TRANSGENIC TECHNOLOGY Plant transformation getting DNA into a

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TRANSGENIC TECHNOLOGY

TRANSGENIC TECHNOLOGY

Plant transformation ügetting DNA into a cell ügetting it stably integrated ügetting a plant

Plant transformation ügetting DNA into a cell ügetting it stably integrated ügetting a plant back from the cell

Requirement 1. a suitable transformation method 2. a means of screening for transformants 3.

Requirement 1. a suitable transformation method 2. a means of screening for transformants 3. an efficient regeneration system 4. genes/constructs ü Vectors ü Promoter/terminator ü reporter genes ü selectable marker genes ü ‘genes of interest’

Transformation methods DNA must be introduced into plant cells Indirect - Agrobacterium tumefaciens Direct

Transformation methods DNA must be introduced into plant cells Indirect - Agrobacterium tumefaciens Direct - Chemical method - Electrical method - Physical methods Chemical Method 1. Use of PEG (Polyethylene glycol (PEG)-mediated ) 2. Protoplasts are incubated with a solution of DNA and PEG

Electrical method 1. Electroporation (electropermeabilization) 2. Cells or protoplast are subjected to short electrical

Electrical method 1. Electroporation (electropermeabilization) 2. Cells or protoplast are subjected to short electrical pulse Physical Methods 1. Particle bombardment 2. Microinjection 3. Silicon Carbide whiskers

Agrobacterium-mediated transformation n n A natural genetic engineer 2 species • A. tumefaciens (produces

Agrobacterium-mediated transformation n n A natural genetic engineer 2 species • A. tumefaciens (produces a gall) • A. rhizogenes (produces roots) n Oncogenes (for auxin and cytokinin synthesis) + Opines n In the presence of exudates (e. g. acetosyringone) from wounded plants, Virulence (Vir) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.

BACTERIAL GALL DISEASES n Galls: overgrowth or proliferation of tissue, primarily due to increased

BACTERIAL GALL DISEASES n Galls: overgrowth or proliferation of tissue, primarily due to increased cell division (hyperplasia) and increased cell size (hypertrophy). n Bacterial Galls: induced by bacteria in 3 different genera. • Agrobacterium • Pseudomonas • Clavibacter n Genes for plant hormone production found on bacterial plasmids!

Crown Gall Disease: Agrobacterium tumefaciens n n n Gram Dicots Worldwide

Crown Gall Disease: Agrobacterium tumefaciens n n n Gram Dicots Worldwide

Disease Cycle

Disease Cycle

Agrobacterium tumefaciens n Characteristics • Plant parasite that causes Crown Gall Disease • Encodes

Agrobacterium tumefaciens n Characteristics • Plant parasite that causes Crown Gall Disease • Encodes a large (~250 kbp) plasmid called Tumor-inducing (Ti) plasmid n Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Tumor DNA)

Agrobacterium tumefaciens üT-DNA integrates stably into plant genome üSingle stranded T-DNA fragment is converted

Agrobacterium tumefaciens üT-DNA integrates stably into plant genome üSingle stranded T-DNA fragment is converted to ds. DNA fragment by plant cell Then integrated into plant genome n 2 x 23 bp direct repeats play an important role in the excision and integration process n

Agrobacterium tumefaciens n n n Tumor formation = hyperplasia Hormone imbalance Caused by A.

Agrobacterium tumefaciens n n n Tumor formation = hyperplasia Hormone imbalance Caused by A. tumefaciens • Lives in intercellular spaces of the plant • Plasmid contains genes responsible for the disease n Part of plasmid is inserted into plant DNA n Wound = entry point 10 -14 days later, tumor forms

Agrobacterium tumefaciens n What is naturally encoded in T-DNA? • Enzymes for auxin and

Agrobacterium tumefaciens n What is naturally encoded in T-DNA? • Enzymes for auxin and cytokinin synthesis n n Causing hormone imbalance tumor formation/undifferentiated callus Mutants in enzymes have been characterized • Opine synthesis genes (e. g. octopine or nopaline) n n Carbon and nitrogen source for A. tumefaciens growth Insertion genes • Virulence (vir) genes • Allow excision and integration into plant genome

Ti plasmid of A. tumefaciens

Ti plasmid of A. tumefaciens

1. Auxin, cytokinin, opine synthetic genes transferred to plant 2. Plant makes all 3

1. Auxin, cytokinin, opine synthetic genes transferred to plant 2. Plant makes all 3 compounds 3. Auxins and cytokines cause gall formation 4. Opines provide unique carbon/nitrogen source only A. tumefaciens can use!

Agrobacterium tumefaciens n How is T-DNA modified to allow genes of interest to be

Agrobacterium tumefaciens n How is T-DNA modified to allow genes of interest to be inserted? • In vitro modification of Ti plasmid n n n n T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) Insertion genes are retained (vir genes) Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance geneticin (G 418) or hygromycin] Ti plasmid is reintroduced into A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) Antibacterial agents (e. g. chloramphenicol) added to kill A. tumefaciens G 418 or hygromycin added to kill non-transgenic plant cells Surviving cells = transgenic plant cells

Agrobacterium and genetic engineering: Engineering the Ti plasmid

Agrobacterium and genetic engineering: Engineering the Ti plasmid

Co-integrative and binary vectors LB RB Co-integrative Binary vector

Co-integrative and binary vectors LB RB Co-integrative Binary vector

Agrobacterium-mediated transformation Agrobacterium tumefaciens cause ‘Crown gall’ disease Agrobacterium is a ‘natural genetic engineer’

Agrobacterium-mediated transformation Agrobacterium tumefaciens cause ‘Crown gall’ disease Agrobacterium is a ‘natural genetic engineer’ i. e. it transfers some of its DNA to plants

Expose wounded plant cells to transformed agro strain Electroporate TDNA vector into Agrobacterium and

Expose wounded plant cells to transformed agro strain Electroporate TDNA vector into Agrobacterium and select for tetr Induce plant regeneration and select for Kanr cell growth

Electroporation ü Explants: cells and protoplasts ü Most direct way to introduce foreign DNA

Electroporation ü Explants: cells and protoplasts ü Most direct way to introduce foreign DNA into the nucleus

Diagram of one technique

Diagram of one technique

Microprojectile bombardment • uses a ‘gene gun’ • DNA is coated onto gold (or

Microprojectile bombardment • uses a ‘gene gun’ • DNA is coated onto gold (or tungsten) particles (inert) • gold is propelled by helium into plant cells • if DNA goes into the nucleus it can be integrated into the plant chromosomes • cells can be regenerated to whole plants

n n In the "biolistic" (a cross between biology and ballistics )or "gene gun"

n n In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot into the plant cell with a pulse of helium. Once inside the cell, the gene comes off the bead and integrates into the cell's genome.

n Model from Bio. Rad: Biorad's Helios Gene Gun

n Model from Bio. Rad: Biorad's Helios Gene Gun

Microinjection ü Most direct way to introduce foreign DNA into the nucleus ü Achieved

Microinjection ü Most direct way to introduce foreign DNA into the nucleus ü Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast ü Labour intensive and slow ü Transformation frequency is very high, typically up to ca. 30%

Silicon Carbide Whiskers ü Silicon carbide forms long, needle like crystals ü Cells are

Silicon Carbide Whiskers ü Silicon carbide forms long, needle like crystals ü Cells are vortex mixed in the present of whiskers and DNA ü DNA can be introduced in the cells following penetration by the whiskers

Gene construct

Gene construct

Gene construct Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’.

Gene construct Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’.

Vectors ü Ti-plasmid based vector a. Co-integrative plasmid b. Binary plasmid ü Coli-plasmid based

Vectors ü Ti-plasmid based vector a. Co-integrative plasmid b. Binary plasmid ü Coli-plasmid based vector a. Cloning vector b. Chimeric Plasmid ü Viral vector a. It is normally not stably integrated into the plant cell b. It may be intolerant of changes to the organization of its genome c. Genome may show instability

Promoter 1. 2. 3. 4. 5. 6. A nucleotide sequence within an operon Lying

Promoter 1. 2. 3. 4. 5. 6. A nucleotide sequence within an operon Lying in front of the structural gene or genes Serves as a recognition site and point of attachment for the RNA polymerase It is starting point for transcription of the structural genes It contains many elements which are involved in producing specific pattern and level of expression It can be derived from pathogen, virus, plants themselves, artificial promoter

Types of Promoter ü Promoter always expressed in most tissue (constitutive) -. 35 s

Types of Promoter ü Promoter always expressed in most tissue (constitutive) -. 35 s promoter from Ca. MV Virus -. Nos, Ocs and Mas Promoter from bacteria -. Actin promoter from monocot -. Ubiquitin promoter from monocot -. Adh 1 promoter from monocot -. p. EMU promoter from monocot ü Tissue specific promoter -. Haesa promoter -. Agl 12 promoter ü Inducible promoter -. Aux promoter ü Artificial promoter -. Mac promoter (Mas and 35 s promoter)

Reporter gene easy to visualise or assay - ß-glucuronidase (GUS) (E. coli) -green fluorescent

Reporter gene easy to visualise or assay - ß-glucuronidase (GUS) (E. coli) -green fluorescent protein (GFP) (jellyfish) - luciferase (firefly)

GUS Cells that are transformed with GUS will form a blue precipitate when tissue

GUS Cells that are transformed with GUS will form a blue precipitate when tissue is soaked in the GUS substrate and incubated at 37 o. C this is a destructive assay (cells die) The Uid. A gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.

GUS Bombardment of GUS gene - transient expression Stable expression of GUS in moss

GUS Bombardment of GUS gene - transient expression Stable expression of GUS in moss Phloem-limited expression of GUS

HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic

HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic plant expressing a HAESA: : GUS fusion. It is expressed in the floral abscission zone at the base of an Arabidopsis flower. Transgenic plants that harbor the AGL 12: : GUS fusions show rootspecific expression.

Inducible expression

Inducible expression

GFP (Green Fluorescent Protein) ü Fluoresces green under UV illumination ü Problems with a

GFP (Green Fluorescent Protein) ü Fluoresces green under UV illumination ü Problems with a cryptic intron now resolved. ü Has been used for selection on its own. GFP glows bright green when irradiated by blue or UV light This is a nondestructive assay so the same cells can be monitored all the way through

GFP protoplast colony derived from protoplast mass of callus regenerated plant

GFP protoplast colony derived from protoplast mass of callus regenerated plant

Selectable Marker Gene let you kill cells that haven’t taken up DNA- usually genes

Selectable Marker Gene let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance Most common: 1. antibiotic resistance 2. kanamycin, hygromycin 2. herbicide resistance phosphinothricin (bialapos); glyphosate

Only those cells that have taken up the DNA can grow on media containing

Only those cells that have taken up the DNA can grow on media containing the selection agent

Gene of interest Sequence of DNA which will be inserted to the host cell

Gene of interest Sequence of DNA which will be inserted to the host cell and its product will be studied or beneficial for mankind Origin of gene interest: 1. Non plant genes 2. Plant genes

pathogen-derived genes Exogenous genes (non-plant genes) bacterial genes any other organism Endogenous genes (Plant

pathogen-derived genes Exogenous genes (non-plant genes) bacterial genes any other organism Endogenous genes (Plant genes) Enzymes in biochemical pathway Natural resistance genes

Screening technique There are many thousands of cells in a leaf disc or callus

Screening technique There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA therefore can get hundreds of plants back - maybe only 1% will be transformed How do we know which plants have taken up the DNA? Could test each plant - slow, costly Or use reporter genes & selectable marker genes

Screening n n Transformation frequency is low (Max 3% of all cells) and unless

Screening n n Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by nontransformed. Usual to use a positive selective agent like antibiotic resistance. The Npt. II gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.

Screening (selection) n n Select at the level of the intact plant Select in

Screening (selection) n n Select at the level of the intact plant Select in culture • • single cell is selection unit possible to plate up to 1, 000 cells on a Petri-dish. • Progressive selection over a number of phases

Selection Strategies Positive n Negative n Visual n

Selection Strategies Positive n Negative n Visual n

Positive selection n n Add into medium a toxic compound e. g. antibiotic, herbicide

Positive selection n n Add into medium a toxic compound e. g. antibiotic, herbicide Only those cells able to grow in the presence of the selective agent give colonies Plate out and pick off growing colonies. Possible to select one colony from millions of plated cells in a days work. Need a strong selection pressure - get escapes

Negative selection n n Add in an agent that kills dividing cells e. g.

Negative selection n n Add in an agent that kills dividing cells e. g. chlorate / BUd. R. Plate out leave for a suitable time, wash out agent then put on growth medium. All cells growing on selective agent will die leaving only non-growing cells to now grow. Useful for selecting auxotrophs.

Visual selection n n Only useful for coloured or fluorescent compounds Plate out at

Visual selection n n Only useful for coloured or fluorescent compounds Plate out at about 50, 000 cells per plate. Pick off coloured / fluorescent compounds Possible to screen about 1, 000 cells in a days work.

Positive and Visual Selection

Positive and Visual Selection

Regeneration System How do we get plants back from cells? We use tissue culture

Regeneration System How do we get plants back from cells? We use tissue culture techniques to regenerate whole plants from single cells getting a plant back from a single cell is important so that every cell has the new DNA

Regeneration Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro

Regeneration Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana

Somatic embryogenesis in peanut

Somatic embryogenesis in peanut