Plant Material and Growth Conditions
For these studies, C. sativa cv. Suneson (Camelina) was used. The previously described high lauric (20 mol% of total seed fatty acids) Camelina line was used as our primary metabolic engineering platform (Kim et al. 2015a). Two plants per pot (20 cm diameter, 20 cm deep) with soil were grown under greenhouse conditions of approximately 24 °C, 16 h day/18 °C, 8 h night, with supplemental lighting (400–500 μmoles/m2/s) as needed to maintain day length conditions.
Insects and Insect Extracts
The pupae of codling moths were purchased from Andermatt Biocontrol AG (Switzerland). After being sexed, the male and female pupae were kept separately and emerged at 23 ± 1 °C, 17:7 L/D and 70% relative humidity. 2- to 3-day-old adults were used in the experiments. For pheromone extraction, 5–6 pheromone glands of virgin females were dissected 1–2 h into the scotophase, and extracted in 50 µL of n-heptane for 30 min.
Gene Cloning—Preparation of Constructs
The open reading frames (ORFs) of UcTE (Genebank accession number: Q41635.1) and codon optimized-CpoCPRQ were synthesized by Invitrogen. The sequence of three seed-specific promoters for the α’-subunit of β-conglycinin gene (β-con) (Chamberland et al. 1992), β-phaseolin gene (ß-Phaseolin) (van der Geest and Hall 1996), oleosin gene (Oleosin) (Fan et al. 2013), and two terminators for nopaline synthase gene (NOS), and nopaline synthase gene (HSP) were also synthesized by Invitrogen. ORFs of CpoCPRQ (AHW98354), AtWRINKLED1 (AY254038), CvLPAAT (ALM22867), P19 (P69516.1), and sequence of napin gene promoter (Napin), octopine synthase terminator (OCS) were amplified from entry clones. The glycinin gene promoter (Glycinin) was already contained in the final expression vector pBinGlyBar (Nguyen et al. 2013).
To compare different strategies towards production of high amounts of C12 pheromone precursors in Camelina, four plant expression vectors were constructed (Fig. 1). (1) CPRQ1.0 contained four exogenous genes of which UcTE was controlled by Glycinin and Cpo_CPRQ, AtWRINKLED1 and CvLPAAT were controlled by napin promoter (Fig. 1a). (2) CPRQ1.1 contained one exogenous gene Cpo_CPRQ codon optimized for A. thaliana (Arabidopsis) (Cpo_CPRQ_Ath) and controlled by Glycinin (Fig. 1b). (3) CPRQ2.1 contained four exogenous genes of which Cpo_CPRQ_Ath was controlled by Glycinin, UcTE was controlled by β-con, P19 was controlled by Napin and Cpo_CPRQ was controlled by ß-Phaseolin (Fig. 1c). (4) CPRQ2.2 also contained four exogenous genes, which were three copies of Cpo_CPRQ (one without codon optimized, one codon optimized for Arabidopsis, one codon optimized for Oryza sativa) controlled by ß-Phaseolin, Glycinin, Oleosin, respectively, and a P19 controlled by Napin (Fig. 1d). CPRQ1.0 was transformed into wild type Camelina, whereas the other three vectors were transformed into high lauric acid type Camelina.
PCR amplification was performed using the entry clone as template with a pair of degenerate primers, on a Veriti Thermo Cycler, using Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific™) under conditions as follows: start at 98 °C for 30 s, and 38 cycles at 98 °C for 5 s, 55 °C for 10 s and 72 °C for 50 s, followed by a final extension step at 72 °C for 10 min. Subsequently, fusion PCR was performed using phusion®Taq (Thermo Scientific™) (Atanassov et al. 2009) to do truncation and gene fusion for gene assembly, using the same PCR programs as described before. All genes with promoters and terminators were cloned into the plant expression vector pBinGlyBar, which contained a bar marker gene for Basta selection of transformed plants, by using Multisite Gateway® recombination cloning technology (Invitrogen). The constructed expression clones were confirmed by sequencing.
Floral Transformation of Camelina by Agrobacterium
The constructed expression vectors were introduced into Agrobacterium tumefaciens strain GV3101 (MP90RK) by electroporation (1700 V mm−1, 5 ms, Eppendorf 2510). The transformed Agrobacterium cells were grown on solid LB medium supplemented with antibiotics (50 mg/L rifampicin, 50 mg/L gentamicin and 50 mg/L spectinomycin) after incubating at 30 °C for 36 h. Afterwards, a single clone from each expression clone was incubated in 2 mL liquid LB medium with antibiotics as described above at 30 °C for 36 h. The Agrobacterium solution was then transferred to 30 mL medium for a 36 h incubation, and after that, the solution was transferred to 1 L medium for a 24 h incubation. Subsequently, the 5 weeks old Camelina plants were transformed by the floral vacuum infiltration method as described by Lu and Kang (2008) and Liu et al. (2012). A Basta resistance gene was used as a selection marker (Nguyen et al. 2013).
Sampling for Seed Oils Analysis
Seeds harvested from the floral-dipped plants were sown in soil for Basta selection, and the surviving T1 plants were considered as transformants. T2 seeds were harvested from matured T1 plants. Twenty-five T2 seeds from each T1 plant were randomly selected for pooled seeds fatty acid analysis. After the analysis, the four most productive plants from each strategy were identified. Fifteen individual seeds from the identified plants were randomly selected for single seed fatty acid analysis. Then, four plants from the best strategy line were selected for further propagation. For each plant, 25 seeds were sown for producing T3 seeds. Similarly, 25 T3 seeds from each T2 plant were randomly selected for pooled seeds fatty acid analysis, and 15 individual seeds from the three most productive plants were randomly selected for single seed fatty acid analysis. Afterwards, 20 to 45 seeds from the three T3 plants were sown to produce T4 seeds. Likewise, 25 T4 seeds from each T3 plant were randomly selected for pooled seeds fatty acid analysis. As the T4 seeds upon analysis could be considered homozygous, the seeds from all mother plants (T3 plants) were also pooled for fatty acid analysis and subsequently used for isolation of fatty acids for biotests.
Fatty Acid Analysis of Seed Oils
Fatty acids were analyzed as fatty acid methyl esters (FAMEs), which were generated from putative transformants either by grinding 25 pooled seeds (for production analysis of one transformant) or by grinding 15 seeds individually (for variation analysis within one transformant) from each mother plants in 1 mL 2% H2SO4 in methanol in a 4 mL glass vial. After grinding, the samples were incubated for 1 h at 90 °C. After cooling down to room temperature, 1 mL water and 1 mL heptane were added and the vial was vortexed. Then, the heptane phase containing the FAMEs was transferred to a new Agilent vial for GC/MS analysis on an Agilent 5975 mass-selective detector, coupled to an Agilent 6890 series gas chromatograph equipped with a polar column (HP-INNOWax, 30 m × 0.25 mm, 0.25 μm), and helium was used as carrier gas. The oven temperature was set at 80 °C for 1 min, then increased to 230 °C at a rate of 10 °C/min and held for 10 min. Fatty acid compounds were identified by comparison of retention times and mass spectra with those of reference compounds. To determine the position of double bonds in unsaturated fatty acids, DMDS derivatization was performed according to Dunkelblum et al. (1985). The DMDS-adducts were analyzed by GC/MS on a non-polar column (HP-5MS, 30 m × 0.25 mm, 0.25 μm) under the following oven temperature program: 80 °C for 2 min, then increased at a rate of 15 °C/min to 140 °C, and then increased at a rate of 5 °C/min to 260 °C, and held for 30 min.
Isolation and Reduction of Acids for Biotests
T4 seeds from 44 plants of line CPRQ2.2_5-3, 48 plants of line CPRQ2.2_5-12 and 18 plants of line CPRQ2.2_9-26 were harvested. After acid methanolysis similar to that described above, the methanolysis product in heptane was additionally washed with 1 mL of water, and then dried over anhydrous sodium sulfate before GC/MS analysis.
FAME samples were converted into alcohols in different batches. Batch 1 was a pooled FAME sample from 44 plants of line CPRQ2.2_5-3 and 48 plants of line CPRQ2.2_5-12, combined in a total volume of ca. 20 mL, which contained ca. 3% of the E8,E10-12:Me. After solvent evaporation, approximately 140 mg of FAMEs were obtained. To reduce fatty acid methyl esters to the corresponding alcohols, 3.4 mL anhydrous diethyl ether was added in a 50 mL flask under a nitrogen atmosphere and cooled with an ice-salt bath to 0 °C, while lithium aluminum hydride (39.3 mg, 1.04 mmol, ~ 2.0 equiv) was added. After stirring for 10 min, the 140 mg FAME sample dissolved in 1 mL of anhydrous diethyl ether was added into the flask. The mixture was stirred for 15 min and then warmed to room temperature and continued stirring for 2 h. After reaction, the reduction mixture was cooled with ice bath, and treated by successive addition of 10 µL of water, 10 µL of 15% sodium hydroxide solution and 30 µL of water, followed by stirring for 15 min at room temperature. The crude product was dried over anhydrous sodium sulfate and purified by flash chromatography on silica gel 60 (70–230 mesh) before analysis by GC/MS. This batch of plant-derived alcohol product was used in the electrophysiological experiment (see below).
The second batch of FAME samples, extracted from 5.1 g seeds from 48 plants of line CPRQ2.2_5-12, was directly reduced and the crude alcohol product was used in the first field trapping experiment without further purification.
The third batch of FAME samples, from 1.05 g seeds of line CPRQ2.2_5-12, was first fractionated by silver nitrate-silica column (15 cm, 2 mm i.d., 10% AgNO3) before reduction. The column was eluted consecutively with 4 mL mixture of heptane/acetone (98:2) as mobile phase, followed by another 4 mL heptane/acetone (96:4). The fractions that contained the target compound E8,E10-12:Me as the major component (above 85%) were combined and reduced to alcohol as described above.
The antennal electrophysiological responses of male C. pomonella to the plant-derived codlemone, to gland extracts of female adults, as well as synthetic codlemone were recorded on an Agilent 7890 gas chromatograph equipped with a flame ionization detector (FID) (Agilent, Santa Clara, California) and an electroantennographic detector (EAD). An HP-INNOWax column (30 m × 0.25 mm, 0.25 μm) was used in the GC. Hydrogen was used as the carrier gas with a constant flow of 1.8 mL/min, and a 1:1 division of the GC effluent was directed to the FID and EAD. The inlet was set at 250 °C, the transfer line was set at 255 °C and the detector was set at 280 °C. A PRG-2 EAG (10 × gain) probe (Syntech, Kirchzarten, Germany) was used in the recordings. Both tips-cut antennae from a male adult, associated with the head were mounted to the probe using conductive gel (Blågel, Cefar, Malmö, Sweden), and the antennal preparation was put in a flow of charcoal-filtered and humidified air (velocity ca. 25 cm/s). The GC oven was programmed from 80 °C for 1 min, then increased to 210 °C at a rate of 10 °C/min and held for 10 min. Data were collected with the software GC-EAD Pro Version 4.1 (Syntech).
A first round of field trapping experiments was carried out in two apple orchards in southern Sweden, a small orchard (ca. 2 ha, 55° 33.55 N, 14° 18.67 E) and a bigger orchard (> 10 ha, 55° 29.70 N, 14° 17.24 E), and in home gardens in Lund municipality (55° 42.31 N, 13° 11.48 E) from June 9th to 17th, 2021. Delta-traps with sticky inserts (Csalomon, Budapest, Hungary) and red rubber septum (Catalogue no. 224100-020, Wheaton Science Products, Millville, NJ, USA) dispensers were used. Plant-derived codlemone and synthetic codlemone (sample of unknown origin from our laboratory collection of reference compounds; isomeric purity 97.7% EE, 0.4% EZ, 1.3% ZE and 0.6% ZZ) were loaded on rubber septa at a dose of 100 µg/septum of the E8,E10-12:OH (AI), whereas 100 µL of n-heptane alone was loaded as a negative control.
A second round of field trapping was carried out in home gardens in Lund municipality (as specified above) from June 24th to 28th, 2021 to compare the activity of plant-derived codlemone after additional purification (mainly removal of C18 alcohols and improvement of isomeric purity as described above) with the initially obtained plant-derived codlemone. Conventionally produced codlemone (same as in previous experiment) was used as a positive control.
Traps within a replicate were randomly suspended in a row of apple trees in the orchards, at a height of ≈ 2 m above ground, with a distance of 10–12 m between traps and 12 m between rows. For experiments in home gardens, one trap with each treatment (or control) was hung in one tree at ≈ 2 m above ground and separated by at least 2 m. Distance between home gardens (and thus between replicates) was ≥ 100 m. The trapped moths were identified by their morphological characters. Catch data were log(x + 1)-transformed before performing statistical analysis. In the first experiment, catch data were compared using t-test, whereas catch data in the second experiment were compared using Anova followed by the Bonferroni post-hoc test (SPSS ver. 27).
Flight Tunnel Assay
The behavioral response of male C. pomonella to the plant-derived and synthetic pheromone was observed in a Plexiglas flight tunnel size 0.9 × 0.9 × 3 m (Valeur and Löfstedt 1996). The experimental conditions were: temperature 20 ± 1 °C, relative humidity 30 ± 2%, wind speed 0.3 m/s and light intensity 3.6 lx. A rubber septum was used as dispenser on which 100 µg of plant-derived crude or purified codlemone, or synthetic codlemone dissolved in 100 µL n-heptane were loaded (similar to baits used for field experiments). Experiments were conducted 0–1.5 h into the scotophase using 2–3 days old virgin males. A male was introduced in the downwind end of the flight tunnel and held in the pheromone plume for approximately 5–10 s, and allowed to take off and then observed for 3 min. For each treatment group, 15–20 males were tested, and each male was used only once. Differences in proportion of males reaching the odor source were analysed by the Chi-square test (SPSS ver. 27).