Master of Science (MS) in Horticulture – Major in Embryology/ Tissue Culture (UPLB)
OPTIMIZATION OF CALLUS INDUCTION AND SOMATIC EMBRYOGENESIS IN AQUILARIA MALACCENSIS
CHAPTER 1
INTRODUCTION
Background of the Study
Aquilaria malaccensis Lam. is one of the most economically and ecologically significant tree species in the genus Aquilaria, renowned as a primary source of agarwood, a highly valuable resin used in perfumery, traditional medicine, and religious practices. Due to overharvesting, habitat loss, and illegal trade, A. malaccensis has been listed under Appendix II of CITES, necessitating sustainable propagation and conservation strategies.
Conventional propagation of A. malaccensis relies primarily on seeds; however, seed-based propagation faces major constraints such as low viability, seasonal availability, genetic heterogeneity, and inconsistent growth performance. Vegetative propagation through cuttings has shown limited success due to poor rooting and survival rates. These limitations highlight the need for reliable in vitro propagation systems.
Plant tissue culture, particularly callus induction and somatic embryogenesis, offers a powerful platform for mass propagation, genetic conservation, and future biotechnological applications. Somatic embryogenesis enables the regeneration of whole plants from somatic cells, providing uniform, true-to-type plantlets suitable for plantation establishment and conservation programs. However, standardized and efficient protocols for callus induction and somatic embryogenesis in A. malaccensis remain limited, especially under Philippine laboratory conditions.
This study aims to optimize callus induction and somatic embryogenesis protocols for A. malaccensis by evaluating the effects of explant source and plant growth regulator (PGR) combinations. The results are expected to contribute to sustainable agarwood production, conservation, and future research in resin biosynthesis and genetic improvement.
Statement of the Problem
Despite the high economic value and conservation importance of Aquilaria malaccensis, efficient and reproducible in vitro regeneration protocols through somatic embryogenesis are not yet well established. Specifically, there is limited information on the optimal explant type and plant growth regulator combinations necessary to induce high-quality embryogenic callus and regenerate viable plantlets.
This study seeks to answer the following questions:
- Which explant source (leaf, nodal, or internodal tissue) is most responsive to callus induction in A. malaccensis?
- What combinations and concentrations of auxins and cytokinins result in the highest callus induction frequency?
- Which treatments promote embryogenic callus formation and somatic embryo development?
- What is the regeneration efficiency of somatic embryos into complete plantlets?
Objectives of the Study
General Objective
To optimize callus induction and somatic embryogenesis protocols for Aquilaria malaccensis.
Specific Objectives
- To evaluate the effect of explant source on callus induction efficiency.
- To determine the optimal plant growth regulator combinations for callus induction.
- To induce and characterize embryogenic callus formation.
- To regenerate somatic embryos into viable plantlets.
- To assess survival and growth of regenerated plantlets during acclimatization.
Significance of the Study
The findings of this study will benefit the following sectors:
- Agarwood Industry: Provides a scalable propagation method for elite planting materials.
- Conservation Programs: Supports ex situ conservation of an endangered species.
- Scientific Community: Expands knowledge on somatic embryogenesis in woody tropical species.
- Future Research: Serves as a foundation for genetic transformation, metabolite studies, and resin induction research.
Scope and Limitations
This study will focus on in vitro callus induction and somatic embryogenesis of A. malaccensis using selected explants and plant growth regulators under controlled laboratory conditions. Molecular analysis and large-scale field trials are beyond the scope of this research.
CHAPTER 2
REVIEW OF RELATED LITERATURE
Biology and Economic Importance of Aquilaria malaccensis
A. malaccensis is a tropical evergreen tree native to Southeast Asia. Agarwood formation is a defense response triggered by injury or microbial infection, resulting in the accumulation of aromatic resin within the heartwood.
Plant Tissue Culture in Woody Species
Woody plants are generally recalcitrant to tissue culture due to phenolic exudation and slow growth rates. However, advances in media formulation and PGR optimization have improved regeneration success in several tree species.
Callus Induction and Somatic Embryogenesis
Callus induction is typically initiated using auxins such as 2,4-D and NAA, while cytokinins like BAP and TDZ influence cell division and differentiation. Somatic embryogenesis involves the formation of bipolar structures capable of developing into complete plants.
Tissue Culture Studies in Aquilaria Species
Previous studies report variable success in callus induction and regeneration among Aquilaria species, emphasizing the need for species-specific protocol optimization.
CHAPTER 3
MATERIALS AND METHODS
Experimental Site
The study will be conducted at a plant tissue culture laboratory equipped with laminar airflow cabinets, autoclave, growth room, and culture racks.
Plant Material and Explant Preparation
Healthy juvenile A. malaccensis seedlings will serve as donor plants. Explants (leaf, nodal, and internodal segments) will be surface-sterilized using standard aseptic techniques.
Culture Media and Treatments
Murashige and Skoog (MS) basal medium supplemented with varying concentrations of:
- Auxins: 2,4-D, NAA
- Cytokinins: BAP, TDZ
Treatments will be arranged in a completely randomized design.
Callus Induction and Evaluation
Cultures will be incubated under controlled temperature and photoperiod. Callus induction frequency, texture, color, and fresh weight will be recorded.
Somatic Embryogenesis and Plant Regeneration
Embryogenic calli will be transferred to embryo maturation and regeneration media. Somatic embryo stages (globular, heart, torpedo) will be documented.
Acclimatization
Regenerated plantlets will be hardened in a greenhouse and survival rates recorded.
Data Analysis
Data will be analyzed using ANOVA, and mean comparisons will be performed using appropriate post-hoc tests.
CHAPTER 4
EXPECTED RESULTS AND DISCUSSION FRAMEWORK
This chapter will present results on callus induction efficiency, embryogenic potential, and regeneration success. Comparative analysis among explants and PGR treatments will be discussed in relation to existing literature.
CHAPTER 5
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
Summary
This study aims to establish an optimized in vitro regeneration protocol for Aquilaria malaccensis via callus induction and somatic embryogenesis.
Conclusions
Conclusions will be drawn based on treatment performance and regeneration outcomes.
Recommendations
Further studies may include genetic stability analysis, metabolite profiling, and scale-up using bioreactor systems.
REFERENCES
(To be compiled following the prescribed thesis format)