Methods of Gene TransferPravin V Jadhav, PhDAssistant Professor, Biotechnology Centre, Dr. PDKV, AkolaE-mail: jpraveen26@yahoo.co.in

Transgenic versus Cloning

Why transgenic plants ?

Plant Genetic Engineering Process

Methods of genetic transformation/ gene delivery

OUTLINE

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Transgenic versus Cloning Transgenic : creation of transgenic animal or

plant (introduction of foreign gene into organism)

• transgenic organisms produced by introduction of foreign gene into germ line (transgenic offspring!!!)

• introduction of gene into somatic cells -> gene therapy Cloning : obtaining an organism that is

genetically identical to the original organism • such as Dolly the sheep • asexual propagation of plants (taking cuttings)

What is a transgenic?

TransgenicAn organism containing a transgene introduced by technological (not breeding) methods

Transgene The genetically engineered gene added to a species

Why transgenic plants ?

Why do we need transgenic plants ?•Improvement of agricultural value of plant (resistance to herbicides, resistance to insect attack, Bacillus thuringiensis toxin) •living bioreactor, produce specific proteins•studying action of genes during development or other biological processes (knock-out plants, expression down-regulated)

Transgenic PlantsAdvantages & Disadvantages

• Advantages:- Plant cells are totipotent: whole plant can be regenerated

from a single cell (engineered cells - engineered plants)- Plants have many offspring: rare combinations and

mutations can be found- Transposons used as vectors

• Disadvantages:- Large genomes (polypoid - presence of many genomes in

one cell) - plants regenerating from single cells are not genetically

homogenous (genetically instable)

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Genetically modified plants

What About the Term Genetic Engineering?

Genetic engineering is the basic tool set of biotechnologyGenetic engineering involves:Isolating genes Modifying genes so they function better Preparing genes to be inserted into a new species Developing transgenes

Plant Genetic Engineering Process

Cell

Extracted DNA

Cell divisionTransgenic plant

A single gene

Transformation

Plant cell

Production of transgenic plants

Isolate and clone gene of interest

Add DNA segments to initiate or enhance gene expression

Add selectable markers

Introduce gene construct into plant cells (transformation)

Select transformed cells or tissues

Regenerate whole plants

• Prepare tissue for transformation• Tissue must be capable of developing into

normal plants• Leaf, germinating seed, immature embryos

• Introduce DNA• Agrobacterium or gene gun

• Culture plant tissue• Develop shoots & Roots

• Screening of putative transformants• Field test the plants

Developing Transgenic Plant

Introducing the Gene

Selection of

transformants

Introduction of the

gene

Create transforma

tion cassette

STEPS

Plant transformation

• DNA delivery systems must be • Simple • Efficient and preferably inexpensive • The method must be available for use either because

it is in the public domain or because it can be licensed • System of choice depends on

• the target plant • its regeneration system

A. Cell culture and plant regeneration systemB. Cloned DNA to be introduced

1. selectable marker gene• kanamycin or G148 resistance: neomycin, phosphotransferase (NPTII),

hygromycin B: hygromycin phosphotransferase (HygB)gentamicin: gentamicin acetyltransferase

• streptomycin: streptomycin phosphotransferase• Bialophos: BAR

2. promoter (constituitive or inducible), coding regionC. Method of delivery of DNA into the cellD. Proof of transformation of plant

Requirements for plant transformation

Selectable marker geneScreening & Selection of transformant

Positive selectionPMI (phospho- mannose isomerase) Plant cells without this enzyme are unable to survive in a tissue culture medium containing mannose-6-phosphate as a sole carbon source.

Positive selection

Removable selectable marker geneGenes using the Cre-lox system or transposable elements

Selectable marker gene

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Transformation Cassettes

Contains

1. Promoter• Regulatory sequence/initiation site

2. Gene of interest• The coding region and its controlling elements

3. Selectable marker• Distinguishes transformed/untransformed plants

4. Insertion sequences• Aids Agrobacterium insertion

P G M TATA

• Constitutive promoter • CaMV 35S : suitable for expression of foreign genes in dicots:

• The maize ubiquitin promoter, also a constitutive promoter which

• drives strong expression of transgenes in monocots.

• Organ/ tissue specific promoters • Vicilin and phytohemaglutinin, glutenin promoters seed specific expression

• a-amylase promoter for expression in the aleurone of cereal grains;

• Patatin promoter for tuber specific expression in potatoes and the RuBisCo promoter for green tissue specificity

Commonly used promoters

Selectable Markers

•allow the selection of transformed cells, or tissue explants

•by ability to grow in the presence of an antibiotic or a herbicide.

•frequently used - kanamycin and hygromycin

Screen able markers

•encode gene products whose enzyme activity can be easily assayed

•allowing not only the detection of transformants

•also estimation of the levels of foreign gene expression in transgenic tissue

• markers such as GUS, luciferase or -galactosidase allow screening for enzyme activity by

histochemical staining or fluorimetric assay of individual cells

•can be used to study cell-specific as well as developmentally regulated gene expression

Marker genescreen able marker & selectable marker

Plant Transformation Methods

• PEG

• Calcium phosphate

• Artificial lipids

• Proteins

• Dendrimers

ChemicalPhysical

• Agrobacterium Tumefaciens

• Agrobacterium Rhizogenes

• Virus-mediated

Biological & In-planta

• Microinjection

• Biolistics - gene gun/Particle

bombardment

• Electroporation

• Microinjection

• Silica/carbon fibers

• Lazer mediated

Physical

Chemical Biological In planta

Physical Methods of Transformation

•Microinjection

•Biolistics - gene gun/Particle bombardment

•Electroporation

•Silica/carbon fibers

•Lazer mediated

• 1970s, 1990 versatile method – in vivo (skin and muscles)• short pulses of high voltage to carry DNA across the cell

membrane• to assist the uptake of useful molecules such as a DNA vaccine

into a cell• Parameters, electrical field strength [V/cm], pulse length

Electroporation

Drawbacks•Limited effective range of ~1 cm between the electrodes•Surgical procedure is required to place the electrodes deep into the internal organs•High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating electron-avalanche transfection

Electroporation Technique

Duracell

DNA containingthe gene of interest

Plant cell

Protoplast

Power supply

DNA inside the plant cell

The plant cell withthe new gene

This electroporator is for low-current applications such as those using small electrodes

Microinjection

MAJOR LIMITATIONS:shallow penetration of particlesassociated cell damagethe inability to deliver the DNA systemicallythe tissue to incorporate the DNA must be able to regenerate and the expensive equipment .

• Simplest method of direct introduction of therapeutic DNA into target cells

• Looks like a pistol but works more like a shotgun with “Golden pellets”

• First described as a method of gene transfer into plants

• John Sanford at Cornell University in 1987

• Particle bombardment -physical method of cell transformation in which high density and sub-cellular sized particles are accelerated to high velocity in order to carry DNA or RNA into living cells

Particle gun

Particle gun

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For particle bombardment, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. In the early days, the force used to accelerate the particles was a .22 caliber blank. Today, most devices use compressed helium.

The DNA-coated particles can end up either near or in the nucleus, where the DNA comes off the particles and integrates into plant chromosomal DNA.

The particles punch holes in the plant cell wall and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction and the biological incompatibilities associated with Agrobacterium are avoided.

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DNADelivery

Agrobacterium Particle gun

•Agrobacterium Tumefaciens

•Agrobacterium Rhizogenes

•Virus-mediated

2. Biological Methods

Agrobacterium- mediated

• In the laboratory, bacteria are co-

cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells.

• Most of the native transferred bacterial DNA is replaced with genes of interest

• Agrobacterium is a soil borne gram-negative bacterium, that has a unique ability to introduce part of its DNA into plant cells.

Agrobacterium tumefaciens

• Wild type Tk plasmid = 200 kb – too large for cloning• Intermediate shuttle plasmid is used to cut in Gene of Interest• VIR genes must be removed for genetic engineering• LB and RB are required for insertion and recombination with

plant genome

• Insertion into plant host is random (sort of)

• First cloned gene – luciferase in tobacco plant

Mechanism of Agrobacterium- mediated transformation

Mechanism of Agrobacterium- mediated transformation

Mechanism of Agrobacterium- mediated transformation

Chemical Transformation

• PEG

• Calcium phosphate

• Artificial lipids

• Proteins

• Dendrimers

PEG mediated

• It is the oldest (direct DNA) reliable method for plant transformation. In the first report (Krens et al. 1982 Nature 296:72), Agrobacterium Ti plasmid was introduced into petunia protoplasts. Formation of tumors, opine synthesis and Southern blot provided the verification, which is an extensive and complete analysis to show success of transformation.

• The first report of generating transgenic plants using this method was provided by Paszkowski et al. (1984). They regenerated transformed protoplasts into plants that were kanamycin (drug) resistant.

• This method has been very useful and applied to several plant species.

• But it is a tedious procedure!

Meristem transformation

Floral dip method

Pollen transformation

Non-tissue culture basedIn-Planta Transformation

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Vacuum Infiltration

– Plant leaf disks are placed in a suspension of bacteria and vacuum pulled

– Air is release like a sponge being squeezed– Vacuum is released and solution floods tissue– Plant disk is cultured

Floral Dip

– Simple submersion of plant into bacterium suspension

– No vacuum is needed

– Conducted with plants grown until just flowering

– Progeny seeds are harvested and germinated using selective antibiotic

Analysis of T0 plants

Yield characters

Physiology

Morphology

GUS expression

Gene expression

Confirmation with selectable marker, Screenable marker, Negative & Positive control

GFP expression in soybean tissue

Shows variability in expression pattern standard illumination on left – gfp illumination on right

• Few Examples of Transgenic crop

Golden Rice Synthesis

Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes

Phytoene synthase &Lycopene-b-cyclase Carotene desaturaseT DNA

Germ-line transformation with agrobacterium

XCross

T-formed rice with genes T-formed rice with gene

Progeny rice plant with complete b carotene pathway

• Golden rice contains increased levels of pro-vitamin A .

• Traditional rice is white (a).

• The prototype of golden rice was developed in 2000 and is a light yellow color (b). It contains 1.6 mg/g of carotenoid.

• In 2005, new transgenic lines were developed that dramatically increased the amount of carotenoid synthesized, making the rice a deep golden color (c).

• This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene – trial

Golden Rice

World's First Blue Roses On Display In JapanDanielle Demetriou, Daily Telegraph, October 31, 2008, See the rose at

http://www.telegraph.co.uk/news/worldnews/asia/japan/3327043/Worlds-first-blue-roses-on-display-in-Japan.html

Tokyo, Japan - World's first blue roses have been unveiled to the public for the first time at an international flower fair in Japan, following nearly two decades of scientific research. The blue-hued blooms are genetically modified and have been implanted with a gene that simulates the synthesis of blue pigment in pansies.

Its scientists successfully pioneered implanting into the flowers the gene that produces Delphinidin, the primary plant pigment that produces a blue hue but is not found naturally in roses.The world's first genetically modified blue roses were unveiled in the laboratory four years ago, although further research was required to make them safe to grow in nature.

The Blue Rose was developed by Suntory Flowers

Tearless Onion

Dr EadyCrop & Food Research in New Zealand and his collaborators in Japan

As onions are sliced, cells are broken, alliinases - break down aa sulphoxides - generate sulphenic acids - unstable - rearrange into a volatile gas - syn-propanethial-S-oxide – diffuses by air - reaches the eye - reacts with the water to form a diluted solution of sulphuric acid - Tear glands produce tears to dilute and flush out the irritant

Final Test of the TransgenicConsumer Acceptance

RoundUp Ready Corn

Before After

Thank you!