Three cultivars of USA breeding: Jack and Jack x 4 (resistant to biotic stress), and Spencer (sensitive to micropathogens infection), 7 varieties of Kazakhstan breeding (Kazakhstan, Eureka, Vita, Tazhan, Zara, Perizat, Dannaya) were used for introduction of lignification genes. Ten biological replicates of each homozygous transgene and wild-type were grown simultaneously in a random block design, in the same environment in greenhouse. Plants were grown under long-day conditions (16 hours light, 28°C, and 65% humidity) to allow the development of health soybean plants.

Germ-line transformation was performed using A. tumefaciens, strain EHA 105 transformed with following vectors (Table 1).

Table 1.
Vectors used for genetic transformation

*The genetic constructs of lignification genes were kindly presented by Professor J.M. Widholm laboratory, Crop Science Dep., UIUC, USA.

Transformation in A. tumefaciens or E. coli (for glycerol plasmid stocks and plasmid propagation) was performed using electroporation.

Soybean flower petals were separated and flower was cut in the carpel remove 1/3 of stigma with small scissors. About 2 µL of A. tumefaciens suspension transformed with a vector containing a gene of interest was placed in the center of the open flower to the stigma by a microsyringe or a micropippete (Figure 1a). After 30 minutes flowers were inspected and if the suspension was not enough the second pipetting was performed. Treated flowers were marked by color threads correspondent to the genes of interest (Figure 1b). Pipetting time was optimized to introduce transformed A. tumefaciens at 3-4 stages of soybean flower development, when the anthers were fully formed and pollen tubes are growing into mature, but not yet dividing zygote (Figure 1c). To synchronize pipetting time all soybean flowers opened the day before were removed at 8:00. It was determined that the stigma allocation of substances that stimulate the germination of pollen starts at 9:00, at around 9:15 pollen is attached to the stigma and the pollen germination begins and continues till 9:30 (Figure 1d-f). Therefore, the best time for pipetting in a greenhouse conditions (at 28°C) was 9:00 am – 12:00 pm. In the field the time range can be 11:00 – 13:00 the same day, when the flowers have opened and the resulting pollen tubes are able to pass the desired genetic material (at the time of the pollen activity maximum). The timing of pipetting in the field may be dependent on temperature and solar radiation.

Figure 1.
Agrobacterium tumefaciens-mediated soybean germ-line genetic transformation. a The process of soybean flower pipetting with A. tumefaciens - suspension containing constructs with the genes of interest. b Marking of treated flowers by colored threads. c Scheme of stages of soybean flower and stigma development, adapted from https://www.agrodialog.com.ua/biologiya-cveteniya-soi.html. d - f – anatomy of soybean pollination (magnification 400x): d - stigma allocation of substances that stimulate the germination of pollen, 9:00 am; e - pollen attachment to the stigma and the beginning of pollen germination, 9:15 am; f - germination of the pollen tube, 9:30 am

Plasmid DNA was extracted from an overnight culture by using QIAprep Spin Mini prep Kit QIAGEN (Qiagen, Valencia, CA, USA). Genomic DNA was extracted from fresh mature but young transgenic soybean leaves using the method (Weigel & Glazebrook, 2009)⁠ and was used in PCR. Following primers were used (Table 2).

Table 2.
PCR primers used for the study

Transgene presence in plant leaves tissues was confirmed by the PCR reaction using Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA) with denaturation at 94 °C (1 min.), annealing at 48°C or 54°C (40 s) for PtMYB4., PAL5, F5H, CAD1 constructs and extension at 72°C (90 s), for 35 cycles.

Multiplex PCR was performed similarly, with the exception of the temperature of the annealing stage (60°C).

RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) was used for extraction of total RNA from examples. DNA was digested with DNase I, using the TURBO DNA-free kit (Ambion/Life technologies, Carlsbad, CA, USA). cDNA was synthesized using oligo (dT) primer and reverse transcriptase enzyme, Promega GoScript kit, (Madison, WI, USA) for positive control or excluding reverse transcriptase enzyme as a negative control for genomic DNA contamination. To amplify PtMYB4, PAL5, F5H and CAD1 transcripts from cDNA same primers as for genomic DNA detection were used.

Total genomic DNA of transgenic plants was digested with BamHI endonuclease (Fermentas, ThermoFisher, USA). Fragment size was 1,5 kB for PAL5 transformants and 1,2 kb for CAD1 transformants. DNA was precipitated in isopropanol and sodium acetate solution, precipitate was washed with ethanol and dissolved in water. After DNA electrophoresis DNA was transferred to a nylon membrane (Amersham, UK) using denaturation alkaline buffer (1 N NaOH + 0.4 M NaOH + 0.6 M NaCl). Membrane was dried for 30 min., DNA was fixed in UV-light (265 nm) for 5 minutes. Membrane was used for subsequent hybridization with P³²-labeled probes prepared from PAL5 and GUS PCR products (as described in molecular analysis section) using NEBlot® Kit (New England Biolabs, Ipswich, MA, USA). After a prehybridization at 50°C hybridization was performed for 16h at 50°C in hybridization oven (Amersham, UK). Membrane was washed with 2 times in 1X SSC and 0.1% SDS at 50°С for 15 minutes and 3 times in 0.2X SSC and 0.1% SDS at 58°C. The intensity of radioactive label was measured using radioactivity counter. Exposure of the membrane with the X-ray film (Hyperfilm TMMP, Amersham, UK) was carried out with an amplifying screen. Same membrane was hybridized with the gus-probe.

For Northern blot total RNA was extracted from the same plants using guanidinium thiocyanate method. 5 ug RNA was subjected to agarose gel electrophoresis and was transferred to a nylon membrane (Amersham, UK). Membrane was prepared similarly to the membrane used for Southern blotting. Membrane was used for subsequent hybridization with P³²-labeled probes. Probes were prepared from PCR products (as described in the molecular analysis section) using NEBlot® Kit (New England Biolabs, Ipswich, MA, USA). Membrane was washed with 0.2X SSC and 1% SDS at 58°C for 1 hour.

Lignin content and composition was performed as described in (Van Acker et al., 2013). The lignin composition was investigated with thioacidolysis (Robinson & Mansfield, 2009). The monomers involved in β–O–4-ether bonds, released upon thioacidolysis, were detected with gas chromatography (GC) as their trimethylsilyl (TMS) ether derivatives on a Hewlett-Packard HP 6890 Series GC system (Agilent, Santa Clara, CA, USA) coupled with a HP-5973 mass-selective detector. The GC conditions were as described in the protocol. The quantitative evaluation was carried out based on the specific prominent ions for each compound. Response factors for H (hydroxyphenyl), G (guaiacyl), and S (syringyl) units were taken from. The sum of H, G, and S is a good estimate of the total thioacidolysis yield and, thus, the condensation degree of the lignin polymer.

PAL activity was determined by the conversion of L-phenylalanine to trans-cinnamic acid essentually as described previously (Edwards & Kessman, 1992)⁠ with a few modifications: 300 mg of fresh leaves were ground in liquid nitrogen and extracted with 1200 µL of the extraction buffer, containing 0.05 M TrisHCl (pH-8.8), 0.5% ascorbate, 10% glycerol and 10 mM 13-mercaptoethanol. 100 µL of crude enzyme extract was combined with 400 µL of 0.05 M Tris buffer (pH 8.8), containing 0.2 mM phenylalanine as substrate. The reaction mixture was incubated for 30, 60, 120 and 180 min. at 37°C. The reaction was stopped by adding of 100 µL 0.5M HCl. Cinnamic acid was extracted with 1 mL of toluene and the OD was measured at 290 nm, with toluene used as a blank. The calibration curve was obtained using trans-cinnamic acid from Sigma. Total protein was measured using Bio-Rad Protein Assay (Bradford method).

For CAD1 enzyme activity determination plant tissue (300 mg) was ground in liquid nitrogen and crude extract was prepared using the extraction buffer containing Tris HCL 0.05M, pH 8.8, 10% glycerol and 10 mM β-mercaptoethanol. The CAD assay was performed according to (Mansell, Gross, Stöckigt, Franke, & Zenk, 1974)⁠ with a few modifications. The enzyme activity was determined by the conversion of alcohol to aldehyde (reverse reaction). The formation of hydroxycinnamaldehyde at 37°C was monitored at 400 nm using the following molar extinction coefficients: 1.78 x 10⁴ M^-1 cm^-1 for coniferyl aldehyde and 14.7 x 103 M^-1 cm^-1 for sinapyl aldehyde (Hawkins & Boudet, 1994)⁠. The reaction mixture contained in a total volume of 500 µL : 25 mM Tris- HCl, pH 8.8, 200 µl NADP (2mM), 200 µL (2mM) coniferyl alcohol/sinapyl alcohol and 100 µL crude extract. The same mixture, except 100 µL of TrisHCl buffer instead of the crude extract, was used as a blank. Protein concentrations were determined by the Bradford dye-binding assay.

We performed A. tumefaciens-mediated transformation of 1070 flowers of 3 USA and 7 established and zoned in Kazakhstan soybean varieties. Overall we obtained 833 pods (78% efficiency) containing 2000 putative transgenic soybean seeds of zero generation (T₀), expressing the genes of interest (PtMYB4, PAL5, F5H, and CAD1), respectively. Seeds forming efficiency on average was 187%. We did not observe any significant differences by size, shape or mass of pods and seeds between T₀ and control plants (data is not shown). The average number of seeds in a pod at T₀ was 2.5 seeds. T₀ seeds were planted to obtain plants of the first generation (T₁) for the further molecular analysis.

We analyzed the presence of the transgenes in the DNA and the presence of the transgene transcripts in T₁ soybean plants with PCR using genomic DNA and RT-PCR respectively. As a positive control for PCR of transgenic soybean plants, we used vector plasmid DNA. As a negative control for PCR, genomic DNA of untransformed wild parental plants was used.

The results of the standard PCR analysis of PtMYB4 gene insertion into soybean genome of T₁ plants shown in Figure 2a, b, and PAL5 gene insertion into genome of T1 plants – shown in Figure 2c. RT-PCR analysis of transgenic soybean lines of first generation demonstrated expression of PtMYB4 gene in RNA level (Figure 2 d, e).

Figure 2
PCR and RT-PCR analyses of T1 transgenic soybean plants. M – marker, Pl + plasmid, positive control; – wild type, negative control. a: PCR analysis of transgenic lines 1, 2, 3, 4, 21, 22, 23, 24 of Jack cultivar demonstrate PtMYB4 introduction into genome. b: PCR analysis of transgenic lines 4, 24, 25, 66, 6, 9, 11, 12 of Vita cultivar demonstrate PtMYB4 introduction into genome. c: PCR analysis of transgenic lines 118, 120, 121 of Tazhan cultivar demonstrate PAL5 introduction into genome. d: RT-PCR with reverse transcriptase of transgenic lines 4, 24, 25, 26, 66, 6, 9 demonstrate PtMYB4 expression at the RNA level. e: RT-PCR without reverse transcriptase of transgenic lines 4, 24, 25, 26, 66, 6, 9.

Overall, the presence of PtMYB4 gene insertion into genome was confirmed in 45 transgenic soybean lines, presence of PAL5 – in 30 transgenic lines, F5H – in 17 lines, CAD1 – in 20 transgenic lines. From 2000 analyzed putative transgenic soybean seeds we generated 112 T₁ transgenic plants with average efficiency of transformation 5,6 %. Importantly we did not observe any significant difference between the varieties in term of transformation efficiency, which proves that the developed method of germ-line transformation is not genome-specific. Data for other lignification genes is not shown.

Seeds of T₁ transgenic soybean plants were planted to obtain of second-generation plants (T₂) for confirmation of stable transformation by molecular and biochemical analyses.

About 1000 T₂ transgenic soybean plants, obtained from confirmed by PCR T₁ transgenic plants, were selected randomly and screened by PCR and multiplex PCR (Figure 3). As shown in Figure 3 a-c, a large number of T₂ transgenic soybean lines demonstrates the presence of the gene of interest (PtMYB4 and PAL5) (Figure 3 a, b), and marker gene nptII (Figure 3c). PCR of soybean plants of second generation T₂ have confirmed stable insertion of valuable PtMYB4 and PAL5 genes and marker gene nptII (Figure 3 c) into soybean genome.

As shown in Figure 3d, all genetic elements of CsVMV/PtMYB4sens. construct: the valuable gene, reporter LicB - (lichenase), marker nptII, T-nos terminator confirmed to be inserted into soybean genome in second generation (T₂) of transgenic plants. Multiplex PCR shows the main genetic elements included in gene construction through alignment at annealing temperature 48°C for all genetic elements (valuable gene, reporter, marker, t-nos terminator) of the CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1 constructs. Multiplex PCR demonstrated the integration of the whole transgenic construction into genome of transgenic plant. This confirms the stable insertion of whole genetic construction containing the genes of interest into genome of soybean transgenic lines. Overall, we confirmed the stable integration of all elements of genetic constructions of CsVMV/PtMYB4sens., 35S/PAL5, С4H/F5H, Cs/CAD1 genes into soybean genome with the efficiency more than 50%, in second generation T₂ of transgenic soybean lines.

Figure 3.
PCR-analysis (a-c) and Multiplex PCR-analysis (d) of T₂ soybean transgenic plants. a: Samples 6, 7, 15, 16, 27, 30, 32, 39, 81 - transgenic lines T₂, confirmed introduction into genome of PtMYB4 gene (top part of fig.) and marker gene nptII (bottom part of fig.). b: samples 12, 11a, 13, 7, 11, 4, 7, 1 ,9, 10 - transgenic lines T₂, confirmed introduction into genome of the PAL5 gene, and c: marker gene nptII. d: Samples 6, 7, 15, 16, 27, 30, 32, 39, 81 – T₂ transgenic lines, confirmed introduction of the elements of CsVMV/PtMYB4 construct: gene of interest (1000 bp), reporter gene LicB (700 bp), marker gene nptII (500 bp), T-nos terminator (200 bp).

The same putative transgenic plant examples were subjected to the Southern and Northern blot analysis (Figure 4). Total genomic DNA digested with BamHI. Hybridization with the PAL-probe confirmed the presence of the transgenes in all tested lines except PBI 121 PAL5 #1 and #3, which is in agreement with the PCR data (Figure 4a). Further we hybridized the same membrane with the gus-probe allowed seeing the pattern of gene insertion and estimating the transgene copy number (Figure 4b). All tested lines have multiple copies of the transgenes. The fact that bands are much wider in PBI 101 PAL5 line suggests the presence of a tandem repeats. There were no bands in negative control lines.

The same transgenic lines were used for total genomic RNA isolation and a Northern blot analysis. Northern blot with total genomic RNA of PBI 101 PAL5 and PBI 121 PAL5 lines was performed with untreated samples and samples treated with water (Figure 4 c). Hybridization with the pal probe revealed the presence of the genomic PAL in all samples, including control and the presence of transgenic band in PBI 121 PAL5 line #4 (Figure 4 c). Hybridization of the same membrane with the gus probe resulted in a band at the same place, suggesting the presence of bisictronic mRNA due the expression of a transgene at the transcriptional level (Figure 4 d). This band appears much weaker in untreated samples due to the significantly lower amount of RNA. Hybridization with the nptII probe revealed the presence of the nptII band in all samples, except the control as expected (Figure 4 e). Thus, the soybean transgenic lines are capable of the expression of introduced genes at the transcriptional level.

Figure 4.
Southern (a, b) and Northern (c, d, e) blot analyses, using genomic DNA from transgenic soybean plants, transformed with PBI101PAL5 or PBI121PAL5, digested with BamHI, and hybridized with: pal probe (a, c), gus-probe (b, d), nptII probe (d). Dashed line in the blots c and d represents a cut in the membrane.

Further we analyzed the content of lignin in the cell wall in CAD1 and PAL5 transformants. Lignin content in control lines was 23.17% of the dry weight, 28.8% in Cs/CAD1 transformants and 29.99% in 35S/PAL5 transgenic lines. This confirms that lignin content is increased in the presence of the target lignification genes expression.

Furthermore, we analyzed lignin composition in these transgenic lines. We observed an overall increase of lignin monomer content in 35S/PAL5 transgenic lines compared to the control lines. In the Cs/CAD1 transformants we observed an increase of S-monomers content compared to the control lines (Figure 5 a, b).

Additionally, we analyzed the activity of PAL and CAD enzymes expressed in the transgene plants to convert L-phenylalanine to trans-cinnamic acid and the conversion of coniferyl and synapil alcohols to the corresponding aldehydes, respectively. We observed a significant increase of PAL5 activity in PBI 101 PAL5 line #11 (Figure 5 c) as well as an increase of CAD1 activity in PBI 101 CAD1 line #5 (Figure 5 d). Correspondent with the transgene expression this proves the functional activity of the inserted genes of interest. All tested lines have multiple copies of the transgenes.

Figure 5.
Lignin content and enzyme activity in T₂ transgenic lines with introduced lignification genes. a - The lignin content in control and T₂ soybean transgenic plants expressing PAL5 gene. b - The lignin content in control and T₂ soybean transgenic plants expressing CAD1 gene. H - hydroxyphenyl, G - guaiacyl, and S – syringyl monomers of lignin. c - Changes over time of enzyme PAL5 activity in transgenic lines expressing PAL5 gene. d – Changes over time of enzyme CAD activity in transgenic lines expressing CAD1 gene. Error bars represent (n=8) (Weissgerber, Milic, Winham, Garovic, 2015)