Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Nov 1, 2016
Project End Date
Oct 31, 2019
Grant Year
Project Director
Dhir, SA, KU.
Recipient Organization
Performing Department
Plant Science
Non Technical Summary
Pharmacologically important proteins such as edible vaccines, growth factor hormones, and monoclonal antibodies have been expressed in transgenic plants. However, a low level of antigen accumulation in plants is an impediment for plant-based edible vaccination systems. Hyper-expression of foreign proteins (especially of microbial origin, up to 30% of total cellular protein) has been accomplished via chloroplast genetic engineering since chloroplasts are ideal due to their ability to process eukaryotic protein. Cholera is among the top three diseases listed by the WHO, and the mortality rate is estimated to be more than 100,000 deaths annually. Diarrhea is caused by Vibrio cholerae (diarrhea) by colonizing the small intestine and producing enterotoxin, of which the cholera toxin (CT) is considered the main cause of toxicity. The molecular structure of CT involves two subunits: toxigenic A (CTA) and B (CTB). It is now known that CTB is non-toxic and confers protection against diarrhea infection when used for vaccination purposes, unlike CTA, which is toxic.In this study, we propose to develop transgenic alfalfa plants expressing the CTB gene, which can be used in plant-based edible vaccination systems. Alfalfa can be consumed without processing (as sprouts), and vaccines produced in seeds are stable over long storage periods. We will develop a chloroplast transformation vector comprised of the CTB gene, using aadA as the selectable marker gene, a 16S rRNA promoter and a psbA terminator from the chloroplast transformation vector provided by Dr. Henry Daniell, University of Pennsylvania, and Dr. Amit Dhingra, Washington State University. Alfalfa leaves will be bombarded with the vector using the Biolistic PDS 1000/He device. Spectinomycin resistant clones will be analyzed for the presence of the CTB gene using PCR and Southern blot analysis. They will also be analyzed for the presence of CTB protein using polyclonal antibodies raised against cholera holotoxin, using Western blotting experiments.The proposed research project would also enable us to initiate collaborative research with the University of Pennsylvania, Washington State University and USDA-ARS, St. Paul, MN. Minority student participation in the proposed project would provide them "hands-on" experience in biotechnology techniques and prepare them for productive careers in food and agricultural sciences. The project will provide an opportunity to our students to conduct innovative research in one of the frontier areas of agricultural sciences. The production and success of an immuno-modulatory transmucosal carrier molecule, like cholera toxin, in chloroplast will open the way for improving the efficacy of plant based edible vaccines.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
(B) Objectives: This research proposal is based upon the hypothesis that chloroplast genetic engineering can be used to produce transgenic plants with high level of protein expression in transgenic plants as compared to nuclear transgenic plants. We propose, therefore, to insert the CTB gene from Vibrio cholerae into alfalfa that will express the CTB subunit protein in the leaf and sprouts as an edible vaccine source. Molecular evaluation will be used to confirm the integration and stable expression of the foreign protein in the alfalfa plastid genome. This technology will provide a new approach aimed at introducing agronomically and commercially important traits into alfalfa to improve its adaptability and market value. Hence our project's specific objectives are:Objective 1: Develop tissue culture and plant regeneration protocols for alfalfaDiscussion: We will identify optimum factors, such as a source of explants, tissue age, type and concentration of auxins, cytokinins, effect of sugar (sucrose, maltose, glucose) in the culture medium that promote efficient plant regeneration in selected cultivars of alfalfa. Protocols for high frequency plant regeneration via somatic embryogenesis and somatic embryo encapsulation will be developed.Objective 2: Construct chloroplast targeting DNA vectors for expression of cholera toxin-B subunit Discussion: A chloroplast-expression vector with the green fluorescent protein (GFP) marker along with aadA and CTB will be constructed in collaboration with Dr. Henry Daniell, Department of Biochemistry, University of Pennsylvania, Philadelphia, PA and Dr. Amit Dhingra, Department of Horticulture, Molecular Plant Sciences Graduate Program, Washington State University, Pullman, WA. The presence of CTB in the correct orientation to the 5' and 3' signal sequence will be tested by restriction mapping of the vector and the expression of the gene in E. coli will be tested by immuno-blotting procedures.Objective 3: Introduce chloroplast DNA constructs into alfalfa using Biolistic Gene Gun and polyethylene glycol (PEG)Discussion: Preliminary toxicity studies (using streptomycin and spectinomycin) will be performed to determine the level of selective agents to reduce the number of non-transformed cells. Biolistic gene gun (after calibrating for DNA/gold amount, the nature of explants, and the interval distance of particle) will be used for DNA delivery. Optimum conditions will also be determined for protoplast isolation and PEG-mediated transformation for transient expression of genes.Objective 4: Characterization and regeneration of transgenic plantsDiscussion: After introduction of the chloroplast-expressing DNA vector, transformed cells will be observed at different times using the fluorescence stereomicroscope. After the independent transgenic embryos are big enough (3-5 mm in size), they will be transferred onto a regeneration medium. Putative transformants will be further identified and confirmed for DNA integration using the polymerase chain reaction (PCR), Southern hybridization and immunoblot analysis.
Project Methods
The introduction of the CTB gene into alfalfa using a Biolistic gun and characterization of transformed tissue and expression of transgeneswill be carried out, including isolation of CTB DNA fragment and DNA vector construction. The details of protocols and procedures for the proposed experiments, such as isolation of genes/proteins, DNA constructs, DNA delivery approaches and evaluation of transgenic plants are discussed below to achieve the following objectives:Objective 1: Develop tissue culture protocol for alfalfa Experimental approach: Medicago sativa seeds of several cultivars will be provided by Dr. Deborah A. Samac, USDA-ARS, University of Minnesota: surface-sterilized with 70% (v/v) ethanol for 30 s, dipped in 0.1% HgCl2 for 5 min, washed three times in sterile distilled water for 2 min each with shaking and soaked in sterile distilled water for 6 h. The sterile seeds will be placed onto agar-solid half-strength MS medium (Murashige and Skoog, in transparent plastic bottles and cultured for a period of 16 h light and 8 h dark. From seven to ten?day old seedling leaves and hypocotyls will be primarily used for the tissue culture establishment. These explants will be cut into small pieces (0.5-1.0 cm segments) and will be placed on a modified Murashige and Skoog's medium with 30 g/L of sucrose and different concentrations of auxins and cytokinins. Efficiency of shoot induction and somatic embryogenesis will be tested under several hormonal combinations. The selected calli from callus initiation medium will be transferred every two weeks to a plant regeneration medium. The regenerated shoots will be transferred onto rooting medium containing modified MS without any hormones for plant regeneration.Objective 2: Experimental approach: The AMA1 and MSP1 will be synthesized and cloned into the pGEMT Easy Vector (Promega, Madison, WI, USA). The CTB sequence will be amplified using the pLD-5'-UTR-CTB-Pins vector (Ruhlman et al., 2007) as the template. The sequence will be verified to check any errors, fused and sub-cloned into the pBSSK+ (Stratagene, La Jolla, CA, USA) vector. The CTB-AMA1 and CTB-MSP1 expression cassette will be created with tobacco regulatory elements and ligated into the pLDctv tobacco chloroplast transformation vector to obtain pLD CTB-AMA1 and pLD CTB MSP1 (Daniell et. al., 2001). The leaves of tobacco will be bombarded with pLD-CTB-AMA1 and pLD CTB-MSP1 and the transformation will be obtained using the protocol described by Singh et al., 2009; Dhir et al., 2010). The pUC-based alfalfa long flanking plastid sequence will be used to integrate foreign genes into the intergenic spacer region between the trnI (Ile) and trnA (Ala) genes as described by Ruhlman et al. (2007). The native plastid ribosomal operon promoter (Prm) and aadA:rbcL selectable maker gene cassette will be assembled in the pBSSK+ vector containing the Prrn promoter and rbcL 3'-UTR Objective 3: Introduce chloroplast DNA constructs using Biolistic Gene Gun and polyethylene glycol in alfalfa Experimental approach: Fully expanded, dark green leaves of about 6-8 week old plants will be used for bombardment. Leaves will be placed abaxial side down on filter paper laying on MS medium (Dhir et al., 2010) in standard petri-plates for bombardment. Gold (1.0 µm) microprojectile will be coated with plasmid DNA (chloroplast vector) and bombardment will be carried out with the biolistic device PDS 1000/He (Bio-Rad) as described earlier (Lawton et al., 2000). Several Two days after bombardment, the leaves will be cut into small 0.5 cm2 pieces and will be placed onto regeneration medium containing spectinomycin (100-500 mg/L). Similarly, protoplasts will be isolated from leaves and will be transformed using the PEG for transient and stable transformation using electroporation procedures as described earlier (Dhir et al., 2008; Mitchell et al., 1998; Singh et al., 2008; Lopez-Arellano et al., 2015). Objective 4: Regeneration and characterization of transgenic plantsExperimental approach: The first spectinomycin resistant tissues will be identified after 4-6 weeks of selection. Resistant shoots from the second/third culture cycle will be transferred to the rooting medium with spectinomycin (100-500 mg/L). Rooted plants will be transferred to soil and grown to maturity under continuous lighting conditions for further analysis. Polymerase chain reaction (PCR) will be completed using DNA isolated from control and transgenic plants to confirm true chloroplast transformations from nuclear transformants. Primers for testing the presence of the aadA gene (that confers spectinomycin resistance) in transgenic plants will be specific for the aadA coding sequence and 16SrRNA gene. No PCR product will be obtained with nuclear transgenic plants using this set of primers. This screening is essential to eliminate mutants and nuclear transformants and save space and labor while maintaining hundreds of plants.(i) DNA gel blot and probing of total cellular DNA: Total cellular DNA will be extracted by the method of Sambrook et al. (1989). The DNA will be digested using different restriction enzymes. DNA gel blots will be performed as described by Sambrook et al. (1989). Briefly, digested DNA will be electrophoresed in a 1.0% agarose gel and transferred to nylon membrane using the Posiblot apparatus. Random priming will generate labeled probes. DNA gel blots will be hybridized at 65 0C in Rapid Hybridization Buffer. After overnight hybridization at 65 0C, blots will be washed initially in 0.1 X SSPE plus 0.1% SDS and then the blots will be developed. (ii) Detection of GFP fluorescence in alfalfa chloroplast: Confocal fluorescence microscopy will be used to confirm the expression of GFP in chloroplasts. The leaves will be cut into pieces and osmotically treated with 13% (v/v) mannitol for 1 h, and incubated in CPW buffer (27.2 mg KH2PO4/l, 101 mg KNO3/l, 1480 mg CaCl2, 246 mg MgSO4, 0.16 mg KI/l, 0.025 mg CuSO4,pH 5.8) containing 2% (w/v) cellulase and 0.5% (w/v) pectinase for 8 h to release the protoplasts (Winfield et al., 2001; Singh et al., 2008). The protoplast suspension will be dropped onto a cover glass for confocal laser scanning microscopy (FV1000-IX81, Olympus Co., Japan). Images will be scanned and captured at 1000 X 9 magnification using FITC channel and RITC channel for green and red fluorescence imaging, respectively.(iii) Immunoblot Total chloroplast-derived CTB protein from chloroplasts will be extracted in ice-cold phosphate-buffered saline solution containing a 1X proteinase inhibitor cocktail. After centrifugation at 4 0C for 10 min at 14,000 rpm to remove cell debris, the soluble protein extract will be collected and protein concentration will be determined using the Bio-Rad protein assay reagent kit. For immunoblotting, protein extracts will be electrophoresed in 4-20% gradient SDS-PAGE gels and will be transferred to an nitrocellulose membrane using a semi-dry transfer apparatus. Polyclonal antiserum (1:1000 working dilution) to GFP or CTB will be purchased from Bio-design and used to probe the membranes. (iv)Means by which data will be collected and analyzed: The experiments related to somatic embryo production, plant regeneration and marker selection based assays will be replicated and subject to appropriate statistical analysis for mean separation (by least significant difference) and correlation coefficients will be calculated. The transformation experiments will be conducted in replicates and the comparison among the treatments will be computed by the General Linear Model procedure of analysis of variance and significant differences among treatment will be observed and means will be separated by SAS program (PROC GLM procedure).