Plant material
To make a research collection of grapevine in vitro, vines of 22 grapevine genotypes were collected from field-grown mothervines of All-Russian National Scientific Research Institute of Vine And Winemaking "Magarach" (lat.: 44.850984′N, long.: 33.650112′E), vernalized for 1 month at a temperature of + 4°C and germinated in vessels with water. Further in the greenhouse grown green shoots were superficially sterilized by submersion in sodium hypochlorite solution (2.5% NaOCl, 0.1% Tween 20) for 15 min and then fourfold washed with sterile water. The shoots were then cut into single-node cuttings and placed in tubes on modified MS according Zlenko et al. 2017 [described as «plant growth» medium (PG) contained KNO3 (922,0 mg l− 1), NH4NO3 (308,8 mg l− 1), KH2PO4 (82,0 mg l− 1), MgSO4 • 7 H2O (597,0 mg l− 1), CaCl2 (331,0) mg l− 1), vitamins by MS, FeEDTA and microelements halved by MS] medium supplemented with 1% (w/v) sucrose and 0.75% (w/v) agar (Panreac, Spain) and cultivated at 5.7 pH, 25 ± 1 ° C and light intensity of 65 µmol m− 2 s− 1 during 16-h day photoperiod. After 2 months cultivation on PG medium from single-node cuttings (without signs of bacterial or fungal infection), shoots developed, which were cut off and placed on culture vessels (total volume 500 ml) containing 50 ml agarized PG medium supplemented of 0.05 mg l− 1 NAA, 7 plants per each culture vessel.
Development of the basic composition of callus induction and regeneration medium
Two first experiments on callus induction and regeneration in 22 studies grapevine genotypes were performed on Murashige and Skoog (MS) (Murashige and Skoog 1962) and modified MS according Mezzetti et al. 2002 [described as «initiation medium» (IM) contained KNO3 (1050 mg l− 1), NH4NO3 (400 mg l− 1), KH2PO4 (200 mg l− 1), MgSO4 • 7 H2O (400 mg l− 1), CaNO3 (750 mg l− 1), NaH2PO4 (200 mg l− 1), microelements and vitamins by MS] mediums with supplemented with 0.75% (w/v) agar (Panreac, Spain) and various concentrations and combinations of the growth regulators BA or TDZ jointly with IBA:
Experiments no. 1 – MS medium supplemented with 2% (w/v) sucrose and TDZ ranging from 0.5 to 1.5 mg l− 1 in increments of 0.5 mg l− 1, with addition of IВА 0.01, 0.05 or 0.1 mg l− 1 (Zhang et al. 2011). To induce regeneration, the explants were pre-cultivation in dark conditions (3 weeks for leaf fragments and 2 weeks for petiole and internode fragments), at 24 ± 0.5°C.
Experiments no. 2 – IM medium supplemented with 3% (w/v) sucrose and BA ranging from 2.0 to 3.0 mg l− 1 in increments of 0.5 mg l− 1, with IВА ranging from 0.05 to 0.15 mg l− 1 in increments of 0.05 mg l− 1 (Kumsa 2017). To induce regeneration, explants were pre-cultivation in dark conditions (4 weeks for leaf fragments and 3 weeks for petiole and internode fragments), at 24 ± 0.5°C.
Fragments of leaves, petioles and internodes obtained from aseptic in vitro grapevine plants cultivated on PG medium supplemented of 0.05 mg l− 1 NAA were used as explants. The leaves were cut into fragments measuring 5 mm x 5 mm and placed on Petri dishes with the adaxial side. Petioles and internodes were cut into fragments 5 mm long and placed longitudinally on the culture medium. All explants were placed on Petri dishes containing 25 ml culture medium supplemented different combinations of growth regulators (according to experimental conditions no. 1, 2), 10 explants per Petri dish. After pre-cultivation in the dark, the explants were cultured in the light conditions, where the light intensity was 65 µmol m− 2 s− 1 during 16-h day photoperiod and temperature 25 ± 1°C. The duration of both experiments was 2 months. In total, both experiments included 1188 variants in triplicate, 10 explants in each, for a total of 35,640 explants.
Development of a regeneration protocol
To study the influence of various factors on the efficiency of regeneration (cultural media and the duration of the dark period of explant pre-cultivation and shoot elongation) to increase the efficiency of regeneration we using of 2 grapevine genotype with those types of explants cultivated with supplement growth regulators (fragments of petioles cultivated on 2.0 mg l− 1 BA and 0.15 mg l− 1 IBA for the rootstock Kober 5BB and fragments of leaves cultivated on 2.0 mg l− 1 BA and 0.05 mg l− 1 for the cultivar Podarok Magaracha) that showed the highest efficiency of shoot formation in experiments no. 1 and 2. The leaves were cut into fragments measuring 5 mm x 5 mm and placed on Petri dishes with the adaxial side. Petioles and internodes were cut into fragments 5 mm long and placed longitudinally on the culture medium.
To study the influence of cultural media on the regeneration efficiency of the Kober 5BB rootstock and the Podarok Magaracha cultivar, 6 types of media were used: IM, PIV (Franks et al., 1998), NN (Nitsch and Nitsch, 1969), WPM (McCown and Lloyd, 1981), DKW (Driver and Kuniyuki, 1984) and MS. Explants were placed on Petri dishes containing culture medium (IM, PIV, NN, WPM, DKW, MS), supplemented with 2.0 mg l− 1 BA and 0.15 mg l− 1 IBA for rootstock Kober 5BB or 2.0 mg l− 1 BA and 0.05 mg l− 1 for cultivar Podarok Magaracha. All explants were pre-cultivation 2 weeks in dark conditions. They were then transferred for cultivation to lighting conditions with a 16-hour photoperiod (65 µmol m− 2 s− 1,) at 25 ± 1°C. The experiment was performed in duplicate, 15 explants in each, a total of 360 explants were used. The duration of the experiment was 8 weeks. During the experiment regeneration efficiency (E) was calculated as the quotient of dividing the number of explants with developed shoots (Ne) by the total number of explants (No); the results were expressed as a percentage: E = (Ne/No) ∙ 100 (Shvedova et al., 2023).
To study the effect of the duration of the dark phase of cultivation on the efficiency of regeneration, explants were placed in contact with IM medium 3% (w/v) sucrose, 0.75% (w/v) agar, with the addition of growth regulators 2.0 mg l− 1 BA and 0.15 mg l− 1 IBA for rootstock Kober 5BB or 2.0 mg l− 1 BA and 0.05 mg l− 1 for cultivar Podarok Magaracha. Explants were pre-cultured in dark conditions for 0, 1, 2, 3, 4 and 5 weeks. They were then transferred for cultivation to lighting conditions with a 16-hour photoperiod (65 µmol m− 2 s− 1,) at 25 ± 1°C. The experiment was performed in duplicate, 15 explants in each, a total of 360 explants were used. The duration of the experiment was 7 weeks. During the experiment regeneration efficiency and duration period of regeneration was observed. Regeneration efficiency (E) was calculated as the quotient of dividing the number of explants with developed shoots (Ne) by the total number of explants (No); the results were expressed as a percentage: E = (Ne/No) ∙ 100. The duration of the regeneration period was considered the number of days between the appearance of the first and last regenerates.
To study the effect of the dark phase duration of explants cultivation on the elongation efficiency were pre-cultured in dark conditions for 2 weeks in IM medium 3% (w/v) sucrose, 0.75% (w/v) agar, with the addition of growth regulators 2.0 mg l− 1 BA and 0.15 mg l− 1 IBA for rootstock Kober 5BB or 2.0 mg l− 1 BA and 0.05 mg l− 1 for cultivar Podarok Magaracha. They were then transferred for cultivation to lighting conditions in the same mediums. Explants were cultivated in the light for 3 and 4 weeks. Then they were transferred in the same mediums to dark conditions for elongation of regenerated shoots. The experiment was performed in duplicate with 15 explants each, a total of 360 explants were used. The total duration of the experiment was 14 weeks. During the experiment elongation efficiency (E) was calculated as the quotient of dividing the number of explants with elongated (more than 1 cm in length) shoots (Ne) by the total number of explants with shoots (No); the results were expressed as a percentage: E = (Ne/No) ∙ 100.
Elongated plants-regenerates were cut from explants, grown and rooted on PG medium with the addition 0.05 mg l− 1 NAA.
Agrobacterium-mediated transformation
A. tumefaciens strain EHA105 (Hood et al. 1993) harbouring pBin35SGFP binary vector was used for the transformation experiments. The T-DNA of the pBin35SGFP binary vector contained the m-gfp5-ER and the nptII genetic cassette (Fig. 1). Transformation experiments were carried out using pre-cultured leaf explants of the Podarok Magaracha grapevine cultivar.
Transient Transformation
Experiments were conducted by co-cultivation of leaf explants (pre-cultured leaf fragments for three days on IM medium, with the addition of growth regulators 2.0 mg l− 1 and 0.05 mg l− 1 IBA) jointly with Agrobacterium tumefaciens suspension. An overnight culture of A. tumefaciens was prepared in 50 ml of liquid YEB medium [5 g l− 1 beef extract, 5 g l− 1 peptone, 5 g l− 1 sucrose, 1 g l− 1 yeast extract, 0.2 g l− 1 MgSO4, pH 7.5 (Travin et al., 2019)], supplemented with 100 mg l− 1 Km and 50 mg l− 1 Rif, when shaking in an orbital shaker (180 rpm) within 24 hours at 28 ºC in the dark. The leaf explants were inoculated with 5 agrobacterial suspensions, diluted with distilled water to 0.2, 0.4, 0.6, 0.8 and 1.0 units (OD600), per 300 explants to each concentrations of inoculum. The explants were placed into a glasses containing 50 ml of inoculum and kept for 30 min in an orbital shaker (100 rpm) for inoculation. After drying in air flow laminar box the explants were transferred onto filter paper, placed on a solid surface of IM medium supplemented with 2.0 mg l− 1 BA and 0.05 mg l− 1 IBA in Petri dishes and co-cultivated of 60 explants from each concentrations of inoculum for five time periods (24 h, 48, 72, 96, 120 h) in dark conditions at a temperature of 24ºС. After co-cultivation period, the explants were washed with distilled water supplemented with 300 mg l− 1 Tm, and then transferred to the medium for explants cultivation and elimination of Agrobacterium (IM medium containing 2.0 mg l− 1 BA, 0.05 mg l− 1 IBA, 150 mg l− 1 Tm and 50 mg l− 1 Km). Every 2 weeks, explants were transferred to fresh media. A total of 1500 leaf explants were used in the experiment (60 explants for each of 5 inoculum concentrations in 5 time periods of co-cultivation).
Transient gfp expression was detected using an SteREO Discovery V12-1 (Zeiss, Germany), with the optical filter GFP Plus (GFP protein excitation at 450–490 nm). Real-time image capture was performed with a Axiocam 506 color (Zeiss, Germany) camera attached to the fluorescent microscope.
The number of explants expressing GFP was counted on 1, 2, 3, 4, 5, 6, 14, 21, 28, 42, 56 and 70 days after transformation. Transformation efficiency (E) was calculated as quotient of dividing the number of transient expression explants (Ne) by the total number of explants (No); the results were expressed as a percentage: Е = (Ne/No) ∙ 100 (Shvedova et al., 2023).
To optimize the combination of growth regulators for the regeneration of a transgenic shoots we used IM medium, supplemented with growth regulators BA (ranging from 1.5 to 3.0 mg l− 1 in increments of 0.5 mg l− 1) jointly with IBA or IAA (each 0.1–0.4 mg l− 1 with a step of 0.1 mg l− 1). Thus, thirty-two combinations of growth regulators were used in the experiment, with 100 explants in each combination. To carry out transformation, explants were pre-cultured for three days on IM medium supplemented with each of 32 combinations of growth regulators, and then the explants were inoculated and co-cultured on the same media. Genetic transformation was carried out with an inoculum of the agrobacterial strain EHA105 with a concentration at an optical density of OD600 = 0.8. Explants were co-cultivated in dark conditions at a temperature of 24ºС for 72 hours. Further the explants were washed with distilled water supplemented with 600 mg l− 1 Tm (for the first time) and 300 mg l− 1 Tm (a second time), and then transferred to the same medium for shoots regeneration containing grow regulators and antibiotics for elimination of A. tumefaciens (150 mg l− 1 Tm) and selection transgenic tissue (50 mg l− 1 Km). Every 2 weeks, explants were transferred to fresh media. In sum, 3200 explants were inoculated.
PCR analysis
For PCR- analysis, the genomic DNA was isolated from antibiotic-resistant and wild type plants of grapevine using СTAB method (Murray, Thompson, 1980). The respective forward and reverse primer sequences for npt ll were Npt-mf-up: 5'- TCTGATGCCGCCGTGTTCC-3' and Npt-mf-low: 5'- ATGCGCGCCTTGAGCCTG-3' (anticipated amplification fragment of 448 bp); those for gfp were GFP-F: 5'-GGACGACGGGAACTACAAGA-3' и GFP-R: 5'-CATGCCATGTGTAATCCCAG-3' (anticipated amplification fragment of 350 bp); those for virB were virBf: 5'-GGCTACATCGAAGATCGTATGAATG-3‘ and virBr: 5'-GACTATAGCGATGGTTACGATGTTGAC-3' (anticipated amplification fragment of 480 bp). The PCR reactions for npt ll, virB and gfp genes were carried out in a 10 µL volume containing 1.0 µl of a 10х Taq Turbo buffer, 0.2 µl of 10 mM dNTPs, 0.1 µl of two 100 ρM primer, 0.2 µl of 5 U µl− 1 HS-Taq polymerase, 7.5 µl of mQ H2O and 1 µl (≈ 30 ng) of a DNA template. Reactions were carried out in a Mastercycler nexus gradient (Eppendorf) as the follow: for Npt-mf-up – Npt-mf-low and GFP-F – GFP-R, 1 cycle of 5 min at 95°C, followed by 35 cycles of 15sec at 95°C, 30 sec at 60°C, 1 min at 72°C, and one final cycle of 7 min at 72°C; for virBf–virBr, 1 cycle of 5 min at 95°C, followed by 35 cycles of 45 s at 93°C, 45 s at 58°C, 45s at 72°C, and one final cycle of 5 min at 72°C. The products were separated in 2.0% agarose gel using the electrophoresis systems.
Southern blot analysis
Grapevine genomic DNA (20 µg) was digested overnight at 37C with 20 U Hind III. DNA from wild-type grapevine plants digested with HindIII served as a negative control. After agarose gel (0.8%) electrophoresis, the digestion products were transferred to and immobilized on a Hybond N + membrane (GE Healthcare, Little Chalfont, United Kingdom) according to the manufacturer's instructions. The DNA probe was constructed by PCR using plasmid pBINmGFP5ER as the template and primer pairs Bgfg (5'-AGTAAAGGAGAAGAACTTTTCACTGGAGTT-3') and Sagr5 (5'-TTTGTATAGTTCATCCATGCCATGTGT-3') to amplify the sequences of gfp gene. DNA probes (gfp – 711 bp) were labeled with alkaline phosphatase using an AlkPhos Direct Labeling Kit (GE Healthcare). Prehybridization, hybridization (incubated at at 60ºC overnight) with alkaline phosphatase labeled probe and subsequently washings of the membrane were carried out according to the AlkPhos Direct Labeling Kit protocol. Detection was performed using CDP-Star detection reagent following the manufacturer’s directions (GE Healthcare). The signal from the blot was accumulated on X-ray film (Retina XBE blue sensitive, Carestream Health INC., NY, United States) in film cassette at room temperature for 24 h. X-ray films were scanned on Amersham imager 600 (GE Healthcare Life Sciences, Japan) after development.
Statistical analysis
Significant differences between the variants were estimated by analysis of variance (ANOVA) followed by multiple comparisons of individual averages and evaluation by Duncan's test using Statistica 6.1 (StatSoft Inc). Analysis of the morphogenic potential of 22 grapevine genotypes was carried out using the principal component method using the PAST program (PAleontological STatistics, Version 4.05).