Plant Tissue Culture: Experimental Procedure and Key Precautions | 植物组织培养实验操作与注意事项

📚 Plant Tissue Culture: Experimental Procedure and Key Precautions | 植物组织培养实验操作与注意事项

Plant tissue culture is a core technique in modern biotechnology and a frequent practical topic in biology examinations. It uses sterile techniques to grow plant cells, tissues, or organs on a nutrient medium, relying on the remarkable property of cell totipotency.

植物组织培养是现代生物技术中的核心技术之一，也是生物考试中经常出现的实验课题。它利用无菌技术，在人工培养基上培养植物细胞、组织或器官，其理论基础是植物细胞的全能性。

1. The Principle of Totipotency | 植物细胞全能性的原理

Every living, nucleated plant cell contains the complete genetic information of the parent plant. Under suitable conditions, a differentiated somatic cell can dedifferentiate into callus tissue and then redifferentiate to form shoots and roots, eventually producing a complete plant.

每一个具有细胞核的活植物细胞都含有该植物完整的遗传信息。在适宜条件下，已分化的体细胞可脱分化形成愈伤组织，再通过再分化形成芽和根，最终发育成完整植株。

Dedifferentiation means mature cells regain the ability to divide; redifferentiation means callus cells form specific organs such as shoots or roots.

脱分化是指已成熟的细胞重新获得分裂能力；再分化是指愈伤组织中的细胞形成芽或根等特定器官。

Triploblastic? No. Every nucleated plant cell → callus → complete plant

每个含核植物细胞 → 愈伤组织 → 完整植株

2. Essential Materials and Equipment | 必需材料与设备

Successful tissue culture requires strict aseptic conditions. Key equipment includes an ultra-clean bench or laminar flow hood, an autoclave, sterile forceps, scalpels, inoculation loops, alcohol lamps, culture bottles, Petri dishes, and measuring instruments.

成功的组织培养需要严格的无菌条件。关键设备包括超净工作台、高压蒸汽灭菌锅、无菌镊子、解剖刀、接种环、酒精灯、培养瓶、培养皿及测量工具等。

Ultra-clean workbench: supplies filtered sterile air and UV sterilisation.
Autoclave: sterilises media, water, and instruments under high pressure and temperature.
Instruments: forceps, scalpels, needles, and spreaders must be sterilised by flaming.
Culture vessels: bottles or Petri dishes must be autoclave-proof and clear.

超净工作台：提供过滤后的无菌空气并用紫外灯消毒。
高压灭菌锅：在高温高压下灭菌培养基、无菌水和器具。
接种工具：镊子、解剖刀、接种针等需用火焰灼烧灭菌。
培养容器：培养瓶或培养皿必须耐高温并保持透明。

3. Preparation of MS Culture Medium | MS培养基的配制

The most widely used medium is Murashige and Skoog (MS) medium. It contains macro-nutrients, micro-nutrients, iron salts, vitamins, sucrose as an energy source, plant growth regulators, and agar as a solidifying agent.

最常用的培养基是MS培养基。它含有大量元素、微量元素、铁盐、维生素、作为碳源的蔗糖、植物生长调节剂以及用作凝固剂的琼脂。

Component	Main Function
Macro-nutrients	Provide N, P, K, Ca, Mg, S
Micro-nutrients	Provide Mn, Zn, Cu, B, Mo etc.
Sucrose	Carbon source and osmotic regulator
Agar	Solidifying agent
Auxin and cytokinin	Regulate growth, callus induction, rooting

成分	主要功能
大量元素	提供N、P、K、Ca、Mg、S
微量元素	提供Mn、Zn、Cu、B、Mo等
蔗糖	碳源与渗透调节剂
琼脂	培养基凝固剂
生长素和细胞分裂素	调节生长、诱导愈伤组织和生根

4. Adjusting pH and Autoclaving | 调节pH与高压灭菌

Before autoclaving, the medium must be adjusted to pH 5.6 to 5.8 using hydrochloric acid or sodium hydroxide. If the pH is wrong, agar may not solidify properly or nutrient uptake may be abnormal.

高压灭菌之前，培养基的pH必须用盐酸或氢氧化钠调节至5.6至5.8。若pH不当，琼脂可能无法正常凝固，或者植物对营养的吸收会出现异常。

Add the plant growth regulators before sterilisation if they are heat-stable. Some compounds such as certain vitamins may be filter-sterilised after autoclaving if they are heat-sensitive.

耐热的植物生长调节剂可在灭菌前加入；某些热不稳定的维生素则需要用过滤灭菌法在灭完菌后加入。

121 °C + 103 kPa + 15–20 min = complete sterilisation of medium

121 °C + 103 kPa + 15–20分钟 = 培养基彻底灭菌

Dispense the molten medium into culture vessels before autoclaving, cap loosely to allow steam penetration, then autoclave and allow the medium to cool and set at a slanted angle if needed.

应在灭菌前将熔化状态的培养基分装到培养容器中，瓶盖不要拧太紧以利于蒸汽穿透。灭菌后让培养基自然冷却并凝固，必要时摆成斜面。

5. Explant Selection and Surface Sterilisation | 外植体的选择与表面消毒

An explant is any piece of plant tissue used to start a culture. Young, actively growing tissues such as shoot tips, young leaves, or stem segments are preferred because they are less contaminated and have higher totipotency.

外植体是用于启动培养的植物组织块。通常选择幼嫩、生长旺盛的组织，如茎尖、幼叶或茎段，因为它们带菌少且全能性较高。

The standard procedure is: wash with running tap water and detergent, rinse with sterile distilled water, soak in 70% ethanol for about 30 seconds, then treat with a surface disinfectant such as 0.1% mercuric chloride solution or 10–15% sodium hypochlorite solution, and finally rinse three to five times with sterile distilled water.

标准流程为：用自来水加洗涤剂冲洗，再用无菌蒸馏水漂洗，放入70%乙醇中浸泡约30秒，然后用0.1%升汞溶液或10%–15%次氯酸钠溶液进行表面消毒，最后用无菌水冲洗3至5次。

Sterilant	Approx. Time	Note
70% ethanol	30 s	Wetting agent; kills surface microbes fast
0.1% HgCl₂	6–8 min	Very effective; toxic and must be rinsed well
10% NaOCl solution	10–15 min	Safer but may need a longer time

消毒剂	参考时间	注意事项
70%乙醇	30秒	润湿效果好，快速杀死表面微生物
0.1%升汞	6–8分钟	效果强但有剧毒，必须充分冲洗
10%次氯酸钠溶液	10–15分钟	较安全，但需更长处理时间

6. Inoculation Under Aseptic Conditions | 无菌条件下接种

Inoculation is the transfer of the sterilised explant onto the culture medium. This step must be performed inside an ultra-clean bench after 15 to 30 minutes of UV irradiation followed by 10 minutes of air flow.

接种是将消毒后的外植体转移到培养基上的过程。此步骤必须在超净工作台内进行：先开启紫外灯照射15至30分钟，再开启风机吹10分钟。

Key precautions include: spray hands with 70% ethanol, flame-sterilise forceps and scalpels then cool them before touching the tissue, hold the culture bottle cap near the flame, and never reach across an open dish.

关键注意事项包括：用70%乙醇喷洒双手，镊子和解剖刀要灼烧灭菌并放凉后再接触组织，打开培养瓶时瓶口应靠近火焰，绝不能从已打开的培养皿上方伸手横过。

Trim the explant into 0.5 to 1 cm pieces with a sterile scalpel. Remove any dead, brown, or damaged regions, then press the explant gently onto the medium surface so that it remains in close contact with the nutrients.

用无菌解剖刀将外植体切成0.5至1厘米的小块，去除坏死、褐变或受损的部分，然后轻轻将外植体按入培养基表面，使其与培养基充分接触。

Work close to the flame to create an upward sterile air current.
Do not touch the inside of the bottle cap or the rim.
Do not talk, cough, or breathe directly onto the culture.
Close the lid immediately after inoculation and label the vessel.

操作时应靠近酒精灯火焰，利用上升的无菌气流。
不能接触瓶盖内侧或瓶口边缘。
操作时不要说话、咳嗽或正对培养物呼吸。
接种后立即盖好瓶盖，并做好标签记录。

7. Role of Auxin and Cytokinin Ratios | 生长素与细胞分裂素的比例作用

Plant growth regulators are the most important factors controlling morphogenesis in tissue culture. Auxins such as NAA, IAA, and 2,4-D promote root induction and callus growth; cytokinins such as BAP or kinetin promote shoot formation and cell division.

植物生长调节剂是控制组织培养中形态发生的最重要因素。NAA、IAA、2,4-D等生长素促进生根和愈伤组织生长；BAP、激动素等细胞分裂素促进芽的形成和细胞分裂。

High auxin / low cytokinin → root formation

Low auxin / high cytokinin → shoot formation

The auxin-to-cytokinin ratio also affects callus induction and maintenance. A moderate balance of both regulators usually favours undifferentiated callus proliferation.

生长素与细胞分裂素的比例也会影响愈伤组织的诱导和继代培养。两者的比例适中时，通常有利于未分化愈伤组织的增殖。

8. Culture Conditions: Temperature, Light, and Humidity | 培养条件：温度、光照与湿度

Most plant cultures are maintained at 25 ± 2 °C with a 12 to 16 hour photoperiod. Light intensity typically ranges from 1,000 to 3,000 lux for shoot proliferation; root induction may be improved by lower light intensity or a short period of darkness.

多数植物组织培养常在25 ± 2 °C下进行，光周期为12至16小时。芽增殖阶段的光照强度通常为1000–3000勒克斯；诱导生根时适当降低光照或短期暗培养可能更有利。

Humidity inside the culture vessel is usually saturated, but excessively high humidity and poor ventilation can cause vitrification or glassy translucent shoots. Therefore, culture vessels should allow some gas exchange through vented caps.

培养瓶内湿度通常接近饱和，但湿度过高、通风不良会导致玻璃化苗现象，使芽呈半透明水渍状。因此，培养瓶最好使用带有透气孔的瓶盖，以实现一定的气体交换。

During callus induction, some species prefer darkness because phenolic oxidation is reduced. However, after transferring callus to shoot-induction medium, light becomes necessary for chlorophyll synthesis and shoot development.

在诱导愈伤组织阶段，有些植物更喜欢暗培养，因为暗处可减少酚类物质氧化。但将愈伤组织转移到芽诱导培养基后，光照对叶绿素合成和芽的发育就非常必要。

9. Subculturing and Proliferation | 继代培养与增殖

Subculturing means transferring a piece of established culture to fresh medium at regular intervals, usually every 3 to 4 weeks. This provides fresh nutrients and prevents the accumulation of toxic metabolic waste products.

继代培养是指每隔3至4周将已经形成的培养物转移到新鲜培养基上。这样可以补充营养，并防止有害代谢产物积累。

Callus can be divided into small clumps using a sterile scalpel. The best regions to transfer are pale yellow, compact, and actively growing parts; shiny or wet-looking tissue may be suffering from hyperhydration or contamination.

愈伤组织可用无菌解剖刀切成小块。应选择淡黄色、结构致密且生长旺盛的部位进行转移；那些发亮或水渍状的组织可能发生玻璃化或已经被污染。

Careful record-keeping is essential. For each subculture, record the date, medium formula, growth regulator concentration, culture number, and visible changes in colour or morphology.

详细记录非常重要。每次继代都应记录日期、培养基配方、生长调节剂浓度、培养编号以及颜色或形态上的明显变化。

10. Rooting and Acclimatisation | 生根与驯化

When enough shoots have been produced, they are transferred to a rooting medium. This is often half-strength MS medium with an auxin such as NAA or IBA at 0.1 to 1.0 mg/L, which encourages vigorous root emergence.

当产生足够多的不定芽后，可将其转移到生根培养基上。生根培养基常用半量MS，并加入0.1–1.0 mg/L的NAA或IBA等生长素，以促进健壮根的发生。

Once roots are 1 to 2 cm long, the young plantlets must be acclimatised. Gently remove them from the culture vessel, wash away all agar with sterile water, and transplant them into a mixture of vermiculite and sterilised soil.

当根长到1–2厘米时，小植株必须进行驯化。先将小植株轻轻取出，用无菌水洗掉根部附着的琼脂，然后移栽到蛭石与灭菌土壤的混合基质中。

During the first one to two weeks, maintain high humidity by covering the plants with a transparent plastic bag or a mist chamber. Gradually open the cover to reduce humidity, increase light intensity, and finally transfer the plantlets to the greenhouse.

移栽后的前1至2周，可用透明塑料袋或喷雾箱保持高湿度。随后逐步打开覆盖物以降低湿度、增强光照，最后将小植株移到温室中。

11. Common Problems and Solutions | 常见问题与解决方法

Contamination is the most common failure. Bacteria usually appear as slimy white or yellow colonies around the explant, while fungi produce visible mycelia and spores. The most reliable response is to discard the contaminated culture and improve aseptic technique.

污染是组织培养中最常见的问题。细菌通常在外植体周围形成黏滑的白色或黄色菌落，真菌则出现肉眼可见的菌丝和孢子。最可靠的处理方法是弃去污染培养物，并改进无菌操作技术。

Contamination: caused by poor surface sterilisation, dirty workbench, or poor handling.
Browning: caused by oxidation of phenolic compounds released from wounded tissue.
Vitrification: watery, translucent shoots due to excessive cytokinin, high humidity, or weak ventilation.
Callus without organs: incorrect auxin/cytokinin balance or unsuitable light conditions.

污染：由外植体消毒不彻底、工作台不洁或操作不当引起。
褐变：由受伤组织释放的酚类物质被氧化引起。
玻璃化：细胞分裂素过多、湿度太高或通气不良造成芽呈水渍状半透明。
愈伤组织不分化：生长素与细胞分裂素比例不合适或光照条件不适。

To reduce browning, choose young explants, cut cleanly with a sharp scalpel, add activated charcoal or polyvinylpyrrolidone (PVP) to the medium, and subculture frequently to fresh medium.

为减轻褐变，应选择幼嫩外植体、用锋利解剖刀快速切割，在培养基中加入活性炭或聚乙烯吡咯烷酮（PVP），并及时转移到新鲜培养基上。

12. Safety and Examination Key Points | 安全事项与考试要点

Mercuric chloride is highly toxic. Always wear gloves, avoid skin contact, and dispose of mercury-containing waste separately. Instruments that touched HgCl₂ solution must never be used directly on living tissue before rinsing.

升汞有剧毒，操作时务必戴手套，避免皮肤接触，含汞废液要单独处理。接触过升汞溶液的器具必须彻底冲洗后才能接触植物活组织。

In an examination, the most frequently tested points are: the meaning of totipotency, the composition of MS medium, the purpose of auxin and cytokinin, the order of sterilisation steps, aseptic techniques, and the reason why a high auxin/cytokinin ratio favours roots.

考试中最常考的知识点包括：全能性的含义、MS培养基的成分、生长素与细胞分裂素的作用、消毒步骤的先后顺序、无菌操作技术，以及高比例生长素/细胞分裂素促进生根的原因。

Explant → Sterilisation → Inoculation → Callus → Shoot → Root → Acclimatisation

外植体 → 消毒 → 接种 → 愈伤组织 → 芽 → 根 → 驯化

The single most important precaution in plant tissue culture is to keep everything that contacts the tissue sterile, while remembering that the explant itself must be alive and healthy. Master both the theory and the practical sequence, and the exam questions become predictable.

植物组织培养中最重要的一条注意事项是：所有接触组织的物品都必须无菌，同时外植体本身必须保持活性和健康。掌握好理论原理和操作流程，考试题就会变得非常容易判断。

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Plant Tissue Culture: Experimental Procedure and Key Precautions | 植物组织培养实验操作与注意事项