Plant transformation
Embryogenic tissues induced from immature cotyledons of soybean cultivar Bragg were transformed by particle bombardment as described by Droste [16]. The pCL1390-UBQ3-SnOLP vector, a pCAMBIA1390 derivative (Cambia.org), contains the complete SnOLP gene ORF (GenBank accession AF450276) driven by the UBQ3 promoter (UBQ3-P) from A. thaliana and the hygromycin-phosphotransferase marker gene (hpt II) driven by the cauliflower mosaic virus (CaMV) 35S promoter (Figure 1).
Selection of hygromycin resistant embryogenic tissues, embryo histodifferentiation, conversion into plants and acclimation were carried out using the methodology previously described [16],[17].
Hygromycin-resistant embryogenic tissues were visually selected and separately cultured for establishment and proliferation of lines corresponding to putative independent transformation events. Thus, plants were recovered from three putative transformation events: B1, B2 and B3 lines. Plants derived from an independent piece of hygromycin-resistant tissue were noted as being clone plants.
Plants derived from non-transformed embryogenic tissues subjected to the same culture conditions were recovered and used as controls for molecular characterization.
PCR analysis
Genomic DNA was extracted from all recovered putative transformed plants (B1, B2 and B3 lines) and from untransformed soybean plants using the CTAB method described by Doyle and Doyle [18]. PCR analyses were performed using specific primers for the SnOLP gene (PPS1-foward 5’-CGCGGATCCATGGGCTACTTGAGATCT-3’ and PCPT-reverse 5’-CCCAAGCTTTTACTTGGCCACTTCATC-3’ [15]), which amplify a 744-bp DNA fragment, and for the hptII gene (forward 5’-GCGATTGCTGATCCCCATGTGTAT-3’ and reverse 5’-GGTTTCCACTATCGGCGAGTACTT-3’), which amplify a 512-bp DNA fragment. The PCR reaction mixture consisted of 100 ng of template DNA, 0.2 mM dNTPs, 1.5 mM MgCl2, 1X Taq Buffer, 2 units of Taq® DNA Polymerase (Invitrogen), and 0.5 μM of each primer. Reactions were hot-started (3 min at 94°C) and subjected to 25 cycles as follows: 1 min at 94°C, 1 min at 50°C and 2 min at 72°C with a final extension of 72°C for 5 min. All amplification reactions were carried out in a PCR Express Thermal Cycler (Thermo Hybaid, UK). PCR-amplified products were analyzed in 1% agarose gel, stained with ethidium bromide and visualized under UV light.
Protein expression analysis
For protein expression analysis, 0.2 g of fresh leaf tissue was excised from transgenic T0 plants and non-transgenic plants and homogenized in 500 μL of extraction buffer containing 50 mM of 1 M Tris–HCl (pH 6.8), 0.2% (w/v) polyvinylpyrrolidone (PVP-40) and 1% (v/v) β-mercaptoethanol. Samples were stirred for 30 min at 4°C and then clarified by centrifugation at 10,000 g. The protein content in the crude extract was determined by the Bradford method [19], using bovine serum albumin as standard. For each plant, approximately 50 μg of crude protein extract was subjected to 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The presence of the SnOLP protein was detected using polyclonal antibody specific for tobacco osmotin (kindly supplied by Dr. Bernard Fritig and Dr. Pierrette Geoffroy, Institut de Biologie Moléculaire des Plantes du C.N.R.S, France). The protein bands were visualized using the ECL Western Blot Detection and Analysis System (GE Healthcare). To disrupt less-specific interaction more stringent conditions were used by including detergent (0.1% Tween-20) in the wash solution.
Progeny analysis
T1 seeds obtained from self-fertilization of two T0 plants (one representative from B1 line and one from B3) were sown in pots containing soil and maintained in greenhouse. All T1 plants were screened for the presence of the SnOLP and hptII genes by PCR. Subsequent generations were obtained by self-fertilization of transgenic plants. Homozygous plants were detected in T3 generation by progeny tests and confirmed by PCR. Homozygous transgenic condition was monitored up to T7.
T5 homozygous transgenic plants were crossed, as pollen donors, with non-transgenic plants of BRS Fepagro 24 and BRS 211 soybean cultivars. SnOLP positive F1 plants obtained from crosses were self-fertilized to produce the F2 generation. F2 plants were screened for the presence of the SnOLP gene.
Transgene copy number estimation by quantitative Real Time PCR (qPCR)
One T5 homozygous plant from each B1 and B3 transgenic lines was assayed. Transgene copy number was estimated using relative quantification by qPCR standard curve analysis [20]. The curve was determined by the quantification of an endogenous gene in different DNA dilutions (1:100, 1:1,000 and 1:10,000). Lectin was chosen as the endogenous gene. Two lectin-encoding genes are present in soybean genome, this means there are four alleles in the homozygous diploid genome. The dilution 1:10,000 was hypothetically supposed to contain 4 alleles, the 1:1,000 40 alleles and 1:100 400 alleles. The copy number of transgenes in the same DNA dilutions was automatically calculated in proportion to that of the endogenous lectin genes using the StepOne Applied Biosystem Real-time Cycler™ (Quantification – standard curve experiment).
Primer pairs with a Tm at 60°C were designed to amplify gene sequences corresponding to SnOLP (forward 5’-CAACTTCGATGGTGCTGGTA-3’ and reverse 5’-TCA AAG CGT ATT CGG CTA GG-3’), hptII (forward 5’-TGGTTGGCTTGTATGGAGCAGCAG-3’ and reverse 5’-TGGTCAAGACCAATGCGGAGCATA-3’) and a lectin gene (forward 5’-TACCTATGATGCCTCCACCA-3’ and reverse 5’-GAGAACCCTATCCTCACCCA-3’).
qPCR was carried out under the following cycling conditions: 5 min at 94°C; followed by 40 repetitions of 10 s at 94°C, 15 s at 60°C and 15 s at 72°C; and 2 min at 40°C. A melting curve analysis was performed at the end of the PCR run, over the range 55-99°C, increasing the temperature stepwise by 0.1°C every 1 s. Each 25-μL reaction contained 12.5 μL diluted DNA template, 1x PCR buffer (Invitrogen, São Paulo, Brazil), 2.4 mM MgCl2, 0.024 mM dNTP, 0.1 μM each primer, 2.5 μL SYBR-Green (1:100.000, Molecular Probes Inc., Eugene, USA) and 0.3 U Platinum Taq DNA Polymerase (Invitrogen, São Paulo, Brazil). PCRs were performed in technical quadruplicates, and no-template reactions were used as negative controls.
Plant growth, drought treatment and physiological analysis
A preliminary test was performed to analyze the behavior of transgenic soybean plants under drought conditions. Eight non-transgenic Bragg plants and eight T6 homozygous plants from each transgenic line (B1 and B3) were grown in 1-L plastic pots for 26 days in greenhouse. Plants were assessed for tolerance to water deficit stress by withholding irrigation for 10 days. Plants were monitored daily for wilting.
A second experiment was carried out to provide detailed characterization of physiological parameters in T7 transgenic plants subjected to drought stress. Plants were individually grown in PVC columns (100 cm in height and 35 cm in diameter) filled with turf and vermiculite (1:1 v/v), natural phosphate and macronutrients (Terral, TrueMix, Brazil). Plants were maintained in greenhouse at 28 ± 2°C and 60 ± 10% relative air humidity. Photosynthetically active radiation (PAR) was measured using a Quantum Sensor LI-COR (Q-45556) attached to a LI-COR 6400 (LICOR-6400, LI-COR Inc., Lincoln, NE, USA). The photosynthetic photon flux density (PPFD) varied from 647 to 1020 μmol m−2 s−1. The experiment was carried out with a completely randomized design using the two transgenic lines (B1 and B3) and the Bragg wild-type (WT) plants, under two water regimes (watered/always irrigated and stressed/with a water deficit imposed at beginning of pod formation – R3 stage) with five biological replicates. The experimental unit was one soybean plant grown in a PVC column.
Before sowing, the substrate was dried at 105°C. Each PVC column was filled with 43.0 kg of substrate. Six small holes were made in the column bottom to facilitate initial drainage. Subsequently, columns were irrigated with water up to saturation and covered with plastic bags, and excess water was allowed to drain out for 24 hours. Then, drainage holes were sealed, and columns were weighed for field capacity determination. Three seeds were sown per column, leaving one plant per column after thinning on the day 12 after sowing. Every two weeks, 0.5 L of half-strength “Hoagland” solution [21] was applied to each column. The plants were irrigated regularly with water to maintain the substrate at field capacity up to the R3 stage (46 days after emergence; beginning of pod formation). After that, plants were separated into two groups: one continued to receive regular irrigation (watered plants), and the other was subjected to water deficit (stressed plants).
Measurements of water potential were performed as described [22] using an Oregon Corvallis pressure chamber, 97330 (PMS Instrument Company, Albany, OR, USA). Leaves were collected from the upper portion of the middle third of each plant.
The net assimilation rate (Pn), stomatal conductance (gs), and the transpiration rate (E) were measured from 09:00 to 11:00 a.m. under artificial, saturating photosynthetic photon flux (PPF) (900 μmol m−2 s−1), using a portable photosynthesis system infrared gas analyzer (Li-cor 6400XTR, Nebraska, USA.). Measurements were recorded at one, six and twelve days after imposing water stress.
At harvest maturity, 100-grain weight and grain production per plant were determined in five plants of each transgenic line (B1 and B3) and five WT plants.
Statistical analyses
The segregation rates of the T1 progenies of transgenic soybean plants, as well as the segregation ratios in the F2 generation from crosses between T5 homozygous transgenic plants and non-transgenic plants of two commercial cultivars, were analyzed using the chi-square test to confirm the expected Mendelian segregation pattern of 3:1 (transgenic:non-transgenic plants).
Predawn leaf water potential, net CO2 assimilation rate, stomatal conductance, transpiration rate, grain production per plant and 100-grain weight of the transgenic plants grown under two water regimes were compared to those of non-transgenic ones grown in the same environmental conditions. Data were subjected to analysis of variance (ANOVA) and comparison of means was performed with Student’s t-test using SPSS Statistics software. Physiological parameters obtained for B1 and B3 lines were compared to those of WT-plants for each evaluation day and water regime.