BS Agricultural Biotechnology Major in Crop Biotechnology – (UPLB)

CHAPTER 1 – INTRODUCTION

Background of the Study

Aquilaria malaccensis Lam. is a tropical tree species widely known for its ability to produce agarwood, a highly valuable resinous wood used in perfumery, traditional medicine, incense, and religious rituals. Agarwood commands premium prices in international markets, particularly in the Middle East and East Asia, making A. malaccensis an economically important species. However, unsustainable harvesting practices and habitat destruction have resulted in a rapid decline of natural populations, leading to its inclusion in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

Conventional propagation of A. malaccensis through seeds is limited by low germination rates, seasonal availability, and genetic variability. Vegetative propagation methods are also constrained by poor rooting and slow growth. As a result, plant tissue culture has emerged as a promising alternative for mass propagation, conservation, and genetic improvement of this endangered species.

Callus induction is a fundamental process in plant tissue culture and serves as a prerequisite for advanced applications such as somatic embryogenesis, genetic transformation, and secondary metabolite production. In woody plant species, successful callus induction is often influenced by the type of explant, culture medium, and particularly the composition and concentration of plant growth regulators (PGRs). Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthaleneacetic acid (NAA), either alone or in combination with cytokinins like 6-benzylaminopurine (BAP) and kinetin, have been reported to promote callus formation in various plant species.

Despite the growing interest in A. malaccensis tissue culture, standardized and optimized protocols for callus induction remain limited. This study seeks to address this gap by evaluating the effects of different PGR combinations on in vitro callus induction of A. malaccensis, thereby contributing to sustainable propagation and conservation efforts.

Statement of the Problem

The declining population of Aquilaria malaccensis necessitates the development of effective propagation and conservation strategies. However, the lack of a standardized in vitro callus induction protocol limits the application of tissue culture techniques for this species. Specifically, there is insufficient information on the optimal combination and concentration of plant growth regulators required to induce high-quality callus formation in A. malaccensis explants.

This study aims to answer the following questions:

Which plant growth regulator combinations promote the highest callus induction rate in Aquilaria malaccensis?
How do different PGR treatments affect the time required for callus initiation?
What are the morphological characteristics of callus formed under different PGR combinations?

Objectives of the Study

General Objective

To optimize in vitro callus induction in Aquilaria malaccensis using different plant growth regulator combinations.

Specific Objectives

To evaluate the effects of different auxin and cytokinin combinations on callus induction efficiency.
To determine the most effective PGR concentration for callus formation.
To compare callus characteristics such as color, texture, and growth rate among treatments.
To identify the optimal plant growth regulator combination for sustained callus induction.

Significance of the Study

The findings of this study will benefit the following sectors:

Researchers and Students: The study will provide baseline information and a reference protocol for future tissue culture and biotechnology research on Aquilaria malaccensis and other woody species.
Forestry and Conservation Agencies: An optimized callus induction protocol can support conservation programs and reduce dependence on wild populations.
Agarwood Industry Stakeholders: Improved propagation techniques can contribute to sustainable plantation establishment and long-term agarwood production.

Scope and Limitations of the Study

This study will focus on the in vitro induction of callus from selected explants of Aquilaria malaccensis using Murashige and Skoog (MS) basal medium supplemented with different plant growth regulator combinations. Data collection will be limited to callus induction rate, time to callus initiation, and morphological characteristics of the callus.

The study will not include genetic, biochemical, or metabolite analyses of the induced callus. Results obtained under controlled laboratory conditions may not directly reflect responses under field conditions.

CHAPTER 2

REVIEW OF RELATED LITERATURE

Overview of Aquilaria malaccensis

Aquilaria malaccensis belongs to the family Thymelaeaceae and is native to Southeast Asia. The species is best known for producing agarwood as a response to injury or microbial infection. Due to its high economic value, the species has been subjected to overexploitation, leading to population decline and conservation concerns.

Plant Tissue Culture in Woody Species

Plant tissue culture is a technique that enables the growth of plant cells, tissues, or organs under aseptic and controlled conditions. In woody plant species, tissue culture is particularly useful for overcoming limitations associated with conventional propagation methods. However, woody plants often exhibit challenges such as phenolic exudation and low in vitro responsiveness.

Callus Induction and Its Importance

Callus is an unorganized mass of proliferating cells formed from plant tissues when cultured under appropriate conditions. Callus induction is a critical step in micropropagation, somatic embryogenesis, and genetic transformation. The success of callus formation depends on factors such as explant type, nutrient medium, and plant growth regulator composition.

Role of Plant Growth Regulators in Callus Formation

Plant growth regulators play a crucial role in regulating cell division and differentiation in plant tissue culture. Auxins, particularly 2,4-D and NAA, are commonly used to induce callus formation. Cytokinins such as BAP and kinetin are often combined with auxins to enhance cell proliferation and improve callus quality. The balance between auxin and cytokinin concentrations determines the type and growth characteristics of the induced callus.

Previous Studies on Callus Induction in Aquilaria Species

Several studies have reported successful callus induction in Aquilaria species using different explants and PGR combinations. Research has shown that auxin-dominant media, particularly those supplemented with 2,4-D, are effective in inducing callus formation. However, variations in response have been observed depending on explant source and culture conditions, indicating the need for further optimization.

CHAPTER 3

MATERIALS AND METHODS

Research Design

The study will employ an experimental research design using a Completely Randomized Design (CRD) to evaluate the effects of different plant growth regulator combinations on callus induction.

Plant Material and Explant Preparation

Healthy Aquilaria malaccensis seedlings will be used as the source of explants. Young leaves or nodal segments will be excised and washed under running tap water. Surface sterilization will be conducted using 70% ethanol followed by sodium hypochlorite solution, after which the explants will be rinsed thoroughly with sterile distilled water.

Culture Medium and Conditions

Murashige and Skoog (MS) basal medium will be used, supplemented with 3% sucrose and solidified with agar. The pH of the medium will be adjusted to 5.7 ± 0.1 prior to autoclaving. Cultures will be incubated at 25 ± 2 °C under dark conditions to promote callus induction.

Plant Growth Regulator Treatments

Different combinations of auxins and cytokinins will be incorporated into the culture medium as follows:

Control: MS medium without plant growth regulators
2,4-D at 1.0 mg/L
2,4-D at 2.0 mg/L
NAA (1.0 mg/L) + BAP (0.5 mg/L)
NAA (2.0 mg/L) + BAP (1.0 mg/L)
2,4-D (1.0 mg/L) + kinetin (0.5 mg/L)
2,4-D (2.0 mg/L) + kinetin (1.0 mg/L)

Data Collection

The following parameters will be recorded:

Percentage of callus induction
Number of days to callus initiation
Callus fresh weight
Callus color and texture

Statistical Analysis

Data will be analyzed using Analysis of Variance (ANOVA). Significant differences among treatment means will be determined using Tukey’s Honestly Significant Difference (HSD) test at a 5% level of significance.

CHAPTER 4

RESULTS AND DISCUSSION

Overview of the Chapter

This chapter presents the results obtained from the in vitro callus induction experiments conducted on Aquilaria malaccensis using different plant growth regulator (PGR) combinations. The findings are presented in tables and figures and are discussed in relation to the objectives of the study and relevant literature.

Callus Induction Percentage

This section presents the percentage of explants that successfully formed callus under each PGR treatment.

Table 4.1. Percentage of callus induction of Aquilaria malaccensis explants under different plant growth regulator treatments.

(Insert table showing treatments vs % callus induction)

The results showed significant differences in callus induction percentages among the treatments. Explants cultured on media supplemented with auxin-containing PGRs exhibited higher callus induction rates compared to the control treatment.

Discussion:
The enhanced callus induction observed in auxin-supplemented media may be attributed to the role of auxins in promoting cell division and dedifferentiation. Similar findings have been reported in previous studies on Aquilaria species and other woody plants, where auxin-dominant media effectively induced callus formation.

Days to Callus Initiation

This section describes the time required for visible callus formation following culture initiation.

Table 4.2. Mean number of days to callus initiation under different PGR treatments.

(Insert table showing treatments vs days to callus initiation)

Callus initiation occurred earlier in treatments containing higher concentrations of auxins compared to the control and auxin–cytokinin combinations.

Discussion:
The reduced time to callus initiation in auxin-rich treatments suggests that auxins accelerate cellular dedifferentiation. This observation is consistent with previous tissue culture studies involving woody species.

Callus Morphological Characteristics

The induced callus was evaluated based on color and texture.

Table 4.3. Morphological characteristics of callus induced under different PGR treatments.

(Insert table describing callus color and texture per treatment)

Friable and light-colored callus was predominantly observed in selected auxin–cytokinin combinations, while compact or browned callus was more common in other treatments.

Discussion:
Callus morphology is an important indicator of tissue viability and suitability for further applications. Friable callus is often preferred for subsequent regeneration or cell suspension cultures.

Callus Fresh Weight

This section presents the mean fresh weight of callus obtained from each treatment.

Table 4.4. Mean fresh weight of callus induced under different PGR treatments.

(Insert table showing treatments vs callus fresh weight)

Treatments containing optimized auxin concentrations resulted in significantly higher callus biomass compared to the control.

Discussion:
Increased callus biomass reflects enhanced cell proliferation, which is influenced by PGR balance. Similar trends have been reported in earlier studies on callus induction in woody plants.

Summary of Key Findings

Overall, the results indicate that plant growth regulator composition significantly affects callus induction efficiency, initiation time, and morphological quality in Aquilaria malaccensis.

CHAPTER 5

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary of the Study

This study was conducted to optimize in vitro callus induction in Aquilaria malaccensis using different plant growth regulator combinations. Explants were cultured on Murashige and Skoog (MS) medium supplemented with various auxin and cytokinin treatments, and their responses were evaluated based on callus induction percentage, time to callus initiation, callus morphology, and fresh weight.

Conclusions

Based on the results of the study, the following conclusions were drawn:

Plant growth regulator supplementation significantly influenced callus induction in Aquilaria malaccensis.
Auxin-dominant treatments, particularly those containing 2,4-D, resulted in higher callus induction rates and faster callus initiation.
Specific auxin–cytokinin combinations produced friable and actively growing callus suitable for further tissue culture applications.
The optimized PGR combination identified in this study can serve as a foundation for future micropropagation and conservation efforts.

Recommendations

In view of the findings of this study, the following recommendations are proposed:

Future studies may explore the regeneration potential of the induced callus through organogenesis or somatic embryogenesis.
Molecular or biochemical analyses may be conducted to assess the genetic stability and metabolite production of callus tissues.
Anti-browning treatments such as activated charcoal or antioxidants may be incorporated to further improve callus quality.
Scaling up the optimized protocol for large-scale propagation and conservation programs is recommended.