Plant biotechnology (Biot3093)

Ayalnesh S.(MSc)
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 Chapter: 1
What is Biotechnology?

 Bio means life and

 technology means the application of knowledge for practical use.

 Biotechnology is the use of living organisms to make useful products

 i.e the use of cells and biological molecules

Biological molecules include DNA, RNA and proteins

 The term “Biotechnology" was first coined in 1919 by Karl Ereky.

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Biotechnology is defined as any technique that uses living organisms (or parts
of organisms) to make/ modify products, to improve plants and animals or to
develop microorganisms for specific uses.
 It offers efficient and cost-effective means to produce an array of novel,
value-added products and tools.
 It has the potential
 to increase food productivity,
 reduce the dependency of agriculture on chemicals,
 lower the cost of raw materials and
 reduce the negative environmental impacts associated with traditional
production methods
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 Generally biotechnology is the study and manipulation of living things or their
component molecules, cells, tissues or organs for the benefit of humans

 Biotechnology has been used since 1970’s to reflect the application of

existing new technologies to the research and development of products from
plants and animals.

 Biotechnology is currently being used in many areas including agriculture,

bioremediation, food processing and energy production.

 In agriculture, genetic engineering is being used to produce plants that are

resistant to insects, weeds and plant diseases.

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 Applications of biotechnology
Environmental Biotechnology
 Waste management
 Bioremediation: the use of microbes to break down organic molecules
or environmental pollutants.

 Phyto remediation: the use of plants to remove pollutants (e.g. heavy metals)
from the environment.

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 Pollution prevention
 Renewable resources
 Biodegradable products
 Alternative energy sources

Medical biotechnology
 Diagnostics
 Therapeutics
 Vaccines
Agricultural Biotechnology
 Plant biotechnology
 Animal biotechnology
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 Introduction
Plant biotechnology
Plant biotechnology is science of changing the genetic make up of plants
 Plant biotechnology is rapidly expanding field within biotechnology that
chiefly involves the introduction of foreign genes in to economically important
plant species, resulting in crop improvement and the production of novel
products in plants
 Plants are the most important organisms on this planet
 They occupy the bottom most parts of food chain

 Now a days population growth is increasing time to time

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 Almost 85% of the world’s food consumed by humans is plant based

 However crop production is decreasing due to different factors

 like change in environmental conditions that leads to food insecurity

 As food crops are highly dependent on

 Water availability

 Soil and Climatic conditions

 So it is necessary to improve crop production by adapting crops with

environmental stress and making them resistant to diseases

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 This can be achieved by implementing biotechnological techniques (plant
biotechnology) in agriculture.

Plant biotechnology focuses on manipulating plant heredity to develop new and

improved plant types for the benefit of humankind as

 Some plants show diseases resistance

 some varieties are high yielding,

 Some are drought tolerant,

 some give high yield but no drought tolerance

 some give less yield but drought tolerant all these are desirable characteristics that
are used to create desirable plant types that better suited for cultivation

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 Applications of plant biotechnology
A. Crop Improvement
 resistance to pest and diseases.
 tolerance to environmental conditions
 Improved color and quality
B. Pharmaceuticals
 Plants that produce edible vaccines
C. Food
 Improved taste and nutrition
 Improved handling qualities
D. Industrial
 plants that produce plastics, fuels, and other products
 plants for environmental cleanup
E. Other
 pesticides made from naturally-occurring microorganisms and insects
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 Plants are mainly manipulated for
 Improved quality

Increased yield

Increased tolerance to environmental stresses

Herbicide tolerance

Pest resistance

Nitrogen fixing ability

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Higher yield : Improvement in yield can be achieved by evolving high yielding
varieties.

 grain yield,

 fiber yield,

 tuber yield,

 cane yield or oil yield depending upon the crop species

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Improved quality

 grain size,

 milling and baking quality in wheat.

 malting quality in barley,

 color and size of fruits,

 protein content in pulses,

 fibre length, strength and fineness in cotton.

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Increased tolerance to environmental stresses like

 drought,

 soil acidity,

 extreme temperatures,

 Increased tolerance to insect pests

 In general improved varieties can be developed through the use of donor

parents that have desirable trait.

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Technologies applied in plant biotechnology
 Conventional plant breeding

 Tissue culture and micro propagation

 Molecular breeding or marker assisted selection

 Genetic engineering/Recombinant DNA technology

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Tissue culture and micro propagation
 It is a collection of techniques used to maintain or grow plant cells, tissues or
organs under sterile conditions on a nutrient culture medium of known
composition.

 It is widely used to produce clones of a plant in a method known as Micro

propagation

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 Conventional plant breeding

 Conventional plant breeding involves cross-breeding of plants from the

related species to produce new varieties with different traits.

 But it takes many generations to achieve desired result.

 Breeding can only be done between two plants that can sexually mate
with each other

This limits the new traits that can be added to those that already
exist in a particular species and

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  when plants are crossed, many traits are transferred along with the trait/s
of interest

 Another important way of improvement is the introduction of new genetic

material (e.g., genes for biotic and abiotic stress resistance) from other
sources, such as gene bank accessions and related plant species

 For these modern biotechnology provides new tools that can facilitate
development of improved plant breeding methods that can be achieved
faster and it even facilitates to transfer genes from unrelated species

 Genetic engineering and

 Molecular marker assisted selection

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Genetic engineering

 Genetic engineering, also known as genetic modification, enables scientists

to transfer genes from one organism to another

 Genetic engineering is changing the way we produce food by allowing for

rapid increases in crop yields.

 Scientists can use genetic engineering

 to increase crop yields,

 lower food costs,

 improve food quality,

 Generally to over come the problem of food insecurity and medicinal value
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Molecular (DNA) markers
 Are segments of DNA that can be detected through specific laboratory
techniques like Polymerase Chain Reaction(PCR)
 PCR is used to generate/amplify the DNA sequences that are linked to a
heritable trait such as yield or disease resistance.
 DNA marker technologies offer plant breeders potential of making genetic
progress more precisely and more rapidly than phenotypic selection
 With the advent of marker-assisted selection (MAS), a new breeding tool is
now available to make more accurate and useful selections in breeding
populations

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Conventional breeding Marker assisted breeding

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 Summary
 Biotechnology refers to genetic manipulation of organisms for specific
purposes.

 Agricultural biotechnology is the term used in crop and livestock improvement

through biotechnology tools.

 The term plant biotechnology refers to application of engineering techniques

to modify plants for the benefit of humans.

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 Genetic engineering techniques are utilized to produce transgenic plants with
desirable genes like

 disease resistance,

 herbicide resistance,

 increased shelf life of fruits etc.

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 CHAPTER 2

Gene Cloning Strategies

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 Gene Cloning
What is gene?
 Genes are the chemical blueprints that determine an organism's traits
 Gene cloning, also known as molecular cloning.
 Gene cloning strategies is the process of isolating a DNA sequence /gene of
interest for the purpose of making multiple copies of it.
 Gene cloning strategy is a set of techniques adopted for gene cloning for a
particular purpose.
 Strategy depends on the starting information and desired endpoint.
 Techniques for gene cloning enable scientists to prepare multiple copies of apices of
DNA.
 Gene cloning is used to prepare many copies of the gene itself.
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Fundamental steps included in gene cloning are:
1) Selection of the host organism
 the most commonly used host organisms are non-pathogenic laboratory strains of
bacteria (E. coli) which is genetically engineered for optimal performance.

2) Selection of cloning vector

 Bacterial plasmids are by far the most commonly used cloning vectors

3) Preparation of the vector

 The cloning vector can be prepared by restriction digestion

4) Preparation of the insert

 The DNA to be cloned (insert) can come from multiple sources
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5) Generation of the recombinant DNA

 Once, the vector and inserts are prepared, they mixed by using DNA ligase.

6) Introduction of the recombinant DNA into the host organism

 the process of transferring plasmids into new host cells is called transformation.

 the most commonly used technique to introduce recombinant DNA in to the host
cell is exposing cells to a heat-shock, which makes them permeable to the plasmid
DNA

7) Selection of the clones of organisms containing the vectors

 Bacteria are cultured on semi-solid agar-media containing an antibiotic and

 they are selected based on their ability to grow on media containing that antibiotic.

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 Selectable markers allows selection of the organisms that carry this marker
and therefore the desired plasmid.
 After overnight incubation, individual bacteria grow up into visible colonies of
several million cells
8) Screening for clones with the desired recombinant DNA molecules
 DNA sequencing is performed on isolated plasmids and used to confirm the
presence of the insert and its correct sequence.
9) Expansion and isolation of the recombinant DNA
 After screening, the correct bacterial clone carrying the desired recombinant
DNA is expanded in liquid culture to amplify the plasmid.

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 Cloning Vectors

Different types of vectors are available for gene cloning

 plasmids,

 bacteriophages,

 bacterial artificial chromosomes (BACs),

 yeast artificial chromosomes (YACs)

 From these vectors plasmids are commonly used as cloning vectors

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Plasmids  They are self replicating, double
 Bacterial plasmids are the most stranded, circular DNA molecules in
commonly used cloning vectors bacterial cells that are naturally

 Cloning vectors are DNA molecules present in addition to bacteria's

that are used to "transport" cloned chromosomal DNA.

sequences between biological hosts

 Plasmids are tiny circular pieces of

DNA that are commonly found in
bacteria.

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Why plasmids are good cloning vectors

 small size (easy to manipulate and isolate)

 circular (more stable)

 replication independent of host cell

 several copies may be present (facilitates replication)

 have antibody resistance (detection easy)

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Requirements for a cloning vector
 Should be capable of autonomous replication in at least one host
organism.

 Should be of small size, since this aids the preparation vector DNA and
reduces the complexity of analyzing recombinant molecules.

 Should be capable of amplifying the cloned sequence by occurring in

multiple copies.

 There should be a unique cleavage site for a range of restriction

endonucleases.

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 Should possess one or more genetic markers enabling easy selection of
cloned molecules.

 Should permit detection by simple genetic tests

 Should have appropriate transcriptional and translational signals located

adjacent to cloning sites for better expression of cloned DNA sequences.

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 Gene to be cloned can be introduced into the cloning vector at one of the
restriction sites present in multiple cloning site.

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 Vector components
 The vectors used for multiplication of
DNA fragments in a suitable host are
called cloning vectors

Vectors must contain:

 Origin of replication

 Selectable marker and

 Multiple cloning site

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1. Origin of replication
 Origin of replication (ORI) recruits the
DNA replication machinery and allows
the propagation of the plasmid.

 It is a DNA segment recognized by

the cellular DNA-replication
enzymes.

 Without replication origin, DNA

cannot be replicated in the cell.

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Selectable markers selectable marker can survive

 Selectable markers are an antibiotic

resistance genes.

 They allows the selection of

positively transformed cells

 The most commonly used antibiotic

resistance genes are ampicillin, and
kanamycin resistance

 Under the selective conditions, only

cells that contain plasmids with
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Multiple cloning site (MCS).
 Cloning vectors contain a multiple cloning site or polylinker: a DNA segment
with several unique sites for restriction endonucleases(RE) located next to
each other

 MCS are the active sites of the RE enzymes

 Allow insertions of DNA into the vector

 Contain multiple restriction enzyme sites to facilitate the cloning procedure

 Restriction sites of the polylinker are not present anywhere else in the
plasmid.

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Multiple cloning sites
(polylinker)

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Restriction enzymes
 Restriction enzymes: enzymes that
cut DNA in specific places

 Breaks only palindrome sequences

 Important in DNA research

 Companies purify and market

restriction enzymes

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Promoter
 Vector contain the origin of replication, multiple cloning site, and selectable
marker, but it will also need a promoter that can drive the expression of the
gene

 Promoters are found upstream of their target genes

 Promoters are used to drive the transcription of the vector's transgene as

well as the other genes in the vector such as the antibiotic resistance gene.

 promoters play a large role in determining where and when your gene of
interest will be expressed

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 Summary
 The objective of molecular cloning is to obtain multiple copies of specific
DNA fragments

 In molecular cloning restriction enzymes are used as molecular scissors

 Restriction enzymes cut the DNA at specific palindromic sites (sequence that
is the same on both antiparallel DNA strands).

 DNA ligase is the glue of molecular genetics that holds the ends of the DNAs
together

 Origin of replication, selectable marker and multiple cloning site are common
futures of cloning vectors
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Procedures for
gene cloning

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 CHAPTER 3

Gene transfer methods in Plants

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