Module I: Introduction to plant tissue culture

Plant biotechnology involves production of genetically modified plants by removing genetic

information from an organism, manipulating it in the laboratory and then transferring it in into a
plant to change certain characteristics of it.

Plant tissue culture is the technique of growing plant cells, tissues, organs, seeds or other plant parts
in vitro i.e in glass under sterile environment and under controlled physical, chemical and nutritional
conditions.

Concept of Totipotency:
 Totipotency is the ability of a single cell to divide and produce all of the differentiated cells in
an organism.
 In other words, totipotency s the genetic potential of a plant cell to produce the entire plant.
 Plants can be separated into their component parts (organs, tissues, or cells), which can be
manipulated in vitro and then grown back to complete plants.
 Isolated cells from differentiated tissue are generally non-dividing and quiescent; to express
totipotency they undergo de-differentiation and re-differentiation.
 The possibility of regenerating an entire plant from few cells was given by Gottlieb
Haberlandt (Father of plat tissue culture)

Dedifferentiation: Dedifferentiation is the transformation of cells from a given differentiated state to

a less differentiated or stem cell-like state. It increases the developmental potency of cells.

Redifferentiation: The process by which a dedifferentiated cell attains a particular function and loses
its ability to divide again is called redifferentiation.
Media Composition
i. Inorganic Nutrients:
 Macronutrients- N, K, P, Ca, S, Mg
 Micronutrients- Fe, Mn, B, Cu, Zn, I, Mo, Co

ii. Carbon (energy source), vitamins:

 Sucrose or glucose (sometimes fructose), concentration 2-5 %
 Most media contain myo-inositol, which improves cell growth especially in
monocots
 An absolute requirement for vitamin B1 (thiamine)
 Growth is also improved by the addition of nicotinic acid and vitamin B6
(pyridoxine)
 Some media contain pantothenic acid, biotin, folic acid, p-amino benzoic acid,
choline chloride, riboflavin and ascorbic acid (C-vitamin)

iii. Growth regulators

 Auxins:
 induces cell division, cell elongation, swelling of tissues,
 formation of callus, formation of adventitious roots
 inhibits adventitious and axillary shoot formation
 2,4-D, NAA, IAA, IBA, 2,4,5-T, pCPA

 Cytokinins:
 shoot induction, cell division
 kinetin, BA, BAP, zeatin, 2iP

 Gibberellins:
 plant regeneration, elongation of internodes

 Abscisic acid:
 induction of embryogenesis
 Skoog & Miller gave auxin-cytokinin balance for root and shoot development.
high kinetin ---> shoot
high auxin ---> root

iv. Organic supplements

 N in the form of amino acids (glutamine, asparagine) and nucleotides (adenine AdS)
 Organic acids: TCA cycle acids (citrate, malate, succinate, fumarate), pyruvate
 Complex substances: yeast extract, malt extract, coconut milk, protein hydrolysate
 Activated charcoal is used where phenol-like compounds are a problem. It absorbs
toxic pigments and stabilizes pH
 Also PVP (polyvinylpyrrolidone), citric acid, ascorbic acid, thiourea, glutathione and
L-cysteine are used to prevent oxidation of phenols

v. Gelling agent
 Agar
 Agar is the most commonly used gelling agent.
 Marine red algae contain the structural polysaccharide agar, which consists of 2
components, agarose and agaropectin. 
 Agarose and agaropectin readily form gels that contain high amounts of H 2O (up
to 99.5%). 
 When agar is mixed with liquid, it forms a gel that melts at about 100  C and
solidifies at about 45 C.
 Other benefits are that agar does not react with any components of the medium
and it is not digested by enzymes from the plant tissue.
 Agar does not gel well under acidic conditions (pH <4.5).
 The inclusion of activated charcoal in media may also inhibit gelling of agar.
 The agar concentrations commonly used in plant culture media range between
0.5% and 1%; these concentrations yield a firm gel at typical media pHs.
 The concentration of agar may be critical to plant response in culture. Medium
that is too soft may produce hyperhydric (abnormal, glassy-looking) tissue while
medium that is too hard may cause reduced growth.

 Agarose
 When greater purity is needed, agarose may be used.
 Agarose is extracted from agar leaving behind agaropectin and its sulfate
groups. Agarose also has higher gel strength than agar and thus less is required
for solidification of media. Agarose is used in situations where the impurities of
agar are a major disadvantage, such as in protoplast culture.

 Gelrite
 Gelrite™ consists of a polysaccharide produced by the bacterium Pseudomonas
elodea.
 Unlike agar, Gelrite cannot be reheated and gelled successfully. One limitation
of Gelrite is that the concentration of divalent cations such as calcium and
magnesium ions must be within the range of 4-8 mM/liter.
 Concentrations of these two ions either less than or greater than this range
result in the media not gelling.
 Gelrite™ may also produce hyperhydric plants when used at low concentrations.
  Phytagel
 Phytagel™ is an agar substitute produced from a bacterial substrate composed
of glucuronic acid, rhamnose and glucose.
 It produces a clear, colorless, high-strength gel, which aids in detection of
microbial contamination. Phytagel provides an economical alternative to agar as
a gelling agent. It is used at a concentration of 1.5-2.5 g/L.
 To prevent clumping, Phytagel should be added to rapidly stirring culture
medium which is at room temperature.

Types of medium
i. Murashige and Skoog (MS) medium
 This medium was invented by two scientists named Toshio Murashige and Folke K.
Skoog in 1962.
 It is the most used medium in the tissue culture lab.
 Sometimes you may observe some numbers behind the MS, which indicate the
concentration of sucrose in the medium. For example, MS0 indicates sucrose absence
and MS10 indicated the presence of 10g/l sucrose in the medium.
 The formulation is a blend of nutrients like inorganic salts, vitamins, and amino acids.

STOCK A STOCK B STOCK C STOCK D

KNO3, KI, Na2.EDTA, M-inositol,
NH4NO3, MnSO4.4H2O, FeSO4.7H2O nicotinic acid,
MgSO4.7H2O, H3BO3, pyridoxin HCl,
KH2PO4, ZnSO4.7H2O, Thiamine HCl,
CaCl2.2H2O Na2MnO4.2H2O, Glycine
CuSO4.5H2O,
CaCl2.6H2O

Purpose: This medium is used to induce organogenesis, callus culture, micropropagation,

and cell suspension.

ii. Linsmaier and Skoog (LS) medium

 This medium was developed by Linsmaier and Skoog in 1965.
 It was first used to optimize the organic supplements of the tobacco culture.
 The medium has a similar component as Murashige and Skoog with a twist of Linsmaier
and Skoog vitamins.
 He found that the increased concentration of thiamine hypochlorite (0.4mg/l rather
than 0.1mg/l) compensated for the absence of vitamins except for inositol.
 Inositol is an enzymatic cofactor in glycolysis and TCA cycle and it is also involved in
primary and secondary metabolism of the plants.

Purpose: It is used for the purpose of organogenesis, callus culture, cell suspension, and
micropropagation.

iii. Gamborg (B5) medium

 This medium was developed by O. L. Gamborg in 1968.
 He used the media for the callus and cell suspension culture of Glycine max belonging
to the family of Fabaceae.
  This medium is a blend of nutrients like inorganic salts, vitamins, and carbohydrates.
 The medium has a higher concentration of nitrate and potassium and a lower
concentration of ammonia.
 Potassium nitrate is useful in inducing the soybean root callus formation and
ammonium sulfate plays an essential role in cell growth.
Purpose: It is used for the purpose of protoplast culture.

iv. Nitsch and Nitsch Medium

 The medium was developed by J. P. Nitsch in 1969 for the establishment of the in vitro
anther culture of Nicotiana, from family Solanaceae.
 It contains a high concentration of thiamine, biotin, and folic acid that supports anther
callus.
Purpose: To establish the in vitro anther culture.

v. White’s Media
 The medium was developed by P. R. White in 1963 for the establishment of the root
culture of tomato.
 This was the earliest plant tissue culture media developed for root culture.
 It has a lower salt concentration and a higher concentration of MgSO4.
 The concentration of nitrate is 19% lesser than the MS media.
Purpose: White’s medium can be used for the purpose of shoot culture and callus culture.
It is suitable for culture Musa and Daucus species.
Cell Culture
Single cells culture was first obtained from leaves of flowering plants by Haberlandt in 1902.
The in vitro cell culture technique broadly involves the following aspects:
1. Isolation of single cells.
2. Suspension cultures growth and sub-culturing
3. Synchronization of suspension cultures.
4. Measurement of growth of cultures.
5. Measurement of viability of cultured cells.

1. Isolation of Single Cells:

i. From Intact Plant Organ
 Mechanical Method:
- Leaf tissue is the most suitable material for isolation of single cells.
- Surface sterilize the leaves and cut it into small pieces (<1 c m2).
- Place it in a medium and subject it to grinding in a glass homogenizer tube.
- Filter the homogenate and then centrifuge it at a low speed to remove cellular
debris.
- Supernatant is diluted to achieve the desired cell density.
Advantages:
- It eliminates exposure of cells to harmful effects of enzymes.
- The cells are not plasmolysed which is often desirable in physiological and
biochemical studies.
 Enzymatic Method:
- The enzyme macerozyme (under suitable osmotic pressure) is perfectly suited
for isolation of plant cells.
- Macerozyme is isolated from Rhizopus species.
- The enzyme macerozyme used to isolate cells degrades the middle lamella.
Additionally, it also weakens the cell wall.
Advantages:
- In some case, it has been possible to obtain pure preparations of spongy
parenchyma and palisade parenchyma
ii. From cultured tissue
- Cells can also be isolated from cultured tissues (callus culture).
- Repeated subculture on agar medium may also improve the friability of tissue which is
highly desirable for raising a fine cell suspension in liquid medium.
2. Suspension culture growth and subculturing:
 The isolated cells are grown in suspension culture i.e in a continuously agitated liquid
media.
 Agitation of medium serves two function:
- Exerts mild pressure on cell aggregates breaking them into smaller clumps and
single cells.
- Agitation maintains uniform distribution of cells and cell clumps in the medium.
Also provides gaseous exchange between the culture and culture air.
 Cell suspension cultures are maintained by routine sub-culturing.
 For subculturing, the cells are picked up in the early stationary phase and transferred to
a fresh medium.
 The cells divide and enlarge as they are incubated in suspension culture for 21-28 days.
Types of Suspension Culture
There are two types of suspension cultures: i- Batch culture ii- Continuous culture.
i- Batch Culture:
- It is the type of cell suspension culture grown in a fixed volume of nutrient
culture medium.
- During incubation, the biomass of suspension culture increases due to cell
division and cell enlargement until the growth stops due to exhaustion of
some factors or accumulation of certain toxic metabolites.

Fig: Cell number per unit volume vs time

in a batch-grown cell suspension
culture.

- Batch cultures are characterized by a constant change in the pattern of cell

growth and metabolism. For this reason, these cultures are not suited for
studies related to cellular behaviour.
- These cultures are maintained continuously maintained by transferring
small amounts of suspension medium (inoculum) to fresh medium at
regular intervals (2-3 days).
Types of Batch Culture:
i- Slowly rotating cultures:
 Single cells and cell aggregates are grown in a specially designed
flask, the nipple flask.
 Each nipple possesses eight nipple-like projections. The capacity of
each flask is 250ml.
 As the flat disc rotates at the speed of 1-2 rpm, the cells within each
nipple of the flask are alternatively bathed in a culture medium and
exposed to air.
 ii- Shake Culture:
 In this method, single cells and cell aggregates in fixed volume of
liquid medium are placed in a conical flask.
 Conical flasks are mounted with the help of clip on a horizontal large
square plate of an orbital platform shaker.
 The square plate moves by a circular motion at 60-180 rpm.

iii- Spinning Culture:

 Large volume of cell suspension may be cultured in 10lt bottles
which are rotated in a culture spinner at 120 rpm at an angle of 45 ° .

iv- Stirred Culture:

 Used for large scale batch culture.
 The cell suspension inside the vessel is kept dispersed continuously
by bubbling sterile air through culture medium.
 Internal magnetic stirrer is the most convenient way to agitate the
culture medium safely. The magnetic stirrer revolves at 200-600
rpm.
 The culture vessel is a 5-10lt round bottom flask.

ii- Continuous Culture:

 - In continuous cultures, there is a regular addition of fresh nutrient
medium and draining out the used medium so that the culture volume is
normally constant.
- These cultures are carried out in specially designed culture vessels
(bioreactors)
- Continuous cultures are carried out under defined and controlled
conditions- cell density, nutrients, oxygen, pH, etc.
- The cells in these cultures re mostly at exponential phase of growth.

Types of Continuous Culture:

i- Open Type: The addition of fresh medium is balanced by the outflow of

old medium including harvest of cells, which indefinitely maintains a
constant submaximal growth, i.e majority of cells are in similar metabolic
state.
The open system may also be of two different types:
 Chemostats:
- Growth rate and cell density are held constant by a fixed rate of
input of growth limiting nutrient medium.
- The growth limiting is so adjusted that is reflects the increase or
decrease in growth rate of cells.
 Turbidostats:
- Fresh medium flows in response to increase in turbidity so as to
maintain culture at fixed optical density of suspension.

ii- Closed Type:

 Cells are retained and the inflow of fresh medium is balanced by
outflow of corresponding volumes.
 The cells from the out flowing medium are separated mechanically
and same medium is added back.
 In this type of culture cell biomass continues to increase as the
growth proceeds.

3. Synchronization of suspension culture:

  In normal circumstances, the cultured plant cells vary greatly in shape, size, cell cycle, etc
(asynchronous). Due to variations in the cells, they are not suitable for genetic,
biochemical and physiological studies.
 Synchronous culture may be regarded as a culture in which the cell cycles for majority of
cultured cells occurs simultaneously.
 Methods for synchronization: i- Physical Method
ii- Chemical Method

PHYSICAL METHOD
i. Selection by Volume: Synchronization may be achieved by selecting a particular size of
cell aggregates by using cell fractionation technique (extraction, homogenization and
centrifugation)
ii. Temperature shock: Low temperature shocks combined with nutrient starvation are
reported to induce synchronization of suspension cultures.

CHEMICAL METHOD
i. Starvation: Cultures starved of nitrogen, phosphorous or carbonate, result in arrest of
cell growth during G1 or G2 phase. After a period of starvation when these growth
limiting compounds are supplied to the medium, the stationary cell enters in divisional
phase synchronization.
ii. Inhibition: Inhibitors of DNA synthesis like 5-amino-uracil, 5-fluorodeoxypurine,
hydroxyurea, etc in cell culture help to accumulate the cells at G1/S checkpoint.
Removal of inhibitor is followed by synchronous division of cells.
iii. Mitotic arrest: Suspension culture in exponential growth is supplied with 0.02% (w/v)
colchicine for 4-8 hr inhibit the spindle formation. The removal of colchicine will help
to induce the synchrony in the cell suspension culture.

4. Measurement of growth of culture:

It is necessary to assess the growth of cells in the culture. The selected parameters are cell
counting, packed cell volume and increase in weight.
i- Cell counting:
 It a reasonably accurate but tedious and time-consuming process as cells in suspension
culture mostly exist as colonies in varying sizes.
 These cells have to disrupted by treatment of pectinase or chromic acid, and then
counted using a hemocytometer.
ii- Packed Cell Volume (PCV):
 PCV is expressed as ml of pellet per ml of culture.
 The suspension culture is centrifuged at 2000g g for 5 mins and the volume of pellet
(PCV) is to be recorded for determination of PCV.
 The pellet can be washed and dried overnight and weighed for determination of dry cell
weight.
iii- Cell Fresh Weight:
 The wet cells are collected on a pre-weighed nylon fabric filter.
 They are then washed to remove the medium, drained under vacuum and weighed. This
gives fresh weight of cells.
 However, for accuracy larger samples are to be used.
5. Measurement of viability of cultured cells:
The viability of cells can be measured by microscopic examination of cells directly or after
staining.

i- Phase Contrast Microscopy:

 Microscopic assessment of viability is based on cytoplasmic streaming and presence of
healthy nucleus.
 Phase contrast microscopy is thus used for better picture of these features.
ii- Evan’s Blue Staining:
 0.025% solution of Evan’s blue stains the damaged cells while the viable cells remains
unstained.
iii- Fluorescein Diacetate (FDA):
 A 0.01% FDA solution prepared in acetone is added to cell or protoplast suspension.
 As FDA is non-fluorescing and non-polar, it freely permeates inside plasma membrane.
 It is then cleaved by esterase activity in living cells that releases fluorescein.
 Fluorescein then accumulates in cytoplasm of living cells that gives green fluorescence
under UV light.

Single Cell Culture (Single Cell Clones)

 A clone is mass of cells derived from a single cell through mitosis.
 The clones are expected to be identical with regard to genotype and karyotype. However, some
changes in cell may occur after cloning.
 Single cells separated from plant tissues can be cloned under suitable conditions.

Techniques of Single Cell Culture:

i- Filter paper raft-nurse tissue technique
 Single cell is isolated from suspension culture or a friable callus.
 Few days before isolation of single cells, a sterile 8x8 m m2 filter paper is aseptically
placed on an actively growing callus of same or different species.
 The filter paper is wetted by soaking the water and nutrient from callus tissue.
 The isolated single cell is then placed aseptically on wet filter paper.
 The culture system is incubated for 16hr in white light or under continuous darkness at
25℃ .
 The nutrients diffuse from the culture medium through callus tissue and filter paper to
single cell.
The callus tissue on which the single cell grows is called nurse tissue.
 The single cell divides and redivides to form a small colony.
 When the cell colony reaches suitable size, it is transferred to a fresh medium where it
gives rise to callus tissue.

ii- Micro-chamber technique

 The nurse tissue was replaced by a conditioned medium and single cell were grown in
microchamber.
 A drop of medium carrying single cell is isolated from suspension culture aseptically and
placed on sterile microscope slide.
 A drop of paraffin is placed on either side of cultural drop and a cover slip is placed on
each drop.
 A third cover-slip is placed in culture drop bridging the two cover slips and forming a
microchamber thar enclose single cell aseptically within the paraffin oil.
Paraffin prevents loss of water from cell culture but allows gaseous exchange.
 The whole micro-chamber slide is placed in a petri-plate and incubated under white
light for 16 hrs at 25℃ .
 When the cell colony becomes sufficiently large, it is transferred to a fresh solid or semi-
solid medium.
iii- Micro-drop method
 In this method single cells are cultured in a special Cuprak dishes which have two
chambers- a small outer chamber and a large inner chambers.
 The large chamber carries numerous numbered wells.
 Each well of inner chamber is filled with a micro-drop of liquid medium containing
isolated single cell.
 The outer chamber is filled with sterile distilled water to maintain the humidity inside
the dish.
 After covering the dish with lid, the dish is sealed with paraffin.
 The dish incubated under 16 hrs white cool light at 25 ℃ .
 The cell colony derived from the single cell is transferred on to a fresh solid or semisolid
medium in a culture tube for further growth

iv- Bergman’s plating technique

 The cell suspension culture is first filtered aseptically to obtain free cells and small cell
aggregates.
 The suspension of free cells is then mixed with equal volume oof agar medium cooled at
35℃ .
 The mixture is then rapidly dispersed inti petriplate such that the cells are evenly
dispersed in a thin layer (apx. 1mm thick).
 The dishes are then sealed and incubated for cell division, which will give rise to
colonies that can be used to initiate cell clones.
Callus Culture
 Callus is formed by the proliferation of the parent tissue.
 The cells of a callus are parenchymatous, amorphous and unorganised.
 Generally, callus is formed as a result of injury at the cut ends of a stem or a root.
 When tissues on culture produce unorganised mass of callus with no regular form then it is
called callus culture.
 On a culture medium, the explant grows forming undifferentiated tissue or callus.
 By repeated sub-culturing, this callus can be maintained for a prolonged period.
The cell is sub-cultured because:
i- The nutrients may be exhausted
ii- Agar may be desiccated
iii- Cell metabolites that accumulate may cause toxicity.
Callus culture may be initiated from explants of any multicellular plant. The explant may be taken
from stem, root, leaf, flower, fruit or seed.
Callus formation has been recorded from storage parenchyma, pericyclic cells of roots, cambial cells
of vascular bundles, pro-vascular cells, secondary phloem, pith cells, mesophyll cells and cotyledons.

Nutrient Medium Requirement for Callus Culture:

 Some standard media like MS medium can be used for callus culture.
 For initiation and maintenance of callus culture, kinetin is widely used.
 For callus initiation usually an exogenous supply of hormone is required. But explants having
cambial cells do not require exogenous hormone supply.

Depending upon the hormone requirement, there are 5 types of callus

cultures:
i- Auxin Requiring Culture
ii- Cytokinin Requiring Culture
iii- Cultures requiring both auxin and cytokinin
iv- Gibberellin requiring culture (in dicots). Gibberellin inhibits growth of callus tissue in
monocots.
v- Cultures requiring natural extracts like yeast extract, coconut milk, casein
hydrolysate, tomato juice, etc.

Method of Callus Culture:

 Explant from suitable material (such as carrot root, potato tuber, stem of tobacco etc) are
taken.
 The explant is first surface sterilized with 1.6% sodium hypochlorite solution or 0.1% mercuric
chloride solution or 1% aqueous solution of bromine.
 The inner uncontaminated tissue is then excised.
 The tissue is then placed on the culture medium and the culture is incubated at 25 ℃ for 6-8
weeks.
Development of Callus Culture:
i- Induction Stage:
 Metabolism is stimulated and the cells prepare to divide.
 Cell size remains unchanged.
ii- Cell Division Stage
 Cells divide actively and the cell size decreases.
 Cell division is mainly periclinal and occurs towards the periphery giving rise to wound
cambial cells.
iii- Differentiation
 Cells differentiate by expansion and maturation.
 Rapidly growing calluses are more or less alike but as growth rate decreases the callus shows
their characteristic structure and form.
 The cells of callus may differentiate to form root or shoot primordia.

Nature of callus tissue:

 Some cultures are hard and anatomically consists of compactly arranged small cells without
intracellular spaces. Such cells may be composed of lignified cells.
 Other callus culture may be friable consisting of large loosely arranged cells with intercellular
spaces. Such cultures are fragile and breaks easily. They are suitable for suspension culture.

Significance of Callus Culture:

i- Organogenesis, morphogenesis and somatic embryogenesis
ii- Callus is a good source of genetic variability.
iii- Initiation of cell suspension culture
iv- For production of secondary metabolites
v- Several biochemical assays can be performed using callus culture.