BE304 Plant Cell culture

1. BE304 Plant Cell culture Dr. Michael Parkinson, School of Biotechnology Dr. Michael Parkinson

2. ASSESSMENT • One hour open book exam • 2 experimental protocols in plant cell culture. • You should minutely dissect these and make sense of them. You will get 2 marks for every valid point that you make. • You can also get marks for suggesting alternatives that could have been used. Dr. Michael Parkinson

3. Seeds were washed overnight under a running tap, rinsed for 10s in 70% ethanol then sterilised in 20% Domestos + 0.1% v/v Tween20 for 10 minutes followed by 3 rinses in sterile distilled water. Why use seeds? Why wash overnight? Why rinse in 70% EtOH? Why sterilise at all? Why Domestos? Why 20% for 10 mins? Why 0.1% v/v Tween20? Why rinse? Types of points Dr. Michael Parkinson

4. Resources • Powerpoint presentation of lectures • Webpages@dcu.ie/~parkinsm/teaching • Partially worked solution to exam question • Text books • Agriculture 631 • Plant cell and tissue culture 571 • Secondary metabolism 660 • Transformation 572 Dr. Michael Parkinson

5. Lecture outline • Micropropagation • Production of products in cell cultures • Plant transformation • For every item, you will be given an experimental protocol. These will broken down into a number of sections. There will be a series of lectures covering the sections followed by a detailed discussion of another protocol. • We will also try out some of your findings. Dr. Michael Parkinson

6. Micropropagation • Advantages and disadvantages of micropropagation • Methods of micropropagation • Choice of explant • Media • Stage I - Sterilisation • Stage II - Multiplication • Stages III and IV- Rooting, hardening off and transfer to greenhouse Dr. Michael Parkinson

7. Advantages and disadvantages of micropropagation • Speed - roughly a 10X increase every 2 months (possible to produce 106 plants from a single starting plant in on year). • Axenic - provided that the original explant is free of contaminant, the resulting plants will all be uncontaminated. • Clonal propagation • Cost - 0.15€ per explant Dr. Michael Parkinson

8. Historical aspects • First commercially used with orchids - conventional propagation rate of 1 per year. • Through protocorms, 1,000,000 per year. Corm (Swollen stem) Chop up Maturation Dr. Michael Parkinson

9. Axillary branching Adventitious shoot formation Somatic embryogenesis >95% of all micropropagation. Genetically stable Simple and straightforward Efficient but prone to genetic instability Little used. Potentially phenomenally efficient. Methods of micropropagation Dr. Michael Parkinson

10. Axillary Branching Stem Leaf petiole Shoot tip Axillary bud in the axil of the leaf Dr. Michael Parkinson

11. Desirable properties of an explant Easily sterilisable Juvenile Responsive to culture Shoot tips Axillary buds Seeds Hypocotyl (from germinated seed) Leaves Choice of explant Dr. Michael Parkinson

12. When you make an explant like an axillary bud, you remove it from the sources of many chemicals and have to re-supply these to the explants to allow them to grow. Media Shoot tip - Auxins and Gibberellins Leaves - sugars, GAs Roots - water, vitamins mineral salts and cytokinins Dr. Michael Parkinson

13. Medium constituents • Inorganic salt formulations • Source of carbohydrate • Vitamins • Water • Plant hormones - auxins, cytokinins, GA’s • Solidifying agents • Undefined supplements Dr. Michael Parkinson

14. Carbohydrates • Plants in culture usually cannot meet their needs for fixed carbon. Usually added as sucrose at 2-3% w/v. • Glucose or a mixture of glucose and fructose is occasionally used. • For large scale cultures, cheaper sources of sugars (corn syrup) may be used. Dr. Michael Parkinson

15. Photoautotrophic culture • Growth without a carbon source. Therefore need to boost photosynthesis. • High light intensities needed (90-150mMole/m2/s) compared to normal (30-50). • Usually increase CO2 (1000ppm) compared to normal 369.4ppm. • Much reduced level of contamination and plants are easier to transfer to the greenhouse. Dr. Michael Parkinson

16. Inorganic salt formulations • Contain a wide range of Macro-elements (>mg/l) and microelements (<mg/l). • A wide range of media are readily available as spray-dried powders. • Murashige and Skoog Medium (1965) is the most popular for shoot cultures. • Gamborgs B5 medium is widely used for cell suspension cultures (no ammonium). Dr. Michael Parkinson

17. Vitamins • A wide range of vitamins are available and may be used. • Generally, the smaller the explant, the more exacting the vitamin requirement. • A vitamin cocktail is often used (Nicotinic acid, glycine, Thiamine, pyridoxine). • Inositol usually has to be supplied at much higher concentration (100mg/l) Dr. Michael Parkinson

18. Plant hormones (Growth regulators) • Auxins • Cytokinins • Gibberellic acids • Ethylene • Abscisic Acid • “Plant Growth Regulator-like compounds” Dr. Michael Parkinson

19. Auxins • Absolutely essential (no mutants known) • Only one compound, Indole-3-acetic acid. Many synthetic analogues (NAA, IBA, 2,4-D, 2,4,5-T, Pichloram) - cheaper & more stable • Generally growth stimulatory. Promote rooting. • Produced in meristems, especially shoot meristem and transported through the plant in special cells in vascular bundles. Dr. Michael Parkinson

20. Cytokinins • Absolutely essential (no mutants known) • Single natural compound, Zeatin. Synthetic analogues Benyzladenine (BA), Kinetin. • Stimulate cell division (with auxins). • Promotes formation of adventitious shoots. • Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem. Dr. Michael Parkinson

21. Gibberellins (GA’s) • A family of over 70 related compounds, all forms of Gibberellic acid. • Commercially, GA3 and GA4+9 available. • Stimulate etiolation of stems. • Help break bud and seed dormancy. • Produced in young leaves. Dr. Michael Parkinson

22. Ethylene • Involved in wound responses in plants. • Produced in all cells of the plant and causes thickening of stems and leaf abscission. • Reduces adventitious shoot formation. • Interacts with an ethylene-binding protein (EBP) in the cell membrane. Binding of AgNO3 or norbornadiene to EBP antagonises ethylene effects. Dr. Michael Parkinson

23. Abscisic Acid (ABA) • Only one natural compound. • Promotes leaf abscission and seed dormancy. • Plays a dominant role in closing stomata in response to water stress. • Has an important role in embryogenesis in preparing embryos for dessication. Helps ensure ‘normal’ embryos. Dr. Michael Parkinson

24. ‘Plant Growth Regulator-like substances’ • Polyamines - have a vital role in embryo development. • Jasmonic acid - involved in plant wound responses. • Salicylic acid. • Not universally acclaimed as plant hormones since they are usually needed at high concentrations. Dr. Michael Parkinson

25. Undefined supplements • Sources of hormones, vitamins and polyamines. • e.g. Coconut water, sweetcorn extracts • Not reproducible • Do work. Dr. Michael Parkinson

26. Bacteria and fungi will overgrow the explant on the medium unless they are removed. Pre-treatments to clean up the explant Detergents Sterilants and Antibiotics Pre-treatments Transfer plants to a greenhouse to reduce endemic contaminants Force outgrowth of axillary buds. Washing removes endemic surface contaminants. Stage I - Sterilisation Dr. Michael Parkinson

27. Air bubbles on the surface of the explant can protect bacteria and fungi from the liquid sterilant. Mixing should therefore be done in such a way as to reduce air bubble formation Detergents (e.g. Triton, Tween20) reduce the surface tension of the waxy cuticle on the leaf surface and increase wetting. Uses of detergents Air bubble around epidermal hair Leaf surface Dr. Michael Parkinson

28. There are 3 principal ways to kill off surface contaminants. oxidant action Active halogen Heavy metal poisoning *Powerful chemicals such as conc. sulphuric acid may be used on seeds. There is always a trade-off between killing the surface contaminants and killing the explant. As far as possible, cut surfaces should be protected. Sterilants Dr. Michael Parkinson

29. Sterilants used Antibiotics are rarely used since many are bacteriostatic and can cause mass overgrowth of cultures when they are removed. There are no antifungal compounds that are proven to be innocuous. Dr. Michael Parkinson

30. Stage II - Multiplication • Nodal cuttings are made. This removes the inhibitory effect of the shoot apex on bud outgrowth (Apical dominance). • GA’s may be added to promote etiolation, especially in species that form rosettes. • Cytokinins may be used to increase bud growth (antogonises auxin effect). • Multiplication is very labour-intensive. Dr. Michael Parkinson

31. Stages III and IV Rooting and transfer to the greenhouse • Plants must be rooted by using media containing auxin or by dipping explant bases in auxin solutions. • Progressively, the plants must be hardened by increasing the light intensity, and reducing sugar, inorganic salts and humidity. • Medium must be removed prior to transplantation to prevent contamination. Dr. Michael Parkinson

32. Micropropagation by adventitious shoot formation • Adventitious shoot formation is the de-novo development of shoots from cell clusters in the absence of pre-existing meristems. • In some species (e.g. Saintpaulia), many shoots can be induced (3000 from one leaf). • In other species (e.g. coffee), it may be necessary to induce an unorganised mass proliferation of cells (callus) prior to adventitious shoot formation. Dr. Michael Parkinson

33. Control of organogenesis Cytokinin Leaf strip Adventitious Shoot Root Callus Auxin Dr. Michael Parkinson

34. Plant Hygiene • Pathogens affect yield (average 30% reduction) • There are strict plant sanitation requirements for import of plants. • Viruses and bacteria will be multiplied along with the explants and need to be removed prior to plant multiplication. Dr. Michael Parkinson

35. Ways to eliminate viruses • 1 Heat treatment. Plants grow faster than viruses at high temperatures. • 2 Meristemming. Viruses are transported from cell to cell through plasmodesmata and through the vascular tissue. Apical meristem often free of viruses. Trade off between infection and survival. • 3. Not all cells in the plant are infected Adventitious shoots formed from single cells can give virus-free shoots. Dr. Michael Parkinson

36. Elimination of viruses Plant from the field Pre-growth in the greenhouse Active growth Heat treatment 35oC / months Adventitious Shoot formation ‘Virus-free’ Plants Virus testing Meristem culture Micropropagation cycle Dr. Michael Parkinson

37. PRODUCTION OF PRODUCTS • Advantages and disadvantages • Cost of production • Plant cell culture systems • Ways to increase product formation • Commercial production Dr. Michael Parkinson

38. Advantages Can manipulate environment Can feed precursors Possible to select in culture Possible to get all cells in a culture producing. Can continuously extract. Can retain biomass Disadvantages High cost Contamination Low intrinsic production Advantages and disadvantages Dr. Michael Parkinson

39. Cost of production • Plant cells are slow growing. • Full of water (90% - 95%). • Easily contaminated. • Shear-sensitivity means specially modified fermenters necessary • All this puts the cost of production of dry mass to $25 per kilo. Product only a fraction of this. Dr. Michael Parkinson

40. Organised Shoot cultures. ‘Hairy root’ cultures Embryo fermentations. Unorganised Callus Cell suspension culture Plant cell culture systems Dr. Michael Parkinson

41. Shoot cultures • Under conditions of high cytokinin, a culture producing a mass of shoots may be produced by adventitious shoot formation. • For light-associated products, may be much more high yielding. • Sensitive to shear • Illumination a problem for scale up Dr. Michael Parkinson

42. ‘Hairy root’ cultures • ‘Hairy roots’ are produced by infecting sterile plants with a natural genetic engineer, Agrobacterium rhizogenes. • Genes for auxin synthesis and sensitivity are engineered into plant cells leading to gravity-insensitive mass root production. • Very useful for products produced in roots. • Aggregration and shear sensitivity are a major problem for scale-up Dr. Michael Parkinson

43. Embryo Fermentations • Somatic Embryos may be produced profusely from leaves or zygotic embryos. • For micropropagation, potentially phenomenally productive. • Shear sensitivity is a problem. • Maturation in liquid is a problem. Dr. Michael Parkinson

44. Shikonin production in culture • Shikonin production in the intact plant • Introduction into culture • Optimisation of production through medium manipulations • Fermentation Dr. Michael Parkinson

45. Callus • Equimolar amounts of auxin and cytokinin stimulate cell division. Leads to a mass proliferation of an unorganised mass of cells called a callus. • Requirement for support ensures that scale-up is limited (Ginseng saponins successfully produced in this way). Dr. Michael Parkinson

46. Cell suspension culture • When callus pieces are agitated in a liquid medium, they tend to break up. • Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support. • Large scale (50,000l) commercial fermentations for Shikonin and Berberine. Dr. Michael Parkinson

47. ‘Friable’ callus goes easily into suspension. 2,4-D Low cytokinin semi-solid medium enzymic digestion with pectinase blending Removal of large cell aggregates by sieving. Plating of single cells and small cell aggregates - only viable cells will grow and can be re-introduced into suspension. Introduction of callus into suspension Dr. Michael Parkinson

48. Introduction into suspension Sieve out lumps 1 2 Initial high density + Subculture and sieving Pick off growing high producers Plate out Dr. Michael Parkinson

49. Growth kinetics • 1. Initial lag dependent on dilution • 2. Exponential phase (dt 1-30 d) • 3. Linear/deceleration phase (declining nutrients) • 4. Stationary (nutrients exhausted) 3 4 2 1 Dr. Michael Parkinson

50. Large (10-100mM long) Tend to occur in aggregates Shear-sensitive Slow growing Easily contaminated Low oxygen demand (kla of 5-20) Will not tolerate anaerobic conditions Can grow to high cell densities (>300g/l fresh weight). Can form very viscous solutions Characteristics of plant cells Dr. Michael Parkinson