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Agricultural biotechnology companies have been asked to provide intact transgenic seed to regulatory agencies as reference materials for evaluating transgene and protein detection methods (PCR and immunoassay). Due to intellectual-property and product-stewardship considerations, submission of devitalized seed prior to regulatory approval is preferable in any given country. Commonly used devitalization procedures, such as heating or autoclaving, degrade the protein and/or DNA rendering the seed unfit as a reference material for these tests. A novel method for devitalizing seed was developed that involves hydration, freezing in liquid nitrogen, and lyophilization. The devitalization method described here was found to preserve the transgenic DNA and protein in cotton (Gossypium hirsutum) and maize (Zea mays) seed allowing its use as a reference material for evaluating detection methods.
Agricultural biotechnology companies are cognizant of the need to control the distribution of regulated recombinant genes and technology in the environment. Companies are required to provide regulatory agencies around the world with detection methods for transgenic crops to support registration, import, and labeling laws. These analytical techniques are typically needed prior to deregulation, and some countries request that transgenic seed/grain be provided to local laboratories to confirm and validate these assays. Protein- or DNA-based detection methods may be required. Concerns over accidental release of unapproved transgenic crops and the need to protect proprietary germplasm obviate the need to provide devitalized tissues to countries where these transgenic crops are not yet approved. Most regulatory agencies are aware of this concern and accept ground seed as reference material for assay validation. However, a small number of jurisdictions request whole seed for this purpose.
Simple and efficient seed devitalization methods that maintain the integrity of the DNA and native proteins in the whole seeds have not been reported. Currently available devitalization procedures, such as autoclaving, degrade the protein and/or DNA rendering the seed unfit as a reference material for detection assays. Individual seeds may also be cut (quartered) and placed in individual vials, but this procedure is very labor intensive. Reported here is a simple method for devitalizing whole maize (Zea mays) and cotton (Gossypium hirsutum) seed in bulk, along with results of transgenic DNA and protein assays investigating the effects of the procedure on these constituents. Specifically, Herculex I™ maize seed containing the cry1F and pat genes1 and WideStrike™ cotton seed containing the cry1F, cry1Ac and pat genes1 were devitalized with this new procedure and the stability of the DNA and transgenic proteins were evaluated.
Seeds from transgenic (Herculex I and WideStrike) and nontransgenic maize and cotton were soaked in deionized water overnight at 4°C in the dark. The following day, the water was decanted and the hydrated seeds were fully submersed in liquid nitrogen and frozen for 3–5 min. The liquid nitrogen was decanted and the seeds were collected in a vessel and lyophilized (approximately one week). After the treated seeds were dried, treated and nontreated seeds were analyzed for viability, protein, and DNA stability.
Germination tests with 100 seeds per treatment (nontreated maize, devitalized maize, nontreated cotton, and devitalized cotton) were placed on moist, indented germination pads (Seedsburo Equipment Co., Chicago, IL) in plastic petri dishes at 27°C in the dark for up to 7 d. Numbers of germinated and nongerminated seed were counted and recorded. After the treated seeds were shown be devitalized, several seeds were finely ground in an IKA-Werke MF-10 grinder (Staufen, Germany) for subsequent protein and DNA characterization. The remaining seeds were kept at room temperature to assess long-term stability.
Samples of the ground cotton seed tissue (approximately 15 mg) were analyzed for Cry1F, Cry1Ac, and PAT proteins, and samples of the ground maize seed tissue were analyzed for Cry1F, using commercially available ELISA (enzyme-linked immunosorbent assay) kits and validated Dow AgroSciences methods. The PAT protein is nondetectable in Herculex I maize seed, so this ELISA was not conducted on the maize seed. The proteins were extracted from seed samples in microfuge tubes with a PBST (Sigma Chemical, St. Louis, MO) solution and two 1/8 inch steel ball bearings in a bead mill (Geno-Grinder, BT&C/OPS Diagnostics, Bridgewater, NJ) for 3 min at 1500 strokes/min. The extract was centrifuged; the aqueous supernatant was collected, diluted, and assayed using specific Cry1F, Cry1Ac, and PAT ELISA kits developed by Strategic Diagnostics (Newark, DE) for Cry1F and Cry1Ac, and Envirologix (Portland, Maine) for PAT. Serial dilutions of each sample (treated and nontreated seeds of the transgenic and conventional control) were incubated in the wells of an anti-Cry1F, anti-Cry1Ac, or anti-PAT antibody-coated plate. After a washing step, an aliquot of horseradish peroxidase-conjugated anti-Cry1F, Cry1Ac, or anti-PAT antibody was added and incubated in the plate to form an antibody–protein–HRP–antibody conjugate sandwich. At the end of this incubation period, the unbound reagents were removed from the plate by washing with PBST. The presence of the three proteins was detected by incubating the antibody-bound HRP conjugates with TMB (3,3′,5,5′-tetramethylbenzidine), generating a colored product. Since the proteins are bound in the antibody sandwich, the level of color development is related to the concentration of the Cry1F, Cry1Ac, or PAT in the sample (i.e., lower protein concentrations result in lower color development). The absorbance at 450 nm minus 650 nm was measured using a spectrophotometric plate reader, and compared to a standard curve to obtain quantitation of the transgenic proteins in the seed tissue extracts.
Samples for SDS-PAGE were prepared from finely ground seeds from each treatment of the transgenic and conventional controls. Approximately 50 mg of maize seed and 150 mg of cotton seed was extracted in 1 mL of PBST (Sigma, St. Louis, MO) for 3 min in a bead mill at 1500 strokes/min. Three 1/8 inch ball bearings were added to the microfuge tubes to facilitate the grinding process. After grinding, the soluble protein was collected by centrifuging the sample for 5 min at ~10,500 × g. As a positive control, 1–2 ng of microbe-derived Cry1F protein was dissolved in 10 μL of PBST. The samples were mixed with Laemmli buffer containing 2.5% 2-mercaptoethanol, heated for 5 min at ~100°C, and separated on 4–20% polyacrylamide gels from Bio-Rad (Hercules, CA). Two gels were prepared and one SDS-PAGE gel was stained with GelCode Blue (Pierce Chemical, Rockford, IL) total protein stain and the other gel was transferred onto a nitrocellulose membrane (Bio-Rad) for Western blot analysis. The analysis was carried out essentially as described in the Protein Electrophoresis Applications Guide2 from Hoefer Scientific (San Francisco, CA). The blot was probed with an anti-Cry1F polyclonal antibody from Strategic Diagnostics and detected with a horseradish peroxidase-labeled goat anti-rabbit polyclonal antibody from Bio-Rad. Immunospecific bands were visualized by exposing the membrane to CL-XPosure X-ray Film from Pierce.
Genomic DNA was isolated from maize and cotton seeds using a modified CTAB extraction protocol as described by Richards, Reichardt, and Rogers3 and further purified by using Genomic-tips according to the QIAGEN (Valencia, CA) Genomic DNA Handbook. The DNA was quantified using picogreen (Invitrogen, Carlsbad, CA) and 10 μg of genomic DNA from each of the treated and nontreated seed lots was independently digested with Hind III restriction enzyme. Positive control samples for hybridization were prepared by combining plasmid or fragment DNA with genomic DNA from the conventional control and digested using the same procedures and restriction enzyme as the test samples. DNA from the conventional control seeds was digested using the same procedures and restriction enzymes as the negative controls. The digested DNA was resolved on a 0.8% agarose gel and transferred on to positively charged nylon membranes according to Sambrook and Russell.4 DNA probes specific for the cry1F gene and a 1-kb ladder molecular size marker (Invitrogen) were radioactively labeled with [α-32P]dCTP. This was accomplished using Prime-It RmT Random Primer Labeling Kit (Stratagene, La Jolla, CA) and ProbeQuant G-50 Micro Columns (GE Healthcare, Piscataway, NJ) according to the manufacturers’ suggested procedures. Prehybridization and hybridization reactions were carried out according to Sambrook and Russell.4 After hybridization, the membranes were washed and exposed to X-ray film (Pierce) sandwiched between two intensifying screens for 2 d.
The regulatory agencies within some countries are requiring whole seed to test methods of DNA and protein detection. Whole seed is currently being requested to validate claims that transgenic seed can be detected at certain detection limits (e.g., 1 seed in 100 or 1000 seeds). Current methods of devitalizing seed involve subjecting samples to heat which can lead to protein and DNA degradation5–7 or physically cutting the seeds into quarters with sharp utensils which is very labor intensive. The most obvious method used to destroy viability is autoclaving. However, autoclaving is known to degrade proteins8 which can be inappropriate if the material is to be used for protein detection method validation. Another method includes exposing seeds to an inert gas and heat of at least 40°C for a period of up to 45 d to terminate seed respiration.6, 9 This method has been shown to maintain the popping quality of popcorn; however, it may result in protein degradation and is not freely available (patented). In the method described here, maize and cotton seeds were devitalized by soaking in deionized water overnight followed by freezing in liquid nitrogen and lyophilization. Holding the seeds at a cold temperature (~4°C) during the hydration process may help inhibit proteases that could effect protein composition. A germination test demonstrated that 100% of the treated seed was nonviable after this simple procedure (Figure 1). This method kept the seed intact, and allowed for the seed to be stored at room temperature for at least 6 months with no noticeable degradation. To test the stability of the DNA and protein, control and devitalized seed of two different crops were assessed by classical molecular biological and biochemical tools. Southern blot analysis was performed on DNA isolated from maize and cotton seeds to investigate the stability of the genomic DNA. Using a radioactively labeled probe for the cry1F gene, comparisons of the Southern blot hybridization patterns of the treated and untreated seeds were possible using genomic DNA digested with the endonuclease Hind III. Hybridization patterns for cry1F gene were identical for both treated and untreated seeds of both maize and cotton (Figure 2). These data indicate that the devitalization procedure described here maintained the genomic DNA and did not affect the quality of the DNA. As expected, all of the positive plasmid and fragment controls hybridized with the cry1F gene probes at the expected molecular weight. The cry1F probe did not hybridize to the conventional control DNA (Figure 2).
Aqueous protein extracts of the ground seeds were compared by SDS-PAGE, Western blot, and ELISA. The total extractable protein of the devitalized seeds was unchanged as demonstrated by SDS-PAGE analysis (Figure 3). The protein profiles were identical and the Cry1F protein was equivalent in both the devitalized and control seed of both crops. The full-length Cry1F, which is very susceptible to proteases,10 was maintained in the cotton seed (Figure 3). Quantitative ELISA analysis of the Cry1F protein in both maize and cotton was shown to be unaffected. In addition, the Cry1Ac and PAT proteins within the devitalized and viable cotton seed were present at equivalent amounts (Figure 4).
The moisture content of seed is known to affect tolerance to freezing.11–13 Using this knowledge, we developed a novel method to produce intact devitalized seed that preserves the integrity of the protein and DNA in the samples. While lower hydration of seed followed by exposure to higher freezing temperatures (e.g. −20 or −80°C) may devitalize some seed, soaking overnight in water followed by submersion in liquid nitrogen and lyophilization appears to be a robust method of devitalization that maintains the integrity of the protein and DNA. This simple, reproducible process resulted in intact cotton and maize seed that was 100% devitalized and able to be stored at ambient temperature for over 6 months with no apparent degradation of the seed. The resulting devitalized transgenic seed can used as a reference material for evaluating transgenic crop detection methods without the risk of releasing viable, regulated plant material or intellectual property.