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Gene technology makes it possible toalter plants to meet requirements ofagriculture, nutrition, and industry

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• Recent years have witnessed spectacular developments in plant gene technology.

• In 1984 the group of Marc van Montagu and Jeff Schell in Gent and Cologne, and the group of Robert Horsch and collaborators of the Monsanto Company in St. Louis, Missouri (United States) simultaneously published procedures for the transfer of foreign DNA into the genome of plants utilizing the Ti plasmids of Agrobacterium tumefaciens (new nomenclature:Rhizobium radiobacter).

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• The Ti plasmid (tumor-inducing plasmid) of Agrobacterium tumefaciens has been developed as a vehicle for introducing foreign genes into plants. When Agrobacterium infects plants, a region of the Ti plasmid called the T-DNA is taken up by the plant cell and incorporated into one of its chromosomes.

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• This method has made it possible to alter the protein complement of a plant specifically to meet special requirements:- for example, to render plants resistant to pests or herbicides,- to achieve a qualitative or quantitative improvement in the productivity of crop plants,- and to adapt plants so that they can produce defined sustainable raw materials for chemical industry.

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22.1 A gene is isolated

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• Let us consider the case where a transgenic plant A is to be generated, which synthesizes a foreign protein (e.g., a protein from another plant B). For this,

• the gene encoding the corresponding protein first has to be isolated from plant B.

• Since a plant probably contains between 25,000 and 50,000 structural genes, it will be difficult to isolate a single gene from this very large number.

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A gene library is required for the isolation of a gene

• To isolate a particular gene from the great number of genes existing in the plant genome, it is advantageous to make these genes available in the form of a gene DNA library.

• Two different kinds of gene libraries can be prepared.

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• To prepare a genomic DNA library, the total genome of the organism is cleaved by restriction endonucleases into fragments of about 15 to several 100 kbp.

• Digestion of the genome in this way results in a very large number of DNA sequences, which frequently contain only parts of genes.

• These fragments are inserted into a vector (e.g., a plasmid or a bacteriophage) and then each fragment is amplified by cloning, usually in bacteria.

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• To prepare a cDNA library, the mRNA molecules present in a specific tissue are first isolated and then transcribed into correspond-ing cDNAs by reverse transcriptase.

• The cDNAs are inserted into a vector and amplified by cloning.

• The mRNA is isolated from a tissue in which the corresponding gene is expressed to a high extent.

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• In contrast to the fragments of the genomic library, the resulting cDNAs contain entire genes without introns and can therefore, after transfor-mation, be expressed in prokaryotes to synthe-size proteins.

• Since a cDNA contains no promoter regions, such an expression requires a prokaryotic promoter to be added to the cDNA.

• To prepare a cDNA library from leaf tissue, for example, the total RNA is isolated from the leaves, of which the mRNA may amount to only 2%.

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• To separate the mRNA from the bulk of the other RNA species, one makes use of the fact that eukaryotic mRNA contains a poly(A) tail at the 3’ terminus.

• This allows mRNA to be separated from the other RNAs by affinity chromatography.

• The column material consists of solid particles of cellulose or other material to which a poly-deoxythymidine oligonucleotide [poly-(dT)] is linked.

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• When an RNA mixture extracted from leaves is applied to the column, the mRNA molecules bind to the column by hybridization of their poly-(A) tail to the poly-(dT) of the column material, whereas the other RNAs run through (Fig. 22.1).

• With a suitable buffer, the bound mRNA is eluted from the column.

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Figure 22.1 Separation of mRNA from an RNA mixture by binding to poly(dT) sequences that are linked to particles.

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• To synthesize by reverse transcriptase a cDNA strand complementary to the mRNA, a poly-(dT) is used as a primer (Fig. 22.2).

• Subsequently, the mRNA is hydrolyzed by a ribonuclease either completely or, as shown in the figure, only partly.

• The latter way has the advantage that the mRNA fragments can serve as primers for the synthesis of the second cDNA strand by DNA polymerase.

• By using DNA polymerase I, these mRNA fragments are successively replaced by DNA fragments and these are linked to each other by DNA ligase.

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Figure 22.2 Transcription of mRNA to double stranded cDNA.

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• A short RNA section remains, which is not replaced at the end of the second cDNA strand, but this is of minor importance, since in most cases the mRNA at the 5’ terminus contains a non-encoding region.

• The double-stranded cDNA molecules thus formed from the mRNA molecules are amplified by cloning.

• Plasmids or bacteriophages can be used as cloning vectors.

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• Nowadays a large variety of made-to-measure phages and plasmids are commercially available for many special purposes.

• A distinction is made between vectors that only amplify DNA and expression vectors by which the proteins encoded by the amplified genes can also be synthesized.

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A gene library can be kept in phages

• Figure 22.3 shows the insertion of cDNA into the DNA of a λ phage.

• In the example shown here, the phage DNA possesses a cleavage site for the restriction endonuclease EcoRI.

• To protect the restriction sites within the cDNA, the cDNA double strand is first methylated by an EcoRI methylase at the EcoRI restriction sites.

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• DNA ligase is then used to link chemically synthesized double-stranded oligonucleotides with an inbuilt restriction site, (in this case for EcoRI) to both ends of the double-stranded cDNA.

• These oligonucleotides are called linkers.• The restriction endonuclease EcoRI cleaves

this linker as well as the λ phage DNA and thus generates sticky ends at which the comp-lementary bases of the cDNA and the phage DNA can anneal by base pairing.

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• The DNA strands are then linked by DNA ligase, and in this way the cDNA is inserted into the vector.

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Figure 22.3 Insertion of cDNA in a λ-phage insertion vector.

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• The phage DNA with the inserted cDNA is packed in vitro into a phage protein coat (Fig. 22.4), using a packing extract from phage-infected bacteria.

• In this way one obtains a gene library, in which the cDNA formed from the many different mRNAs of the leaf tissue are packed in phages, which, after infecting bacteria, can be amplified ad libitum, whereby each packed cDNA forms a clone.

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Figure 22.4 A recombinant phage DNA is packed into a virus particle. E. Coli cells are infected with the formed phage and plated on agar plates. The cells of the infected colonies are lysed by the multiplying phages and show as transparent spots (plaques) in the bacterial lawn.

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• The bacteria are infected by mixing them with the phages and they are then plated on agar plates containing cultivation medium.

• At first the infected bacteria grow on the agar plates to produce a bacterial lawn, but then are lysed by the phages, which have been multiplied within the bacteria.

• The lysed bacterial colonies appear on the agar plate as clear spots called plaques.

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• These plaques contain newly formed phages, which can be multiplied further.

• It is customary to plate a typical cDNA gene library on about 10 to 20 agar plates.

• Ideally, each of these plaques contains only one clone.

• From these plaques, the clone containing the cDNA of the desired gene is selected, using specific probes as described later.

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A gene library can also be kept in plasmids

• To clone a gene library in plasmids, cDNA is inserted into plasmids via a restriction cleavage site in more or less the same way as in the insertion into phage DNA (Fig. 22.5).

• The plasmids are then transferred to E. coli cells.

• The transfer is brought about by treating the cells with CaCl2 to make their membrane more permeable to the plasmid.

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• The cells are then mixed with plasmid DNA and exposed to a short heat shock.

• In order to select the transformed bacterial cells from the large majority of untransformed cells, the transformed cells are provided with a marker.

• The plasmid vector contains an antibiotic resistance gene, which makes bacteria resistant to a certain antibiotic, such as ampicillin or tetracycline.

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• When the corresponding antibiotic is added to the culture medium, cells containing the plasmid survive and grow, whereas the other non-transformed cells die.

• After plating on an agar culture medium, bacterial colonies develop, which can be recognized as spots.

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Figure 22.5 cDNA can be propagated via a plasmid vector in E. coli. An antibiotic resistance gene on the plasmid enables the selection of the transformed cells.

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• In order to check whether a plasmid actually contains an inserted DNA sequence (insert), plasmid vectors have been constructed in which the restriction cleavage site for insertion of the foreign DNA is located inside a gene, which encodes the enzyme β-galactosidase (Fig. 22.6).

• This enzyme hydrolyzes the colorless compound X-Gal into an insoluble blue product.

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• When X-Gal is added to the agar plate culture medium,

• all the clones that do not contain a DNA insert, and therefore contain an intact β-galactosidase gene, form blue colonies.

• If a DNA segment is inserted into the cleavage site of the β-galactosidase gene, this gene is interrupted and is no longer able to encode a functional β-galactosidase.

• Therefore the corresponding colonies are not stained blue but remain white (blue/white selection)

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Figure 22.6 To check whether the plasmid of a bacterial colony carries a DNA insert, the cleavage site of the plasmid vector is contained within a β-galactosidase gene.

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A gene library is screened for a certain gene• Specific probes are employed to screen the

bacterial colonies or phage plaques for the desired gene.

• A blot is made of the various agar plates by placing a nylon or nitrocellulose membrane on top of them.

• Some of the phages contained in the plaques, or the bacteria contained in the colonies, bind to the blotting membrane, although most of the contents of the plaques and the colonies remain on the agar plate.

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• Two kinds of probes can be used to screen the phage or bacterial clones bound to the blotting membrane:

1. Specific antibodies to identify the protein formed as gene product of the desired clone (Western blot); and

2. Specific DNA probes to label the cDNA of the desired clone by hybridization (with radioactivity).

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A new plant is regenerated following transformation of a leaf cell

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1. Transformation by A. Tumefaciens

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• Agrobacterium tumefaciens attacks plants at wounds.

• Leaf discs therefore, with their cut edges, are a good target for performing a transformation (Fig. 22.16).

• The leaf discs are immersed in a suspension of A. tumefaciens cells transformed by the vector.

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• After a short time, the discs are transferred to a culture medium containing agarose, which, besides nutrients, contains the phyto-hormones cytokinin and auxin to induce the cells of the leaf disc to grow a callus.

• The addition of the antibiotic kanamycin kills all plant cells except the transformed cells, which are protected from the antibiotic by the resistance gene.

• The remaining agrobacteria are killed by another antibiotic specific for bacteria.

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• The cut edges of the leaf discs are the site where the calli of the transformed cells develop.

• When the concentrations of cytokinin and auxin are appropriate, these calli can be propagated almost without limit in tissue culture on agarose culture media.

• In this way transformed plant cells can be kept and propagated in tissue culture for very long periods of time.

• If required, new plants can be regenerated from these tissue cultures.

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• To regenerate new plants, cells of the callus culture are transferred to a culture medium containing more cytokinin than auxin, and this induces the callus to form shoots.

• Root growth is then stimulated by transferring the shoots to a culture medium containing more auxin than cytokinin.

• After plantlets with roots have developed and grown somewhat, they can be transplanted to soil, where in most cases they develop to normal plants, capable of being multiplied by flowering and seed production.

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• The pioneering work of Jeff Schell, Marc van Montagu, Patricia Zambryski, Robert Horsch, and several others has developed the A. tumefaciens transformation system to a very easy method for transferring foreign genes to cells of higher plants.

• Nowadays it is often possible even for students to produce several hundred different transgenic tobacco plants with no great difficulty.

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• Using this method, more than 100 different plant species have been transformed successfully. Initially, it was very difficult or even impossible to transform monocot plants with the Agro-bacteriumsystem.

• Recently, this transformation method has been improved to such an extent, that it also can now be successfully applied to transform several monocots, such as rice.

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2. physical gene transfer

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• An alternative way to transform plant cells is a physical gene transfer, the most successful being the bombardment of plant cells by microprojectiles.

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Plants can be transformed by a modified shotgun

• Transformation by bombardment of plant cells with microprojectiles was developed in 1985. The microprojectiles are small spheres of tungsten or gold with a diameter of 1 to 4mm, which are coated with DNA.

• A gene gun (similar to a shotgun) is used to shoot the pellets into plant cells (Fig. 22.17).

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Figure 22.17 Transformation of a plant by a gene gun. Gold or tungsten spheres arecoated with a thin DNA layer by a deposit of CaCl2. The spheres are inserted in front of a plastic projectile into the barrel of the gun.

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• Initially, gunpowder was used as propellant, but nowadays the microprojectiles are often accelerated by compressed air, helium, or other gases.

• The target materials include calli, embryonic tissues, and leaves.

• In order to penetrate the cell wall of the epidermis and mesophyll cells, the velocity of the projectiles must be very high and can reach about 1,500 km/h in bombardments with a gene gun in a vacuum chamber.

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• The cells in the center of the line of fire may be destroyed during such a bombardment but, because the projectiles are so small, the cells nearer the periphery survive.

• The DNA transferred to the cells by these projectiles can be integrated not only in the nuclear genome, but also in the genome of mitochondria and chloroplasts.

• This makes it possible to transform mito-chondria and chloroplasts.

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• In some plants, the gene gun works especially well.

• Thus, by bombardment of embryonic callus cells of sugarcane, routinely 10 to 20 different transformed plant lines are obtained by one shot.