Papers by Bhavani Balakrishnan
Universiti Sains Malaysia, Dec 1, 2013
CHAPTER 1.0 INTRODUCTION 1 1.1 Research objectives 4 CHAPTER 2.0 LITERATURE REVIEW 5 2.1 Eurycoma... more CHAPTER 1.0 INTRODUCTION 1 1.1 Research objectives 4 CHAPTER 2.0 LITERATURE REVIEW 5 2.1 Eurycoma longifolia: Locality and tree morphology 5 2.2 Ethnobotanical practices in native communities 6 2.3 Elucidation of compounds and pharmacological properties 6 2.3.1 Malaysian Ginseng 7 2.3.2 Antiparasitic activites 9 2.3.3 Cytotoxic properties 11 2.3.4 Preventing bone loss and osteoporosis 12 iv 2.3.5 Other properties 2.4 The consequences of fame on Eurycoma longifolia 2.5 Current in vitro propagation methods of Eurycoma longifolia 2.6 Agrobacterium rhizogenes: Nature's genetic engineer 2.7 Hairy root phenotype 2.8 Agrobacterium rhizogenes-mediated transformation of Eurycoma longifolia 2.9 Potential applications for hairy root cultures of Eurycoma longifolia 2.9.1 Genetic and metabolic engineering 2.9.2 Molecular farming: Phytochemicals, proteins and bioreactors 2.9.3 Functional gene analysis 2.9.4 Phytoremediation CHAPTER 3.0 MATERIALS AND METHODS 3.1 Micropropagation of Eurycoma longifolia 3.1.1 Callus induction from in vivo and in vitro leaf explants 3.1.2 Induction and multiplication of somatic embryos 3.1.3 Propagation of in vitro shoot cultures 3.1.4 Surface sterilisation of seeds and growth of in vitro plantlets 3.2 Microscopy observations of somatic embryos 3.2.1 Sample preparations for histological analysis 3.2.2 Scanning electron microscopy (SEM) observations v 3.3 Agrobacterium rhizogenes chemotaxis and attachment to somatic embryos 3.3.1 Preparation of Agrobacterium rhizogenes strains 3.3.2 Medium preparation and protocol for chemotaxis assay 3.3.3 SEM observations of bacterial attachment 3.4 Agrobacterium rhizogenes-mediated transformation of Eurycoma longifolia 3.4.1 Plant materials 3.4.1.1 In vivo leaves and rachis 3.4.1.2 In vitro leaves, stems, roots and somatic embryos 3.4.2 Bacterial preparation for transformation 3.4.3 Hairy root induction from in vivo leaves, rachis and in vitro leaves, stems and roots of Eurycoma longifolia 3.4.4 Optimisation of transformation efficiency in Eurycoma longifolia somatic embryos 3.4.4.1 Experimental design: Optimisation of parameters 3.4.4.2 GUS histochemical assay 3.4.4.3 Induction of hairy roots in somatic embryos of Eurycoma longifolia vi 3.4.5 Genomic DNA extraction and detection of transformants using polymerase chain reaction (PCR) CHAPTER 4.0 RESULTS 4.1 Micropropagation of Eurycoma longifolia 4.1.1 Callus induction from in vivo and in vitro leaf explants 4.1.2 Induction and multiplication of somatic embryos 4.1.3 Propagation of in vitro shoot cultures 4.1.4 Surface sterilisation of seeds and growth of in vitro plantlets 4.2 Microscopy observations of somatic embryos 4.2.1 Histological observations 4.2.2 SEM observations 4.3 Interaction between Agrobacterium rhizogenes and Eurycoma longifolia 4.3.1 Chemotaxis movement assay 4.3.2 SEM observation of bacterial attachment 4.4 Agrobacterium rhizogenes-mediated transformation of Eurycoma longifolia 4.4.1 Hairy roots induction from in vivo leaves, rachis and in vitro leaves, stems and roots of Eurycoma longifolia vii 4.5 Optimisation of transformation efficiency in Eurycoma longifolia somatic embryos 4.5.1 Virulence of Agrobacterium rhizogenes strains 4.5.2 Indole-butyric acid (IBA) concentrations 4.5.3 Dithiothreitol (DTT) concentrations 4.5.4 Polyvinylpyrrolidone (PVP) concentrations 4.6 Hairy root induction in somatic embryos of Eurycoma longifolia 4.7 Detection of transformants using polymerase chain reaction (PCR) CHAPTER 5.0 DISCUSSION 5.1 Micropropagation of Eurycoma longifolia 5.1.1 Callus induction from in vivo and in vitro leaf explants 5.1.2 Induction and multiplication of somatic embryos 5.1.3 Propagation of in vitro shoot cultures 5.1.4 Surface sterilisation of seeds and growth of in vitro plantlets 5.2 Microscopy observations of somatic embryos 5.2.1 Histological observations 5.2.2 SEM observations 5.3 Interaction between Agrobacterium rhizogenes and Eurycoma longifolia 5.3.1 Chemotaxis movement assay viii 5.3.2 SEM observation of bacterial attachment 5.4 Agrobacterium rhizogenes-mediated transformation of Eurycoma longifolia 5.4.1 Hairy roots induction from in vivo leaves, rachis and in vitro leaves, stems and roots of Eurycoma longifolia 5.5 Optimisation of transformation efficiency in Eurycoma longifolia somatic embryos 5.5.1 Virulence of Agrobacterium rhizogenes strains 5.5.2 Indole-butyric acid (IBA) concentrations 5.5.3 Dithiothreitol (DTT) concentrations 5.5.4 Polyvinylpyrrolidone (PVP) concentrations 5.6 Hairy root induction in somatic embryos of Eurycoma longifolia 5.7 Detection of transformants using polymerase chain reaction (PCR) CHAPTER 6.0 CONCLUSION 6.1 Conclusion of investigation 6.2 Suggestions for future explorations 100 xi
AFRICAN JOURNAL OF BIOTECHNOLOGY, 2012
The study was conducted on the storage of Dendrobium sonia-28 protocorm-like bodies (PLBs) at var... more The study was conducted on the storage of Dendrobium sonia-28 protocorm-like bodies (PLBs) at various temperatures (5, 10, 15, 20, and 25°C) and under mineral oil layer. Biochemical analyses were conducted on the PLBs by determining the chlorophyll, total soluble protein and peroxidase activities. In addition, histological analyses of cell structure, rate of survival and shoot growth after the recovery process were carried out. This study was performed in two conditions; three (3) weeks and twelve (12) days of growth recovery period after 3 weeks of storage treatment. The PLBs were transferred to half strength Murashige and Skoog (MS) medium containing 1.0 mg/L benzylaminopurine (BAP) and kept at 25±2°C for twelve (12) days during recovery period. Overall, PLBs stored under 5°C and mineral oil layer produced the lowest values in chlorophyll analysis, rate of survival and shoot growth. However, total soluble protein content was increased during twelve (12) days of growth recovery period. Histological analysis has shown that the cell structure of these PLBs sustained a high degree of damage due to the treatment. PLBs stored under 5°C and mineral oil layer recorded the highest peroxidase activity attributed to higher stress conditions.
The effect of strain virulence on Agrobacterium rhizogenes
Proceedings of the Annual International Conference Syiah Kuala University Life Sciences Engineering Chapter, Nov 30, 2012
Eurycoma longifolia, known locally as 'Tongkat Ali' in Malaysia, is popularly sought out as herba... more Eurycoma longifolia, known locally as 'Tongkat Ali' in Malaysia, is popularly sought out as herbal remedy in many parts of South East Asia. Consequently, this has resulted in the indiscriminate collection of the taproot from the wild, leading to fears of extinction among conservationists. Agrobacterium rhizogenes is a naturally occurring soil bacterium that infects injured plants and causes a massive proliferation of roots, known as hairy roots. The objective of this study is to determine the transformation efficiency of 2 different strains of Agrobacterium rhizogenes on somatic embryos of Eurycoma longifolia using transient GUS expression as an indicator of successful transformation. Somatic embryos cultured in vitro for 4 to 5 weeks were used as explants and were pre-cultured for 2 days in full strength MS medium containing 0.5 mg/L IBA added with 1% PVP and 2mg/L DTT. The explants were transformed using Agrobacterium rhizogenes strains AR12 and AR14. GUS assay was carried out 1 week after transformation and observed. Observations indicate that AR12 is more effective in transforming somatic embryos of Eurycoma longifolia compared to AR14. Therefore, Agrobacterium rhizogenes strain AR12 is a potential candidate for the successful transformation of Eurycoma longifolia somatic embryos, leading to the induction of sustainable hairy root cultures.
Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental... more Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental structures that are remarkably similar to seeds found in the wild. The primary components of a growing somatic embryo are its shoot and root apical meristems indicated by dense layers of rapidly growing cells. The increased understanding of In vitro culture systems and anatomical changes provide information into cellular processes that govern genetic transformation of E. longifolia with Agrobacterium rhizogenes. The presence of meristematic regions on cultured somatic embryos suggests that they are suitable for genetic transformation as genetic elements could be transported to these regions where growth and differentiation are centered. This allows the successful integration and expression of transferred DNA in the host organism, leading the way for an efficient A. rhizogenes-mediated transformation protocol.
African Journal of Biotechnology
The study was conducted on the storage of Dendrobium sonia-28 protocorm-like bodies (PLBs) at var... more The study was conducted on the storage of Dendrobium sonia-28 protocorm-like bodies (PLBs) at various temperatures (5, 10, 15, 20, and 25°C) and under mineral oil layer. Biochemical analyses were conducted on the PLBs by determining the chlorophyll, total soluble protein and peroxidase activities. In addition, histological analyses of cell structure, rate of survival and shoot growth after the recovery process were carried out. This study was performed in two conditions; three (3) weeks and twelve (12) days of growth recovery period after 3 weeks of storage treatment. The PLBs were transferred to half strength Murashige and Skoog (MS) medium containing 1.0 mg/L benzylaminopurine (BAP) and kept at 25±2°C for twelve (12) days during recovery period. Overall, PLBs stored under 5°C and mineral oil layer produced the lowest values in chlorophyll analysis, rate of survival and shoot growth. However, total soluble protein content was increased during twelve (12) days of growth recovery period. Histological analysis has shown that the cell structure of these PLBs sustained a high degree of damage due to the treatment. PLBs stored under 5°C and mineral oil layer recorded the highest peroxidase activity attributed to higher stress conditions.
Pakistan Journal of Botany
Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental... more Histological analysis conducted on somatic embryos of Eurycoma longifolia shows the developmental structures that are remarkably similar to seeds found in the wild. The primary components of a growing somatic embryo are its shoot and root apical meristems indicated by dense layers of rapidly growing cells. The increased understanding of In vitro culture systems and anatomical changes provide information into cellular processes that govern genetic transformation of E. longifolia with Agrobacterium rhizogenes. The presence of meristematic regions on cultured somatic embryos suggests that they are suitable for genetic transformation as genetic elements could be transported to these regions where growth and differentiation are centered. This allows the successful integration and expression of transferred DNA in the host organism, leading the way for an efficient A. rhizogenes mediated transformation protocol.
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Papers by Bhavani Balakrishnan