Tissue culture is a technique that grows plant cells, tissues or organs in an artificial nutrient medium under sterile conditions. The basic requirements for tissue culture include various inorganic and organic nutrients, vitamins, amino acids, sucrose as a carbon source, and plant growth regulators. The inorganic nutrients include macro and micronutrients that provide minerals for growth. The medium is solidified with a gelling agent like agar and adjusted to pH 5.8 before autoclaving. Stock solutions of ingredients are prepared in advance and mixed as needed to prepare the culture medium under sterile conditions.

1. Basic Requirement For
Tissue Culture

2. TISSUE CULTURE
Def;
Tissue culture is a technique that uses an
artificial medium to grow and maintain cell
and tissue or an organ under controlled
environmental conditions.

3. Explant
• Explant is living tissue removed from an organism and placed
in a medium for tissue culture.
Totipotency
Plant cell have a unique property call totipotency.
That is , most of the plant cell have the capacity to regenerate
into the entire plant when placed in proper environment with
nutrient and hormonal support.

4. Basic Requirement For Tissue Culture
1. Media Constituents
The basic requirements of mineral elements such as
potassium, calcium, magnesium, nitrogen, phosphorous
and sulphur, which are required in large quantities
(macronutrients/macroelements/ major elements) and
others, like iron, manganese, copper, zinc, boron and
molybdenum, are required in small quantities
(micronutrients/ microelements/minor elements) for the
growth of plant tissues are fulfilled by providing their
common salts in the medium.

5. • One type of ion may be contributed by
more than one salt in the medium. For
example, in MS medium, NO3– ions are
contributed by NH4NO3, as well as
KNO3, and K+ ions by KNO3 and
KH2PO4.

6. 2. Inorganic Nutrients
(i) Macronutrients.
• The macronutrients are required in millimolar (mM)
quantities.
Calcium (Ca2+), potassium (K+), magnesium (Mg2+),
nitrogen (NO3-), sulphur (SO42-) and phosphorous
(PO43-) are the macroelements and the essential
ingredients of plant tissue culture media.

7. • These are added to the medium as calcium
nitrate, potassium dihydrogen phosphate and
magnesium sulphate. Alternately, potassium and
calcium may be provided as KCl or KNO3 and
CaCl2.2H2O, respectively. Nitrogen is one of the
vital elements required for the growth of plants
in cultures as also in nature.

8. • Inorganic nitrogen is usually supplied in the form
of ammonium (NH4+) and/or nitrate (NO3-) ions.

9. (ii) Micronutrients
• Some micronutrients, although required in small
quantities, are essential for tissue growth in cultures.
They act as cofactors of enzymes. Micronutrients
typically include boron (BO33-; from H3BO3),
manganese (Mn2+; from MnSO4.

10. Organic Nutrients
• In order to achieve the best growth of plant tissues in
cultures, the medium needs to be supplemented with
organic compouApparently, the cultured plant cells
synthesize them in sub-optimal quantities. Sucrose, a
source of carbon or energy, is a very important
constituent of plant tissue culture media. nds, such as
vitamins and amino acids.

11. (i) Vitamins and amino acids.
• Vitamins that act as coenzymes are required to be
added to the medium for healthy growth of tissue
cultures. The most widely usedvitamins are those of B
group, viz., thiamine (vitamin B1), nicotinic acid (also
known as niacin or vitamin B3), pyridoxine (vitamin
B6) and myo-inositol (sometimes referred to as meso-
inositol). Glycine, the simplest amino acid, is a
common constituent of plant tissue culture media.

12. (ii) Carbon source
• Most plant tissue cultures are unable to
photosynthesize because of the absence of chlorophyll
or poorly developed chloroplasts, limited CO2 in the
culture vessel due to poor gaseous exchange and
absence of optimum light intensity. It is, therefore,
obligatory to add to the culture medium an utilizable
source of carbon necessary for various metabolic
activities.

13. • The most commonly used carbon source is sucrose at a
concentration of 2–5 % (w/v). Generally, sucrose
autoclaved along with the medium supports better
growth of tissues than filter-sterilized sucrose.
Autoclaving causes hydrolysis of sucrose into more
efficiently utilizable sugars, such as glucose and
fructose.

14. Plant Growth Regulators
• Addition of growth regulators to an otherwise complete
medium, containing inorganic and organic nutrients and
sucrose called Basal medium (BM), is invariably necessary to
trigger various types of growth and differentiation. Although
the explants may have certain levels of endogenous growth
hormones (naturally synthesized growth regulators), it is often
necessary to supplement them exogenously to evoke certain
responses.

15. • The growth regulators are generally required in very
minute quantities (0.001–10 lM). The nature and
quantity of growth regulators in the medium is varied
according to the variety of plant, nature of the tissue
and also the stage of culture (initiation of callus,
induction of somatic embryogenesis, shoot
differentiation or multiplication, rooting of shoots,
etc.).

16. • In order to develop a tissue culture protocol for a
new plant species, various types and
concentrations of growth regulators in several
permutation and combination need to be tested.
The concentrations of growth regulators used are
very often reported in mg L-1.

17. (i) Auxins
• Auxins are involved with many developmental
processes in plants, including elongation of stem and
internodes, tropisms, apical dominance, abscission
and rooting. In tissue cultures, auxins have been used
to induce cell division, cytodifferentiation and
organogenic and embryogenic differentiation.
Generally, auxin at low concentration favours root
initiation, whereas at higher concentration induces
callus formation.

18. (ii) Cytokinins
• This is another major group of plant hormones.
Natural cytokinins are N6substituted adenine
derivatives and occur in plants as nucleosides
and nucleotides. Roots are the possible sites of
their synthesis.

19. • In nature, cytokinins are concerned with cell
division, modification of apical dominance and
shoot differentiation. Incorporation of these
compounds in culture media is mainly to trigger
cell division, and to induce differentiation of
adventitious shoots from callus and organs, and
shoot proliferation by the release of axillary
buds from apical dominance

20. (iii) Gibberellins
• These are less commonly used in plant tissue culture.
There are over 20 known gibberellins, of which GA3 is
used most often.

21. (iv) Ethylene and Abscisic acid
• Ethylene (ethane, C2H4) is an unusual, gaseous
plant hormone. It is produced by ageing and
stressed tissues.

22. Other Supplements
• (i) Polyamines, derived through decarboxylation of
amino acids, have been used to promote
organogenesis/somatic embryogenesis (Mengoli and
Bagni 1992; Rajam et al. 1998). Of the three
polyamines (putrescine, spermidine and spermine),
putrescine has proved most effective (Altman 1990;
Litz 1993).

23. • (ii) TIBA (2,3,5-triiodobenzoic acid) is an inhibitor of
auxin polar transport. In carrot cultures, it arrests
development of somatic embryos at the globular
stage.
• (iii) Phloroglucinol has been shown to promote rooting
in rosaceous fruit trees such as apple.
• (iv) Activated charcoal is usually added at 0.5 % to
culture media to promote rooting and/or to adsorb
toxic exudates from cultured tissues.

24. Undefined Supplements
• In order to promote growth of certain calli and evoke a
desired morphogenic response in cultures, several
complex, undefined organic supplements such as
banana homogenate, CH, CM, corn milk, ME, TJ and YE
have been used in the medium. These crude natural
plant products are generally used when no other
combination of defined components yield the desired
results.

25. Gelling Agents
• In static liquid cultures, the tissue would get
submerged and die of anaerobic conditions. To
circumvent this problem, the medium is solidified with
a suitable gelling agent. The most desirable properties
of a gelling agent are that it should: (i) be inert (ii)
withstand sterilization by autoclaving and (iii) be liquid
when hot so that the medium could be dispensed in
culture vessels in desired quantities.

26. • (i) Agar. Agar, obtained from red algae, especially
Gelidium amansii, is the most commonly used gelling
agent.
• (ii) Agarose. Agarose, comprising b-D(1-3) and 3,6-
anhydro-a-L(1-4) galactopyranose molecules, is
obtained by further purifying agar to remove
agaropectins with its sulphate groups.

27. • (iii) Gellan gum. A linear polysaccharide obtained from
the bacterium Pseudomonas elodea and marketed
under different trade names, such as gelrite (C.P. Kelco
& Co.) and phytagel (Sigma-Aldrich Chemical Co.), has
become a popular substitute of agar.
• (iv) Isubgol. Indian scientists have shown that the husk
of Plantago ovata seeds (Isubgol), used at 3 %, could be
a cheaper alternative to gel plant tissue culture media
(Jain and Babbar 1998).

28. pH of the Medium
• The pH of medium greatly influences the uptake
of ingredients by the tissue in culture, solubility
of salts and gelling efficiency of agar. The pH of
medium is usually set at 5.8 as at this pH all the
salts are maintained in a near buffered form.

29. • In general, a pH higher than 6.0 gives a fairly hard
medium and a pH below 5.0 does not allow
satisfactory gelling of the agar due to its hydrolysis
during autoclaving. The pH of culture medium
generally drops by 0.3–0.5 units after it is autoclaved
and then changesthroughout the period of culture
both due to oxidation and differential uptake of ions
such as NH4+ and NO3- and secretion of substances by
the growing tissue.

30. Media Preparation
• Preparation of culture media is a critical step in tissue
culture work wherein great precision is required on the
part of the investigator. There are two possible ways to
prepare medium. One method is to weigh and dissolve
the required quantities of the ingredients separately
and mix them every time a medium is to be prepared.
But this is a cumbersome method and chances of
committing error are more.

31. • A more convenient and widely practiced method
is to prepare, in advance, a series of
concentrated stock solutions and store them in a
refrigerator. Wheneveramediumistobeprepared,
the required amounts are drawn from the stock
solutions and mixed.For example,to prepare MS,
four stock solutions are prepared .

32. Reference
Auther;
Sant Saran Bhojwani Prem Kumar Dantu
Book;
Plant Tissue Culture: An Introductory Text