Regulation of Resveratrol Production in Vitis ...

Appl Biochem Biotechnol DOI 10.1007/s12010-014-1384-2

Regulation of Resveratrol Production in Vitis amurensis Cell Cultures by Calcium-Dependent Protein Kinases O. A. Aleynova & A. S. Dubrovina & A. Y. Manyakhin & Y. A. Karetin & K. V. Kiselev

Received: 13 July 2014 / Accepted: 10 November 2014 # Springer Science+Business Media New York 2014

Abstract Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of transresveratrol to human health and plant protection against fungal pathogens are well-established, little is known about the molecular mechanisms regulating stilbene biosynthesis in plant cells. It has been recently shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased resveratrol accumulation in cell cultures of Vitis amurensis. It is possible that calcium-dependent protein kinases (CDPKs) play an important role in the regulation of resveratrol biosynthesis. In the present work, we investigated the effects of overexpression of other members of the CDPK multigene family (VaCPK9, VaCPK13, VaCPK21, and VaCPK29) on resveratrol accumulation and growth parameters of grape cell cultures. The obtained data show that overexpression of VaCPK29 increased resveratrol content 1.6–2.4-fold and fresh biomass accumulation 1.1–1.4-fold in the four independently transformed cell lines of V. amurensis compared with that in the empty vector-transformed calli. However, overexpression of the VaCPK9, VaCPK13, and VaCPK21 genes did not considerably affect resveratrol content and fresh/dry biomass accumulation in the independently transformed cell lines of V. amurensis. VaCPK29-transformed calli were capable of producing between 1.02 and 1.39 mg/l of resveratrol, while the control calli produced 0.48 to 0.79 mg/l of resveratrol. The data indicate that the VaCPK9, VaCPK13, and VaCPK21 genes are not involved in the regulation of stilbene biosynthesis in grape cells, while the VaCPK29 and VaCPK20 genes are implicated in resveratrol biosynthesis as positive regulators.

O. A. Aleynova : A. S. Dubrovina : K. V. Kiselev Laboratory of Biotechnology, Institute of Biology and Soil Science, Far East Branch of Russian Academy of Sciences, Vladivostok 690022, Russia A. Y. Manyakhin Mountain-Taiga Station, Far East Branch of Russian Academy of Sciences, Posyolok Gornotaezhnoe, Primorsky krai, Ussuriisky Region 692533, Russia Y. A. Karetin Laboratory of Embryology, A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Palchevsky St. 17, 690059 Vladivostok, Russia

Appl Biochem Biotechnol

Keywords Resveratrol . CDPKgene . Calcium . Callus culture . Vitisamurensis . Overexpression

Introduction Trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a naturally occurring plant phenol displaying a wide range of biological activities, including anti-inflammatory, antioxidant, and platelet antiaggregatory properties, and modulation of lipoprotein metabolism [1]. Resveratrol has been shown to possess chemopreventive properties against certain cancers and cardiovascular diseases and to have positive effects on age longevity [2, 3]. Trans-resveratrol is one of the major phytoalexins accumulated in plants in response to various stresses, and it acts to enhance plant disease resistance to both biotic and abiotic stresses [4, 5]. For example, in leaves and berries, it acts as an antimicrobial phytoalexin that is produced in response to stresses, such as wounding or pathogen attack [4, 6, 7]. It has been shown that resveratrol acts as an important regulator of initiation of the cell death during the plant hypersensitive reaction [8]. Thus, resveratrol presents a considerable interest in pharmacological studies and for plant science due its beneficial effects on human health and its importance in plant protection against various stresses. Studying the regulatory mechanisms and signaling pathways controlling resveratrol biosynthesis in plant cells is of a great interest for plant physiology and biotechnology. Currently, little is known about the regulatory mechanisms controlling biosynthesis of resveratrol and other stilbenes in plants, and the study of the mechanisms regulating resveratrol biosynthesis in plant cells is of a great interest for plant physiology and biotechnology. Resveratrol is synthesized in a number of plant species from different plant families, such as Vitaceae (grapevine), Fabaceae (peanut), Polygonaceae (knotweed), or Ericaceae (cranberry) [1]. It is known that a variety of resveratrol-producing plant cell cultures of different species contain low resveratrol levels (up to 0.03 % dry wt.) and the treatment of the cultures with UV irradiation, elicitors, or other agents did not result in a considerable increase in resveratrol production [1, 9, 10]. Stilbenes, including resveratrol, are synthesized via the phenylpropanoid pathway [11]. Phenylalanine ammonia lyase (PAL, EC 4.3.1.5), the first enzyme in this pathway, catalyzes monooxidative deamination of phenylalanine to produce cinnamate. Stilbene synthase or resveratrol synthase (STS, EC 2.3.1.95) is the key enzyme for the final step of resveratrol formation, as it condenses three molecules of malonyl-CoA and one molecule of cumaryl-CoA to form resveratrol [12]. A variety of transcription factors regulating biosynthesis of phenylpropanoids at the transcription levels have been characterized (reviewed in [13, 14]). Two transcription factors, Myb14 and Myb15, positively regulating biosynthesis of resveratrol and other stilbenes have been recently identified and characterized [15, 16]. It has recently been shown that DNA methylation is involved in the regulation of resveratrol biosynthesis in Vitis amurensis [17, 18]. Experimental evidence indicated that active resveratrol accumulation in grape cell cultures is Ca2+ dependent [19, 20]. Treatment of a V. amurensis callus culture containing low levels of resveratrol with the calcium ionophore A23187 increased resveratrol levels to 0.03 % dry cell wt. [20], while treatment with the calcium channel blockers (LaCl3, verapamil, and niflumic acid) and an antagonist of calcium-dependent protein kinase (CDPK), W7 (N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide), significantly reduced the accumulation of resveratrol in cell cultures of V. amurensis with high resveratrol content [19–21]. In plants, CDPKs are currently considered as the key calcium sensor proteins in calciummediated signaling [22] and exist as a multigene family [23–25]. Since CDPKs are known to


be of great importance for the activation of defense reactions in plants [10, 26], we proposed that some CDPKs could be involved in the regulation of resveratrol biosynthesis in V. amurensis. Recently, we obtained the data indicating that one of the CDPKs of V. amurensis, VaCPK20, is involved in the signaling pathway regulating resveratrol biosynthesis [27]. VaCPK20 overexpression under the control of double CaMV 35S promoter in V. amurensis increased resveratrol production in the five independently transformed callus cell lines 9–68 times compared with the control cells. Comparison of the deduced amino acid sequence of VaCPK20 with Arabidopsis CPKs revealed the highest homology with AtCPK1 (81 % positives, AK1 isoform, GenBank acc. no. NM_120569, L14771). Notably, the phenylalanine ammonia lyase (PAL, EC 4.3.1.5) enzyme was identified as one of the possible substrates for phosphorylation of AtCPK1 [28]. Since CDPK is a multigene family of enzymes, which are known to play both redundant and distinct biological functions in plant metabolism, it would be interesting to study whether there are other members of the CDPK superfamily positively or negatively regulating biosynthesis of resveratrol, besides VaCPK20. It has previously been shown that expression of VaCPK9 (1e), VaCPK13 (2a), VaCPK21 (1d), and VaCPK29 (1a) was up-regulated in transgenic cell cultures of V. amurensis with high resveratrol content [19]. The purpose of the present study was to examine the effects of overexpression of other VaCPKs (VaCPK9, VaCPK13, VaCPK21, and VaCPK29) belonging to different CDPK subfamilies on resveratrol content and biomass accumulation in cell cultures of V. amurensis.

Materials and Methods Plant Materials and Growth Conditions The V2 callus culture of wild-growing grapes V. amurensis Rupr. (Vitaceae) was established in 2002 as described previously [29]. The KA-0 empty vector-transformed cell line was obtained in 2012 by co-cultivation of the V2 cell suspension with Agrobacterium tumefaciens GV3101::pMP90 strain containing pZP-RCS2-nptII vector [30], which contained only the kanamycin (Km) resistance gene nptII, as described previously [31]. The VaCPK9, VaCPK13, VaCPK21, and VaCPK29-overexpressing cell lines of V. amurensis were obtained in 2013 by transformation of the V2 cell suspension with A. tumefaciens strain GV3101::pMP90 containing pZP-RCS2-VaCPK9-nptII, pZP-RCS2-VaCPK13-nptII, pZP-RCS2-VaCPK21-nptII, or pZP-RCS2-VaCPK29-nptII, respectively, as described [27, 31]. The VaCPK9-, VaCPK13-, VaCPK21-, and VaCPK29-transgenic cell lines were independently transformed to obtain three to four independent transgenic cell lines overexpressing each CDPK and were designated as KA08-I, KA08-II, KA08-III, and KA08-IV (VaCPK9); KA15-I, KA15-II, KA15-III, and KA15-IV (VaCPK13); KA07-I, KA07-II, and KA07-III (VaCPK21); and KA10-I, KA10-II, KA10-III, and KA10-IV (VaCPK29). In total, we obtained 15 CDPK-transgenic cell lines. The grape cell cultures were cultivated at 30-day subculture intervals in the dark at 24–25 °C in test tubes with 15 ml of the solid Murashige and Skoog-modified WB/A medium supplemented with 0.5 mg/l 6-benzylaminopurine (B) and 2 mg/l α-naphthaleneacetic acid (A) [29]. Isolation and Sequencing of VaCPK9, VaCPK13, VaCPK21, and VaCPK29 Full-length complementary DNA (cDNA) coding sequences of VaCPK9 (a.k.a VaCDPK1e, GenBank (GB) acc. no. KC488319), VaCPK13 (VaCDPK2a, GB acc. no. KC488320), VaCPK21 (VaCDPK1d, GB acc. no. KC488318), and VaCPK29 (VaCDPK1a, GB acc. no.


KC488317) genes were amplified based on the known VaCPK9, 13, 21, and 29 sequences using primers 5′ATG GGT ATT TGT CTA AGC AAA, 5′CTA AAA AAC CTT CAA TCC CTG (VaCPK9); 5′ATG GGG AAC TGT TGC AGA T, 5′TTA CTC ATT CCC CAA GTT TAG (VaCPK13); 5′ATG GGT TGT TTT AGC AGT AAG, 5′AAA TAG CTT GAC TGG CGG TT (VaCPK21); 5′ATG GGT TTC TGC TTC TCC AG, 5′TCT CTG CTT CMA CTC GGT ATC (VaCPK29) and RNA isolated from inflorescences or leaves of wild-growing V. amurensis as reported [32]. The target amplicons were isolated from the gel using the Cleanup Mini kit (Evrogen, Moscow, Russia). The cDNAs of VaCPK9 (1611 bp), VaCPK13 (1584 bp), VaCPK21 (1638 bp), and VaCPK29 (1572 bp) were subcloned into the pTZ57R/T plasmid (Fermentas, Vilnius, Lithuania) and sequenced. Multiple sequence alignments and the phylogenetic tree based on pairwise alignment were done using ClustalX program [33]. Overexpression of VaCPK9, VaCPK13, VaCPK21, and VaCPK29 in Cell Cultures of V. amurensis To generate the construction for plant cell transformation, the full-length cDNA of VaCPK9 (GB acc. no. KC488319) was amplified by PCR using the forward primer 5′GCT CGA GCT CAT GGG TAT TTG TCT AAG CAA A and the reverse primer 5′TCG AGG ATC CCT AAA AAA CCT TCA ATC CCT G from pTZ57-VaCPK9 using the cDNA synthesized from the total RNA of the V. amurensis inflorescence. The forward primer contained a SacI restriction site, and the reverse primer contained a BamHI restriction site, which are underlined. The full-length cDNA of VaCPK9 was cloned into the pSAT1 vector [30] by the SacI and BamHI sites under the control of the double cauliflower mosaic virus (CaMV 35S) promoter. Then, the expression cassette from pSAT1 with the VaCPK9 gene was cloned into the pZP-RCS2-nptII vector [30] using the PalAI (AscI) sites. The pZP-RCS2-nptII construction also carried the nptII gene under the control of the double CaMV 35S promoter. The used restriction enzymes were obtained from SibEnzyme (Novosibirsk, Russia). Plasmid DNA samples (pSAT1 and pZP-RCS2-nptII; [30]) and the A. tumefaciens GV3101::pMP90 strain were kindly provided by Professor Alexander Krichevsky (State University of New York, Stony Brook, USA). The full-length cDNA of VaCPK13 (GB acc. no. KC488320) was amplified by PCR using the forward primer 5′GCT CGA GCT CAT GGG GAA CTG TTG CAG AT and the reverse primer 5′TCG AGG TAC CTT ACT CAT TCC CCA AGT TTA G from pTZ57-VaCPK13 using the cDNA synthesized from the total RNA of the V. amurensis leaves. The forward primer contained a SacI restriction site and the reverse primer contained a KpnI restriction site, which are underlined. The full-length cDNA of VaCPK13 was cloned into the pSAT1 vector by the SacI and KpnI sites under the control of the double CaMV 35S promoter. Then, the expression cassette from pSAT1 with the VaCPK13 gene was cloned into the pZP-RCS2-nptII vector using the PalAI sites. The full-length cDNA of VaCPK21 (GB acc. no. KC488318) was amplified by PCR using the forward primer 5′GCT CCT CGA GAT GGG TTG TTT TAG CAG TAA and the reverse primer 5′TCG AGG ATC CAA ATA GCT TGA CTG GCG GT from pTZ57-VaCPK21 using the cDNA synthesized from the total RNA of the V. amurensis leaves. The forward primer contained an XhoI restriction site, and the reverse primer contained a BamHI restriction site, which are underlined. The full-length cDNA of VaCPK21 was cloned into the pSAT1 vector by the XhoI and BamHI sites under the control of the double CaMV 35S promoter. Then, the expression cassette from pSAT1 with the VaCPK21 gene was cloned into the pZP-RCS2-nptII vector using the PalAI sites. The full-length cDNA of VaCPK29 (GB acc. no. KC488317) was amplified by PCR using the forward primer 5′ACT CGA GCT CAT GGG TTT CTG CTT CTC CAG and the reverse


primer 5′TCG AGG ATC CTT ATC TCT GCT TCA CTC GGT ATC from pTZ57-VaCPK29 using the cDNA synthesized from the total RNA of the V. amurensis leaves. The forward primer contained a SacI restriction site, and the reverse primer contained a BamHI restriction site, which are underlined. The full-length cDNA of VaCPK29 was cloned into the pSAT1 vector by the SacI and BamHI sites under the control of the double CaMV 35S promoter. Then, the expression cassette from pSAT1 with the VaCPK29 gene was cloned into the pZPRCS2-nptII vector using the PalAI sites. The overexpression constructs of VaCPK9, VaCPK13, VaCPK21, and VaCPK29 (pZPRCS2-VaCPK9-nptII, pZP-RCS2-VaCPK13-nptII, pZP-RCS2-VaCPK21-nptII, or pZPRCS2-VaCPK29-nptII) and empty vector (pZP-RCS2-nptII) were introduced into the A. tumefaciens strain GV3101::pMP90 and transformed into the V. amurensis suspension culture V2 by co-cultivation with the bacterial cells as described [27, 31]. After transformation, the calli were cultivated for a 3-month period in the presence of 10–15 mg/l of Km to select transgenic cells and for a 4-month period in the presence of 250 mg/l of Cf to suppress the bacteria. The absence of A. tumefaciens was confirmed by PCR of the VirB2 gene using primers 5′ATG CGA TGC TTT GAA AGA TAC CG and 5′TTA GCC ACC TCC AGT CAG CG as described [27, 31]. The expression of the nptII gene was verified by PCR using primes 5′ ATG TGG ATT GAA CAA GAT GG and 5′TCA GAA GAA CTC GTC AAG AA, Ta 55 °C, and elongation time of 50 s. For analyzing the total VaCPK9 expression (expression of the endogenous VaCPK9 gene and the exogenous VaCPK9), we used primers S1 5′ATG CGC TTT TGA AGG CAA CA and A1 5′CTA CAT AGT AAG CAC TCC CAA designed to the kinase domain of VaCPK9. For analyzing expression of the exogenous VaCPK9 (transgene), we used primers S2 5′GTC AAT AGG CAC AAG CTC GA designed to the 3′ end of the protein coding region of the VaCPK9 gene and A2 5′GAG AGA CTG GTG ATT TTT GCG designed to the CaMV 35S terminator in the pSAT1 vector. For analyzing expression of the endogenous VaCPK9 gene, we used primers S2 and A3 5′CTG TGC CGA GTG CAA CAG AAA designed to the 3′UTR of the VaCPK9 messenger RNA (mRNA). For analyzing the total VaCPK13 expression, we used primers S1 5′TAT TCT TCA AGC CAG GTG AGA and A1 5′CCA TAA TTC CGC TTG AGG AC designed to the kinase domain of VaCPK13. For analyzing expression of the exogenous VaCPK13 (transgene), we used primers S2 5′CTT CTA GGC ATT ATT CAA GAG G designed to the 3′ end of the protein coding region of the VaCPK13 gene and A2 5′GAG AGA CTG GTG ATT TTT GCG and A2 designed to the CaMV 35S terminator in the pSAT1 vector. For analyzing expression of the endogenous VaCPK13 gene, we used primers S2 and A3 5′CTT GTG TGG ATG AAC AAA AGA C designed to the 3′UTR of the of VaCPK13 mRNA. For analyzing the total VaCPK21 expression, we used primers S1 5′TCA GGG GCA TTA CTC TGA GA and A1 5′TGC TTC ATC CTT GCT GGA CAA designed to the kinase domain of VaCPK21. For analyzing expression of the exogenous VaCPK21 (transgene), we used primers S2 5′ATG GCA ATG GAA CGA TTG AC designed to the 3′ end of the protein coding region of the VaCPK21 gene and A2 5′GAG AGA CTG GTG ATT TTT GCG designed to the CaMV 35S terminator in the pSAT1 vector. For analyzing expression of the endogenous VaCPK21, we used primers S2 and A3 5′ACA TTC AGC AAC CAT CAC TGA designed to the 3′UTR of the of VaCPK21 mRNA. For analyzing the total VaCPK29 expression, we used primers S1 5′CGG CGA AAG GTA GTT ATT CC and A1 5′TTC TCA GGC TTC AAG TCC CT designed to the kinase domain of VaCPK29. For analyzing expression of the exogenous VaCPK29 (transgene), we used primers S2 5′GAATGG GGG ATG AAG CGA CT designed to the 3′ end of the protein coding region of the VaCPK29 gene and A2 5′GAG AGA CTG GTG ATT TTT GCG


designed to the CaMV 35S terminator in the pSAT1 vector. For analyzing expression of the endogenous VaCPK29, we used primers S2 and A3 5′TTA AAC TTA TCT CTG CTT CCA C designed to the 3′UTR of the of VaCPK29 mRNA. Total RNA Isolation and Quantitative RT-PCR Total RNA isolation was performed using the cetyltrimethylammonium bromide-based extraction developed in [34] with slight modifications [31]. Complementary DNAs were synthesized as described previously [35, 36]. The cloned reverse transcription (RT)-PCR products were sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA), as described [18]. cDNAs of VaCPK9, VaCPK13, VaCPK21, VaCPK29, GAPDH, and Actin1 were amplified using EvaGreen Real-time PCR (Biotium, Hayward, USA). The qRT-PCRs were performed using a Real-Time PCR Kit (Syntol) in a thermocycler supplied with Multicolor Real-Time PCR Detection System (DNA Technology, Moscow, Russia). Expression was calculated by the 2−ΔΔCT method [37]. Scaling options are the highest (the highest expressing sample accrued the value 1 in the relative mRNA calculation). VaActin1 (GB acс. no. DQ517935) and VaGAPDH (GB acс. no. GU585870) genes were used as endogenous controls to normalize variance in the quality and the amount of cDNA used in each real-time RT-PCR experiment. qRT-PCR data shown were obtained from at least two independent experiments and are averages of eight technical replicates for each independent experiment (four qPCR reactions normalized to VaActin1 and four qPCR reactions normalized to VaGAPDH expression). High-Performance Liquid Chromatography The dried and powdered callus culture samples (100 mg) were extracted with 96 % EtOH (3 ml) for 2 h at 60 °С. An ethanolic solution of resveratrol (3,4′,5-trihydroxy-trans-stilbene approx. 99 % GC; SIGMA-ALDRICH; St. Louis, USA) was used as the standard to calculate the resveratrol content in V. amurensis extracts. The analytical HPLC was carried out using a Shimadzu 10 series HPLC system equipped with UV-vis detector (Tokyo, Japan). Extracts and fractions were analyzed using a 5 μm, 250×4.6 mm Phenomenex Luna C18 column (Phenomenex, Torrance, CA) thermostated at 30 °C as described [38]. Statistical Analysis The statistical analysis was carried out using the Statistica 10.0 program (StatSoft Inc, Boston, USA). The data are presented as mean±standard error (SEM) and were tested by paired Student’s t test. The 0.05 level was selected as the point of minimal statistical significance in all analyses.

Results Genetic Transformation of V. amurensis with the VaCPK9, VaCPK13, VaCPK21, and VaCPK29 Genes and Selection of the Transgenic Cell Lines Since it has previously been shown that expression of VaCPK9 (1e), VaCPK13 (2a), VaCPK21 (1d), and VaCPK29 (1a) was up-regulated in transgenic cell cultures of V. amurensis with high resveratrol content [19], we decided to investigate the effect of overexpressing these VaCPKs


on the content of resveratrol and biomass accumulation in cell cultures of V. amurensis. For the overexpression experiments, we used the members from the subfamily II (VaCPK9, VaCPK21, and VaCPK29) and the subfamily III (VaCPK13) of the CDPK multigene family (Fig. 1). To establish grape cell cultures overexpressing the VaCPK9, VaCPK13, VaCPK21, and VaCPK29 genes, the V2 suspension culture was incubated with A. tumefaciens strains, bearing constructs pZP-RCS2-nptII (empty vector, KA-0 cell line), pZP-RCS2-VaCPK9-nptII (four independently transformed KA08 cell lines), pZP-RCS2-VaCPK13-nptII (four independently transformed KA15 cell lines), pZP-RCS2-VaCPK21-nptII (three independently transformed KA07 cell lines), or pZP-RCS2-VaCPK29-nptII (four independently transformed KA10 cell lines) where the VaCPK genes were cloned under the control of the double CaMV 35S promoter. GV3101::pMP90 strain of A. tumefaciens was inoculated in multiple separate flasks with cell suspensions of V. amurensis to establish independently transformed KA-0, KA07-I, KA07-II, KA07-III, KA08-I, KA08-II, KA08-III, KA08-IV, KA10-I, KA10-II, KA10-III, KA10-IV, KA15-I, KA15-II, KA15-III, and KA15-IV cell lines. We selected transgenic cell

Fig. 1 Phylogenetic tree showing the relationships between the four used CDPKs (in ovals) of V. amurensis and other CDPKs from V. amurensis and 34 CDPKs of A. thaliana. CDPK genes with investigated functions on cell biomass accumulation and resveratrol biosynthesis (in italic). Predicted amino acid sequences of the VaCDPKs were aligned using the ClustalX program with the amino acid sequences of 34 Arabidopsis CDPKs retrieved from the Arabidopsis Information Resources (TAIR) database (GenBank acc. no. NM_120569, NM_111902, NM_118496, NM_117025, NM_119697, NM_127284, NM_121286, NM_121950, NM_112932, NM_101746, NM_103271, NM_122264, NM_115044, NM_129750, NM_001203865, NM_127343, NM_121256, NM_001204003, NM_104875, NM_129449, NM_116710, NM_116709, NM_001203743, NM_128707, NM_129148, NM_001204020, NM_116708, NM_126019, NM_202421, NM_106132, NM_148230, NM_115613, NM_103952, and NM_121941). Multiple sequence alignments and a phylogenetic tree based on pairwise alignment were done with ClustalX program [33]. The branch lengths are proportional to divergence with the scale of 0.1 representing 10 % change


aggregates in the presence of 10–15 mg/l Km for 3 months and established several Kmresistant lines. Previously, Km sensitivity of the parent V2 culture was tested, and it was shown that these calli completely ceased to grow during the second month of cultivation at such low Km concentration as 10 mg/l. Therefore, cell selection in the presence of 10–15 mg/l Km for 3 months was used to establish the transgenic cell lines. During the first month after transformation, we selected the fast-growing calli from separate primary small aggregates, which appeared in the presence of Km and established several Km-resistant independent clonal lines KA-0, KA07-I, KA07-II, KA07-III, KA08-I, KA08-II, KA08-III, KA08-IV, KA10-I, KA10-II, KA10-III, KA10-IV, KA15-I, KA15-II, KA15-III, and KA15-IV. The KA-0 calli reproduced morphological, growth, and biosynthetic characteristics of the parent V2 culture. This indicated that transformation by the empty vector did not cause significant perturbations in transformed cells. The KA-0 cell line was used as a control in all further experiments, including the present investigation. The fast-growing KA07-I, KA07-II, KA07-III, KA08-I, KA08-II, KA08-III, KA08-IV, KA10-I, KA10-II, KA10-III, KA10-IV, KA15-I, KA15-II, KA15-III, and KA15-IV cell lines were used for further investigation. The formation of stable callus phenotype of the used cell lines was observed 2 months after the removal of Km from the nutrient medium. These calli represented friable vigorously growing homogenous tissues, which did not appear to have undergone differentiation. We did not observe the formation of any differentiated structures, such as root- or shoot-like structures, in the KA07, KA08, KA10, and KA15 transformed cell lines on the WB/A medium with standard hormone composition and on the medium with decreased rates of 6-benzylaminopurine and 2 mg/l α-naphthaleneacetic acid in the dark. The semiquantitative RT-PCR has shown that the nptII gene was transcribed in all obtained transgenic cell lines (data not shown). The absence of A. tumefaciens was confirmed using PCR to control the presence or absence of the VirB2 gene (data not shown). Using different primer sets, we analyzed total, transgenic, and endogenous VaCPK9, VaCPK13, VaCPK21, and VaCPK29 expression. The four independently transformed transgenic cell lines KA08-I, KA08-II, KA08-III, and KA08-IV actively expressed the exogenous VaCPK9 (transgene), and its expression was approximately at the same level in the analyzed KA08 lines (Fig. 2a).

Fig. 2 Quantification of the transgene, endogenous, and total VaCPK9 (a, b, c), VaCPK13 (d, e, f), VaCPK21 (g, h, i), and VaCPK29 (j, k, l) mRNAs performed by real-time PCR in the obtained transgenic cell lines. qRT-PCR data were obtained from at least two independent experiments and are averages of eight technical replicates for each independent experiment (four qPCR reactions normalized to VaActin1 and four qPCR reactions normalized to VaGAPDH expression) and presented as mean±standard error. *p