Methods of Gene Transfer in plants Table of Contents • Introduction • Indirect Methods Agrobacterium-mediated genetic transformation •Direct Method Microprojectile/particle Bombardment

Methods of Gene Transfer in plants
Table of Contents
• Introduction
• Indirect Methods
Agrobacterium-mediated genetic transformation
•Direct Method
Microprojectile/particle Bombardment (biolistics)
ElectroporationMicroinjection
Chemical mediated gene transfer
Liposome mediated gene transfer
Silicon carbide method
Introduction
Foreign genes are introducedartificially into crops by overcoming the fertility barriers.This process, also known asgenetic transformation,is a very important step in genetic engineering. Horizontal vs vertical gene transfer The natural transfer of genetic material from one organism to another is referred to as horizontal gene transfer or the lateral gene transfer. The foreign DNA is either randomly inserted into the host genome or recombines if there is sequence homology between the two genomes. This is different from the vertical gene transfer where the genetic material is transferred from the parents to the offsprings, through sexual reproduction.

Horizontal gene transfer is facilitated by various mechanisms. In prokaryotes mainly transformation (intake of genetic material from surrounding), conjugation (exchange of genetic material with the physical union of two cells) and transduction (transmission of DNA through bacteriophages from one cell to another) are responsible for the transfer of the gene within organisms. In eukaryotes, the presence of the outer cell membrane and the nuclear membrane makes transfer of DNA difficult between organisms. Horizontal gene transfer plays important role in evolution of both prokaryotes and eukaryotes.

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Plant transformation systems generally include following steps,
• Introduction of a DNA segment into totipotent cells.
• Its integration into host cells genome. • Subsequent regeneration from transformed cellto produce whole plant.
Plant transformation methods therefore require an efficient wayto introduce DNA into cell and the regeneration of the transformed cells or tissues into whole plants.
TheDNA segment which is introduced in this process contains the gene of interestanda cassette containing additional genetic material. Additional genetic material includes
• A promoter which determines the site and timing of expression of the introduced gene
• A terminator to identify the end of transcription and
• Amarker gene which allows selection of plants having the introduced gene.
Various desirable traits have been efficiently introduced and stably expressed in almost 150 plant species.

Different methods are available to achieve genetic transformation of plants i.e. the delivery of the foreign DNA into the host plant. These are divided into two main groups
• Indirect methods: In this case vector is needed for insertion of the foreign DNA into the host genome.
• Direct methods: This method is vector independent. The DNA is directly inserted into the host genome.

Figure: Types of methods for gene transfer in plants
In-direct Methods
Agrobacterium-mediated genetic transformation
The method of genetictransformation, which employs bacteria as a vector to introduce the gene construct into the target cell,isknown as indirect method. Thismethod uses Agrobacterium (a gram-negative soil bacteria which causes crown gall disease in many plants) for the plant transformation experiments. The most commonly studied species of this genus is Agrobacterium tumefaciens, which forms an efficient delivery system for genetic transformationin plants.These bacteria harbor a large plasmid called Ti plasmid (tumour inducing) having tumor-inducing genes (T-DNA) and other genes involved in integration of T-DNA into host genome.Wounded plants secrete a sap with high content of phenolic compounds which serve as chemical attractants for Agrobacteria and stimulateexpression of virgenes. It results ininfection of plantby Agrobacterium, insertion of T-DNA region at a random site inhost genome and proliferation of plant cells to form crown gall growth.

Another commonly used species is Agrobacterium rhizogens, which induces hairy roots in plants.It contains Ri plasmid (root inducing plasmid). The genus Agrobacterium has a wide host range and can infect a number of dicots and some monocots.

Figure: Crown gall disease caused by Agrobacterium on rose stem.

Structure of Ti Plasmid
In transformation experiments, vector is the genetic vehicle needed to transport the gene of interest, promoter, terminator and selectable marker genes to DNA of host plant. Virulence of Agrobacterium is conferred by Ti plasmid having genes for tumor-induction, T-DNA integration and synthesis of plant hormones and opines.

Figure: Ti plasmid with T-DNA region
• Origin or replication
This region is responsible for the replication of Ti plasmid independent of the bacterium cell.

• Virulence region
This region contains genes called vir genes whose products enable processing and transfer of the T-DNA from bacterium to plant cells. Their expression is triggered by certain phenolic compounds like acetosyringone, which are secreted by plants in response to wounding.

• T-DNA region
It is a region of Ti plamid which contains genes from induction of tumour. It is flanked by 25 bp direct repeat sequences on both sides. These repeats are called as Left border (LB) and Right border (RB). The different genes present in this region are – iaaM and iaaH genes – responsible for synthesis of indole acetic acid (an auxin), ipt gene – responsible for synthesis of an enzyme isopentenyl adenine (a cytokinin) tml gene – another gene involved in formation of tumours. Opine biosynthesis genes–lead to synthesis of opines.

• Region of opine catabolism
It contains several other genes involved in metabolism of opines. This region of the plasmid is not transferred to the plant cells during infection.

Use of Ti plasmid in genetic transformation
For its use in genetic transformation as a vector, most of the T-DNA region of bacterial plasmid is replaced with the gene of interest while leaving the left and right border sequences. The T-DNA region is defined not by its sequence but by its borderswhich enables itsinsertion into host plant genome.

Direct Methods
Direct methods are those methods which do not use bacteria as mediators for integration of DNA into host genome.

These methods include microprojectile bombardment,electroporation and microinjection.
Microprojectile/particle Bombardment (biolistics)
Biolistics is a method where cells are physically impregnated with nucleic acids or other biological molecules.Abiolistic particle delivery system is a device for plant transformation where cells are bombarded with heavy metal particles coated with DNA/RNA. This technique was invented by John Stanford in 1984 for introduction of DNA into cells by physical means to avoid the host-range restrictions of Agrobacterium.Agrobacterium-mediated genetic transformation system works well for dicotyledonous plants but has low efficiency for monocots.Biolistic particle delivery system provides an effective and versatile way to transform almost all type of cells. It has been proven to be a successful alternative for creating transgenic organisms inprokaryotes, mammalian and plant species.

Figure: A biolistic microprojectile gun
In this process, construct having gene of interest is coated on the surface oftiny particles of gold or tungsten (0.6 – 1 mm in size). Prior to coating, DNA is precipitated with calcium chloride, spermidine and polyethylene glycol.These coated microparticles are loaded on to the macrocarrier and accelerated to high speed by using pressurized helium gas.Plant cell suspensions, callus cultures, or tissues could be used as the target of thesemicroprojectiles. As the microprojectiles penetrate the plant cell walls and membranes to enter the cells, coated DNAis released from its surface and incorporated into the plant’s genome.In biolistics, use of binary vectors with T-DNA border sequences is not required.

This method is especially importantformonocots, for which efficiency of othertransformation methods is not satisfactory. A wide range of tissues such as apical and floral meristems, embryos, seedlings, leaves, cultured cells and floral tissues could be used as target in this method.

Figure: Particle bombardment method of Plant transformation (1) Isolation of protoplasts. (2)Injection of DNA-coated particles using particle gun. (3) Regeneration of transformed protoplasts into plantlets. (4) Acclimatizationof regenerated plantlets in a greenhouses.

A number of parameters should be carefully considered before using particle bombardment. These can be classified under three categories:
• Physical parameters
Nature, chemical and physical properties of the metal particles utilized to carry the foreign DNA. The nature and preparation of DNA,binding of DNA on the particles and target tissues.
• Environmental parameters
Variables such as temperature, photoperiod and humidity of donor plants, explants, and bombarded tissues affect physiology of tissues and influence receptiveness of the target tissue.
• Biological parameters
Choice and nature of explants, pre- and post bombardment culture conditions, osmotic pre- and post-treatment of explants.
Advantages of particle bombardment over Agrobacterium-mediated DNA transfer:
• This system is species independent and can been used successfully for a wide range of organisms.
• Many species which are recalcitrant to other direct transfer methods or are not readily amenable to Agrobacterium-mediated transformation have been transformed by this technique.
• Introduced DNA does not need sequences necessary for T-DNA replication and transfer as complex interaction between bacterium and plant tissue does not take place.

• Transformation of organelle DNA (mitochondria and chloroplasts) has also been achieved by this method.

• Multiple genes can be introduced in a single plant.

• Particles can be coated with DNA/RNA/siRNA/large fragments of nucleic acids.
Limitations of particle bombardment method : • Limited regeneration capacity of tissue being bombarded
• Efficiency of stable integration of DNA.
• Insertion of multiple copies of the gene
• Integration of rearranged and/or truncated DNA sequences
• Damage to the cellular tissue.

• Specialized and expensive equipments are required
Electroporation
Electoporation is a method of transformation via direct gene transfer. In this technique mixture containing cells and DNA is exposed to very high voltage electrical pulses (4000 – 8000 V/cm) for very brief time periods (few milliseconds). It results in formation of transient pores in the plasma membrane, thorough which DNA seems to enter inside the cell and then nucleus.

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Figure: Electroporation (A) Diagram showing formation of transient pores in cell membrane on applying electrical pulse, entry of DNA inside thecell and sealing of pores afterwards.

A suspension of cells with plasmid DNAis taken in an electroporation cuvette placed between electrodesand electrical pulses are applied.Temporary micropores are formed in cell membranes whichallow cells to take up plasmid DNA leading to stable or transient DNA expression.

Figure: (A) Main components of an electroporator. (B) Cuvettes used for electroporation. These are plastic cuvettes with lid and aluminium electrodes, having a maximum capacity of 400 ?l.

Cells which are arrested at metaphase stage of cell cycle are especially suitable for electroporation as these cells have absence of nuclear envelope and an unusual permeability of the plasma membrane. Protoplasts are used for electroporation of plant cells as thick plant cell walls restrict movement of DNA. The electroporation method was originally developed for protoplasts, but has given equally good results with cells and even tissues with easy recovery of regenerated plantlets. Immature zygotic embryos and embryogenic calli have also been used for electroporation to produce transgenic maize. Transformation of protoplast is associated with low transient expression of transgenes as compared to organized tissues and low regeneration frequencyespecially in monocotyledonous plants. The electrical field and chemicalsubstancesapplied to disorganize cell walls strongly reducethe viability and capability of divisionof protoplasts.

Electoporation as a transformation method is fast, convenient, simple, and inexpensive and has low cell toxicity. The disadvantage associated with this technique is difficulty in regenerating plants from protoplasts, if protoplast is used for electroporation.

Microinjection
The process of using a fine glass micropipette to manually inject transgene at microscopic or borderline macroscopic level is known as microinjection.The transgene, in the form of plasmids, cosmids, phage, YACs, or PCR products, can be circular or linear and need not be physically linked for injection.

Microinjection involves direct mechanical introduction of DNA intothe nucleus or cytoplasm using a glass microcapillary injectionpipette. The protoplasts are immobilized in low melting agar, while working under a microscope, using a holding pipette and suction force. DNA is then directly injected into the cytoplasm or the nucleus. Theinjected cells are then cultured invitro and regenerated into plants. Successful examples of this process has been shown in rapeseed, tobacco and various other plants.

Stable transformants can be achieved through this method but it requires technical expertise and is atime consuming process. Also,microinjection has achieved only limited success in plant transformationdue to the thick cell walls of plants and a lack of availability of a singlecell-to-plant regenerationsystem in most plant species.
In this technique a traditional compound microscope (around 200X magnification) or aninverted microscope (around 200x magnification) or a dissecting stereomicroscope (around 40-50x)is used.Under the microscope target cell is positioned and cell membrane and nuclear envelope are penetrated with the help of two micromanipulators.One micromanipulator holds the pipette and another holds the microcapillary needle.

Figure: Illustration of microprojectile method
There are two types of microinjection systems; constant flow system and pulsed flow system.

• Inthe constant flow system the amount of sample injected is determined by the duration for which needle remains in the cell.The constant flow system is relatively simple and inexpensive but outdated.

• The pulsed flow systemhas greater control over thevolume of substance delivered, needle placement and movement and has better precision. This technique results in less damage to the receiving cell, however, the components of this system are quite expensive.

Chemical mediated gene transfer
Cells or protoplasts can be stimulated to take up foreign DNA using some chemicals. Polyethylene glycol (PEG) is the most commonly used chemical for this purpose. It helps in precipitation of DNA, which can then be taken up by the calls through the process of endocytosis.

Liposome mediated gene transfer
Plasmid containing foreign desired gene can be enclosed in small lipid bags called lipososmes, which can then be fused with protoplasts using chemicals like PEG.
Silicon carbide method
In this method, fibres of organic material like silicon carbide are used for gene transfer. These fibres, when mixed with plasmid DNA and plant tissue or cells, help in penetration of the foreign DNA into the plant tissue.

Figure: An illustration showing different techniques used for transformation of tree species. Source: Castellanos-Hernández, Osvaldo A., et al. “Genetic Transformation of Forest Trees”.

References/Bibliography
• Chawla, H. S. Introduction to plant biotechnology. Science Publishers, Inc., 2000.
• Gelvin, Stanton B. “Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool.” Microbiology and molecular biology reviews 67.1 (2003): 16-37.

• Mantell, Sinclair H., John A. Matthews, and R. A. McKee. Principles of plant biotechnology: an introduction to genetic engineering in plants. Blackwell Scientific Publications, 1985.
• Primrose, Sandy B., and Richard Twyman. Principles of gene manipulation and genomics. John Wiley ; Sons, 2009.
• Slater, Adrian, Nigel W. Scott, and Mark R. Fowler. “Genetic manipulation of plants.” Plant Biotechnology Oxford, England: Oxford University Press (2003).

Web links
http://mmbr.asm.org/content/67/1/16.full.pdf+html http://tnau.ac.in/eagri/eagri50/GPBR311/lec23.pdf http://www.academia.edu/3617256/Gene_Transfer_Technologies_in_plants_Roles_in_improvi ng_Cropshttp://www.slideshare.net/Thirusangu/agrobacterium-mediated-gene-transfer http://www.slideshare.net/USrinivasa/gene-transfer-in-plants-008 http://www.slideshare.net/saugatbhatt/methods-27443684 http://www.slideshare.net/JirainneSerra/gene-transfer-by-physical-methods http://www.slideshare.net/mohammedsami31508/plant-transformation-methods