Kodiswaran Kandasamy Tissue Culture Unit Forest Biotechnology Division FRIM kodiswaran@frim.my

1. PLANT PROPAGATION (AN OVERVIEW) Kodiswaran Kandasamy Tissue Culture Unit Forest Biotechnology Division FRIM kodiswaran@frim.gov.my

2. PLANT PROPAGATION - conventional - tissue culture

3. CONVENTIONAL PROPAGATION 1. Cuttings (leaf, stem, root) 2. Layering (stem) 3. Marcotting 4. Grafting 5. Seed

4. TISSUE CULTURE 1. Micropropagation 2. Somatic embryogenesis 3. Suspension cell Culture 4. Protoplast fusion 5. Micrografting 6. Polyploidisation 7. Haploid Cell Culture 8. Mutation Breeding 9. Artificial Seeds

6. plant tissue culture some basics

7. Description Growing surface sterilised plant tissue in a nutrient medium under aseptic conditions in a controlled environment

8. Hormonal control of growth shoots AUXIN CYTOKININ Low High BAP Kinetin 2iP Zeatin IBA NAA IAA Low High roots

9. Effect of IAA and Kinetin on Proliferation and morphogenesis: Auxin - 3 3 0.03 Cytokinin 0.2 0.2 0.02 1 (mg/l)

10. MICROPROPAGATION (commercial)

11. Key factors • Laboratory design • Defined culture procedures • Scheduling & Stock keeping • Calculations • Human resource

12. Laboratory design • Appropriate size of each section • Media Prep & Washing • Autoclave & Media room • Transfer Room • Growth Rooms • Work Flow (Layout) • From non-clean to clean • Utilities & Specification • Clean water • Reliable power supply • Floor & Wall finish • Equipment layout

13. Micropropagation cycle Laboratory Greenhouse

14. Culture procedures • Define Explant Type & Size • Define Developmental Stages • Define Media for Each Stage • Define Growth Room Conditions • Define Number of Explant/Vessel • Define Sub-Culture Interval (cycle) • A Complete Step-by-Step guide Nodal explant Shoot-tip Plantlets ready for next sub-culture

15. Scheduling & stock keeping • Continuous production • Multiplication cycle - numbers • Rooting Stages - numbers • Ready for Sale (ex-lab) - numbers • Allocation of resources • Equipment • Manpower • Raw material • Stock keeping (database) • Production record & estimation • Plan production to meet targets (demand) • Tracking & able to trace stock

16. Calculations • Production estimations • Input requirement & cost • Raw material • Labour • Production efficiency • Cost estimations • Budget estimation • Business plan • Cash flow (P&L) • Marketing • Delivery schedule

17. Human resource • Smooth running of production line • Skilled & semi-skilled • Strict job specification • Production Manager • Production Supervisor • Operators • Support staff • Monitor & evaluate performance • Healthy and safe workplace

18. Planning production Y = An x B x F1 x F2 x F3 Y = No. of plantlets produced A = No. of shoots produced at each subculture B = No. of initial explants available n = No. of subculture passages F1 = Proportion of usable shoots after Stage II F2 = Proportion of usable shoots after Stage III F3 = Proportion of usable shoots after Stage IV

19. work @ frim at a brief

20. Genetic resources Forests are the main home of Earth's species. While covering only 6% of the planet's land surface, tropical forests contain at least 50%, possibly 70% and conceivably 90% of all species. ½ km2 of Malaysia's forests can feature as many tree and shrub species as the whole of the United States and Canada, while a single bush in Peruvian Amazonia has revealed as many ant species as in the British Isles. Norman Myers

21. Growing more crops and trees per acre leaves more land for Nature • We cannot choose between malnourished children & saving endangered wild species • Without higher yields, peasant farmers will destroy the wildlands and species to keep their children from starving • Sustainably higher yields of crops and trees are the only visible way to safe both • Dr. Norman Borlaug

22. TISSUE CULTURE LABORATORY (MAKMAL KULTUR TISU) Introduction R&D Services Products Staff Contacts

23. Facilities & Infrastructure We are fully equipped to undertake a complete range of in vitro studies, i.e. micropropagation, suspension cell culture, somatic embryogenesis, etc. Transfer Room Culture Racks Media Preparation Room Growth Rooms Holding Area Weaning Chamber Misting Area Growing Area

24. Research & Development Development of suitable, economically viable mass propagation protocol for various forest species. Selected Parent Explant Material Culture Initiation Multiplication Weaned Plantlet Cell suspension Embryogenic callus Somatic Embryos S.E. germination Shorea (Rita System)

25. Shorea leprosula

28. Acacia hybrids Wood density (AB6 x MB20) Harvest (pods/seeds) Transfer to laboratory Surface sterilisation Fiber Length (AB3 x MB22 Confirmation (DNA/Isozyme) In vitro germination Non-hybrids Discarded Hybrids Clonal Trial Transplant Multiplication

29. IMMATURE PODS MATURE PODS

30. In vitro germination & plant production • Crosses > 70,000 • Seed Pods > 380 • Seeds > 2,000 • Seedlings (ca 70%) > 1,400 • Selected clones = 400 • Plantlets (for clonal trial) = 20,000

31. Neobalanocarpus heimii (Chengal) Culture initiation Explant source Regeneration Shoot formation Callus proliferation Callus initiation Shoot multiplication for Somatic Embryogenesis for Micropropagation

32. Germination (in vitro) Seed dissected Seed explants Seedling (in vitro) in vitro germination Germination (ex vitro) Seeding (ex vitro) ex vitro germination Culture Establishment from Seed Explant

33. Tongkat Ali (somatic embryogenesis)

34. Mangrove species

36. Weaning Chamber Patent Pending: (# PI 2005 2865)

37. Teak (Tectona grandis)

38. Acacia (Acacia hybrids)

39. Jelutong (Dyera costulata)

40. Sesenduk (Endospermum diadenum)

41. Karas (Aquilaria malaccensis)

42. Sungkai (Peronema canescens)

43. Sample Received MAIN ACTIVITY FLOW-CHART Reject sample (Sample again) PROTOCOL DEVELOPMENT Record information R & D No Sterilisation Method Prepare for production Order (received) No Culture Initiation Shoot multiplication No Multiplication (culture) Sales (ex-lab) Rooting of shoots No Rooting (of shoots) Weaning (ex-nursery) Delivered ( to client) No Weaning

44. other experiences a quick recap

46. Somatic embryogenesis

47. Suspension cell culture

48. Artificial seed production

49. Anther & pollen culture

50. Protoplast culture