1. Plant Tissue Culture

2. What is it?
Tissue culture is the term used for “the process of growing cells artificially in the laboratory”
Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)

3. Why does it work? Plant cells – Dedifferentiate
Plant cell division- Somatic cells are diploid
Mitosis – Chromosomes duplicate and form clones
Totipotency

4. What’s the history?
Cell theory, suggesting totipotentiality of cells. Schleiden M. J., Arch. Anat., Physiol. U. wiss. Med. (J. Muller), 1838: ; Schwann T., W. Engelman, No. 176 (1910)

5. What’s the history?
First but unsuccessful attempt of tissue culture using monocots. Haberlandt G., Sitzungsber Akad. Wiss. Wien, Math.-Naturwiss. Kl., 111:
FW Went demonstrated that there were growth substances in coleoptiles from Avena

6. What’s the history?
White generated continuously growing culture of meristematic cells of tomato on medium containing salts,yeast extract and sucrose and 3 vitamins (pyridoxine, thiamine, nicotinic acid) – established the importance of additives

7. What’s the history?
Successful continuously growing cambial cultures of carrot and tobacco. Gautheret R. J., C. R. Acad. Sci. (Paris), 208: ; Nobecourt P., C. R. Soc. Biol. (Paris), 130: ; White P. R., Am. J. Bot., 26: 59-64

8. What’s the history?
Tumor-inducing principle of crown gall tumors identified. Braun A. C. Phytopathol. 33: & P. N. A. S. USA 45:
Formation of adventitious shoots and roots in tobacco. Skoog F. and Tsui C., Am. J. Bot., 355:

9. What’s the history?
Virus-free Dahlia through meristem culture. Morel G. and Martin C., C. R. Hebd. Seances Acad. Sci. (Paris), 235:
First successful micro-grafts. Morel G. and Martin C., C. R. Acad. Sci. (Paris), 235:

10. What’s the history?
Discovery that root or shoot formation in culture depends on auxin : cytokinin ratio. Skoog F. and Miller C. O., In vitro Symp. Soc. Exp. Biol., No. 11:
Pro-embryo formation in callus clumps and cell suspension of carrot. Reinert J. and Steward F. C., Naturwiss., 45:

11. What’s the history?
Enzymatic degradation of cell wall for protoplast formation. Cocking E. C., Nature, 187:
Vegetative propagation of orchids by meristem culture. Morel G., Am. Orchid Soc. Bull., 29:

12. What’s the history?
Development of MS medium. Murashige T. and Skoog F., Physiol. Plant., 15:
To be continued….

13. What is needed?
Appropriate tissue (some tissues culture better than others)
A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid
Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture

14. What is Needed Growth regulators – discussed in depth later
Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites

15. Nutrient Media for Plant Tissue Cultures

16. Functions of medium Provide water Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas exchange
Removal of plant metabolite waste

17. Major Components Salt Mixtures Organic Substances Natural Complexes
Inert Supportive Materials
Growth Regulators

18. Mineral Elements

19. Macronutrient salts Function of nutrients in plant growth
Nitrogen – Influences plant growth rate, essential in plant nucleic acids (DNA), proteins, chlorophyll, amino acids, and hormones.
Phosphorus – Abundant in meristimatic and fast growing tissue, essential in photosynthesis, respiration,
Potassium – Necessary for cell division, meristematic tissue, helps in the pathways for carbohydrate, protein and chlorophyll synthesis.

20. Macroelements Nitrogen (N) nitrate ion (NO3- oxidized)
ammonium ion (NH4+ reduced)
25-60 mM
organic

21. Amino Acids -The most common sources of organic nitrogen used in culture media are amino acid mixtures, (e.g., casein hydrolysate), L-glutamine, L-asparagine, and adenine. When amino acids are added alone, they can be inhibitory to cell growth. Tyrosine has been used to stimulate morphogenesis in cell cultures but should only be used in an agar medium. Supplementation of the culture medium with adenine sulfate can stimulate cell growth and greatly enhance shoot formation. L-tyrosine - stimulates shoot formation.

22. Macroelements Potassium (K) 20 -30 mM Phosphorous (P) 1-3 mM
Calcium (Ca) 1-3 mM
Magnesium (Mg) 1-3 mM
Sulfur (S) 1-3 mM

23. Calcium - Involved in formation of cell walls and root and leaf development. Participates in translocation of sugars, amino acids, and ties up oxalic acid (toxin)
Magnesium - Involved in photosynthetic and respiration system. Active in uptake of phosphate and translocation of phosphate and starches.
Sulfur - Involved in formation of nodules and chlorophyll synthesis, structural component of amino acids and enzymes.

24. Micronutrients
Iron (Fe) 1 m M - Involved in respiration , chlorophyll synthesis and photosynthesis. FeNaEDTA = sodium salt of EDTA sequesters iron, making it available to plants.
Manganese (Mn) 5-30 m M - Involved in regulation of enzymes and growth hormones. Assists in photosynthesis and respiration.

25. Micronutrients Zinc (Zn) Boron (B) Copper (Cu) 0.1 m M
Molybdenum (Mo) 1 m M
Cobalt (Co) 0.1 m M
Iodine (I) Nickel (Ni), aluminum (Al), and silicon (Si)

26. Organic Compounds Sugar – carbon source sucrose
Others – fructose,glucose
20 to 40 g/l, usually

27. Organic Compounds Vitamins
thiamine (vitamin B1) - essential as a coenzyme in the citric acid cycle
nicotinic acid (niacin) and pyridoxine (B6)
myo-inositol - part of the B complex, in phosphate form is part of cell membranes, organelles and is not essential to growth but beneficial

28. Still other organics Organic Acids Phenolic compounds
Citric acid (150 mg/l) typically used with ascorbic acid (100 mg/l) as an antioxidant.
Can also use some of Kreb Cycle acids
Phenolic compounds
Phloroglucinol - Stimulates rooting of shoot sections

29. Charcoal
Activated charcoal is used as a detoxifying agent. Detoxifies wastes from plant tissues, impurities
Impurities and absorption quality vary
Concentration normally used is 0.3 % or lower
Charcoal for tissue culture
acid washed and neutralized
never reuse

30. Natural Complexes - Undefined
-Coconut endosperm
-Fish emulsion
-Protein hydrolysates
-Tomato juice
-Yeast extracts
-Potato agar

31. Growth regulators - Hormones
-auxin - roots
-cytokinin - shoots
-gibberellin – cell enlargement
-abscisic acid – plant stress hormone
-ethylene – BAD!

32. Support Systems Agar (from seaweed) Agarose
Gelrite (Phytagel) (from bacteria)
Mixtures (Phytagar)
Mechanical (bridges, rafts)
Sand

33. Media Formulations Many available Differ in salt concentrations
Differ in presence or absence of salts
M&S most widely used by far