Effect of Plant Growth Regulators on In vitro Shoot Multiplication and Rooting in Bacopa monnieri

Introduction

Bacopa monnieri (L.) Pennell, belonging to the family Plantaginaceae, is an important medicinal plant known for its neuropharmacological properties, particularly in enhancing memory and treating neurological disorders. It is widely distributed in wetlands and marshy areas of tropical and subtropical regions. The increasing demand for herbal medicines has led to excessive harvesting of natural populations, resulting in depletion of this valuable species. Conventional propagation through seeds is limited due to poor germination rates and slow growth. Therefore, plant tissue culture techniques offer a promising alternative for rapid and large-scale propagation. Plant Growth Regulators (PGRs), including cytokinins and auxins, play a vital role in regulating plant growth and morphogenesis in tissue culture. Cytokinins are primarily responsible for shoot proliferation, whereas auxins promote root formation. The present study aims to investigate the effect of different PGRs on in vitro shoot multiplication and rooting in Bacopa monnieri.

Materials and Methods

Plant Material and Explant Preparation

Healthy and disease-free plants of Bacopa monnieri were collected from natural wetland habitats and used as the source material for in vitro studies. Nodal segments measuring approximately 1-2 cm in length were excised from actively growing shoots and selected as explants due to their high regenerative potential and ability to produce multiple shoots under controlled culture conditions.

Surface Sterilization

The explants were first washed thoroughly under running tap water for 15-20 minutes to remove surface contaminants such as dust and debris. This was followed by treatment with 1-2% Teepol (a liquid detergent) for 5 minutes to eliminate adhering impurities, after which the explants were rinsed with distilled water. Subsequently, surface sterilization was carried out under aseptic conditions using 70% ethanol for 30 seconds, followed by 0.1% mercuric chloride (HgCl₂) treatment for 2-3 minutes. Finally, the explants were rinsed 3-4 times with sterile distilled water to remove any residual traces of sterilizing agents.

Culture Medium

Murashige and Skoog (MS) basal medium was used as the nutrient medium for culture establishment and growth. The medium was supplemented with 3% (w/v) sucrose as a carbon source and solidified with 0.8% (w/v) agar. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C and 15 psi pressure for 15-20 minutes to ensure sterility.

Shoot Multiplication

For shoot induction and multiplication, the sterilized nodal explants were inoculated onto MS medium supplemented with varying concentrations of cytokinins such as 6benzylaminopurine (BAP) ranging from 0.5 to 2.0 mg/L and kinetin from 0.5 to 1.5 mg/L, either alone or in combination with auxins like indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA). The cultures were incubated in a growth room maintained at 25±2°C under a photoperiod of 16 hours light and 8 hours dark. Observations on shoot initiation, number of shoots per explant, and shoot length were recorded at regular intervals.

Root Induction

Well-developed shoots obtained from multiplication cultures were carefully excised and transferred to fresh MS medium supplemented with different concentrations of auxins for root induction. Auxins such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthalene acetic acid (NAA) were used in the range of 0.5-1.5 mg/L. The cultures were maintained under the same environmental conditions, and observations on root number, length, and percentage of rooting were recorded.

Acclimatization

The rooted plantlets were gently removed from the culture vessels, washed thoroughly to remove adhering agar, and transferred to small pots containing a sterilized mixture of soil, sand, and vermiculite in the ratio of 1:1:1. The plantlets were initially maintained under high humidity conditions, often by covering with transparent polyethylene bags, and gradually acclimatized to ambient environmental conditions. After successful hardening, the plants were transferred to field conditions for further growth and development.

Results

Effect of Cytokinins on Shoot Multiplication

BAP was found to be more effective than kinetin in inducing shoot proliferation.

Effect of Auxins on Rooting

Acclimatization

Approximately 80% of plantlets survived after transfer to soil under greenhouse conditions.

Discussion

The results clearly indicate that cytokinins, particularly BAP, play a crucial role in shoot multiplication. The synergistic effect of BAP and IAA significantly enhanced shoot proliferation, which is consistent with earlier findings. Auxins were essential for root induction, with IBA being more effective for root elongation and IAA for root initiation. The variation in response may be attributed to differential uptake and metabolism of plant growth regulators. The success of acclimatization demonstrates the practical applicability of the protocol for large-scale propagation.

Conclusion

• BAP (1.5 mg/L) is optimal for shoot multiplication

References

1. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
2. George EF, Hall MA, De Klerk GJ, editors. Plant propagation by tissue culture. 3rd ed. Dordrecht: Springer; 2008.
3. Tiwari V, Singh BD, Tiwari KN. Shoot regeneration and somatic embryogenesis from different explants of Brahmi (Bacopa monniera). Plant Cell Rep. 1998;17(67):538-543. https://doi.org/10.1007/s002990050438
4. Sharma S, Kamal B, Rathi N, Chauhan S, Jadon A, Vats S, et al. Highly efficient shoot regeneration of Bacopa monnieri (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD. Acta Physiol Plant. 2010;32(3):443-452. https://doi.org/10.1007/s11738009-0421-4
5. Khan MA, Abbasi BH, Ahmed N, Ali H. Effects of plant growth regulators on cell growth and bacoside A production in callus cultures of Bacopa monnieri. Afr J Biotechnol. 2012;11(36):8849-8854. https://doi.org/10.5897/AJB11.4116