SUMMARY OF PROPOSED RESEARCH INCLUDING KEY GOALS

1. For scientifically qualified assessors:

Taxol is a natural diterpenoid alkaloid first isolated from the inner bark of pacific yew, Taxus brevifolia (Wani et al., 1971) and widely use in the pharmaceutical industry as the antitumor agents. However, low taxol content, high cost of extraction and the destruction of yew trees spark a major environmental controversy and cause a problem to fulfil its growing demand. The application of plant tissue culture technique has appeared as an eco-friendly method and feasible approach for commercial supply, but the production rate of this secondary metabolite are still very low. Only 10% of taxol secreted in the extracellular medium of T x media suspension cultures (Wickremesinhe and Arteca, 1993). These problems prompted a great effort to discover and identify the alternative means to enhance the mass production of taxol in plant cell cultures. In this study, elicitation using coronatine (Cor) and ultrasound (US) will be used for synthesis and transport of taxol. Cor is a toxin produced by Pseudomonas syringae and can act as a plant growth regulator and stimulates the defence mechanism in plants. Meanwhile, Low-intensity US is a physical or mechanical stimulus that can enhance the cellular transporter activity and trigger the pathway of defence-related secondary metabolites. It is hypothesized that the taxol accumulation in Taxus x media culture is higher than control (unelicited) and single treatment after treated with Cor and low- energy US. The experiment will use two-stage suspension culture and longitudinally young stem as the explant. The effect of these two elicitors on biomass production and taxol production will be analysed. At the end of this study, it is expected that the taxol production and cell growth will show positive result after elicited with Cor and low-energy US. This study will be a significant endeavour in enhance the release of taxol through elicitation approach.

2. For lay readers:

Secondary metabolites are chemicals produced by plants and do not have any physiological functions, but use as flavour, food colours, fragrances and pharmaceutical study. Taxus species are one of the medicinal plants that can produce natural anti-cancer agents known as taxol. However, over exploitation of natural sources of taxol from bark, stem, roots and needles put yew trees as an endangered species. Besides, the low yield and slow growth of yew trees limit its potential as a viable source of taxol. Thus, it is important to find alternative method to increase the taxol production in Taxus sp. The production of taxol from Taxus x media can be greatly enhanced through elicitation. Elicitation is a method to stimulate the gene expression of enzymes responsible for the synthesis of secondary metabolites. The substance used in stimulating the production of exuded compound is called elicitor. Elicitor is formed inside or outside of plant cells and imitate as a stress to activate the plant defence systems and cause qualitative and quantitative changes in the secondary metabolite content. Two elicitors that will be used in this study are Coronatine (Cor) and low-energy ultrasound (US). Plants use Cor to build defensive compounds and as growth regulators. US, on the other hand can stimulated the cell membrane permeabilization and encourage the release of taxol to culture medium. Plant cell cultures will be used to retrieve the secondary metabolite on demand by maintain the growth of plant cells in aseptic condition with appropriate supply of nutrients. It is expected that the accumulation of taxol treated with elicitors is higher than the untreated culture. This study will provide a good understanding about the effect of elicitors in activate the chemical defence system in plants and directly stimulate the production of taxol.

RESEARCH Outcomes / Products

1. What is the proposed outcome of your project:

It is expected that all treatments will give significant increase of taxol accumulation when compared with control. Combination between Cor and US exposure will improve the extracellular and cell-associated taxol production. The expected taxol production from the treatment of both Cor and US is 20 to 50% higher than control. Elicitation of Taxus x media using Cor and US also will enhance the cell growth. The use of suspension culture will be one of the reasons that improve the accumulation of taxol and can be a good strategy for the continous production of taxol.

2. Why is it important?

Taxol is well known anti-cancer drug derived from natural source of Taxus species. However, direct extraction from yew trees has subjected them as an endangered species. On the other hand, the process of extraction and purification of taxol is also time consuming and not economically feasible. Thus, plant tissue culture is a promising approach to obtain this secondary metabolite. However, one of the main problems in increase the taxol production is instability in the synthesis of secondary metabolite. In order to maximize the yield of taxol, the combination of biotic and abiotic elicitor can be a good strategy. Besides, the elicitation approach is also more economical beneficial compare with other methods such as precursor feeding, phytohormone regulation and biotransformation. US is a simple but effective elicitor while Cor is expected has the similar biological activity as methyl jasmonate (MeJA), the most common elicitor used in stimulate secondary metabolites. The results from this study can provide a fundamental understanding of the effects of US in combination with Cor on the production of secondary metabolite and plant cell culture. Understanding how Taxus x media cell culture react with these two elicitors can provide a a beneficial in link the growth rate with the production of taxol.

DETAILS OF RESEARCH PROJECT

1. Introduction and Background:

Taxus species are medicinal plants and members of the Taxaceae family that can produce taxol, a chemotheraphy drug used widely in cancers treatment. FDA approval of taxol in 1992 and a series of success of taxol in clinical test for various cancers led to massive demand for this active substance. The increase of demand was expected since taxol also useful to combact non-canceorus disorders, such as polycystic kidney disease (Woo et al., 1994). However, it is difficult to perform large-scale culture because of the difficulty to gain this active compound from natural source. Three mature, century-old yew trees would produce only 1 g of taxol. It is estimated that about 10 tons of bark or felling of 3000 trees was needed to yield 1 kg of purified taxol to treat 500 cancer patients. Besides, the natural source of taxol from yew trees is only about 0.01% to 0.06% (Sung et al., 2005). All these concerns have prompted extensive efforts in find the method that can increase the mass production taxol. Taxus x media is choose for this study since it is good starting materials for the taxol production via biotechnological technique. Besides, the amount of taxol produce by this cultivar is the highest among other Taxus species (Table 1).

The elicitation approach has been one of the most promising strategies in improve the mass production of taxol and other secondary metabolites in cell cultures (Roberts ND Shuler, 1997). The idea of using elicitor arise from the defence pathway utilize by plant in deal with environmental stress, pathogenic microorganisms, insects and higher herbivorous predators by stimulate the production of offensive chemicals like secondary metabolites. One of the most commonly studied and effective biotic elicitor is methyl jasmonate (MeJA). However, the report that Cor resemble the biological activity of the jasmonic acid gives an idea that the mode of action between these two compounds could be similar. The chemical structure of Cor is more stable than MeJA because of the rigid cis-orientation in its bi-cyclic skeleton. This lead to the higher levels of induction in the production of secondary metabolites by plant cells treated with Cor when compare with those treated with natural JA. Although Cor stimulated paclitaxel production with less efficiency than MeJA, it resulted in higher paclitaxel excretion to the liquid phases of culture medium. Besides that, there are not many reports about the influence of Cor on the production of secondary metabolite in plant cell suspension culture.

Table 1: Content (wt %) of selected taxanes in different species of Taxus plants (Lin Lidong, 2003)

The combination of biotic and abiotic elicitor had synergistic effects to taxol production in many reports. Thus, it is expected that the combine use of Cor with a physical stimulus like US might increase the yield of taxol in the Taxus culture. Generally, the abiotic elicitors use in experimental studies is chemical agents, heavy metals and biochemical agents like polysaccharides and organic acid. The use of US to biotechnology is relatively new and not many reports can be found on the effects of US in increase the useful secondary metabolites. This simple and effective elicitor can activate cells or enzymes and induced plant defence metabolism by ultrasonic waves. High intensity US waves can bring detrimental effect to cells, enzymes and other biological materials. Meanwhile, low intensity US waves can change the cellular metabolism, improve the mass transfer of reagents and enhance the release of intracellular products from cultured cells by increase the membrane permeability. The use of US will not disrupt the culture process and allow continuous use of cell’s biosynthetic capacity (Kilby and Hunter, 1990). US deserve an extensive research regarding its effects in plant secondary metabolite due to its wide application in biological field. The combination of Cor and US has great prospect and economic benefits in enhance the taxol production at low cost. Until now, there is no study or information about the effect of Cor and US and their combination on the Taxus species.

2. Work which has led up to the project:

The production of taxol using cell cultures were first reported by Christen et al., in 1989. Cell suspension cultures can emit secondary metabolites into growth medium. For instance, the production of resveratrol in the medium was higher than in the cell (Zamboni et al., 2009). Work by Kajani et al., in 2010 on Taxus baccata L cell culture showed that 74.9 % of total taxanes was exuded to growth medium (5. 584 mg/ L). Roberts et al., in 2003 reported that suspension culture of Taxus canadensis secreted more than 90% of the total taxol to the medium after treated with cell wall enzymes. Cell culture of Taxus chinensis emitted 90% of the total paclitaxel in medium with chitosan (Zhang et al., 2007). Komaraiah et al., in 2003 reported that up to 70% of the plumbagin in Plumbago rosea cell culture were released into the medium. Taxol accumulate at high level (1.5 mg/L) in the second phase of growth

Previous research by J. Wu and L. Lin in 2003 on the application of US as a physical stimulus and MeJA in increase the mass production of taxol in Taxus chinensis becomes a fundamental reference for this study. Their study showed that the taxol yield was increased about 1.5- to 1.8-fold with 2 minutes US exposure once or twice during a 4 week culture period. Meanwhile, the total taxol after treated with MeJA increase about 5-fold. The percentage of extracellular taxol and cell-associated taxol also showed significant increase. The taxol production from combination between US and MeJA treatment was 20 to 50% higher than each of the treatment used alone. Another research by Wu and Lin in 2002 found that low energy US can play a role as abiotic elicitor to trigger the plant defence responses and induce the production of plant metabolite in cell cultures. Apart from that, US also can increase the cell membrane permeabilization to increase the efficiency of intracellular product release. In their previous study, they found that the combination of US stimulation and in situ solvent extraction in a Lithospermum erythrorhizan cell culture led to 2 to 3-fold increases in the yield of shikonin.

Tamogami and Kodama reported in 2000 that Accumulation of flavonoid phytoalexins in rice occurred when treated with Cor. This shown that the effect of Cor on flavonoid production was greater than that of JA or its precursor, 12-oxo-phytodienoic acid (OPDA). Cor also gave positive result in the alkaloid production in Eschscholzia californica cell cultures (Haider et al., 2000). The previous work by also showed the greater increase in the accumulation of glyceollins, the phytoalexins of soybean (Glycine max. L.), in soybean cell suspension cultures after treated with Cor. Besides, the effect of Cor in induced the phytoalexin productin was greatly better when compared with JA and MeJA. (Fliegmann et al., 2003; Lauchli et al, 2002).

3. Aims of the project:

• To study the biological and synergistic effects of Cor and US on the taxol production of Taxus x media cell culture.

(d) Experimental design and methods to be used in investigating this problem:

1. Cell culture establishment

Approximately 1.0 cm long section of longitudinal young stem of Taxus x media will be wash with distilled water and soak in 70% (v/v) ethanol for 30 to 60 seconds. After that, the stem will be surface sterilized by immersion in 1.5% sodium hypochlorite with a few drops of Tween 20 for 20 to 30 minutes. After sterilization, the explants will be rinsed 3 times with sterile distilled water. Next, the longitudinally halved stem sections will be placed with the inner cut surface in contact with the solid Gamborg’s B5 media to induce calli. The media supplemented/ fortified with 3.0% sucrose (w/v), 0.8% agar (w/v), 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-Benzyladenine (BA), at pH 5.5. All of the cultures will be placed in darkness at 25^oC for 25 days. To obtain a homogenous callus, several subcultures of calli will be done every 3 weeks. The snow, friable and massive callus will be used as the inoculums for cell suspension culture.

2. Cor treatment

The same media without agar will be used for cell suspension culture. About 10 g of fresh weight of callus will be used as the inoculums and inoculated into 300 mL conical flasks containing 100 mL liquid growth B5 medium and placed on orbital shakers (120 rpm) at 25^oC in darkness. 1 uM Cor will be added to the shake-flask culture on day 7 post inoculation since the Taxus cell culture is expected to be in the middle of a rapid growth phase, which is usually a favourable time for elicitor treatment in most plant cell cultures.

3) US treatment

An ultrasonic cleaning bath will be used to sonicate the cells in Erlenmeyer flasks from the shake-flask culture (Lin et al., 2001). The bath will had a fixed frequency of 38.5 KHz and variable power levels. The bath temperature wwill be maintained at 25±0.5^oC. The culture will be sonicated at level 2 US power (corresponding to 13.7 mW/ cm³ in the culture) once (on day 5, 9, or 16) or twice (day 5 and day 9) for 2 min each time (total US energy of each exposure = power x period = 1.64 J/cm³). The US power and exposure period will be selected based on the study by Lin et al., (2001) and Lin and Wu in 2002.

3. Determination of cell density

The cells in the shake-flasks will be separated from the medium by filtration through a Whatman filter paper under vacuum. The wet cell mass will be rinsed thoroughly with deionized water and then dried at 50^oC in an oven to constant weight.

4. Taxol extraction and determination

The taxol content in powdered dried cell and medium in the culture flasks will be extracted by methods previously described by Wu and Lin (2003). The dried cells will pulverized and suspended in 10 mL methanol, filtered and air-dried. After that, the cells will be re-dissolved in dichloromethane: water (1:1, v/v) followed by centrifugation at 5000 rpm. Dichloromethane phase will be collected, air-dried and re-dissolved in 100 µL methanol (HPLC grade) and filtered passing through a 0.45 µm syringe filter, before being injected to HPLC. The taxol content in the extract solution will be quantified by HPLC system, equipped with a C-18 column of 25 x 4.6 mm dimension. Taxol will be eluted with a linear gradient of acetonitrile and water (45: 55, v/v)/ at a flow rate of 1 mL/min with UV detection at 227 nm. Identification of taxol will be accomplished by comparison of retention times with authentic standard.

5. Statistical analysis

The results will be analyzed statistically by one-way ANOVA analysis and Duncan multiple range tests. The data will be presented as the mean value ± standard deviation (SD) of three independent experiments.