Improving Plants for the 21 st Century

1. Improving Plants for the 21st Century Cell and Tissue Culture Technology and Applications

2. PLANT CELL AND TISSUE CULTURE • Introduction • Procedures Utilizing Tissue Culture Techniques • Tissue Culture Techniques • Plantlet Regeneration

3. Introduction to Plant Tissue Culture • Plant cell and tissue culture includes a wide range of cultural techniques for regeneration of functional plants from embryonic tissues, tissue fragments, calli, isolated cells, or protoplasts • Basic Concept is Totipotency :each living sell of a multicellular organism would be capable of developing independently if provided with the proper external condition (white, 1954)totipotent cell is one that is capable of developing by regeneration into a whole organism (morgan, 1901)

4. Micropropagation Laboratory • Establishment of aseptic culture • Proliferation • Rooting • Acclimatization

5. Basic Tissue Culture Procedures

6. What are explants and how are they obtained ? • Plant tissue cultures are generally initiated from multicellular tissue fragments, called explants, obtained from living plants. • Explants may originate from a wide range of plant tissues, such as… • leaf, • stem, • root, • petiole, • hypocotyl, • cotyledon, • embryo, or • meristem

7. From “explants” to “callus” on a solid medium • The explant is commonly cultured on a nutrient medium solidified in agar. Explants from most species of plants may be induced to divide in an unorganized manner on specifically formulated nutrient media • An undifferentiated mass of cells, known as callus (plural, calli), is formed within 4 to 8 weeks. • The callus may be divided, with clusters of cells transferred to fresh agar media to form subcultures. Repeated subculturing of the callus permits rapid multiplication of the cultured material. • Plant regenerability may decline, and genetic stability of the plant material may be altered, with successive subculturing. • Callus cultures are incubated under aseptic conditions, normally in dim light, with temperatures around 25°C.

8. Nutrient medium and the role of growth hormones? • The nutrient medium commonly contains • inorganic salts, sugar as a source of carbon, and vitamins to maintain high growth rates • Phytohormones such as auxins and cytokinins may be added to control cell growth and division • The ratio of auxin to cytokinin has an important role in the initiation of shoot and root primordia. • a low auxin: cytokinin ratio stimulates initiation of shoot buds and suppresses root initiation; • a high auxin: cytokinin ratio leads to dedifferentiation and favors root initiation; • an intermediate ratio favors continued division of cells as undifferentiated callus • The optimum culture medium may vary with the species, the genotype within the species, and the origin and age of the cultured tissue. • The preferred physical state of the culture medium, whether a liquid medium or a solid agar gel, may vary with the species and the culture environment.

9. Tissue Culture Manipulations

10. Plantlet Regeneration • Plantlets can be initiated • Indirectly from callus via • adventitious shoots • somatic embryos OR • Directly from explants such as • Axillary buds • the culture is transferred to a rooting medium to induce root initiation and subsequently plantlets • Somatic embryos have both root and shoot apices present and can develop directly into plantlets. • Adventitious shoot initiation (organogenesis) occurs with a wider range of plant species than initiation of somatic embryos; few major field crop species can be routinely induced to form somatic embryos.

11. “Acclimatization” of regenerated plantlets • Acclimatization: It refers to the successful growth and development of plantlets regenerated through tissue culture techniques in soil. • The establishment of a healthy plantlet in soil with minimum mortality is as essential for success in tissue culture propagation as obtaining a high frequency of plantlet regeneration. • Difficulty in establishment: Species differ in their capability of adjusting to the new environment. During this period the plantlet must change from the heterotrophic state to the autotrophic state, where it synthesizes its own organic food requirements. • Water loss from the regenerated plantlet is high, due to inadequacy of the root system formed in culture to maintain the plant in soil, and a reduced presence of epicuticular wax on leaves and stems of regenerated plantlets. The regenerated plantlet must be protected from desiccation and hardened to attain some tolerance to moisture stress. • the new plantlets, which have been developed under aseptic conditions, should be protected from soil pathogens so that they can grow and develop into healthy plants.

12. Factors Affecting Tissue Culture Efficiency • Plant regeneration from tissue culture varies with the following parameters: • plant species, • genotype within the species, • source of the cultured tissue, • age and health of the donor plant, • nutrient medium, • other factors

13. Meristem tip culture

14. Anther Culture Procedures