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Generation and Selection of Transgenic Olive Plants
转基因橄榄植物的产生与筛选   

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Abstract

Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation protocol using olive somatic embryos as explants (Torreblanca et al., 2010). Embryogenic cultures derived from radicles of matured zygotic embryos are infected with Agrobacterium tumefaciens, AGL1 strain, containing a binary plasmid with the gene of interest and the nptII selection gene. After a meticulous selection procedure, carried out using solid and liquid media supplemented with paromomycin, the putative transformed lines are established. A preliminary confirmation of their transgenic nature is carried out through PCR amplification. Afterwards, plants can be obtained through an efficient regeneration protocol, whose main characteristics are the use of a low-ionic-strength mineral formulation, a phase in liquid medium for synchronization of cultures and the use of semi-permeable cellulose acetate membranes for embryo maturation (Cerezo et al., 2011). Final confirmation of transgene insertion is carried out through Southern or Northern analysis using leaf samples of regenerated plants.

Keywords: Olea europaea(油橄榄), Genetic transformation(遗传转化), Agrobacterium tumefaciens(根癌农杆菌), Somatic embryogenesis(体细胞胚胎发生)

Background

The protocol developed by Torreblanca et al. (2010) differs from the previous olive transformation protocol, developed by Rugini et al. (2000), in several aspects; mainly, kind of explant, Agrobacterium strain and the selection method used. Rugini et al. (2000) used embryogenic masses as explants, which were incubated in a bacterial suspension of LBA4404 Agrobacterium strain for 48 h. After the infection, the explants were rinsed in water and cultured in embryogenic medium supplemented with 250 mg/L cefotaxime; however, the selection of transgenic embryos was not started until 30 days after the infection, with the addition of 100 mg/L kanamycin. To speed up the process, the explants were transferred to liquid medium in light, and the embryos which turned green were selected and cultured in isolation on solid multiplication medium with 150 mg/L kanamycin. Later on, the plant regeneration process was carried out without kanamycin. In contrast, Torreblanca et al. (2010) used globular somatic embryos as explants and the AGL1 Agrobacterium strain, with an incubation period of only 2 h and 2 days co-culture. Afterwards, the explants were transferred to selection medium with 200 mg/L paromomycin, and re-cultured onto fresh selection medium weekly during the first month and bi-weekly thereafter. In addition, a 3 weeks selection period in liquid medium supplemented with 50 mg/L paromomycin was included. The selection process and the use of somatic embryos as explants solved the problems of chimaeric transgenic embryos appearance, and higher transformation efficiencies were obtained. The protocol for olive plant regeneration published by Cerezo et al. (2011) improved whole plant recovery (shoots and roots) from 1.5% up to 50%. Both protocols together, have allowed the development of a reliable regeneration and transformation procedure in olive, recently used in flower induction studies (Haberman et al., 2017). Indeed, these protocols have been employed to analyze the effect of overexpression of MtFT1 gene in olive.

Materials and Reagents

  1. Biological material
    1. Embryogenic olive cultures, formed by callus and globular embryo structures of yellow-creamed colour
    2. Agrobacterium tumefaciens AGL1 strain harbouring a binary vector, containing the gene of interest and the nptII selection gene

  2. Chemicals and materials
    1. Sterile filter paper cut 10 x 10 cm (Filtros Anois, FILTER-LAB®, catalog number: RM13054252 )
    2. Petri plates (90 cm) (J. D. CATALAN, S. L.)
    3. Mesh, 3 x 3 (ALBUS Suministros de Laboratorio)
    4. Active charcoal (Sigma-Aldrich, catalog number: C9157 )
    5. Assay tubes (25 x 150 mm) (Kimble Chase Life Science and Research Products, catalog number: 73500-20150 )
    6. Dialysis tubing cellulose membrane (Sigma-Aldrich, catalog number: D9777-100FT )
    7. Jiffystrips 5-50 peat pots square, 4.5 x 4.5 cm (Jiffy, catalog number: 110007 )
    8. Plant pots (12.5 and 20 cm)
    9. 1:1 peat moss:perlite substrate (Projar professional)
    10. Agrobacterium liquid growth medium (LB medium) (AppliChem, catalog number: 414753 )
    11. 10 mM magnesium sulphate (MgSO4) (AppliChem, catalog number: 131404 )
    12. Antibiotics:
      1. Paromomycin (Duchefa Biochemie, catalog number: P0141 )
      2. Cefotaxime (PhytoTechnology Laboratories, catalog number: C380 )
      3. Timentin (Duchefa Biochemie, catalog number: 011258 )
    13. ¼ OM (Cañas and Benbadis, 1988) macroelements
    14. ¼ MS (Murashige and Skoog, 1962) microelements
    15. ½ OM Vitamins
    16. Myo-inositol (Sigma-Aldrich, catalog number: I5125 )
    17. Sucrose (D(+)-Saccharose) (VWR, catalog number: 27478.467 )
    18. L-Glutamine (Biowest, catalog number: P1012 )
    19. Casein hydrolysate (N-Z-Amine® A) (Sigma-Aldrich, catalog number: C0626 )
    20. Mannitol (Sigma-Aldrich, catalog number: M9647 )
    21. Plant hormones:
      1. N6-2-Isopentenyladenine (2iP) (Duchefa Biochemie, catalog number: D0934 )
      2. N6-benzyladenine (BA) (Duchefa Biochemie, catalog number: B0904 )
      3. Indole-3-butyric acid (IBA) (Sigma-Aldrich, catalog number: I5386 )
      4. Zeatin riboside (ZR) (Duchefa Biochemie, catalog number: Z0917 )
    22. Olive cyclic embryogenesis medium (ECO) (see Recipes)
    23. Germination medium (see Recipes)
    24. Shoot proliferation medium (see Recipes)
    25. Plant rooting medium (see Recipes)

Equipment

  1. Culture flasks (125 ml) (Nalgene)
  2. Autoclave (JP SELECTA, model: Autester MOD 437-G )
  3. Constant temperature/orbital shaker incubator (Optic Ivymen System)
  4. Laminar flow hood (Telstar, model: BH-100 )
  5. Laboratory centrifuge (Sigma Laborzentrifugen, model: 3K30 )
  6. Spectrophotometer (JP SELECTA, model: UV-2005 )
  7. Walk in plant growth cabinet with controlled light and temperature conditions

Procedure

Note: All this protocol must be conducted under strictly sterile conditions in a laminar flow hood; except B7 step and molecular analysis.

  1. Agrobacterium-mediated transformation of olive embryogenic callus
    1. Obtain an embryogenic olive culture, derived from radicle of mature seed (Orinos and Mitrakos, 1991). Embryogenic friable callus (Figure 1A), containing globular embryos, is maintained and multiplied on ECO medium (see Recipes), transferring to fresh medium at 4-week intervals in darkness at 25 ± 2 °C (Pérez-Barranco et al., 2009).
    2. Grow Agrobacterium tumefaciens AGL1 strain, containing a binary plasmid harbouring the gene of interest and the selection gene nptII (pBINUbiGUSInt as example; containing uidA and nptII genes), in LB medium at 28 °C and 250 rpm for 24 h (with suitable antibiotics for the plasmid used), to obtain a culture of 40 ml at 0.5 OD600 nm. Then, centrifuge the culture at 4,000 x g, wash the pellet with 10 mM MgSO4 without shaking and dilute it in liquid ECO medium, keeping a final 0.5 OD600 nm.
    3. Inoculate globular somatic embryos 1-2 mm diameter, isolated from embryogenic callus, in the diluted Agrobacterium suspension for 20 min under mild agitation; about 20 embryos into 10 ml of Agrobacterium suspension with a total of 80 embryos, approximately (Figure 1B). Then, dry the embryos out on sterile filter paper (Figure 1C).
    4. Co-culture the infected somatic embryos on ECO solid medium for 48 h in darkness, 15-20 embryos per plate (Figure 1D).
    5. Afterwards, wash the somatic embryos with ECO liquid medium supplemented with 250 mg/L cefotaxime and 250 mg/L timentin (Figure 1E). Blot the embryos dry on filter paper (Figure 1F) and transfer them onto selection medium; i.e., ECO solid medium supplemented with 250 mg/L cefotaxime, 250 mg/L timentin and 200 mg/L paromomycin (Figure 1G). Re-culture the explants onto fresh selection medium weekly during the first month and bi-weekly thereafter.
    6. When the explants show proliferation of paromomycin resistant embryogenic callus on selection medium (Figure 1H), after 3 months of culture approximately, transfer those calli individually to 250 ml culture flasks with 40 ml of ECO liquid medium supplemented with 25-50 mg/L paromomycin (Figure 1I). Incubate the suspensions in an orbital shaker at 120 rpm for 3 weeks in darkness.
    7. Afterwards, sieve the suspensions through a 3 x 3 mm screen (Figure 1J) and culture the small globular embryos separately in a plate on ECO selection medium with 200 mg/L paromomycin. After two months, proliferating callus is transferred to test tubes (Figure 1K).
    8. Calli growing on selection medium can be analysed through PCR to obtain a preliminary verification of transformation (Figure 1L).


      Figure 1. Sequence of olive somatic embryos transformation via Agrobacterium tumefaciens. A. Isolated somatic embryos; B. Inoculation of somatic embryos in a diluted Agrobacterium tumefaciens suspension; C. Somatic embryos drying onto sterile paper after inoculation; D. Co-culture of somatic embryos with Agrobacterium on solid medium; E. Somatic embryos washing in liquid medium supplemented with antibiotics; F. Somatic embryos drying onto filter paper after washing; G. Culture of somatic embryos onto selection medium, supplemented with antibiotics; H. Growth of resistant embryogenic callus on selection medium; I. Incubation of the resistant embryogenic callus in liquid selection medium; J. Obtainment of the fine fraction of the embryogenic callus (1-3 mm); K. Growth of putative transgenic callus on solid selection medium for multiplication; L. PCR verification in an electrophoresis gel. Scale bars = 10 mm.

  2. Regeneration of transformed olive plants
    1. Once the transgenic nature of the paromomycin resistant embryogenic calli has been confirmed by PCR, the process of transgenic plants regeneration from these calli can be started, following the protocol described by Cerezo et al. (2011). Firstly, grow each independent embryogenic calli in ECO liquid medium to obtain globular embryos of small size, 1-3 mm, e.g., culture 0.5 g of callus into 50 ml of medium at 120 rpm for 4 weeks in darkness and later filter through a 3 x 3 mm screen (Figures 2A-2C).
    2. Culture the isolated small globular embryos onto ECO maturation medium for a 4 weeks period (without hormones or cefotaxime and supplemented with 1 g/L active charcoal) in Petri dishes. Incubate the embryos in darkness, 20 globular embryos per plate (Figure 2D).
    3. Transfer the embryos directly on top of 4 x 4 cm dialysis tubing cellulose membrane sections lied onto fresh ECO maturation medium and incubate them for another 4 weeks period in darkness (Figure 2E). These membranes are prepared according to the manufacturer’s instructions and sterilized by autoclave.
    4. Afterwards, culture mature embryos at cotyledonary stage in germination medium (see Recipes) under light conditions (16 h photoperiod, 40 µmol m-2 sec-1 irradiance level). (Figure 2F)
    5. Transfer the shoots obtained to medium supplemented with 5.6 μM ZR (Figure 2H) for further proliferation.
    6. To induce rooting, transfer shoots with two or three nodes (2-3 cm) to DKW rooting medium (see Recipes) supplemented with 0.5 μM IBA (Figures 2G-2I).


      Figure 2. Sequence of regeneration of olive transformed plants. A. Growth of transformed embryogenic callus on selection medium; B. Incubation of the transgenic callus in liquid medium; C. Obtaining a fine fraction embryogenic culture (1-3 mm); D. Culture of small transformed isolated embryos onto ECO maturation medium; E. Culture of transformed pre-maturated embryos onto cellulose acetate membrane lied on ECO maturation medium; F. Culture of transformed maturated embryos in germination medium; G. Transformed olive plantlets; H. Transformed plant growing in multiplication medium; I. Transformed olive plant in rooting medium. Scale bars = 10 mm.

    7. Acclimate rooted plants to ex vitro conditions in a greenhouse. Wash roots with distilled water and transfer the plants to a jiffy tray with 1:1 peat moss:perlite substrate and cover the tray with a translucent plastic top. Gradually, adapt the plants to ex vitro conditions by increasing exposure to ambient humidity opening the cover. Transfer the plants to larger pots as they grow (Figures 3A-3D).
    8. Carry out a Southern or Northern analysis to confirm the transgenic nature of regenerated plants.


      Figure 3. Sequence of recovery of transformed olive plants in a greenhouse. A. A well-developed transformed olive plant; B. Transformed olive plants acclimated into a jiffy tray with 1:1 peat moss:perlite substrate; C. Transformed olive plants in 12.5 cm pots; D. Transformed olive plants in 15 cm pots. Scale bars = 25 mm.

Data analysis

At least 50 explants were used per each transformation experiment, 3 replicates of each one. Transformation efficiency was estimated as the percentage of explants growing on selection medium, in the presence of paromomycin, after selection in liquid medium.

Notes

The selection pressure used after the infection is genotype dependent; e.g., the concentration of paromomycin may vary between 150-200 mg/L in solid medium and 25-50 mg/L in liquid medium. In addition, for some genotypes, an increasing selection pressure is advisable, starting with 50 mg/L paromomycin in solid medium and increasing up to 100, 150 and finally, 200 mg/L.

Recipes

Note: The pH of all media has been adjusted to 5.7 using NaOH or HCl.

  1. Olive cyclic embryogenesis medium (ECO) (Pérez-Barranco et al., 2009)
    ¼ OM (Cañas and Benbadis, 1988) macroelements
    ¼ MS (Murashige and Skoog, 1962) microelements
    ½ OM Vitamins
    0.05 g/L myo-inositol
    20 g/L sucrose
    0.550 g/L glutamine
    1 g/L casein hydrolysate
    0.5 µM 2iP
    0.44 µM BA
    0.25 µM IBA
    0.42 µM cefotaxime
  2. Germination medium (Cerezo et al., 2011)
    Modified MS medium
    ⅓ macroelements
    0.1 g/L myo-inositol
    10 g/L sucrose
  3. Shoot proliferation medium (Vidoy et al., 2012)
    Modified RP medium (Roussos and Pontikis, 2002)
    1 g/L myo-inositol
    20 g/L mannitol
    1.2 g/L glutamine
    5.6 µM ZR
  4. Plant rooting medium (Revilla et al., 1996)
    Half-strength DKW medium (Driver and Kuniyuki, 1984) salts with no vitamins or amino acids
    2% sucrose
    0.5 μM IBA

Acknowledgments

This protocol was developed from the following published papers: Torreblanca et al., 2010 and Cerezo et al., 2011. This work was supported by grant P11-AGR-7992-Junta de Andalucía, Spain. The authors have no conflict of interest.

References

  1. Cañas, L. A. and Benbadis, A. (1988). In vitro plant regeneration from cotyledon fragments of the olive tree (Olea europaea L.). Plant Sci 54: 65-74.
  2. Cerezo, S., Mercado, J. A. and Pliego-Alfaro, F. (2011). An efficient regeneration system via somatic embryogenesis in olive. Plant Cell Tiss Organ Cult 106 (2):337-344.
  3. Driver J. A. and Kuniyuki A. H. (1984). In vitro propagation of Paradox Walnut Rootstock. HortScience 19 (4): 507-509.
  4. Haberman, A., Bakhshian, O., Cerezo-Medina, S., Paltiel, J., Adler, C., Ben-Ari, G., Mercado, J.A., Pliego-Alfaro, F., Lavee, S. and Samach, A. (2017). A possible role for flowering locus T-encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction. Plant Cell Environ 40 (8): 1263-1280.
  5. Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473-497.
  6. Orinos, T. and Mitrakos, K. (1991). Rhizogenesis and somatic embryogenesis in calli from wild olive (Olea europaea var. sylvestris (Miller) Lehr) mature zygotic embryos. Plant Cell Tiss Organ Cult 27 (2): 183-187.
  7. Pérez-Barranco, G., Torreblanca R., Padilla, I.M.G., Sánchez-Romero, C., Pliego-Alfaro, F. and Mercado, J. A. (2009). Studies on genetic transformation of olive (Olea europaea L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for nptII selection. II. Transient transformation via particle bombardment. Plant Cell Tiss Organ Cult 97: 243-251.
  8. Revilla, M. A., Pacheco, J., Casares, A. and Rodríguez, R. (1996). In vitro reinvigoration of mature olive trees (Olea europaea L.) through micrografting. In Vitro Cell Dev Biol Plant 32 (4): 257-261.
  9. Roussos, P. A. and Pontikis, C. A. (2002). In vitro propagation of olive (Olea europaea L.) cv. Koroneiki. Plant Growth Regulation 37: 295-304.
  10. Rugini, E., Rita, B. and Rosario, M. (2000). Olive (Olea europaea var. sativa) transformation. In: Jain, S. M. and Minocha, S. C. (Eds.). Molecular Biology of Woody Plants (vol 2). Kluwer Academic Publishers pp: 245-279.
  11. Torreblanca, R., Cerezo, S., Palomo-Ríos, E., Mercado, J. A. and Pliego-Alfaro, F. (2010). Development of a high throughput system for genetic transformation of olive (Olea europaea L.) plants. Plant Cell Tiss Organ Cult 103 (1): 61-69.
  12. Vidoy-Mercado, I., Imbroda-Solano, I., Barceló-Muñoz, A. and Pliego-Alfaro, F. (2012). Differential in vitro behaviour of the Spanish olive (Olea europaea L.) cultivars ‘Arbequina’ and ‘Picual’. Acta Horticulturae 949: 27-30.

简介

橄榄油(Olea europaea L.)是地中海盆地最重要的石油作物之一。生物技术的改善受到橄榄组织在体外再生的顽固性质的阻碍。在以前的研究中,我们小组开发了一个可靠的土壤杆菌介导的转化方案,使用橄榄体细胞胚作为外植体(Torreblanca et al。,2010)。含有具有感兴趣基因和nptII选择基因的二元质粒的根瘤农杆菌(Agrobacterium tumefaciens)AGL1菌株感染源自成熟合子胚胎胚根的胚性培养物。经过细致的选择程序,使用补充巴龙霉素的固体和液体培养基进行,建立推定的转化系。其转基因性质的初步确认是通过PCR扩增进行的。之后,可以通过有效的再生方案获得植物,其主要特征是使用低离子强度的矿物制剂,在液体培养基中用于培养物同步的阶段以及使用半渗透性乙酸纤维素膜进行胚胎成熟(Cerezo et al。 ,2011)。转基因插入的最终确认通过Southern或Northern分析使用再生植物的叶样品进行。

【背景】由Torreblanca等人开发的协议。 (2010)与Rugini等人(2000)开发的先前的橄榄转化协议有所不同,主要是外植体的种类,土壤杆菌菌株及其选择方法。 Rugini等人(2000)使用胚性质量作为外植体,将其在LBA4404农杆菌菌株的细菌悬浮液中温育48小时。感染后,将外植体在水中漂洗并在补充有250mg / L头孢噻肟的胚发生培养基中培养;然而,转基因胚胎的选择直到感染后30天才开始,加入100mg / L卡那霉素。为了加快这一过程,将外植体轻度转移到液体培养基中,选择转绿的胚,在含有150mg / L卡那霉素的固体增殖培养基上分离培养。之后,植物再生过程在没有卡那霉素的情况下进行。相比之下,Torreblanca et al。(2010)使用球形体细胞胚作为外植体和AGL1农杆菌菌株,其培养时间仅为2小时和2天共培养。之后,将外植体转移到具有200mg / L巴龙霉素的选择培养基中,并在第一个月每周和之后每两周重新培养到新鲜的选择培养基上。另外,在补充有50mg / L巴龙霉素的液体培养基中选择3周。体细胞胚的选择过程和使用方法解决了嵌合体转基因胚胎外观的问题,获得了较高的转化效率。由Cerezo等人(2011)发表的橄榄植物再生方案将整株植物恢复(芽和根)从1.5%提高到50%。这两种方案一起使橄榄最近用于花诱导研究(Haberman等人,2017)中开发了可靠的再生和转化程序。事实上,这些方案已经被用来分析橄榄中MtFT1基因过表达的效果。

关键字:油橄榄, 遗传转化, 根癌农杆菌, 体细胞胚胎发生

材料和试剂

  1. 生物材料
    1. 胚芽橄榄培养物,由黄色奶油色的愈伤组织和球状胚胎结构形成
    2. 含有感兴趣基因和nptII选择基因的双元载体的农杆菌AgL1菌株

  2. 化学品和材料
    1. 无菌滤纸切成10 x 10厘米(Filtros Anois,FILTER-LAB ,产品目录号:RM13054252)
    2. 培养皿(90cm)(J.D.CATALAN,S.L。)
    3. 网格,3×3(ALBUS Suministros de Laboratorio)
    4. 活性炭(Sigma-Aldrich,目录号:C9157)
    5. 测定管(25×150mm)(Kimble Chase生命科学和研究产品,目录号:73500-20150)
    6. 透析管纤维素膜(Sigma-Aldrich,目录号:D9777-100FT)
    7. Jiffystrips 5-50泥炭盆平方米,4.5 x 4.5厘米(Jiffy,目录号:110007)
    8. 植物盆(12.5和20厘米)
    9. 1:1泥炭苔:珍珠岩基材(Projar专业)
    10. 液体生长培养基(LB培养基)(AppliChem,目录号:414753)
    11. 10mM硫酸镁(MgSO 4)(AppliChem,目录号:131404)
    12. 抗生素:
      1. 巴龙霉素(Duchefa Biochemie,目录号:P0141)
      2. 头孢噻肟(PhytoTechnology Laboratories,目录号:C380)
      3. 特美汀(Duchefa Biochemie,目录号:011258)
    13. ¼OM(Cañas和Benbadis,1988)macroelements
    14. ¼MS(Murashige和Skoog,1962)微量元素
    15. ½OM维生素
    16. Myo肌醇(Sigma-Aldrich,目录号:I5125)
    17. 蔗糖(D(+) - 蔗糖)(VWR,目录号:27478.467)
    18. L-谷氨酰胺(Biowest,目录号:P1012)
    19. 酪蛋白水解产物(N-Z-胺)(Sigma-Aldrich,目录号:C0626)
    20. 甘露醇(Sigma-Aldrich,目录号:M9647)
    21. 植物激素:
      1. N6-2-异戊烯腺嘌呤(2iP)(Duchefa Biochemie,目录号:D0934)
      2. N6-苄基腺嘌呤(BA)(Duchefa Biochemie,目录号:B0904)
      3. 吲哚-3-丁酸(IBA)(Sigma-Aldrich,目录号:I5386)
      4. 玉米素核苷(ZR)(Duchefa Biochemie,目录号:Z0917)
    22. 橄榄循环胚胎发生培养基(ECO)(见食谱)
    23. 发芽介质(见食谱)
    24. 拍摄增殖介质(见食谱)
    25. 植物生根培养基(见食谱)

设备

  1. 培养瓶(125毫升)(Nalgene)
  2. 高压灭菌器(JP SELECTA,型号:Autester MOD 437-G)
  3. 恒温/轨道摇床培养箱(光学常春藤系统)
  4. 层流罩(Telstar,型号:BH-100)
  5. 实验室离心机(Sigma Laborzentrifugen,型号:3K30)
  6. 分光光度计(JP SELECTA,型号:UV-2005)
  7. 走在植物生长柜内,控制光照和温度条件。

程序

注:所有这些协议必须在严格的无菌条件下在层流罩中进行;除了B7步骤和分子分析。

  1. 农杆菌介导的橄榄胚发生愈伤组织的转化
    1. 获得来自成熟种子胚根的胚发生橄榄培养物(Orinos和Mitrakos,1991)。将含有球形胚的胚胎发生脆性愈伤组织(图1A)维持并在ECO培养基上繁殖(见食谱),在25±2℃的黑暗中以4周的时间间隔转移至新鲜培养基中(Pérez-Barranco等人。,2009)。
    2. 培养根癌农杆菌AgL1菌株,其含有携带目的基因和选择基因的二元质粒(pBINUbiGUSInt作为实例;含有uidA 和在LB培养基中28℃和250rpm下培养24小时(对于使用的质粒,使用合适的抗生素),在0.5OD 600nm处获得40ml的培养物,子>。然后,将培养物在4,000×g离心,用10mM MgSO 4洗涤沉淀物而不摇动,并将其稀释在液体ECO培养基中,保持最终的0.5OD 600 nm 。
    3. 在稀释的土壤杆菌悬浮液中温育搅拌20分钟,从球形胚愈伤组织中分离1-2mm直径的球形体细胞胚;将约20个胚胎移植到约10ml含有总计80个胚胎的农杆菌悬液中(图1B)。然后,在无菌滤纸上干燥胚胎(图1C)。
    4. 在ECO固体培养基上共同培养感染的体细胞胚在黑暗中48小时,每个平板15-20个胚胎(图1D)。
    5. 之后,用补充有250mg / L头孢噻肟和250mg / L特美汀的ECO液体培养基洗涤体胚(图1E)。在滤纸上将胚胎干燥(图1F)并将其转移到选择培养基上;即补充250mg / L头孢噻肟,250mg / L特美汀和200mg / L巴龙霉素的ECO固体培养基(图1G)。在第一个月和第二个星期每周重新培养外植体到新鲜的选择培养基上。
    6. 当外植体在选择培养基上显示巴龙霉素抗性胚发生愈伤组织的增殖(图1H)时,大约培养3个月后,将这些愈伤组织分别转移到250ml培养瓶中,加入40ml ECO液体培养基,补充25-50mg / L巴龙霉素(图1I)。将轨道摇床中的悬浮液在黑暗中以120rpm的速度培养3周。
    7. 之后,通过3×3mm的筛子筛分悬浮液(图1J),并在具有200mg / L巴龙霉素的ECO选择培养基的平板上分别培养小球形胚。两个月后,增殖愈伤组织转移到试管中(图1K)。
    8. 在选择培养基上生长的愈伤组织可以通过PCR进行分析以获得初步的转化验证(图1L)。


      图1.经由根癌农杆菌转化橄榄体细胞胚的序列A.分离的体细胞胚; B.在稀释的根癌土壤杆菌悬浮液中接种体细胞胚胎; C.接种后在无菌纸上干燥体细胞胚; D.体细胞胚与土壤杆菌在固体培养基上的共培养; E.在补充有抗生素的液体培养基中洗涤体细胞胚胎; F.洗涤后将体细胞胚在滤纸上干燥; G.将体细胞胚培养到选择培养基上,补充抗生素; H.选择培养基上抗性胚发生愈伤组织的生长; I.在液体选择培养基中温育抗性胚发生愈伤组织; J.获得胚胎发生愈伤组织的细小部分(1-3毫米); K.在固体选择培养基上增殖推定的转基因愈伤组织以增殖; L.电泳凝胶中的PCR验证。比例尺= 10毫米。

  2. 转化的橄榄植物的再生
    1. 一旦巴龙霉素抗性胚发生愈伤组织的转基因性质已经通过PCR得到证实,按照Cerezo等人描述的方案,可以开始从这些愈伤组织再生转基因植物的过程。 (2011年)。首先,在ECO液体培养基中培养各种独立的胚性愈伤组织,以获得1-3mm大小的球形胚,例如将0.5g愈伤组织培养于50ml培养基中,120rpm,4周黑暗,然后通过3×3毫米的筛网过滤(图2A-2C)。
    2. 将离体的小球状胚胎在培养皿中培养4周(不含激素或头孢噻肟并添加1g / L活性炭)到ECO成熟培养基上。在黑暗中孵育胚胎,每个平板20个球状胚胎(图2D)。
    3. 将胚直接转移到4×4cm透析管顶部的纤维素膜部分上,将其置于新鲜的ECO成熟培养基上,并在黑暗中再孵育4周(图2E)。这些膜根据制造商的说明书制备并且通过高压灭菌器消毒。
    4. 之后,在光照条件下(16h光周期,40μmolm -2 s -1辐照度水平),在发芽培养基的子叶阶段培养成熟胚胎(参见食谱)。 (图2F)
    5. 将获得的芽转移到补充有5.6μMZR(图2H)的培养基中进一步增殖。
    6. 为了诱导生根,将具有2-3个节点(2-3cm)的枝条转移到补充有0.5μMIBA(图2G-2I)的DKW生根培养基(参见食谱)。


      图2.橄榄转化植物的再生序列A.转化的胚性愈伤组织在选择培养基上的生长; B.在液体培养基中培养转基因愈伤组织; C.获得细胚胎发生培养物(1-3毫米); D.将小转化的分离的胚胎培养到ECO成熟培养基上; E.转化的预成熟胚胎到ECO成熟培养基上的醋酸纤维素膜上培养; F.在发芽培养基中培养转化的成熟胚胎; G.转化的橄榄苗; H.转化的植物在增殖培养基中生长; I.在生根培养基中转化橄榄植物。比例尺= 10毫米。

    7. 在温室中使生根的植物适应外部条件。用蒸馏水清洗根部,并将植物转移到1:1泥炭苔:珍珠岩基质上,并用半透明塑料盖覆盖托盘。逐渐地,通过增加暴露于环境湿度以打开盖子来使植物适应<体外条件。
      植物转移到更大的盆栽(图3A-3D)

    8. 进行Southern或Northern分析以确认再生植物的转基因性质

      图3.在温室中回收转化的橄榄植物的序列。 :一种。一个发展良好的转化橄榄植物; B.转化的橄榄植物适应1:1泥炭苔:珍珠岩基质的jiffy托盘; C.在12.5厘米盆中转化的橄榄植物; D.在15厘米盆中转化橄榄植物。比例尺= 25毫米。

数据分析

每个转化试验使用至少50个外植体,每个转化试验3个重复。在液体培养基中选择后,在巴龙霉素的存在下,将转化效率估计为在选择培养基上生长的外植体的百分比。

笔记

感染后使用的选择压力是基因型依赖性的;例如,巴龙霉素的浓度可以在固体培养基中150-200mg / L和液体培养基中25-50mg / L之间变化。此外,对于一些基因型,选择压力增加是可取的,从固体培养基中的50毫克/升巴龙霉素开始,增加到100,150和最终200毫克/升。

食谱

注意:使用NaOH或HCl将所有介质的pH值调整到5.7。

  1. 橄榄循环胚胎发生培养基(ECO)(Pérez-Barranco等人,2009)
    ¼OM(Cañas和Benbadis,1988)macroelements
    ¼MS(Murashige和Skoog,1962)微量元素
    ½OM维生素
    0.05克/升肌醇 - 肌醇
    20克/升蔗糖
    0.550克/升谷氨酰胺
    1克/升酪蛋白水解物
    0.5μM2iP
    0.44μMBA
    0.25μMIBA
    0.42μM头孢噻肟
  2. 萌发培养基(Cerezo et al。 ,2011)
    修改MS媒体
    ⅓宏元素
    0.1克/升myo-inositol
    10克/升蔗糖
  3. 拍摄增殖介质(Vidoy et。,2012)
    改良的RP介质(Roussos和Pontikis,2002)
    1克/升myo -inositol
    20克/升甘露醇
    1.2克/升谷氨酰胺
    5.6μMZR
  4. 植物生根培养基(Revilla等人,1996)
    半强度DKW培养基(Driver and Kuniyuki,1984)盐类,不含维生素或氨基酸。
    2%蔗糖
    0.5μMIBA

致谢

该协议是从以下发表的论文中发展出来的:2011年的Torreblanca等人和Cerezo等人。2011年的这项工作得到了P11-AGR-7992-军政府安达卢西亚,西班牙。作者没有利益冲突。

参考

  1. Cañas,L.A。和Benbadis,A。(1988)。 橄榄树子叶碎片( > Olea europaea L.)植物科学 54:65-74。
  2. Cerezo,S.,Mercado,J.A。和Pliego-Alfaro,F。(2011)。 通过橄榄体细胞胚胎发生的高效再生系统
    。植物细胞Tiss器官邪教 106(2):337-344
  3. Driver J.A.和Kuniyuki A.H。(1984)。 Paradox Walnut 砧木的体外繁殖 > HortScience 19(4):507-509。
  4. Haberman,A.,Bakhshian,O.,Cerezo-Medina,S.,Paltiel,J.,Adler,C.,Ben-Ari,G.,Mercado,JA,Pliego-Alfaro,F.,Lavee,S。和Samach,A.(2017)。 开花位点T编码基因在解释影响橄榄的环境和内部线索中的可能作用> Olea europaea L.)花诱导。 Plant Cell Environ 40(8):1263-1280。
  5. Murashige T.和Skoog F.(1962)。 烟草组织培养快速生长和生物测定的修正培养基。 Physiol Plant 15:473-497。
  6. Orinos,T.和Mitrakos,K.(1991)。 野生橄榄愈伤组织的根际发生和体细胞胚胎发生( Olea europaea var。sylvestris( Miller)Lehr)成熟的合子胚胎。植物细胞Tiss器官Cult 27(2):183-187。
  7. Pérez-Barranco,G.,Torreblanca R.,Padilla,I.M.G.,Sánchez-Romero,C.,Pliego-Alfaro,F.和Mercado,J.A。(2009)。橄榄(橄榄油橄榄)体细胞胚胎的遗传转化研究:一,评价(1)橄榄油(橄榄油橄榄油)不同的氨基糖苷类抗生素用于选择 nptII 。 II。通过粒子轰击的瞬时转化。植物细胞Tiss器官邪教 97:243-251。
  8. Revilla,M.A.,Pacheco,J.,Casares,A。和Rodríguez,R.(1996)。 体外成熟橄榄树的恢复活力( Olea europaea L.)通过微接枝移植。体外细胞开发生物植物 32(4):257-261。
  9. Roussos,P.A。和Pontikis,C.A。(2002)。 橄榄的体外传播( Olea europaea L。)cv。 Koroneiki。 植物生长调节 37:295-304。
  10. Rugini,E.,Rita,B.和Rosario,M。(2000)。 Olive( Olea europaea
  11. Torreblanca,R.,Cerezo,S.,Palomo-Ríos,E.,Mercado,J.A。和Pliego-Alfaro,F.(2010)。 开发用于橄榄遗传转化的高通量系统(Olea europaea L.植物植物细胞Tiss器官崇拜103(1):61-69。
  12. Vidoy-Mercado,I.,Imbroda-Solano,I.,Barceló-Muñoz,A.和Pliego-Alfaro,F.(2012)。西班牙橄榄(体外行为) em> Olea europaea L.)品种'Arbequina'和'Picual'。 Acta Horticulturae 949:27-30。
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引用:Palomo-Ríos, E., Cerezo, S., Mercado, J. A. and Pliego-Alfaro, F. (2017). Generation and Selection of Transgenic Olive Plants. Bio-protocol 7(22): e2611. DOI: 10.21769/BioProtoc.2611.
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