Biotechnology

1. • The term Biotechnology was coined by Karl Ereky, a Hungarian engineer
in 1919
• An important aspect of all plant biotechnology processes is the culture of
either the microorganisms or plant cells or tissues and organs in artificial
media.
• The term ‘‘plant tissue culture” broadly refers to the in vitro cultivation of
plants, seeds, plant parts (tissues, organs, embryos, single cells, protoplasts
etc.) on nutrient media under aseptic conditions.
• In 1902 well-known German plant physiologist, Gottlieb Haberlandt
(1854–1945), attempted to cultivate plant cells in vitro. He is regarded as
the father of plant tissue culture.

2. • The differentiation of whole plants in tissue cultures may occur via shoot and
root differentiation, or alternatively the cells may undergo embryogenic
development to give rise to somatic embryos.
• Differentiation of plants from callus cultures has often been suggested as a
potential method for rapid propagation.

3. • The release of protoplasts from root tip cells using a fungal cellulase in 0.6M
sucrose was reported by Cocking in 1960.
• The most universally used high-salt medium was developed by Skoog and his
students (Murashige and Skoog, 1962).
• Another landmark in the development of Plant tissue culture has been the
discovery of haploids, when Guha and Maheshwari (1964) obtained haploid
embryos from Datura innoxia.

4. • The basic nutritional requirements of cultured plant cells are very similar to
those utilized by plants.
• A nutrient medium is defined by its mineral salt composition, carbon source,
vitamins, growth regulators and other organic supplements which include
organic nitrogen, acids and complex substances.
• When reference is made to a particular medium, the intention is to identify
only the salt composition unless otherwise specified.
• Any number and concentration of amino acids, vitamins, growth regulators or
organic supplements can be added in an infinite variety of compositions to a
given salt composition to achieve the desired results.

5. • The MS medium of Murashige and Skoog (1962) salt composition is very widely used in
different culture systems.
• The B5 medium of Gamborg et al. (1968) or its derivatives have been useful for cell and
protoplast culture.
• This medium was originally designed for callus and suspension cultures but it has also
been effectively used for plant regeneration.
• It differs from MS medium in having much lower amounts of nitrate in the form of
ammonium.
• The N6 medium was developed for cereal anther culture and is used successfully with
other types of cereal tissue culture (Chu, 1978)

6. • The medium generally consists of inorganic nutrients, carbon and energy sources, vitamins,
phytohormones (growth regulators), and organic supplements which include organic
nitrogen, acids and complex substances.
Inorganic nutrients
• Mineral elements are very important in the life of a plant.
• For example, calcium is a component of the cell wall, nitrogen is an important part of amino
acids, proteins, nucleic acids, and vitamins, and magnesium is a part of chlorophyll
molecules.
• Similarly iron, zinc and molybdenum are parts of certain enzymes.
• Besides, C, H, N, and O, 12 other elements are known to be essential for plant growth.

7. • According to the recommendations of International Association for Plant Physiology, the
elements required by plants in concentration greater than 0.5 mmol/l are referred to as
macro/major elements and those required in concentration less than that are micro/ minor
elements.
• The following are required in macro- or millimole quantities: N, K, P, Ca, S, and Mg.
• The essential micronutrients which are required in micromolar concentrations include iron
(Fe), manganese (Mn), boron (B), copper (Cu), zinc (Zn), iodine (I), molybdenum (Mo)
and cobalt (Co).
• When mineral salts are dissolved in water, they undergo dissociation and ionization.
• The active factor in the medium is the ions of different types rather than the compounds.

8.  For most purposes a nutrient medium should contain from 25 to 60 mM inorganic nitrogen.
 The cells may grow on nitrate alone, but often there is a distinct beneficial effect and
requirement for ammonium or another source of reduced nitrogen.
 Besides, nitrate alone in the medium drifts the pH towards alkalinity.
 Adding a small amount of an ammonium compound together with nitrate checks this drift.
 Nitrate is used in the range of 25–40 mM and ammonium in the range of 2–20 mM.
 Potassium is required at concentrations of 2 to 26 mM. Potassium is generally supplied as
the nitrate or as the chloride form and sodium cannot be substituted for potassium.
 A concentration of 1–3mM calcium, sulphate, phosphate and magnesium is usually
adequate.


9. • Iron is generally added as a chelate with ethylene diamine tetra acetic acid (EDTA).
• In this form iron remains available up to a pH of 8.0.
Carbon and Energy Source
• The standard carbon source is sucrose but plant tissues can utilize a variety of carbohydrates
such as glucose, fructose, lactose, maltose, galactose and starch.
• In the cultured tissues or cells, photosynthesis is inhibited and thus carbohydrates are needed
for tissue growth in the medium.
• The sucrose in the medium is rapidly converted into glucose and fructose.
• Sucrose is generally used at a concentration of 2–5%.

10. • Most media contain myoinositol at a concentration of ca. 100mg/l, which improves cell
growth.
Vitamins
• Normal plants synthesize the vitamins required for growth and development. But plant cells
in culture have an absolute requirement for vitamin B1 (thiamine), vitamin B (nicotinic
acid) and vitamin B6 (pyridoxine).
• Some media contain pantothenic acid, biotin, folic acid, p-amino benzoic acid, choline
chloride, riboflavine and ascorbic acid.
• The concentrations are in the order of one mg/l.
• Myo-inositol is another vitamin used in the nutrient medium with a concentration of the
order of 10–100mg/l.

11. Growth regulators
• Hormones now referred to as growth regulators are organic compounds that have been
naturally synthesized in higher plants, which influence growth and development.
• Apart from natural compounds, synthetic compounds have been developed which
correspond to the natural ones.
• There are two main classes of growth regulators that are of special importance in plant
tissue culture.
• These are the auxins and cytokinins, while others viz. gibberellins, abscisic acid, ethylene,
etc. are of minor importance.
• Some of the naturally occurring growth regulators are the auxin indole acetic acid (IAA)
and the cytokinins zeatin and isopentenyladenine (2 iP), while others are synthetic growth
regulators.

12. Auxins
• auxins induce cell division, cell elongation and formation of callus.
• They also help in the induction of adventitious roots.
• It often inhibits adventitious and axillary shoot formation.
• At low auxin concentrations, adventitious root formation predominates, whereas at high
auxin concentrations, root formation fails to occur and callus formation takes place.
• Auxin is present in sufficient concentration in the growing shoot tips or flowering tips of
plants to ensure multiplication and elongation.
• Auxin circulates from the top towards the base of the organs with a polarity strongly
marked in young organs.

13. • The compounds most frequently used are highly effective 2,4-dichlorophenoxy acetic acid
(2,4-D), naphthalene acetic acid (NAA), indole acetic acid (IAA), indole butyric acid
(IBA), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), p-chlorophenoxy acetic acid (pCPA)
and picloram (4-amino-3,5,6-trichloropicolinic acid).
Cytokinins
• Cytokinins are derivatives of adenine and have an important role in shoot induction.
• Cytokinins also have a clear effect on cell division.
• These are often used to stimulate growth and development.
• They usually promote cell division if added together with an auxin.
• Cytokinins have a clear role in organogenesis where they stimulate bud formation.

14. • At higher concentrations (1 to 10 mg/l), adventitious shoot formation is induced but root
formation is generally inhibited.
• Cytokinins promote axillary shoot formation by decreasing apical dominance.
• The most frequently used cytokinins are 6-benzyl amino purine (BAP) or 6-
benzyladenine (BA), N-(2-furfurylamino) 1-H-purine-6-amine (kinetin), 6-(4-hydroxy-
3methyl-trans-2-butylamino) purine (zeatin), and isopentenyladenine (2 iP).
• Zeatin and 2-iP are naturally occurring cytokinins while BA and kinetin are synthetically
derived cytokinins.
• Stock solutions of IAA and kinetin are stored in amber bottles or bottles covered with a
black paper and kept in dark since they are unstable in light

15. Other growth regulators
• Gibberellins are normally used in plant regeneration.
• GA3 is essential for meristem culture of some species.
• In general, gibberellins induce elongation of internodes and the growth of meristems or
buds in vitro.
• Gibberellins usually inhibit adventitious root as well as shoot formation.
• They seem to oppose the phenomenon of dedifferentiation.

16. • Abscisic acid is an important growth regulator for induction of embryogenesis.
• This is a growth inhibitor, which seems to be synthesized when a plant is under difficult
conditions.
• It has a favorable effect on abscission.
• Ethylene is a gaseous compound produced by cultured cells, but its role in cell and organ
culture is not known.
• The practical use of ethylene, which is difficult in a gaseous state, made great progress
after the discovery of 2- chloroethane phosphoric acid. This product, when applied in a
powder form, penetrates the tissue where it liberates ethylene.

17. Organic Supplements
Organic nitrogen
• Cultured cells are normally capable of synthesizing all the required amino acids, but it is
often beneficial to include organic nitrogen in the form of amino acids such as glutamine
and aspargine and nucleotide such as adenine.
• For cell culture, it is good to add 0.2 to 1.0 g/l of casein hydrolysate or vitamin-free
casamino acids.
• In some cases L-glutamine (up to 8 mMor 150 mg/l) may replace the casein hydrolysate.
• The amino acids included in the media and amount in mg/l are: glycine (2), aspargine
(100), tyrosine (100), arginine (10) and cysteine (10).
• Sometimes adenine sulphate (2–120 mg/l) is added to the agar media for morphogenesis.

18. Organic acids
• Plant cells are not able to utilize organic acids as a sole carbon source. Addition of TCA
cycle acids such as citrate, malate, succinate or fumarate permits growth of plant cells on
ammonium as the sole nitrogen source.
• The cells can tolerate a concentration of upto 10mM of the acid.
• Pyruvate may also enhance growth of cells cultured at low density.
Complex substances
• A variety of extracts viz. protein hydrolysate, yeast extract, malt extract, coconut milk,
orange and tomato juice have been tested. With the exception of protein hydrolysate and
coconut milk, most others are used as the last resort.

19. • If used at higher concentration, the complex substances may adversely affect cell growth.
• Coconut milk is commonly used at 2–15% (v/v).
• The present trend is, however, towards fully defined media and the use of complex
mixtures is losing favor.
Anti browning compounds
• Many plants are rich in polyphenolic compounds. After tissue injury during dissection,
such compounds will be oxidized by polyphenol oxidases and the tissue will turn brown or
black.
• The oxidation products are known to inhibit enzyme activity, kill the explants, and darken
the tissues and culture media, a process which severely affects the establishment of
explants

20. • Activated charcoal is generally used at concentrations of 0.2 to 3.0% (w/v) in the medium
where phenol like compounds are a problem for in vitro growth of cultures.
• It can adsorb toxic brown/black pigments and stabilizes pH.
• It also helps to reduce toxicity by removing toxic substances produced during the culture
and permits unhindered cell growth.
• Besides activated charcoal, polyvinylpyrrolidone (250–1000 mg/l), citric acid and ascorbic
acid (100 mg/l each), thiourea or L-cysteine are also used to prevent oxidation of phenols.
• Phloroglucinol, a phenolic compound, is sometimes added to inhibit the enzyme IAA
oxidase responsible for the breakdown of IAA.

21. Gelling Agents
• Agar, a seaweed derivative, is the most popular solidifying agent.
• It is a polysaccharide with a high molecular mass and has the capability of gelling media.
• Solublized agar forms a gel that can bind water and adsorb compounds.
• The higher the agar concentration, the stronger is the binding of water.
• Plant tissue culturists often use Difco Bacto agar at a concentration of 0.6 to 1.0% (w/ v),
although other forms of agar (agarose, phytagar, flow agar, etc.) are also becoming
popular.
• In vitro growth may be adversely affected if the agar concentration is too high.

22. • With higher concentrations, the medium becomes hard and does not allow the diffusion of
nutrients into the tissues.
Besides agar, the following alternatives are also available.
i. Alginate can be used for plant protoplast culture.
ii. Gelrite at 0.2% can be used for solidification of media.

23. pH
• Nutrient medium pH ranges from 5.0 to 6.0 for suitable in vitro growth of explant.
• pH higher than 7.0 and lower than 4.5 generally stops growth and development.
• The pH before and after autoclaving is different. It generally falls by 0.3 to 0.5 units after
autoclaving.
• If the pH falls appreciably during plant tissue culture (the medium becomes liquid) then a
fresh medium should be prepared.
• It should be known that a starting pH of 6.0 could often fall to 5.5 or even lower during
growth.
• pH higher than 6.0 give a fairly hard medium and a pH below 5.0 does not allow
satisfactory gelling of the agar.

24. PROTOCOLS
General Methodology for Medium Preparation
Preparation of macro- and micronutrient stock solutions
• Stock solutions of macro- and micronutrients, vitamins and growth regulators are prepared
in distilled or high purity demineralized water.
• Chemicals should be of the highest grade.
• Usually the stock solutions of macro and micronutrients are prepared as 10x and 100x
concentrations.
• These nutrient solutions canbe dispensed in bottles and stored frozen
• While dissolving the nutrients in water, one compound is added at a time to avoid
precipitation. For macronutrient stock solution, calcium chloride is dissolved separately in
water and then added to the rest of the solution to avoid precipitation.

25. Preparation of stock solutions of Murashige and Skoog (MS) medium
Constituent Concentration in MS
medium (mg/l)
Concentration in the stock solution
(mg/l)
Volume to be taken/
liter of medium
Macronutrients (10x)
NH4NO3 1650 16500 100
KNO3 1900 19000
MgSO4·7H2 O 370 3700
CaCl2·2H2O 440 4400
KH2PO4 170 1700
Micronutrients (100x)
H3 BO3 6.2 620 10
MnSO4·4H2 O 22.3 2230
ZnSO4·7H2O 8.6 860
Na2MoO4·2H2O 0.83 83
KI 0.25 25
CuSO4·5H2O 0.025 2.5
CoCl2 ·6H2O 0.025 2.5

26. Constituent Concentration in MS
medium (mg/l)
Concentration in the stock solution
(mg/l)
Volume to be taken/
liter of medium
Iron source
Fe·EDTA- Na salt 40 Added fresh
Vitamins
Nicotinic acid 0.5 50 mg/100 ml 1
ThiamineHCl 0.1 50 mg/100 ml 0.2
Pyridoxine HCl 0.5 50 mg/100 ml 1
Myo-inositol 100 Added fresh
Others
Glycine 2 50 mg/100 ml 4
Sucrose 30,000 Added fresh
Agar 8000 Added fresh
pH 5.8

27. Preparation of vitamin and growth regulator stock solutions
• All the growth regulators are not soluble in water.
• Solubility of different growth regulators is given in Table2.
• The compound should be dissolved in few-ml of solvent and then water slowly added to
make up to the requisite volume.
• Concentrations of compounds can be taken as mg/l or in molarity

28. Table 3.5: Preparation of stock solutions of growth regulators
Compound Abbreviation mg/100 ml (1mM)
Auxins
2,4-Dichlorophenoxy aceticacid 2,4-D 22.1
Indole-3 acetic acid IAA 17.5
Indole-3 butyric acid IBA 20.32
α-Naphthalene acetic acid NAA 18.62
β-Naphthoxy acetic acid NOA 20.23
2,4,5-Trichlorophenoxy acetic acid 2,4,5-T 25.56
p-Chlorophenoxy acetic acid pCPA 18.66
Picloram PIC 24.12
Concentration in mg/l: It is preferable to dissolve 50 mg/100 ml to give a concentration of
0.5 mg/ ml.

29. Compound Abbreviation mg/100 ml (1mM)
Cytokinins
Adenine Ade 18.91
Benzyl adenine or benzyl amino purine BA or BAP 22.52
N-isopentenylamino purine 2-iP 20.33
Kinetin KIN 21.52
Zeatin ZEA 21.92
Others
Gibberellic acid GA3 34.64
Abscisic acid ABA 26.43
Colchicine Col 39.94

30. • Add each component according to the list, including growth regulators, by using correctly
sized graduated cylinders or pipettes or balance.
• Add water to just below the final volume
• Adjust the pH of the medium to the required value (e.g. pH 5.8 for MS) by drop-wise
addition of 1N KOH or 1N HCl with constant stirring.
• Add the needed amount of agar (e.g. 6–8 g/l) or any other gelling agent.
• Heat the solution while stirring until the agar is dissolved.
• The solution becomes transparent when the agar is completely dissolved.
• Distribute the medium in glass or polypropylene vessels and plug it with cotton or cover it
with aluminium foil.

31. • The culture medium is sterilized in an autoclave for 20 min at 121°C at 15 p.s.i (105 kPa).
• If filter-sterilized hormones/compounds are to be added, then these are added to the
autoclaved medium.
• Medium is cooled in laminar airflow to 50–60° C, the hormones are added by Millipore or
any other filter assembly using 0.22 mm filter, and then dispensed into sterile vessels.
• After cooling, the media are preferably stored at 4–10°C.
• It is advisable to mark each vessel to show the precise medium and date of preparation.
• The culture medium should be used 3–4 days after preparation, so that if it is not properly
sterilized, contamination will start appearing and this medium can be discarded.