Plant biotechnology

Plant biotechnology is a set of techniques used to adapt plants for

specific needs or opportunities. Situations that combine multiple
needs and opportunities are common.
Micropropagation
Micropropagation is the artificial process of producing plants vegetatively
through tissue culture or cell culture techniques. In this artificial process of
propagation, plants are produced invitro by asexual means of reproduction or by
vegetative propagation.
Micropropagation is a method of plant propagation using extremely small pieces
of plant tissue taken from a carefully chosen and prepared mother plant, and
growing these under laboratory conditions to produce new plants. It is widely
used in commercial horticulture.
 Plant tissue culture:
Plant tissue culture is defined as culturing plant seeds, organs,
explants, tissues, cells, or protoplasts on a chemically defined
synthetic nutrient media under sterile and controlled
conditions of light,temperature, and humidity.

Plant Tissue culture is the in vitro aseptic culture of cells, tissues,

organs, whole plant under controlled nutritional and
Environmental,conditions often to produce the clones of plants.
Principle of Plant Tissue Culture
• Plant tissue culture relies on the fact that many plant cells have the
ability to regenerate a whole plant (totipotency).
• Single cells, plant cells without cell walls (protoplasts), pieces of
leaves, stems or roots can often be used to generate a new plant on
culture media given the required nutrients and plant hormones.
• The controlled conditions provide the culture an environment
conducive for growth and multiplication.
• These conditions include the proper supply of nutrients, pH
medium, adequate temperature, and proper gaseous and liquid
environment.
Plant Tissue Culture Technique;
• Preparation of plant tissue for tissue culture is performed
under aseptic conditions under HEPA filtered air provided by a laminar flow
cabinet.
• The tissue is grown in sterile containers, such as Petri dishes or flasks in a
growth room with controlled temperature and light intensity.
• Living plant materials from the environment are naturally contaminated on
their surfaces (and sometimes interiors) with microorganisms, so their
surfaces are sterilized in chemical solutions (usually alcohol
and sodium or calcium hypochlorite) before suitable samples (known
as explants) are taken.
• The sterile explants are then usually placed on the surface of a sterile solid
culture medium but are sometimes placed directly into a sterile liquid
medium, particularly when cell suspension cultures are desired.
• Solid and liquid media are generally composed of inorganic salts plus a few organic
nutrients, vitamins, and plant hormones. Solid media are prepared from liquid media with
the addition of a gelling agent, usually purified agar.
• The composition of the medium, particularly the plant hormones and the nitrogen source
(nitrate versus ammonium salts or amino acids) has profound effects on the morphology of
the tissues that grow from the initial explant.
• For example, an excess of auxin will often result in a proliferation of roots, while an excess
of cytokinin may yield shoots.
• A balance of both auxin and cytokinin will often produce an unorganized growth of cells
or callus, but the morphology of the outgrowth will depend on the plant species as well as
the medium composition.
• As cultures grow, pieces are typically sliced off and subcultured onto new media to allow
for growth or to alter the morphology of the culture. The skill and experience of the tissue
culturist are important in judging which pieces to culture and which to discard.
• As shoots emerge from culture, they may be sliced off and rooted with auxin to produce
plantlets which, when mature, can be transferred to potting soil for further growth in the
greenhouse as normal plants.
Steaps of Plant tissue culture
Types of Plant tissue culture
• Seed Culture
• Meristem Culture
• Embryo Culture
• Callus Culture
• Organ Culture
• Protoplast Culture
Seed Culture:
Seed culture is a type of tissue culture that is extensively
employed in the cultivation of orchids and other plants. Plant
tissues are extracted from an in-vitro grown plant and put in an
artificial environment where they may develop for this process.
Methods of Culture:
Seed:
Plant tissue culture is unique in that it allows you to produce full
plants from any component of the plant, even the seed. You can grow
plants straight from seeds in this form of culture. These plants will be
consistent and grow at a much faster rate than in the field. When
seeds have a hard outer shell and take longer to germinate in field
circumstances, this is a good method to use.
The orchid is a common example of a plant for which seeds can be
used as explants.
Meristem Culture:
Plants have fascinating tissues at the top of their developing system, called
meristems. Meristematic cells are cells that do not yet have a specific
function in these organs. These cells have the power to grow into any plant
organ and fulfill the duties that go along with it.
In plant tissue culture, we take use of meristematic cells’ potential to
create plant parts and, eventually, a fully functional plant. Meristem
culture is tissue culture that uses meristem tissues.
Protoplast Culture:
The cell wall is a hard covering that surrounds each plant cell. This cell wall
makes it more difficult for other elements to quickly enter the plant cell. A
protoplast is a plant cell that has had its cell wall removed. Researchers can
more easily conduct cell investigations and understand how biochemical
events occur within a cell using protoplasts.
Protoplast culture is used to develop plants such as arabidopsis,
rice, lettuce, tobacco, and others. These are the most prevalent
techniques for growing plants in vitro.
Embryo Culture:
Embryo culture is the sterile isolation and in vitro growth of an
immature or mature embryo with the goal of creating a viable plant.
In 1904 Hannig, who obtained healthy plants from in vitro isolated
embryos of two crucifers, Cochleria and Raphanus, made the first
attempt to generate angiosperm embryos.
In 1924, Dietrich grew embryos of numerous plant species and
observed that mature embryos developed properly, but embryos
taken from immature characteristics of seeds failed to attain mature
embryo organisation.
 Embryo
The embryo is a part inside a plant seed that contains the undeveloped
forms of that plant's roots, stem, and leaves. The embryo develops after
the process of fertilization takes place in the flowers. The seed is the
starting point for the plant as the embryo activates in the right conditions
and eventually becomes a seedling.
They can be two types of:
A-Zygotic Embryogenesis
Fertilization is a process where the pollen (male part of the flower) fuses
with the ovule (female part of the flower) to form an embryo. This is an
important step in plant sexual reproduction. When fertilization happens,
the fused cells form a 'zygote'. This zygote undergoes constant cell division
and finally forms an embryo. We call this process of developing an embryo
'embryogenesis'.
B-Somatic embryogenesis (SE)
We can define SE as a process in which embryo-like structures are
produced using somatic tissues. These embryo-like structures then
develop into a whole plant using different culture mediums. The key to
remember for you is that this method's success depends on the
composition of the culture medium.
Four important steps in SE:
• Induction of pre-embryogenic callus;
• Maintenance of pre-embryogenic callus;
• Development of embryogenic cultures; and
• Regeneration of embryogenic cultures.
Plant regeneration via SE occurs in five steps:
• Initiation of embryogenic cultures;
• The proliferation of embryogenic cultures;
• Prematuration of somatic embryos;
• Maturation of somatic embryos; and
• Plant development on nonspecific media.
Types of somatic embryogenesis
Indirect Somatic Embryogenesis
Indirect somatic embryogenesis is a process where a callus is first
produced from the explant, and then embryos are formed from the callus
tissue or a cell suspension culture. This technique is commonly used in
transgenic experiments.
Direct Somatic Embryogenesis
In direct somatic embryogenesis, embryos are formed directly from a cell
or small group of cells without an intervening callus production. However,
direct somatic embryogenesis is generally a rare event in tissue culture.
The biological mechanism of how direct SE occurs is not clear and it
happens at random for some tested plants.
Phase of Somatic embryogenesis
SE can be split into four phases:
• The first phase is where embryogenic masses (callus retaining cells
with embryogenic competence) initiate from vegetative cells or
tissues.
• In the second phase, embryogenic cell lines (a group of cells with an
embryogenic fate) are maintained and developed.
• The third phase involves somatic embryo formation (embryo
undergoes globular, heart shaped, torpedo and cotyledonary
stages), and maturation (accumulation of reserve substances).
• Somatic embryos are converted (germinated) into viable plantlets.
Organogenesis
In plant tissue culture, organogenesis is the formation of
organs from the cultured explants (plant material such as
roots, leaves and flowers).The organogenesis process is
where the plant organs, either shoots or roots, are
developed.
Types of Organogenesis:
Indirect Organogenesis
The process in which plant organs are derived from a calli mass (tissue
formation occurring on a plant wound or at the site of a cut) in the
explant is termed indirect organogenesis.
Indirect organogenesis has been used to produce transgenic plants in
two ways:
• A plant can be regenerated from a transformed callus.
• The initial explant is transformed, and the callus and shoots are
developed from the modified explants.
Typically, indirect organogenesis is more critical for transgenic plant
production.
Direct Organogenesis
The production of direct buds or shoots from tissue with no
intervening callus stage is called direct organogenesis.

Direct organogenesis has been used to propagate plants with

improved multiplication rates (high number of plant per explant) thus
reducing operational costs. It has also been used to produce more
transgenic plants. And, most importantly, it has been used for clonal
propagation (genetic multiplication of a cultivar without sexual
reproduction) since it ensures the production of uniform planting
material without genetic variation.
Phases of organogenesis:
Organogenesis
Organogenesis can be split into three phases:
• In the first phase, cells in the explants acquire 'competence' which is
defined as the ability to respond to hormonal signals of organ induction.
During this phase, the process leading to organogenic competence is called
dedifferentiation where differentiated cells become undifferentiated.
• During the second phase, competent cells in cultured explants are destined
and determined for specific organ formation under the influence of the
phytohormones balance.
• Lastly, the third phase is where morphogenesis proceeds independently of
the exogenously supplied phytohormones. It means, plant organs take the
shape of roots or shoots when phytohormones are removed from the
culture med
Steap of embryo Culture
Organ Culture:
Organ culture is a type of tissue culture in which an organ is
separated and cultured in a laboratory setting. Any organ plant
can be used as an explant in the culture phase shoot, root, leaf,
and flower.
The process is used to preserve the shape or function of
organs, as well as their many tissue components, allowing the
organ to resemble and retain its qualities in vivo.
New growth differentiated structures occur here as long as the
physiological properties of the organ are retained. As a result,
an organ assists in the conveyance of information concerning
patterns of growth, differentiation, and development.
Callus Culture:
Calluses are unspecialized, disorganized, and dividing masses of
cells. A callus forms when explants are cultured in the
appropriate medium. Tumor tissue that forms from wounds in
differentiated tissues/organs is a good illustration of this.
Cell suspension culture
A cell suspension or suspension culture is a type of cell culture
in which single cells or small aggregates of cells are allowed to
function and multiply in an agitated growth medium, thus
forming a suspension. Suspension culture is one of the two
classical types of cell culture, the other being adherent culture.
Plant hormones:
Plant hormones regulate plant growth, development, reproductive
processes, longevity, and death. Six main types: Auxins, abscisic acid,
cytokinins, ethylene gibberellin, and brassinosteroids.

Auxins:
The Auxins facilitate cell division and root differentiation. Auxins
induce cell division, cell elongation, and formation of callus in cultures.
For example, 2,4-dichlorophenoxy acetic acid is one of the most
commonly added auxins in plant cell cultures. The Cytokinins induce
cell division and differentiation.
Cytokinin:
The plant hormones cytokinin are critical for plant regeneration in
tissue culture, with cytokinin playing an instrumental role in shoot
organogenesis. Type-B response regulators govern the transcriptional
output in response to cytokinin and are required for plant
regeneration.
Abscisic acid (ABA):
Abscisic acid (ABA) plays a significant role in the regulation of many
physiological processes of plants. It is often used in tissue culture
systems to promote somatic embryogenesis and enhance somatic
embryo quality by increasing desiccation tolerance and preventing
precocious germination.
Gibberellins:
The plant hormone gibberellin (GA) controls major aspects of plant
growth such as germination, elongation growth, flower development,
and flowering time.
Organogenesis
In plant tissue culture, organogenesis is the formation of organs from
the cultured explants (plant material such as roots, leaves and
flowers).The organogenesis process is where the plant organs, either
shoots or roots, are developed.
APPLICATIONS OF CELL AND TISSUE CULTURE
•Micropropagation
•Production of virus-free plants
•Production of artificial seeds
•Embryo rescue
•Haploids and triploid culture
•Somatic hybrids and cybrids
•Production of plant secondary metabolites
•Somaclonal variation
• In vitro plant germplasm conservation
Cloning Tissue culture

The identical offspring created by asexual reproduction are Tissue culture, on the other hand, involves producing a
clones. It requires only the division of mitotic cells and is large number of plantlets in a short period of time, as seen
also known as somatogenic propagation. in micropropagation.

A fragment of the donor is extracted and used as an

Clones have the same genetic material as their ancestors explant, which is then cultured on a sterile medium and
and without any difference between them. divided into different parts, each of which grows into a new
plantlet.

Anther culture, suspension culture, single cell culture,

They have 100 percent identical genetic makeup as their
pollen culture, and somatic embryogenesis are some of the
ancestors, they are the carbon copy of their parents.
other types.

Example -Dolly, a sheep (female), was the first animal to be

cloned from adult somatic cells by the nuclear transfer
The phases of tissue culture are initiation, multiplication,
process. Tetra was the first rhesus macaque developed by
root formation, shoot formation, and acclimatization.
embryo splitting. The ox, mule, cat, wolf, dog, rat and
rabbit are the cloned animals.
Biotechnology biofertilizer
Biofertilizers are biological preparations of efficient
microorganisms that promote plant growth by improving
nutrient acquisition. They enhance soil productivity by fixing
atmospheric nitrogen, solubilizing soil phosphorus, and
stimulating plant growth.
Biofertilizers such as Rhizobium, Azotobacter, Azospirilium and
blue green algae (BGA) have been in use a long time.
Rhizobium inoculant is used for leguminous crops. Azotobacter
can be used with crops like wheat, maize, mustard, cotton,
potato and other vegetable crops.
Benefits of biofertilizers
• Biofertilizers are means of fixing the nutrient availability in the soil. Generally
Nitrogen deficiencies.
• Since a bio-fertilizer is technically living, it can symbiotically associate with
plant roots. Involved microorganisms could readily and safely convert
complex organic material into simple compounds, so that they are easily
taken up by the plants
• It has also been shown that to produce a larger quantity of crops,
biofertilizers with the ability of nitrogen fixation and phosphorus solubilizing
would lead to the greatest possible effect.
• They advance shoot and root growth of many crops versus control
groups.This can be important when implementing new seed growth.
• Biofertilizers also promote healthy soil, leading to greater farming
sustainability.
Biological nitrogen fixation
Biological nitrogen fixation (BNF) can be defined as the conversion of
atmospheric dinitrogen (N2) to ammonia (NH3) under the combined
action of biological and chemical activities.
Different Ways of Nitrogen Fixation.
Plants are the main source of food. The nutrients obtained from plants
are synthesized by plants using various elements which they obtain
from the atmosphere as well as from the soil. This group of elements
includes nitrogen as well. Plants obtain nitrogen from the soil and
utilise it in the process of protein synthesis. Unlike carbon dioxide and
oxygen, atmospheric nitrogen cannot be obtained through the stomata
of leaves. Because the nitrogen gas present in the atmosphere can not
be directly used by plants. There are certain bacteria and some natural
phenomenon that help in Nitrogen fixation.
Biological Nitrogen Fixation:

Certain bacteria or prokaryotes are capable of converting

atmospheric nitrogen to ammonia. This process is called biological
nitrogen fixation. The enzyme nitrogenase converts dinitrogen to
ammonia. Nitrogen-fixing bacteria may be free-living or symbiotic.
Some of the free-living nitrogen fixers are Azotobacter, Beijernickia,
Rhodospirillum, cyanobacteria, etc. Examples of symbiotic nitrogen
fixers are Rhizobium (in the root nodules of legumes) and Frankia (in
the root nodules of non-leguminous plants), etc.
Symbiotic Nitrogen Fixation
A species of bacteria called Rhizobium, help in nitrogen fixation. These
bacteria live in the roots of leguminous plants (e.g., pea and beans
plants) and using certain types of enzymes, they help in fixing nitrogen
in the soil. During this biological process, they convert the non-
absorbable nitrogen form into a usable form. This form of nitrogen gets
dissolved in the soil, and plants absorb the modified nitrogen from the
soil. This is the reason behind farmers implementing crop rotation,
where leguminous plants help to replenish nitrogen content in the soil
without the necessity of fertilizers.
Nitrogen fixation by bacteria is an example of the symbiotic
relationship between Rhizobium and leguminous plants. While bacteria
fix nitrogen in the soil, plants provide them food.
 Transgenic plant
A transgenic plant is a modified organism where genes are transferred
from one organism to another through genetic engineering techniques.
The purpose of producing a transgenic plant is to obtain a species that
has ideal traits, high yield and quality.
Methods Used for Gene Transfer
There are two methods majorly which are used to transfer genes in
plants. The methods include:
1. Agrobacterium mediates gene transfer
Agrobacterium tumifaciens is a plant pathogen. It is known to cause
crown gall disease, which is swelling in plants just above the soil level.
After infecting the plants, they transfer their genetic material to them,
which eventually gets incorporated into the plant genome.
For genetic engineering, the bacterium is incorporated with a
Ti plasmid with desirable genes and made to infect the plant.
The Ti plasmid is a tumour inducing circular plasmid, that transfers the
host chromosomes to the plants and is also responsible for causing
the swelling.
2. Particle bombardment / Gene gun method

As the name suggests, in this method, the desired gene is coated in a

gold or tungsten particle and bombarded into the plant cells. Once
bombarded, the sequence is incorporated into the plant cells, which
can be proliferated by tissue culture methods.
3.Polyethylene glycol (PEG)
Polyethylene glycol (PEG) can induce genetic transformation in both
bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae)
without cell wall removal. PEG-mediated transformation of E. coli is
technically simple and yields transformants with an efficiency of
10(6)-10(7) transformants/microgram
4.Microinjection
Microinjection is the process of transferring genetic materials into a
living cell using glass micropipettes or metal microinjection needles.
Glass micropipettes can be of various sizes with tip diameters ranging
from 0.1 to 10 µm. DNA or RNA is injected directly into the cell's
nucleus.
Applications of Transgenic Plants
• Resistance to biotic and abiotic stress: Biotic stress is imposed on plants
as a result of the action of living beings such as viruses, bacteria, pests
and pathogens. To relieve the plants from such stress they are
incorporated with disease-resistant genes, which gives a better yield and
quality to the crops.
• Abiotic stress, as a result of changes in the environment, causes great
damage to the plants. Soil composition, humidity, water level, and
temperature are important factors for plant growth. Due to changes in
the climate, all the factors seem to be altered. Thus, plants are
incorporated with stress-tolerant genes for better production.
• Increased nutritional value: Biofortification is the process of increasing
the nutritional value of a crop. Malnutrition is a common problem in
developing countries. As a solution, plants are engineered to produce
crops of better nutritional value.
• Factories for production of recombinant proteins: Recombinant human
proteins have been produced using animal and microorganism systems,
but due to some shortcomings it has been shifted to the plant system.
Vaccines and antibiotics have been obtained from transgenic plants.
However, this application is still in the development stage and has not
been commercialised yet.
Examples of Transgenic Plants
• Golden rice: Golden rice was produced to overcome the deficiency of
vitamin A in children. Using the gene gun methods, rice species were
incorporated with the phytoene synthase genes, which increases the
vitamin A content of the rice grains.
• Bt cotton: Bt cotton is a genetically modified crop that is resistant to pest
bollworm.
• Flavr Savr: Flavr Savr is a genetically modified tomato crop that has a
longer shelf life due to delays in ripening and softening.
 Bt Crops
Bt crops are transgenic crops that are genetically engineered from
the DNA of bacterium Bacillus thuringiensis.
• Bt Crops are transgenic crops that produce the same toxin as the
bacterium Bacillus thuringiensis in the plant cell, thereby, protecting
the crops from pests. The bacterium secretes specific proteins known
as “cry proteins” that are toxic to insects. A few of the Bt crops
include cotton, brinjal, corn, etc.

• When an insect feeds on the transgenic plants, the toxic cry protein
present in the plants crystallizes the digestive system of insects,
eventually leading to its death. However, it has no harmful effects on
the human digestive system.
Bacillus thuringiensis:
• Bacillus thuringiensis is a gram-positive, spore-forming bacteria which
is mainly found in the soil. As stated above, it produces proteins that
are toxic to insects. Organic farmers use this bacterium in a solution
and spray it on the plants to protect them from pests.
• The practice of using Bacillus thuringiensis began in the year 1996
with small quantities of genes from the bacterium. This facilitated the
production of cry proteins in plant cells that helped to kill pests. Pests
like European and southwestern corn borer, tobacco and cotton
budworm, pink bollworm and Colorado potato beetle largely
destroyed the crop yields. Bacillus thuringiensis protected the crops
against such pests.
Types of Bt crops
The following types of Bt crops were produced by the researchers:
Bt Cotton
The Bt cotton variety is genetically transformed with the Bt gene to protect
the plants from bollworm, a major pest of cotton. The worms present on
the leaves of Bt cotton become lethargic and sleepy and thus, cause less
damage to the plants. When the worms consume the plant, the toxic
proteins produced by the crops are ingested, thereby, killing them.
Bt Brinjal
Bt brinjal is also produced by genetic transformation of a crystal protein gene cry
1 Ac from the bacterium Bacillus thuringiensis. Bt brinjal was developed to
provide resistance against lepidopteron insects. The proteins produced by Bt
genes bind to the receptors present on the insect’s membrane, resulting in the
formation of pores on the membranes. This disrupts the digestive process and
leads to the death of the insect.
Advantages of Bt Crops
Following are the major advantages of Bt crops:
• It helps in improving the crop yield, thereby, raising the farmer’s
income. This results in increased farm production.
• They help in controlling soil pollution as the use of synthetic pesticides
is reduced.
• Bt crops help in protecting beneficial insects.
• It can easily feed an increasing population due to increased yields in a
short time.
• It leads to the production of disease-free crops owing to the reduction
of pesticides.
• It leads to more productivity in a small area of land.
Disadvantages of Bt Crops
• Bt crops have a few disadvantages as well:
• Bt crops are costlier than naturally grown crops.
• It can disrupt the natural process of gene flow.
• The pests might become resistant to the toxins
produced by these crops and the crop production
might decline.
FLAVR SAVR-TM
"FLAVR SAVR™ tomato was developed through the use of antisense RNA to
regulate the expression of the enzyme polygalacturonase (PG) in ripening
tomato fruit. This enzyme is one of the most abundant proteins in ripe
tomato fruit and has long been thought to be responsible for softening in.
Benefits of genetically modified tomatoes
1 Delayed ripening.
2 Environmental stress tolerance.
3 Pest resistance.
4 Improved nutrition.
5 Improved taste.
6 Vaccines.
Plant Products of Biotechnology
Plant products of biotechnology approved for food use have
been modified to contain traits such as:
• Insect resistance
• Disease resistance
• Herbicide tolerance
• Altered nutritional profile
• Enhanced storage life
Product Trait

Alfalfa Herbicide tolerance, altered lignin production

Apple Non-browning

Bean Virus disease resistance

Canola Herbicide tolerance, modified oil/fatty acid, pollination control system, phytase production

Cotton Herbicide tolerance, insect resistance, low gossypol

Eggplant Insect resistance

Flax, Linseed Herbicide tolerance

 Abiotic stress tolerance, altered growth/yield, herbicide
tolerance, insect resistance, modified product quality (modified
Maize
alpha amylase, lysine boost, phytase production), pollination
control system

Melon Delayed ripening

Papaya Disease resistance

Pineapple Delayed ripening, modified fruit color

Plum Disease resistance

Disease resistance, herbicide tolerance, insect resistance,

Potato modified product quality (modified starch, reduced acrylamide
potential, non-bruising), fungal disease resistance
 Agricultural biotechnology
Agricultural biotechnology is a collection of scientific techniques used
to improve plants, animals and microorganisms. Based on an
understanding of DNA, scientists have developed solutions to increase
agricultural productivity.
Examples of Biotechnology in Agriculture
• Genetically Modified Crops.
• Developing of Biofuels.
• Improving Plant Growth.
• Improving Plant Seed Quality.
• Improve Animal Health and Breeding.
• Learn More at Fruit Growers Supply.