Type of plant tissue cultures:

→ Tissue culture is commonly used to describe all kinds of in vitro plant cultures including cultures of
unorganized aggregates of cells. The following kinds of cultures are most generally recognized in practice:
1-Callus culture:
- Callus is a largely unorganized, amorphous mass of loosely arranged parenchyma cells, it is initiated and
maintained on nutrient media in vitro. Unorganized means not have organ.
- It is produced by cultivating explant on a static culture (solid or semisolid media) in darkness to suppress
organization.
- A callus may appear yellowish or white, or green or pigmented. Light colored friable tissue is generally most
desirable form for optimum growth.
- The formation of callus with an explant marks the beginning of successful plant cell culture.
-The cells although undifferentiated contain the genetic information present in the normal plant.
Callus Induction:
1. Explants are sterilized with 2% Na hypochlorite solution for 10 M. And /or 70% ethanol solution for 2-3M
2. The explants should be rinsed with water.
3. The stem or any part of plants thus sterilized is cut to approximately 1 Cm in length using a sterilized scalpel
4. Each piece is transferred with tweezers to a solid medium in a flask or a petti-dish.
5. The plant material is incubated aseptically at around 25 C on the solid medium for several weeks or more and
a callus is produced
Within two or three weeks explants show masses of undifferentiated cells known as callus.
Sign of aging in callus:
A. Slow of growth b. Necrosis and browning
Aging is usually due to:
. 1. Exhaustion of nutrients 2. Inhibition of nutrient diffusion, 3. Accumulation of metabolites
Temperature, pH. Light and Oxygen:
A temperature of 17-25 ° C is normally used for induction of callus tissues and growth of cultured cells.
-The medium pH is usually adjusted to between 5 and 6 before autoclaving and extremes of pH are avoided.
- Callus grows in darkness to prevent organogenesis.
If the conditions are right, a small "forest" of plants will develop in the tissue culture container.

2-Cell suspension culture:

-It means isolation and deposition of single cell or small groups of cells which are normally derived from a
callus tissue or from plant in liquid medium provided with continuous supply of air and continuous agitation.
-The growth rate of the suspension cultured cells is generally higher than that of the solid culture.
-The cell suspension is more desirable particularly in production of useful metabolites in a large-scale.
Preparation of Cell-Suspension Culture:
1. Cultures are initiated by simply placing fleshy cut section from surface-sterilized plant organs on medium
containing suitable hormones (generally auxins and cytokinins) are used.
2. On such a medium the explant exhibits callusing which usually starts at the cut ends and gradually extends
over the entire surface of the tissue.
3. The callus is separated from the parent explant and transferred to a fresh medium of the same composition to
build up a reasonable amount of tissue.
4. Repeated subculture on the agar medium may also improve the friability of the tissue which is highly
desirable for raising a fine cell suspension on liquid medium
5. After two months a very friable callus was formed which on transfer to the liquid medium give fine
suspension (suspension culture).
Importance of agitation of suspension cultures:
A. It exerts mild pressure on cell aggregates breaking them into smaller clumps and single cells.

B. It maintain distribution of cells in the medium.

C. It provides gaseous exchange between the culture medium and culture air.

Advantages of suspension culture:

1- Production of biochemical in large scale is more rapid

2- Control of nutritional and environmental condition is easier

3- It permits the selection of specific cells for secondary metabolites 4-it is more convenient for
biotransformation research work

3-Protoplast cultures 4-Root culture:

-Plant protoplast refers to plant cells in which cell wall has been removed Root culture can be established
via enzyme digestion or mechanical process. in vitro from explants of the
-Isolation process of protoplast occurs by dipping plant tissue into root tip of either primary or
hypertonic solution in which the plasma membrane shrinks away from cell lateral roots and can be
wall and then cell wall can be removed by enzymatic digestion (pectinase cultured on fairly simple
and cellulose) or by mechanical methods. media.
- Protoplast cultures have an important role in plant biotechnology.
-Protoplasts can be plated out on to solid medium and callus produced. - The growth of root in vitro is
Whole plants can be regenerated by organogenesis or somatic unlimited.
embryogenesis from this callus.
-Protoplasts are ideal targets for transformation by a variety of means. It is not used in plant
Isolation of protoplast: transformation.
1- Mechanically: Use of fine knife in isolation of protoplast from higher
plants. (yield is low, not useful in isolation of protoplast from meristematic
and less vacuolated cells) due to damaged cells.
2- Enzymatic isolation: using a concentrated solution of cellulose enzyme
(prepared from some fungus species) in a simple salt solution with a high
osmotic potential to maintain the cells (Isolation of protoplast from every
plant tissue is possible).
 5-Shoot tip and -The tip of the shoot which contains the shoot apical meristem can be cultured in
meristem culture: vitro producing clumps of shoots from either auxiliary or adventitious buds

-This used for cereals regeneration as they are less genotype-dependent (seedlings
can be used as donor material),
6-Embryo culture: -Embryos can be used as explants to generate callus cultures or somatic embryos.
- Embryo culture is a type of plant tissue culture that is used to grow embryos from
seeds and ovules in a nutrient medium.
-In embryo culture, the plant develops directly from the embryo or indirectly
through the formation of callus and then subsequent formation of shoots and roots.
-The technique has been developed to break seed dormancy, test the vitality of
seeds, production of rare species and haploid plants.
7-Haploid -The tissue culture techniques enable to produce homozygous plants in relatively short time
period through the protoplast, anther and microspore cultures (pollen contains the male
production: gametophyte, which is termed the microspore) instead of conventional breeding ------
Haploids are sterile plants having single set of chromosomes which are converted into
homozygous diploids by spontaneous or induced chromosome doubling
-The doubling of chromosomes restores the fertility of plants resulting in production
of double haploids with potential to become pure breeding new cultivars.
-The term androgenesis refers to the production of haploid plants from young pollen
cells without undergoing fertilization.
Elicitation: An Elicitor is a substance which, when introduced in small concentrations to a
living cell system, initiates or improves the biosynthesis of specific compounds
Classification of Elicitors
A) Biotic clicitors From biological origin B) Abiotic clicitors are the substances of non-
biological origin.

They include: 1. Chemicals such as inorganic salts, heavy metals,

1. Enzymes and polysaccharides derived from some chemicals that disturb membrane integrity.
microorganisms 2. Physical factors like mechanical wounding, UV
2. Phytochemicals produced by plants in response irradiation, high salinity, high or low osmolarity,
to physical damage, fungs or bacteria attack. extreme temperature (freezing, thawing), high pressure.

Plant tissue culture techniques:

1. Micropropagation or clonal propagation:
Micropropagation is the technique of multiplication and vegetative growth of plants tissues or cells in aseptic
and favorable in vitro conditions.
-In conventional cultivation, many plants have long periods of growth and multiplication or do not germinate
under certain climatic conditions.
- Micropropagation insures rapid multiplication and good regular supply of these plants within a minimum
space and time.
-Micropropagation produces clones with desired characteristics. higher rate of multiplication, controlling or
alteration of environment to meet specific needs and availability of plants all year round.
 Other advantages of micropropagation are rapid and mass propagation of plants, production of genetically
engineered plant, secondary metabolites, pathogen-free material, conservation of threatened plant species
and preservation of genetic material by cryopreservation.

Stages of micropropagation
Micropropagation involves four main stages:
Stage 1: Establishment of in vitro culture:
This stage involves the introduction of selected explants into culture, avoiding contamination and providing an
environment that promotes shoot production.
Stage is affected by various factors such as choice of explant, elimination of contamination and culture
condition including ingredients, light and temperature.
Stage II: Shoot multiplication:
The main goal of this stage is to increase the number of propagules by repeated subcultures until the required
number of shoots is attained.
Stage III: Root formation stage:
Aims to produce rooting of shoots and conditioning of the plants to increase their ability onsurvival and
acclimatization during transplanting.
Stage IV: Acclimatization of tissue-cultured plantlets:
Stage IV is called also as weaning or hardening stage. Acclimatization stage involve transplantation of plants in
vitro to soil under greenhouse.
During hardening, plantlets transfer gradually from high to low humidity and from low light intensity to high
intensity conditions.
Plants are then potted in an appropriate substrate under greenhouse and gradually hardened.
[Link] 3. Somatic embryogenesis:
-Organogenesis is one major path It is an in vitro method of plant regeneration widely used as an important
for the in vitro regeneration of biotechnological tool for sustained colonal propagation.
plants. It is a scientific term -Somatic embryogenesis is a nonsexual process of plant regeneration by
which means the production of which embryos are formed from somatic cells or tissues and developed
organs, either shoot or root. into a whole plant.
Somatic embryogenesis can generate plants either directly from the
-It occurs via two ways, direct explants or indirectly by the formation of callus.
and indirect.
-Direct somatic embryogenesis is generally rare in comparison with
-Direct organogenesis is the indirect son embryogenesis.
formation of direct buds or shoots In indirect somatic embryogenesis, callus is first produced from the
from a tissue with no intermediate explarr. Embryos can then be produced from the callus tissue or from a
callus stage: cell suspension produced from that callus.
➤ Callus induction, embryo formation and proliferation, embryo
-However indirect organogenesis maturation and embryo germination are the four stages of somatic
has the callus as an intermediate embryogenesis.
stage. ➤ During these stages, the embryos may pass through globular, heart,
torpedo and cotyledonary forms.
 -Organogenesis is affected by the ➤ The applications of somatic embryogenesis are mass multiplication,
balance of auxin and cytokinin production of artificial seeds and embryogenic protoplasts.
and the response of the tissue to - Somatic embryos may develop from single cells or from a small group
phytohormones during culture. of cells.
-Mass multiplication and Repeated cell divisions lead to the production of a group of cells that
conservation of germplasm at develop into an organised structure known as Globular stage embryo.
either normal or sub-zero Farther development results in heart- and torpedo stage embryos, from
temperatures are the applications which plants can be regenerated.
of organogenesis in plant -Zygotic embryos undergo a fundamentally similar development through
improvement. the globular which is formed after the 16- ell stage), heart and torpedo
stages.
-polarity is established early in embryo development. Signs of tissue
differentiation become apparent at the lobular stage and apical meristems
are apparent in heart-stage embryos.

Uses and applications of tissue culture:

 Plant tissue culture have been used to study basic problems related to growth and differentiation under high
reproducible conditions.
 It has also acquired many practical applications in agriculture and horticulture.

1- Plant micro-propagation: II- Production of secondary metabolites:

It is the most common application involves the A- Production of native plant constituents:
micropropagation of the medicinal plants. 1. The rate of cell growth and biosynthesis in culture
horticulture crops, ornamental and fruits via initiated from a very small amount of plant material is
plant regeneration. quite high and the final product may be produced in a
Aims of use of (tissue culture)in short time.
micropropagation:
2. Plant cell cultures are maintained under controlled
A. Elimination of disease and production of
condition both environmental and nutritional which
diseases free plants.
ensure the continuous yields of metabolites.
B. Rapid and continuous production of
3. Cell suspension culture offers a more effective
genetically modified plants.
mechanism of incorporating precursors into cells than is
C. Introduction of new varieties of plants.
found in whole plant
b- Biotransformation of extremely supplied chemical
compounds (will be studied later

Problems in plant tissue culture:

-Browning, contamination and vitrification are the common problems that take place during procedures of plant
tissue culture.
-Sometimes, plant tissue culture called "black art" because there are many differences that make it difficult to
solve problems when they arise. As a result, many laboratories define the cause of the problems by diagnosing
the symptoms and accurately tracking each step in the culture process.
1. Browning of tissue  -Browning or blackening occurs as a defensive mechanism to wounding of
and media: tissues during explants excision from mother plants or during subcultures.
 -Tissue injuries stimulate the release of phenolic compounds which are
oxidized by enzymes into quinines. Quinones lead to production of dark
pigments that cause black or brown colour of tissue and media.
Accumulation of quinones also may cause eventual death of the tissue in
some plants.
 -The browning can be reduced by Washing of explants for 2-3 hours in
sterile water, incubation of excised explants in darkness and frequent
subcultures in liquid medium at 2-3 days interval or in a new culture media
on regular intervals. Addition of activated charcoal (AC) or
polyvinylpyrrolidone (PVP) is effective in reducing blackening of media by
adsorbing toxic compounds released by plant tissues.
 However, growth regulators, minerals and different organic compounds in
media can be adsorbed to activated charcoal. Antioxidants such as ascorbic
acid, citric acid, L-systeine mercaptoethanol and glutathione can be used to
prevent browning
2. Contamination:  -Microbial contamination is a permanent problem that threatens development of all
in vitro techniques. Bacteria and fungi are the common surface contaminants in
plant tissue culture Therefore, accurate sterilization of tools and media which are
used in culturing is a must before culture.
 -Different sterilization techniques are applied in the process of in vitro culturing of
plants. Steam sterilization (autoclaving at 121 C, 15 psi for 20-40 min) is used for
nutrient media, culture vessels, glassware and plastics" sterilization. Dry heat (160-
180°C for 3h) can sterilize instruments (scalpel, forceps, needles etc.), glassware,
pipettes and other plastics Flame sterilization for sterilization of mouth of culture
vessel and instruments (scalpel, forceps, needles etc.). Filter sterilization can
sterilize thermolabile substances such as growth factors, amino acids, vitamins and
enzymes. Alcohol sterilization (70% ethanol) is applied for sterilization of
worker's hands, working surfaces and laminar flow cabinet.
 -Sometimes, surface sterilization is not enough for effective control of bacteria, as
a result antibiotics must added to media. The type of sterilizing agent used, its
concentration and the time of sterilization are dependent on the type of tissue; for
example, seeds with tough seed coat will require a longer sterilization time than
softer tissues, such as leaves or stems.
3. Vitrification:  Called hyperhydricity, indicates water soaking, hyperhydration or glass formation.
 Vitrification is a serious problem, particularly in micropropagation, due to poor
survival of plants when they are transferred from in vitro to ex vitro. Vitrification
is a physiological disorder which leads to the appearance of transparent watery
structures in plant tissue cultures.
 Hyperhydricity makes tissues in vitro to exhibit higher water content, lower
chlorophyll content, lack of cell wall lignification and leaves with large
intercellular spaces in the spongy mesophyll and palisade cells.
High concentration of cytokinin, high water retention capacity when the container is
too tightly closed and a low conc. of gelling agent are the reasons for this phenomenon.
 Hyperhydricity can be controlled through decreasing water content by increasing
agar concentration and effective ventilation or by bottom cooling of the container
vessels. Reducing the ammonium ion concentration in culture media can also help
to prevent this problem.