PLANT REGENERATION THROUGH PROTOCORM-LIKE BODIES DERIVED FROM STEM THIN LAYER OF Anubias barteri var. Nana Petite

Article Highlights
- Effective induction of PLBs from stem thin layer and shoot regeneration using specific BAP and auxin combinations.
- Significant role of potato extract in enhancing shoot regeneration.
- Highest shoot regeneration rate achieved with 3 mg/L BAP concentration.

Abstract
Anubias barteri var. nana Petite, a highly valued ornamental aquatic plant in the Araceae family, faces challenges in vegetative propagation due to its slow multiplication rate. This study established a robust micropropagation protocol for rapid and efficient multiplication of A. barteri var. nana Petite. Explants were subjected to thorough sterilization, and shoot induction and multiplication were optimized using varying concentrations of Benzyl adenine (BA) on Murashige and Skoog (MS) medium. Protocorm-like bodies (PLBs) were induced from transverse thin-layer explants using combinations of BAP and Naphthaleneacetic acid (NAA). PLB multiplication and shoot regeneration were achieved through a sequential culture protocol. In addition, the influence of potato extract on shoot regeneration and the role of Indole-3-butyric Acid (IBA) in root development were explored. This study demonstrated a 100% survival rate of regenerated shoots in aquariums. Ongoing research is focused on further enhancing PLB multiplication. This optimized micropropagation protocol holds promise for the large-scale production of A. barteri var. nana Petite, addressing the limitations of natural propagation.

INTRODUCTION

Anubias barteri var. nana Petite holds significant commercial importance as a highly sought-after decorative aquatic plant within the Araceae family. This cultivated variant, renowned for its slow growth and cluster formation, typically ranging from 1 cm to 5 cm in height, has garnered popularity in both aquatic and ornamental settings(George et al., 2015).

Despite its aesthetic appeal, the natural propagation of A. barteri has proven inefficient, leading to numerous studies on micropropagation techniques, including in vitro multiplication(George et al., 2015);(Huang et al., 1994);(Kanchanapoom et al., 2012);(Rittirat et al., 2021), in vitro organogenesis(Surendra et al., 2019);(Rittirat et al., 2023), and hydroponic culture(Sholichah et al., 2020). However, these efforts have resulted in an average shoot production ranging between 2 shoots to 5 shoots from a single shoot.

To address this limitation, the utilization of protocorm-like bodies (PLBs) presents a promising approach for mass production because of their rapid formation, uniform characteristics, disease-free nature, and sustainability(Cardoso et al., 2020);(Sheelavanthmath et al., 2005). Noteworthy success has been achieved in the utilization of PLBs for the proliferation and multiplication of ornamental plants within the Araceae family. For example, Anthurium andreanum cv. CanCan exhibited 97.8% rate of PLB induction, averaging 120 PLBs for each explant over a period of 50 days in cultivation(Gantait et al., 2012);(Yu et al., 2009). Similarly, TDZ was shown effective for inducing PLBs from leaf explants of Cattleya tigrine with 17.5% induction rate, and GA₃significantly increases shoot regeneration from PLBs with approximately 220 developed plantlets per culture flask(Fritsche et al., 2022).

Despite these successes, there is currently no documented report on regeneration from PLBs on Anubias barteri var. nana Petite. Given the documented success of PLBs in various plant species, this study aimed to identify the optimal concentrations of plant growth regulators and potato extract for propagating this species through shoot tips and PLBs. The development of this micropropagation protocol provides a methodical approach to augment the propagation of Anubias barteri var. nana Petite, effectively overcoming the limitations of natural propagation methods.

MATERIALS AND METHODS

Shoot Sterilization

Shoots of Anubias barteri var. nana Petite were subjected to surface sterilization using soap for 15 minutes, followed by exposure to an antifungal agent (Mancozeb, India) for 10 minutes, washed with running water, and further rinsed with 70% alcohol for 30 seconds. Subsequently, explants were soaked in a commercial bleach solution (5% sodium hypochlorite) at concentrations of 10%, 20%, 30%, and 40% (v/v) for 15 minutes, followed by a 30-minute soak with an antibiotic solution (1 mg/mL). After disinfection, 3-5 mm shoot tip explants were isolated and placed on MS (Murashige and Skoog, 1962) basal medium.

Shoot Multiplication from Shoot Tips

Shoot tips free of microbial infection were cultured in MS media supplemented with (0, 1.0, 2.0, 3.0, 4.0 mg/L) BAP for 6 weeks for shoot induction. In a subsequent experiment, a single shoot (approximately 0.8 cm in height) was transferred to MS medium supplemented with various BAP concentrations (2.0, 3.0, 4.0 mg/L) for multiplication over 4 weeks. Shoot multiplication parameters, including the number of shoots, shoot height, number of leaves per shoot, and leaf length were recorded.

Protocorm-like Bodies (PLBs) Induction, Proliferation and Shoot Regeneration

For PLB induction, in vitro stems were separated from the cluster, leaves were removed, and the stem was cut into 1-2 mm transverse slices. These slices were placed on MS medium containing 10% (v/v) coconut water (CW), 2% (w/v) sucrose, and complemented with (0.5-3.0 mg/L) BAP along with (0.5-2.0 mg/L) NAA. The percentage of PLB induction and the number of PLBs per explant were recorded after 6 weeks of dark culturing. PLB morphology and counting were performed at 2X to 4X magnification under a stereo microscope (Olympus SZ51, Japan).

Secondary PLB Induction and Shoot Regeneration

For PLB proliferation, PLB clusters were separated into individuals and placed on MS medium containing 10% (v/v) CW, 2% (w/v) sucrose, added (0.5-1.5 mg/L) BA and (0.2-0.5 mg/L) IAA. After 3 weeks, the secondary PLB number and diameter, indicating PLB growth speed, were recorded.

To regenerate shoots from PLBs, tuber-shaped PLBs were separated and transferred to MS medium containing 10% (v/v) CW, 2% (w/v) sucrose, 1.0 g/L activated charcoal, 2 g/L peptone, and 1-3 mg/L BA and 0.5 mg/L IBA. After 3 weeks, shoot height was measured, and shoot quality was determined by leaf color, stem diameter, and leaf length.

Effect of Potato Extract on Shoot Proliferation

To assess the impact of potato extract on shoot proliferation, PLBs were cultured on MS medium complemented with 1.5 mg/L BAP and 0.5 mg/L NAA ranging from 10 g/L to 50 g/L of PE. Data on shoot number and individual shoot height were collected after the designated culture period.

Rooting

In vitro plantlets (1-1.5 cm) regenerated from PLBs were cultured on MS medium supplemented with 0.1% (w/v) activated carbon, 10% (v/v) CW, and Indole-3-Butyric Acid (IBA) at different concentrations (0.5, 1.0, 1.5, 2.0 mg/L). After 4 weeks, the number of roots per shoot, root length, shoot height, and number of leaves per shoot were recorded.

Experimental Design and Statistical Analysis

All experiments were conducted in a Completely Randomized Design (CRD) with three repetitions for each treatment, and each repetition included five jars, each containing at least one explant. The data underwent basic statistical analysis and were examined using

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