Accomplishments
Developed soybean lines carrying an antigenic protein that is produced in the seed which will serve as a candidate vaccine against a highly economic viral disease, Porcine Reproductive and Respiratory Syndrome (PRRS), of swine. This disease is considered to be the most significant animal health problem that currently faces the swine industry in Illinois and Missouri, and throughout the US, Canada, and Europe. Currently, there is no effective vaccine against this disease. The virus attacks 60-70% of swine in the US and at an estimated cost of $5 to $10 per finished pig which inflicts a significant economic blow to the swine industry. Therefore, this soybean-based candidate vaccine will have tremendous impact on protecting swine against this virus, and will be delivered orally to animals, thereby reducing cost of vaccination.
Executive Summary
The value of soybean as an important agricultural commodity and as a major source of protein (~40% protein content in soybean seed0, renders the soybean as a model crop for production of valuable proteins such as antigenic proteins that can be used as plant-based oral vaccines to prevent animal diseases. This medicinal use of soybeans will generate a new utilization industry for this specialty crop, and will increase the market share of soybeans. This new specialty soybean will possess a new valuable medicinal trait that goes beyond the known benefits of the health-promoting compounds present in the soybean seed. In this project, we have first developed an efficient gene transfer system for soybean which significantly increases the frequency and recovery of transgenic lines. This report has been recently published in a scientific journal (see publications list below), and has been highlighted by AgBiotechNet (http://www.Agbiotechnet.com) under their Hot Topics section. We have been able to use this transformation system to transfer genes into 13 different soybean cultivars. Then, we have introduced a sequence that codes for an antigenic protein, and recovered several transgenic soybean lines that express this protein in their seed. These soybean lines have now been increased, and the seed carrying this high-value protein will serve as a candidate vaccine against PRRS.
Project Highlights
A highly-efficient soybean transformation system using cotyledonary explants and a partially disarmed strain of Agrobacterium tumefaciens was established.
A total of 25 putative transgenic soybean lines carrying the open reading frame (ORF) 5 of PRRSV have been developed using Agrobacterium-mediated transformation of immature zygotic cotyledons of soybean. Among those, four lines have been confirmed for stable integration of the ORF5 transgene following Southern blotting.
Expression of the ORF5 antigenic protein in these plants was determined following western blot analysis using polyclonal antibodies synthesized for ORF5. The above four lines expressed the highest levels of the antigenic protein. Therefore, T1 seed was harvested from these four lines, and T2 plants were germinated, and plants were grown in the greenhouse.
Seed from these T2 plants will be collected within the next month, analyzed for segregation of the transgene, and seed will be analyzed for antigenic protein levels. Seeds from those lines expressing the highest level of the antigenic protein will be used in mice feeding studies to assess the efficacy of this soybean-based antigenic protein in inducing an immune response in immunized mice.
A total of 40 transgenic lines of potato carrying two different ORFs, ORF4 and OPRF5, of PRRSV have been also established. Among these, two lines carrying each of the two ORFs have been identified to express the highest level of the corresponding antigenic. The expression of the antigenic protein of one of the ORFs has been detected using immunofluorescence.
Publications
- Refereed articles
Ko, T-S., S. Lee, S. Krasnyanski, and S.S. Korban. 2003. Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explant. Theor. Appl. Genet. 107: 439-447.
Ko, T-S., S. Lee, S.K. Farrand, and S.S. Korban. 2003. A partially disarmed vir helper plasmid pKYRT1 promotes somatic embryogenesis following transformation of immature cotyledons of soybean. Planta (submitted).
Ko, T-S. and S.S. Korban. 2003. Effects of explant orientation on location and frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature zygotic cotyledons of soybean [Glycine max (L.) Merrill.]. J. Exp. Bot. (submitted).
Ko, T-S., S. Lee, R.L. Nelson, and S.S. Korban. 2003. Screening multiple soybean genotypes (MG 00 to MG VIII) for somatic embryogenesis and Agrobacterium-mediated transformation. Theor. Appl. Genet. (submitted).
- Abstracts
Korban, S.S. 2003. Perspectives on plant-based vaccines. Crop Sci. (in press).
Ko T., S. Lee, R.L. Nelson, and S.S. Korban. 2003. Efficient screening of soybean genotypes for competence for transformation. Crop Sci. (in press).
Recognition
Outstanding Researcher Award, American Society for Horticultural Science, 2002.
Although the above award is a career award, nevertheless the work undertaken by this IMBA-funded research project obviously contributed to receiving this recognition.
Dr. Korban bas been asked to convene a Symposium on "Genetic and Metabolic Engineering for Value-Added Traits" at the upcoming ASA-CSSA-SSSA Annual Meetings, Nov. 2-6, 2003, Denver, CO.
