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Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens
小立碗藓中原丝体的组织培养和收集   

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Abstract

Moss spores germinate to form an alga-like filamentous structure called the protonemata. Protonemata are the earliest stage (the haploid phase) of a bryophyte life cycle and eventually give rise to a mature gametophyte. Protonemata of the moss Physcomitrella patens (P. patens) are important not only in their life cycle, but also for research. Protonemata are used for various things such as RNA/DNA extractions and protoplast isolation. We can obtain high yield of intact protoplasts from protonemata. Protoplasts can be used to study a variety of cellular processes, such as subcellular localization of proteins, isolation and analyses of intact organelles and DNA transformation. In addition, the completed sequence of the P. patens genome facilitates the use of genetic and molecular approaches to identify genes and the ability of the moss to undergo homologous recombination at appreciable frequency offers a powerful way to determine gene function. Therefore, culture of P. patens protonemata is critical.

Materials and Reagents

  1. A vigorously growing tissue which is about 10 d old (the earliest stage of gametophyte; Physcomitrella patens subspecies patens (Gransden) was used as the tissue and it was obtained from Ralph S. Quatrano (Department of Biology, Washington University in St. Louis, MO 63130, USA)
  2. 200 ml sterile distilled water
  3. 70% alcohol in a spray bottle (for surface sterilization)
  4. Growth medium (see Recipes)

Equipment

  1. 1 L flask in which to prepare the growth medium
  2. Sterile petri dishes (90-mm)
  3. Sterile cellophane discs
  4. Sterile tweezers
  5. Sterile test tubes (25 x 150 mm)
  6. Micropore surgical tape
  7. Sterile pipettes (1 ml)
  8. Sterile tips (1 ml)
  9. Dispensing instrument (e.g., IKA T 10 basic ULTRA-TURRAX®)
  10. Laminar flow cabinet
  11. Autoclave (e.g., Sanyo, model: MLS-3780 )

Procedure

Note: Steps 2-12 should be carried out under sterile conditions.

  1. BCDA medium is prepared according to Table 1 and sterilized by an autoclave for 20 min at 121 °C;
  2. Surface sterilization is performed with 70% alcohol in the Laminar flow hood;
  3. Media was cooled to 60 °C and approximately 30 ml BCDA medium was poured into sterile petri dishes (90 mm). This was allowed to cool until the media solidified; Place a piece of sterile cellophane discs onto the BCDA medium (Video 1);

    Video 1. Place a piece of sterile cellophane discs onto the BCDA medium

  4. Place one petri dish tissue (vigorously growing tissue) into one test tube (Video 2);

    Video 2. Place one petri dish tissue into one test tube

  5. Add 6-8 ml of H2O to one sterile test tube (containing one petri dish tissue), and cut it into fragments using dispensing instrument for 1-2 min at a speed of about 15,000 rpm;
  6. Pipette 1-2 ml of the protonemata suspension from step 6 onto each petri dish from step 4. Spread the suspension evenly by gently swirling the plates (Video 3);

    Video 3. Pipette 1-2 ml of the protonemata suspension from step 6 onto each petri dish from step 4. Spread the suspension evenly by gently swirling the plates

  7. Seal the Petri dishes with micropore surgical tape;
  8. Incubate these fragments for 6-7 d under standard conditions (25 °C) with a light cycle of 16 h of light/8 h of darkness and a light intensity of 70-80 μmol/s/m2;
  9. Transfer 1-2 dishes of the tissue (over cellophane) onto BCD solid media (Table 2) growth 3-4 d under the same culture conditions to step 9 for next cycle (this is a vigorously growing tissue which is about 10 d old tissues; Video 4);

    Video 4. Transfer 1-2 dishes of the tissue onto BCD solid media growth 3-4 d under the same culture conditions to step 9 for next cycle

  10. Harvest the tissue (protonemata of P. patens from step 9) for downstream analysis by scraping them from the cellophane using sterile tweezers.
  11. For tissue stock, take a tiny amount (about 2 mm2; using sterile tweezers) of 10 d old tissues into sterile test tubes with BCD medium. Wrap the tubes in foil and store them at 4 °C. They remain viable for about six months.

Recipes

  1. Growth medium

    Table 1. BCDA medium recipe
    Reagent
    Quantity (for 1 L)
    Final concentration
    Solution B
    10 ml
    1 mM MgSO4
    Solution C
    10 ml
    1.84 mM KH2PO4
    Solution D
    10 ml
    10 mM KNO3
    CaCl2
    111 mg
    1 mM
    FeSO4.7H2O
    12.5 mg
    45 μM
    (NH4)2C4H4O6
    0.92 g
    5 mM
    Agar
    7.5 g
    0.75% (w/v)
    Glucose
    5 g
    0.5% (w/v)
    Hoagland’s A-Z trace
    1 ml
    Trace element solution
    H2O
    To 1 L


    Table 2. BCD medium recipe
    Reagent
    Quantity (for 1 L)
    Final concentration
    Solution B
    10 ml
    1 mM MgSO4
    Solution C
    10 ml
    1.84 mM KH2PO4
    Solution D
    10 ml
    10 mM KNO3
    CaCl2
    111 mg
    1 mM
    FeSO4.7H2O
    12.5 mg
    45 μM
    Agar
    7.5 g
    0.75% (w/v)
    Glucose
    5 g
    0.5% (w/v)
    Hoagland’s A-Z trace
    1 ml 
    Trace element solution
    H2O
    To 1 L


    Table 3. Recipe of Solution B, C, D and Hoagland’s A-Z trace

    Reagent
    Quantity (for 1 L)
    Final concentration
    Solution B
    MgSO4.7H2O
    25 g
    0.1 M
    H2O
    To 1 L

    Solution C
    KH2PO4
    25 g
    184 mM
    H2O
    To 1 L
    Adjust the pH to 6.5 using KOH
    Solution D
    KNO3
    101 g
    1 M
    H2O
    To 1 L

    Hoagland’s A-Z trace
    Al2(SO4)3.K2SO4.24H2O
    55 mg
    0.006% (w/v)
    CoCl2.6H2O
    55 mg
    0.006% (w/v)
    CuSO4.5H2O
    55 mg
    0.006% (w/v)
    H3BO3
    614 mg
    0.061% (w/v)
    KBr
    28 mg
    0.003% (w/v)
    KI
    28 mg
    0.003% (w/v)
    LiCl
    28 mg
    0.003% (w/v)
    MnCl2.4H2O
    389 mg
    0.039% (w/v)
    SnCl2.2H2O
    28 mg
    0.003% (w/v)
    ZnSO4.7H2O
    55 mg
    0.006% (w/v)
    H2O
    To 1 L

    Solution B, C, D and Hoagland’s A-Z trace are sterilized and then stored at 4 °C or room temperature.

Acknowledgments

This work was supported by grants from Beijing Natural Science Foundation (No. 5132004), China Postdoctoral Science Foundation and State Education Ministry Scientific Research Foundation for the Returned Overseas Chinese Scholars to Dr. Wang.

References

  1. Ashton, N. W., and Cove, D. J. (1977). The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants in the moss Physcomitrella patens. Mol Gen Genet 154, 87-95.
  2. Boyd, P. J., Hall, J., and Cove, D. J. (1988). An airlift fermenter for the culture of the moss Physcomitrella patens. In: Glime, J. M. (ed). Methods in bryology. Hattori Botany Laboratory, 41-45.
  3. Cove, D. J., Perroud, P. F., Charron, A. J., McDaniel, S. F., Khandelwal, A. and Quatrano, R. S. (2009). The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harb Protoc 2009(2): pdb emo115.

简介

苔藓孢子发芽形成称为质子发芽的藻状丝状结构。原生质是苔藓植物生命周期的最早期(单倍体阶段),并最终产生成熟配子体。苔藓小立碗藓(Physcomitrella patens)( patens )不仅在其生命周期中很重要,而且对于研究也很重要。质子被用于各种事物,例如RNA/DNA提取和原生质体分离。我们可以从质子发芽获得高产量的完整原生质体。原生质体可用于研究多种细胞过程,例如蛋白质的亚细胞定位,完整细胞器的分离和分析以及DNA转化。此外,完成的 P的序列。 patens基因组促进使用遗传和分子方法来鉴定基因,并且苔藓在可感知的频率下进行同源重组的能力提供了确定基因功能的有效方式。因此,培养p。 patens protonemata是至关重要的。

材料和试剂

  1. 使用大约10天(作为配子体的最早期的子叶小立碗藓(Physcomitrella patens subspecies patens)(Gransden))的生长旺盛的组织作为组织,并且从Ralph S.Quatrano(Department of Biology,Washington University in St.Louis, MO 63130,USA)
  2. 200毫升无菌蒸馏水
  3. 喷雾瓶中70%的酒精(用于表面灭菌)
  4. 生长培养基(参见食谱)

设备

  1. 1L烧瓶中,其中制备生长培养基
  2. 无菌培养皿(90毫米)
  3. 无菌玻璃纸碟
  4. 无菌镊子
  5. 无菌试管(25×150mm)
  6. 微孔手术胶带
  7. 无菌移液器(1 ml)
  8. 无菌提示(1 ml)
  9. 配药仪器(,例如,IKA T 10基本ULTRA-TURRAX®)
  10. 层流柜
  11. 高压灭菌(例如,Sanyo,型号:MLS-3780)

程序

注意:步骤2-12应在无菌条件下进行。

  1. 根据表1制备BCDA培养基,并在121℃下用高压灭菌器灭菌20分钟;
  2. 在层流罩中用70%酒精进行表面灭菌;
  3. 将培养基冷却至60℃,并将约30ml BCDA培养基倒入无菌培养皿(90mm)中。 使其冷却直至介质固化; 将一块无菌玻璃纸碟放在BCDA培养基上(视频1);

    视频1.将一块无菌玻璃纸光盘放在BCDA介质上
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  4. 将一个陪替氏培养皿组织(强力生长的组织)放入一个试管(视频2);

    视频2.将一个培养皿组织放入一个试管中
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  5. 向一个无菌试管(含有一个培养皿组织)中加入6-8ml的H 2 O,并使用分配仪器以约15,000rpm的速度将其切成片段1-2分钟;
  6. 吸取1-2毫升来自步骤6的质子凝胶悬浮液到步骤4的每个培养皿上。通过轻轻地旋转平板(视频3)将悬浮液均匀地扩散;

    视频3.吸取1-2毫升来自步骤6的质子悬浮液到步骤4的每个培养皿上。通过轻轻地旋转平板
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  7. 用微孔手术胶带密封培养皿;
  8. 将这些片段在标准条件(25℃)下孵育6-7天,光周期为16小时/8小时黑暗,光强度为70-80μmol/s/m 2 >;
  9. 在相同的培养条件下,将组织(玻璃纸上的)1-2块(在玻璃纸上)转移到BCD固体培养基(表2)上3-4天,以进行下一个循环的步骤9(这是大约生长的组织, ;视频4);

    视频4.将1-2碟的组织转移到BCD固体培养基上3-4天,在相同的培养条件下进行下一个周期的步骤9
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  10. 收获来自步骤9的组织(来自步骤9的马铃薯的电子照相),用于使用无菌镊子从玻璃纸刮下它们用于下游分析。
  11. 对于组织原料,取10d旧组织的微量(约2毫升,使用无菌镊子)到具有BCD培养基的无菌试管中。 包装在铝箔管和存储在4°C。 它们保持活力约六个月。

食谱

  1. 生长培养基

    表1. BCDA培养基配方
    试剂
    数量(1 L)
    最终集中
    解决方案B
    10 ml
    1mM MgSO 4
    解决方案C
    10 ml
    1.84mM KH 2 PO 4
    解决方案D
    10 ml
    10 mM KNO 3
    CaCl <2>
    111 mg
    1 mM
    FeSO 4 7H <2> O
    12.5 mg
    45μM
    (NH 4)2 2 CH 4 H 4 O 6 6 0.92克
    5 mM
    Agar
    7.5克
    0.75%(w/v)
    葡萄糖
    5克
    0.5%(w/v)
    Hoagland的A-Z跟踪
    1 ml
    微量元素溶液
    H sub 2 O
    到1 L


    表2. BCD介质配方
    试剂
    数量(1 L)
    最终集中
    解决方案B
    10 ml
    1mM MgSO 4
    解决方案C
    10 ml
    1.84mM KH 2 PO 4
    解决方案D
    10 ml
    10 mM KNO 3
    CaCl <2>
    111 mg
    1 mM
    FeSO 4 7H <2> O
    12.5 mg
    45μM
    Agar
    7.5克
    0.75%(w/v)
    葡萄糖
    5克
    0.5%(w/v)
    Hoagland的A-Z跟踪
    1 ml 
    微量元素溶液
    H sub 2 O
    到1 L


    表3.解决方案B,C,D和Hoagland的A-Z跟踪的配方

    试剂
    数量(1 L)
    最终集中
    解决方案B
    MgSO 4。 。 O
    25克
    0.1 M
    H sub 2 O
    到1 L

    解决方案C
    KH 2 PO 4
    25克
    184 mM
    H sub 2 O
    到1 L
    用KOH
    调节pH至6.5
    解决方案D
    KNO 3
    101克
    1 M
    H sub 2 O
    到1 L

    Hoagland的A-Z跟踪
    Al <2>(SO 4 ) 3 K sub 2 SO SO 4 24H 2 O
    55毫克
    0.006%(w/v)
    COCl <2> 6H <2> O
    55毫克
    0.006%(w/v)
    CuSO 4 5H sub 2 O
    55毫克
    0.006%(w/v)
    H 3 BO 3
    614 mg
    0.061%(w/v)
    KBr
    28 mg
    0.003%(w/v)
    KI
    28 mg
    0.003%(w/v)
    LiCl
    28 mg
    0.003%(w/v)
    MnCl 2 4H O
    389 mg
    0.039%(w/v)
    SnCl 2 2H <2> O
    28 mg
    0.003%(w/v)
    ZnSO 4 。 7H O
    55毫克
    0.006%(w/v)
    H sub 2 O
    到1 L

    将溶液B,C,D和Hoagland的A-Z痕迹灭菌,然后在4℃或室温下贮存。

致谢

这项工作得到了北京自然科学基金(编号:5132004),中国博士后科学基金和国家教育部侨务科学研究基金会对王博士的资助。

参考文献

  1. Ashton,N.W。,和Cove,D.J。(1977)。 在青苔Physcomitrella patens中分离和初步表征营养缺陷型和类似物抗性突变体 。 Mol Gen Genet 154,87-95。
  2. Boyd,P.J.,Hall,J.,and Cove,D.J。(1988)。用于培养小立碗藓的空气发酵罐。 In:Glime,J.M。(ed)。 Hattori植物学实验室,41-45。
  3. Cove,D.J.,Perroud,P.F.,Charron,A.J.,McDaniel,S.F.,Khandelwal,A.and Quatrano,R.S.(2009)。 青苔Physcomitrella patens :一种用于植物发育和基因组的新型模型系统 Cold Spring Harb Protoc 2009(2):pdb emo115。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X. and He, Y. (2015). Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens. Bio-protocol 5(15): e1556. DOI: 10.21769/BioProtoc.1556.
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