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Gentiobiose Feeding in Gentian in vitro Overwintering Buds or Plantlets
在龙胆越冬芽及越冬苗上体外施加龙胆二糖   

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Abstract

To study the functions of sugars in plants, feeding experiment is one of the most common and easy methods. However, the traditional method, e.g., a floating of leaf discs on sugar-containing solution seems to have an insufficient efficiency of sugar uptake, despite of high osmotic and injury effects. This is a protocol to feed oligosaccharide gentiobiose into in vitro cultured tissues of gentian. This protocol enables to incorporate gentiobiose into intact tissues without exposure to osmotic stress and may be useful to other plant species that are able to propagate by shoot tip culture.

Keywords: Gentian(龙胆), Gentiobiose(龙胆), Overwintering bud(越冬芽), Plantlet(苗), Budbreak(萌芽)

Materials and Reagents

  1. Gentian (Gentiana triflora) tissue culture plantlets
  2. Sucrose
  3. Gentiobiose (Sigma-Aldrich, catalog number: G3000-5G )
  4. Gellan gum (Wako USA, catalog number: 075-03075 )
  5. Liquid nitrogen
  6. Milli Q grade water
  7. MS vitamins (see Recipes)
  8. Propagation medium (see Recipes)
  9. IOWB induction medium (see Recipes)
  10. Gentiobiose medium (see Recipes)

Equipment

  1. Sterile magenta boxes (sterilized by autoclaving)
  2. Surgical tape (3M, catalog number: 1530-0 )
  3. Glass culture tubes (sterilized by autoclaving)
  4. Silicon plugs (sterilized by autoclaving)
  5. Sterile 15 ml plastic tubes (such as Greiner Bio-One GmbH, catalog number: 188271 )
  6. Sterile syringe filter (Millipore, catalog number: SLGP033RS )
  7. Sterile petri dishes (IWAKI PUMPS, catalog number: SH90-20 )
  8. Cutoff filter (Millipore, catalog number: UFC5003BK )
  9. Growth chambers
  10. Clean bench
  11. Scalpel
  12. Forceps
  13. Ball miller
  14. Centrifuge
  15. Freeze dryer

Procedure

  1. Preparation and gentiobiose feeding of gentian IOWB and plantlets
    1. In vitro shoot cultures of gentian were prepared according to the method of Hosokawa et al. (1996).
    2. Transfer shoot tips (approximately 1 cm) to magenta boxes containing 70 ml of propagation medium and culture at 20 °C for 1 to 2 months under a 16/8 h light/dark photoperiod (Figure 1A).
    3. Transfer 3 to 5 shoot tips (approximately 1 cm) to a magenta box containing 70 ml of IOWB induction medium (Figure 1B) and culture at 20 °C for over 6 months under a 16/8 h light/dark photoperiod (Figure 1C).
    4. Harvest the produced IOWBs and cut into approximately 2 cm length pieces using a scalpel and forceps on a plastic petri dish (Figure 2A).


      Figure 1. IOWBs induction from gentian plantlets. A. Cutting shoot tips from plantlet. B. Shoot tips placed on IOWB induction medium. C. IOWBs produced from gentian plantlets cultured for 6 months on IOWB induction medium. Arrows indicate IOWBs. Bar: 2 cm

    5. Transfer IOWBs or plantlets to glass culture tubes containing 2 ml of a gentiobiose medium and seal the top of the tubes with silicon plugs (Figure 2B).


      Figure 2. Gentiobiose feeding into gentian IOWB and plantlet. A. Harvest of produced IOWB. B. Placement of IOWB and shoot tip on gentiobiose medium. C. IOWB cultured on gentiobiose medium. D. Shoot tip cultured on gentiobiose medium. Bar: 1 cm

    6. Culture at 20 °C under a 16/8 h light/dark photoperiod for appropriate period (Figure 2 C-D).
    7. Harvest parts of the samples not in contact with the medium.

  2. Detection of gentiobiose in IOWBs or plantlets
    1. Freeze-dry and pulverize IOWBs or plantlets in a ball-miller.
    2. Homogenize 10 mg dry weight of samples with 500 μl of 50% (v/v) methanol.
    3. Centrifuge the homogenates at 20,000 x g for 5 min and filtrate the supernatants through a cutoff filter.
    4. Subject the filtrates to thin layer chromatography (Figure 3) or other analyses to detect gentiobiose.


      Figure 3. Example analysis of gentiobiose incorporated in gentian plantlets. Extracts from leaves of plantlets cultured on normal MS medium (Control) or MS medium containing 0.05% gentiobiose labeled with rhodamine (Treated) for 3 days were subjected to TLC analysis. STD, 0.01% gentiobiose labeled with rhodamine.

Notes

  1. Propagation and feeding steps should be aseptically performed using a clean bench.
  2. Labeled gentiobiose should be used only for confirmation of incorporated gentiobiose.
  3. For details about gentian IOWBs and plantlets, see Imamura et al. (2014).
  4. For details about TLC analysis, see Takahashi et al. (2014).
  5. For gentiobiose labeling, see the following publications, Hase et al. (1978) and Fry (1997).

Recipes

  1. MS vitamins (100 ml)
    10 g myo-inositol
    50 mg nicotinic acid
    50 mg pyridxine hydrochloride
    10 mg thiamine hydrochloride
    200 mg glycine
  2. Propagation medium (1L)
    4.6 g MS salt
    1 ml MS vitamins
    30 g sucrose
    2 g gellan gum
    Adjust pH 5.7 with KOH and autoclave for 15 min
  3. IOWB induction medium (1L)
    4.6 g MS salt
    1 ml MS vitamins
    60 g sucrose
    2 g gellan gum
    Adjust pH 5.7 with KOH and autoclave for 15 min
  4. Gentiobiose medium (1L)
    4.6 g MS salt
    1 ml MS vitamins
    10 g gentiobiose or labeled gentiobiose*
    2 g gellan gum
    Adjust pH 5.7 with KOH and autoclave for 15 min
    *Add to autoclaved MS medium after decrease in temperature to approximately 60 °C
    *Labeled gentiobiose is used for confirmation of incorporated gentiobiose

Acknowledgments

This protocol is adapted from the following publications, Imamura et al. (2014) and Takahashi et al. (2014). This research was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology.

References

  1. Fry, S. C. (1997). Novel ‘dot-blot’ assays for glycosyltransferases and glycosylhydrolases: optimization for xyloglucan endotransglycosylase (XET) activity. Plant J 11(5): 1141-1150.
  2. Hase, S., Ikenaka, T. and Matsushima, Y. (1978). Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem Biophys Res Commun 85(1): 257-263.
  3. Hosokawa, K., Nakano, M., Oikawa, Y. and Yamamura, S. (1996). Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana. Plant Cell Rep 15(8): 578-581.
  4. Imamura, T., Higuchi, A., Sekine, K. T., Yamashita, T. and Takahashi, H. (2014). High concentration of sucrose induces overwintering bud formation in gentian plantlets cultured in vitro. Plant Biotech 31(2): 97-104.
  5. Takahashi, H., Imamura, T., Konno, N., Takeda, T., Fujita, K., Konishi, T., Nishihara, M. and Uchimiya, H. (2014). The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial gentiana. Plant Cell 26(10): 3949-3963.

简介

为了研究植物中糖的功能,饲养实验是最常见和最容易的方法之一。 然而,传统方法,例如,在含糖溶液上漂浮叶盘似乎具有不充分的糖吸收效率,尽管具有高的渗透和损伤效应。 这是一种将寡糖龙胆二糖供给到体外培养的龙胆的组织中的方案。 该方案能够将龙胆二糖掺入完整组织而不暴露于渗透胁迫,并且可以用于能够通过芽尖培养繁殖的其它植物物种。

关键字:龙胆, 龙胆, 越冬芽, 苗, 萌芽

材料和试剂

  1. 龙胆( Gentiana triflora )组织培养苗
  2. 蔗糖
  3. 龙胆二糖(Sigma-Aldrich,目录号:G3000-5G)
  4. 结冷胶(Wako USA,目录号:075-03075)
  5. 液氮
  6. Milli Q级水
  7. MS维生素(见配方)
  8. 传播介质(参见配方)
  9. IOWB感应培养基(参见配方)
  10. 龙胆中等(见配方)

设备

  1. 无菌洋红色盒(高压灭菌)
  2. 手术胶带(3M,目录号:1530-0)
  3. 玻璃培养管(高压灭菌)
  4. 硅胶塞(高压灭菌)
  5. 无菌15ml塑料管(例如Greiner Bio-One GmbH,目录号:188271)
  6. 无菌注射器过滤器(Millipore,目录号:SLGP033RS)
  7. 无菌培养皿(IWAKI PUMPS,目录号:SH90-20)
  8. 截止滤波器(Millipore,目录号:UFC5003BK)
  9. 生长室
  10. 清洁长椅
  11. Scalpel
  12. 镊子
  13. 球磨机
  14. 离心机
  15. 冷冻干燥器

程序

  1. 龙胆IOWB和小植物的制备和龙胆二烯喂养
    1. 根据Hosokawa等人的方法(1996)制备龙胆的体外苗培养物。
    2. 传送拍摄提示(约1厘米)到包含的洋红色盒子   70ml的繁殖培养基并在20℃下培养1至2个月 在16/8小时光/暗光周期下(图1A)
    3. 转移3到5   (约1cm)加入到含有70ml水的品红盒中 IOWB诱导培养基(图1B),并在20℃下培养6个月以上   在16/8小时光/暗光周期下(图1C)
    4. 收获 产生IOWB并使用a切成约2cm长的片 解剖刀和镊子在塑料培养皿(图2A)

      图1。 IOWB从龙胆苗感应。 A.切割茎尖 苗。 B.将枪头放置在IOWB诱导培养基上。 C. IOWBs 其由在IOWB诱导下培养6个月的龙胆子植物产生 中。 箭头表示IOWB。 酒吧:2厘米

    5. 转移IOWBs或 植物到含有2ml龙胆二糖培养基的玻璃培养管   并用硅插塞密封管的顶部(图2B)

      图2.将龙胆醇喂养到龙胆IOWB和小植物中。 A.收获   的IOWB。 B.IOWB和芽尖放置在龙胆二糖上 中。 C.IowB在龙胆二糖培养基上培养。 D.拍摄提示培养   龙胆二糖培养基。 酒吧:1厘米

    6. 在20℃,16/8h光/暗光周期下培养适当的时间(图2C-D)。
    7. 收获部分样品不与介质接触。

  2. 检测IOWB或小植物中的龙胆二糖
    1. 在球磨机中冷冻干燥并粉碎IOWB或小植物
    2. 用500μl50%(v/v)甲醇匀化10mg干重的样品
    3. 将匀浆在20,000×g离心5分钟,并通过截止滤器过滤上清液。
    4. 将滤液进行薄层色谱(图3)或其他分析以检测龙胆二糖

      图3.掺入龙胆中的龙胆二糖的实施例分析 苗。 从正常MS培养的小植株叶片中提取 培养基(对照)或含有0.05%标记的龙胆二糖的MS培养基 罗丹明(处理)3天进行TLC分析。 STD, 0.01%用罗丹明标记的龙胆二糖。

笔记

  1. 繁殖和喂养步骤应使用干净的工作台进行无菌操作。
  2. 标记的龙胆二糖只能用于确认并入的龙胆二糖
  3. 关于龙胆IOWB和小植物的细节,参见Imamura等人(2014)。
  4. 有关TLC分析的详细信息,请参见Takahashi等人(2014年)。
  5. 对于龙胆二糖标记,参见以下出版物Hase et al。(1978)和Fry(1997)。

食谱

  1. MS维生素(100ml)
    10g肌醇-6 50mg烟酸
    50mg盐酸吡哆酮
    10mg盐酸硫胺素 200mg甘氨酸
  2. 传播介质(1L)
    4.6克MS盐
    1 ml MS维生素
    30克蔗糖 2g结冷胶
    用KOH调节pH 5.7并高压灭菌15分钟
  3. IOWB诱导培养基(1L)
    4.6克MS盐
    1 ml MS维生素
    60克蔗糖 2g结冷胶
    用KOH调节pH 5.7并高压灭菌15分钟
  4. 龙胆生物培养基(1L)
    4.6克MS盐
    1 ml MS维生素
    10 g龙胆二糖或标记的龙胆二糖 *
    2g结冷胶
    用KOH调节pH 5.7并高压灭菌15分钟
    * 在温度降至约60℃后,加入高压灭菌的MS培养基中。
    * 标记的龙胆二糖用于确认掺入的龙胆二糖

致谢

该协议改编自以下出版物,Imamura等人(2014)和Takahashi等人(2014)。这项研究得到了教育,文化,体育,科学和技术部的青年科学家助学金(B)的支持。

参考文献

  1. Fry,S.C。(1997)。 糖基转移酶和糖基水解酶的新型"斑点印迹"测定:木葡聚糖的优化内切半乳糖苷酶(XET)活性。植物J(11):1141-1150。
  2. Hase,S.,Ikenaka,T。和Matsushima,Y。(1978)。 用荧光化合物标记还原性末端糖对寡糖的结构分析。生物化学通讯(Biochem Biophys Res Commun)85(1):257-263。
  3. Hosokawa,K.,Nakano,M.,Oikawa,Y.and Yamamura,S。(1996)。 来自Gentiana的商业栽培品种的叶,茎和根外植体的不定生殖再生>。 Plant Cell Rep 15(8):578-581。
  4. Imamura,T.,Higuchi,A.,Sekine,K.T.,Yamashita,T.and Takahashi,H。(2014)。 高浓度的蔗糖诱导龙胆苗中越冬芽形成 培养。植物生物技术 31(2):97-104。
  5. Takahashi,H.,Imamura,T.,Konno,N.,Takeda,T.,Fujita,K.,Konishi,T.,Nishihara,M.and Uchimiya,H。 龙胆低聚糖龙胆二糖功能在草本多年生龙胆的芽休眠调节中的作用。 植物细胞 26(10):3949-3963。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Takahashi, H. and Nishihara, M. (2015). Gentiobiose Feeding in Gentian in vitro Overwintering Buds or Plantlets. Bio-protocol 5(12): e1499. DOI: 10.21769/BioProtoc.1499.
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