Plant tIssue culture technIques—Tools In plant mIcropropagatIon Ahmet Onay, Department of Biology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey EUROPEAN BIOTECHNOLOGY CONGRESS September 28 – October 1, 2011 İstanbul – TURKEY

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

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 Worldwide industry multibillion Euros

FUNDAMENTAL ABILITIES OF PLANTS HOW 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.

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 engineered potatoes Gottleib Haberlandt

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

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

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

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

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

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

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

WHAT IS MICROGRAFTING?

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

PHOTOAUTOTROPHIC CULTURE

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.

WHAT’S IN THE FUTURE 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

ACKNOWLEDGEMENTS All photos in this talk were taken by Ahmet Onay and Engin Tilkat, except the ones on the slayt 12 and 15 which was obtained from the WEB pages. TUBITAK TOVAG - 3355 has granted part of these studies… And the technical assistance of all co-authors are gratefully acknowledged. Ahmet Onay, Department of Biology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey

THANK YOU！