Embryogenic cultures of peanut were initiated from mature zygotic embryos (McKently, 1991) of the commercial cultivar, Georgia Green. Seeds were sterilized by shaking twice for 20 min each in 20% Clorox (1% w/v NaOCl), followed by three rinses with sterile deionized water. The plumule part of the embryo axis was excised and cultured on embryogenesis medium, which consisted of FN Lite medium (Samoylov et al. 1998) supplemented with 2.4% sucrose, 3 mg/L (12.42 µM) picloram, 1 g/L glutamine (filter-sterilized) and 0.8% agar. The pH was adjusted to 5.8 prior to autoclaving. Embryogenic cultures were grown in the dark at 26 ± 2 °C for up to 12 months and bombarded approximately 2 weeks after subculture under the assumption that the cultures were between lag and stationary phases of growth.

The vector (pBI101) contained the cpo-p gene from P. pyrrocinia (Wolfframm et al. 1993). The cpo-p coding region with a double CaMV 35S promoter and nos terminator (1.8 kb) was excised from the pBI101-derivative with HindIII/EcoRI digestion and subcloned into HindIII/EcoRI-digested pRT66, which contains the hygromycin phosphotransferase (hph) coding region under the control of the CaMV 35S promoter (Topfer et al. 1993). The resulting plasmid containing cpo-p and hph is referred to as pRT66 cpo-p (Fig. 1).

Twenty clusters of somatic embryos, with 3–5 embryos per cluster, were arranged in the central 2-cm area of a petri dish. Particle bombardment was carried out using a PDS 1000/helium-driven biolistic device (Bio-Rad Laboratories, Hercules, CA) as described previously (Ozias-Akins et al. 1993; Yang et al. 1998), except that 0.6 µm gold particles were used. Tissues were desiccated by uncovering the plates for 2–3 h prior to bombardment in the laminar flow hood. Selection was initiated in liquid embryogenesis medium containing 10 mg/L hygromycin 3 days post-bombardment in the dark. The medium was changed weekly for the first two subcultures to eliminate loose cell debris and agar medium after which it was changed every two weeks and the level of hygromycin was increased to 20 mg/L. A three-step regeneration protocol was followed to obtain plants from stably transformed somatic embryo cell lines (Ozias-Akins et al. 1993).

PCR was performed on hygromycin-resistant cell lines or regenerated putative transgenic plants to check for the presence of the cpo-p gene. Genomic DNA was isolated by the CTAB method (Murray and Thompson, 1980). A 582-bp fragment of the open reading frame of cpo-p was amplified using the sense primer S67 (5′-CAG CCC ATC GTC TTC CAT CAT GGC-3′) and an antisense primer A648 (5′-CGA CTT CAG GTC CTC GGT CTG ATC-3′). The PCR amplification was carried out using the following program: 94 °C for 5 min followed by 40 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min. The same PCR-amplified 582-bp fragment was also used as a probe for Southern and Northern blot hybridization.

For Southern blot analysis, peanut genomic DNA (10 µg) was digested to completion with HindIII or EcoRI and electrophoresed on a 0.8% agarose gel. DNA was transferred to GeneScreen Plus nylon membrane (PerkinElmer, Waltham, MA) using 0.4 N NaOH. The 582-bp cpo-p probe was labeled with α-³²P-dCTP using a random priming DNA labeling kit (Roche Applied Science, Indianapolis, IN) and hybridization was carried out overnight at 65 °C in the solution of Church and Gilbert (Church and Gilbert, 1984). Following hybridization, the membrane was washed at 65 °C for 15 min in 25 mL 2× SSPE/1% SDS, 15 min in 100 mL of 0.5× SSPE/1% SDS, and 15 min in 100 mL 0.1× SSPE/1% SDS. Hybridization signal was detected using a Cyclone Imaging System equipped with OptiQuant software (PerkinElmer, Waltham, MA).

Total plant RNA was isolated from selected embryogenic callus or young leaves (50–100 mg) using the Qiagen RNeasy Minikit (Qiagen, Valencia, CA). Total RNA (10 µg) was electrophoresed on a 1.0% denaturing formaldehyde (2.2 M) gel using 1× MOPS running buffer as described (Sambrook et al. 1989). RNA was transferred to GeneScreen Plus membrane using 7.5 mM NaOH and hybridized with the PCR-amplified 582-bp ³²P-labelled cpo-p gene fragment.

The method for Western blot analysis of CPO-P was modified from Rajasekaran et al. (2000). Leaf tissue was ground to a fine powder in liquid nitrogen and extraction buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) was added (0.5 mL per g of leaf). The homogenate was centrifuged twice at 13,000 g at room temperature for 5 min each to remove cell debris. The supernatant was collected and total protein in each sample was determined using the BioRad Protein Assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard. Aliquots of the resulting supernatants (40 µg crude protein) were denatured by heating with Laemmli buffer at 100 °C for 4 min and separated on a 12% polyacrylamide gel along with protein molecular weight standards (14.4–97.4 kDa Low-Range, Bio-Rad, Hercules, CA), and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA) with an electroblotter (Bio-Rad Laboratories, Hercules, CA). After blocking with 0.5% non-fat dry milk in PBST buffer, the membranes were probed with CPO-P polyclonal antiserum at 1∶1000 dilution and goat anti-rabbit alkaline phosphatase secondary antibody conjugate (Sigma-Aldrich, St. Louis, MO) diluted at 1∶1000. Nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate reagents were used for signal detection.

The in vitro analysis of antifungal activity of transgenic peanut extract to A. flavus was based on the method described previously (DeLucca et al. 1997; Rajasekaran et al. 2000). For tissue extract preparation, 1–2 g of embryogenic callus or peanut leaves were frozen in liquid nitrogen and ground without buffer. The sample extract was collected by centrifuging ground tissues at 10,000 g for 10 min at 4 °C. A conidial suspension (10⁵ cells/mL) of A. flavus NRRL3357 was pre-germinated for 8 h in potato dextrose broth (Voight Global Distribution, Lawrence, KS) and 17 µL were added to 153 µL of plant extract and incubated for 1–4 h at 30 °C. Three 50 µL aliquots were taken from each sample and spread onto three potato dextrose agar plates. The number of colony forming units was counted after incubation at 30 °C for 24–36 h. Antifungal assays were repeated at least three times for each transgenic sample and non-transgenic control Georgia Green. One-way ANOVA was used to analyze the effect of transgenic callus or leaf extracts on conidia germination. Mean separation was performed using the method of Tukey.

The EGFP (Enhanced Green Fluorescent Protein) gene (Clontech Laboratories, Mountain View, CA) was placed under the control of a constitutively expressed A. nidulans glyceraldehyde phosphate dehydrogenase (gpdA) gene promoter and the A. parasiticus N-myristoyl transferase (nmt-1) gene transcriptional terminator. All of these elements were subcloned into the plasmid vector pBlueScript-SK (Stratagene) to produce the vector gpd-EGFP (Rajasekaran et al. 2008). Plasmid gpd-EGFP was cotransformed with the vector pSL82 harboring the A. parasiticus nitrate reductase (niaD) gene into the niaD mutant of A. flavus 70. One isolate stably expressing high levels of GFP, designated A. flavus 70-GFP, was used in all experiments.

Mature transgenic peanut cotyledons and controls were inoculated with A. flavus 70-GFP to evaluate fungal spread in situ (Rajasekaran et al. 2005, 2008). Cotyledons were soaked overnight in water and sliced (10 mm × 6 mm × 6 mm) to keep the explants uniform. The A. flavus GFP strain was grown for 7 days at 30 °C on malt extract agar medium (Sigma-Aldrich, St. Louis, MO) before assay. Conidia were harvested by flooding a single plate with 9 mL of 0.01% (v/v) sterile Triton X-100 solution and scraping the surface of mycelium with a sterile pipette. Conidia were diluted to 2 × 10⁴ per mL for seed inoculation studies. About 100 conidia (5 µL) were introduced into cotyledon segments through a needle wound of approximately 3–4 mm depth. A minimum of 10 cotyledon segments per plate and three replicate plates were used per transgenic T4. To maintain high humidity, cotyledons were placed on three layers of Whatman filter papers soaked with water and the petri dishes were sealed with two layers of parafilm. After ten days of incubation at 30 °C the cotyledons were examined for GFP fluorescence of mycelium using the Olympus SZH10 GFP-stereomicroscope and the fungal colonization was quantitatively assessed as follows. Cotyledons were ground in liquid nitrogen, and 500 µL of 50 mM phosphate buffer, pH 7.2, was added to 500 mg of powdered cotyledons and mixed well by vortexing. The samples were centrifuged at 10,000 rpm for 10 min in a swinging bucket rotor. One hundred µL aliquots of supernatant from each sample were placed in a 96-well HP Viewplate, and the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using the Perkin-Elmer HTS 7000 fluorometer. Average fluorescence values were obtained for each sample and were subjected to non-parametric ANOVA using the GraphPad Prism software.

Most bombarded embryogenic tissues of Georgia Green showed rapid necrosis in medium containing 20 µg/mL hygromycin, and only a few tissues showed growth after ∼ 8 weeks on liquid selection. Tissues that were actively growing on liquid selection medium were transferred to agar medium containing 20 µg/mL hygromycin, and individual pieces were assigned a unique cell line number. Thirteen bombardment experiments with 107 plates yielded 147 hygromycin-resistant cell lines for an average of 1.37 cell lines per plate, which is within the reported peanut transformation efficiency range of 0.8 to 4.6 hygromycin resistant callus lines per bombardment (Higgins et al. 2004; Ozias-Akins et al. 1993; Wang et al. 1998; Yang et al. 1998). It is difficult to get accurate transformation efficiency data based on somatic embryo numbers bombarded since clusters of somatic embryos contain variable numbers, and some cell lines may have originated from the same transformation event but were separated due to agitation during liquid selection. In our estimation, there were 60–100 embryos present in each bombarded plate; therefore, the transformation efficiency for Georgia Green on a per embryo basis ranged from 1.37–2.28%. Plants were regenerated from 75 hygromycin-resistant cell lines and grown in the greenhouse.

The presence of the cpo-p gene was verified by PCR. A 582-bp fragment of the cpo-p gene was amplified from all 24 of the hygromycin-resistant cell lines tested but not from untransformed Georgia Green (data not shown). This result indicated that the hygromycin selection system was very effective for recovery of cpo-p transgenic cell lines from Georgia Green using the pRT 66 cpo-p construct.

Out of 78 putative T0 transgenic plants tested, 67 (86%) of them amplified the 582-bp fragment of cpo-p. As expected, hygromycin selection for transgenic peanut was not 100% efficient for recovery of a linked gene. Similar results were previously shown by Yang et al. (1998), who reported that 92% of cell lines PCR-positive for the hygromycin resistance gene also showed PCR amplification of the nucleocapsid protein gene of tomato spotted wilt virus, and Wang et al. (1998) who reported that 92% of hygromycin-resistant and PCR-positive cell lines showed PCR amplification of the vspB promoter-driven reporter gene, β-glucuronidase. It is likely that the non-selected gene linked with hph was disrupted in some instances during plasmid DNA integration.

Southern blot analysis with HindIII-digested DNA from hygromycin-resistant cell lines that were also confirmed to be PCR-positive for cpo-p, showed that most had multiple (2–4) hybridizing bands (Fig. 2A). Since the pRT66 cpo-p plasmid has only a single HindIII recognition site, these results indicated that multiple insertions were prevalent in the transgenic material. The particle bombardment method often results in the integration of multiple copies of transgenes (Meyer, 2000), which has frequently been observed in peanut particle bombardment experiments (Higgins et al. 2004; Livingstone and Birch, 1999; Yang et al. 1998). In addition, the band patterns revealed that some cell lines, such as 93-5-5 and 93-5-6 that originated from the same container during liquid selection, represented the same transformation event. Reducing the liquid selection time or replacing it with selection on agar medium could reduce the chance that multiple lines would originate from one event. However, selection on agar is more laborious for large-scale experiments and might increase the chance of recovering escapes. Southern blot analysis with transgenic T0 and T1 progeny plants from line 104-4-8 confirmed stable integration and transmission of the cpo-p gene (Fig. 2B).

To examine the bacterial cpo-p gene expression in peanut, and to efficiently select useful transgenic cell lines for regeneration, Northern blot analysis was conducted on hygromycin-resistant embryogenic cell lines. Eighteen out of twenty cell lines showed transcripts of cpo-p indicating that a high (90%) percentage of hygromycin-resistant somatic embryos expressed the gene (data not shown). The presence of the cpo-p transcript in transgenic plants regenerated from the cell lines expressing cpo-p was confirmed by Northern blot analysis of total RNA from leaf tissue (Fig. 3).

The cpo-p gene product, a 32 kD protein (Wolfframm et al. 1993), was detected by Western blot analysis in transgenic plants (Fig. 4). The result demonstrated that this bacterial cpo-p gene is efficiently expressed in transgenic peanut. One regenerated line, J6-2-2 T0-3, which was verified by PCR to be an escape, was also included in the Western blot analysis as a negative control and showed no immunoreactive band thus confirming the PCR data. In summary, 18/20 lines tested by Northern blot analysis showed cpo-p transcription, whereas six of those transcribing cpo-p were tested and shown to express CPO-P at the protein level. Only two lines were subjected to all molecular assays plus bioassay due to their vigor (and thus availability of leaf material) or ability to set seed.

To assay the potential antifungal activity of CPO-expressing transgenic plants was a major objective of this study. Crude protein extracts from embryogenic tissues of two Northern-blot-positive cell lines significantly inhibited (P<0.05) the growth of pre-germinated A. flavus conidia compared with non-transformed Georgia Green (Fig. 5A). Leaf extracts from mature transgenic peanut plants also showed significant antifungal activity for T1 progenies of line 104-4-8 (Fig. 5B). The presence of cpo-p transcript (Fig. 3) and CPO-P protein (Fig. 4) confirmed that the antifungal property of the transgenic progeny plants was due to cpo-p gene expression. Variation in expression and in vitro antifungal activity among individual plants was observed in this study and also was reported in CPO-expressing transgenic tobacco (Jacks et al. 2000; Rajasekaran et al. 2000).

Although little data were collected to compare the antifungal activity of extracts from embryogenic tissues with leaf tissues, we did observe that one cell line (71-7-2) with callus extract reduced the number of A. flavus colonies by 50% compared with the control, whereas with leaf extract, the number of A. flavus colonies was reduced by only 20%. Further experiments would be necessary to conclude whether the reduced antifungal activity in leaf extracts might be due to differences in protein accumulation resulting from the physiological or developmental status of the plant or to proteases or other substances that might not be as abundant in callus extracts. Our results do support, however, the use of embryogenic tissue extracts to predict antifungal activity of plant tissue extracts.

Cotyledon segments from both controls and transgenic groups inoculated with A. flavus 70-GFP strain turned yellow 4–5 days after inoculation due to colonization of cotyledons by the fungus. At the inoculation site the 70-GFP strain showed extensive growth and sporulation. Cotyledons from transgenic seeds showed significantly less (50–80%) fluorescence than the controls indicating a reduction in colonization of transgenic peanut cotyledons by A. flavus 70-GFP strain (Fig. 5C).