1. PLANTS (PHG 407) DR. DALIA RASHEED LECTURE (4)
BIOTECHNOLOGY OF MEDICINAL
PLANTS (PHG 407) DR. DALIA RASHEED LECTURE (4)

2. Timeline of Plant Tissue Culture
1839: Schwan expressed the view that each eukaryote living cell should be capable of independent development if provided with the proper external conditions.
1902: Haberlandt attempted to culture the mesophyll tissue and root hair cells.
This was the first attempt of in vitro culture.
Unfortunately; Haberlandt failed to grow a whole plant.
1904: Haning attempted to culture excised embryos from mature seeds.
1922: Kotte was successful in obtaining growth from isolated root tips on
inorganic media.
1934: White used yeast extract (vit B) with inorganic salts to culture root tips of tomato.
1937: Discovery of the auxins role in plant tissue culture.
1939: White & Guatheret established callus culture using Tobacco. Callus obtained was termed as tobacco tumor (crown gall or Plant Cancer).
1950s: Establishment of cell suspension cultures and identification of PGR.

3. Components of plant tissue culture
1. Explant
3. Aseptic conditions
2. Growth media
4. Culture conditions
Explant could be: part of leaf, apical bud, axillary bud, part of fruit, flower, seed, root, part of the node segment. cotyledons etc. (generally any part cut from the plant).
Chances of success increase with young actively grown tissues (e.g. Meristem)

4. Growth Media Salt Mixtures Macro-nutrient Organic Substances
N, P, K, Ca,Mg,Fe
Micro-nutrient
Zn, Mo, Cu
Organic Substances
Carbon Sources
Sucrose
Vitamins
Adenine
Thiamine
Gelling agents
Agar, agarose
Growth Regulators

5. Growth (nutrition) media
Composition of Growth Media
Inorganic Macro and micronutrients.
Vitamins.
Carbon source
Phyto-hormones.
Gelling or solidfying agents (if required)
other additives

6. Nutrition Medium Composition
Inorganic (macro & micro) nutrients:
Macronutrients: present in realtively large quantities
( mM, in mg(s)or g(s))
e.g., K, Nitrogen, Ca, P, Mg S.
They are essential for plant growth and develpment and formation of proteins and enzymes. Added in form of inrorganic salts, e.g., KCl, K2NO3, sodium phosphate,
Micronutrients: present in relatively small quantities
(µM, in mg(s)-µg(s))
e.g. CO, Fe, Zn, Cu, B, Mo, Mn.
Are required for healthy plant cell growth and cellular functions.

7. Nutrition Medium Composition
Vitamins: (B1 , B3, B6, B12)
Thiamine (B1) is required by all cells for growth. Other vitamins are commonly added.
Energy source or carbon source: Sugar
sucrose and glucose are mostly used than other
mono- and disaccharides.
Myo-inositol is a sugar alcohol not a vitamin. It is important for cell division as it gets in as phosphatidyl-inositol.

8. Nutrition Medium Composition
Undefined organic supplemets:
Coconut milk, casein hydrolysate are needed for some cultures.
Activated charcoal: added to some media to adsorb toxicants which could kill the explants and or cause its browning. (1% activated charcoal)
Gelling agents (Agar)
pH range from
>7 and <4.5 stops growth
>6.0 gives a hard medium,
<5.0 no gelling of the agar.

9. Nutrition Medium Composition (Growth regulators:
Auxins, were the first of the major plant hormones to be discovered. Auxin (namely IAA) is present in all parts of a plant, although in very different concentrations. Endogenous auxins include IAA, NAA (Naphthalene acetic acid) , phenylacetic acid, and indole-3-butyric acid.
Synthetic auxin analogues include 2,4-dichlorophenoxyacetic acid (2,4-D) used as herbicides.
Auxins, are responsible for:
Callus formation, rooting of cuttings, and the induction of adventive embryogenesis
IAA
IBA
NAA
2,4-D
2,4,5-T

10. Nutrition Medium Composition
Growth regulators:
Cytokinins, are involved primarily in cell growth and shoot differentiation, Cytokinins participate in local and long-distance signalling.
There are two types of cytokinins:
Adenine-type cytokinins represented by kinetin, zeatin, and 6- benzylaminopurine (benzyl adenine – BA or BAP).
Phenylurea-type cytokinins like diphenylurea and thidiazuron (TDZ).
Gibberellins: Cell Enlargement
Abscisic acid: Enables more normal development of embryos

11. Auxin and Cytokinin Ratio

12. Sterilization (aseptic conditions), WHY?
Plant tissue culture media are rich in nutrients, that support the growth of many micro-organisms (bacteria , fungi)
They grow much faster than the cultured tissue &
finally kill it.
The contaminants may also give out metabolic wastes which are toxic to the plant tissue.

13. Sources of Contamination
The Media itself.
The culture vessels.
The explant.
The environment of the transfer area.
The environment of the culture room.
The instruments used in handling the plant
material during inoculation and subculture.

14. Sterilization Techniques
Steam sterilization (Autoclave)
Use of steam at 121ºC with pressure 15 psi for 20 min.
Dry sterilization
Heat at ºC for 3 hours.
Filter sterilization
Membrane filters of size μm
Flame Sterilization
Chemical Sterilization

15. Environmental Factors affecting the plant tissue culture
Temperature
The room for incubating cultures must be maintained at a controlled temperature around (10-32) ± 2ºC. For higher or lower temp. special incubators can be used.
Light
Cultures are generally grown in diffused light, i.e. Photoperiods. Fluorescence tubes and a timing device are used to set light/dark regimens.
Humidity
Relative humidity must be maintained around 50%
>50% Cotton plugs become damp & chances of culture contamination
increases
<50% the nutritive media are liable to drying.

16. Laboratory Requirements & Organization for Tissue Culture
Autoclave
Culture Area
Media room

17. Incubation

18. Young Plantlets
Subculture: is a new cell culture made by transferring some or all plants (or cells) from a previous culture to fresh growth medium

19. Green house (Adaptation, Acclimatization), for adjusting to the environmental changes

20. Plants in the Field