CYTODIFFERENTIATION•In plant tissue culture, during growth and maturation of the callus tissue or

free cells in suspension culture, few dedifferentiated cells un-dergo cytoquiescence and
cytosenescence and these twin phenomena are mainly associated with differentiation of vascular
tissue.Cytodifferentiation occurs ei-ther spontaneously or under the stimulus of spe-cific nutritional
or hormonal factors

. intracellular degradative changes are associated with Cytodifferentiation. Auto-destruction of

cellular or-ganelles due to autolysis by acid phosphatase

How does environment affect the cell differentiation in plants

1.LIGHT: Short-day” plants will flower only when exposed to extended periods of darkness. • “Long-
day” plants will flower only when exposed to short periods of darkness.

2.TEMPERATURE: High temperature(35 degree C) stimulates the cellular differentiation. Low

temperature(10-15 degree C) inhibit the cyto differentiation.

3.SUGAR LEVEL: It serves as an energy source in cyto differentiation • Dual purpose: -production &
deposition of cellulose - deposition of lignin in secondary wall • Essential for trachied development
production • The ratio of xylem to phloem is also varied with sucrose levels in media. Concentration
above 1.5-2.5% of only xylem and above 4% stimulates both xylem and phloem.

4.HORMONE: Auxin :cytokinine ratio in the media affect the differentiation of cells in cultured plant.
Ethylene-Stimulation or suppression of cytodiff. depends upon the degree of ethylene
concentration.

Totipotency: The ability of an individual cell to develop into a whole plant is referred as cellular
totipotency

Importance of Totipotency in Plant Science

1. reconstruction of plants from the totipotent cell.

2. vegetative propagation of many economical, medicinal and agriculturally important species
3. genetic modification of plants, production of homozygous diploid plants through haploid cell
culture, somatic hybridization, mutation.
4. germplasm preservation of totipotent cells of endangered plant species
5. production of secondary metabolites
6. Helps in production of those plants whose seeds very minute and difficult to germinate .

APPLICATIONS OF CELLULAR TOTIPOTENCY

1. Production of large number of haploids from microspores
2. Plant breeding- Give rise to triploids from endosperm cells
3. Large scale production of plants in bioreactors and their conversion into synthetic seeds
4. Nucellar embryogeny- to raise virus-free clones of plants.
5. Improvement of crop plants through manipulations at cellular level

Morphogenic potential-It is the ability of a single cell to divide and development of all those
differentiated cells into an organism
LOSS OF MORPHOGENIC POTENTIAL IN LONG-TERM CULTURES
1. due to repeated subculturing of the single culture which increases non potent cells
2. complex multicellular explants are used to initiate culture
3. only a small number of cells are totipotent i.e. capable of yielding regenerative culture,
remaining cells are nontotipotent in a callus culture
4. In long term cultures, the number of non-totipotent cells get increased due to
cytological instability of the culture cells

Morphogenic potential of some cultures can also be restored by-

1.Altering harmonal balance within cells

2. By giving cold treatments
3. By altering media composition
4. 1-4% of activated charcoal is added to the proliferation medium which supports
the development of embryos and restoring media

Callus and suspension culture

Callus: It is defined as undifferentiated or unorganized mass of plant cell.it can lead
to the formation of root shoot and somatic embryo from which whole plant can be
synthesized.
Types of callus
1.compact callus-cells are densely aggregated
2.friable callus-cells are loosely aggregated.callus become soft and breaks apart
easily.It provides inoculum for suspension culture.

De-differentiation: The phenomenon of mature cells , reverting to meristmatic state

to produce callus is called de-differentiation.
Re-differentiation- The ability of callus cells to differentiate into plant organ or a
whole plant is regarded as re-differentiation.

Stages of callus culture

1.INDUCTION-cells in explant dedifferentiate and begin to divide
2.PROLIFERATIVE-rapid cell division
3.MORPHOGENESIS-differentiation and formation of organised structures through
organogenesis or somatic embryogenesis

FACTORS AFFECTING CALLUS CULTURE FORMATION

1. Explant  All plants show different response to the culturing. Embryogenic,
reproductive and meristematic tissue are preferred as they can be readily
cultured.
2. Composition of medium- MS media  Macronutrients- N,P,K,Mg,Ca
Micronutrients- Mn,Mo, Co,Fe,Cu,Zn  Vitamins- Thiamine essential for growth
3. Physical factors- Temperature A temperature of 18 to 25 °C is normally used for
induction of callus culture.  Light Availability Exposure to higher light
intensities destroys the explants. Blue light (shoot formation) and red light(root
formation). Photochemical period : 16-24 hours.  pH The medium ph is
adjusted between 5 and 6 before autoclaving and extremes of pH are avoided.
4. Growth Regulators: Phytohormones  Auxins :for inducing callus culture
formation and root formation NAA ,IAA and IBA  Cytokines: basically Kinetin
 for shoot formation.  Gibberellins: for stimulating shoot apices formation. 
Absicsic Acid: induces embryogenesis.

Suspension culture: A suspension culture consist of single cells, small cell groups and
larger cell aggregates dispersed in a liquid medium and actively growing under
agitation and aeration.it is initiated from friable callus

TYPES OF SUSPENSION CULTURES

1.BATCH CULTURE- Cell suspensions are grown in 100-250 ml flasks each containing
20-75 ml of fixed culture medium. Subculturing- Increase in biomass due to cell
division and cell enlargement lead to nutrient exhaustion and acumulation of toxic
substances. Cell dispersion and cell aggregates taken and subcultured.
2.CONTINOUS CULTURE-culture are grown under steady state by continuously
adding fresh media and draining out used media
a.open continuous- - Inflow of the medium is accompanied by outflow of medium
containing cells which are added back to culture.
b.closed continuous- cell biomass continues to increase as the growth proceeds

types of open continuous culture

1.chemostat cultures, a steady state of cell growth is maintained by a constant
inflow of the fresh medium in which the concentration of a chosen nutrient
(nitrogen, phosphorus or glucose) has been adjusted so as to be growth limiting.
2.turbidostat cultures, the input of fresh medium is intermittent, controlled by an
increase in the turbidity of the culture from cell growth

PROTOPLAST
DEFINITION- It is a plant cell that has its cell wall completely removed using either
mechanical or enzymatic means.

ISOLATION OF PROTOPLAST-
1.Mechanical method- plant tissue> Cells Plasmolysis> Microscope Observation of
cells> Cutting cell wall with knife> Release of protoplasts> Collection of protoplast
Drawbacks
Used for vacuolated cells like onion bulb scale, radish and beet root tissues. • Low
yield. • Laborious and tedious process. • Low viability

2.Enzymatic method-
one step method- Leaf sterilization & Removal of epidermis> Plasmolysed
cells.>addition of Pectinase +cellulase> Protoplast released> Isolated Protoplast

1.Incubation of leaf segments over night, 2. Treated with enzymes to liberate

protoplasts, Mixture is filtered, 3. Centrifugation, 4. Protoplast forms pellet, 5.
These are washed with sorbitol 3X, Centrifugation, 6. Cleaned protoplast float

two step method- Leaf sterilization & Removal of epidermis.>plasmolysed cells>

Pectinase> Release of isolated cells> cellulase> Protoplast released.
Purification of protoplast
1. Protoplasts are separated after digestion period from enzymes and cellular
debris, transfer to the suitable medium.
2. A concentrated solution of mannitol, sorbitol and sucrose
3. centrifugation at low speed followed by filtration through nylon mesh
4. Again followed by centrifugation or by density gradient centrifugation step.
5. This pallet is dissolved

Protoplast viability
1. Fluorescein diacetate: It accumulates only inside the plasma lemma of viable
protoplasts, can be detected with fluorescence/UV microscopy.
2. Evans blue: Intact viable protoplasts, exclude the Evans blue stain.
Impermeability of the cell to Evans blue indicates a living cell.
3. Cyclosis: or protoplasmic streaming can be a measure of viability

Culture of protoplast

Optimal culture conditions:

1. Optimal density to the culture. at low density protoplasts will lose soluble cell
components.
2. Optimal auxin to cytokynin ratio, glucose and sucrose.
3. Maintain the osmoprotectant in the medium until the cell wall has reformed.
4. 20-28 °C, pH5.5-5.9, 0.25% casein hydrolysate, BAP and NAA
5.MS &B5 media witj suitable modification
Culturing of protoplast
 Protoplasts are cultured either in semisolid agar or liquid medium.  Sometimes,
protoplasts are first allowed to develop cell wall in liquid medium, and then
transferred to agar medium.
Liquid culture medium
 1. It is easy to dilute and transfer.  2. Density of the cells can be manipulated as
desired.  3. For some plant species, the cells cannot divide in agar medium,
therefore liquid medium is the only choice.  4. Osmotic pressure of liquid medium
can be altered as desired.
Osmoticum
refers to the reagents/ chemicals that are added to increase the osmotic pressure of
a liquid.  The isolation and culture of protoplasts require osmotic protection until
they develop a strong cell wall. addition of an osmoticum is essential for both
isolation and culture of protoplast to prevent their rupture.
NON IONIC OSMOTICA-The non-ionic substances most commonly used are soluble
carbohydrates such as mannitol, sorbitol, glucose, fructose, galactose and sucrose.
Mannitol, being metabolically inert, is most frequently used
IONIC OSMOTICA- Potassium chloride, calcium chloride and magnesium phosphate
are the ionic substances in use to maintain osmotic pressure.

Steps for culturing

 Development of a cell wall around the membrane of the protoplast.  This is
followed by the cell divisions that give rise to a small colony.  With suitable
manipulations of nutritional and physiological conditions, the cell colonies may be
grown continuously as cultures or regenerated to whole plants.

Methods for culturing

1.HANGING DROP CULTURE-cultivation of protoplast in droplet on feeder layer>
.Inverted Suspending on the lid of a Petri dish, with sterile water in the base to
provide humidity>cell aggregate formation

2.MICRO DROP CULTURE-Specially designed dishes namely cuprak dishes with outer
and inner chambers are used for micro drop culture.  The inner chamber carries
several wells wherein the individual protoplasts in droplets of nutrient medium can
be added.  The outer chamber is filled with water to maintain humidity.

3.FEEDER LAYER-For culture of protoplasts at low density feeder layer technique is

preferred.  This method is also important for selection of specific mutant or hybrid
cells on plates. FEEDER LAYER CELLS usually consist of adherent growth-arrested, but
viable and bioactive cells which acts as substratum to provide nutition to growing
cells

4.ON THE WELLS OF MICROTITRE PLATE

5.ON SEMI SOLID AGAR MEDIUM

SOMATIC HYBRIDISATION
DEFINITION-Somatic hybridization broadly involves in vitro fusion of isolated
protoplasts to form a hybrid cell and its subsequent development to form a hybrid
plant.
 no barriers of incompatibility (at interspecific, inter-generic or even at
interkingdom levels) for the protoplast fusion.
 Protoplast fusion that involves mixing of protoplasts of two different
genomes can be achieved by spontaneous, mechanical, or induced fusion
methods
SPONTANEOUS FUSION-Protoplasts fuse spontaneously during isolation
process mainly due to physical contact.Eg; egg fertilization.it results in
homokaryons
MECHANICAL FUSION-The protoplasts can be pushed together mechanically
to fuse. may damage protoplasts by causing injuries
INDUCED FUSION-Freshly isolated protoplasts can be fused by induction. 
There are several fusion-inducing agents which are collectively referred to as
fusogens. NaN03 , high pH/Ca2+ , polyethylene glycol, polyvinyl alcohol,
lysozyme, concavalin A, electro fusion.

Advantages of somatic hybridisation

1. Production of novel interspecific and intergeneric hybrids. Pomato
(Hybrid of potato and tomato).
2. Production of fertile diploids and polyploidy from sexually sterile
haploids and triploids.
3. Transfer gene for disease resistance, a biotic stress resistance, herbicide
resistance and many other quality characters
4. Formation of cytoplasm containing organelles of two different parents

Limitation of somatic hybridisation

1. Poor regeneration of hybrid plants.
2. Non-viability of fused products
3. Not successful in all plants.
4. Production of unfavorable hybrids.
5. Lack of an efficient method for selection of hybrids

EMBRYOGENESIS
DEFINITION:Refers to development of plant embryo through sexual or
asexual means of reproduction.embroyogenesis that occurs naturally
through sexual reproduction is called zygotic embryo & those that
occurs in plant culture lab through induction of plant cell are called
somatic embryo

Somatic embryogenesis: Somatic embryogenesis is an artificial

process in which a plant or embryo is derived from single somatic cell or
group of somatic cells. • Somatic embryos are formed from plant cells
that are not normally involved in the development of embryos i.e.
ordinary plant tissue. • No endosperm or seed coat is formed around a
somatic embryo

DIRECT EMBRYOGENESIS • The embryos initiate directly from explants in

the absence of callus formation. • Embryos are formed due to PEDCs
cells.
INDIRECT EMBRYOGENESIS Callus from explants take place from which
embryos are developed . Embryos are formed due to IEDCs cells

APPLICATIONS OF SOMATIC EMBRYOGENESIS:-

• Clonal propagation of genetically uniform plant material.
• Elimination of viruses.
• Provision of source tissue for genetic transformation.
• Generation of whole plants from single cells called protoplasts.
• Development of synthetic seed technology
Limitations
• Difficult method.
• Loss of regenerative ability.
• High percentage of albino shoots during regeneration.
• Not possible with all plant species

ARTIFICIAL SEEDS
DEFINITION-It consists of a bead of gel containing somatic embryo along
with nutrients, growth regulators, pesticides, antibiotics, all the
necessary requirements needed by the embryo to develop into a new
plantlet. It is produce by mainly two systems
Desiccated system: This involves hardening of the somatic embryo in
the maturation phase by adding polymer or treating them with abscisic
acid. This is followed by drying or desiccation to produce a desiccated
system.
Hydrated system: This involves coating the embryos in a gel with
materials similar to sodium alginate. The corresponding process involves
allowing sodium alginate to fall into a solution of calcium chloride. The
drop before falling is inserted with embryos thus it falls into the solution
forming a gel coat around the embryo.

ANTHER CULTURE
 Haploid plants are characterized by possessing only a single set of
chromosomes (gametophytic number of chromosomes i.e. n) in the
sporophyte.
 1.Monoploids (monohapioids): • These are the haploids that possess
half the number of chromosomes from a diploid species e.g. maize,
barley.
 2. Polyhaploids: • The haploids possessing half the number of
chromosomes from a polyploid species are regarded as polyhaploids e.g.
wheat, potato.
Invivo techniques for haploid production
1.Androgenesis: Development of an egg cell containing male nucleus to
a haploid is referred to as androgenesis. the egg nucleus has to be
inactivated or eliminated before fertilization
2. Gynogenesis: An unfertilized egg can be manipulated (by delayed
pollination) to develop into a haploid plant
3. Distant hybridization: Hybrids can be produced by elimination of one
of the parental genomes
4. Irradiation effects: Ultra violet rays or X-rays may be used to induce
chromosomal breakage and their subsequent elimination to produce
haploids.
5. Chemical treatment: Certain chemicals (e.g., chloramphenicol,
colchicine, nitrous oxide, maleic hydrazide) can induce chromosomal
elimination in somatic cells

Invitro techniques for haploid production

1.Androgenesis: Haploid production occurs through anther or pollen
culture, and they are referred to as androgenic haploid
2. Gynogenesis: • Ovary or ovule culture that results in the production
of haploids, known as gynogenic haploids.

ANDROGENESIS-In androgenesis, the male gametophyte (microspore or

immature pollen) produces haploid plant.  The basic principle is to stop
the development of pollen cell into a gamete (sex cell) and force it to
develop into a haploid plant.  There are two approaches in
androgenesis— • anther culture and • pollen (microspore) culture.

Anther culture
1. The selected flower buds of young plants are surface sterilized and
anthers removed along with their filaments.
2. The anthers are excised under aseptic conditions, and crushed in 1%
acetocarmine to test the stage of pollen development.
3. If they are at the correct stage, each anther is gently separated
(from the filament) and the intact anthers are inoculated on a
nutrient medium
4. The anther cultures are maintained in alternating periods of light
(12-18 hr.) and darkness (6-12 hrs.) at 28°C
5. As the anthers proliferate, they produce callus which later forms an
embryo and then a haploid plant

Microscope culture

1. The pollen can be extracted by pressing and squeezing the anthers

with a glass rod against the sides of a beaker
2. The pollen suspension is filtered to remove anther tissue debris
3. Viable and large pollen (smaller pollen do not regenerate) are
concentrated by filtration, washed and collected
4. pollen are cultured on a solid or liquid medium
5. The callus/embryo formed is transferred to a suitable medium to
finally produce a haploid plant and then a diploid plant (on
colchicine treatment)
 Endomitosis is the phenomenon of doubling the number of
chromosomes without division of the nucleus.

Factors affecting androgenesis

1. Genotype of donar plants

2. Stage of microspore or pollen-microspores ranging from tetrad
to bi-nucleate stages are more responsive.
3. Physiological status of a donar plant-The plants grown under
best natural environmental conditions (light, temperature,
nutrition, CO2 etc.) with good anthers and healthy microspores
are most suitable as donor plants. • Flowers obtained from
young plants, at the beginning of the flowering season are highly
responsive
4. Pretreatment of anthers