1. Tissue culture and application in plant propagation & improvement Plant tissue culture: involves the culture of all types of cells, tissues, & organs under asceptic conditions The position of tissue culture in relation to molecular biology and breeding & genetics Molecular Biology Breeding & Genetics Tissue culture

2. Steps in producing plants by tissue culture Tissue culture :The growth in an artificial medium of cells derived from living tissue 1.Explantation The asceptic surgical removal of a suitable tissue or organ (the explant) Examples: shoot tips, stem sections, leaves, petals, cotyledons, roots, fruits, embryos, shoot apical meristems Virus free explant is usually taken from the true shoot meristem (the apical meristematic dome) which is less than 0.1mm in height

3. 2.Proliferation Multiplication of the propagules Growth & differentiation of explant depends mainly on the culture medium Varied formulas are used. These are made of inorganic salts, sugars, vitamins, amino acids, purine basis, organic complexes such as coconut ‘water’ (liquid endosperm) or yeast extract & growth regulators

4. 3.Acclimatization & Establishment Preparation of the propagule for an independent existence outside of culture by hardening and acclimatization

5. Advantages of tissue culture Large scale multiplication in lesser time & space Production of virus free plants Year round production of plants Highly beneficial for plants where other vegetative methods are difficult In dioecious fruit plants, production of female plants is possible through micro- propagation e.g papaya

6. Disadvantages of tissue culture Requires very expensive facilities Requires technical skill Requires very close and prompt identification and control of pathogens Lab produced plants need to be properly hardened for them to establish in field

7. Tissue culture techniques 1.Embryo culture: involves the removal of the embryo from the seed & subsequent growth in vitro until developing plant can be transplanted to the soil & grown to maturity Used mainly to recover plants (embryos) during attempts at wide hybridization by sexual crosses between distantly related plants Also used to break dormancy in seeds and promotion of germination of important seeds that would have lost viability during storage

8. 2.Meristem culture Meristem: a clone of actively dividing cells about 0.1mm in diameter 0.25mm long Involves removal of the meristem with 2 or 3 leaf premordia & subsequent culture on a nutrient medium Has successfully been used for elimination of viruses from plants Virus moves within the vascular bundles and in the meristems there are no vascular bundles thus viruses can be eliminated

9. 3. Micropropagation An extension of more traditional methods of plant propagation aimed at the rapid clonal multiplication of superior genotypes of disease free and pest free plants. Multiplication is achieved by sub culturing It has several advantages over traditional methods 4. Somatic embryogenesis The development of embryo -like structures from cells of somatic (non sexual) origin

10. 5. Somaclonal variation Involves the ability to change one or a few characteristics of an otherwise outstanding cultivar without altering the remaining and often unique part of the genotype 6. Invitro selection A tool that facilitates the identification of plants which are resistant or tolerant to stress produced by phytotoxins form pathogens, herbicides, cold, temp, Al, Mn & salt toxicity

11. Invitro contd Selection usually involves subjecting population of cells to a suitable selection pressure, recovering any variant lines which would have developed stress or tolerance to stress Plants are then regenerated from the selected cells

12. 7. Anther culture A means of producing haploid plants that involves removal of anthers and/or immature pollen from a plant & placing them in a suitable culture medium which will induce regeneration from the haploid tissue of the microspores or pollen 8.Protoplast culture Protoplasts: cells from which the cell wall has been removed by mechanical &/or enzymatic methods Its therefore amenable to a number of techniques that are not possible with plant cell cultures. Regeneration of plants from protoplasts is generally more difficult to accomplish than from cells & callus cultures

13. Examples of tissue culture application in horticulture 1.Embryo culture for cucurbit improvement Problem : Papaya ringspot virus Type W (PRV-W) causes a serious viral disease of commercial cucurbit varieties. In Queensland, Australia production losses have reached $8m annually Solution : C.ecuadrensis is highly resistant to isolates of PRV- W. Interspecific hybrids were produced between C. maxima, C. moschata & C.ecuadrensis (wild species), by hand pollination followed by embryo culture 2. Meristem culture for virus elimination

14. 3.Meristem culture combined with heat treatment has played an important role in obtaining virus free planting materials in a number of countries 4.Micropropagation for rapid increase of new varieties for early release – Ginger – Pineapple 5.Microprogation as a source of planting material – Papaya/paw paw – Banana 6.The use of somaclonal variation & mutation breeding for the genetic improvement of banana

15. Genetic variations in vegetatively propagated plants 1.Mutation -occurs when a DNA gene is damaged or changed in such a way as to alter the genetic message carried by that gene -affects chromosomes, cytoplasmic genes (mitochondria, plastids) 2. Chimera- An organism, organ, or part consisting of two or more tissues of different genetic composition, produced as a result of organ transplant, grafting, or genetic engineering.

16. a rare chimera: Gala A graft chimera

17. Chimera Chimera - a plant or plant part composed of genetically different layers. The most common example is a "variegated" plant where different regions or layers of the leaf are yellow or white due to no chlorophyll development, i.e. these are chlorophyll mutants Peridemal chimera Most common type Mutated tissue is relatively thin layer completely covering a core of non mutated tissue Relatively stable chimera

18. b) Meridemal chimera- - Similar to peridemal, only a portion of the mutated tissue is involved -A very common chimera c) Sectional chimera- - Arise from a mutation affecting all cells of a shoot - Its rare, only from plants whose apical meristems arise from very few cells N/B Look up examples of these online, books

19. Plant protection in vegetative propagation concepts The major aim is to produce disease free or pathogen free plants Pathogens of importance 1.Fungi- saprophytes, parasites Example : facultative or obligate – Phytopthora spp, Pythium spp, Rhizoctonia spp Control : fumigation, pasteurization, sanitation, environmental control or use pathogen free propagules

20. 2. Bacteria Contaminated stock plants are the major source Invade plants through wounds Control : difficult- eliminate source of contamination, sterilize tools, pasteurize soils 3. Viruses Obligate parasites Most are spread by vectors (aphids, nematodes, thrips etc) Produce symptoms which are not detectable at higher temp Control vectors, programs for production of virus tested plant material

21. 4. Mycoplasma like organisms Extremely small one-celled parasitic organisms intermediate between bacteria & viruses in size Live in phloem of susceptible plants Leaf hoppers & psylla are some of the vectors 5. Fastidious Bacteria Single celled bacteria like organisms that do not have a nucleus Cannot be cultured in standard media Occur in the xylem & phloem of susceptible plants and in the bodies of leaf hoppers

22. 6. Viroids A single free RNA molecule of low molecular weight about a tenth size of a small virus Causes citrus exocortis & chrysanthemum mottle 7. Nematodes Are ectoparasitic or endoparasitic Control : fumigation, sanitation (rotation) 8. Insects & mites Sucking & tissue eating insects

23. Methods for detecting pathogens in propagation material 1. Culture indexing 2. Virus indexing- take suspect plant & graft it on a very susceptible plant 3. Serology- ELISA 4. Electropheresis- obtain suspect extract, purify it & obtain unique RNA and use electrophoresis to read RNA type 5. Molecular biology