Direct Methods for Transformation of Plants or Plant Cells

In this research paper on gene technology I hope to share some understanding in the process of

- Pesticides can be held within the soil for many years after it has been sprayed, effecting the growth and development of crops. Transgenic methods for altering crops allow the soil to stay cleaner, allowing the plants that grow there to be healthier. The safer method would be the transgenic method as far as soil is concerned.

In this experiment we were meant to observe the transferring of DNA. There are many ways in which DNA can be transferred into an organism, for example; transformation, transduction, and conjugation. In our experiment we used

Next, the transformation efficiency was calculated by dividing the total amount of DNA on the agar plate in μl by the number of cells growing on the +pGLO LB/amp/ara plate.

Certain grasses are subject to genetic transformation for example, according to Xinlu Chen. In her experiment using the heat shock method, the significant increase of temperature increased the amount of time a reporter gene was expressed. The addition of different thiol compounds during the process also increased the short time expression.

In 1928, Fred Griffith first discovered genetic transformation by infecting mice with unencapsulated and non-pathogenic pneumococci (Lacks 2003). This was the start that opened up the field of biotechnology. Genetic transformation is a process where foreign DNA crosses a membrane of another cell and then alters the genetic material (Encyclopædia Britannica 2015). Genetic transformation can occur in a few different ways: projectile bombardment, electroporation, and heat shock (Weedman 2015). Heat shock is defined by increasing the temperature of cells environment making the plasma membrane become more permeability allowing new DNA to transfer into the cell (Weedman 2015). The cell receiving the new DNA, also known as competent cells, can either amplify the DNA or clone the DNA (JoVE 2015). The most common type of DNA used to perform genetic transformations is plasmids: small, round DNA molecules that still contains two strands of DNA that has the ability

Genetic engineering is used in health treatments, agricultural applications, and environmental solutions. Genetic transformations incorporate foreign genetic material into the DNA of a different organism via a vector, which carries the genetic material. Plasmid DNA is small, round, and autonomous, due to its origin of replication. In biotechnology, plasmids carry beneficial genes, such as antibiotic resistance, and also a reporter protein, in this case, Green Fluorescent Protein

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

Purpose: The purpose of this experiment is to teach the students step by step on how the genetic transformation process works using pGLO. Genetic transformation is when an individual cell’s genetic material is changed by exogenous DNA.

Technology now allows us to transfer genes between organisms. For example, the tomato plant 's beetle resistance relies on a gene from a bacterium (Bacillus thuringiensis), which scientists inserted into the tomato plant 's genome. This gene, called cry1Ac, encodes a protein that is poisonous to certain types of insects, including the beetle. How is this done? Gene transfer technology is simply a sophisticated version of a cut-and-paste operation. Once the desired gene is identified in the native organism 's genome, it can be cut out, transferred to the target plant, and pasted into its genome… Once the new gene has been introduced, the plant can be bred to create a new strain that passes the gene from generation to generation. (pp 8,9)

These chemical signals set off a cascade of gene activity in the A. tumefaciens which direct a series of events required for the transfer of tDNA from the plasmid into the plant’s cells through the wounds of the plant. The tDNA then moves into the nucleus of the plant cell and becomes integrated into the plant chromosome (Understanding GMOs).

Transformation is the “process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome” (MedicineNet.com, “Definition of Genetic transformation”). Transformation in bacterial cells occurs when the cell incorporates DNA into its genetic material. Bacteria cells that have the ability to take up DNA are called “competent.” In a lab setting, this is encouraged by placing the mixtures of transformation solution and plasmid DNA on ice, then rapidly transferring them to a hot water bath for about fifty seconds, and then placing them back on ice again. This procedure is called heat shock and increases the permeability of the cell membrane to DNA. The agent which the new genetic material is incorporated into is the bacterial plasmid.

During this lab, genetic transformation was looked at. Genetic transformation is the insert of foreign DNA into a host cell. Competent cells are cells that can take up genetic transformation through the vector, which is the transfer mechanism of a gene. In lab, the competent cell that was used was E. coli and the vector is pGLO. The method heat shock was used, which is the sudden changing of temperature that increases the absorption of the plasma membrane, making the cells then have to pick up the DNA from other medium. E coli was the first gene to be heat shocked and is known as a good heat shock protein, it was first uncovered in 1991. (Lai et al. 2014) Here, the bacteria E. coli is being transformed by the gene encoding green fluorescent protein (GFP).

Instead of transferring large blocks of genes from donor plant to recipient, small isolated blocks of genes are put into the plant chromosome through biolistics, vectors, or protoplast transformation (Horsch 1993). Biolistics is a technique that shoots the gene block into the potential host cell. In order for the process to succeed, the microscopic particles and DNA must enter the cell nuclei and combine with the plant chromosome. Biolistics is commonly used but has a slight failure risk since the breeder has little control over the destination of the gene block (Mooney & Bernardi 1990). Bacteria or viruses can also carry the gene blocks into a new cell. Common vectors in gene transfer between plants are Agrobacterium tumefaciens and Agrobacterium rhizogenes. In the soil, the bacteria will infect the plants with their own plasmid, transferring the desired gene that was placed in the bacteria's DNA. Vector gene transfer is a preferred method of transformation since this modification already occurs naturally in the environment (Rudolph & McIntire 1996). Last is protoplast transformation, which uses enzymes to dissolve the cellulose in the plant wall that leaves a protoplast. Once a specific gene block is added to the protoplast, the cell wall will re-grow into a transgenic plant.

The coding region of the gene is usually fused to a promoter, most commonly used is the 35S promoter from cauliflower mosaic virus (CMV), in order to promote higher expression levels. (Snow et. al, 1997) The popular method for genetic engineering of crop plants is natural gene transfer via an Agrobacterium tumefaciens vector, a bacterium normally found in soils. The transfer-DNA (T-DNA) vector is made by inserting the desired gene fragment in between specific 25bp repeat domains in the bacterium. The vector is then inserted into the Agrobacterium and "the virulence gene products of Agrobacterium actively recognize, excise, transport, and integrate the T-DNA region into the host plant genomes." (Conner et. al, 1999) The amount of DNA transferred is only about 10kb and the nature of the gene is usually well understood. The expression of the gene introduced can also be controlled by adding additional sequences that might allow the gene to be constitutively expressed, expressed only in certain cell types, or expressed as a result of different environmental changes. This method of gene transfer, however, will only work for the natural host range of the bacterium and therefore other methods are used for additional crop plants. Such methods are uptake of naked DNA by electroporation or particle gun bombardment. The use of genetic markers, as mentioned previously, allows for the preferential growth of cultures that contain the new genetic