1. Background

The use of plant cell culture technology i.e. cell- suspension, hairy root or organ cultures by using bioreactors for producing compounds valuable to the industry has long been considered as an attractive solution to the problem of extracting the compounds from the whole plants whose availability may be limited. Under the bioreactor system, the cell can be cultured in large quantities under control conditions. A lot of interest has been focused on the potential of plant cell culture system for natural product synthesis. Currently, 3 large -scale process have been established in Japan (Shikonin), Korea (Ginseng) and United States (anti-cancer drug, Taxol). Michelia (Cempaka) species is known to produce high value essential oils.The popularity of the fragrance has resulted in many species of Michelia being grown all over the world. It is the oil of Michelia that is the essence of JOY and J'adore, among the world's most expensive perfumes. Most of the companies, such as Natural Scents, (USA) sell the absolute essential oil of Michelia sp in a range of USD 20-USD30 for ¼ fl. ounce. The world fragrance and flavour market was estimated around USD14.15 Billion in 1999 with a projected growth to USD18.4 Billion by 2004 (Source: Leffingwell & Associates, 2001). As a starting material for valuable perfumes, this essential oil is expensive and low yield in preparation from the whole plants. It is very desired that the essential oil of Michelia and species can be producible from cultures under bioreactor system.

2. Status of Research
Callus culture from petal of Michelia alba has already been initiated in MARDI as initial work to this project. So far, 17 compounds have been found from floral parts of intact plant including the major compounds, i.e. linalool, alpha-terpineol, geraniol and cineole, whereas only 3 compounds (linalool, geraniol and cineole) have been found in callus cultures. Research works initiated by Euyama has already identified 100 compounds from the flower parts and leaves of Michelia sp.(Euyama et al, 1992). In Taiwan, callus cultures of Michelia sp have been established by several groups of researchers. Investigation on regulatory controls for key steps in biosynthetic pathway of the major fragrance compounds of Michelia alba has been proposed by MARDI as a separate project in collaboration with Dutch scientist.

3. Objectives
a. To develop culture system for the production of biomass and fragrance compounds from Michelia alba ('cempaka putih')
b. To investigate the probable biosynthetic pathway leading to the accumulation of major fragrance compounds i.e. linalool and 1,8 cineole
c. To establish a suitable bioreactor system for the production of fragrance compounds using small-scale bioreactors

4. Problems
Currently, only few systems seem to be economically viable due to the lack of technological innovation in techniques and culture conditions leading to the low yield of the product. Advancement in molecular biology, biochemistry and bioprocess technology seems to have some answers to this predicament. This proposed project could bring together experts and pool resources from the participating institutions i.e.. MARDI, MINT and UPM as well as establish linkages and strategic alliances with other research institutions at local and abroad. The achievements from this project would be a preliminary important step in realizing the ultimate goal, that is to be able to commercially produce valuable products from plant cell cultures.

5. Expected Output
a. The technology based on the culture of plant cells in bioreactors can lead to the development of commercially feasible system for the production of various fine chemicals of natural origin. From the perspective of the public at large, the technology will lead to a change of focus from the consumption of artificial compounds towards natural products.
b. Information on the physiological responses of cultured tissue to different chemical and physical regimes, and factors involve in biosynthesis and accumulation of the fragrance compounds
c. Linkages with other research institution and universities at local and abroad as well as development of human resource (e.g M.sc and Phd graduates) with new skill, technology and knowledge.

 

EXECUTIVE SUMMARY

Project Title:
Development of Plant Bioreactor Technology for the Production of Fragrance
Compounds from Michelia alba ('Cempaka putih')

 

6. Methodology

In order to characterize the compounds produced by the flower of intact plants of the selected species, extraction, separation and identification techniques shall first be developed. Extraction shall be carried out from different stages of Michelia alba flowers collected from designated trees. Tissue and cell cultures in the form of callus, somatic embryo and cell suspension shall then be developed from the various stages of flower development such as flower bud, half and full-bloom flowers and also from different parts of flower.

Investigation shall be carried out on the basic requirements for nutrient, phytohormone and physical conditions; light, pH and temperature on cultures. Emphasis will be given on the influence of hormones or growth regulator during the initiation and growth of cultures. Production of essential oils from these cultures shall then be investigated. Potentially high-yielding cell lines shall be obtained through recurrent selection and screening of these cultures.

As a prerequisite for bioreactor studies, studies on growth and product accumulation, first, need to be carried out under shake flask conditions. Simultaneously, studies on the biosynthesis of compounds in cultures shall be carried out to determine the probable biosynthetic pathway leading to the formation of major fragrance compounds and to determine the rate limiting steps. Factors which could enhance product yield shall also be investigated. These include precursor feeding, cell immobilization and elicitation. Studies on growth and production kinetics shall also be carried out to determine its pattern.

Finally, cultures shall be grown in bioreactors. Investigation shall be carried out on basic design requirement: shear stress, kinetics of growth and production and culture parameters such as, mixing, aeration and oxygen demand under small-scale bioreactors.

Period of the project : 3 Years (2002 - 2005)

Achievements

Progress/Achievements for 2002

New product
-

New process
· Protocols for the development of callus and somatic embryos with different floral explants using MS Media in combination with growth hormone BAP and 2,4-D.
· Developed a simple technique of differentiating somatic embryos and non-somatic embryos using staining technique with 2 types of dyes, Acetocarmine and Evan Blue.
· Extraction was done at MINT using simple distillation technique. Compound profiling was carried out using GC/MS at MINT but quantification of essential oils cannot be done since the amount of oil extracted was very little.
· Successfully extracted the flowers and leaves of M.alba using Simultaneous Distillation Extraction unit (SDE) with organic solvents: dichloromethane at ratio 7:3.
· Some parts of partial purification work on linalool synthase, 1,8 cineole synthase and limonene synthase by using ultracentrifugation, ammonium sulphate precipitation, gel filtration and dialysis technique are in progress, before these enzymes can be determined for their specific activities and molecular masses by SDS-PAGE technique.

Patents filed - 0

Commercialization - 0

Discovery - Determine the compound profiling from full bloom flowers,

Publication - 0

Human Resource Development - 0