Plant materials, growth conditions, and vector
seeds were thrice surface-sterilized with 20% (v/v) bleach for 15 min; the seeds were then washed with sterile distilled water thrice before plating onto MS [34
] medium supplemented with 2% (w/v) sucrose for 5 d. The seedlings were then transferred into 15-cm-diameter pots filled with sterilized soil. Camelina
plants were then grown in a growth chamber (150 μmol photons m-2
; 75% RH) under a16 h light 22°C/8 h dark 18°C temperature/photoperiod regimen and watered every week with distilled water. Collected seeds were dried at 37°C in an incubator one week before use. Total RNA was isolated from fresh leaves by using Trizol reagent (Invitrogen, HK.). To generate full-length cDNA for RT-PCR, reverse transcription was performed using M-MLV reverse transcriptase (Promegra, HK). For Camelina
transformation, full length AtPAP2
(At1g13900, TAIR Database) cDNA was amplified with Platinum® Pfx
(Invitrogen, HK) and subcloned into the plant transformation vector pBa002 with Xhol and Sacl.
Six-week old WT Camelina
plants that first started flowering in the growth chamber were used for transformation following methodology of Lu et al. [14
]. After first transformation, new flowers in inflorescences were used for second and third transformations to improve the transformation ratio. After the third transformation, new inflorescences were cut to reduce background.
Generation of Camelina overexpression lines and null-lines
Homologous AtPAP2 transgenic lines of Camelina were selected on Basta plates (5 g/l Basta, Riedel-deHaen, Germany, 500 g/l carboncillin, 100 seeds/plate); WT Camelina was used as the negative control. Resistant plants were transferred to soil for growth to maturity. Their transgenic status was confirmed by genomic PCR and western blotting analyses using anti-AtPAP2 antibodies. Homozygous T3 seeds of the transgenic plants were used for further analyses. When there was 3:1 segregation of T1 transgenic seeds on Basta plates, null-lines were selected by their shorter hypocotyl lengths on MS plates. Identities of the three null-lines were confirmed by genomic PCR.
Seed viability, root and hypocotyl length measurements
Camelina seeds were surface sterilized with 20% (v/v) bleach for 15-20 min, washed with sterile distilled water and then plated on MS medium (50 seeds/plate). The experiment was carried out in a growth room (~100 μmol photons/m2/s) under a 16 h light (22°C)/8 h dark (18°C) temperature/photoperiod regimen. The lengths of roots and hypocotyls were measured 5 d after germination. Viability tests on Camelina seeds were performed in 10-cm diameter Petri plates each containing 20 ml of MS medium supplemented with 2% (w/v) sucrose; results were recorded after 5-6 d.
Extraction of plant genomic DNA and genomic PCR
Genomic DNA was extracted from young leaves using a CTAB-based method [35
]. The 25 μl reaction mixture contained ~200 ng of genomic DNA, 0.5 μl of each primer (15 mmol/l), 0.5 μl of dNTP mix (10 mmol/l each), 5 μl of green GoTaq reaction buffer and 0.25 μl of GoTaq DNA polymerase (Promega, HK). Cycling began with one cycle at 95°C for 2 min, followed by 30 cycles at 95°C for 30 s, 50°C for 30 s, 72°C for 150 s and a final extension at 72°C for 10 min. PCR products were sequenced using the primers AtPAP2-f (5'-TGCACTCGAGATGATCGTTAATTTCTCTTTC-3') and AtPAP2-r (5'-GTACGAGCTCTTATGTCTCCTCGTTCTTG-3'), and analyzed by electrophoresis in 1.0% (v/v) agarose gels.
Western blot analysis
Camelina leaves were finely ground in 1.5-ml Eppendorf tubes each containing 200 μl of ice-cooled extraction buffer (50 mMTris-HCl, pH 7.4 containing 150 mMNaCl, 1 mM EDTA, and 0.2 mM PMSF) and incubated on ice for 30 min with occasional mixing. The protein extract was separated by centrifugation at 4°C for 30 min at 10,000 × g. The supernatants were transferred to new 1.5-ml Eppendorf tubes and protein concentrations were determined with a Bio-Rad (Hercules, CA, USA) Protein Assay Kit. Proteins separated on 8% (v/v) SDS-PAGE were transferred to Hybond C-Extra membranes (GE Healthcare, HK) (400 mA, 1 h). Membranes were blocked with 5% (w/v) non-fat milk in TBST buffer (20 mMTris-HCl, pH 7.6, 137 mMNaCl, 0.1% (v/v) Tween 20) for about 2 h and probed with specific antiserum for 3 h at room temperature or overnight at 4°C. The membrane was rinsed with TBST, and HRP-labeled secondary antibody (1:10,000) in TBST was added for one more hour. The proteins were visualized with the Enhanced Chemiluminescence method (GE Healthcare, HK).
Measurement of photosynthetic rates and stomatal conductances
Photosynthetic rates and stomatal conductances were measured using a portable photosynthesis system (LI-COR, LI-6400, Nebraska, USA) in the morning (08:30-12:30) under a fixed blue-red light-emitting diode (LED) light source. Ten measurements were made on each fully expanded leaf collected from the shoot tips of 35-37-d old plants; at least 3 plants from each line were used for measurements. The leaf area of the standard broadleaf chamber was 6 cm2 (2 × 3 cm) and a fixed light source was provided by an array of red and blue LEDs. P vs. I curves were plotted using the instrument's auto-program function. Measurements were taken at light intensities of 200, 400, 600, 800 and 1000 μmol photons/m2/s.
Photosynthetic rate (A) was calculated as A = F(Cr-Cs)/100S-CsE, where A = net assimilation rate (μmol CO2/m2/s1), F = molar flow rate of air entering the leaf chamber (μmol/s), Cr = mole fraction of CO2 in the reference IRGA (infra-red gas analyzer) (μmol CO2/mol air), Cs = mol fraction of CO2 in the sample IRGA (μmol CO2/mol air), S = leaf area (cm2), and E = transpiration rate (mol H2O/m2/s).
Stomatal conductance is the rate at which water evaporates from stomata; it is directly related to the relative size of the stomatal aperture. Conductance is calculated from gsw = 1/(1/gtw-Kf/gbw), where gsw = stomatal conductance to water vapor (mol H2O/m2/s), gtw = total conductance to water vapor (mol H2O/m2/s), Kf = stomatal ratio, and gbw = boundary layer conductance to water vapor (μmol H2O/m2/s).
HPLC analysis of sugar content
For measurement of sucrose and hexose of plant tissues, 0.1 g aliquots of freeze-dried tissue powder were resuspended in 1 ml of 70% (v/v) ethanol, incubated at 70°C for 90 min and centrifuged at 13,000 × g
for 10 min. After passing through a 0.22 mm filter, a 10 μl sample was injected into a CarboPac PA 1 column (4 × 250 mm) connected to a Dionex (Sunnyvale, CA, USA) LC 20 Chromatography system and sugar contents were analyzed by high performance anion exchange chromatography with pulsed amperometric detection [36
]. Standard curves were prepared with 0-0.1 mg/ml sucrose, fructose and glucose solutions in 70% (v/v) ethanol.
SPS activity assay
SPS activity was assayed by the anthrone test [22
]. Samples were incubated for 20 min at 25°C in 50 μl of pre-balanced buffer (50 mM HEPES-KOH pH 7.5, 20 mMKCl, and 4 mM MgCl2
) containing (a) (for Vmax
assay) 12 mM UDPG and 10 mM Fru6P (in a 1:4 ratio with glucose-6-phosphate (Glc6P), and (b) (for Vlimiting
assay) 4 mM UDPG and 2 mM Fru6P (in a 1:4 ratio with Glc6P) and 5 mM KH2
Implementation of SimaPro software for life cycle analysis of green diesel from unaltered Camelina and its genetically modified counterparts
SimaPro was used to construct a LCA (life cycle analysis) of bio-diesel from wild type and genetically modified plants with different seed yields. The general processes for initiating a LCA study in SimaPro include: i. Determining the goal and scope of the specific study, which define the LCA system boundary. ii. Building of the life cycle flow tree, which specifies each stage involved in the life cycle. All environmental inflow and outflow inventory data are collected at this stage to build the life cycle processes; relevant life cycle assembly is then undertaken, followed by final construction of life cycles. Waste scenarios may be added into the life cycle flow tree, if available. iii. Choosing appropriate life cycle impact assessment methodology to calculate prospective results. iv. Interpreting results.
Life cycle analysis was conducted for biofuel derived from unmodified Camelina seeds (Group 1) and for biofuels derived from genetically modified Camelina with enhanced seed yields. We assumed that all input parameters were fixed and that the outputs of genetically modified Camelina seed yield increased by 10% (Group 2), 20% (Group 3), 30% (Group 4), 50% (Group 5) and 100% (Group 6) over output from the unmodified Camelina farming process. The whole life cycle was divided into several stages, including Camelina farming, seed oil production, refined oil production and end use oil output, which also set the system boundary defined for the study. The life cycle stages were used to organize data for software calculations.
Functional unit and life cycle inventory
The function unit in this study was one MJ of energy content in the fuel output. The choice was appropriate since energy content is the fundamental measurement for all environmental and energy flows in the life cycle analysis. The life cycle inventory for every stage came from the public Camelina
database for Camelina
in Montana, USA and the database in SimaPro. The input data for Camelina
farming, seed oil extraction, refined oil production and transportation are shown in Additional file 3
As shown in Additional file 3
Group 1 represented the original Camelina
seed yield, and Groups 2, 3, 4, 5 and 6 represented the yield increases in genetically modified plants, with seed yields increasing by 10%, 20%, 30%, 50% and 100% per unit area, respectively. The cumulative inputs of potassium (as K2
O), thomas meal fertilizer (as P2
) and urea-N were fixed for the one-hectare farming input. The quantity of fossil fuel diesel used remained unchanged on a unit area basis but changed with changing Camelina
seed output. As for GHG emissions, methane and dinitrogen monoxide emissions per kilogram of seed output remained unchanged. Carbon dioxide emissiona from diesel changed linearly with changing fossil diesel consumption, but at the same time remained fixed per unit area farmland output.
Growth phenotypes measurement, seed morphology, flowering time, and data analysis
Duration of time to first bud opening in the primary inflorescence was used as a measure of flowering time in Camelina. Flowering times were recorded in the T1, T2 and T3 generations (n = 4-6). Seed morphology of Camelina was observed microscopically. In Tables , , , , , statistical differences (P < 0.05) in the same row for each line were based on one-way ANOVA analysis followed by Tukey's Honestly Significant Differences (HSD) test using statistical program SPSS 18.