14 Genetically Modified Crops – Methods of Gene Transfer

Dr. Sunil Mittal

1. Introduction

2. Vectors for Plants

2.1 Plasmid based vectors

3.Vectorless or direct DNA transfer methods

3.1 Physical gene transfer methods

3.2 Chemical Methods of gene transfer

4. Applications

5. Advantages of Genetically Modified Crops

6. Disadvantages of Genetically Modified Crops

7. Environmental Problems Concluding remarks

1.Introduction

Definition: Gene transfer is a process that involves the transfer of foreign DNA into host cell so as to obtain desired product. Gene transfer via sexual and vegetative production is well established. However, it takes long time. Therefore, scientists are currently using genetic engineering techniques for gene transfer in plants.

The methods for the production of genetically engineered plants/crops require the involvement of various vectors. Some of them are discussed briefly in the upcoming sections.

Vector is a round shaped deoxyribonucleic acid that has capacity to independently exist as well as replicate. It acts as an artificial vehicle to carry genetic information from donor to receptors. Two types of vectors are used specially in plants such as Agrobacterium Ti and Agrobacterium Ri plasmids. Both Ti and Ri plasmids have almost same general characters like T region that can be transferred in any species and then integrated in genome. Vectors have some unique features such as low molecular weight, genetic marker and unique restriction enzymes. There are different types of gene transfer processes in plants that are either vector mediated or vectorless. All these processes are discussed in detail in the upcoming sections.

2.Vectors for Plants

Generally, Plasmid and viral genome based vectors are used for plants.

2.1 Plasmid based vectors

Ti plasmid based vectors

Ti plasmid is found in the Agrobacterium tumefaciens. This strain is naturally occurring gram negative soil bacteria. It has two species like A. tumefaciens and A. rhizogenes. Both act as natural genetic engineers because they have ability to transform plants. A. tumefaciens is responsible for crown gall disease in plants by inserting T – DNA from plasmids with size up to 200kb. Size of plasmids always varies in range of 15 to 30 kb according to strain.

Fig 1: Different regions of T-DNA (https://en.wikipedia.org/wiki/Ti_plasmid)

Genes required for the transfer of T-DNA alongwith the mechanism

Vir genes are required for the transfer of T-DNA into host cells. Vir genes are also known as virulence genes and conatin virA, virB, virC, virD, virE, virG and virH.
These genes are involved only in the DNA transfer. They do not get transferred alnwith T-DNA.
The process of T-DNA transfer is initiated with the plant wounding i.e., introducing the bacteria into plant wound.
Wounding leads to the production of some substances such as acetosyringone. These compounds are identified by vir genes.
VirA gene is a member kinase that senses phenolic compounds and gets autophosphorylated on histidine residue on the binding of acetosyringone.
Phosphate group of VirA is transferred to aspartate residue of virG protein.
VirA and VirG act together for the activation of other vir genes.
T-DNA is nicked from Ti plasmid by virD1 and virD2 (endonuclease).
VirD2 remains attached to the nicked T-DNA at its 5’ end.
Nicked DNA gets placed from the plasmid and produces single stranded T-DNA.
VirE2 binds to the single stranded T-DNA so as to protect it from the attack of nucleases.
Vir E2 helps in the transport of T-DNA into the nucleus of plant cell.
during the transfer of T-DNA is required and that energy is provided by virB11 protein (ATPase activity).
VirF is also in T-DNA delivery.
The process of T-DNA transfer is a polar process that begins at right border and abolishes at left border.
The single stranded T-DNA is converted into double stranded and gets incorporated inside the plant genome via non-homologous recombination.

3.Vectorless or direct DNA transfer methods

Direct transfer of genes into receptor has been proved it is simple and effective method for the transfer of foreign. This method is further divided in three categories. Those are mentioned below

Physical gene transfer methods
Chemical gene transfer methods

3.1 Physical gene transfer methods

In this method, there is no natural vector for the transfer of the DNA to receptor plant cell and therefore, DNA is introduced directly without the help of any vehicle. This process is also referred to as DNA mediated gene transfer. Various physical gene transfer methods are discussed in detail.

a) Electroporation

Electroporation is one of the most popular physical genetic transformation methods because of its simplicity, cheapness and fast nature. In this method, pulse electrical field is applied to introduce the DNA inside the plant cells. When electric pulse is applied across the membrane, it leads to the formation of temporal pores ranging from 40-120nm. Before, the resealing of pores target material is introduced in plant cells. Some factors affect the efficiency of electroporation such as temperature, electric field strength, ionic concentration, DNA conformation and DNA concentration. Recently, this technique has applicability in sugarbeet and rice plants.

Steps for the electroporation process

Harvest the cells during the logarithmic phase
Centrifuge the 500g sample (2000 rpm) at 40C for 5 mintues
Resuspend the cells in growth medium at conc. of 1X 10 cells/ ml.
Electric filed applied by 300V/ 1050 µf for 1-2 minutes
Introduce the electroplated cells to culture dish and grow the cells.
Analyze the DNA, RNA and culture cells continuously to get positive cell line

Fig 3: Steps involved in the gene transfer during electroporation process

Benefits

Simple and quick method. Low cell toxicity.
Inexpensive than others.
Small amount of DNA is required than others.

Limitations

Difficult process to regenerate plants from protoplasts.
High intensity of pulse can destroy the protoplasts.
Some pores of larger size may be formed during electroporation process.
Transformation protocol is laborious.

b) Particle Bombardment

The method developed by Sanford is a physical method of cell transformation in which high density genetic material is transferred with high velocity for introduction of DNA into receptor plant cells. During the process, gene gun shoots a piece of DNA into recipient plant cells. This DNA piece is coated with tungsten or gold and fired across the stopping screen, accelerated by Helium into plant cell. This method is applied for the wounding of the plants so as to increase the Agrobacterium transformations. Particle bombardment method is also known with various names like particle acceleration, particle inflow gun and gene gun.

Fig 4: Gene transfer via particle bombardment process

Advantages

Binary vector is not required.
Simple transformational protocol.
Applicable on all plant species.

Disadvantages

Equipments are costly.
During the DNA transformation, there are chances of damage.
Leads to non homologous integration of chromosomes.
Difficult in getting single copy transgenic events.

c) Macroinjection

In this method, DNA solution is injected into developing plant parts like shoots, with the help of micropipettes. The injection volume of genetic material that has to be introduced into receptor plants is 5-10 µl.

d) Microinjection

In this method, DNA is introduced directly into the target cells under microscopic control. This method has a unique property that it can even penetrate the intact cell walls. However, in this method, the cells have to be immobilized first. The cells are immobilized by agarose embedding, agar embedding, suction holding pipettes and poly-lysine treated glass surfaces methods. This method of gene transfer has been applied in tobacco and Brassica napus.

Disadvantages

Slow process
Production of chimaeric plants
Expensive method
Highly skilled and experienced staff is required.

e) Liposome- mediated transformation

This method employs the fusion of protoplasts using PEG. DNA enters into the protoplasts by endocytosis of liposomes. This technique is useful in tobacco and carrot.

The process involves number of steps such as:

Liposomes get adhered to the surface of the protoplasts.
Lipososmes get adhered at the site of adhesion.
Plasmids get released inside the cells.

Advantages

Low level cell toxicity
No chances of DNA damage
Applicable on wide variety of cells High reproducibility
Stable and safe storage

f) Silicon carbide fiber-mediated transformation

This method employs the use of silicon carbide fibres that have great intrinsic hardness with sharp edges. The process involves the vortexing of silicon carbide fibers, plasmid DNA and tissue in the eppendorf tube. The fibres have 10-80 µm length and 0.6 µm diameter. During the mixing process, DNA gets penetrates into plant cells through silicon carbide fiber. This technique has been demonstrated in maize and tobacco plants.

Advantages

Simple procedure
Affordable
few types of equipment are needed.

Disadvantages

Silicon carbide is carcinogenic in nature

g) Ultrasound-mediated DNA transformation

Ultrasound- mediated DNA transformation technique is also known as sonoporation. In this technique, plasmid DNA and plant cells are mixed together with buffer in sonicator. Ultrasonic pulses are applied at 0.5 w/cm2 intensity for 30 minutes. Thereafter, samples are rinsed with buffer and cultured for further growth and differentiation. This technique was applied in tobacco.

Advantages

Inexpensive
Multifunctional
Easy to standardize

Disadvantage

High intensity of ultrasonic pulse can cause damage to cells

3.2Chemical gene transfer methods

These methods involve destabilization of plasma membrane with chemicals. Plant cells are incubated with foreign DNA in buffers having poly L-ornithine, polyvinyl alcohol and polyethylene glycol.

a) Polyethylene glycol (PEG) mediated gene transfer

For the first time, this method was tested on Petunia and tobacco in presence of polyethylene glycol and poly L- ornithine.

Steps involved in the PEG mediated transfer

Protoplasts are isolated.
Plasmid DNA is mixed with protoplast suspension.
To this mixture, 40% PEG is added. This solution should be added slowly since it has high viscosity.
The mixture is incubated for 5 minutes.
Efficiency of PEG method is affected by magnesium concentration and calcium ions in the presence of donor DNA during incubation period.

Advantages

Completely controlled experiment.
Intermediate vector not required.

Disadvantage

Protoplast is required.

b) Calcium phosphate co-precipitation method

This method involves the mixing of donor DNA with calcium chloride and isotomic phosphate buffer so as to form DNA-CaPO4
The precipitates thus formed react for several hours with actively dividing cells.
The precipitates are washed and incubated in culture medium.

DMSO enhances the transformation efficiency. The success of transformation depends upon the DNA concentration.

c) The polycation DMSO technique

Polycation and polybrene are used to increase the surface area for the adsorption of foreign DNA. Thereafter, 25% DMSO treatment is given to accelerate membrane permeability and increase the uptake of DNA.

Advantage

Less toxic as compared to other polycations.
Small quantities of plasmid DNA are required.

d) DEAE dextran procedure

This method involves the use of diethyl aminoethyl dextran (DEAE). This chemical forms a complex with DNA and then causes the transformation of plant cells. DMSO shock increases the efficiency of transformation upto 80% but all the transformations are unstable.

Virus mediated gene transfer: Some viruses have been explored for their potential to be used as vectors. Three vectors that are commonly used as are caulimoviruses, geminiviruses and RNA viruses. Out of these, RNA viruses do not have potential to be used as vector.

4.Applications

Insecticidal Resistance: Major reason behind the reduction in crop production is insect attack. To handle this problem, some insect resistant plants have been developed based on insecticidal proteins. During the sporulation process, Bacillus thuringiensis (Bt) produces δ-endotoxins (cry 1, cry 2, cry 3 & other proteins). These cry proteins are toxic in nature. After ingestion of δ-endotoxins, these proteins bind with midgut epithelial cells and cause the damage of the epithelial surface. This is an effective method to control the insects in various crops such as cotton, tobacco, tomato, potato, maize, canola and rice.

Virus Resistant plants:

Virus resistant plants have been developed through several approaches such as ribozyme mediated technology, introduction of coat protein (cp) gene and antisense RNA technology. The best approach amongst these is the introduction of cp gene. First transgenic virus resistant plant was tobacco produced in 1986 containing Tobacco Mosaic Virus cp gene. After tobacco many other plants have been produced using this technology such as papaya, watermelon.

Uncoating of virus particles is a necessary process that is required for the expression and replication of viral genome. However, cp gene blocks this process and provides resistant against virus.

Herbicidal Resistance: Weeds are unwanted plants that grow with cultivated plants and compete for the nutrients and space. When herbicides are applied for weed killing, the crops also get affected. The herbicides also inhibit the process of photosynthesis in crop plants. However, this process is an essential process. Therefore, some crops have been engineered genetically for herbicide resistance.

Let us take an example of glyphosate. This is a non- selective herbicide and competitive inhibitor of 5- enolphyruvylshikimate 3-phosphate synthase (EPSP). EPSP is very important enzyme that is required for the synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) in plants. Shikimate and phosphoenol pyruvate are converted into EPSP. Therefore, two approaches have been used to produce resistance against the glyphosate herbicide.

First approach leads to the overproduction of EPSP. Overproduction will provide resistance as number of enzymes will be available to the cell. This technique has been used in tobacco, tomato, soyabean, canola, sugarbeat and cotton.
In the second approach, mutant version of EPSP synthase gets expressed. This mutant version possesses resistance to the herbicide.

Modification of Plant Nutritional Content: Genetic engineering plays a crucial role in the improvement of the nutritional value of crops. Applying this process, lipids, vitamins, amino acids and iron content can be increased. The famous example is the production of vitamin A enriched golden rice. In rice, vitamin A is very low. However, A. Potrykus and Bayer developed a new rice variety that is known as golden rice through genetic engineering. This variety is enriched with pro vitamin A. For the development of golden rice, three genes i.e., phytoene synthase, phytoene synthase and lycopene synthase, were introduced. These three genes are involved in the biosynthesis of carotenoids. The carotenoids are the precursors of vitamin A.

Plants as Bioreactors: Transgenic plants can be used for the production of antibodies (plantibodies), chemicals and proteins on commercial scale since they can grow easily and produce large biomass. Nowadays, biopharmaceutical sector is trying to eliminate animal derived proteins by plantibodies since there are more chances of contamination of animal derived proteins and that can prove pathogenic.

Edible vaccines: Production and packaging of commercial vaccines is very expensive. Moreover, trained persons are required to administer the injections. The solution of this problem is the production of edible vaccines as fruit or vegetable. The edible vaccines do not require elaborated packaging, sterilization, purification and delivery. Suitable parts of plants are consumed by humans to enhance the immunity of body. Examples include potatoes, bananas, tomatoes, lettuce, carrot etc.

Bioplastic Production: Plastic has become a biggest threat to whole the world because of its non-biodegradable nature. To solve this problem, genetic engineering technique is in use these days for the production of bioplastic (biodegradable) from plants.

For example, polyhydroxybutyrate (PHB), a biodegradable and renewable biopolymer is produced from a bacteria Alcaligenes eutrophus.

are consumed by humans to enhance the immunity of body. Examples include potatoes, bananas, tomatoes, lettuce, carrot etc.

For example, polyhydroxybutyrate (PHB), a biodegradable and renewable biopolymer is produced from a bacteria Alcaligenes eutrophus.

Delayed Ripening: Softening of fruits due to ripening process is a major problem as it causes economic losses. The process of ripeneing can be delayed using genetic engineering approach. Basically, polygalacturoanse is the enzyme that is responsible for ripening of fruit. This enzyme acts on the polygalacturonic acid of the cell wall and results in gradual softening of fruit. Expression of polygalacturonase (PG) enzyme has been reduced through antisense technology. With the help of this technology, a permanent copy of antisense for the PG gene has been introduced using Agrobacterium tumefaciens. In this technology antisense RNA binds with opposite sense mRNA and forms a stable duplex. This duplex prevents the formation of normal amount of polygalacturonase and interferes with the process of translation. This technique has been demonstrated effectively in tomato.

Development of Stress Resistant Plants: There are several types of stress such as low water availability, flooding, high salt concentrations, high and low temperature that are having negative effects on the growth and production of plants. Metabolic genetic engineering has been used for the synthesis of salt and cold tolerant rice variety.

5.Advantages of Genetically Modified Crops

Better flavor and high nutritional value.
More productivity and high yield.
Elimination of unwanted characters.
GM crops act as vaccine carrier. For example, banana.
GM crops show delayed ripening and can be stored for long time.
Virus and herbicide resistant.
Growth of GM crops is faster than others.
High capacity to tolerate stress.

6.Disadvantages of Genetically Modified Crops

GM plants decrease the diversity if grown for long time.
Cross pollination of GM plants with other plants bring unwanted change in plants producing negative effects on quality and quantity.
Sometimes transferred genes have some negative effect on human body by causing allergies.
Pollens of GM plants have some undesired properties such as respiratory and skin disease.

7.Environmental Problems

Gene pollution is transfer of gene form GM plants to weeds and pests to make them super weed and super pests.
Leakage of GM proteins in soil causes soil pollution and proves to be dangerous for soil flora and fauna.
GM plants are harmful for natural pollinators such as insects and butterflies. For example, Bt corn produces pollen that are highly toxic to Monarch larvae and black swallowtail.
GM proteins are responsible for the surface and ground water pollution.Helpful in the conservation of water, soil and energy.
GM crops are eco-friendly.

Economic Problems

Expensive technology
Controlled by multinational companies
Disruption of current farming and less food production
Depletion of traditional practices

Concluding remarks

The consumption and growth of GM crops is having a great dispute. The reason behind is the linking of ethical issues with the suitability of GM crops for human beings. The main disadvantage of genetic engineering is the elimination of diversity and has wide array of technical and ethical concerns.

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