TRANSGENIC PLANTS: Creation

Transgenic plants can be produced by Microprojectile bombardment: shooting DNA-coated tungsten or gold particles into plant cells. Electroporation: use of a short burst of electricity to get the DNA into cells. Agrobacterium tumefaciens-mediated transformation: The plant cells are totipotent, therefore a genetically modified single cell can regenerate into a new plant.

A. tumefaciens is a soil bacterium responsible for crown gall disease of dicotyledonous plants. The galls or tumors are formed at the junction between the root and the stem of infected plants. After the infection by A. tumefaciens, the plant cells begin to proliferate and form tumors, and they begin to synthesize an arginine derivative called opine. The opines synthesized are usually either nopaline or octopine depending on the strain of A. tumefaciens. These opines are used as energy sources by the infecting bacteria.

The ability of A. tumefaciens to induce crown galls in plants is controlled by genetic information carried on Ti plasmid (tumor-inducing plasmid). Ti plasmid has two components the T-DNA (Transferred DNA) and the vir region, which are essential for the transformation of plant cells. During the transformation process, the T-DNA is excised from the Ti plasmid, transferred to a plant cell, and integrated into the DNA of the plant cell. The integration of the T-DNA occurs at random chromosomal sites. In some cases, multiple T-DNA integration events occur in the same cell. Structure of Ti plasmid

Structure of the nopaline Ti plasmid pTi C58: ori, origin of replication; Tum, genes responsible for tumor formation;Nos, genes involved in nopaline biosynthesis; Noc, genes involved in the catabolism of nopaline; vir, virulence genes.

T DNA: In nopaline-type Ti plasmids the T-DNA is a 23,000-nucleotide-pair segment that carries 13 known genes including genes encoding enzymes that catalyze the synthesis of phytohormones (the auxin indoleacetic acid and the cytokinin isopentenyl adenosine). These phytohormones are responsible for the tumorous growth of cells in crown galls. The T-DNA region is bordered by 25-nucleotide-pair imperfect repeats, one of which must be present in cis for T-DNA excision and transfer. The deletion of the right border sequence completely blocks the transfer of T-DNA to plant cells.

The vir (for virulence) region: It contains the genes required for the T-DNA transfer process. These genes encode the DNA processing enzymes required for excision, transfer, and integration of the T-DNA segment. They are expressed at very low levels in A. tumefaciens cells growing in soil. However, exposure of the bacteria to wounded plant cells or exudates from plant cells induces enhanced levels of expression of the vir genes. This induction process is very slow for bacteria, taking 10 to 15 hours to reach maximum levels of expression. Phenolic compounds such as acetosyringone act as inducers of the vir genes, and transformation rates can often be increased by adding these inducers to plant cells inoculated with Agrobacterium.

Ti plasmid vector for creating transgenic plants Since T-DNA region of the Ti plasmid of A. tumefaciens is transferred to plant cells and becomes integrated in plant chromosomes, foreign genes could be inserted into the T-DNA and then transferred to the plant. In the modified Ti plasmid the genes responsible for tumor formation are deleted and selectable markeris added along with appropriate regulatory elements. The kanr gene from the E. coli transposon Tn5 has been used extensively as a selectable marker in plants; it encodes an enzyme called neomycin phosphotransferase type II (NPTII). NPTII is one of several prokaryotic enzymes that detoxify the kanamycin. The NPTII coding sequence are provided with a plant promoter and plant termination and polyadenylation signals. Such constructions with prokaryotic coding sequences flanked by eukaryotic regulatory sequences are called chimeric selectable marker genes.

One widely used chimeric selectable marker gene contains the cauliflower mosaic virus (CaMV) 35S promoter, the NPTII coding sequence, and the Ti nopaline synthase (nos) termination sequence; this chimeric gene is usually symbolized 35S/NPTII/nos. The Ti vectors used to transfer genes into plants have the tumor-inducing genes of the plasmid replaced with a chimeric selectable marker gene such as 35S/NPTII/nos. A large number of sophisticated Ti plasmid gene- transfer vectors are now used routinely to transfer genes into plants.

Ti Plasmid-Derived Vector Systems

A desired DNA sequence can be inserted into the T-DNA region of Ti plasmid and then the Ti plasmid contained in A. tumefaciens is used to deliver and insert the gene(s) into the genome of plant cell. The Ti plasmids have several limitations as routine cloning vectors.  The production of phytohormones by transformed cells growing in culture prevents them from being regenerated into mature plants. Therefore, the auxin and cytokinin genes must be removed from any Ti plasmid-derived cloning vector.  A gene encoding opine synthesis is not useful to a transgenic plant and may lower the final plant yield by diverting plant resources into opine production. Therefore, the opine synthesis gene should be removed.  Ti plasmids are large (approximately 200 to 800 kb). Therefore, a large segment of DNA that is not essential for a cloning vector must be removed.  Because the Ti plasmid does not replicate in Escherichia coli, therefore it cannot be cloned in E. coli.  Transfer of the T-DNA, which begins from the right border, does not always end at the left border. Rather, vector DNA sequences past the left border are often transferred.

To overcome these constraints, recombinant DNA technology was used to create a number of Ti plasmid-based vectors. These vectors contain the following components:

 A selectable marker gene, such as neomycin phosphotransferase, that confers kanamycin resistance on transformed plant cells. This gene is put under the control of plant (eukaryotic) transcriptional regulation signals, including both a promoter and a termination–polyadenylation sequence.  An origin of DNA replication that allows the plasmid to replicate in E. coli .  The right border sequence of the T-DNA region. This region is absolutely required for T- DNA integration into plant cell DNA. Most cloning vectors include both a right and a left border sequence.  A polylinker (multiple cloning site) to facilitate insertion of the cloned gene into the region between T-DNA border sequences.  A “killer” gene encoding a toxin downstream from the left border to prevent unwanted vector DNA past the left border from being incorporated into transgenic plants. If this incorporation occurs, and the killer gene is present, the transformed cells will not survive.

These cloning vectors lack vir genes; therefore, they cannot by themselves affect the transfer and integration of the T-DNA region into the genome of recipient plant cells. Two different approaches have been used to achieve this. 1. Binary vector system: The binary cloning vector contains either the E. coli and A. tumefaciens origins of DNA replication or a single broadhost range origin of DNA replication. All the cloning steps are carried out in E. coli before the vector is introduced into A. tumefaciens. The recipient A. tumefaciens strain carries a modified (defective or disarmed) Ti plasmid that contains a complete set of vir genes but lacks the T-DNA region, so that this T-DNA cannot be transferred. With this system, the defective Ti plasmid synthesizes the vir gene products and acts as a helper plasmid. This enables the T-DNA from the binary cloning vector to be inserted into the plant chromosomal DNA. Since transfer of the T-DNA is initiated from the right border, the selectable marker, is usually placed next to the left border. A few binary vectors have been designed to include two plant selectable markers, one adjacent to the right border and the other adjacent to the left border.

2. Cointegrate vector system: The cointegrate vector has a plant selectable marker gene, the target gene, the right border, an E. coli origin of DNA replication, and a bacterial selectable marker gene. The cointegrate vector recombines with a modified (disarmed) Ti plasmid that lacks both the tumor-producing genes and the right border of the T-DNA within A. tumefaciens. The entire cloning vector becomes integrated into the disarmed Ti plasmid to form a recombinant Ti plasmid

The cointegrate cloning vector and the disarmed helper Ti plasmid both carry homologous DNA sequences for homologous recombination. Following recombination, the cloning vector becomes part of the disarmed Ti plasmid, which provides the vir genes. In this cointegrated configuration the genetically engineered T-DNA region can be transferred to plant cells.

A practical problem that arises when using binary vectors is that their relatively large size (usually >10 kb) often makes it difficult and inconvenient to manipulate them in vitro. In addition, larger plasmids tend to have fewer unique restriction sites for cloning purposes. For these reasons, it is advantageous to develop and use smaller binary vectors. Based on the DNA sequence of a commonly used binary vector, pBIN19, it was predicted that more than half of the DNA could be deleted and the vector would still be completely functional.

Minivector: A mini-binary vector (pCB301) of size 3.5-kb was constructed which can be used to clone DNA fragments to be transferred into the plant genome. However, this cannot be introduced into A. tumefaciens by conjugation because certain regions of DNA required for conjugal transfer have been deleted. Therefore, this is introduced by electroporation. A number of derivatives of minivector pCB301 were constructed.

Although A. tumefaciens-mediated gene transfer systems are effective in several species, monocot plants such as rice, wheat, and corn are not readily transformed by A. tumefaciens. However, by refining and carefully controlling conditions, protocols have been devised for the transformation of corn and rice by A. tumefaciens carrying Ti plasmid vectors. For example, immature corn embryos were immersed in an A. tumefaciens cell suspension for a few minutes and then incubated for several days at room temperature in the absence of selective pressure. The embryos were then transferred to a medium with a selective antibiotic that allowed only transformed plant cells to grow. These cells were maintained in the dark for a few weeks. Finally, the mass of transformed plant cells was transferred to a different growth medium that contained plant hormones to stimulate differentiation and incubated in the light, which permitted regeneration of whole transgenic plants.