57) Methods of genetic engineering: agrobacterial genetic transformation

The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants.^[7][8] A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome (1983).

The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.

Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in anAgrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die.

Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.

58)Methods of genetic engineering: bioballistics methods

A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.

This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.

The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into a plantchromosome.

Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots.

Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits.

The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species.

Design [edit]

The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. It was invented by John C Sanford, Ed Wolf and Nelson Allen at Cornell University,[1][2][3] and Ted Klein of DuPont, between 1983 and 1986. The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with a marker gene.[4] Genetic transformation was then proven when the onion tissue expressed the gene.

The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 caliber nail gun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on the bullet and shot down the barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk and the genetic information blasted into the sample in the dish with a doughnut effect (devastation in the middle, a ring of good transformation and little around the edge). The gun was connected to a vacuum pump and was under vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually improve the machine for mass production to the scientific community. Biorad contracted with Dupont to manufacture and distribute the device. Improvements include the use of helium propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold and silver are also used. Gold may be favored because it has better uniformity than tungsten and tungsten can be toxic to cells, but its use may be limited due to availability and cost.

59)Cryopreservation of plant cell and tissue Cryopreservation - freezing at very low temperatures. Usually it is carried out in liquid nitrogen at-196oC.

Freezing Plant cells from animals differs mainly presence preculture stage.

Cryoprotectants - substances that reduce the damaging effects of physical and chemical factors in cryopreservation. These include sucrose, dextran, ethylene glycol, polyvinyl pyrrolidone, dimethylsulfoxide (DMSO), glycerol. To determine the toxicity of the cryoprotectant cell was kept at room temperature in its various concentrations for 30 - 50 minutes, then determine their viability.

Typically, cooling is carried out in two phases (Fig. 26):

Stage 1: from-28oC to +20 at a rate of 1 degree per minute (plant cell freezing rate of 0.5 degrees per minute to -35 ° C), maintained at this temperature for 15 minutes.

2nd stage: immersion in liquid nitrogen (instantaneous cooling to - 196oC).

Freezing produced in special vehicles. In their absence, - an alcohol bath (0.5 - 1 liter alcohol is poured into the metal thermos flask was immersed in it for 15 minutes vial was added with stirring and liquid nitrogen or dry ice, the temperature is brought to-32oC (temperature should not exceed -28 or below-32C.) Next transferred into liquid nitrogen ampoule.

When thawing ampules forceps transferred into a water bath at 37 - 40 ° C, an ampoule of 1 mL were thawed for 0.5 - 1 minutes.

After thawing, the cells were washed in either growth medium (animal) or supportive environment. Plant cells can also be laundered 3 - 10% sucrose solution.

The cells were checked for viability using the vital dye staining dead cells. The final criterion is a clear return to growth on standard culture media used for the culture.

Immortalized mammalian cells after thawing have an increased susceptibility to viruses, which manifests itself in the first two passages. Next, the sensitivity of returns to the original.

The slowdown

Slowdown can be achieved by the following methods:

1. Storage under a layer of mineral oil (for fungal and bacterial cultures).

2. Change in gas composition and atmospheric pressure inside the culture vessel.

3. Changing light conditions.

4. Cooling stop temperature to active growth.

5. Hormonal and osmotic inhibitors. From hormonal inhibitors most often used hlorholinhlorida (for plant cells) of osmotic - mannitol in a concentration of 3-6%.

6. Replacement of CaCl2 to Ca (NO3) 2 in the culture media.

For the potato as a method allowing to keep the gene pool, it is recommended nodulation in test tubes.