Title of Invention	A PROCESS OF PRODUCTION OF SECONDARY METABOLITES BY CULTURING PLANT PARTS
Abstract	The present invention relates to a process of plant tissue culture for the production of secondary metabolites using solid state fermentation technique.

Full Text

The present invention relates to a process for plant tissue culture using solid state fermentation, technique. More particularly the present invention relates to a process involving incubation of plant parts by solid state fermentation technique without or with feeding of nutrients. BACKGROUND OF THE INVENTION Plant tissue culture can be defined as the culture of plant cells tissue and organs under aseptic conditions. It is supporting the growth and/or development of isolated plant cells, tissues or organs on an artificial, nutritive medium. Generally such cultures are axenic, which means there is no other life form present on the culture medium than the plant tissue of interest. Plant tissues are maintained on the culture medium for a specified period of time and may be transferred to fresh medium periodically, or to a different medium to alter the path of development. The tissue or organ that is removed from a plant and placed into culture is called the explant. Tissue culture media are nutritive solutions presented to the explant in a liquid or semi-solid state. Explants may be immersed in a liquid medium as might occur in root culture, or explants may be positioned above the medium, but in contact with the medium, as provided by a Heller support or a raft (e.g. Magenta's Membrane Raft or Osmotek!s Life Raft). Alternatively, the medium may be gelled by the addition of agar; a heteropolysaccharide derived from algae. The medium then has a consistency similar to Jell-O. This gelled agar will support the explant and keep it from submerging in the medium, yet allow diffusion of the medium ingredients into the plant tissues. There are many different formulations of media, and the one selected will depend largely upon the plant species and the cell, tissue or organ selected for culture. One of the most widely employed media is the medium developed by Toshio Murashige and Folke Skoog (Murashige, T. and F. Skoog. 1962. Physiologia Plantarum 15:473-497). The Murashige and Skoog medium is so well known to the scientific community that it is often abbreviated simply as the MS medium. Other important media formulations include Gamborg's B-5 medium and McCown's Woody Plant medium. There are others too numerous to mention that are used for specific purposes. Basically a nutritive medium contains macronutrients (minerals required in rather large concentrations such as N, P, K, Mg, S, Ca), micronutrients (also called traGe elements because they are required in low concentrations such as » Fe, Zn, B, I, Mn, Mo, Co, Cu), vitamins (thiamine, nicotinic acid, riboflavin most commonly), sugars (sucrose and glucose are common) and sugar alcohols (myo-inositol in particular). A medium may also include amino acids, nucleic acid bases, or other organic molecules. Phytohormones or plant growth regulators may be added to the basal medium to stimulate the growth and development of the explant in a particular fashion. There Plant tissue culture has an important role to play in the production and isolation of several insecticides, antifungal, anticancer, antidiabetic and several other pharmaceutically important ingredients. » Most reports available disclose plant tissue or cell culture utilizing the skills of submerged or semi solid fermentation and not the use of solid substrate fermentation. A Japanese patent discloses an apparatus for solid plant tissue 3 1 or cell culture comprises a culture medium support, formed using an assembly of fibers with a density of 0.005-0.150 g/cu cm. Using this device, the growth rate is higher than in conventional apparatus where agar is used in the culture medium. As an example of propagation by cutting, there has been known to propagate shoots of asparagus by culturing small lateral branches or nodes as an explant, followed by rooting to produce plants (Chee-kok Chin, HortScience 7(4): 590-591, 1982; Takayama S. et al., Japanese Patent Application Laid-Open-to-Public , Publication N o. 09722/1988). There has also been reported a method of propagation by cutting a plant derived from apical meristem culture of garlic on:a propagation medium (Bhojwani S., ScientiaiHorticulturae 13:47-52, 1980). Similar method has been reported about onion (Hussey G., Sci. Hortic., 9: 227-226, 1978). Furthermore, there have been many reports on other plants, for example, caladium (Hartman R. D., Phytopathology 64(2): 237-240, 1971), cordyline (Kunisaki J. T., HortScience 10 (6): 601-602, 1975), pineapple (Mathews V. H. et al, Scientia Hort. 11: 319-328. 1979), carnation (Fujino M. et al,, Summary of Lectures at 1971 Spring Meeting of » Japan Society of Horticulture, p 302-303), etc. SUMMARY OF THE INVENTION The present invention is summarized in that a plant tissue/cell culture is carried out using solid state fermentation technique. The fermentation is also carried out in a closed bioreactor, Plafractor. The fermentation is also carried out by a technique called Fed-batch. The objects of this invention therefore are: • to provide a bioreactor for a contained solid state fermentation such that the fermenting solid plant tissue or bell culture and the fermentation products it produces are kept isolated from the outside environment during the course of fermentation • to reduce solvent consumption required in large quantities for extracting from fermentation broth or the aqueous residue • to produce higher yield and purity of the desired metabolite from the plant tissue or cell culture. • to assure full safety for the cytotoxic fermentation of solid plant tissue or cell culture. • to reduce the period for fermentation and to control the temperature easily. The contained bioreactor is 'PlaFractor' and is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, in situ extraction of the end product, recovery of the solvents and post harvest sterilization (US 6,197,573). The ! PlaFractor ', a novel bioreactor for solid state fermentation is a unique invention of Biocon India Limited. The modular construction of this bioreactor provides multiple modules stacked on top of} one another, each with a base connected to frame for holding the solid medium in isolation from the exterior environment. The construction of the bioreactor allows solid state fermentation to be carried out in a manner such that the fermenting material and the fermentation products it produces are kept isolated from the outside environment during the course of the fermentation. This containment of the fermentation process is of significant importance when working with microbial metabolites and solid plant tissue or cell culture, which are cytotoxic in nature. An important aspect of the bioreactor is a mechanism of heat removal resulting in stringent temperature control of the fermentation process. In comparison, maintaining a constant temperature of growth in solid state fermentation using tray cultures is not efficient. The base plate of the bioreactor has multiple channels called non-communicating channels that carry heating and cooling fluids sandwiched between two sheets. Heat is transferred to and from the modules by conduction. In this way the temperature of the module is precisely maintained to meet the specific requirement of different fermentation. The base of the module contains a second set of channels, the communicating channels to deliver sterile air as supply of oxygen into the solid substrate bed for optimum growth conditions. Moisture loss because of » passage of sterile! air is significantly reduced by regularly reversing the direction of airflow every few hours. Using this, homogeneity in moisture content is maintained throughout the bioreactor. These aspects provide ample convenience over previous SSF methodologies that require multiple manipulations at each step of the fermentation process. To achieve the said objectives this invention provides a method for the solid plant tissue or cell culture comprising: » Fresh parts from a plant are surface sterilized with mercuric chloride and or in combination with hypochlorite. Then these are repeatedly rinsed with sterile water, cut into small pieces and placed on Murashige Skoog (MS) medium along with, Rhizobium rhizogenes for the induction of hairy roots. The bacterial culture is then removed by keeping these leaf samples on MS medium in the presence of an antibacterial agent. On incubation, hairy roots appear and these hairy roots can be repeatedly subcultured onto fresh medium and thus obtained hairy roots can be made to grow on a supporting substrate for the generation of metabolites. This procedure is carried out in a closed bioreactor. The bioreactor has provisions to feed the nutrients intermittently. The grown culture is further processed by techniques comprising extraction, chromatography, precipitation, crystallization and drying. The present invention has the following advantages over the other reported methods (i) Fermentation in a bioreactor, which is fully contained as a result assuring full safety for the plant tissue or cell culture (ii) Easier control of fermentation parameters like temperature, pressure, air supply, moisture and fed batch technique making the process economically attractive. » (iii) Fewer steps for the isolation and purification to get the pure product, thus saving processing time and additional expenses. EXAMPLES Example 1 Fresh leaves of Coleus forskholii were repeatedly washed with sterilized water and then surface sterilized with mercuric chloride followed by sodium hypochlorite solution. Then these were washed with sterilized water and cut into small pieces. These are placed on MS medium along with Rhizobium rhizogenes culture. Later these leaves are placed on fresh MS medium with carbenicillin for the removal of bacterial culture. On incubation, hairy roots appeared and these hairy roots were cut into small pieces and subcultured on to fresh MS medium. Well-grown hairy roots were cut into small pieces and transferred on to fresh medium and grown for 3 to 4 weeks. Into 9cm petri plate, one layer of porcelain beads are placed and liquid MS medium was added such that all the beads are sufficiently wet with the medium. This is sterilized by autoclaving and cooled to room temperature. Small pieces of hairy roots were put on to these beads and grown for 4 to 5 weeks at 24 deg C. The roots were harvested and estimated for forskholin content. 1.8mg forskholin was obtained per g root dry wt. » Example 2 Hairy roots were grown as mentioned in Example 1 One layer of porcelain beads is loaded onto a bioreactor having 22600-cm2 surface area. Liquid MS medium is added such that all the beads are sufficiently wet with the medium. The bioreactor was sterilized at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 24 degree C. Small pieces of hairy roots were aseptically put on to these beads and grown for 4 to 5 weeks at 24 deg C. Liquid MS medium was sprayed onto the growing hairy roots as a nutritional feed on a weekly basis. The roots were harvested and estimated for forskholin content, 1.5mg forskholin was obtained per g root dry wt. Example 3 Fresh leaves of Nothapodytes foetida were repeatedly washed with sterilized water and then surface sterilized with mercuric chloride followed by sodium hypochlorite solution. Then these were washed with sterilized water and cut into small pieces. These are placed on MS medium along with Rhizobium rhizogenes culture. Later these leaves were placed on fresh MS medium with carbenicillin for the removal of bacterial culture. On incubation, hairy roots appeared and these hairy roots were cut into small pieces and subcultured on to fresh MS medium. Well-grown hairy roots were cut into small pieces and transferred on to fresh medium and grown for 3 to 4 weeks. Into 9cm petri plate, one layer of porcelain beads are placed and liquid MS medium was added such that all the beads are sufficiently wet with the medium. This is sterilized by autoclaving and cooled to room temperature. Small pieces of hairy roots were put on to these beads and grown for 4 to 5 weeks at 24 deg C. The roots were harvested and estimated for camptothecin content. 0.12mg camptothecin was obtained per g root dry wt. » Example 4 Hairy roots were grown as mentioned in Example 1 One layer of porcelain beads is loaded onto a bioreactor having 22600-cm2 surface area. Liquid MS medium is added such that all the beads are sufficiently wet with the medium. The bioreactor was sterilized at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 24 degree Centigrade. Small pieces of hairy roots were aseptically put on to these beads and grown for 4 to 5 weeks at 24 deg C. Liquid MS medium was sprayed onto the growing hairy i roots as a nutritional feed on a weekly basis. The roots were harvested and estimated for camptothecin content. 0.1 mg camptothecin was obtained per g root dry wt. WE CLAIM 1. A process of production of secondary metabolites by culturing plant parts using solid state fermentation technique. 2. A process in claim 1, wherein the plant parts comprises cell, tissue or organ of a plant 3. A process as in claim 1, wherein the culturing technique comprises hairy root culture, callus culture, cell culture or tissue culture. 4. A process as in claim 1, wherein the solid state fermentation is carried out in a contained bioreactor, 5. A process as in claim 4, wherein the fermentation parameters comprising temperature, pressure, humidity and oxygen supply in the bioreactor are controlled. 6. A process as in claim 4, wherein the bioreactor is 'Plafractor1. 7. A process as in claim 1, wherein the plants used is selected from a group comprising Azadirachta indica, Mappia foetida (Nothapodytes foetida), Hypericum perforatum, Coleus forskholii, Podophyllum peltatum, Podophyllum emodi, Gymnema sylvestre, Catharanthus roseus (Vinca rosea), Cephalotaxus harringtonia, Taxus brevifolia, Taxus buccata, Betula alba 8. A process as in claim 1, wherein the solid state fermentation is done by fed-batch technique. 9. A process of production of secondary metabolites by culturing plant parts using solid state fermentation technique substantially as herein described with reference to the foregoing examples.

Documents:

109-mas-2002- abstract.pdf

109-mas-2002- claims duplicate.pdf

109-mas-2002- claims original.pdf

109-mas-2002- correspondence others.pdf

109-mas-2002- correspondence po.pdf

109-mas-2002- description complete duplicate.pdf

109-mas-2002- description complete original.pdf

109-mas-2002- description provisional.pdf

109-mas-2002- form 1.pdf

109-mas-2002- form 19.pdf

109-mas-2002- form 26.pdf

109-mas-2002- form 3.pdf

109-mas-2002- form 5.pdf