Agrobacterium-mediated Transformation of Strawberry

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New Phytologist
Jan 2013



Traditional breeding for improvement of strawberry (Fragaria x ananassa) is difficult because strawberry is an octoploid, hybrid species. Genetic modification of strawberry would though be a promising alternative for obtaining the desired improvements in existing elite strawberry cultivars (Schaart et al., 2011). The availability of suitable genes for trait improvements in strawberry has however been a rate-limiting step until recently, but with the completion of the sequencing of the genome of woodland strawberry (F. vesca) (Shualev et al., 2011), we now have access to a treasure chest with valuable candidate genes. For strawberry, methods for genetic transformation have originally been described by Nehra et al. (1990) and James et al. (1990) and success of transformation was shown to be highly cultivar dependent. The latest progress in strawberry transformation is reviewed by Husaini et al. (2011). In our lab transformation of strawberry is based on the method for shoot regeneration described by Passey et al. (2003) and the use of the supervirulent Agrobacterium strain AGL0 (Lazo et al., 1991). We mainly make use of the strawberry transformation as a tool for functional analysis of candidate genes. For this the cultivar Calypso is a very suitable genotype because of its high transformation efficiencies (up to 100%) and ever-bearing fruiting characteristic, which provides a continuous supply of strawberry fruits once the plants start flowering.

Keywords: Strawberry (草莓), Agrobacterium (农杆菌), Transformation (转化), Genetic modification (遗传修饰)

Materials and Reagents

  1. Sucrose (Duchefa Biochemie BV, catalog number: S0809 )
  2. Daishin agar (Duchefa Biochemie BV, catalog number: D1004 )
  3. 6-Benzylaminopurine (BAP) (Duchefa Biochemie BV, catalog number: B0904 ) Indole-3-butyric acid (IBA) (Duchefa Biochemie BV, catalog number: I0902 )
  4. Murashige and Skoog salts including vitamins (MS) (Duchefa Biochemie BV, catalog number: M0221 )
  5. Gelrite (Duchefa Biochemie BV, catalog number: G1101 )
  6. 3’,5’Dimethoxy-4’-hydroxy-acetophenone (Acetosyringone) (Sigma-Aldrich, catalog number: D134406 )
  7. LB broth (LB) (Sigma-Aldrich, catalog number: L3022 )
  8. Rifampicin (Duchefa Biochemie BV, catalog number: R0146 )
  9. Dimethyl sulfoxide (DMSO) (Merck KGaA, catalog number: 802912 )
  10. MN615 filter paper 82 mm (MACHEREY-NAGEL GmbH & Co. KG, catalog number: MN615)
  11. Thidiazuron (TDZ) (Duchefa Biochemie BV, catalog number: T0916 )
  12. 1-Naphthalene acetic acid (NAA) (Duchefa Biochemie BV, catalog number: N0903 )
  13. Cefotaxime sodium salt (cefotaxime) (Duchefa Biochemie BV, catalog number: C0111 )
  14. Kanamycin monosulphate monohydrate (kanamycin) (Duchefa Biochemie BV, catalog number: K0126 )
  15. D-glucose monohydrate (glucose) (Duchefa Biochemie BV, catalog number: G0802 )
  16. Shoot multiplication medium (SMM) (see Recipes)
  17. Shoot propagation medium (SPM) (see Recipes)
  18. Shoot regeneration medium (SRM) (gluc.) (see Recipes)
  19. SRM + AS (see Recipes)
  20. SRM SEL (see Recipes)
  21. MS-liquid + AS (see Recipes)
  22. 1 mg/ml NAA (see Recipes)
  23. 0.22 mg/ml TDZ (see Recipes)
  24. 1 mg/ml IBA (see Recipes)
  25. 1 mg/ml BAP (see Recipes)
  26. 100 mM AS (see Recipes)
  27. 125 mM Cefotaxime (see Recipes)
  28. 50 mg/ml Kanamycin (see Recipes)
  29. 50 mg/ml Rifampicin (see Recipes)
  30. 30 g/100 ml Glucose (see Recipes)


  1. 50 ml tube (Greiner Bio-One GmbH, catalog number: 210261 )
  2. Petri dish (Greiner Bio-One GmbH, catalog number: 633180 )
  3. Rotary shaker (C24 incubator shaker, New Brunswick Scientific)
  4. Centrifuge Multifuge 3LR (Heraeus Holding)


  1. For transformation of strawberry we made use of ‘in vitro’ grown plants. These plants were alternated cultured under sterile conditions on shoot propagation medium (SPM), which is Murashige and Skoog macro and micro elements and vitamins (Murashige and Skoog, 1962) (MS) (2.2 g/L), sucrose (30 g/L) Daishin agar (9 g/L), supplemented with BAP and IBA both at concentrations of 0.1 mg/L (pH 5.8) and shoot multiplication medium (SMM) which is MS (2.2 g/L), sucrose (30 g/L) Daishin agar (9 g/L), supplemented with BAP at concentration of 0.5 mg/L (pH 5.8).
  2. The plants were subcultured every 4-6 weeks by transferring side-shoots to fresh SPM and were grown in the light (16 h/8 h light/dark) in a culture room at 25 °C.
  3. Transformations were performed under sterile conditions in a biological safety down flow cabinet. For the transformation leaf discs of 7 mm in diameter were cut from ‘in vitro’ leaf material grown for four weeks on SPM. Leaf discs were collected in pertidishes containing cocultivation medium which is SRM+AS [SRM; MS (2.2 mg/L) with glucose (30 g/L), NAA (0.2 mg/L), TDZ (1.0 mg/L) and gelrite (4 g/L) (pH 5.8) supplemented with acetosyringone (100 μM)].
  4. The Agrobacterium strain AGL0, which contains the supervirulent Ti plasmid pTIBo542 (Lazo et al., 1991) as well as a binary vector harbouring a kanamycin resistance gene (nptII) as plant selectable marker, was cultured overnight in 50 ml tubes containing 10 ml liquid LB medium supplemented with kanamycin (50 mg/L) and rifampicin (50 mg/L). The bacteria were incubated in a rotary shaker at a temperature of 28 °C.
  5. After overnight growth, the bacteria were pelleted by centrifugation at 2,000 x g (3,000 rpm) for 10 min and the bacterium pellet was resuspended in 40 ml of filter-sterilized liquid MS-medium containing glucose (30 g/L) and acetosyringone (100 μM) (pH 5.2).
  6. For transfection 20 ml of resuspended Agrobacterium was poured on the leaf discs which were cut and collected before in a petri dish containing cocultivation medium.
  7. After 10-20 min the leaf discs were blotted dry on a sheet of filter paper and were subsequently placed on fresh cocultivation medium that was covered by a disc of MN615 filter paper. The leaf discs were placed with the adaxial side in contact with the filter paper (so, upside-down) and cocultivated with the Agrobacterium cells under sterile conditions, for four days at 21 °C in the dark.
  8. After cocultivation the leaf discs were transferred to SRM supplemented with cefotaxim (250 mg/L) for the elimination of Agrobacteria and kanamycin (100-150 mg/L) for the selective regeneration of shoots. In case overgrowth of the leaf discs with Agrobacteria is experienced, the leaf discs may be wash in a solution containing 500 mg/L cefotaxime prior to transfer to SRM. The leaf discs were cultured in a growth room at 21 °C and at 16 h/8 h light/dark conditions.
  9. The SRM medium was refreshed every 4 weeks and shoots that regenerated on the leaf discs were harvested and transferred to jars containing shoot propagation medium (SPM) with kanamycin (50 mg/L) and cefotaxime (250 mg/L) for further selection and propagation under sterile conditions, at 24 °C and at 16h/8h light/dark conditions. In a typical transformation experiment 50-100% of the leaf discs give transgenic shoots.
  10. Shoots that propagated well and produced roots on this medium were transferred to the same medium but now without kanamycin and cefotaxime to show absence of Agrobacterium and after sufficient roots were formed, the plants were transferred to the greenhouse for further evaluation.


Note: All media containing antibiotics or IBA (which is light-sensitive), should be prepared freshly. All other media may be stored for a maximum of 14 days.

  1. Media for Propagation of Strawberry
    1. Shoot Multiplication Medium (SMM)
      for 1,000 ml
      4.4 g
      30 g
      Agar Daishin
      9 g
      0,5 ml BAP-stock (1mg/ml), final concentration 0.5 mg/L
      pH = 5.8
      Autoclave (hormones are coautoclaved)
    2. Shoot Propagation Medium (SPM)
      for 1,000 ml
      4.4 g
      30 g
      Agar Daishin
      9 g
      BAP 0,1 ml BAP-stock (1mg/ml), final concentration 0.1mg/L)
      IBA 0,1 ml IBA-stock (1mg/ml), final concentration 0.1mg/L)
      pH= 5.8
      Autoclave (hormones are co-autoclaved)
      Plants are cultured in alternating cycles on SMM for 4-6 weeks and then on SPM for 4-6 weeks. For transformation, leaves of plants which are grown for 4 weeks on SPM medium are used.

  2. Media for strawberry transformation
    1. Shoot Regeneratoon Medium (SRM) (1,000 ml)
      4.4 g MS
      4 g gelrite
      into 900 ml MilliQ water
      0.2 ml NAA-stock (1 mg/ml) (final conc. = 0.2 mg/L)
      4.5 ml TDZ-stock (0.22 mg/ml) (final conc. = 1 mg/L)
      adjust pH to 5.8 with 0.1 N KOH
      after autoclaving add 100 ml filter sterilised glucose-stock (30 g/100 ml)
    2. For SRM + AS (1,000 ml)
      Add to SRM (after autoclaving)
      1,000 μl acetosyringone-stock (100 mM) (final concentration 100 μM)
    3. For SRM SEL (1,000 ml)
      Add to SRM (after autoclaving)
      1,000 μl kanamycin-stock (50 mg/ml) (final concentration 100 mg/L)
      2,000 μl cefotaxime-stock (125 mg/ml) (final concentration 250 mg/L)
    4. MS-liquid + AS (1,000 ml)
      4.4 g MS-salts+vit.
      30 g Glucose
      into 1,000 ml MilliQ
      1,000 μl AS-stock (100 mM)
      adjust pH to 5.2 (!) with 0.1 N KOH
      filter-sterilise and store at 4 °C in 50 ml tubes

  3. Stock solutions
    1. NAA (1 mg/ml)
      Dissolve 50 mg NAA in 1 ml 0.1 N KOH and adjust volume to 50 ml with MilliQ; stored at 4 °C
    2. TDZ (0.22 mg/ml)
      Dissolve 55 mg TDZ in 1 ml 0.1 N KOH and adjust volume to 250 ml with MilliQ; stored at 4 °C
    3. IBA (1 mg/ml)
      Dissolve 50 mg IBA in 1 ml 0.1 N KOH and adjust volume to 50 ml with MilliQ; stored at 4 °C
    4. BAP (1 mg/ml)
      Dissolve 50 mg BAP in 1 ml 0.1 N KOH and adjust volume to 50 ml with MilliQ; stored at 4 °C
    5. AS (100 mM)
      Dissolve 98 mg Acetosyringone in 300 μl DMSO and adjust volume to 5 ml with Ethanol absolute; stored at 4 °C
    6. Cefotaxime (125 mg/ml)
      Dissolve 1,000 mg Cefotaxim in 8 ml MilliQ; filter sterilise; stored at 4 °C
    7. Kanamycin (50 mg/ml)
      Dissolve 400 mg Kanamycin in 8 ml MilliQ; filter sterilise; stored at 4 °C
    8. Rifampicin (50 mg/ml)
      Dissolve 400 mg Rifampicin in 100 μl 0.1N HCl and adjust volume to 8 ml with MilliQ; filter sterilise; stored at -20 °C
    9. Glucose (30 g/100 ml)
      Dissolve 300 g Glucose in 1,000 ml MilliQ; filter sterilise; store at room temperature in 50 ml tubes


This protocol was adapted from the method for shoot regeneration described by Passey et al. (2003) and the use of the supervirulent Agrobacterium strain AGL0 (Lazo et al., 1991).


  1. Husaini, A.M., Mercado J.A., Teixeira da Silva J.A., Schaart J.G. (2011). Review of factors affecting organogenesis, somatic embryogenesis and Agrobacterium tumefaciens-mediated transformation of strawberry. In: Husaini AM, Mercado JA (Eds), Genomics, Transgenics, Molecular Breeding and Biotechnology of Strawberry. Genes, Genomes and Genomics 5 (Special Issue 1), Global Science Books Ltd., Isleworth, United Kingdom: 1-11
  2. James, D.J., Passey, A.J., Barbara, D.J. (1990). Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria x ananassa Duch.) using disarmed binary vectors. Plant Sci 69, 79-94.
  3. Lazo, G. R., Stein, P. A. and Ludwig, R. A. (1991). A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology (N Y) 9(10): 963-967.
  4. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.
  5. Nehra, N. S., Chibbar, R. N., Kartha, K. K., Datla, R. S., Crosby, W. L. and Stushnoff, C. (1990). Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Rep 9(6): 293-298.
  6. Passey, A. J., Barrett, K. J. and James, D. J. (2003). Adventitious shoot regeneration from seven commercial strawberry cultivars (Fragaria x ananassa Duch.) using a range of explant types. Plant Cell Rep 21(5): 397-401.
  7. Schaart, J. G., Kjellsen, T. D., Heggem, R., Iversen, T. H, Schouten, H. J. and Krens, F. A. (2011). Towards the production of genetically modified strawberries which are acceptable to consumers. In: Husaini AM, Mercado JA (Eds) Genomics, Transgenics, Molecular Breeding and Biotechnology of Strawberry. Genes, Genomes and Genomics 5 (Special Issue 1), Global Science Books Ltd., Isleworth, United Kingdom: 102-107.
  8. Shulaev, V., Sargent, D. J., Crowhurst, R. N., et al. (2011). The genome of woodland strawberry (Fragaria vesca). Nat Genet 43(2): 109-116.


由于草莓是八倍体,杂种物种,用于改善草莓( Fragaria x ananassa )的传统育种是困难的。草莓的遗传修饰虽然是获得现有的优良草莓栽培品种所期望的改进的有希望的替代方法(Schaart等人,2011)。然而,随着草地草莓基因组测序的完成(Shualev ),用于草莓中性状改善的合适基因的可用性是速率限制步骤 et al。,2011),我们现在可以获得宝贵的候选基因宝库。对于草莓,遗传转化的方法最初由Nehra等人(1990)和James等人(1990)描述,并且转化的成功被证明是高度的品种依赖。草莓转化的最新进展由Husaini等人审查(2011)。在我们的实验室中,草莓的转化是基于由Passey等人(2003)描述的苗再生方法和使用超强毒力土壤杆菌菌株AGL0(Lazo et al。,1991)。我们主要利用草莓转化作为候选基因功能分析的工具。为此,栽培品种Calypso是非常合适的基因型,因为其高转化效率(高达100%)和永不结实的特性,一旦植物开始开花,其提供草莓果实的连续供应。

关键字:草莓, 农杆菌, 转化, 遗传修饰


  1. 蔗糖(Duchefa Biochemie BV,目录号:S0809)
  2. Daishin琼脂(Duchefa Biochemie BV,目录号:D1004)
  3. 6-苄基氨基嘌呤(BAP)(Duchefa Biochemie BV,目录号:B0904)吲哚-3-丁酸(IBA)(Duchefa Biochemie BV,目录号:I0902)
  4. Murashige和Skoog盐,包括维生素(MS)(Duchefa Biochemie BV,目录号:M0221)
  5. Gelrite(Duchefa Biochemie BV,目录号:G1101)
  6. 3',5'-二甲氧基-4'-羟基 - 苯乙酮(Acetosyringone)(Sigma-Aldrich,目录号:D134406)
  7. LB肉汤(LB)(Sigma-Aldrich,目录号:L3022)
  8. 利福平(Duchefa Biochemie BV,目录号:R0146)
  9. 二甲基亚砜(DMSO)(Merck KGaA,目录号:802912)
  10. MN615滤纸82mm(MACHEREY-NAGEL GmbH& Co.KG,目录号:MN615)
  11. 噻苯隆(TDZ)(Duchefa Biochemie BV,目录号:T0916)
  12. 1-萘乙酸(NAA)(Duchefa Biochemie BV,目录号:N0903)
  13. 头孢噻肟钠盐(头孢噻肟)(Duchefa Biochemie BV,目录号:C0111)
  14. 卡那霉素一硫酸盐一水合物(卡那霉素)(Duchefa Biochemie BV,目录号:K0126)
  15. D-葡萄糖一水合物(葡萄糖)(Duchefa Biochemie BV,目录号:G0802)
  16. 拍摄倍增介质(SMM)(参见配方)
  17. 拍摄传播介质(SPM)(参见配方)
  18. 射击再生介质(SRM)(葡萄糖)(见配方)
  19. SRM + AS(请参阅配方)
  20. SRM SEL(参见配方)
  21. MS-liquid + AS(参见配方)
  22. 1 mg/ml NAA(参见配方)
  23. 0.22 mg/ml TDZ(参见配方)
  24. 1 mg/ml IBA(见配方)
  25. 1mg/ml BAP(参见配方)
  26. 100 mM AS(参见配方)
  27. 125 mM头孢噻肟(见配方)
  28. 50mg/ml卡那霉素(见Recipes)
  29. 50 mg/ml利福平(见配方)
  30. 30克/100毫升葡萄糖(见配方)


  1. 50ml管(Greiner Bio-One GmbH,目录号:210261)
  2. 培养皿(Greiner Bio-One GmbH,目录号:633180)
  3. 旋转振荡器(C24培养摇床,New Brunswick Scientific)
  4. 离心机Multifuge 3LR(Heraeus Holding)


  1. 对于草莓的转化,我们利用'in vitro '生长的植物。将这些植物在无菌条件下在作为Murashige和Skoog macro和微量元素和维生素(Murashige和Skoog等人,1962)(MS)(2.2g/L)的枝条繁殖培养基(SPM) ,添加浓度为0.1mg/L(pH5.8)的BAP和IBA和作为MS(2.2g/L)的枝条繁殖培养基(SMM)的蔗糖(30g/L)Daishin琼脂(9g/,添加了浓度为0.5mg/L(pH 5.8)的BAP的蔗糖(30g/L)Daishin琼脂(9g/L)。
  2. 通过将侧枝转移至新鲜的SPM,将植物每4-6周传代培养,并在25℃的培养室中在光照(16小时/8小时光/暗)中生长。
  3. 在无菌条件下在生物安全下流橱中进行转化。对于转化,从在SPM上生长四周的"体外"叶材料切下直径为7mm的叶圆片。将叶圆片收集在含有共培养培养基的腐皮中,其为SRM + AS [SRM; (100μM)的葡萄糖(30g/L),NAA(0.2mg/L),TDZ(1.0mg/L)和gelrite(4g/L)(pH5.8)的MS(2.2mg/]。
  4. 包含超强毒力Ti质粒pTIBo542(Lazo等人,1991)的土壤杆菌属菌株AGL0以及含有卡那霉素抗性基因的二元载体(ptII)作为植物选择性标记)在含有10ml补充有卡那霉素(50mg/L)和利福平(50mg/L)的液体LB培养基的50ml管中培养过夜。将细菌在28℃的旋转振荡器中孵育
  5. 过夜生长后,通过在2,000xg(3,000rpm)离心10分钟使细菌沉淀,将细菌沉淀重悬于40ml含有葡萄糖(30g/l)的过滤灭菌的液体MS-培养基中, L)和乙酰丁香酮(100μM)(pH 5.2)
  6. 为了转染,将20ml重悬浮的土壤杆菌倒在叶盘上,在切片并收集之前,在含有共培养培养基的培养皿中。
  7. 在10-20分钟后,将叶圆片在一张滤纸上吸干,随后放置在由MN615滤纸圆盘覆盖的新鲜共培养基上。将叶盘放置成使其正轴侧与滤纸接触(因此,上下颠倒),并在无菌条件下与农杆菌细胞共培养4天,在黑暗中21℃。
  8. 在共培养后,将叶圆片转移到补充有头孢噻肟(250mg/L)的SRM中,用于消除土壤杆菌和卡那霉素(100-150mg/L)以选择性再生枝条。在经历具有农杆菌的叶盘过度生长的情况下,可以在转移到SRM之前在含有500mg/L头孢噻肟的溶液中洗涤叶盘。将叶圆片在21℃和16小时/8小时光/暗条件下的生长室中培养
  9. 每4周更新SRM培养基,收获在叶圆片上再生的芽,并转移到含有具有卡那霉素(50mg/L)和头孢噻肟(250mg/L)的枝条繁殖培养基(SPM)的罐中用于进一步选择和繁殖在无菌条件下,在24℃和16h/8h光/暗条件下。在典型的转化实验中,50-100%的叶盘产生转基因苗
  10. 在该培养基上繁殖良好并产生根的枝条转移到相同的培养基中,但现在没有卡那霉素和头孢噻肟,显示不存在土壤杆菌,并且在形成足够的根之后,将植物转移到温室中进一步 评价


注意:所有含抗生素或IBA(对光敏感)的培养基应该新鲜制备。 所有其他媒体最多可存储14天。

  1. 草莓传播的媒介
    1. 拍摄倍增介质(SMM)
      Agar Daishin
      0.5ml BAP-储备液(1mg/ml),终浓度0.5mg/L
      pH = 5.8
    2. 拍摄传播介质(SPM)
      Agar Daishin
      BAP 0.1ml BAP-原液(1mg/ml),终浓度0.1mg/L) IBA 0.1ml IBA-原液(1mg/ml),终浓度0.1mg/L) pH = 5.8
      植物在交替循环中在SMM上培养4-6周,然后在SPM上培养4-6周。 为了转化,使用在SPM培养基上生长4周的植物叶。

  2. 草莓转化的媒体
    1. 芽再生培养基(SRM)(1,000ml)
      4.4 g MS
      4 g gelrite
      加入到900ml MilliQ水中
      0.2ml NAA-原液(1mg/ml)(最终浓度= 0.2mg/L)
      4.5ml TDZ-原液(0.22mg/ml)(最终浓度= 1mg/L)
      用0.1N KOH将pH调节至5.8
    2. 对于SRM + AS(1,000 ml)
    3. 对于SRM SEL(1,000 ml)
      1000μl卡那霉素原液(50mg/ml)(终浓度100mg/L) 2,000μl头孢噻肟 - 原液(125mg/ml)(终浓度250mg/L)
    4. MS-液体+ AS(1,000ml)
      加入到1,000ml MilliQ中
      用0.1N KOH将pH调节至5.2(!)

  3. 库存解决方案
    1. NAA(1mg/ml) 将50mg NAA溶解在1ml 0.1N KOH中,并用MilliQ调节体积至50ml; 储存在4℃下
    2. TDZ(0.22mg/ml) 将55mg TDZ溶于1ml 0.1N KOH中并用MilliQ调节体积至250ml; 储存在4℃下
    3. IBA(1mg/ml) 将50mg IBA溶解在1ml 0.1N KOH中并用MilliQ调节体积至50ml; 储存在4℃下
    4. BAP(1mg/ml) 将50mg BAP溶解在1ml 0.1N KOH中并用MilliQ调节体积至50ml; 储存在4℃下
    5. AS(100mM)
      将98mg乙酰丁香酮溶于300μlDMSO中,用乙醇绝对调节体积至5ml; 储存在4℃下
    6. 头孢噻肟(125mg/ml)
      溶解1000毫克头孢噻肟的8毫升MilliQ; 过滤灭菌; 储存在4℃下
    7. 卡那霉素(50mg/ml)
      将400mg卡那霉素溶解在8ml MilliQ中; 过滤灭菌; 储存在4℃下
    8. 利福平(50mg/ml)
      将400mg利福平溶于100μl0.1N HCl中,用MilliQ调节体积至8ml; 过滤灭菌 储存于-20°C
    9. 葡萄糖(30g/100ml)
      将300g葡萄糖溶解在1,000ml MilliQ中; 过滤灭菌; 在室温下储存在50ml管中


该方案改编自Passey等人(2003)描述的芽再生方法和超强毒农杆菌菌株AGL0(Lazo等人,1991)的使用, 。


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引用:Schaart, J. G. (2014). Agrobacterium-mediated Transformation of Strawberry. Bio-protocol 4(1): e1022. DOI: 10.21769/BioProtoc.1022.