.,Hewezi,T.,Carpenter,S.C.,Loria,R.,Baum,T.J.and Wang,X.(2013)。 从植物寄生线虫分泌的泛素羧基延伸蛋白 在植物中裂解以促进植物寄生。植物J 74(2):185-196。
  3. Jung,C.S.,Griffiths,H.M.,De Jong,D.M.,Cheng,S.,Bodis,M。和De Jong,W.S.(2005)。 马铃薯 P 位点编码类黄酮3',5'-羟化酶 。 Theor Appl Genet 110(2):269-275。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chronis, D., Chen, S., Lang, P., Tran, T., Thurston, D. and Wang, X. (2014). Potato Transformation. Bio-protocol 4(1): e1017. DOI: 10.21769/BioProtoc.1017.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。