Problems in tissue culture

1. Problems in tissue culture • Culture contamination • Vitrification • Acclimatization • Post culture behavior

2. Culture Contamination • Two sources: • Carry over of microorganism on the surface or in tissue of explants • Through faulty procedures in the laboratory

3. Culture Contamination • Cause economic losses, by overrunning the culture either killing the explant or rendering it for the subculture • Affect the productivity both in vitro and of the progeny plant

4. Organisms associated with plant surface • Fungi including yeast • Bacteria • Mollicutes (mycoplasmas, spiroplasmas and related organism)

5. Endophytic microorganisms • Intercellular endophytic microorganism Virus Viroid Fastidius prokaryotes • Intracellular endophytic microorganism L-forms of common plant associated bacteria Fastidius bacteria associated with plant vascular tissue vector transmitted and may be spread by contact between infected and healthy plant they are capable of clonal propagation

6. Aspect of quality control • Awareness of the range and natural history of possible contaminant of the crop, including specific pathogen • Adequate preparation of the donor plant including treatment to reduce or eliminate pathogens • Confirmation of the status of culture in stage I following employment of strategies to obtain healthy cultures and again based on reliable screening methods

7. Aspect of quality control • Rigorous monitoring of production to confirm the status of the cultures. In large scale production this will necessitate sampling production and is dependent on an appropriate sampling protocol • An awareness that the spectrum of contaminating microorganism may alter with time in culture • Monitoring of progeny based on sampling of production

8. Detection and identification methods

9. Vitrification • Translucency • Hyperhydration • Succulency • Glassines Change into a glassy appearance Produce fragile plants which have a glassy and hyperhydrous appearance

10. Vitrification • The cells were surrounded by thin walls and contained a relatively poor and largely vacuolated cytoplasm • Increased cellular space due to extra protoplastic water • Many of the chloroplasts lacked of the normal organization into grana and stroma • Chloroplasts contained large starch grains whereas the chlorophyl content was lower • Defective epidermal tissue • Faulty deposition if epicuticular waxes • The guard cell did not function properly • Abnormal stomata • Reduced lignifications

11. Factors associated to vitrification Physical and chemical state of the medium • Type of culture media • Gelling agents • Organic component • Inorganic component • Growth regulators • Relative humidity • Environmental condition • Various addition

12. Acclimatization • Process during which plants or other organisms become adjusted or accustomed to a new climate or situation as a result of natural processes • Hardening-off • Moving the complete plants to greenhouse or field • Not unique to micro-propagation

13. Why acclimatization is important? The greenhouse and field have substantially • lower RH • higher light levels • autotrophic growth • septic environment  stressful to micro-propagated plants compared to in vitro condition

14. Common issue • Agar has to be thoroughly washed from the root • Pesticide may be phytotoxic to some species of micro-propagated plantlet • Gradually reduce the RH or amount of mist • Maintain light level in the greenhouse at 50% shade before plants are transplanted to the field

15. Acclimatization • Control environment acclimatization • Specific place that all environmental conditions can be control either automatically or manually • Direct field acclimatization • Transferring the rooted plantlet directly to the field

16. Important aspect in the control environment acclimatization • Humidity • Light • Soil and container • Diseases • Temperature • Nutrient

17. Control humidity • Avoid the use of an automatic mist system due to mist leaches nutrients, causes the medium to become too wet, allows the plantlets to dry, creates an environment favorable for the growth of algae and some fungi and bacteria • Fogging • The use of a humidifier • Placing plantlets in an enclosed area that will water vapor • The use of anti-transpirants to reduce water losses

18. Light • While in vitro, plantlet has been exposed to relatively low level of light and their leaves are thin and thus resemble shade leaves • Leaves of plantlet place under too high a light level will become chlorotic and necrotic • Shading up to four weeks under up to 90% will reduce transpirational demand and excessive light that can destroy chlorophyll molecules • Following a period of shading, plantlet should be gradually moved to the light level under which they will be grown • Control of photoperiod is also important to prevent dormancy or to control plant development

19. Soil and containers Requirement A uniform medium that adequately supports the plants, has suitable pH, well buffered and sufficiently porous • Inhibitors or dramatic shift in pH in medium can adversely affect root growth • Larger container is better • Peat plugs or small foam blocks are recommended

20. Diseases • Very essential • Plantlet is generally suitable to diseases- causing organisms • High humidity is conducive to the growth of many plant diseases causing fungi and bacteria • An integrated approach of sanitation and application of pesticides is generally used: • Disinfested medium, new or disinfested container and benches • New poly-ethylene covers • Clean hand • Clean and disinfested instruments

21. Temperature • The temperature of the air and growing medium are generally controlled • Adjusting the amount of shading and humidity can aid in temperature control • Ventilation and fan system • Fog and air condition • The temperature of the root zone is important to encourage root growth

22. Nutrient • Nutrient can be originally from the Media, if the media consists of soil, sand and compost • Fertilizer may be incorporated or top dressed in slow release form • A soluble complete fertilizer diluted to ¼ to ½ the recommended rate is recommended

23. Direct field acclimatization • It is possible in some species • Vanilla, Teak, Potato • A covering of 40 mesh screen • Only 6 – 14% survived • Survival and yield varied among clones

24. Rooting and acclimatization • In vitro rooting • Ex vitro rooting Disadvantage: • Lack root hair • Died and collapsed after plantlet was removed from culture, however new lateral and adventitious root formed during acclimatization • The transition zone between root and shoot was abnormal • The vascular connection were poorly form • Restricted water uptake • Labor intensive and expensive In vitro rooting

25. Ex-vitro rooting Direct rooting during acclimatization • Attention must be paid to humidity, light and temperature • Treatment with root inducing growth regulators may be required prior to acclimatization • No agar adheres to the base of the cutting

26. Post Culture Behavior • Dwarfs • Color changes or mosaic pattern (Chimera) • Growth habit changes • Change in productivity Cause: • When shoots are derived from dedifferentiated cell • Rapid proliferation of single cells or multi-cellular primordia through organogenesis or embryogenesis • In vitro process or by added biochemical and stress agent • Temporary or heritable deformities

27. Cause • Variation that existed in the source plant • Chimera • Non chimeric chromosomal variation • Genetic changes • Mitotic abnormality • Somatic crossing over • polyploidy • Epigenetic or physiological effect • A non heritable change in phenotype that occurs in a substantial percentage of the propagated population through an inducible directed and reversible process

28. Chimera • Plant or plant part composed of genetically different sort of cells as a result of mutation or grafting • Plant with two or more distinct genotypes

29. Chimera • Cell origin • Mericlinal chimera a section of one or two of the histogenic layers are different • Sectorial chimera all histogens in a sector are different • Periclinal chimera one histogen is different from the others • Mimicked by variegated or mosaic forms • Due to en-even distribution of viruses in plant tissue

30. Histogen • Cell layers in all higher plant tissue that trace back to distinct layers in the apical meristem • 3 layers in angiosperm • LI an outer epidermal layer • LII an internal tunica layer • LIII a cortical layer • LII layer produce gametic tissue and some surrounding maternal tissue • The remaining maternal tissue is also formed by LIII and LI • Root derived from LII and LIII layers

32. GROWING POINT (APICAL MERISTEM)

33. Non chimeric chromosomal variation • Breakage in heterochromatic region • Somatic crossing over (mitotic exchange between homologous chromosomes) • Gene amplification due to mutagenic agent

34. Permanent genetic change • Somaclonal variation • Genetic change • Polyploidy  Aneuploidy and breakage • Micronucleus formation  Bi- or multi nucleate cells • Duplication  Recombination • Inversion  Amplification • Simple base pair change  Organelle genome variation • Isozyme differences • Expression of cryptic transposable element • Change in chromosome structure

35. Definition • Euploidy An even increase in number of genomes (entire chromosome sets) • Aneuploidy An increase in number of chromosomes within a genome

36. Euploid

37. Aneuploid

38. Plant variation from dedifferentiated cell • Mitotic asynchrony caused by growth regulator effect on DNA biochemistry (2,4,5-T; 2,4-D; antibiotic; alkaloid; physical mutagen) • Disorientation or dysfunction of the mitotic apparatus (spindle fiber) • Selection pressure due to the change in plant’s environment

39. 2,4-D • Increase growth and reduced cell cycle time • Stimulate DNA synthesis • Endo-reduplication lead to nuclear fragmentation • Increased mitotic crossing over • Increase poly-ploid

40. Temporary alterations • Altered flowering, sex expression, fertility and yield • Increased vigor and root-ability • Increased branching • Expression of off-type and off-color phenotypes • Alter susceptibility to diseases and biochemical including herbicide • Rejuvenation

41. Rejuvenation • Bring back to youthful appearance (juvenile) • Juvenility: The condition of a seedling plant that prevents flowering or sexual gameto-genesis