Plant tIssue culture technIques — Tools In plant mIcropropagatIon

1. PlanttIssueculturetechnIques—ToolsInplantmIcropropagatIon Ahmet Onay, Department of Biology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey EUROPEAN BIOTECHNOLOGY CONGRESS September 28–October 1, 2011 İstanbul –TURKEY

2. WHAT IT IS? • In vitro propagation? Or • Micropropagation?Or • In vitro culture? “… THE ASEPTİC CULTURE OF PLANT…” Implies- regeneration - multiplication

3. IMPORTANCE OF PLANT TISSUE CULTURE TECHNIQUES True-to-type clones A single explant can be multiplied into several thousand Year-round production Rare and endangered plants can be cloned safely To produce virus free plants Long-term germplasm storage with ‘tissue banks’ Plant cultures easier to export than are soil-grown plants Production of difficult-to-propagate species Worldwideindustry multibillion Euros

4. FUNDAMENTAL ABILITIES OF PLANTSHOW CAN A PLANT CELL OR TISSUE DEVELOP? • Totipotency • Dedifferentiation • Competency Therefore, tissue can be regenerated from explants such as cotyledons, hypocotyls, leaf, ovary, protoplast, roots, anthers, etc.

5. WHAT’S THE BACKGROUND 1902 – Haberlandt – The Concept 1920s – Knudson – Simple Orchid Germination– First commercial use 1930s – Thimann & Went – Auxin 1930s – White/Gautheret/Nobecourt –Root Cultures 1950s – Skoog’s group – Cytokinins, –The discovery of the structure of DNA by Crick and Watson 1960s –Morel-Orchid micropropagation, thermotherapy 1970’s – Genetic engineering took off 1990s –by Calgene –genetically engineeredpotatoes Gottleib Haberlandt

6. WHAT IS NEEDED? • Appropriate tissue • A suitable growth medium • Aseptic (sterile) conditions • Growth regulators:The ratio of auxins and cytokinins • Frequent subculturing

7. TYPES OF PLANT TISSUE CULTURE TECHNIQUES • Culture of intact plants (Seed orchid culture) • Embryo culture (embryo rescue) • Organ culture:Micropropagation A.Organogenesis in solid or semi solid medium 1. Meristem and shoot tip culture 2. Bud culture 3. Root culture 4. Leaf culture 5. Anther culture B. Somatic embryogenesis C. Organogenesis and somatic embryogenesis in bioreactors D. In vitro micrografting E. Thin cell layer technology (TCLs) F. Photoautotrophic culture 4. Callus culture 5. Cell suspension and single cell culture 6. Protoplast culture, somatic hybridization

8. Culturing (micropropagating) Plant Tissue - the steps • Stage 0 – Selection & preparation of the mother plant • sterilization of the plant tissue takes place • Stage I  - Initiation of culture • explant placed into growth media • Stage II - Multiplication • explant transferred to shoot media; shoots can be constantly divided • Stage III - Rooting • explant transferred to root media • Stage IV - Transfer to soil • explant returned to soil; hardened off

9. ORGANOGENESIS OF PISTACHIO Fresh apical tip (a) or nodal bud (b) 1 2 3 Culture initiation Lignified shoots Hardened plantlets 6 5 4 Rooting Shoot proliferation

10. 1 2 3 5 4 6 ADVENTITIOUS ORGANOGENESIS IN PISTACHIO Adventitious Buds 2 Elongation and 2 2 3 from Single Leaflet Regeneration of Plantlets 4

11. Isolated kernels Immature fruits Ebryogenic tissue Ebryogenic tissues in liquid medium Maturation of SE Ebryogenic tissues Development of SEs Swollen SEs Isolated SEs for germination Germinated SEs Acclimatised somatic seedlings 6 months after transplanting somatic seedlings SOMATIC EMBRYOGENESIS IN PISTACHIO

12. Bioreactor;tissue culture containers Automated culture system The design of separate compartments MICROPROPAGATION IN BIOREACTORS

13. WHAT IS MICROGRAFTING?

14. The thin cell layer (TCL) system consists of explants of small size excised from different plant organs either longitudinally (lTCL) or transversally (tTCL)

15. PHOTOAUTOTROPHIC CULTURE

16. APPLICATIONS OF MICROPROPAGATION • Through micropropagation, it is now possible to provide clean and uniform planting materials in plantations for several plant species such as oil palm, plantain, pine, banana, abaca, date, rubber tree; field crops – eggplant, jojoba, pineapple, tomato; root crops – cassava, yam, sweet potato; and many ornamental plants such as orchids and anthuriums (Alfonso, A. 2007; Singh et al. 2011). • Bioreactor cultures are being established in several commercial laboratories for micropropagation of ferns, spathiphylum, philodendron, banana, potato, lilies, poinsettia, sugar-cane, and some forest tree species such as eucalyptus, poplar, and early stages of conifer somatic embryos (Aitkin-Christie, 1991; Mehrotra et al 2007;Gross and Levin, 1999; Cervelli and Senaratna 1995).And plant products, pharmaceuticals, food ingredients and cosmetics(Perulllini et al., 2007; Vongpaseuth and Roberts 2007; Pavlov et al. 2007) • Micrografted seedlings are commercialised to avoid the serious crop loss caused by infection of soil-borne diseases for fruit trees and several vegetables (Navarro et al., 1975;Navarro 1981;Jung-Myung Lee et al. 2010). • Transverse thin layer section technology may be ideal for large scale micropropagation of ornamental plants (Jain et al. 1998). • Photoautotrophic flow-through systems for enhanced micropropagation for Gerbera; Hypericum, Myrtus communis, Momordica grosvenori, Eucalyptus (Nguyen and Kozai 2005; Xiao et al.2011) • The development of transgenic methods and the growth of agricultural biotechnology started during the 1980s and the global biotech crophas increased hundred million of hectares area • Palmer et al. 2005; Thomas et al. 2003 • Efficient doubled haploid technology enables breeders to reduce the time and the cost of new cultivar development relative to conventional breeding practices.

17. WHAT’S IN THEFUTURE • Adaptation of tissue culture technology to more species • Fast and mass propagation of transformed plants with “designer genes” • Chloroplast transformation methods • Efficient, computer-controlled flow-through systems to cut down the labor cost • Mass production of plant constituents • New technologies

18. ACKNOWLEDGEMENTS All photos in this talk were taken by Ahmet Onay and Engin Tilkat, except the ones ontheslayt 12 and 15 which was obtainedfromthe WEB pages. TUBITAK TOVAG - 3355 has grantedpart of thesestudies… And the technical assistance of allco-authorsaregratefully acknowledged. Ahmet Onay, Department of Biology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey

19. THANK YOU！