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

2. 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

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
Worldwide industry multibillion Euros

4. 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.

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

10. ADVENTITIOUS ORGANOGENESIS IN PISTACHIO6 5 1 2 3 4
Adventitious Buds 2
Elongation and 2 2
from Single Leaflet 3
Regeneration of
Plantlets 4

11. 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

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

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 ).
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 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

18. 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 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, Diyarbakir, Turkey

19. THANK YOU！