Cactus micropropagation in vitro

Regeneration pathways

In vitro means growth of biological material outside the organism in a controlled vessel. In practice, it may include shoot multiplication from existing buds, direct organogenesis from an explant, regeneration after a callus phase or somatic embryogenesis. These routes differ in variation risk, efficiency and requirements. [Bouzroud et al. 2022]

The older book described tissue propagation as maintaining undifferentiated tissue by spinning test tubes. This is not an accurate model of modern cactus micropropagation. Explant properties, medium composition, growth regulators, light, temperature and time determine development. Vessel movement may matter in selected liquid systems, but is not required for every culture. [Bouzroud et al. 2022]

Explant origin and properties

An explant is the piece introduced into culture. It may contain an areole with a bud, a shoot tip, part of a seedling or another selected region. Age, position on the plant, health and previous conditions affect its response. Two equally sized pieces need not have the same developmental competence. [Bouzroud et al. 2022]

Axillary-bud activation uses existing meristem organization and usually maintains clonal fidelity better than regeneration through prolonged callus. It does not eliminate all risk of change. Starting material may already be a cellular mosaic, and successive subcultures impose selection pressure. The number of subcultures and lineage provenance should therefore remain in the documentation. [Bouzroud et al. 2022]

Micropropagation stages

Initiation establishes a growing culture without visible contamination. Multiplication increases shoot number through successive divisions and subcultures. Shoots are then prepared for independence, often by rooting and changing medium composition. The final stage is acclimatization outside the vessel. [Bouzroud et al. 2022]

Boundaries are not always sharp. Some cacti may form roots during multiplication, while others can be rooted directly outside culture. Separating stages still helps identify where losses occur. A high multiplication coefficient is not success if most shoots fail to become functional plants after acclimatization. [Bouzroud et al. 2022]

Medium composition and preparation

The medium supplies macronutrients, micronutrients and usually vitamins. It often contains sucrose as a carbon source, with agar or another agent giving it consistency. pH affects ion availability and gel properties. Common MS medium is the starting point for many studies, but is not optimal for every species and stage. [Bouzroud et al. 2022] [Szychowski 2010]

The recipe must account for water, reagent purity and sterilization method. Preparation conditions affect repeatability, so records include composition, pH, solidification method and sterilization procedure. Accurate measurement and process control are fundamental laboratory practices. Comparing two media is valuable when other culture conditions are described and as similar as possible. [Bouzroud et al. 2022] [Szychowski 2010]

Growth regulators and tissue responses

Cytokinins may favour bud activation and shoot multiplication, while auxins participate in processes including root initiation. They do not act as two simple switches. Compound type, concentration, relative proportions, exposure time and endogenous tissue state matter. The same dose may give a different result in another explant. [Cortés-Olmos et al. 2025] [Bouzroud et al. 2022]

Studies of Gymnocalycium ‘Fancy’ showed differences between material types and regulator treatments. Unexpected morphotypes appeared in some cultures, which the authors associated with possible somaclonal variation and TDZ effects. One experiment's result does not justify transferring its concentrations to the entire family. [Cortés-Olmos et al. 2025] [Bouzroud et al. 2022]

Photosynthesis and carbon balance in the vessel

In vitro shoots can photosynthesize. The contribution depends on light, carbon dioxide concentration, vessel ventilation, sugar amount and stomatal condition. In a closed vessel containing sucrose, the plant often functions mixotrophically. It uses both organic carbon in the medium and its own assimilation. [Bouzroud et al. 2022]

Insufficient gas exchange favours carbon dioxide and ethylene fluctuations. High humidity and medium composition may cause hyperhydricity. Tissues become glassy, excessively hydrated, mechanically underdeveloped and less photosynthetically effective. A large, rapidly growing shoot is not always good-quality material for subsequent acclimatization. [Bouzroud et al. 2022]

Asepsis and contamination sources

Sugar- and mineral-rich media also support bacterial and fungal growth. Sources include the explant surface, plant interior, tools, vessel, air and operator's hands. The greatest risk arises during opening and subculturing, when the sterile interior contacts the surroundings. [Bouzroud et al. 2022] [Szychowski 2010]

Asepsis means controlled work in a designated space, equipment preparation and procedure validation. An additive inhibiting microorganisms does not replace this organization and may also affect plant tissue. Absence of visible growth does not establish absence of microorganisms. Contamination may appear only after another subculture. [Bouzroud et al. 2022] [Szychowski 2010]

Pathogen control

Some procedures using very small meristems may reduce particular viruses, especially when combined with other treatments. Effectiveness requires confirmation by diagnostic testing. Ordinary multiplication of an areole-bearing piece may reproduce infected material even if the culture looks clean. [Bouzroud et al. 2022]

Healthy material should mean the outcome of a defined procedure and tests, not merely absence of symptoms. This matters for exchanges between collections and protection of rare lines. Aseptic describes working conditions, while free of a particular pathogen describes a diagnostic result. These are different claims. [Bouzroud et al. 2022]

Clonal fidelity and somaclonal variation

Multiplication from existing buds usually limits developmental reorganization and is therefore preferred when preserving a clone. Regeneration after a callus phase and prolonged culture increase the possibility of genetic and epigenetic changes. Somaclonal variation may be valuable for breeding, but is an error when producing faithful copies. [Cortés-Olmos et al. 2025] [Bouzroud et al. 2022]

Appearance after removal from the vessel does not establish complete fidelity. Some changes arise from the in vitro environment itself and may disappear. Others persist in subsequent growth. Control includes comparing morphology after acclimatization and trait stability, with cytological or molecular methods if needed. Several identical plants do not demonstrate a uniform entire batch. [Cortés-Olmos et al. 2025] [Bouzroud et al. 2022]

Acclimatisation physiology

The vessel has high humidity, reduced gas exchange, constant sugar access and gentle light. After transfer, the plant must limit water loss, increase its own photosynthetic contribution and activate roots in the new substrate. Stomata and the protective surface layer do not always immediately function as in a plant grown outside culture. [Bouzroud et al. 2022]

Lower humidity gradually and increase light without abrupt overheating. The substrate must maintain contact with fine roots while providing oxygen. Hyperhydric shoots are particularly vulnerable to wilting and damage. Losses at this stage do not invalidate earlier regeneration quality, but show that the protocol did not yet cover the full cycle. [Bouzroud et al. 2022]

Assessing whole-cycle efficiency

Compare multiplication coefficient with the percentage of uncontaminated cultures, shoot quality, rooting success and survival after acclimatization. Subculture number, time, cost and fidelity to the mother plant also matter. The fastest treatment need not be best if it produces abundant callus, hyperhydricity or unstable morphotypes. [Bouzroud et al. 2022]

Konrad Szychowski's 2010 series remains a valuable record of interest in micropropagation among Polish collectors. The modern account retains its four stages and questions about equipment, media and sterility. Post-acclimatization monitoring adds persistence of the resulting traits and actual plant survival. Only this record allows comparison of different protocols' efficiency. [Bouzroud et al. 2022]

Documenting and scaling cultures

In one culture, hidden contamination or a labelling error affects few shoots. After several subcultures, the same error may affect hundreds of plants. Each line requires a permanent code, date and record of the medium used. Material of uncertain provenance should not be combined merely because it looks similar. [Bouzroud et al. 2022] [Szychowski 2010]

Control batches and limited subculture numbers help detect trait drift. Retain the mother plant and material from an earlier stage as references. Micropropagation is efficient precisely because it amplifies both a correct process and an unnoticed mistake. [Bouzroud et al. 2022] [Szychowski 2010]

Two regeneration routes in studies

Blossfeldia liliputana (B. liliputiana in the paper's title) regenerated shoots by organogenesis through callus. For the monstrous form of Copiapoa humilis subsp. tenuissima (C. tenuissima in the paper), somatic embryo development was studied. A comparison of the experiments discusses the effects of light, growth regulators and sucrose at individual stages. [Ivannikov et al. 2022][Lema-Rumińska et al. 2013]

Micropropagation of Stenocactus coptonogonus

Pérez Molphe Balch and co-authors obtained an average of 16.75 ± 2.47 shoots per explant in Stenocactus coptonogonus on MS medium with 1 mg/l BA, a cytokinin promoting areole-bud development. Explants came from aseptic seedlings, measured 3–10 mm and contained areoles after removal of the shoot tip and base. Cultures were kept at 25 ± 2°C with 16 hours of light and a photon flux of 54 µmol m⁻² s⁻¹. The dispersion value is the standard error. The experiment included three replicates with 5–20 explants per treatment, without a separate count given for this species.[Pérez Molphe Balch et al. 1998, pp. 131–133, Table 1]

On medium with 1 mg/l IBA, 90% of 20 S. coptonogonus shoots rooted in five weeks. In separate substrate-transfer trials, after four weeks, 80% of 20 shoots previously rooted in vitro and 75% of 20 shoots directed to rooting outside culture with Raizone survived. These stages have different denominators and conditions. The table contains no data for S. boedekerianus, and the entry Echinofossulocactus sp. remains separate material unidentified to species.[Pérez Molphe Balch et al. 1998, pp. 132–134, Table 2]

Regenerating yellow pitahaya from cotyledon tissue

Pelah and colleagues compared cotyledon, hypocotyl and epicotyl fragments from ten-day-old Selenicereus megalanthus seedlings. The proximal part of the cotyledon showed the strongest response. On MS medium, 200 µM thidiazuron stimulated organogenic callus and bud formation. Other tissues retained different regeneration capacities. Treatments comprised twelve explants in each of four replicates. Cultures were maintained at 24 ± 1°C with sixteen hours of light and eight hours of darkness. [Pelah et al. 2002, p. 81–83, ryciny 1–2]

After thirty days, bud-bearing callus was transferred to growth-regulator-free MS medium to allow shoot elongation. Fifty isolated shoots were rooted on MS containing 5.3 µM naphthaleneacetic acid. All had rooted after three weeks. Rooted plants were transferred to a greenhouse in a mixture of peat, perlite and vermiculite in equal parts by volume. Survival was 90% after two months. Rooting and acclimatization results describe successive stages of the protocol, each with its own assessment time. [Pelah et al. 2002, p. 82–83]