1. Welcome to Plant Tissue Culture
(an experimental course)

2. Instructor Dr. Chandrama P. Upadhyaya 220, Life Sciences Building,

3. Teaching Assistant Mrs. Moon So Yeon 209 Life Sciences Building

4. (Second Edition) Plant Tissue Culture: Techniques and Experiments
By Roberta H. Smith

5. Evaluation
Two examinations counting 40% each, (20% Exam & 20% Experiments).
- Mid term Exam (date to be announce)
- Final Exam (date to be announce)
Attendance 20%

6. Class and experiment schedule
One hour class in the 451 and then move to lab no 209 or 214.
Students absent in the experiments may be punished by several home work and many experiments.
Students asking questions in the class or during experiments may get extra score in the examination.

7. THE PRINCIPLES OF PLANT TISSUE CULTURE
Chapter 1
THE PRINCIPLES OF PLANT TISSUE CULTURE

8. Definition
The culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

9. How is Plant Tissue Culture Done?
It is grown on a special culture medium which supports its growth and development.
The medium can be either semisolid, such as agar, or liquid, such as purified water.

10. Basic in vitro propagation ...

11. Three Fundamental Abilities of Plants
Totipotency
The potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated.
It implies that all the information necessary for growth and reproduction of the organism is contained in the cell.
Dedifferentiation
Capacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganise into new organ.
Competency
the endogenous potential of a given cells or tissue to develop in a particular way.

12. HISTORY OF PLANT TISSUE CULTURE
cellular theory (Cell is autonom and totipotent)
Schleiden-Schwann
1902
First attempt of plant tissue culture
Harberlandt
1939
Continuously growing callus culture
White
1946
Whole plant developed from shoot tip
Ball
1950
Organs regenerated on callus
1954
Plant from single cell
Muir
1960
Protoplast isolation
Cocking

13. HISTORY OF PLANT TISSUE CULTURE
1962
MS media
Murashige - Skoog
1964
Clonal propagation of orchids
Morel
Haploids from pollen
Guha
1970
Fusion of protoplasts
Power
1971
Plants from protoplasts
Takebe
1981
Somaclonal variation
Larkin

14. Factors Affecting Plant Tissue Culture
Growth Media
Minerals, Growth factors, Carbon source, Hormones
Environmental Factors
Light, Temperature, Photoperiod, Sterility, Media
Explant Source
Usually, the younger, less differentiated the explant, the better for tissue culture
Genetics
Different species show differences in amenability to tissue culture
In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization

15. Basic tools of Plant Tissue culture
Culture medium ( Murashige and Skoog medium)
pH meter
Autoclave
Laminar flow bench
Petri-dish
Forceps and scalpel blade
Pipettes
Culture racks or growth chambers

16. Laminar Flow HEPA (High Efficiency Particulate Air) filters
• They consist of a thin pleated sheet of boron silicate microfibres with aluminum separators
• They are particulate filters which retain airborne particles and microorganisms (gases pass
freely through)
• Filtration occurs by five distinct methods
1) sedimentation
2) electrostatic attraction
4) inertial impaction*

17. Autoclave Unit

18. BASIC MEDIA COMPONENTS FOR PLANT CELL CULTURE
Plants growing in natural habitat (require nutrition)
In vitro plant cultures (require nutrition)
Nutrient Media:
-Essential elements -Organic supplements -Source of carbon

19. Essential Nutrients for the plant growth medium
Macronutrients (required content in the plant - 0.1% or % per dry weight) - C, H, O, P, K, N, S, Ca, Mg
Micronutrients (requirement - ppm/dry weight) - Fe, Mn, Zn, Cu, B, Cl, Mo
Na, Se and Si are essential for some plants

20. BASIC MEDIA COMPONENTS FOR PLANT CELL CULTURE
Macronutrients or inorganic nutrients: for example Nitrates, Phosphorus, Potassium, Magnesium, Sulphate,
Calcium – present as salts in the various media (g/l; or mM)
The source of nitrogen in the media: nitrates (NO3 in the form of Ammonium Nitrate or Potassium Nitrate)
The source of potassium ions (K+) from potassium nitrate or potassium dihdrogen phosphate

21. Media components:
Micronutrients or inorganic nutrients: For example EDTA,
Ferrous, Zinc, Boron, Iodide, Manganese, Molybdenum,
Cobalt, Copper (µg/l; mg/l; or µM).
Iron seems more critical; chelated forms of iron or zinc
are commonly used in culture media rather than iron tartrate/citrate as they are difficult to dissolve and ppt in medium.
EDTA-Fe chelate: as EDTA is not stable in medium
(EDTA complexes with Fe)

22. Media components:
Carbon or Energy source
Sugars, e.g. Sucrose, Glucose, Raffinose, Celibiose,
Mannitol, Fructose, Myo-inositol, Arabinose,
Rhamnose, Trehalose, Xylose, Xylitol, Sorbitol (g/l; %).
-Plant Cells / tissues lack autotrophic ability
-Sucrose is the most preferred carbon source. -Sucrose on autoclaving is converted into glucose and fructose (Partial hydrolysis).
-Glucose supports equally good growth (but fructose is less efficient).

23. Media components:
Amino acids for example Glycine, Casein hydrolysate (CH), Argnine, Asparagine, Leucine, Serine, Proline, Glutamine (mg/l; or mM)
Taken up rapidly by plant cells for stimulating cell growth

24. Media components: Organic supplements
Vitamins for example, Pyridoxine.HCl (Vitamin B6), Nicotinic acid, Thiamine.HCl (Vitamin B1), Biotin, Folic Acid, Calcium Pantothenate (Vitamin B5), Riboflavin, Ascorbic Acid (mg/l; µM)
Generally the vitamins are added in the range of 1- 10mg/l

25. Media components: Other Organic supplements
A variety of organic extracts are used: coconut water,
yeast, malt, banana extract and tomato extract
Significant effects rendered by coconut water (5-20%)
and casein ( %) for plant cell growth
Generally the use of natural extracts is avoided.
Quality and quantity of growth-promoting constituents vary (age of tissue from which the extract is derived,
thereby affecting reproducibility of results).
L-asparagine could effectively replace yeast extract.

26. Media components: Activated Charcoal (AC)
AC is generally acid-washed and neutralized before addition to culture medium (0.5-3%) .
AC also helps to reduce toxicity by removing toxic compounds (e.g. phenolics) produced during culture and permits unhindered cell growth.
Sometimes the darkening of tissues occur during
culture, AC helps to reduce this effect couple with the adsorption of inhibitory compounds.

27. Media components: Growth Regulators
Four broad classes of growth regulators are used:
auxins, cytokinins, gibberrellins and abscisic acid
The growth and organogenesis of tissues become
feasible only on the addition of one or more of these classes
of hormones to a medium .
The ratio of hormones required for root or shoot
induction varies considerably with the tissue, which
seems directly correlated to the amount of hormones synthesized at endogenous levels within the cells of the
explant

28. Media components: Auxins
Media are supplemented with various auxins:
Indole 3-cetic acid (IAA) 1-napthaleneacetic acid (NAA) Indole-3-butyric acid (IBA) 2,4-Dichlorophenoxyacetic acid (2,4-D) 1-napthoxyacetic acid (NOA)
They are generally dissolved in ethanol or NAOH
Common feature of auxin: includes cell division, and in nature, this group of hormones are involved with such activities as elongation of stem, internodes, apical
dominance abscission and rooting.

29. Media components: Cytokinins
These are adenine derivatives, mainly concerned with cell division, modification of apical dominance and shoot differentiation in tissue culture.
Most frequently used cytokinins:
6-benzylaminopurine (BAP), 6-gama-gama-dimethylaminopurine (2-ip), Kinetin and Zeatin.
Zeatin and 2-ip are naturally occurring cytokinins while BA and kinetin are synthetically derived cytokinins. They are generally dissolved in dilute HCl or NAOH.
The ratio of auxins and cytokinins is important with respect to morphogenesis on the cell culture medium.
For callus initiation, embryogenesis and root initiation the requisite ratio of auxins to cytokinin is high, while the reverse leads to axillary and shoot proliferation.

30. Auxin : Cytokinin ratio
Hormone Balance
Auxin Cytokinin
High Low
Low High
Root formation on cuttings
Embryogenesis
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth

31. Media components: Gibberellins and Abscisic acid
GA3 is the most common gibberellin used of more than 20 known gibberellins. -It promotes callus growth and induces embryogenesis. -It also induces stunted plants to elongate.
Abscisic acid (ABA) stimulates or inhibits callus growth depending on the species, also promotes distinct developmental pathways (somatic embryogenesis).

32. Jasmonates (jasmonic acid and its methyl ester), play a
Ethylene
Ethylene is a gaseous, naturally occurring, plant growth regulator.
Associated with controlling fruit ripening.
Some plant cell cultures produce ethylene, which can inhibit
the growth and development of the culture.
Jasmonates (jasmonic acid and its methyl ester), play a
role in embryogenesis, differentiation, root formation and
breaking of seed dormancy.
Salicyclic acid promotes flowering and inhibits ethylene
biosynthesis.
Brassinosteroids play a role in shoot elongation, inhibition of root growth.

33. Media components: Solidyfying Agents & pH
Commonly used for preparing semi-solid or solid tissue culture media: Agar, a polysaccharide from seaweeds.
Normally 0.5 to 1.0 % agar is used in the medium to firm gel at required pH.
Others used are: phytagel and gel-rite (clear gelling agents are valuable aids for detecting contamination)
Plant Cells and tissues require optimum pH for growth and development.
The pH of a medium can be adjusted to the requirement of the experiment.
The pH affects uptake of the ions and for most of the culture media, pH 5.0 to 6.0 before sterilization is considered optimal.