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Development and Implementation of an in vitro Culture System for Intact Detached Grape Berries
完整分离的葡萄果实体外培养系统的开发与实现   

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Abstract

Grape composition depends on the metabolites accumulated and synthesized during grape development. It is of paramount importance for grape growers because of its major role in shaping wine quality. Therefore, understanding the regulation mechanisms that control the accumulation of quality-related metabolites in grape is of both scientific and agronomical interests. The composition of grape berry at harvest is under complex regulation and can be affected by many factors (Conde et al., 2007). The study of the effects of these factors on berries still attached to intact plants can be highly challenging because of the large size of the plants, interplant, intercluster and interberry variability; and because it is complicated to precisely control the nutrients and hormones imported by the berries, and the environment. Therefore, in vitro cultured grape berries are a good model system, which better represents berry anatomy structure (skin and flesh) than grape cell suspensions and nevertheless largely reduces the system complexity compared to whole plant (Bravdo et al., 1990; Pérez et al., 2000; Gambetta et al., 2010). To this end, an in vitro culture system of intact detached grape berries has been developed by coupling greenhouse fruiting-cuttings production and in vitro organ culture techniques (Dai et al., 2014). The cultured berries are able to actively absorb and utilize carbon and nitrogen from the culture medium, and exhibit fruit ripening features such as color changing and softening. This in vitro system may serve to investigate the response of berry composition to environmental and nutrient factors.

Keywords: Vitis(葡萄), Grape quality(葡萄质量), In vitro(体外), Fruit quality(果实品质), Fleshy fruit(fleshy水果)

Materials and Reagents

  1. Grapevine berries from greenhouse-grown fruiting-cuttings of Vitis vinifera L. cv. Cabernet Sauvignon at various developmental stages (e.g. pea size, green berry, veraison, or later stages). The fruiting-cuttings (i.e. only one primary shoot axis with a single cluster per plant) were prepared as described in Mullins and Rajasekaran (1981) and grown in a naturally illuminated and semi-regulated greenhouse (mean seasonal temperature amplitude 20-35 °C) with fungicide treatments every two weeks (Figure 1).
    Note: Efficient fungicide treatments are essential to maximally exclude pathogen infection to grape berries and minimize the sterilization step for the in vitro culture. For example, we continuously provide sulfur contained in sulfur diffusion hotbox (Nivola Sulphur fl 220 v 3 m, Figure 1B) in the greenhouse and treated the vines with armicarb® at 5 g/L every two weeks.


    Figure 1. Fruit-bearing cuttings grown in a semi-controlled greenhouse (A) and the sulfur diffusion hotbox (B)

  2. 70% ethanol
  3. NaClO with available chlorine 2% (Sigma-Aldrich, catalog number: 425044-1L )
  4. MS (Murashige & Skoog) medium (Duchefa Biochemie, catalog number: M 0221 )
  5. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  6. N-Z-Amine A (Sigma-Aldrich, catalog number: C7290 )
  7. Vitamines
    Myo-inositol 100 mg/L (Sigma-Aldrich, catalog number: I5125 )
    Nicotinic acid 1 mg/L (Sigma-Aldrich, catalog number: N0765 )
    Pantothenic acid 1 mg/L (Duchefa Biochemie, catalog number: C0604 )
    Biotin 0.01 mg/L (Sigma-Aldrich, catalog number: B4639 )
    Pyridoxine HCl 1 mg/L (Sigma-Aldrich, catalog number: P9755 )
    Thiamine HCl 1 mg/L (Sigma-Aldrich, catalog number: T4625 )
  8. 0.5 M NaOH
  9. Agar (Kalys, catalog number: HP 696 )
  10. EDTA (Sigma-Aldrich, catalog number: E5134 )
  11. Sterile water
  12. MS medium with sucrose and vitamins (see Recipes)
  13. 200x vitamin (see Recipes)
  14. 20 mM EDTA (see Recipes)

Equipment

  1. Laminar flow cabinet (Steril-Helios, Figure 2A)
  2. Shaker (Dragon lab, sk-330-pro, Figure 2A)
  3. Autoclave machine
  4. Growth room with constant temperature of 26 ± 0.5 °C, light period 16 h/8 h day/night, and light ~50 μmol m-2 s-1 (Figure 4H)
  5. Sterilized 6-well plates (Dutscher, catalog number: 353046 , Figure 5B)
  6. Sterilized plastic boxes with filter (Dutscher, catalog number: E 1650 , Figures 3F, 4G, and 5A)
  7. Tip plate (Figure 3A)
  8. Polystyrene (Figure 3C)
  9. Culture dish (145/20 mm, Greiner Bio-One GmbH, Figure 4E)
  10. Sterilization solution container (Figure 2C-D)
  11. Colander, forceps, scissors, and blade (Figure 2B)


    Figure 2. Equipment used in grape berry culture. A. Laminar flow cabinet with a shaker. B. colander, forceps, scissors, and blade. C. solution container, culture boxes, and small materials just after autoclave. D. Sterilized containers for ethanol and NaClO.

Procedure

  1. Preparation of the floater for liquid medium
    1. Collect tip plates and cut them into suitable size for the culture box (Figure 3A and B).
    2. Prepare polystyrene cuboids corresponding to the size of tip plates (Figure 3C).
    3. Put the polystyrene cuboids on the two sides of the tip plates to form a floater (Figure 3D-F).
    4. Sterilize floaters with 10% NaClO and 90% ethanol during 20 min each following three rinses with sterile water and then install them in the tissue culture box containing liquid medium.


      Figure 3. Preparation of the floater for liquid medium. A. Boxes of micropipette tips. B. Cut tip plates to a suitable size for the culture box. C. Prepare two polystyrene cuboids. D. Assemble polystyrene cuboids with tip plate to form the floater. E. Vertical view of the floater inside of a culture box. F. Side view of the floater inside of a culture box.

  2. Berry in vitro culture
    1. Fruit-bearing cuttings were selected and brought to culture room after removing all leaves (Figure 4A).
    2. Grape clusters were excised from the mother plant, and berries were subsequently excised from peduncle with berry pedicel (about 3 mm) and dropped immediately into tap water (Figure 4B).
    3. Keep tap water running for 15 min to clean the berries.
    4. Put berries into 70% ethanol for 2 sec and transfer immediately into NaClO with 2% available chlorine with a sterilized colander (Figure 4D).
      Note: The volume ratio between berry and sterilization solution is important for an efficient sterilization, and a minimum ratio 1:3 (berry/solution) is recommended.
    5. Shaking the container of NaClO with a shaker at 350-450 rpm for 2 min (Figure 4C).
      Note: Dependent on the status of the greenhouse-grown berries, this step can be extended to 3-5 min. Longer sterilization will hurt berry skin.
    6. Transfer berries into sterilized de-ionized water with colander (Figure 4D) and shake at 350-450 rpm for 2 min.
    7. Repeat step B6 three times.
    8. Transfer fully rinsed berries into 20 mM EDTA solution, which is contained in a big culture dish (Figure 4E).
    9. Cut berry pedicel again to about 2 mm under EDTA solution (Figure 4E), in order to exclude cavitations and to prevent plugging of sieve tubes by callose synthase, a strictly calcium-dependent enzyme.
    10. Gently open the culture boxes and quickly put the berries on the solid or liquid culture medium with berry pedicel rooting into culture medium (see Recipes, Figure 4F and Figure 5).
    11. Gently put the cover of culture box (Figure 4G) and transport all boxes to culture room with constant temperature of 26 ± 0.5 °C, light period 16 h/8 h day/night, and light ~50 μmol m-2 s-1 (Figure 4H).


      Figure 4. Berry in vitro culture procedure. A. Bring fruit-bearing cuttings to the lab after removing all leaves. B. Excise berries from grape cluster and drop them immediately into tap water. C. Sterilize berries with ethanol and bleach solutions with a shaker. D. Transform the sterilized berries to water for rinsing. E. Recut berry pedicel in 20 mM EDTA. F. Install berries into culture medium. G. Put the cover of tissue culture box. H. Install tissue culture boxes into culture room.
    12. Alternative culture methods depending on the desired experiment might be chosen (Figure 5).
    13. The berry culture can be maintained as long as 3 months. Since the medium volume is much greater than the berries, there is no need for change of medium. However, it will be very easy to change the medium composition of liquid medium with syringe injection.
      Note: Berries are not very tightly fixed on the culture medium, so any movement and transport should be done gently to avoid berry slanting.


      Figure 5. Different types of berry in vitro culture. A. Culture with liquid medium and floater. B. Culture with solid medium in a 6-well plate. C. Culture with liquid medium and floater in a small container.

Recipes

  1. MS medium with sucrose and vitamins
    4.3 g M0221
    20 g sucrose
    0.25 g N-Z-AmineA
    Dissolve in 800 ml deionized water with vortex
    5 ml vitamin 200x (see below)
    Adjust pH to 5.8 with 0.5 M NaOH
    Complete to 1 L with deionized water
    Add 9 g agar for solid medium
    Autoclave (120 °C, 20min)
    For medium containing agar, pour 4 ml medium to each well of the 6-well plate (Figure 5B); or 50 ml medium to the tissue culture box with special filters allowing gas exchange (Figure 4G), before medium concretion and inside of the laminar flow cabinet.
    For liquid medium, pour 150 ml medium the tissue culture box with special filters (Figure 5A).
    Note: A minimum 2% of sucrose is needed to prevent berry crack for green Cabernet Sauvignon berries. Different cultivars may have different sensitivity and therefore a suitable sucrose concentration has to be established for other cultivars.
  2. 200x vitamin
    Dissolve 8 mg biotin into 1 ml de-ionized water with one drop of KOH
    25 µl biotin solution
    2 g myo-inositol
    20 mg nicotinic acid
    20 mg pantothenic acid
    20 mg pyridoxine HCl
    20 mg thiamine HCl
    Dissolve and complete to 100 ml with deionized water
    Stored at -20 °C
  3. 20 mM EDTA
    Dissolve 7.44 g EDTA in 1 L de-ionized water
    Autoclave (120 °C, 20 min)
    Stored at room temperature

Acknowledgments

This work was supported by a specific grant from the Environment and Agronomy division (EA) of the Institut National de la Recherche Agronomique (INRA), France. This protocol was developed based on our previous paper (Dai et al., 2014).

References

  1. Bravdo, B., Shoseyov, O., Ikan, R. and Altman, A. (1990). Monoterpene glycoside biosynthesis in detached grape berries grown in vitro. Physiol Plant 78(1): 93-99.
  2. Conde, C., Silva, P., Fontes, N., Dias, A. C. P., Tavares, R. M., Sousa, M. J., Agasse, A., Delrot, S. and Gerós, H. (2007). Biochemical changes throughout grape berry development and fruit and wine quality. Food 1, 1-22.
  3. Dai, Z. W., Meddar, M., Renaud, C., Merlin, I., Hilbert, G., Delrot, S. and Gomes, E. (2014). Long-term in vitro culture of grape berries and its application to assess the effects of sugar supply on anthocyanin accumulation. J Exp Bot 65(16): 4665-4677.
  4. Gambetta, G. A., Matthews, M. A., Shaghasi, T. H., McElrone, A. J. and Castellarin, S. D. (2010). Sugar and abscisic acid signaling orthologs are activated at the onset of ripening in grape. Planta 232(1): 219-234.
  5. Mullins, M. G., and Rajasekaran, K. (1981). Fruiting cuttings: revised method for producing test plants of grapevine cultivars. Am J Enol Vitic, 32: 35-40.
  6. Pérez, F. J., Meza, P., Berti, M. and Pinto, M. (2000). Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro. Plant Cell Tiss Org 61(1): 37-40.

简介

葡萄组成取决于葡萄发育过程中积累和合成的代谢物。这对于葡萄种植者是至关重要的,因为它在塑造葡萄酒品质方面起着重要作用。因此,理解控制质量相关代谢物在葡萄中的积累的调节机制具有科学和农业利益。收获时葡萄浆的组成受到复杂调节,并且可能受到许多因素的影响(Conde等人,2007)。这些因素对仍然附着于完整植物的浆果的影响的研究可能是高度挑战性的,因为植物的大尺寸,植物间,群集间和互花间变异性;并且因为精确地控制由浆果和环境进入的营养物和激素是复杂的。因此,体外培养的葡萄浆果是一种良好的模型系统,其比葡萄细胞悬浮液更好地代表浆果解剖结构(皮肤和肉),并且与整株植物相比大大降低了系统复杂性(Bravdo et al。,1990;Pérezet al。,2000; Gambetta et al。,2010)。为此,通过将温室果枝切割生产和体外器官培养技术相结合,开发了完整脱离的葡萄浆果的体外培养系统(Dai等人al。,2014)。培养的浆果能够积极地吸收和利用来自培养基的碳和氮,并且表现出果实成熟特征,例如变色和软化。这种体外系统可用于研究浆果组合物对环境和营养因素的响应。

关键字:葡萄, 葡萄质量, 体外, 果实品质, fleshy水果

材料和试剂

  1. 来自温室栽培果实的葡萄树浆果。赤霞珠在各种发育阶段(例如豌豆大小,绿色浆果,veraison或后期阶段)。如Mullins和Rajasekaran(1981)中所述制备果实切割物(即每个植物仅具有单个簇的一个主轴线),并在自然光照和半受控温室中生长(平均季节温度幅度为20-35°C),每两周进行杀真菌剂处理(图1) 注意:有效的杀真菌剂处理是最大限度地排除病原体感染到葡萄浆果和最小化体外培养的灭菌步骤所必需的。例如,我们在温室中连续提供硫扩散热箱(Nivola Sulfur fl 220 v 3m,图1B)中包含的硫,并且每两周以5g/L用armicarb处理葡萄树。


    图1.在半控制温室(A)和硫扩散热箱(B)中生长的水果切割

  2. 70%乙醇
  3. NaClO,有效氯2%(Sigma-Aldrich,目录号:425044-1L)
  4. MS(Murashige& Skoog)培养基(Duchefa Biochemie,目录号:M 0221)
  5. 蔗糖(Duchefa Biochemie,目录号:S0809)
  6. N-Z-胺A(Sigma-Aldrich,目录号:C7290)
  7. 维生素
    肌醇100mg/L(Sigma-Aldrich,目录号:I5125) 烟酸1mg/L(Sigma-Aldrich,目录号:N0765)
    泛酸1mg/L(Duchefa Biochemie,目录号:C0604)
    生物素0.01mg/L(Sigma-Aldrich,目录号:B4639) 盐酸吡哆醇1mg/L(Sigma-Aldrich,目录号:P9755)
    盐酸硫胺1mg/L(Sigma-Aldrich,目录号:T4625)
    NaClO,有效氯2%(Sigma-Aldrich,目录号:425044-1L)
  8. MS(Murashige& Skoog)培养基(Duchefa Biochemie,目录号:M 0221)
  9. 蔗糖(Duchefa Biochemie,目录号:S0809)
  10. N-Z-胺A(Sigma-Aldrich,目录号:C7290)
  11. 维生素
    肌醇100mg/L(Sigma-Aldrich,目录号:I5125) 烟酸1mg/L(Sigma-Aldrich,目录号:N0765)
    泛酸1mg/L(Duchefa Biochemie,目录号:C0604)
    生物素0.01mg/L(Sigma-Aldrich,目录号:B4639) 盐酸吡哆醇1mg/L(Sigma-Aldrich,目录号:P9755)
    盐酸硫胺1mg/L(Sigma-Aldrich,目录号:T4625)
    ...
  12. 20 mM EDTA (see Recipes)

Equipment

  1. Laminar flow cabinet (Steril-Helios, Figure 2A)
  2. Shaker (Dragon lab, sk-330-pro, Figure 2A)
  3. Autoclave machine
  4. Growth room with constant temperature of 26 ± 0.5 °C, light period 16 h/8 h day/night, and light ~50 μmol m-2 s-1 (Figure 4H)
  5. Sterilized 6-well plates (Dutscher, catalog number: 353046, Figure 5B)
  6. Sterilized plastic boxes with filter (Dutscher, catalog number: E 1650, Figures 3F, 4G, and 5A)
  7. Tip plate (Figure 3A)
  8. Polystyrene (Figure 3C)
  9. Culture dish (145/20 mm, Greiner Bio-One GmbH, Figure 4E)
  10. Sterilization solution container (Figure 2C-D)
  11. 滤嘴,钳子,剪刀和刀片(图2B)


    图2.用于葡萄浆果培养的设备 A.具有振动器的层流柜。 B.漏勺,镊子,剪刀和刀片。 C.溶液容器,培养箱和刚刚高压灭菌后的小材料。 D.用于乙醇和NaClO的灭菌容器

程序

  1. 液体介质的浮子的制备
    1. 收集端板并将其切成适合培养箱的尺寸(图3A和B)
    2. 准备对应于尖端板尺寸的聚苯乙烯长方体(图3C)
    3. 将聚苯乙烯长方体放在端板的两侧形成浮子(图3D-F)
    4. 用10%NaClO和90%乙醇灭菌漂浮物,每次20分钟   然后用无菌水冲洗三次,然后将其安装在 组织培养箱含有液体培养基。


      图3.准备 的液体介质的浮子。 A.盒的微量移液器吸头。 B.切割 尖端板到用于培养箱的合适尺寸。 准备两个 聚苯乙烯立方体。 D.用尖头板装配聚苯乙烯长方体 形成浮动。 浮游物的垂直的看法在文化里面的 框。 浮子的侧视图里面一个文化箱子。

  2. Berry体外培养
    1. 选择带有水果的插条,并在取出所有叶子后送到培养室(图4A)
    2. 从母本切下葡萄簇,并且浆果 随后从具有浆果花梗(约3mm)的花梗上切除 立即滴入自来水(图4B)。
    3. 保持自来水运行15分钟以清洁浆果。
    4. 将浆果放入70%乙醇中2秒,立即转入   NaClO与2%有效氯与无菌漏勺(图 4D)。
      注意:浆果和灭菌溶液之间的体积比   对于有效的灭菌是重要的,并且最小比例为1:3 (浆果/溶液)。
    5. 用振荡器以350-450rpm摇动容器NaClO 2分钟(图4C) 注意:取决于温室种植的浆果的状态,这 步骤可以延长3-5分钟。 更长的灭菌会伤害浆果 皮肤。
    6. 将浆果转移到带有漏斗的灭菌去离子水中(图4D),并以350-450rpm摇动2分钟。
    7. 重复步骤B6三次。
    8. 将完全漂洗的浆液转移到20mM EDTA溶液中,其包含在大培养皿中(图4E)
    9. 在EDTA溶液下再次将浆果花梗切成约2mm 4E),以排除空穴并防止筛子堵塞 管由胼lose质合酶,一种严格的钙依赖性酶
    10. 轻轻打开培养箱,迅速将浆果放在固体上 或液体培养基与浆果花生根培养基 (参见食谱,图4F和图5)
    11. 轻轻把盖子 培养箱(图4G),并将所有箱运输到培养室 恒温26±0.5℃,光照16小时/8小时昼/夜, 和光〜50μmolm -2 -2 -1 (图4H)。

      图4.浆果体外培养程序。A.将带有水果的切片带到实验室后 去除所有叶子。 B.从葡萄簇中取出浆果并将其丢弃 立即进入自来水。 C.用乙醇和漂白剂消毒浆果   解决方案。 D.将消毒的浆果转化为水 用于冲洗。 E.在20mM EDTA中的浆果花梗。 F.安装浆果 进入培养基。 G.把组织培养箱的盖子。 H.安装 组织培养箱进入培养室
    12. 可以选择取决于所需实验的替代培养方法(图5)。
    13. 浆果培养可以保持长达3个月。 自从 中等体积比浆果大得多,没有必要 改变培养基。 然而,改变介质将是非常容易的 注射器注射液体介质的组成 注意:浆果 不是非常紧密地固定在培养基上,所以任何运动和 运输应该轻轻地做,以避免浆果倾斜。


      图5。 不同类型的浆果在体外培养。 A.用液体培养基培养   和浮动。 B.在6孔板中用固体培养基培养。 C.文化   与液体介质和浮子在一个小容器。

食谱

  1. MS培养基与蔗糖和维生素 4.3 g M0221
    20克蔗糖 0.25克N-Z-胺A/B 用涡流
    溶解在800ml去离子水中 5毫升维生素200x(见下文)
    用0.5 M NaOH将pH调节至5.8 用去离子水完成至1 L / 加入9克固体培养基的琼脂
    高压灭菌(120°C,20分钟)
    对于含有琼脂的培养基,将4ml培养基倒入6孔板的每个孔中(图5B);或50ml培养基与具有允许气体交换的特殊过滤器(图4G)一起,在培养基凝结之前和层流室内部。 对于液体培养基,用特殊过滤器倒入150ml培养基的组织培养箱(图5A) 注意:需要至少2%的蔗糖来防止对于绿色赤霞珠浆果的浆果裂解。不同的品种可能有不同的敏感性,因此必须为其他品种建立合适的蔗糖浓度。
  2. 200x维生素
    用1滴KOH将8mg生物素溶解在1ml去离子水中 25μl生物素溶液
    2 g肌醇-6 20mg烟酸
    20毫克泛酸
    20mg吡哆辛HCl
    20mg盐酸硫胺素 用去离子水溶解并加至100ml 储存于-20°C
  3. 20 mM EDTA
    将7.44g EDTA溶解在1L去离子水中
    高压灭菌(120℃,20分钟)
    在室温下贮存

致谢

这项工作得到了来自法国国家农业研究所(INRA)环境和农学司(EA)的具体拨款的支持。 该协议是基于我们以前的论文(Dai等人,2014)开发的。

参考文献

  1. Bravdo,B.,Shoseyov,O.,Ikan,R。和Altman,A。(1990)。 单独的葡萄浆果中的单萜糖苷生物合成在体外培养。 Physiol Plant 78(1):93-99。
  2. Conde,C.,Silva,P.,Fontes,N.,Dias,A.C.P.,Tavares,R.M.,Sousa,M.J.,Agasse,A.,Delrot,S.andGerós, 整个葡萄浆果的生化变化,水果和葡萄酒的质量。 /em> 1,1-22。
  3. Dai,Z.W.,Meddar,M.,Renaud,C.,Merlin,I.,Hilbert,G.,Delrot,S.and Gomes,E。(2014)。 长期的体外培养葡萄浆果的培养及其应用评估糖供应对花色素苷积累的影响。 J Exp Bot 65(16):4665-4677。
  4. Gambetta,G.A.,Matthews,M.A.,Shaghasi,T.H.,McElrone,A.J.and Castellarin,S.D。(2010)。 糖和脱落酸信号直向同源物在葡萄成熟开始时被激活。 em> Planta 232(1):219-234。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dai, Z., Meddar, M., Delrot, S. and Gomès, E. (2015). Development and Implementation of an in vitro Culture System for Intact Detached Grape Berries. Bio-protocol 5(12): e1510. DOI: 10.21769/BioProtoc.1510.
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