Agrobacterium-mediated transformation on maizeMaria TharumalingamYork Mills CI, 490 York Mills Rd, Toronto, ON M3B 1W6

Abstract:

Bacteria have been observed, time and again, to be extremely useful to not only humans, but many other organisms and the environment. Some bacteria however are known to be pathogens and parasites to animals and plants. A specific genus of bacteria known as Agrobacterium tumefaciens has different strains that cause mutations and diseases such as galls and tumors in plants [4]. The interesting aspect of this is that the T-DNA found in this species, is transferred into the genome of the plant cell. Only after the genes become incorporated in the plant genome do they become active and cause diseases. Agrobacterium-mediated transformation is the process of removing these virulent genes and instead, using this natural process, transfer non-virulent, advantageous genes to the plant [5]. This journal will analyze the possibility of agrobacterium-mediated transformation leading to increased growth/yield of maize.

Introduction:

Every habitat on earth is home to the well-studied microbes commonly known as bacteria. Bacteria occupy habitats such as soil, rock, oceans, and arctic soil; in addition, some live on other organisms such as plants and animals. In fact, there are more bacterial cells in the human body than there are somatic cells [5]. This not only describes the importance of bacteria, it also gives an indication as to how extensive and progressive the evolution of bacteria has become. Scientists use taxonomy to classify all living organisms- the end result is that all organisms can be defined as a distinct species. Due to the diversity and complexity of bacteria, it is given its own domain according to taxonomic ranks [6]. These single celled microbes have an extremely simplistic cell structure- they have no nucleus, or other membrane bound organelles. Their genetic information is contained in a single loop of DNA. Some bacteria may contain additional genetic information in the form of plasmid, an extra circle of genetic information that may give some bacteria an advantage over others [6]. Most bacteria are incredibly useful and have many different functions. Some of these functions include the cycling of nutrients and the production of fermented foods such as soy sauce. [7]However, some bacteria are parasites and pathogens that cause harm to humans and plants. Naturally, many individuals are fully aware of the virulent bacteria that cause harm to humans such as Salmonella and certain strains of E.coli bacteria. One sophisticated bacterium that has been observed to cause severe tumors in plants, is known as Agrobacterium tumefaciens. The genus Agrobacterium, has been divided into multiple species which reflect the disease symptomology and host range [4]. The most important fact is that the sophistication and complexity of the bacterial genes being transferred into the plant to cause tumors, is a process that occurs naturally. A well-defined segment of DNA from the bacterial tumor inducing (Ti) plasmid is transferred to the genome of the host cell. The genes transferred are activated and expressed only after being transferred to the plant [5]. This leads to the production of opines and amino acid derivatives. The production of opines and amino acid derivatives provide a source of carbon and nitrogen that the Agrobacterium tumefaciens can use,

allowing them to thrive [4]. This process has been observed and studied and scientists have found a way to use it to their advantage. Manipulating the genes in the plasmid before being transferred into the plant has opened up many doors in the growing field of biotechnology. This type of genetic engineering is more commonly known as Agrobacterium- mediated transformation. To summarize the process, the virulent genes on the plasmid are removed, and scientists manipulate the remaining genes that will be transferred into the plant, so that they will not cause harm to the plant, but rather be beneficial [3]. The objective of this article is to show how the genetic engineering of A.tumefaciens can affect the growth of genetically modified crops such as maize. If A.tumefaciens is genetically engineered so that when T-DNA is transferred into plants, the genes that are expressed induce plant growth, then there should be further investment into biotechnology because it will have a positive economic impact in society. A major advantage of Agrobacterium-mediated transformation is that a small number of copies of relatively large segments of T-DNA with defined ends are integrated into the plant genome with minimal rearrangement [4]. The expected outcome of this proposal is that more individuals become aware of how advantageous biotechnology is to our society and why more money should be invested into this research. The increase in the growth of genetically modified crops can help boost the economy. Investing money for research and experiments on A. tumefaciens can have a positive impact in the future. Possible ideas that can stem from this proposal is the effect of this same proposed experiment on other genetically modified crops such as barley, soy bean, wheat… etc. Or, ways to simplify agrobacterium-mediated transformation to reduce costs. The objective is that more ideas, opinions and questions arise as result of reading this proposal.

Rationale:

Molecular biology is a very fast growing field and it is extremely important that we continue to invest money into the research that can be conducted with it. Society can progress and evolve when we take time to understand the significance of genetics. As a high school student, molecular genetics was by far the most interesting unit that was covered. Not only was it a fascinating subject, being educated on it was very beneficial. For example, one major concept that applies to healthcare right now is antibiotic resistance. By understanding why antibiotics are important and the significance of antibiotic resistance, students can go around educating others about its importance. Molecular biology is also a very diverse field. Through studying it, it is possible to explore astrobiology which is the application of molecular biology on extraterrestrial planets in the universe. For example, analyzing certain bacteria and its capability to thrive within the soil on Mars may be the start to inhabiting Mars and creating a viable ecosystem. Furthermore, the general concept of using A.tumefaciens and genetically engineering it improve plant health, as opposed to destroy it by causing tumors, is a sophisticated and complicated process. The more research put into it, the more that can be learned from it. This might inspire others to come up with more complicated research topics that could lead to major breakthroughs in this field.

Methodology and Materials:

Extensive amounts of research will be conducted to determine the most efficient way to carry out this experiment. Many academic journals will be referred to when considering the materials and equipment that should be used for the experiment. Some of the materials needed would be the A. tumefaciens strain carrying appropriate T-DNA-containing vectors (for plant growth) and the maize seeds of inbreds. The equipment needed would be a scalpel blade, surgical tape made of unwoven fabric, parafilm (Pechiney Plastic Packaging), one 270 mm pot (270 mm in diameter and 270 mm in height) for maize cultivation and, one 230 mm pot (230 mm in diameter and 190 mm in height) for maize cultivation [2,3].

Transgenic maize plants were first obtained from protoplasts by an electroporation method. For the last two decades, dicot plants were transformed using the soil phytopathogen A. tumefaciens. A. tumefaciens is first transformed with the DNA construct of interest (T-DNA); this modified bacterial strain is then used to introduce the T-DNA into plants. For the successful production of transgenic plants in any species, foreign genes must be delivered to undifferentiated, dedifferentiated or dedifferentiating cells that are actively dividing or about to divide and that are capable of regenerating plants. In maize, the material of choice is immature embryos. When the Agrobacterium strain with the appropriate vectors is transferred to the maize embryos, the primary determinants of a successful transformation are the response of immature embryos in tissue culture, the types of cells that grow from immature embryos and characteristics in growth and regeneration [1]. Agrobacterium strain AGL1 is used together with appropriate pBract vectors which contain the hpt gene. The pBRACT vectors are based on pGreen, a small, versatile vector designed for easy manipulation in E. coli with a high copy number. To enable the small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is separated into its‟ two distinct functions. The replication origin is present on pGreen, and the trans-acting replicase gene is present on an additional vector, named pSoup. Both vectors are required in Agrobacterium for pGreen to replicate. Three different basic plant tissue culture media are used during the transformation and regeneration process; callus induction, transition and regeneration media. After the Agrobacterium strain is prepared, the culture is used to inoculate the barley embryos. The plates containing the barley embryos treated with the Agrobacterium strain are sealed with MicroporeTM surgical tape and incubated at 23-24 degrees Celsius for three days. After three days co-cultivation, the embryos are transferred to fresh callus induction plates containing hygromycin as the selective agent and timentin to remove the Agrobacterium from the cultures. The embryos are transferred to selection plates a total of three times with a two week time interval in-between each selection. After six weeks selection on callus induction medium, the embryo derived callus is transferred to transition medium, again containing hygromycin and timentin, for two weeks at 24 degrees Celsius under low light. After the two weeks on transition medium, the embryo derived material is transferred one final time to regeneration medium, in deep Petri dishes, without any growth regulators but still with the same levels of hygromycin and Timentin. Once plants have grown 2-3 cm in length and roots are formed, they are transferred correctly into soil where leaf samples can be collected for further analysis to confirm the presence of the introduced genes [3].

Expected outcome / Discussion:

The transformation experiment on Maize [1] suggests that the mechanism for the transformation still requires improvement. There was a high frequency of transformation in some of the genotypes within the maize culture; however, the other genotypes were not expected to be transformed efficiently [1]. –The barley transformation experiment [3] displayed positive results. The genes were expressed within the barley after the embryos were allowed to grow. This shows that the results from that experiment were conclusive and that the transformation occurred effectively and immediately without complications. The gene that was expressed was the gus gene in leaf samples [3]. Overall the concept that was learned from these results is that two different mechanisms of Agrobacterium-mediated transformation were used; one method was extremely effective and the other was not. If this proposal was funded and the experiment was carried out, the results would be positive. Immediate growth of the maize would occur which would show that Agrobacterium-mediated transformation should be funded further. If this proposal is funded, the money would be used to ensure that all materials are present and that all the crops grown are put in ideal conditions so that the data is accurate, and reliable. The main objective is to prove that Agrobacterium-mediated transformation will result in the increased growth rate of maize. Biotechnology based approaches targeting increased yield and better crop production need to be extended to more food crops. The core idea in this proposal can be extrapolated to other crops and the approach described herein has potential to be a valuable alternate approach in crop biotechnology.

References:

1. Ishida, Y., Hiei, Y., and Komari, T. (2007) Agrobacterium-mediated transformation of maize. Nat Protoc Nature Protocols 2, 1614–1621.

2. Tzfira, T., and Citovsky, V. (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current Opinion in Biotechnology 17, 147–154.

3. Harwood*, W. A., Bartlett, J. G., Alves, S. C., Perry, M., Smedley, M. A., Leyl, N., and Snape, J. W. (2008) Barley Transformation Using Agrobacterium-Mediated Techniques. Methods in Molecular Biology™ Transgenic Wheat, Barley and Oats 137–147.

4. Gelvin, S. B. (2003) Agrobacterium-Mediated Plant Transformation: the Biology behind the "Gene-Jockeying" Tool. Microbiology and Molecular Biology Reviews 67, 16–37.

5. Analyzing Plant Gene Expression with Transgenic Plants. Analyzing Plant Gene Expression with Transgenic Plants.

6. Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J. R. (2007) Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Applied and Environmental Microbiology 73, 5261–5267.

7. Ireland, K. (2015) Good Uses for Bacteria. LIVESTRONG.COM. LIVESTRONG.COM. 8. Wusirika, R. Genetics, genomics and breeding of maize.