Doctor of Philosophy (PhD) in Horticulture (UPLB)
CELLULAR, MOLECULAR, AND BIOTECHNOLOGICAL REGULATION OF SOMATIC EMBRYOGENESIS AND DEFENSE‑INDUCED METABOLITE BIOSYNTHESIS IN WOODY TROPICAL PLANTS: AQUILARIA MALACCENSIS AS A MODEL SYSTEM
Doctor of Philosophy (PhD) Dissertation Proposal
CHAPTER 1
INTRODUCTION
Background of the Study
Aquilaria malaccensis Lam. is a tropical hardwood species valued as a primary source of agarwood, a resinous wood formed as a defense response to biotic and abiotic stress. Agarwood is among the most expensive non-timber forest products globally, with applications in perfumery, traditional medicine, and religious practices. Due to extensive exploitation and habitat loss, A. malaccensis is listed under Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), necessitating sustainable production and conservation strategies.
Plant cell and tissue culture technologies offer a powerful framework for addressing both conservation and industrial challenges associated with agarwood production. Somatic embryogenesis (SE) enables clonal propagation, genetic conservation, and serves as a platform for cellular-level studies of development and metabolism. In woody tropical species, however, SE remains highly recalcitrant, with embryogenic competence being strongly influenced by explant type, hormonal balance, stress signaling, and epigenetic regulation.
Agarwood formation is intrinsically linked to plant defense responses, involving complex plant–microbe interactions and secondary metabolite biosynthesis pathways. Understanding how defense signaling intersects with cellular totipotency and differentiation under in vitro conditions remains a critical knowledge gap. This dissertation positions A. malaccensisas a model woody species to elucidate the cellular, molecular, and biochemical mechanisms governing somatic embryogenesis and defense-induced metabolite production, with broader implications for tropical tree biotechnology.
Research Problem
Despite advances in plant tissue culture, reproducible somatic embryogenesis systems for Aquilaria malaccensis remain poorly understood at the mechanistic level. The cellular determinants of embryogenic competence, the molecular regulation of SE initiation, and the integration of defense signaling with secondary metabolite biosynthesis in vitro are largely unexplored. This limits both large-scale propagation and the development of cell-based platforms for sustainable agarwood production.
Research Questions
- What cellular and hormonal factors determine embryogenic competence in A. malaccensis callus cultures?
- How do auxin–cytokinin interactions regulate somatic embryo initiation and development?
- What molecular and epigenetic signatures distinguish embryogenic from non-embryogenic tissues?
- Can defense-related elicitation enhance resin-associated metabolite biosynthesis in embryogenic cultures?
- How can optimized somatic embryogenesis systems be scaled for biotechnological applications?
Objectives of the Study
General Objective
To elucidate the cellular, molecular, and biotechnological mechanisms regulating somatic embryogenesis and defense-induced secondary metabolite biosynthesis in Aquilaria malaccensis.
Specific Objectives
- To optimize explant selection and plant growth regulator regimes for stable embryogenic callus induction.
- To characterize morphological and histological features of embryogenic tissues.
- To identify key gene expression and epigenetic markers associated with embryogenic competence.
- To evaluate the effects of abiotic and biotic elicitors on secondary metabolite production in embryogenic cultures.
- To develop a scalable somatic embryogenesis platform applicable to woody tropical plants.
Significance of the Study
This research will contribute to fundamental plant developmental biology by clarifying mechanisms of totipotency in woody species. Practically, it will support sustainable agarwood production, conservation initiatives, and the development of cell-based biofactories for high-value metabolites. The findings will have relevance beyond agarwood, informing tissue culture strategies for other recalcitrant tropical trees.
Scope and Delimitations
The study focuses on in vitro systems of A. malaccensis. Field performance trials, full genome sequencing, and large-scale industrial deployment are beyond the scope of this dissertation.
CHAPTER 2
REVIEW OF RELATED LITERATURE
This chapter synthesizes literature on:
- Biology and defense mechanisms of Aquilaria species
- Somatic embryogenesis in woody plants
- Hormonal regulation of plant totipotency
- Epigenetic control of cell fate in plant tissue culture
- Secondary metabolite biosynthesis and elicitation strategies
- Biotechnological applications of embryogenic cultures
CHAPTER 3
MATERIALS AND METHODS
Experimental Design Overview
The dissertation will be conducted through interconnected experimental modules designed to generate multiple publishable studies.
Plant Material and Explant Source
Juvenile A. malaccensis seedlings and selected elite donor plants will be used. Explants will include leaves, nodal segments, and internodes.
Callus Induction and Somatic Embryogenesis Optimization
Murashige and Skoog (MS) medium supplemented with varying concentrations of 2,4‑D, NAA, BAP, and TDZ will be tested under a factorial experimental design.
Histological and Cytological Analyses
Embryogenic and non-embryogenic calli will be examined using histochemical staining and light microscopy to identify embryogenic structures.
Molecular and Epigenetic Characterization
- RNA extraction and qPCR analysis of embryogenesis-related genes
- DNA methylation assessment using methylation-sensitive markers
Elicitation and Metabolite Analysis
Embryogenic cultures will be subjected to abiotic (methyl jasmonate, salicylic acid) and biotic (fungal-derived elicitors) treatments. Crude metabolite profiles will be analyzed using chromatographic techniques.
Scale-Up and Biotechnological Assessment
Temporary immersion and liquid culture systems will be evaluated for embryo proliferation efficiency and stability.
Statistical Analysis
Data will be analyzed using ANOVA, multivariate analysis, and correlation modeling.
CHAPTER 4
EXPECTED RESULTS AND DISCUSSION FRAMEWORK
This chapter will present integrated analyses of embryogenic competence, molecular regulation, and metabolite biosynthesis. Results will be contextualized within broader woody plant biotechnology literature.
CHAPTER 5
SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS
Summary
The dissertation aims to establish A. malaccensis as a model for understanding somatic embryogenesis and defense-induced metabolism in woody tropical plants.
Conclusions
Conclusions will synthesize findings across cellular, molecular, and biotechnological levels.
Future Directions
Recommendations include genome-level studies, metabolic engineering, and translation into conservation and industrial frameworks.
REFERENCES
(To be compiled following the prescribed doctoral dissertation format)