In vitro regeneration protocol for commercially important Banana cultivar through meristem culture

Introduction

Meristem culture-the excision and in vitro growth of a tiny dome of undifferentiated cells at the shoot apex-was originally developed to eliminate viruses from clonally propagated crops and has since become the backbone of commercial banana micropropagation worldwide [1] . Kenya is a major producer of banana in East Africa, with an annual output exceeding 1.5 million tonnes, yet the demand for high-quality, disease-free planting material far outstrips supply, particularly for the Cavendish subgroup cultivar Grand Naine that dominates export markets [2] . Conventional propagation through sword suckers yields only 5-15 plants per mother plant per year and risks transmitting Fusarium wilt (TR4), banana bunchy top virus, and bacterial soft rot [3] . In vitro multiplication can amplify this rate several hundred-fold while ensuring pathogen-free stock, but success depends on the precise combination of growth regulators, subculture frequency, and acclimatisation conditions, which vary across genotypes and laboratory environments [4] . BAP is the most commonly used cytokinin for banana shoot multiplication, yet optimum concentrations reported in the literature range from 2 to 8 mg L⁻¹ depending on cultivar and explant age [5] . This research aimed to standardise a reliable, reproducible meristem culture protocol for cv. Grand Naine, optimise BAP and IAA concentrations for maximum shoot multiplication, evaluate rooting media, and assess plantlet survival during hardening under the sub-humid conditions of central Kenya.

Material and Methods

Material

Healthy, virus-indexed mother plants of cv. Grand Naine were obtained from a certified tissue culture nursery in Nairobi. Sword suckers (15-20 cm height) were collected, trimmed to a 3 cm corm block, and surface-sterilised by sequential treatment with 70% ethanol (1 min), 0.1% mercuric chloride (8 min), and three rinses in sterile distilled water. Shoot-tip explants (3-5 mm, including 1-2 leaf primordia) were excised under a laminar air flow cabinet and placed on initiation medium. The work was carried out from March to October 2023 [6] .

Methods

Initiation medium was MS basal salts with 30 g L⁻¹ sucrose, 8 g L⁻¹ agar, and 3.0 mg L⁻¹ BAP + 0.5 mg L⁻¹ IAA (pH 5.8). After 4 weeks, initiated cultures were transferred to multiplication media containing BAP at 0, 2, 3, 4, 5, and 6 mg L⁻¹ combined with 0. Comprehensive Interpretation BAP at 4.0 mg L⁻¹ combined with 0.5 mg L⁻¹ IAA produced the highest multiplication ratio (5.7 at cycle 4), and multiplication peaked at the fourth subculture before declining slightly in the fifth cycle. IBA at 1.0 mg L⁻¹ on half-strength MS gave the best balance of rooting percentage (92.3%) and root quality. Hardened plantlets showed 88.6% survival with vigorous growth after 30 days in greenhouse conditions.

Discussion

The results of this research can be summarised as follows: 4.0 mg L⁻¹ BAP was the optimum cytokinin concentration; multiplication improved progressively through four subculture cycles before plateauing; and 1.0 mg L⁻¹ IBA was best for rooting. The literature broadly agrees with these findings. Ali et al. [4] reported peak multiplication at 4-5 mg L⁻¹ BAP for Grand Naine in Bangladesh, and Vuylsteke and De Langhe [7] placed the optimum at 4.5 mg L⁻¹ for AAA cultivars.

Where the present data diverge is in the decline at 6 mg L⁻¹ BAP, which other authors have attributed to vitrification (hyperhydricity) and the formation of stunted, glassy shoots that fail to elongate [8] . The possible explanation is that supra-optimal cytokinin levels disrupt the auxin-cytokinin ratio needed for normal shoot organogenesis, diverting meristematic activity towards callus formation rather than axillary bud proliferation [5] . The high hardening survival (88.6%) is encouraging for commercial adoption under semi-arid conditions. The soilsand-vermicompost mix likely provided adequate aeration and moisture retention, and the controlled humidity (80%) during the first two weeks prevented desiccation-a common cause of hardening failure in tropical tissue culture [9] .

Conclusion

The semi-arid plains of western Kiambu County demand large volumes of disease-free banana planting material that conventional sucker propagation cannot supply. This research established that MS medium supplemented with 4.0 mg L⁻¹ BAP and 0.5 mg L⁻¹ IAA supports a multiplication ratio of 5.7 shoots per explant by the fourth subculture, while half-strength MS with 1.0 mg L⁻¹ IBA achieves 92.3% rooting. Hardened plantlets survived at 88.6% in greenhouse conditions. On a practical level, this protocol can generate over 3,000 uniform plantlets from a single meristem within 8-10 months, making it suitable for scaling up through institutional or commercial tissue culture laboratories. Looking ahead, testing this protocol under field conditions over a full crop cycle and evaluating its cost-effectiveness relative to sucker-based planting would strengthen the case for large-scale adoption.

References

1. Hwang SC, Ko WH. Cavendish banana cultivars resistant to Fusarium wilt acquired through somaclonal variation. Plant Disease. 2004;88(6):580-588.
2. FAO. Banana market review: preliminary results 2023. Food and Agriculture Organization, Rome. 2024.
3. Ploetz RC. Fusarium wilt of banana. Phytopathology. 2015;105(12):1512-1521.
4. Ali A, Sajid A, Naveed SH. Micropropagation of banana cv. Grand Naine through meristem culture. Pakistan Journal of Botany. 2011;43(5):2291-2297.
5. Israeli Y, Reuveni O, Lahav E. Qualitative aspects of somaclonal variation in banana propagated by in vitro techniques. Scientia Horticulturae. 1991;48(1-2):71-88.
6. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 1962;15(3):473-497.
7. Vuylsteke D, De Langhe E. Feasibility of in vitro propagation of bananas and plantains. Tropical Agriculture (Trinidad). 1985;62(4):323-328.
8. Ziv M. In vitro hardening and acclimatization of tissue culture plants. Plant Cell, Tissue and Organ Culture. 1991;24(1):1-8.
9. Preece JE, Sutter EG. Acclimatization of micropropagated plants to the greenhouse and field. Micropropagation. 1991;71-93.
10. Dhed'a D, Dumortier F, Panis B. Plant regeneration in cell suspension cultures of cooking banana cv. Bluggoe. Fruits. 1991;46(2):125-135.
11. Arinaitwe G, Rubaihayo PR, Magambo MJS. Proliferation rate effects of cytokinins on banana cultivar. African Crop Science Journal. 2000;8(1):8188. International Journal of Agriculture and Food Science https://www.agriculturaljournals.com
12. Strosse H, Van den Houwe I, Panis B. Banana cell and tissue culture: review. Banana Improvement: Cellular, Molecular Biology, and Induced Mutations. 2004;1-12.
13. Bairu MW, Aremu AO, Van Staden J. Somaclonal variation in plants: causes and detection methods. Plant Growth Regulation. 2011;63(2):147-173.
14. Kulkarni VM, Suprasanna P, Bapat VA. Plant regeneration through multiple shoot formation and somatic embryogenesis in banana. Current Science. 2006;91(9):1210-1214.
15. Rahman MZ, Nasiruddin KM, Amin MA, Islam MN. In vitro response and shoot multiplication of banana. Biotechnology. 2004;3(1):36-40.