Plant culture media provide inorganic salts, carbon sources, vitamins, and a plant growth-regulatory environment for isolated tissues, and their composition directly affects cell proliferation, callus formation, organogenesis, and rooting. In research applications, basal systems such as MS, B5, N6, and WPM should be selected according to plant material, culture stage, and experimental purpose, with targeted optimization of salt concentration, carbon source, and plant growth regulator conditions.

Keywords: plant culture medium; MS medium; plant tissue culture; plant growth regulator; callus; shoot induction; rooting culture

1 Functional Positioning of Plant Culture Media

1.1 Nutritional and Regulatory Requirements of In Vitro Culture

After plant tissues are isolated, they lose the complete root system, vascular transport, and hormonal regulation provided by the parent plant. The culture medium therefore needs to continuously supply N, P, K, Ca, Mg, Fe, and trace elements while also providing vitamins, a carbon source, and suitable plant growth regulators. The medium thus creates a relatively controllable artificial physiological environment in which ionic composition, osmotic conditions, and regulatory signals jointly determine cell proliferation, dedifferentiation, organogenesis, and plant regeneration.

1.2 Major Components of Culture Media

(1) Inorganic Salt System

Macronutrients mainly include N, P, K, Ca, Mg, and S and participate in nitrogen metabolism, energy conversion, osmotic regulation, cell-wall stability, and multiple enzymatic reactions. Trace elements such as Fe, Mn, Zn, B, Cu, and Mo are required in smaller amounts, but either deficiency or excess can markedly alter tissue growth. Important differences among basal media mainly involve total salt concentration, ionic ratios, and nitrogen-source composition such as NO₃⁻/NH₄⁺.

(2) Carbon Source and Osmotic Environment

Callus, young shoots, and isolated materials cultured under low-light conditions generally cannot completely rely on photosynthesis to meet carbon requirements, so sucrose is a commonly used exogenous carbon source in plant tissue culture. Sucrose also participates in regulation of medium osmotic potential, and changes in its concentration can affect tissue proliferation, water status, somatic embryo development, and organ differentiation. Therefore, when comparing formulations, it should be clearly determined whether sucrose has already been included in the medium.

(3) Vitamins and Organic Components

Organic components such as thiamine, nicotinic acid, pyridoxine, inositol, and glycine participate in coenzyme metabolism, cell division, and tissue growth. Vitamin systems differ markedly among basal media. For example, B5 medium has characteristic thiamine and nitrogen-source compositions, so tissue responses produced by different media cannot be explained solely by total salt concentration.

(4) Plant Growth Regulators

Auxin and Cytokinin are the core regulatory signals affecting the direction of organogenesis in tissue culture. The basal medium determines the nutritional and ionic background, while plant growth regulators further influence dedifferentiation, callus proliferation, shoot formation, and root formation. Optimization of a culture system therefore usually requires simultaneous consideration of basal salt composition and the type, concentration, and treatment stage of growth regulators.

2 Composition Characteristics and Application Positioning of Common Plant Culture Media

2.1 MS Medium

(1) Composition Characteristics

Murashige and Skoog (MS) medium has relatively high total inorganic salt levels and abundant nitrogen supply. NO₃⁻ and NH₄⁺ together constitute the major nitrogen sources, while the formulation also contains K, Ca, Mg, P, S, trace elements, Fe-EDTA, and a vitamin system. This relatively high nutritional strength supports rapidly dividing and proliferating in vitro tissues and is a major basis for the broad applicability of MS medium.

(2) Culture Applications

MS medium is widely used for callus induction, shoot differentiation, shoot proliferation, regeneration after genetic transformation, and aseptic seedling culture in herbaceous plants. It is also commonly used as a basal control when establishing tissue culture systems for new plant materials. For tissues with high nutritional requirements and active cell division, full-strength MS provides a relatively stable basal nutritional environment.

(3) Concentration Adjustment

1/2 MS and 1/4 MS reduce basal salt and nitrogen levels to create a lower-ionic-strength environment and are commonly used for rooting, seedling culture, and salt-sensitive materials. If root formation, tissue morphology, or Hyperhydricity improves markedly after reducing MS concentration, this generally suggests that the original culture system may have had a relatively high salt load and that the phenotype should not be explained solely by plant growth regulator concentrations.

2.2 B5 Medium

(1) Composition Characteristics

Gamborg B5 medium has nitrogen-source and vitamin systems distinct from those of MS. Its ammonium nitrogen level is relatively low, nitrate nitrogen accounts for a larger proportion, and the medium contains relatively high thiamine levels. The essential difference between B5 and MS lies in nutrient ratios and the organic-component background rather than simply total salt concentration.

(2) Culture Applications

B5 is mainly used for plant cell suspension culture, callus culture, and some specific plant materials. It is suitable for maintaining continuous cell proliferation and for studies of cell metabolism, stress responses, and secondary metabolism. For systems primarily focused on cell expansion, metabolic analysis, or callus maintenance, B5 has a relatively clear application positioning.

(3) Concentration and System Comparison

MS and B5 can be used to compare the effects of different nitrogen-source structures and vitamin backgrounds on cell proliferation, callus texture, and regeneration capacity. 1/2 B5 further reduces basal salt strength and can be used to distinguish the effects of medium type from those of total nutrient concentration.

2.3 N6 Medium

(1) Composition Characteristics

N6 medium is a basal system developed for tissue culture of gramineous plants. Its inorganic salt and nitrogen-source composition differs markedly from MS and is more suitable for acquisition and maintenance of embryogenic tissues in some cereal materials. Compared with general-purpose MS medium, N6 has a more specific positioning in embryogenic tissue culture of gramineous plants.

(2) Callus and Embryogenic Culture

N6 is commonly used for immature embryos, mature embryos, anthers, and callus cultures of rice, maize, wheat, and other gramineous plants and is often combined with Auxins such as 2,4-D to establish embryogenic callus induction systems. Experimental evaluation should focus on the proportion of embryogenic callus, tissue proliferation status, and subsequent regeneration capacity rather than recording only the callus-formation rate.

(3) Evaluation of Regeneration Systems

Regeneration studies in gramineous plants can compare callus induction rate, embryogenic maintenance capacity, differentiation rate, and regenerated plant quality under N6 and MS conditions. When the two basal media produce clear differences, nitrogen-source composition, salt concentration, and nutritional structure should be further analyzed for their effects on embryogenic acquisition and organogenesis.

2.4 WPM Medium

(1) Composition Characteristics

Woody Plant Medium (WPM) is an independent basal medium developed for woody plant culture. Its overall inorganic salt environment is relatively mild, but it is not simply a proportionally diluted MS formulation. WPM has its own ionic composition and is more suitable for the requirements of some woody materials for lower-salt environments and long-term in vitro culture.

(2) Culture Applications

WPM is commonly used for shoot tips, axillary buds, stem segments, and adventitious shoots of woody plants and is particularly suitable for shoot proliferation, micropropagation, and long-term subculture. For some woody materials showing abnormal leaves, restricted growth, or unstable long-term subculture under MS conditions, WPM can serve as an important basal-medium comparison condition.

(3) Application Boundaries

Responses of woody plants to basal salts and plant growth regulators vary markedly among species and genotypes. WPM should not be regarded as a universally optimal medium for all woody materials. Practical applications still require optimization according to species, explant type, culture stage, and Cytokinin/Auxin conditions.

2.5 White Medium

(1) Composition Characteristics

White medium is a classical low-salt plant culture system with overall mineral nutrient levels lower than those of MS. In the early development of plant tissue culture, it was mainly used for root tissues and some isolated plant materials. Its relatively low salt concentration creates a clearly different nutritional environment from high-salt MS medium.

(2) Culture Applications

White medium can be used for root culture, tissues with low nutrient requirements, and comparative studies of basal salt concentrations. It is also suitable for analyzing the effects of lower mineral nutrient strength on root elongation, tissue growth, and morphology of in vitro materials.

2.6 MT Medium

(1) System Characteristics

Murashige and Tucker (MT) medium is a modified system developed according to the requirements of in vitro plant culture. Although it has some formulation relationship with MS, its composition is more focused on the culture requirements of specific plant materials and therefore should not simply be treated as an MS concentration variant.

(2) Culture Applications

MT is representative in in vitro culture of citrus and related plants and can be used for shoot culture, tissue proliferation, and plant regeneration. Its application illustrates the material specificity of plant culture media: even among woody plants or fruit trees, a basal nutritional system different from WPM or standard MS may be required.

2.7 NLN Medium

(1) Culture Positioning

NLN medium is mainly used for microspore culture and related embryogenesis systems. Its culture objective is to promote conversion of microspores from the gametophytic developmental pathway to an embryogenic pathway, which has a physiological basis different from conventional shoot proliferation and callus culture.

(2) Research Applications

NLN can be used for isolated microspore culture, embryo-like structure formation, and haploid and doubled-haploid research. Microspore embryogenesis is highly sensitive to inorganic salt composition, sugar concentration, osmotic environment, and culture temperature. Therefore, NLN usually needs to be optimized together with pretreatment methods and culture conditions.

2.8 NS, NB, and DPD Media

(1) System Positioning

NS, NB, and DPD are culture systems used for specific plant cells, tissues, or culture procedures. Their general applicability is usually lower than that of MS, B5, N6, and WPM, but they can provide distinct nutritional, carbon-source, or osmotic environments for specific materials.

(2) Experimental Applications

These media are more appropriately selected according to specific plant materials and experimental workflows. Products without sucrose or agar allow carbon-source type, carbon-source concentration, and gelling-agent conditions to be independently adjusted for medium reconstruction and single-variable studies.

2.9 Hoagland Nutrient Solution

(1) System Positioning

Hoagland nutrient solution is mainly used for hydroponics and mineral nutrition studies of plants with intact root systems and has a different experimental positioning from aseptic in vitro tissue culture media such as MS, B5, and N6. Its major advantage is the ability to establish a chemically defined and controllable rhizosphere mineral nutrient environment.

(2) Nutrient-Deficiency Research

Hoagland variants deficient in N, P, S, Cu, Zn, or other nutrients can be used to establish controlled mineral nutrient-deficiency models for studying root morphological remodeling, elemental uptake and transport, deficiency responses, and nutrient recovery. They are therefore more suitable for nutritional physiology studies of intact plants and should not be directly treated as liquid substitutes for tissue culture media such as MS.

Table 1 Composition and Research Positioning of Common Plant Culture Media

3 Regulation of Organogenesis by Auxin and Cytokinin

3.1 Auxin Regulators

Auxin-type plant growth regulators mainly include 2,4-D, IAA, IBA, and NAA. These molecules differ markedly in chemical stability, activity, and suitable culture stages.

(1) 2,4-D and Dedifferentiation

2,4-D has strong effects on dedifferentiation and induction of cell division and is a commonly used Auxin regulator for callus and embryogenic tissue induction. Sustained high levels of 2,4-D can maintain tissues in an undifferentiated state for prolonged periods and may reduce subsequent organogenic capacity. Therefore, when cultures transition from callus induction to differentiation, 2,4-D usually needs to be reduced or removed.

(2) IBA and NAA in Root Formation

IBA and NAA are widely used for adventitious root induction. IBA can produce relatively stable rooting responses in many plants, whereas NAA is relatively strong and may increase basal callus formation at excessive concentrations. Rooting systems should be evaluated using rooting rate, root number, root length, root morphology, and subsequent transplantation performance rather than judging treatment effects solely by the appearance of root primordia.

(3) IAA and Natural Auxin Responses

IAA is a natural Auxin and more closely reflects the endogenous auxin-signaling environment of plants, but its chemical stability is relatively low. It has high application value in Auxin physiology, natural hormone responses, and receptor-signaling studies, while long-term culture experiments need to consider how degradation affects the actual effective concentration and experimental reproducibility.

3.2 Cytokinin Regulators

Cytokinins mainly promote cell division and shoot formation. Common components include 6-BA, Kinetin, Zeatin, and TDZ, and these molecules are not equivalent in shoot-induction efficiency, shoot quality, or subsequent elongation capacity.

(1) 6-BA

6-BA is widely used for shoot induction and subculture proliferation. Increasing 6-BA levels can increase shoot formation in some materials, but excessive stimulation may cause shortened internodes, dwarf shoots, Hyperhydricity, and reduced subsequent rooting capacity. Therefore, both the number of effective shoots and shoot quality should be evaluated.

(2) Zeatin

Zeatin is a natural Cytokinin and can effectively promote shoot formation in some woody plants and difficult-to-differentiate materials. When 6-BA treatment results in low differentiation rates or reduced shoot quality, comparison with Cytokinin types such as Zeatin can be used to evaluate material responses to differences in cytokinin structure.

(3) TDZ

TDZ has strong Cytokinin-like activity and can markedly promote shoot primordium formation and organogenesis even at relatively low concentrations. Higher concentrations or prolonged exposure may cause shoot clustering, vitrification, and restricted elongation. Therefore, experimental design with TDZ needs to control both concentration and treatment duration.

3.3 Auxin/Cytokinin Balance

A relatively high Auxin environment generally favors dedifferentiation, callus formation, or root formation, whereas a relatively high Cytokinin environment generally favors shoot formation, but this relationship is not a fixed concentration ratio. Species, genotype, explant type, tissue age, and endogenous hormone status can all alter sensitivity to exogenous plant growth regulators. Hormone combinations therefore need to be screened using concentration gradients and functional evaluation according to the specific material.

4 Medium Conversion During Different Culture Stages

4.1 Callus Induction

The callus-induction stage requires differentiated cells to re-enter active division, and the culture system usually contains a relatively strong Auxin signal. Experimental evaluation should further distinguish ordinary callus from embryogenic callus with regeneration potential and assess culture quality using tissue color, texture, growth status, embryogenic characteristics, and subsequent regeneration rate rather than using callus induction rate alone to represent regeneration-system efficiency.

4.2 Shoot Induction and Proliferation

During shoot induction, the culture objective shifts from maintaining undifferentiated cell proliferation to establishing new meristems. This usually requires reducing strong Auxin stimulation and increasing an appropriate Cytokinin signal. Shoot induction rate, number of effective shoots, shoot length, leaf morphology, and subsequent elongation capacity should be evaluated together to distinguish normal organogenesis from abnormal shoot-cluster proliferation.

4.3 Rooting Culture

The rooting stage commonly uses basal media with lower salt concentrations together with IBA or NAA to promote root primordium formation, and 1/2 MS is a commonly used basal system. Root primordium induction and root elongation have different physiological requirements. For some plants, short-term Auxin induction followed by transfer to a low-hormone or hormone-free medium can improve root elongation, branching, and subsequent transplantation adaptability.

4.4 Somatic Embryo Culture

Somatic embryogenesis usually includes embryogenic induction, embryogenic tissue proliferation, embryo formation, and maturation. Auxin is often required during the early stages to maintain the embryogenic state, whereas Auxin needs to be reduced during embryo formation and maturation, together with regulation by ABA, sugar concentration, and osmotic conditions. Continuous use of the same culture conditions can readily cause persistent proliferation of embryogenic tissue while preventing maturation.

5 Key Parameters for Medium Optimization

5.1 Basal Salt Concentration

Basal salt concentration directly affects ionic strength, nutrient supply, and tissue water status. When slow growth, Hyperhydricity, restricted rooting, or leaf abnormalities occur, different salt-strength systems such as MS, 1/2 MS, 1/4 MS, and WPM can be compared to distinguish effects of the basal nutritional environment from those of plant growth regulators.

5.2 Nitrogen-Source Composition

Nitrogen in culture media is mainly supplied as NO₃⁻ and NH₄⁺, and their ratio affects nitrogen metabolism, ionic balance, and pH changes during culture. Different materials have different tolerances to ammonium nitrogen. Therefore, differences among MS, B5, N6, and other culture systems involve not only total nitrogen concentration but also nitrogen-source composition and its balance with other nutrients.

5.3 Sucrose Concentration

Sucrose functions as both an exogenous carbon source and an osmotic regulator. Tissue culture, anther culture, somatic embryogenesis, and embryo maturation have different requirements for sucrose concentration. When using premixed media, it is therefore necessary to confirm whether sucrose is already included and account for it in the final formulation to avoid duplicate addition that would cause the actual carbon-source and osmotic conditions to deviate from the experimental design.

5.4 pH and Gelling Conditions

Medium pH affects the availability of phosphate, iron, and other ions and also changes gel-forming characteristics. The concentration of gelling agents such as Agar directly affects medium mechanical strength and tissue-available water. Abnormal phenotypes such as Hyperhydricity should be analyzed together with Cytokinin levels, gel strength, total salt concentration, and ventilation of the culture vessel.

6 Common Culture Abnormalities and Interpretation of Causes

6.1 Callus Proliferation With Restricted Differentiation

Continued callus proliferation without shoots or embryogenic structures usually indicates that dedifferentiation conditions can maintain cell proliferation but that organogenic signals are insufficient. Evaluation should focus on whether exposure to Auxins such as 2,4-D remains excessively high, whether long-term subculture has reduced embryogenic capacity, and whether Cytokinin conditions during the differentiation stage are appropriate.

6.2 Increased Shoot Proliferation With Restricted Elongation

Formation of large numbers of short shoots, shoot clusters, or shoot primordia is commonly associated with excessive Cytokinin stimulation, particularly the concentration and treatment duration of 6-BA and TDZ. In such cases, the number of effective shoots, shoot elongation, and normal leaf development should be used as major evaluation indicators rather than pursuing shoot proliferation rate alone.

6.3 Reduced Rooting Efficiency

Low rooting rates may be associated with excessively high basal salt concentration, inappropriate IBA/NAA conditions, poor physiological status of shoots, or persistent effects of previous Cytokinin treatment. Evaluation of rooting systems should simultaneously consider medium concentration, Auxin type, and treatment duration to distinguish limitations caused by the nutritional environment from those caused by hormonal responses.

6.4 Hyperhydricity

Hyperhydricity is characterized by abnormal tissue water content, translucent and fragile leaves, and impaired development of normal structures and is commonly associated with high Cytokinin levels, high humidity, insufficient medium mechanical strength, restricted vessel ventilation, and relatively high salt concentrations. This phenotype should be analyzed comprehensively as an abnormality of water balance in the culture system rather than attributed to a single component.

6.5 Tissue Browning

Tissue browning is mainly caused by the release and oxidation of phenolic compounds after explant injury and is particularly common in woody plants and materials rich in phenolics. Experimental control should focus on reducing mechanical damage, shortening initial subculture intervals, improving antioxidant and adsorption conditions, and optimizing light and culture environments according to material characteristics.

Table 2 Common Culture Abnormalities and Priority Troubleshooting Directions

7 Experimental Design for Medium Optimization

7.1 Basal Medium Screening

When establishing a culture system for new materials, basal media with clearly different nutritional characteristics should be compared first. Herbaceous materials can be compared using MS and its lower-concentration variants, woody plants can be compared using MS and WPM, and callus and embryogenic cultures of gramineous plants can be compared using MS and N6. Determining the basal salt background before optimizing plant growth regulators improves the interpretability of experimental variables.

7.2 Optimization of Plant Growth Regulators

Callus induction can use concentration gradients of 2,4-D or other Auxins, shoot formation can compare Cytokinins such as 6-BA, Zeatin, and TDZ, and the rooting stage can focus on IBA and NAA. Each round of experiments should preferably restrict the major variables so that results can be clearly attributed to basal salts, regulator type, or regulator concentration rather than simultaneous changes in multiple culture conditions.

7.3 Evaluation of Culture Outcomes

Culture-system screening should use functional indicators corresponding to the culture objective. Callus cultures should be evaluated for embryogenic capacity and subsequent regeneration, shoot cultures should record the number of effective shoots, shoot length, and abnormal shoot proportion, and rooting cultures should evaluate rooting rate, root number, root length, and root morphology. A single induction rate is insufficient to reflect the stability and actual regeneration value of a culture system.

8 Plant Culture Medium-Related Products

8.1 Common Basal Media for Plant Tissue Culture

8.2 MS, B5, and N6 Medium Formulation Variants

8.3 Hoagland Nutrient Solutions and Nutrient-Deficiency Culture Systems

Plant culture medium selection should match the material type, culture stage, and research objective. The basal salt system determines the nutritional background, plant growth regulators control the major developmental direction, and low-salt, nutrient-deficient, and reconstructable formulations can be used for culture-condition optimization and plant nutrition mechanism research.

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