Whole Plants can be grown from Single Cells
When a plant is wounded mechanically, a patch of soft cells called a callus grows over the wound
Removal of the callus into a culture medium containing appropriate nutrients and plant growth hormones will allow the cells to grow and divide as a suspension culture. It is sometimes necessary to use other cells as a nurse culture.
The callus will redifferentiate into shoots and roots, and ultimately a whole flowering plant will be produced.
Differentiation of cells in a callus depends on the relative auxin/cytokinin concentration. If auxin>cytokinins, roots develop. If auxins<cytokinins, shoots develop.
Due to the cellulose wall, these cells cannot directly take up DNA. The wall can be removed by treatment with fungal cellulase enzymes. The resulting protoplast is enclosed only by a plasma membrane and is much more amenable to experimental manipulation - they will take up macromolecules like DNA and are capable of regenerating whole plants via the formation of calli (multiple calluses)
Leaf disks are an important target for Gene Transfer
Plant cells must be wounded to be targets for Ti gene transfer, and pieces of roots and stems have been used as targets. Leaves are a good source of regenerating cells, the cells coming from small disks cut from a leaf. The cells at the edge of the disk begin to regenerate, and when these disks are ultured briefly in a medium containing agrobacteria, these cells are efficiently exposed to the transfecting agent. These disks are transferred for several days to nurse cultures containing medium with antibiotic (kanamycin), and an antibiotic like cefotaxime to kill agrobacterium. Shoots develop within a few weeks, and these shoots are transfered to medium that induces root formation.
Ti Plasmid of Agrobacterium causes Crown Gall tumors
Ti plasmids are large, circular dsDNA molecules of about 200kb, existing as plasmids within Agrobacterium cells.
Ti plasmids are maintained in Agrobacterium because T-DNA (a part of the Ti Plasmid) carries genes coding for unusual AAs called opines. The infected plant cell is induced to synthesize these amino acids, but the plant cannot use them. Instead, the Ti plasmid is believed to carry genes coding for enzymes that can degrade opines, so they may act as a nutrient for Agrobacterium. This subversion of the plant's metabolism could provide a selective advantage for Agrobacterium
A second set of genes in T-DNA causes the unregulated growth of the plant cell. Two of these genes iaaM and iaaH (indole acetic acids) code for enzymes that lead to the production of an auxin. The third gene, iptZ codes for an enzyme that causes production of a second phytohormone. These two hormones cause the infected plant cell to divide; they also affect the neighbouring cells
T-DNA, Part of the Ti Plasmid is transferred to Plant Cells
Three components are involved in Ti plasmid tumor induction
T-DNA is transferred to the host cell and is a form of mobile element
In addition, vir(ulence) genes are present elsewhere and are needed for the production of trans-acting proteins that are essential for/enhance plant cell transformation.
Genes on the agrobacterium chromosome are also responsible for binding the bacterial cells to the plant.
The virulence genes in agrobacterium are switched on by chemicals produced by wounded plant cells. Following vir activation, the T-DNA element is excised from the plasmid DNA. The T-DNA is flanked by Ti plasmid "border" sequences 25bp long, and are excised with the right border first followed by the second. This is analogous to the process of bacterial conjugation. Other vir genes may be associated with the T-DNA itself and involved in the transfer process. Usually, multiple copies of T-DNA integrate at a single random site in the plant chromosome, but little is known of the mechanism.
T-DNA has been modified to act as a Gene Vector
The binary vector system consists of the 25-bp borders of TDNA, with the phytohormone genes removed from the T-DNA and replaced with foreign DNA, at the same time preventing uncontrolled growth of recipient cells. Other genes required are the vir genes in the Ti plasmid, and can act in trans when supplied on a separate helper plasmid. Selectable markers are also required such as nptII (kanamycin) and dihyodrofolate reductase.
Extra notes can just be inferred, really
>> GUS expression and presence on the T-DNA
T-DNA requires at least
a) bacterial selectable marker
b) LB/RB
C) DNA from 5' region of interest
D) CaMV promoter
E) E.coli GUS
F) nptII gene for selection
Gene guns can be used to transfer DNA - beads one micron in size attaches to DNA, destroys first layer of cells it comes in contact with however only just penetrates the next layers and therefore DNA can integrate.
Plants expressing viral coat protein (tobacco mosaic virus, expressed using cDNA of viral gene) resist infection from tobacco mosaic virus for up to 30 days ( control = 3 to 4 days ) however symptoms occur significantly earlier once infected. The protection agent seemed to be the viral coat protein, and this protection also transferred to other related viruses in some cases. Transgenic expression of TMV replicase cDNA also produced resistance, but suprisingly through the RNA molecules and not the protein
Bacillus Thuringiensis producee crystallized protein that is toxic to insect larvae but not vertebrates. Expression through direct gene transfer gave low protection against insects sensitive to the toxin but not other insects that were more resistant; the conjugation of a synthetic 453AA toxin (modified for greater production in plants attached to the gene fragment encoding 454 to 615 of the bacterial portion produced a 100x more effective toxin
Roundup inhibits the chloroplast enzyme EPSPS which produces important aromatic amino acids; transfer of EPSPS cDNA icnreased the level of enzymatic activity to 20x the norm; this allowed plants to survive treatment with roundup. Another technique is the use of transgenes to introduce enzymes that break down toxins and herbicides, preventing modification of the levels of important enzymes within the cells and possibly affecting cell function.