Plant material
Tomato seeds (Solanum lycopersicum L. cv. Rekordsmen) were kindly provided by the All-Russian Research Institute of Irrigated Vegetable, Melon and Ground Growing (Astrakhan oblast, Kamyziyak, Russia). The seeds were surface sterilized in 96% ethanol for 30 s and in 20% solution (v/v) of the commercial bleach ‘Ace’ with few drops of Tween-20 for 7 min, then rinsed with sterilized distilled water five times for 1 min each. After surface sterilization, the seeds were cultured on Murashige and Skoog (MS) basal medium [28] without growth regulators supplemented with 3% (w/v) sucrose and 0.7% (w/v) agar. The pH was adjusted to 5.8 before autoclaving. The cultures were maintained under 25 ± 1 °C, with fluorescence light (65 μmol m−2 s−1) during the long-day photoperiod (16 h light/8 h dark). The previously developed transgenic tobacco plants of the N. tabacum cv. Petit Havana SR1 line expressing full sized CYP11A1 cDNA of cytochrome P450SCC from the bovine adrenal cortex [29], the wild type plants and the plants transformed with an empty pGreen0229 vector [30], of new generations Т3, T4 and Т5 were cultivated at 25 °C with an illumination intensity of 1000–2000 lx and a photoperiod of 16 h.
Phenotype analysis of transgenic tobacco plants
For comparative phenotypic analysis, we used Т4 and T5 generation plants. Seeds were sterilized at the surface and couched in a Murashige and Skoog medium [28] containing selective agent phosphinotricin. Phosphinotricin resistant plants were open field planted. All of the plants were tested for gene integration and expression by molecular genetic methods. During the course of the plants growth and development, the start time of flowering and different morphological parameters were checked. The study was performed on 15–25 independent plants of each line.
Estimates of the speed of tobacco seeds sprouting and germination
Seeds germinated in Petri dishes on a filter paper. A Petri dish with slits was put on the grill with the tray. A strip of filter paper was passed through the slots at the bottom of the Petri dish providing the necessary hydration of the seeds. On the top Petri dishes was covered with caps to prevent seeds drying. Trays were incubated in a thermostat at 25 °C. In each variant there were 4 replications of 50 seeds. To determine the speed of germination number of germinated seeds was taken into account every day for 10 days. Seeds with the germ root of about 1 mm were counted as sprouted. Evaluation of germination was performed on the 10th day. Normally developed seedlings, the abnormal ones and not germinated seeds were taken into account. Statistical data processing was performed using software Microsoft Excel.
Testing CYP11A1 transgenic tobacco plants for resistance to fungal phytopathogen Botrytis cinerea
The phytopathogenic fungus of the species Botrytis cinerea causing gray mold was used as the test culture. In the provoking conditions, isolated leaves of medium age and of the same size were placed in Petri dishes on filter paper moistened with water. 10 μl suspension of spores of the pathogen containing 2000 spores/ml applied at 6 points on the sheet, after which the Petri dishes with leaves were tightly closed to create a high humidity and were placed and maintained in climate chamber at a temperature of 24 °C and 16-h photoperiod. The leaves of untransformed plants and plants containing only the empty vector plasmid (with no CYP11A1 cDNA) were used as controls. The degree of damage of the leaves lamina, inversely proportional in relation to the relative resistance of the plant to Botrytis cinerea phytopathogen, were evaluated in points according to the number and size of necrotic spots on the 10th days after infection. Testing was also carried out by a different approach, i.e. by the method of counting the number of germinated spores in a drop of the juice from the leaves. 20 μl suspension of spores of Botrytis cinerea (105 spores/ml) and 20 μl of untreated protein extracts from control (untransformed and transformed with an empty vector) and the CYP11A1 transgenic plants were applied into microcuvettes that were subsequently incubated for 24 h at 24 °C. Counting of the number of germinated spores was carried out under the microscope.
Agrobacterium strain and plasmid
Agrobacterium tumefaciens strain AGL0 [31], carrying the binary vector plasmid pBI121mod-P450SCC was used for tomato transformation. The T-DNA of plasmid pBI121mod-P450SCC containing CYP11A1 cDNA, encoding cytochrome P450SCC from the bovine adrenal cortex, driven by the Cauliflower Mosaic Virus 35S promoter (CaMV 35S), as well as the neomycin phosphotransferase II (nptII) gene which confers resistance to kanamycin for selecting of putative transformants (Fig. 1). The plasmid pBI121mod-P450SCC was introduced into A. tumefaciens strain AGL0 using the freeze-thaw method [32].
Agrobacterium-mediated transformation of tomato
Cotyledons with petioles from 10 to 12-day-old tomato seedlings were used as explants for transformation. Prior to co-cultivation, the cotyledon explants were precultured for 3 days on agar-solidified MS medium containing 2 mg/l zeatin and 0.1 mg/l indole-3-acetic acid (IAA) with the abaxial surface in contact with the medium. Such hormonal treatments induced the highest shoots regeneration frequency from cotyledons of tomato cv. Rekordsmen [33].
Agrobacterium tumefaciens AGL0 harboring pBI121mod-P450SCC binary vector was grown in a 250-ml capacity conical glass flask overnight at 28 °C in 50 ml of YEB liquid medium [34] containing 25 mg/l rifampicin and 50 mg/l kanamycin in a rotary shaker (150 rpm). Agrobacterium tumefaciens was resuspended at an OD600 = 0.6–0.7 with MS liquid medium. Then, pre-cultured explants were immersed in the above bacterial suspension for 20 min, blotted with sterile filter paper and transferred on Petri dishes containing agar-solidified MS medium, on the surface of which filter paper disks was placed. Co-cultivation was carried out at 16 °C in the dark for 72 h. After co-cultivation, the explants were washed with MS liquid medium supplemented with 300 mg/l timentin followed by rinsing five times to remove the bacterial overgrowth. Then the disinfected cotyledon explants were cultured on agar-solidified MS selection medium for callus induction containing 2 mg/l zeatin, 0.1 mg/l IAA, 25 mg/l kanamycin and 300 mg/l timentin. The explants were subcultured to fresh medium every 15 days. The explants survived in the selection medium generated calli from their cut ends. These calli were cultured in 300 cm3 glass cultures vessels containing medium for shoot regeneration (agar-solidified MS medium supplemented with 2 mg/l zeatin, 0.1 mg/l IAA, 25 mg/l kanamycin and 150 mg/l timentin). When regenerated kanamycin-resistant shoots were about 1.5-cm long, they were detached from the callus and transferred to root induction medium (half strength agar-solidified MS medium containing 0.2 mg/l indole-3-butyric acid and 100 mg/l kanamycin). Rooted independent transgenic lines of tomato were clonally multiplied in vitro and adapted to soil. Transgenic and non-transgenic tomato plants were grown in plastic pots filled with sterilized soil, at standard agricultural conditions (22–25 °C day time temperature and 18–19 °C night time temperature, humidity 60–70%, illumination 2500 lx). Plants were propagated by grafting of lateral shoots formed on the adult plants.
DNA/RNA extraction, PCR analyses and other molecular genetic techniques
Total genomic DNA was isolated from leaves of putative transformants and wild-type (WT) tomato plants according to Edwards et al. [35] with additional extraction with saturated phenol. To determine the quality of the isolated DNA for PCR analysis, the sequence of the Tom52 actin gene of tomato (NCBI, U60482) was amplified. The amplification was also carried out on the sequence of the selective (nptII) and target (CYP11A1) genes, as well as the virulence genes of the Agrobacterium tumefaciens (virB). Primer sequences, PCR conditions and the expected size of amplified products are presented in Additional File 2. The PCRs were carried out in a 25-μl volume containing 2.5 μl of 10X PCR buffer, 0.5 μl of 10 mM dNTPs, 1 μl of forward and reverse primers at 10 qM, 1 μl of 5 Uμl−1 Taq polymerase, 17 μl of deionized water and 2 μl (~60 ng) of a DNA template. PCR products were carried out in a MJ Mini Personal ThermalCycler (Bio-Rad). The products were separated in 1.0% agarose gel in an electrophoresis system (Amersham Electrophoresis Power Supply—EPS 301 + Hoefer HE 33 Mini Horizontal Submarine Unit).
The expression of CYP11A1 cDNA in transgenic tomato plants was assessed using RT-PCR technique (PCR coupled with reverse transcription). Isolation of total cell RNA from leaves of 5-week-old plants grown in a phytotron was carried out according to [36] or by using kit Total RNA Purification Kit (Jena Bioscience). cDNA was synthesised on the RNA template using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). PCR with synthesized cDNA was performed with primers complementary to the sequences of the target gene CYP11A1, the size of the expected PCR fragment was 520 bp [37]. A fragment (709 bp) of the gene encoding large ribosomal subunit of tomato (25S rRNA) [38] was used as a “housekeeping” gene. cDNA isolated from the original Rekordsmen variety was used as negative control. Standard procedures of molecular cloning were used for recombinant plasmid constructions [34].
Transmission electron microscopy (TEM)
The seeds of wild-type tobacco plants (Nicotiana tabacum cv. Petit Havana SR1) and two transgenic homozygous tobacco lines (TR-2 and TR-7) of T4 generation expressing CYP11A1 cDNA, encoding cytochrome P450SCC from the bovine adrenal cortex, were aseptically germinated on the MS basal medium [28] supplemented with 3% (w/v) sucrose and 0.7% (w/v) agar. The middle sections of cotyledons from the seedlings were fixed for 24 h in 2.5% glutaraldehyde (Merck, Germany) dissolved in 0.1 M Sorensen’s phosphate buffer (pH 7.2) with 1.5% sucrose. Then the samples were washed, postfixed in 1% ОsО4 (Sigma-Aldrich, USA), dehydrated in ethanol of increasing concentrations (30, 50, 70, 96, and 100%) and in propylene oxide (Fluka, Germany). The samples were embedded in mixture of Epon-812 and Araldite (Merck, Germany) according to the standard procedure. For TEM the embedded samples were sectioned with diamond knife using ultramicrotome LKB-V (LKB, Sweden), placed on formvar coated grids and stained with uranyl acetate and lead citrate [39]. The ultrathin sections were examined and photographed with electron microscope Н-500 (Hitachi, Japan). Ultrastructure of mitochondria in mesophyll cells was studied. The average cross-sectional area of mitochondria was determined using the Cell-A software (Olympus, Japan). At least 200 mitochondria were scored for each treatment.
The study of the transgenic tomato plants resistance to drought
It consisted of two stages: study of the reaction of samples on the long absence of irrigation and investigation of the recovery of plants after the resumption of irrigation. The experiment was carried out in the conditions of the glass box in the absence of irrigation, accompanied by high day (35–40 °C) and night (14–16 °C) temperatures. Plants in the development stage of 3–4 true leaves were grown in the absence of irrigation for 3 weeks, biometric indicators were recorded at 7th, 14th and 21th days. Biometric parameters (plant height; mean increment; the number of plants, wilted on 50 and 100%) of transgenic lines No. 4 and No. 7 were compared with each other and with the parent variety Recordsmen grown in the same conditions. Sample size: 15 plant of the variety Recordsmen, 60 plants of T2 generation of the transgenic lines No. 4 and No. 7.
Isolation of steroid fractions from tobacco and tomato leaves
Tobacco and tomato leaves were frozen in liquid nitrogen and lyophilized. Total lipids were extracted from 0.1 g of dry plant material by a CHCl3–MeOH–H2O mixture (5: 10: 4) 3 times for 4 ml. Extracts were joined, dried using a rotary evaporator at 85 °C, and separated by TLC using plates with silica gel 60 F 254 by Merck (200 × 200 mm, 0.5 mm thick) in a CHCl3–EtOAc system (4:1). One part of the plate was colored by a reagent with anisaldehyde (0.5 ml of anisaldehyde in 10 ml 98% H2SO4, 85 ml MeOH, and 5 ml 96% H2SO4). Zones that coincided by chromatographic mobility with cholesterol, pregnenolone, and progesterone were scratched out of the uncolored part of the plates, extracted by CHCl3, and dried using a rotary evaporator. The dry residue was dissolved in MeOH.
Determination of the progesterone content in transgenic plants
It was carried out using a combination of the TLC method and immunoenzymatic assay. Progesterone enzyme immunoassay was applied to leaf extracts from transgenic tobacco and tomato plants, wild-type plants, and to fractions, the chromatographic mobility of which under purification by TLC coincides with the chromatographic mobility of progesterone, according to the service instructions (the recommendations of the kit manufacturer) for the PROGESTERONE EIA kit. The kit was developed at the Institute of Bioorganic Chemistry, the National Academy of Sciences of Belarus, and is intended for quantitative progesterone determination in human blood serum by enzyme linked immunosorbent assay. The kit was kindly provided by A.G. Pryadko.