1. PLANT TISSUE CULTURE PRINCIPLES AND APPLICATIONS Dr. SALAMMA SUGALI POST DOCTORAL FELLOW BIODIVERSITY CONSERVATION DIVISION, Dept of Botany, Sri Krinadevaraya University

2. The whole gamut of discoveries in biology and allied sciences can be grouped together under a single term biotechnology. Manipulation of biological systems via modern technology may solve practical problems. E.g. Plant tissue culture DNA recombination Genetic engineering

3. Plant Tissue Culture? The aseptic culture of plant protoplasts, cells, tissues or organs under conditions which leads to cell multiplication or regeneration of organs or whole plants. It is a blanket term used for all types of cultures under aseptic conditions which leads to the growth and differentiation of explants.

4. PRINCIPLES AND APPLICATIONS PTC IMPORTNACE LABORATORY-EQUIPMENT MEDIA FORMULATION STERILIZATION METHODS PRECAUTIONS

5. The theoretical basis for the PTC was established by Schleidon and Schwann in 1838-39. Haberlandt in early 1900’s proposed the concept of totipotency. First studies: Callus cultured from tree cambium (Gautheret, Nobecourt, Whire in the 1930s.)… cells kept alive but did not develop

6. Principles PTC mainly depends upon the four principles TOTIPOTENCY Genetic potential of plant cell to produce the entire plant. GROUND STATE It refers the normal state of cell. Cell may already be competent or incompetent COMPETENCY Cells retain ability for differentiation and morphogenesis DETERMINISM Ability of a cell to respond to the stimulus that initiates a developmental process leading to morphogenesis.

7. PTC is not a subject but it is cluster of techniques which are useful for multiplication of plantlets. If any body wants to get number of plantlets with in a short period of time PTC is the best way, which requires some specific requirements

8. Laboratory facilities Laboratory comprise 1. A space for preparing media This area has to be equipped with. Analytical balance. p H meter. Autoclave. Refrigerator 2. A chamber for transformations This area is meant for all transformations. LAF cabinet with HEPA filters 3. An area to grow cultures Culture room has to provide 2000 lux light intensity, temperature 25°±1°C

9. CULTURE ROOM

10. MEDIA COMPOSITION PTC medium should provide PTC medium should provide Macro elements Macro elements Mg, Na, Cl, K, P etc. Mg, Na, Cl, K, P etc. Micro elements Zn, Co, I, Ni etc.

11. Organic supplements. Vitamins in the form of Thiamin, Myo inosital,. Amino acids. E.g. Glycine, Aspartic acid, glutamic acid, arginine.. Additives. Casein hydrolysate, Proline, yeast extract coconut milk etc Stable carbon source. Sucrose is easy soluble and readily assimilated

12. . Gelling agents For PTC the medium should be semi solid or liquid. So it is necessary to have a gelling agent. Sodium alginate, Agar-agar are mostly used gelling agents. Plant growth regulators. These are synthetic analogues 1. Auxins The main objective of this PGR is cell division, growth. E.g. IAA, 2 4-D, NAA

13. . Cytokinins The main function of this PGR is cell division. These are purin derivatives. Ex. Zeatin, 2 ip, BAP, KN, TDZ. Gibberellins This PGR regulates the cell elongation, and, GA 3 is commonly used. Abscisic acid Inhibits the cell division. Normally used in Somatic embryogenesis

14. SOURCE OF EXPLANTS The excised piece of tissue or plant fleshy storage organs shoots buds and base of apical meristems root, floral parts leaves and cotyledons. But disease free explants has to be used for PTC The following factors influence the growth of explants. Season, physiology, quality of source plant, size.

15. STERILIZATION OR SANITATION Sanitation is meant to avoid contaminations Washing with running tap water Cleaning with liquid detergents Teepol, Dettol, Tween- 20, Sterilization by Sterilents like 70% alcohol, Hgcl 2, Naocl etc

16. Out line of general cell and tissue culture.

17. INOCULATION All the inoculations must be carried out in LAF cabinet U.V light to be used for 30-60 minutes to kill surface born contaminants. HEPA filters has to use throughout the time of inoculations which may eliminate the entry of pathogens.

18. PRECAUTIONS 1. Moistured hands with alcohol should be away from contact with flame 2. Switch off the UV lamp at least 20-30 min. before starting the inoculations to avoid exposure of UV to skin 3. Very hot instruments may cause deleterious effects on growth. So hot instruments should not be used 4. Care must be taken that the explants should be in contact with medium

19. ACCLIMATIZATION The well in vitro grown plantlets are to be hardened for introducing into the natural and allied habitats of plants. All the controlled conditions which previously provided has to be reduced, so that it can be acclimatized it self for the environmental conditions. V ermiculate is a soil can be used, peat mass enhances the ability of soil fertility.

20. Acclimatized plants In green house conditions In culture conditions Hardened plants

21. TYPES OF CULTURES. Organ culture. Callus culture. Protoplast culture All the cultures like ovule, ovary, anther, embryo, endosperm, comes under these categories

22. ORGAN CULTURE Single cell cultures Suspension cultures CALLUS CULTURE Ovule culture, Ovary culture, anther culture, embryo culture endosperm culture PROTOPLAST CULTURE

23. APPLICATIONS OF PTC 1. HAPLOIDS 2. CLONAL PROPAGATION- MICROPROPAGATION 3. SOMATIC HYBRIDIZATION 4. REDUCED BREEDING CYCLE. 5. CRYOPRESERVATION 6. PATHOGEN FREE PLANTS- SOMATIC EMBRYOGENESIS 7. PRODUCTION OF SECONDERY METABOLITES 8. TRANSGENIC PLANTS 8. SYNTHETIC SEED PREPARATION

24. 1. Haploid production. Mutants can be established E.g. Potato mutants against Black shank disease Rice variety against blast disease 2. Rapid growing of plantlets from elite germ plasm is known as clonal propagation. It is possible through Micro propagation E.g. Potato, dahlia, strawberry, carnations etc 3. Breeding cycle can be reduced Embryo culture. E.g. Apple, peach, pear, citrus.

25. 4. Cryopreservation Plant material can be stored at very low temperatures for virtually indefinite period of time with out any gene manipulations 77k or -196°C in liquid nitrogen Hybrids, suspensions, pollen grains also preserved through this method. 5. Pathogen free plants can be produced by Somatic embryogenesis. Soma clonal variants can be produced by mutating somatic embryoids

26. . Secondary metabolites can be extracted through callus cultures Eg. Shiconin Digitoxin. Transgenic plants Transgenics helps in identifying the genes that regulates the specific characters of trait and moving copies of the same to different plant species. Synthetic seed preparation. Any somatic part of the plant can be made into a seed coated with artificial seed coat having the capacity to produce whole plant in any conditions.

27. . Genetic recombination's. This can be achieved through PTC during seed germination, otherwise forming of multiple shoots in vitro. Fusion of protoplasts of different plant species gives genetically modified plants.. Cell wall studies

28. LIMITATIONS Contaminations Problems in predicting the exact composition of medium to every plant. Exudation of phenolic compounds rather than the callus cultures arrests the plant growth. Vitrification or hyper hydration ( glassy, water soaked development of explants ) In vitro wilting

29. The high success rates will be achieved by successfully integrated practices in the laboratory with the practices acclimatization period Thank you for your attention