Genetic Engineering in Genetic Engineering in PlantsPlants

Genetic engineering, is the direct human manipulation of an organism's genome using modern DNA technology. It is also called genetic modification.

It involves the introduction of foreign DNA or synthetic genes into the organism of interest or by altering the sequence of a gene to convert it to a different gene or deletion of an undesirable gene.

The resultant DNA is called recombinant DNA. Thus it is also called recombinant DNA Technology.

The introduction of new DNA does not require the use of classical genetic methods, however traditional breeding methods are typically used for the propagation of recombinant organisms.

Genetic Engineering: Genetic Engineering:

In genetic engineering:

*The gene that is transferred into a new host is known as transgene [

*The organisms developed after successful gene transfer are known as transgenics

*The plants which carry the stably integrated foreign gene is called transformed plants.

When a foreign gene is inserted into an organism it is called transgenic organism.

When an undesirable gene is deleted from an organism it is known as genetically modified organism (GMO) but not a transgenic organism

Thus, all transgenics are GMOs but all GMOs are nor transgenics

Marker gene(s)Marker gene(s)

Marker gene(s) are used for monitoring and detection of plant transformation systems in order to know whether the DNA has been successfully transferred into recipient cells.

Types of Marker gene(s)Types of Marker gene(s)Reporter gene: A gene that encodes a product that can be readily assayed. Eg. Β-glucuronidase (GUS), luciferase, green flourescent protein (GFP) etc.

Selectable marker: A gene whose expression allows to identify a specific trait or gene in an organism. Eg.Neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase (hpt) etc.

Gene transfer methodGene transfer method 1. Vector mediated gene transfer

A. Agrobacterium mediated gene transferB. Virus mediated gene transfer

2. Vectorless or direct gene transfer methodA. ElectroporationB. Particle bombardment/microprojectileC. MacroinjectionD. MicroinjectionE. Liposome mediated transformationF. SonicationG. PEG mediated gene transfer

AgrobacteriumAgrobacterium mediated mediated Genetic transformationGenetic transformation

► A. Tumefaciens induces tumors called crown gall.► A. Rhizogenes causes hairy root disease. ► In addition to the nuclear DNA, this bacterium

contains a small circular DNA called plasmid. ► Large plasmids are called tumor inducing plasmids

(ti plasmids) and root inducing plasmids (ri plasmids)

Crown Gall – Plant tumorCrown Gall – Plant tumor

Fig. Ti plasmid

Agrobacterium tumefaciensAgrobacterium tumefaciens

Agrobacterium Infection and T-DNA Transfer into Plant Cells

Tzfira and Citovsky (2002) Trends Cell Biol 12:121-129

AgrobacteriumAgrobacterium mediated Genetic mediated Genetic transformationtransformationcan be modified:1.Agrobacterium plasmids are disarmed by deleting naturally occurring T-DNA encoded oncogenes and replacing them with foreign genes of interest2.Restriction enzyme sites are also added. ►The modified plasmid with desired gene(s) can be transferred to any plant for subsequent integration and expression. The engineering capacity of Agrobacterium has been exploited in the Agrobacterium mediated transformation

Steps: A: Isolation of desired gene and place into a plasmid.

B: Transfer of plasmid into bacterium.

C: Co-cultivation of plant cell with bacterium so that bacterium can infects plant cells transferring desired gene into chromosome.

D: In vitro culture of hybrid. E: Regeneration of new plantlet.

F: Transgenic plant carring desired gene.

Steps of Steps of AgrobacteriumAgrobacterium mediated mediated Genetic transformationGenetic transformation

Fig. Agrobacterium mediated gene transfer method

Particle BombardmentParticle BombardmentThe Particle bombardment device, well

known as the gene gun, was developed to enable penetration of the cell wall so that genetic material containing a gene of interest can be transferred into the cell. This physical direct gene transfer method, gene gun is used for genetic transformation of several organisms to introduce a diverse range of desirable traits. Plant transformation using particle bombardment follows the same steps as in Agrobacterium mediated transformation method:

 i. Isolation of desired genes from the source organism

ii. To develop a functional transgenic construct including the selected gene of interest; promoters to drive expression; modification of codon, if needed, to increase successful protein production; and marker genes to facilitate tracking of the introduced genes in the host plant

iii. Insertion of transgenic construct into a useful plasmid

iv. Introduce the transgenes into plant cells v. Regenerate the plants cells, andvi. Test the performance of traits or gene expression

under in vitro, greenhouse and field conditions.

In particle bombardment method, 1-2 µm tungsten or gold particles (called micro-projectiles) coated with genetically engineered DNA are accelerated with air pressure at high velocities and shot into plant tissues on a Petri-plate, as shown in Figure . This is the second most widely used method, after Agrobacterium mediated transformation, for plant genetic transformation.

The device accelerates particles in one of the two ways: (1) by means of pressurized helium gas or (2) by the electrostatic energy released by a droplet of water exposed to high voltage. The earlier devices used blank cartridges in a modified firing mechanism to provide the energy for particle acceleration, and thus, the name particle gun. It is also called Biolistics, Ballistics or Bioblaster.

The microcarriers (or microprojectiles), the tungsten or gold particles coated with DNA, are carried by macrocarriers (macro projectiles) which are then inserted into the apparatus and pushed downward at high velocities. The Macro-projectile is stopped by a perforated plate, while allowing the microprojectiles to propelled at a high speed into the plant cells on the other side. As the micro-projectiles enter the plant cells, the transgenes are free from the particle surface and may inserted into the chromosomal DNA of the plant cells. Selectable markers help in identifying those cells that take up the transgene or are transformed. The transformed plant cells are then regenerated and developed into whole plants by using tissue culture technique.

Figure 24.2: Diagrammatic illustration of gene transfer using Gene Gun meth

The Hand-Held Gas Gun

Purpose:Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue)

One interesting use:Making DNA Vaccines

The technique has many advantages and can be used to deliver DNA into virtually all the tissues, like immature and mature embryos, shoot-apical meristem, leaves, roots etc. Particle bombardment methods are also useful in the transformation of organelles, such as chloroplasts, which enables engineering of organelle-encoded herbicide or pesticide resistance in crop plants and to study photosynthetic processes. Limitations to the particle bombardment method, compared to Agrobacterium-mediated transformation, include frequent incorporation of multiple copies of the transgene at a single insertion site, rearrangement of the inserted genes, and insertion of the transgene at multiple insertion sites. These multiple copies can be associated with silencing of the transgene in subsequent progeny. The target tissue may often get damaged due to lack of control of bombardment velocity.

TRANSGENIC PLANTSTRANSGENIC PLANTS In 2004, 120 million acres were planted to transgenic crops.

BT production (pest resistant) Crops with added nutrients (3rd world) Crops that grow faster and use less water Crops with greater yield Herbicide resistance Altered flower color Virus resistance Fungal resistance Parasite resistance Resistance to cold and freezing Flowers that smell pretty (again!) GENETICALLY MODIFIED FOODS

Virus resistance

Source: Monsanto

Herbicide resistant crops current: soybean, corn, canola, cotton, alfalfa coming: sugarbeet (on hold), lettuce, strawberry, wheat (on hold), Turf grass resistance gene from bacteria is source

papaya, squash, potato resistance gene from a virus

Insect ResistanceInsect Resistance

B. thuringiensis (commonly known as 'Bt') is an insecticidal bacterium, marketed worldwide for control of many important plant pests - mainly caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae, and simuliid blackflies that vector river blindness in Africa. Bt products represent about 1% of the total ‘agrochemical’ market (fungicides, herbicides and insecticides) Bt cotton

Genetically Modified Genetically Modified FoodFoodCan animal genes be jammed into

plants? Would tomatoes with catfish genes taste fishy? Have you ever eaten a genetically modified food? The answers are: “yes”, “no” and almost definitely “yes”

Despite dire warnings about "Frankenfoods“, there have been no reports of illness from these products of biotechnology.

First biotech plant product – Flav’r Sav’r tomato

In 1994, the Flavr SavrFlavr Savr tomato was introduced as the first GM food. It is “tastier, firmer and fresher” than the average tomato.

Bt Corn ReducesBt Corn Reduces::Insecticide MycotoxinInsecticide MycotoxinApplication ProductionApplication Production

Bt BrinjalBt BrinjalThe Bt brinjal is a suite of transgenic brinjals (also known as an eggplant) created by inserting a crystal protein gene (Cry1Ac) from the soil bacterium Bacillus thuringiensis into the genome of various brinjal cultivars. The insertion of the gene, along with other genetic elements such as promoters, terminators and an antibiotic resistance marker gene into the brinjal plant is accomplished using Agrobacterium-mediated genetic transformation. The Bt brinjal has been developed to give resistance against lepidopteron insects, in particular the Brinjal Fruit and Shoot Borer (Leucinodes orbonalis)(FSB). When fruit and shoot borer larvae feed on Bt brinjal plants, they ingest the Bt protein Cry1Ac along with plant tissue. In the insect gut, which is alkaline with a pH >9.5, the protein is solubilized and activated by gut proteases. The Bt protein binds to specific receptor proteins present in the insect membrane, resulting in pore formation in the membranes. This leads to disruption of digestive processes, paralysis, and subsequent death of the fruit and shoot borer larvae.

Strawberry plants developed to tolerate cold temperatures & withstand unexpected frost that could destroy seedlings

+

A strawberry resistant to frost

= 5

Arctic fish DNA

Golden RiceGolden RiceGolden rice produces beta-carotene, a precursor of vitamin A, in the grain. The idea is to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A.Published in Science (2000) an eight-year project by Ingo Potrykus & Peter BeyerFirst field trials 2004Potrykus- golden rice to be distributed for free to subsistence farmers.

A US$10,000 cut off between humanitarian and commercial use was set. So if a farmer of golden rice makes less than $10,000 per year, no royalties need to be paid. In addition, farmers are permitted to keep and replant seed.

Image: International Rice Research Institute (IRRI)

Edible Vaccines – A Biopharming DreamBiotech Plants Serving Human Health Needs

• A pathogen protein gene is cloned• Gene is inserted into the DNA of plant (potato, banana, tomato)• Humans eat the plant • The body produces antibodies against pathogen protein• Human are “immunized” against the pathogen• Examples:

DiarrheaHepatitis BMeasles

pGlo – GfppGlo – GfpGreen fluorescent proteinGreen fluorescent protein

FluorescentFluorescentIn the laboratory, fluorescence is easily achieved by exposing the protein to long range UV light or “ black" light. The fluorophore absorbs light in the UV-B region (395 nm.. plus a smaller absorbance peak at 470 nm) It emits light (fluoresces) at 509 nm, which is in the green part of the visible spectrum

Gfp and Land MinesGfp and Land Mines Neal Stewart at the

University of North Carolina is developing plants that can detect land mines

Plants could be ideal biosensors for land mines as seeds would be spread widely and evenly in a suspect field

The gene that can announce the presence of land mines is gfp

The gene will be expressed in the presence of a land mine

Biotechnology Biotechnology BreakthroughsBreakthroughs

Potatoes with higher solid contentGarlic that lowers cholesterolFruits and vegetables that reduce

risks of cancer and heart disease

14.

Great loves and great achievements involve

great risk

When you lose, don’t lose the lessons…………..

When you realize you’ve made a mistake, take immediate steps to

correct it