Biotechnology Research at FRIM

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  • A Quarterly of the Forest research InstItute MalaysIaMS ISO 9001:2008 DECEMBER 2011Issn 1394-5467

    1. UP-SCALING THE TONGKAT ALI HAIRY ROOT CULTURE2. TONGKAT ALI HAIRY ROOT GENE DISCOVERY3. SYNTHETIC SEEDS: A NEW FORM OF PLANTING MATERIAL4. IDENTIFICATION OF MALAYSIAN TIMBER USING DNA BARCODES

    HIGHLIGHTS OF THE ISSUE

    FRIM in Focus

    Biotechnology Research at FRIM

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    AdvisorsDato Dr Abdul Latif MohmodDr Mohamed Nor Mohd Yusoff

    FRIM in Focus (FIF) is distributed free of charge upon request. We welcome feedback on any of the FIF articles. Address comments and enquiries to:

    Synthetic seeds (background), steps involved in preparation of synthetic seeds (inset)

    The Editor & Writer of FRIM In FocusForest Research Institute Malaysia (FRIM)52109 Kepong, Selangor DE, Malaysia

    EditorialBoard Telephone : 603-6279 7501 | Facsimile : 603-6273 1076E-mail : idasuraini@frim.gov.my | Website : www.frim.gov.my

    Design & Printing : DeezStudio

    Technical EditorNorhara Hussein

    Editor & WriterIda Suraini Abd Shukor

    UP-SCALING THE TONGKAT ALI HAIRY ROOT CULTUREDr Nor Hasnida Hassanhasnida@frim.gov.my

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    Eurycoma longifolia or locally known as tongkat ali is probably the most popular medicinal plant in Malaysia. Its roots are widely used in the manufacturing of herbal and food products. Most of the plant roots required for making health food products are collected from the forest. As such, uncontrolled harvesting of tongkat ali from the forest may decrease the tree population leading to depletion of the species. Thus, there is a need to ensure sufficient

    supply of tongkat ali to sustain the herbal industries as well as to conserve the species.

    The over-dependance of tongkat ali roots from the forest can be addressed indirectly by using biotechnology tools. In this case, plant cell and organ cultures can also be sources of valuable secondary metabolites.

    Cell and organ cultures have a higher rate of metabolism compared to field grown plants because the initiation of cell and organ growth invariably leads to their fast proliferation. In addition, the growth of plant cells or organ cultures is not limited by external factors such as environmental, ecological and climatic conditions.

    For tongkat ali, hairy roots were found to be an attractive source of secondary metabolites. These roots were the results of a genetic transformation using the bacteria Agrobacterium rhizogenes. Hairy roots are advantageous because they can grow faster than plant cell cultures. They are also plagiotropic, or having the tendency to grow horizontally and do not require the external supply of growth hormones as in other types of cell cultures. In

    addition, plagiotropic also increases the aeration in liquid medium and boosts the accumulation of roots biomass.

    Hairy root culture of tongkat ali is presently the best alternative to ensure a sustainable supply of the plants active secondary metabolites. This in turn will sustain the herbal industry as well as safeguard the plant population.

    At Forest Research Institute Malaysia (FRIM), the up-scaling of tongkat ali hairy root culture is being carried out using the bioreactor technology whereby two types of bioreactors; the air lift and the modified stirred tank are used. Using these systems it is now possible to achieve a large scale production of hairy root culture and the bioactive compounds.

    ABOUT THE AUTHORDr Nor Hasnida Hassan is the Head of Tissue Culture Laboratory, Forest Biotechnology Division, Forest Research Institute Malaysia (FRIM). She joined FRIM in August 1996. Her research interest is on plant biotechnology, particularly in tissue culture and plant genetic transformation. Currently, her research focuses on the up-scaling of tongkat ali and kacip fatimah root cultures using the bioreactor technology, in addition to their respective production of bioactive compunds.

    1. Air lift: bubble column bioreactor (5 liter)2. Modified stirred tank bioreactor (7.5 liter)

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    compounds in addition to current studies which are investigating the similarities of bioactive compounds in both types of roots. In both cases, transcriptome sequence data serves to further support the assumption.

    Molecular and genomic study is also required to support the research on the isolated transcripts from the cultured hairy roots of tongkat ali in the bioreactor. In future, information produced from the study will be used to investigate genes involved in the production of bioactive compound in tongkat ali roots.

    Preliminary results have shown that the bioinformatics analysis on transcriptome data of tongkat ali hairy root have assembled 72,275 DNA sequences. Analysis of the biological processes has categorised the sequences into 26 groups and most of the DNA sequences are in the cellular and metabolite process categories.

    ABOUT THE MAIN AUTHORDr Norlia Basherudin joined FRIM in October 1996. Her research interest is on plant biotechnology, particularly in gene isolation and genetic engineering. Currently, her reseaches are on isolation of potential genes involved in biosynthesis of active compounds in tongkat ali, and floral gene isolation from teak.

    ulcer, cytotoxic and aphrodisiac properties and that the active ingredients are concentrated at the taproots. Because of its multiple curing abilities, the demand on tongkat ali for healthcare products has increased, thus exerting pressure on its natural resources from the forest.

    Plant transformation has been used to increase the supply of tongkat ali, thus easing the pressure on its natural sources. The technique used for plant transformation is based on the natural ability of Agrobacterium (a naturally root-infecting bacterial species) to insert specific parts of the DNA into the genome of many host plants. There are two species of Agrobacterium normally used for this purpose. A. tumefaciens is the causal agent in crown gall disease while A. rhizogenes is related to the hairy root disease.

    At Forest Research Institute Malaysia (FRIM), the genetic transformation of tongkat ali hairy root culture was successfully conducted using the A. rhizogenes. Due to its fast growing ability, the hairy root is able to produce a much higher amount of valuable secondary metabolites in a relatively shorter time.

    Previous studies using chemical marker compound have indicated that both wild type and cultured hairy roots produce similar marker

    Transcriptome is a study of RNA pool which is derived from DNA that controls all cells in the organism. The study identifies the genes that are responsible for a specific character in a particular cell type at any given time. The use of the next generation gene sequencing technology such as Solexa now offers a novel and rapid way of profiling the transcriptome that would significantly accelerate the gene discovery study.

    Identification of genes involved in the synthesis of bioactive compounds in tongkat ali has been conducted through transcriptome using the next generation sequencing and bioinformatics analysis.

    Tongkat ali or scientifically known as Eurycoma longifolia from the family of Simaroubaceae, is a slow growing tree with a wide distribution in the Malaysian forest. Tongkat ali has a long history as a traditional medicine and its uses vary between different localities within the country. In the past decade, numerous studies on the chemical composition of tongkat ali have ascertained the presence of compounds such as 10-hydroxycanthin-6one, 9-methoxycanthin-6-one, eurycomalactone and eurycomanol.

    Biological studies which were conducted on tongkat ali root has confirmed its anti-malarial, anti-

    locomotion (36)cell proliferation (85)carbon utilisation (2)

    cell wall organisation or biogenesis (428)cellular component organisation (1824)

    development process (2988)pigmentation (19)

    viral reproduction (24)death (202)

    sulfur utilisation (2)growth (640)

    immune system process (323)biological regulation (3491)response to stimulus (4650)

    cellular process (11,020)

    cell killing (3)multicellular organismal process (2889)cellular component biogenesis (623)rhythmic process (74)biological adhesion (37)nitrogen utilisation (14)multi-organism process (1067)localisation (2523)metabolic process (10,305)signaling (1168)reproduction (1587)

    Biological process categories of cDNA from tongkat ali hairy root culture. Number of genes involved in each category is indicated in brackets

    TONGKAT ALI HAIRY ROOT GENE DISCOVERY Dr Norlia Basherudin | norlia@frim.gov.myDr Norwati Adnan & Dr Mohd Rosli Haron

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    SYNTHETIC SEEDS A New Form of Planting MaterialNor Asmah Hassan | norasmah@frim.gov.myNashatul Zaimah Noor Azman

    procedure for Acacia hybrid (FRIM ID 5/11) and Eurycoma longifolia (tongkat ali). The Acacia hybrid plants have been re-introduced to the natural environment by culturing them onto soil and vermiculite before the direct sowing method could be established. FRIM is also in the process of developing procedures for other species including Aquilaria malaccensis (karas), Endospermum diadenum (sesend