1. ERT211 BIOCHEMICAL ENGINEERING

2. Course Outcome Ability to describe the usage and methods for cultivating plant and animal cell culture Ability to discuss the technologies available in bioconversion

3. Chapter 5 Considerations in Using Plant and Animal Cell Cultures

4. Topic Outline  Usage of plant cell cultures  Potential product from plants cell cultures  Approaches to increase productivity  Difference of plant cells from microbes - implications for bioreactor design

5. What is plant cell culture? Plant cell culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant.plantsclones

6. The purpose of practicing plant and cell culture includes for regeneration/propagation, embryogenesis and secondary metabolites production. In this technique, culture medium components, explant source and plant growth regulator is the important factors. Procedures should be follows includes laboratory set up, medium preparation, sterilization, initiation and maintenance of cultures.

7. Plant cell culture techniques

8. Laboratory requirements for plant cell culture Laminar air flow cabinets Autoclave Oven for dry sterilization Water distillation apparatus Incubator Shakers Fermenters or bioreactors

9. Materials and methods Plants – any part obtained from any plant species can be employed to induce callus tissue Media - inorganic salt - Carbon sources - Vitamins - phytohormones - organic supplements Or ready made medium – Murashige and Skoog, Gamborg, B5, Nitsch etc

10. Establishment of plant cell culture Callus and suspension cultures have been established from hundreds of different plants A callus can be formed from any portion of the whole plant containing dividing cells Callus – tissues arising by proliferation from explants on agar medium/Mass of undifferentiated cells produced in tissue culture is called callus. The callus is highly vacuolated and unorganised cells. Suspension cultures – cells or small cells aggregates growing dispersed in liquid medium

12. Basic protocols for suspension culture

13. Excised plant material is placed on solidified medium Callus forms can be quite large (> 1 cm) Callus transfer to liquid medium (for propagation) Establishment of suspension culture from callus is straightforward if callus is friable A piece of callus is placed in liquid medium in shake flask. With moderate agitation, cells or small aggregates of cells will slough off A platform shaker is used to give a circular/orbital motion in a variable speed control (a range form 30-150 rpm).

14. Cond. 25°C, pH5.5 in the dark These suspended cell then replicate After 2/3 weeks, suspended cells are transferred to fresh medium For large scale suspension cultures, bioreactors system is being used for the purpose of producing high value plant products.

15. Implication for bioreactor design

16. Plant cell vs microbes

17. CharacteristicsMicroorganismPlant Cell Size Shear stress Water content Duplication time Aeration Fermentation time Product accumulation Product phase Mutation Medium cost ($) 2  Insensitive 75% <1 hour 1-2 vvm Days Medium Uncoupled Possible 8-9/m3 >10  Sensitive >90% Days 0.3 vvm Weeks Vacuole Often growth linked Required haploids 65-75/m3

18. Implication for bioreactor design Many of the diff between plant cell and microbes have direct implication for the design and scale up of suspension culture DifferencesImplication for bioreactor design More shear sensitiveMay require operation under low-shear condition Lower growth rateMay need large bioreactor volume Lower respiration rateLower O2 transfer rates required

19. 1. Shear Stress Plant cells are large compared to microorganism When they are expose to high shear stress, the cell will damage Reactors with high shear must be avoided Plant cells may require operation under low-shear conditions.

21. 2. Aeration In aerobic fermentation processes, microorganisms require high aeration (have high metabolic actv) compare to plant cells Require to change the bioreactor system that provide high aeration rate. system can be equipped with mechanical devices used to agitate the liquid broth or by air compressor. Eg airlift reactor

22. Airlift reactor Mixing in airlift bioreactors usually accomplished without any mechanical agitation The bioreactors are used for tissue culture because the tissues are shear sensitive and normal mixing (aeration plus mechanical agitation) is not possible

23. 3. Size Plant cells are large compared to microorganisms. Cell often grow as aggregates or clumps. Cell often grow as aggregates or clumps and may have mass transfer limitations that limit the availability of nutrients to cells within the aggregate. In some case, aggregates are necessary to achieve a mix of cell types essential for good secondary-metabolites formation

24. 4. Duplication times Duplication time in microorganisms is within days compare to plants within weeks. To contain the rapid multiplication of cells, larger bioreactor volumes is needed and usually in the range of 10,000 – 100,000 L. Products are produce within shorter period. Shorter period in the culture systems means less contamination and less cost for the maintenance.

25. Advantages of using plant cell cultures for factory production of chemicals

26. 1. Control of supply of product independent of availability of the plant itself 2. Cultivation under controlled and optimized condition 3. Strain improvement with programs analogous to those used for microbial systems. 4. With the feeding of compounds analogous to natural substances, novel compounds not present in nature can be synthesized

27. 1) Control of supply of product independent of availability of the plant itself Many drugs contained active ingredients source from plant parts. Paclitaxel (taxol) - anticancer agent - isolated from bark of Pacific yew tree (Taxus brevifolia) - Three 100-year-old trees is needed to supply enough paclitaxel for one patient Supply problems greatly impeded the clinical development of this drug

28. Production of paclitaxel in large-scale bioreactors provides a product with fewer contaminants, highly controllable and reproducible. Recent, commercial production of paclitaxel in stirred tank vessel 30000 l

29. 2) Cultivation under controlled and optimized condition The accumulation substances extracted from plants affected by many factors etc. condition of the plant and environment Production of saponin via tissue culture technique from Panax ginseng was affected by the concentration of hormones used. 2,4-D (mg/l) 510 Kinetin (mg/l) 11 Saponin (%) 3.628.78

30. Callus cultures of Oxalis reclinata are yellow in the light, but when they are transferred to the dark, they start producing red anthocyanin pigments.

31. 3) Strain improvement with programs analogous to those used for microbial systems The physiological characteristics of individual plant cells are not always uniform In tissue culture, selection of cell lines for specific compound production will increase in yield. Selection using radioimmunoassay. Similar method to monocolony isolation of bacteria radioimmunoassay (RIA), highly sensitive laboratory technique used to measure minute amounts of substances including antigens, hormones, and chemicals present in plant cells

32. A strain of Euphorbia milli was recognized to accumulate about 7 times higher amounts of anthocyanins than that of the parent strain after 24 selections.

33. 4) With the feeding of compounds analogous to natural substances, novel compounds not present in the nature can be synthesized Addition to the culture media of appropriate precursors or related compounds sometimes stimulates secondary metabolite production Precursors can be synthetic or natural substances This approach is advantages if the precursors are inexpensive The production of limonene is improved with the addition of mevalonic acid (primer precursor)

34. A diagram of limonene biosynthetic pathway.

35. Potential products from plants cultures Pharmaceuticals Food additives

36. Morphinan alkaloids Codeine is an analgesic & cough-suppressing drug Morphine from Papaver somniferum L. (opium poppy) is a traditional commercial source of codein. Morphine can be converted to codeine Thebaine from P.bracteatum also can be converted to codeine

37. Capsule of Papaver somniferum showing latex (opium) exuding from incisionopium

38. Little success has been achieved from culture of undifferentiated cells of these plants for codiene production After many attempts, researchers still failed to get direct codeine and morphine from cell culture Later, Japanese researchers (Furuya et al), studied the biotransformation of codeinone to codeine using immobilized cells of P.somniferum The conversion yield was 70.4% and about 88% of codeine converted was excreted into the medium

39. Shikonin compounds Shikonin and its derivatives (e.g. acetyl shikonin & isobutyl shikonin) are reddish purple pigments accumulated in roots of Lithospermum erythrorhizon They have been used in traditional dying and as a herbal medicine Because of shortage of this plant, Mitsui Petrochemical company in Japan investigated mass cultivation of L. erythrorhizon cells to produce shikonin compounds.

40. Two-stage culture for shikonin production

41. Approaches to increase productivity a) Optimization of cultural condition b) Selection of high-producing strains c) Addition of precursors d) Biotransformation e) Elicitor treatment

42. In order to obtain products in high concentration, many effort have been made to stimulate or restore biosynthetic activities of cultured cells using various methods. several typical approaches that may increase productivity of cultured plant cells : a) Optimization of cultural condition b) Selection of high-producing strains c) Addition of precursors d) Biotransformation e) Elicitor treatment

43. 1) Optimization of cultural conditions Medium -The most important is the medium that influence both the growth of cells and yield of desirable products -Various basal medium have been used and Murashige & Skoog (MS) is among the most widely applicable -Sucrose and glucose are carbon source for plant tissue cultures and affects cell growth and yield of products - Phytohormones such as auxins and cytokinin have shown the most remarkable effects on growth and productivity of plant metabolites.

44. Surrounding environment (temp,pH,light & O 2 ) - Generally, a temperature of 17-25 ° C is normally used for induction of callus tissues and growth of cultured cells - The pH is usually adjusted between 5-6 before autoclave - The use of light depends on the type of culture and the desire products - Oxygen is not critical for plant cultures but still has an effect on the growth and production

45. Cell density - The use of high cell density cultures in a suitable bioreactors found to increase yield in some cultures TWO-STAGE CULTURE - Some plant cells needs different media for the cell growth and secondary metabolite production. - This cultural condition is called two-stage culture. It means that 2 types of culture medium are used. both culture medium may differ in the concentration or types of nutrient, hormone or vitamins used.

46. two types of medium were used because the medium used for promoting growth is not suitable for promoting metabolites production. - In such system, the first medium is used to promote the growth of cells in the culture system. - the second culture is to promote the production of metabolites in the culture. - Cells of plant will be first cultivate in the first medium and after certain period, they will be transferred into the second growth until harvesting period.

47. Two-stage culture for shikonin production

48. 2) Selection of high-producing strains The physiological characteristics of individual plant cells are not always uniform Therefore a rapid assay method is crucial in the selection of a high yielding cell line The specific cell line is obtained from the selection a number of strains producing high level of desirable product The strains then were subjected to further cell cloning to increase the level of secondary metabolites

50. 3) Addition of precursors Precursor – a compound that participate in the chemical reaction that produce another compound Addition of precursors to the culture media sometimes stimulates secondary metabolite production This approach is advantageous if the precursors are inexpensive

51. For exp : Phenylalanine is one of the biosynthetic precursors of rosmarinic acid. Addition of this amino acid to Salvia officialis suspension cultures stimulated the production of rosmarinic acid and shortened the production time as well.

52. 4) Biotransformation  A suitable substrate compound may be biotransformed to a desired product using plant cell  Biotransformation has been extensively applied in the fermentation using microorganisms and their enzymes  For example, L-aspartic acid and L-malic acid can be biotransformed from fumaric acid using microorganisms

53.  Using plant cells, for exp. the biotransformation of  -methyldigitoxin to  -methyldigoxin using Digitalis lanata has been investigated - Digoxin has a large market as a cardiac glycoside  This approach beside precursors feeding are the most commercially realistic approaches because of economic reasons

54. 5)Elicitor treatment Elicitor is an agent of microbial infections on intact plants that cause the synthesis of specific secondary metabolite. Some studies reviewed possible correlations between stress and secondary metabolism in cultured cells. Some suggested that upon infection, plants shows their defense mechanism by secreting secondary metabolite.

55. Elicitors that have been used in plant cell cultures are yeast extract, chitosan, inorganic and organic molecules and many more  Plants grow under stress condition also show elicitation effects. Phosphate limitation in hairy root cultures of Hyoscyamus muticus had increased production of the sesquiterpene solavetivone  Examples of inorganic compounds used are sodium chloride, potassium chloride, sorbitol and abscisic acid  For economical use of the elicitors, they should be cheap and easy to obtain.