Topic. Bench to Batch: Advances in Plant cell culture for producing useful products

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1 Topic Bench to Batch: Advances in Plant cell culture for producing useful products Submitted BY: Quratulain Iram

2 Bench to Batch: Advances in Plant Cell Culture for Producing Useful Product Plants have huge natural and chemical diversity and nowadays 400,000 species of higher plants have been recognized which produce a vast array of natural products. These products include commercially interesting proteins such as vaccines, antibodies and therapeutic proteins. There has been a long standing interest on commercially viable production of secondary metabolites using inlictro cultures. The General Strategy for Producing Plant Products Plant products are generally produced within the plant cells or organs. As the number of plant cells and organs is greater, the greater will be the production of useful products. In other words, high amounts of plant biomass are generally required. If the high productivity of both biomass and product is achieved, then the process will have potential to be commercialized. Approaches to Achieve Consistent High Productivity There are some complex approaches to achieve consistent high productivity. 1) Biomass Optimization:- In vitro plant materials used for the production of useful products are of three main types. 1. Suspension 2. Hairy roots 3. Micro propagated plantlets Regardless of tissues or cells used, the growth and production of biomass should be optimized. This optimization is achieved by following approaches. a) Using different concentrations of nutrients and phytohormones at a time. b) Modified simlex method to asses the importance of independent variables. c) Structural nutritional model to describe and predict growth and nutritional responses of hairy roots and cell cultures.

3 d) Testing key nutrient or nutrient group individually instead of using batch cultures, while keeping the conc. of that particular key nutrient constant at that time. Using these approach, both biomass and products can be maximized. 2) Increasing Production of Plant Products Plant products are mainly of two types. i. Secondary Metabolites Production: Secondary metabolites are one of the major foci as many products are of commercial interest. Recent developments to increase production of secondary metabolites have used metabolic engineering. This approach has been used by a group at caliphornia university, Berkely to produce plant terpenoid products in microbial production systems e.g. E-coli and yeast. This approach involves codon optimization of the artimisinin cyclase gene, amorphadiene synthase, that when co-expressed with on engineered movelonic acid pathway resulted in a fold improvement inamorphadiene production. Elicitation: It is a process in which an elicitor (external stimuli) turn on the biosynthesis of molecules that may only be produced in small amounts or even new compounds. Secondary metabolites can be produced in high quantities in response to external stimuli. Elicitors functions differently in different species. Many of the elicitor are lethal to plant cell cultures as they induce decline in vitality of invitro culture, so should be used in optimum concentrations. Types of Elicitors: There are two types of elicitors. 1) Abiotic 2) Biotic Both elicitor can work individually or in combination to turn or biosynthesis of molecules.

4 Abiotic Elicitors: These include metal ions, other inorganic compounds and electric current. Biotic Elicitors: These include microbial cells or products e.g. clutin or clitosan (a component of fungal cell walls). ii. Transgenic Proteins Production: The transgenic proteins involves vaccines, antibodies antigens, immunomodulators and therapeutic proteins. In vitro systems are used for the production of these proteins. In Vitro Plant Cultures Used to Produce Transgenic Proteins 1. Suspension Cells: Biologically human α-mterferon, human interlukin-12 and hepatitis β surface antigen have been produced in tobacco BY-2 and NT-1 cells. 2. Hairy Roots: Immunomodulators like ricin-b, murine interlukin-12 and monoclonal antibodies have been produced in tobacco hairy roots. The main problem using this technology is low yield of these proteins. Strategies to Improve Protein Yield These are two strategies to improve protein yield. 1. Expression of recombinant proteins in cells using a sucrose inducible RAmy3D promoter. 2. Expression of recombinant proteins fused with fused with a lydroxyprotein (Hyp) rich glycoprotein (HRGP) tag. Application of the above two strategies improve protein yield up to many folds.

5 Use of Novel Plant Expression Systems: To obtain high quantity of pant products, novel expression systems have been used, that include: Duckweed: is a small, edible, aquatic plant that offers an attractive system for the production of recombinant proteins and other molecules. Moss: It is capable of producing successfully many pharmaceutical proteins like human VEGF. It also do post transcriptional modification of many mammalian proteins. 3. Bioreactors: Several types of bioreactors have been used for growing plant cells to produce useful products. These bioreactors include stirred tank, airlift, bubble column, gas-phase, balloon type bubble bioreactor, temporary immersion bioreactors, temporary root zone immersion bioreactors, rotating drum bioreactors, and wave reactors. The suitable selection of bioreactors is dependent upon whether the goal is growing plant cells in suspension cells, hairy roots or micro propagate pantalets. 4. Use of Co-cultures for Over-production of Secondary Metabolites:- Co-culture is a process in which two cultures are grown within some culture device. It includes: Two species of hairy roots Shoots + roots of single or different species Microbes/Insects + some pant cells/tissues or organs. This approach was firstly used for the over production of scopolamine. Several other products have been over-produced by in vitro plant co-culture such as Furanocoumarins, Ginsenosides, Padctaxet, podophyllotoxin, phytoestrogen Isoflavones and non chemical products like aphids, nematodes and viruses etc. This approach increases the production of products many times.

6 5. Use of Disposable Reactors:- Now disposable reactors have been used for plant cell, tissue or organ cultures, usually plastic bags. The advantage of using these disposable bags is low cost of these disposable culture systems than the usual stainless steel tanks. Some types of disposable bioreactors available fro pant cell and tissue culture includes: Bag liner in a vessel, cultife ask, mechanically driven membrane, life reactor, Ebb and Flow bioreactor etc. Conclusion: Using these approaches mentioned above have significantly more the reality of pant produced products closer to commercialization.