PLANT TISSUE CULTURE TECHNIQUES—TOOLS IN PLANT MICROPROPAGATION

Ahmet Onay, Department of Biology, Faculty of Science, University of Dicle,

21280 Diyarbakir, Turkey

EUROPEAN BIOTECHNOLOGY CONGRESSSeptember 28 – October 1, 2011

İstanbul – TURKEY

WHAT IT IS?

• In vitro propagation? Or

• Micropropagation ? Or

• In vitro culture?

“… THE ASEPTİC CULTURE OF PLANT…” Implies - regeneration

- multiplication

• 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

IMPORTANCE OF PLANT TISSUE CULTURE TECHNIQUES

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.

WHAT’S THE BACKGROUND

1902 – Haberlandt – The Concept1920s – Knudson – Simple Orchid Germination – First commercial use1930s – Thimann & Went – Auxin1930s – White/Gautheret/Nobecourt –Root Cultures1950s – Skoog’s group – Cytokinins, – The discovery of the structure of DNA by Crick and Watson1960s – Morel-Orchid micropropagation, thermotherapy1970’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 TECHNIQUES1. Culture of intact plants (Seed orchid culture)2. Embryo culture (embryo rescue)3. Organ culture:Micropropagation

A. Organogenesis in solid or semi solid medium1. Meristem and shoot tip culture2. Bud culture3. Root culture4. Leaf culture5. Anther cultureB. Somatic embryogenesisC. Organogenesis and somatic embryogenesis in bioreactorsD. In vitro micrograftingE. Thin cell layer technology (TCLs)F. Photoautotrophic culture

4. Callus culture5. Cell suspension and single cell culture6. 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 proliferationRootingHardened plantlets

Fresh apical tip (a) or nodal bud (b)1 2 3

6 5 4

Lignified shoots

ADVENTITIOUS ORGANOGENESIS IN PISTACHIO

Adventitious Buds

Regeneration of Plantletsfrom Single Leaflet2

2

Elongation and 3

4

2

6 5

1 2 3

4

SOMATIC EMBRYOGENESIS IN PISTACHIO

Isolated kernels

Swollen SEs Maturation of SE

Ebryogenic tissue Ebryogenic tissuesin liquid medium

Development of SEs

Germinated SEs Acclimatised somatic seedlings

Immature fruits

Isolated SEs for germination

6 months after transplanting somatic seedlings

Ebryogenic tissues

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

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 offerns, 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.

APPLICATIONS OF MICROPROPAGATION

• 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

WHAT’S IN THE FUTURE

ACKNOWLEDGEMENTSAll 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 ！