Introduction

Aloe vera (L.) Burm.f., often listed in older tissue-culture papers as Aloe barbadensis Mill., supplies gel, latex-associated compounds, and processed ingredients to cosmetic, nutraceutical, and pharmaceutical industries [13]. Farmers can multiply selected plants through offsets, but offset production is slow and uneven. Micropropagation offers a faster route when large numbers of uniform plants are required [24]. For a nursery or grower, though, the relevant product is not a shoot inside a vessel; it is a plant that roots, survives acclimatization, and establishes under production conditions.

Aloe vera (L.) Burm.f. is a high-value industrial crop widely utilized across the pharmaceutical, cosmetic, and functional food sectors. The therapeutic and commercial properties of A. vera are primarily localized in the colorless, mucilaginous gel of the inner leaf pulp and the exudates from the outer foliar layers [43]. Global industrial demand for uniform, high-quality planting material has placed significant pressure on conventional agricultural supply chains [63, 52, 40, 77, 21, 9, 26, 8, 64, 11, 37, 80]. Traditional propagation via natural rhizomatous offsets is severely limited by low multiplication coefficients, seasonal constraints, and susceptibility to soil-borne pathogens. In vitro micropropagation provides a scalable biotechnological strategy for the mass production of uniform, pathogen-free clonal stock within compressed operational timelines.

Despite extensive research into Aloe tissue culture systems over past decades, establishing high-frequency micropropagation protocols capable of maintaining genetic true-to-type conformity remains technically challenging [4, 39]. Two primary physiological obstacles impede commercial production: high mortality from culture medium browning caused by the oxidation of exuded phenolic compounds from damaged explant tissues and the risk of somaclonal variations induced by synthetic plant growth regulators (PGRs) [68, 55, 25, 12, 36, 30]. Furthermore, plant regeneration pathways that rely on an intermediate callus phase frequently suffer from chromosomal instabilities, leading to genetic off-types. Developing an auxin-free direct organogenesis protocol using natural organic biostimulants offers an efficient pathway for mass propagation while safeguarding long-term genetic integrity.

Materials and Methods

Testing different basal nutrient concentrations (full-strength, two-thirds strength, one-third strength MS) alongside variable AvG concentrations (0.0% to 40.0% v/v) indicates that reduced inorganic salt levels combined with organic supplementation optimize adventitious root development. Nutrient-free water-agar produces a minimal rooting response of 20.0% (2.7 ± 0.33 roots per shoot). Reducing inorganic salts to one-third strength MS containing 1.0% sucrose elevates rooting efficiency to 76.7% (5.6 ± 0.26 roots per shoot). Incorporating 20.0% (v/v) AvG into one-third strength MS medium achieves 100.0% rooting efficiency within 18 days, yielding a maximum root density of 9.8 ± 0.29 roots per shoot and an average root length of 3.1 ± 0.10 cm.

Rooted plantlets (6 to 8 cm) transferred to an inorganic substrate (Soilrite) under controlled environmental conditions (25 ± 2°C, 90–99% relative humidity) for 25 to 30 days achieved a 95.0% survival rate (76 of 80 plantlets). Acclimatized plantlets transferred to poly-greenhouse conditions (30 ± 2°C, 60–65% relative humidity) in a soil-vermicompost mixture (3:1 ratio) record a 100.0% survival rate over a 3-month hardening period. Long-term ex vitro field monitoring under full solar radiation confirms robust growth, with 84.2% of micropropagated plants exhibiting normal flowering after 18 to 20 months.

The complete propagation process follows a streamlined multi-stage development pipeline:

Explant Collection & Sterilization: Rhizomatous stem node excision (≈ 8 mm), Thiram (2.0%, 25 min), Tween-20 (2.5%, 3 min), and HgCl₂ (0.15%, 12 min) surface sterilization.

Shoot Proliferation Phase: Inoculation on MS medium supplemented with 2.5 mg/L BAP and 10.0% (v/v) AvG across 8-week subculture cycles.

Auxin-Free Rooting Phase: Separation of individual microshoots (≥ 2.0 cm) and culture on one-third strength MS medium with 1.0% sucrose and 20.0% (v/v) AvG.

Primary Acclimatization: Establishment in earthen pots containing sterile Soilrite under 90–99% relative humidity for 25 to 30 days.

Secondary Hardening: Transfer to a soil and vermicompost mixture (3:1 ratio) under poly-greenhouse conditions for 3 months.

Field Integration: Final transplanting into field soil under full sunlight, resulting in 100% survival and normal inflorescence development after 18 to 20 months.

Cytogenetic and Molecular Fidelity Assessment

Establishing genetic stability in micropropagated perennial crops is essential prior to industrial deployment. High shoot proliferation rates driven by synthetic growth regulators can induce somaclonal variation through gene mutations, chromosome structural alterations, or ploidy changes. Evaluating somatic mitosis, pollen microsporogenesis, and genomic marker profiles across two-year-old field-established regenerants validates long-term genetic fidelity.

Somatic Mitosis and Meiotic Cytogenetics: Karyotypic analysis of root-tip squashes confirms that regenerated plants maintain a stable diploid constitution of 2n = 2x = 14, matching the donor mother plant. The somatic karyotype displays a distinct bimodal distribution consisting of four pairs of long acrocentric chromosomes (14.4 to 17.9 µm) and three pairs of short sub-metacentric chromosomes (4.6 to 5.4 µm). No structural alterations, translocations, or ploidy shifts were observed across the evaluated metaphase plates.

 Results and Discussion

Meiotic behavior in microsporocytes proceeds normally from leptotene through tetrad formation. Analysis confirms homologous chromosome pairing, displaying seven distinct bivalents at diakinesis and metaphase-I. Normal 7:7 chromosome disjunction occurs at anaphase-I, followed by chromatid separation at anaphase-II. Low rates of minor meiotic irregularities such as single or double chromosomal bridges and lagging chromosomes occur in both regenerated plants (7.8%) and donor mother plants (8.5%). These minor aberrations represent natural background variation inherent to A. vera rather than tissue culture-induced somaclonal mutations.

Genomic Profiling via RAPD Markers: Randomly Amplified Polymorphic DNA (RAPD) marker analysis provides a genome-wide assessment of sequence stability. Genomic DNA extracted from ten randomly selected two-year-old field regenerants and the donor parent plant was screened using 32 decamer primers. Fifteen primers produced clear, reproducible amplification profiles yielding three or more bands per primer.

The 12 selected primers generated 65 monomorphic bands across all samples, averaging 5.5 loci per primer. Fragment sizes ranged from 200 to 2500 base pairs. Primers OPA-16 and OPM-06 produced the highest band density (10 bands each), whereas OPL-05 and OPAC-07 produced 3 bands each. Zero polymorphic bands were detected among the regenerated plants or between regenerants and the mother plant. The 100.0% monomorphism observed across all evaluated loci confirms genomic uniformity, validating that direct organogenesis using BAP supplemented with AvG carries minimal risk of somaclonal variation [6,59,62].

Commercial Implications and Conclusions

This micropropagation protocol provides a scalable, cost-effective framework for the commercial propagation of Aloe vera. Eliminating synthetic auxins across all regeneration stages reduces chemical input costs while avoiding callusing-related growth abnormalities. Furthermore, incorporating Aloe vera leaf gel (AvG) as a natural organic supplement offers significant physiological advantages: it accelerates shoot multiplication rates and suppresses medium browning caused by phenolic exudation. Combining 2.5 mg/L BAP with 10.0% (v/v) AvG yields 38.5 ± 0.44 shoots per explant by the third subculture cycle.

Transferring micro-shoots to one-third strength MS medium containing 20.0% (v/v) AvG achieves 100.0% root induction without exogenous auxins. Long-term molecular cytogenetic evaluations over two years ex vitro confirm complete genetic stability, showing stable diploid karyotypes (2n = 14), normal meiotic pairing, and 100.0% monomorphic RAPD profiles. Similar approaches using RAPD and ISSR markers have been employed to assess genetic stability in tissue-culture-propagated Aloe vera [62]. This validated culture system provides a reliable, high-yield methodology for the commercial production of genetically true-to-type Aloe vera planting stock.

Cost analysis needs the right denominator.

Costing is where many “low-cost” protocols become difficult to interpret. Aloe vera studies have tested alternative carbon sources, gelling agents, simpler media, and other lower-cost inputs [19,16].

Evidence from other commercial micropropagation systems shows why the accounting boundary matters. In a 2024 comparison of agar culture with a temporary-immersion system, labour accounted for 43% of variable costs in the agar method, and rooting remained a substantial expense in both systems [56]. The numerical values cannot simply be transferred to Aloe vera, but they demonstrate why reagent-only costing misses much of the process. A broader recent synthesis reaches the same conclusion: reducing a single input can shift cost or failure downstream rather than reduce total cost [51].

A credible economic analysis should keep direct consumables, labour, culture-room occupancy, energy, water, vessel handling, and acclimatization space within view. A simple sensitivity analysis for labour rate, contamination, multiplication quality, and hardening survival would show whether the claimed saving is robust.

Field performance and medicinal quality require direct evidence.

Field follow-up remains much thinner than the in vitro literature. The studies available here show that initial performance is modified by acclimatization substrate, spacing, fertilization, and local conditions [7,29,67]. A highly productive culture treatment may offer no field advantage, while a moderate multiplication treatment can still be useful if it gives uniform plants with low post-transfer loss. Claims of superiority should therefore specify the comparator and the observation period.

Medicinal quality requires a separate line of evidence. Gel yield does not establish chemical equivalence. Aloin, acemannan and related polysaccharides, phenolics, antioxidant activity, and mineral composition all depend on analytical method, sampling position, plant age, and growing conditions. Studies that compare in vitro-derived and conventional plants have shown the value of developmentally matched sampling and biochemical analysis [22,54,67]. Visual conformity or greater biomass cannot substitute for these measurements.

Clonal fidelity and authentication

For clonal production, fidelity is part of product identity rather than an optional final test. Aloe studies have used RAPD and ISSR markers for callus-derived material, SCoT and SSR markers for directly regenerated plants, and cytogenetic assessment after field establishment [62,29,27]. Each method answers a limited question. A small marker set cannot establish whole-genome identity; cytology may miss sequence-level change; morphology may miss both.

Conclusion

Direct shoot proliferation relies on balancing explant tissue architecture, basal salt composition and exogenous growth regulator regimes. Nodal segments (≈ 8 mm) isolated from rhizomatous stems containing axillary shoot buds serve as superior explant material compared to foliar tissue, initiating direct bud development without an intervening callus phase. Evaluating shoot organogenesis on Murashige and Skoog (MS) basal medium supplemented with variable concentrations of cytokinins reveals significant performance differences between 6-benzylaminopurine (BAP) and Kinetin (KIN). Across all tested concentrations, BAP outperforms KIN in promoting shoot bud proliferation. Inclusion of 2.5 mg/L BAP yields an average of 14.5 ± 0.31 shoots per explant during the initial 8-week regeneration cycle, which increases to 27.6 ± 0.53 shoots per explant by the third subculture cycle. In contrast, optimal KIN treatments (4.0 mg/L) produce only 9.7 ± 0.29 shoots per explant in the first cycle and 20.3 ± 0.33 shoots in the third cycle. Increasing BAP concentrations beyond 2.5 mg/L reduces shoot output, illustrating the inhibitory effect of supra-optimal cytokinin levels on shoot elongation.

Incorporating homogenized fresh Aloe vera leaf gel (AvG) as a natural organic supplement substantially enhances shoot multiplication while controlling phenolic exudation. AvG contains over 75 active compounds, including carbohydrates (5.43% w/w total sugar, comprising glucose, fructose, maltose, and sucrose), amino acids, enzymes, vitamins, and trace minerals. Combining 10.0% AvG with 2.5 mg/L BAP increases shoot formation to 17.8 ± 0.35 shoots per explant (2.0 cm) in the first cycle, reaching 38.5 ± 0.44 shoots per explant in the third cycle.

Beyond its nutritional contributions, AvG functions as an effective antioxidant matrix.
Unsupplemented explant cultures often experience severe media browning and tissue necrosis caused by polyphenol oxidase activity on released phenolics. Supplementing media with AvG neutralizes secondary phenolic leaching, stabilizing the culture microenvironment without requiring synthetic antioxidants such as ascorbic acid, citric acid, or polyvinylpyrrolidone.

Auxin-Free Rhizogenesis, Hardening, and Field Establishment. In vitro root induction usually requires synthetic auxins such as indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). However, exogenous auxins can alter endogenous hormonal balances, frequently inducing basal callusing and vascular anomalies that reduce ex vitro survival rates. Using AvG as a natural root-inducing supplement offers a reliable alternative that completely avoids synthetic auxins.

Direct regeneration from organized tissues is currently the best-supported route for clonal multiplication, but it is not a guarantee of uniformity. Callus-mediated regeneration has legitimate experimental uses and can also support production when culture history and fidelity are examined with appropriate care.

Progress now depends less on maximizing a single in vitro count and more on connecting the stages. Clean establishment, usable-shoot yield, rooting conversion, survival with new growth, and cost per hardened plant should be reported with stable denominators. Field performance, medicinal composition, pathogen status, and clonal fidelity should be measured whenever those claims are made. Used in this way, the proposed framework offers a practical basis for comparing protocols and for deciding which one’s merit nursery or field evaluation.

Declarations

Funding

No specific external funding was received for this work.

Conflict of interest

The authors declare that they have no competing interests.

Data availability

No new experimental datasets were generated. The literature considered in the review is identified in the reference list.

Author contribution statement

Sapna Kumari: conceptualization and writing original draft. Anindya Pattanayak: conceptualization and writing original draft, Mohammad Danish Masroor: conceptualization, data analysis and writing revised draft. S. N. P. Yadav Deen: supervision and writing review and editing.

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