Plant Transformation

Indirect and direct methods

Indirect method: bacterial mediators are employed.

• Agrobacterium tumefaciens (leaf tissues)

• Agrobacterium rhizogenes (root tissues)

Direct methods: no mediator are used.

• protoplast transformation (microinjection, chemical or electroporation)

• microprojectile bombardment (gene gun)

Proplast transformation

Microinjection Protoplasts attached to slide by embedding in agarose using a holding pipet DNA injected using injection pipet

Proplast transformation

The disadvantages of protoplast transformation are:

• Regeneration can be problematic

• Transient expression level is low

• Viability of protoplasts are strongly reduced after transformation

• Procedures were performed

• Lead to somaclonal variation

Microparticle bombardment: gene gun

Using a gene gun directly shoots a piece of DNA into the recipient plant tissue.

Tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. Most devices use compressed helium as the force to accelerate the particles.

The particles punch holes in the plant cell and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction.

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

Microparticle bombardment: mechanism

DNA-coated microcarriers are loaded on microcarrier.

Micro-carriers are shot towards target tissues during helium gas decompression.

A stopping screen placed allowing the coated microprojectiles to pass through and reach the target cells.

Microprojectile bombardment: gene gun

Advantage:

• This method can be use to transform all plant species.

• No binary vector is required.

• Transformation protocol is relatively simple.

Disadvantage:

• Difficulty in obtaining single copy transgenic events.

• High cost of the equipment and microcarriers.

• Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.).

• Transfer DNA is not protected.

• Lead to somaclonal variation

Agrobacterium-mediated transformation

Used to overcome limitations of conventional breeding

• Slow and uncertain

• Requires sexual crosses followed by repeated backcrosses

• Restricted to plants that can sexually hybridize

• Other genes are transferred also

First published in 1984 by Schell et al. and Horsch et al.

For Agrobacterium-mediated transformation, although the transformation for monocots is still unsatisfactory, it has become the method of choice for years because it is easier to handle and rather inexpensive to perform.

Agrobacterium-mediated transformation

The unique advantages of Agrobacterium-mediated transformation are:

1. Simple and low cost (‘poor man’s vector’)

2. DNA was transferred with defined ends

3. Transgene is linked with the transformation marker

4. Higher frequency of stable transformation with higher ratio of single copy insertions

5. Lower incidence of transgene silencing

6. Capable of transferring long stretches of T-DNA (>150 kb)

Key steps for traditional Agrobacterium-mediated transformation

Agrobacterium tumefaciens

Gram negative bacterium

Soil borne

Naturally attacks certain plants at wound site causing crown gall

Infection results in formation of galls on lower trunk, soil level, roots

Galls produce opines - specialized nitrogenous compounds

Opines used by Agrobacterium as source of nutrients

In 1897, Fridiano Cavara identified a flagellate, bacilloid bacterium as a casual agent of crown gall of grape.

crown gall

The initiation of Agrobacterium infection

Agrobacteria usually infects plants from their wounds, which occurred quite frequent after frost.

In practice, protection from subfreezing winter temperatures and control of chewing insects and nematodes can prevent infection by Agrobacteria.

Crown gall disease is not generally fatal, but it will reduce plant vigor and crop yield, and crown galls will attract other phytopathogens or pests.

In some cases, necrosis or apoptosis is observed after Agrobacterium infection.

Agrobacterium causes crown galls by transforming plants

Ti (Tumor inducing) plasmid T-DNA: transfer DNA

plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g. Saccharomyces cerevisiae).

Ti plasmid of Agrobacteria

Transformation involves in transferring a segment of DNA from Ti plasmid of Agrobacteria into

plant genome.

T-DNA

T-DNA carries genes involved in the synthesis of plant growth hormones (auxin, auxin synthesis;

cyt, cytokinin synthesis) and the production of low molecular weight amino acid and sugar

phosphate derivatives called opines (ocs, octopine; mas, mannopine; and ags, agropine).

Agrobacteria are usually classified based on the type of opines specified by the bacterial T-DNA.

(200~800 kb in size)

25 bp repeat 25 bp repeat

Three components are required for Agrobacterium transformation

Vir (virulence) genes: includes seven major loci, A, B, C, D, E, G, H in Ti plasmid

Chv (chromosome virulence) genes: involving in chemotaxis and attachment in Agrobacteria chromosome

T-DNA: 10~30kb (less than 10% of the Ti plasmid)

Why Agrobacterium is being used for producing transgenic plants?

• T-DNA element is defined by its borders but not the sequences within.

• So researchers can substitute T-DNA coding region with any DNA sequence without any effect on its transfer from Agrobacterium to the plant.

• Although right border and left border are required to delimit the transferred segments, the T-DNA itself has no effect on the efficiency of transfer.

• Therefore, researchers put DNA of interest to replace most of the T-DNA, making Agrobacterium becoming vectors for genetic transformation of plants.

Steps of Agrobacterium-plant cell interaction

1. Plant cell – Agrobacterium cell recognition

2. Signal transduction and transcriptional activation of vir genes

3. Conjugal DNA metabolism

4. Intercellular transport

5. Nuclear import

6. T-DNA integration into plant chromosomal DNA

Cell-cell recognition

acetosyringone

T-pilus

NLS: nuclear localization signal NPC: nuclear pore complex

Plant signals

Wounded plants secrete sap with acidic pH (5.0 to 5.8) and a high content of various phenolic compounds (lignin, flavonoid precursors) serving as chemical attractants to Agrobacteria and stimulants for vir gene expression.

• phenolics, sugars, acidity

Among these phenolic compounds, acetosyringone (AS) is the most effective.

Sugars like glucose and galactose also stimulate vir gene expression when AS is limited or absent.

Low opine levels further enhance vir gene expression in the presence of AS.

acetosyringone

Results of vir gene expression

Expression of vir genes result in

1. Production of T-strands

2. Formation of a bacterium-to-host cell channel (T-pilus)

3. Exportation of the T-complex into the plant cell

Structure of the T-DNA

Induction of vir gene expression ultimately results in the production of a T-DNA copy that is capable of genetically transforming plant cells.

The borders of a T-DNA element are defined as conserved 25-bp sequences that delimit the transferred segments.

The existence and orientation of right border is absolutely required for Agrobacterium pathogenicity but not the left border. Inversion of the right border leads to reduced virulence and transfer of nearly the entire Ti plasmid instead of the T-DNA region.

Transfer of the T-DNA is polar from right to left.

Nuclear import

• Because the large size of T-complex (50,000 kD, ~13 nm in diameter), the nuclear import of T-complex requires active nuclear import.

• The T-complex nuclear import is presumably mediated by the T-complex proteins, VirD2 and VirE2. Both of them have nuclear-localizing activities.

T-DNA integration is not highly sequence-specific

There is no apparent preferences for T-DNA integration. However, sequences flanking

centromere are being integrated less frequently.

About 40% of the integrations are in genes and more of them are in introns.

Deletions of LB and RB after integration are common. LB deletions are more common than RB.

Generally only 24% of LB and 19% of RB are integrated as full-length sequences.

Transgene integration

Comparing with direct DNA transfer methods, Agrobacterium transformation usually resulted in a

lower copy number of integrated transgenes.

However, Agrobacterium transformation can result in tandem copies of a few T-DNA integrated

at a single locus.

Also, about 30% of T-DNA integration events would result in activation of a promoterless

reporter transgene, suggesting that T-DNA may preferentially integrate into transcriptionally

active regions of the genome.

Nester, E.W. Univ . of Washington ; Nobel prize in near future

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of dicot. Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome

Agrobacterium tumefaciens

Ti Plasmid induced Crown gall

Impacts of Agrobacterium in Agriculture

Agrobacteria was used in genetically modified crop (GM)

1) Insect-resistant cotton, corn, and soybean (Bacillus thuringiensis(Bt) cotton)

2) Herbicide tolerant corn 3) Herbicide tolerant soybean

1) 14 millions farmers in 25 countries since 1995 2) Yields increases from 16% to 85% 3) 25%-58% decreases in the number of tillage operation 4) Decreases in the amount of insecticide or number of insecticide

applications 5) Reductions of 83% and 100% in the use of herbicide toxicity

classes II and III 6) Monsanto develops seeds that would double the yields of corn,

soybeans and cotton by 2030 and would require 30 percent less water, land and energy to grow

Reports in Nature Biotechnology, 28, 2010,319-321 indicate positive impact of GM crops

Agroinfiltration (Agrotransformation)

-Modified Ti Plasmid -Maintain membrane transport protein VirD, VirE

Binary Vector (Modify Ti Plasmid)

Agroinfiltration

Nucleus

Ti

35 S GFP Cloning Sites NOS

Binary Vector GFP mRNA

Protein Synthesis

Choices of binary vectors

Binary vectors like pCAMBIA series were constructed from pPZP with more choices of selection markers (nptII, bar, or hpt) and reportors (gus or gfp).

Newer versions of binary vectors have positioned their selection markers close to the left border to ensure that the selection marker is the last to be transferred into plants. Therefore, all plants selected will have a complete T-DNA.

Plant Transformation II

Successful transformants

Herbicide tolerance: bacterial EPSP

Virus resistance: viral coat protein

Insect resistance

• BT toxin insertion (Bacillus thuringiensis)

• Lepidoptera

Bacterial and fungal resistance

Postharvest qualities

• Delay ripening: ethylene

• Delay flower senescence

Improve nutritional quality

• High methionine and high lysine seeds

• Golden rice: beta-carotene

Chapter 18-Genetic Engineering of Plants: Applications

The general principle of producing a GMO is to add new genetic material into an organism's genome.

This is called genetic engineering and was made possible through the discovery of DNA and the creation of the first recombinant bacteria in 1973; an existing bacterium E. coli expressing an exogenic Salmonella gene.[

Herbert Boyer then founded the first company to use recombinant DNA technology, Genetech, and in 1978 the company announced creation of an E. coli strain producing the human protein insulin.

In the United States the United States Department of Agriculture (USDA) reports on the total area of GMO varieties planted. According to National Agricultural Statistics Service, the states published in these tables represent 81- 86 percent of all corn planted area, 88 - 90 percent of all soybean planted area, and 81- 93 percent of all upland cotton planted area (depending on the year).

Genetically Modified Organisms (GMO)

Chapter 18-Genetic Engineering of Plants: Applications

• 80 genetically engineered plants approved in the US

• 132 genetically engineered plants approved in the world

• Insect-, pathogen-, and herbicide-resistant plants

• Stress- and senescence-tolerant plants

• Genetic manipulation of flower pigmentation

• Modification of plant nutritional content

• Modification of plant food taste and appearance

• Plant as bioreactors

• Edible vaccines

• Renewable energy crops

• Plant yield

Genetically Modified Organisms (GMO)

Bacillus thuringiensis (Bt)

Bacillus thuringiensis (Bt) is a gram positive soil bacterium that produces an insecticidal crystal protein from the cry genes (over 100 genes in different strains)

Bt crystal proteins are toxic to insects such as the ECB (European Corn Borer) and other related pests.

When ingested, the Bt crystal toxin is activated by enzymes in the insect's gut. The activated toxin attaches to specific gut receptors, destroys cells in the gut wall, puncturing the gut and poisoning the insect.

Lepidopteran insects like corn rootworm, cotton bollworm, tobacco budworm, etc., cause combined damages of over $7 Billion dollars yearly in the US.

Solution: "In Plant Protection"

Isolate the cry genes from bacteria, and introduce into corn plants = transgenic Bt-corn, followed by Bt-cotton, etc.

These genetically modified plants make the cry proteins in each and every cell of the plant.

Reduces or eliminates the traditional spraying of Bt in the field.

Genetically engineered Papaya to resist the Papaya Ringspot-Virus by overexpression of the virus coat protein

Development of stress- and senescence-tolerant plants: genetic engineering of salt-resistant plants

• Overexpression of the gene encoding a Na+/H+ antiport protein which transports Na+ into the plant cell vacuole.

• This has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)

Development of stress- and senescence-tolerant plants: genetic engineering of flavorful tomatoes

• Fruit ripening is a natural aging or senescence process that involves two independent pathways, flavor development and fruit softening..

• Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow for safe shipping of the fruit.

• Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and contribute to fruit softening.

• In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping of the fruit, polygalacturonase gene expression was inhibited by introduction of an antisense polygalacturonase gene and created the first commercial genetically engineered plant called the FLAVR SAVR tomato.

Methods Used in Plant Transgenesis

Polygalacturonase

Genetic manipulation of flower pigmentation

New pathway in

petunia created by

the maize DFR gene

Manipulation of the anthocyanin biosynthesis pathway

Introduction of maize dihydroflavonol 4-reductase (DFR) into petunia produces a brick red-orange transgenic petunia

Novel flower colors in the horticultural industrial are big money makers.

World’s first blue rose

Blue rose

While blue roses do not exist in nature, falsely-named blue roses were traditionally created by dyeing white roses, since the flower lacks the specific gene that has the ability to produce "true blue" colors.

After thirteen years of collaborative research by an Australian company - Florigene, and a Japanese company - Suntory, a blue rose was created in 2004 employing metabolic engineering.

Years of research resulted in the ability to insert a gene for the plant pigment delphinidin cloned from the petunia and thus inserted into an Old Garden Cardinal de Richelieu rose.

Obtaining the exact hue was difficult because amounts of the pigment cyanidin were still present, so the rose was darker in color than "true blue“.

Recent work using RNAi technology to depress the production of cyanidin has produced a mauve colored flower, with only trace amounts of cyanidin.[

World’s first blue rose

delphinidin gene

joint venture between the Australian based Florigene and the Japanese Suntory company (2004)

Modification of plant nutritional content: increasing the vitamin E content of plants

• Plants make very little vitamin E (alpha-tocopherol) but do make gamma-tocopherol; they do not produce enough of the methyltransferase (MT).

• The MT gene was identified and cloned in Synechocystis (Cyanobacteria) and then in Arabidopsis.

• The Arabidopsis MT gene was expressed under the control of a seed-specific carrot promoter and found to produce 80 times more vitamin E in the seeds.

The Golden Rice Story: -carotene production

Vitamin A deficiency is a major health problem

• Causes blindness

• Influences severity of diarrhea, measles

124 million children worldwide are deficient in vitamin A, which leads to death and blindness.

For many countries, the infrastructure doesn’t exist to deliver vitamin pills

Improved vitamin A content in widely consumed crops an attractive alternative

Mammals make vitamin A from β-carotene, a common carotenoid pigment normally found in plant photosynthetic membranes.

Here, the idea was to engineer the β-carotene pathway into rice.

The transgenic rice is yellow or golden in color and is called “golden rice”

Golden Rice: -Carotene Pathway in Plants

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:

Rice lacks

these enzymes

Normal

Vitamin A

“Deficient”

Rice

The Golden Rice Solution: -Carotene Pathway Genes Added

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;

performs both functions

Daffodil gene

Vitamin A

Pathway

is complete

and functional

Golden

Rice

Golden Rice