Plant Cell Tissue Organ Culture 151, 221233. Figure6 Representative images of shoots or shoots-derived structures developed after 9 weeks from agro-infection on Thompson Seedless (AD), Lambrusco Salamino (E, F), and Ancellotta (G, H) cotyledons, and Thompson Seedless hypocotyls (IL), illuminated by white light (A, C, E, G, IK), and UV light (B, D, F, H, JL). Incubate plates overnight at 37C. Figure8 (A) Amplification of 35S promoter (340 bp) gene fragment from genomic DNA of nine transgenic lines (#A - #L) of Thompson Seedless (Th), one line (#H) of Ancellotta (An) obtained after A. tumefaciens infection, plus the wild-type controls. HortScience 44, 14001406. Privacy Policy. Comparison of regeneration capacity and agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of vitis spp. On M2, among the 53.5% of cotyledons and 32.3% of hypocotyls which showed eGFP fluorescence, only two Lambrusco Salamino green-fluorescent calli developed and proliferated in the selective media, which failed to regenerate eGFP fluorescent shoots (Figures6E, F). (2005). biol. Biol. Rep. 9, 582. doi:10.1038/s41598-018-37335-7, Torregrosa, L., Iocco, P., Thomas, M. R. (2002). In particular, Ancellotta was the less responsive (69%) compared to the other two cultivars, which reached 83% (Lambrusco Salamino) and 86% (Thompson Seedless) of regeneration efficiency. Appl. However, most grapevine cultivars worldwide are considered recalcitrant, and most available genetic transformation protocols involve regeneration by somatic embryogenesis . Scientia Hortic. doi:10.1007/978-3-642-76415-8_21, Ikeuchi, M., Ogawa, Y., Iwase, A., Sugimoto, K. (2016). In addition, contrary to what was observed on MB slices, cotyledons and hypocotyls were able to regenerate more than one putative transgenic shoot from the same explant (data not shown). 39, 6166. (1996). Positive control corresponds to pK7WG2-35S-eGFP-nptII PCR product. TAG. While 7 (14% TE) and 6 (12% TE) fluorescent Thompson Seedless independent lines regenerated from cotyledons (Figures6CF) and hypocotyls (Figures6K, L), respectively, when grown on M2 (13.2 M BAP). Regeneration responses of cotyledons and hypocotyls on each medium and for each genotype were monitored, recording at the ninth week the percentage of regenerating explants [(number of explants regenerating shoots/total explants treated)100] and the average number of shoots calculated exclusively on non-necrotic explants. Not for use in diagnostic procedures. doi:10.1007/S00299-008-0512-2/FIGURES/4, Dhekney, S. A., Li, Z. T., Grant, T. N. L., Gray, D. J. Here, we present a detailed protocol for transformation using DH5-alpha Chemically Competent E. coli cells. Plant regeneration: cellular origins and molecular mechanisms. The frequency of cotyledons/hypocotyls-derived calli showing eGFP fluorescence was reported at 9 weeks after infection, as the [(number of eGFP fluorescent calli/total number of explants treated)100]. (2019). In sterile conditions, mature somatic embryos (SE) at the cotyledonary stage were sliced, separating hypocotyls from cotyledons, and discarding the primary radicle (Figure1A). doi:10.1079/IVP2006770, Li, Z., Jayasankar, S., Gray, D. J. - Plant 42, 220227. Recent and past research works concerning the stable genetic transformation of grapevine cultivars and rootstocks, aiming at transferring selectable marker and reporter genes, were primarily focused on the somatic embryogenesis regeneration system, mainly using pro-embryogenic masses (Dhekney etal., 2008), embryogenic calli (Iocco etal., 2001; Torregrosa etal., 2002; Lpez-Prez etal., 2008; Nakajima etal., 2020), embryogenic cell suspension cultures (Wang etal., 2005), and SEs (Li etal., 2001; Li etal., 2006; Li etal., 2008; Dhekney etal., 2009; Dhekney etal., 2016) as starting explants. Materials 11. Copyright 2023 Capriotti, Ricci, Molesini, Mezzetti, Pandolfini, Piunti and Sabbadini. 18, 92100. PDF DH5-alpha Chemically Competent E. coli cells protocol - GoldBio doi:10.1007/s11240-015-0711-9, Dalla Costa, L., Malnoy, M., Lecourieux, D., Deluc, L., Ouaked-Lecourieux, F., Thomas, M., et al. Regarding Thompson Seedless, it has been demonstrated that MBs have a quite high efficiency in producing new transgenic plants (Mezzetti etal., 2002; Sabbadini etal., 2019b), however, the in vitro regeneration and transformation protocols based on cotyledons and hypocotyls allowed the enhancement of the number of fluorescent shoots produced in this model cultivar. Chimerism in grapevines: implications for cultivar identity, ancestry and genetic improvement. Our study determined that both cotyledons and hypocotyls were proper starting tissues for stimulating the production of adventitious shoots, in contrast with the findings by Vilaplana and Mullins (1989), who did not observe shoot regeneration from cotyledons when cultured on NN-based media with the same type and concentrations of PGRs. via organogenesis. From this set of experiments, it was possible to isolate a single eGFP fluorescent adventitious shoot derived from Ancellotta cotyledons cultured on M2. Horticulturae 7, 511. doi:10.3390/HORTICULTURAE7110511, Franks, T., Botta, R., Thomas, M. R., Franks, J. Genetic transformation of Vitis vinifera via organogenesis. 37, 185194. Physiologia Plantarum 15, 473497. Des. This research was funded by the MIUR-PRIN2017 National Program via grant N.20173LBZM2-Micromolecule. Optimizing agrobacterium-mediated transformation of grapevine. All contact information provided shall also be maintained in accordance with our Vilaplana and Mullins (1989) described an efficient protocol for regenerating adventitious shoots in different cultivars of grapevine, via organogenesis, starting from cotyledons and hypocotyls obtained from flower-derived somatic embryos. The regeneration of independent transgenic lines of Thompson Seedless was observed from cotyledons cultured on both M1 and M2 with a transformation efficiency of 12 and 14%, respectively, and from hypocotyls on M1 and M2 with a transformation efficiency of 6 and 12%, respectively. This innate plasticity competence is the basis of in vitro culture, which has been widely employed in plant mass propagation and biotechnology, as well as to perform functional genetic studies. 62, 305311. Specifically, cotyledons were the most responsive somatic embryo-derived explant in the two substrates tested, and the increase in cytokinin content in the medium had a positive effect on the number of regenerated shoots only in the Thompson Seedless cultivar, while, during the nine weeks following the genetic transformation, the presence of 13.2 M BAP in the selective medium, increased the number of cotyledons and hypocotyls that showed eGFP fluorescence on explants derived from Ancellotta and Lambrusco Salamino. FEREC0112 $170.10 / Each of 1 Qty Check Availability Add to cart Description Specifications Subcloning eciencycells are not suitable for the generation of cDNA libraries. Store the remaining transformation reaction at +4C. doi:10.1186/1472-6750-2-18, Murashige, T., Skoog, F. (1962). Shake at 225 rpm at 37 C for 1 hour. Somatic embryogenesis from stigmas and styles of grapevine. Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat. The acclimatization and the establishment of plantlets in the greenhouse were performed by transferring elongated and rooted in vitro plantlets into plastic propagation trays (60 holes) filled with commercial peat ensuring high humidity content, and then in pots of 14 centimetres in diameter containing commercial soil peat. After the processing of samples by grinding and vortexing, 1.25 L were used as a template in a 50 L PCR reaction. The PCR program consisted of an initial denaturation step at 98C for 5min, followed by 40 cycles of denaturation at 98C for 5 s, annealing at 63.6C for 5 s, and extension at 72C for 20 s, and a final extension at 72C for 1min. Regeneration of plants from somatic tissues, after the first stage of pluripotency acquisition, is generally feasible through de novo shoot organogenesis or somatic embryogenesis, both highly dependent on tissue type and external auxin and cytokinin signals (Ikeuchi etal., 2016). De novo shoot organogenesis occurred under light provided by white fluorescent tubes (16 hours of light at an intensity of 70 mol m-2 s-1) in the growth chamber. Genetic transformation of grape varieties and rootstocks via organogenesis. In this study, we optimized a regeneration protocol, based on the method described by Vilaplana and Mullins (1989), that led to efficient adventitious shoot formation, via organogenesis, from cotyledons and hypocotyls derived from somatic embryos. The cotyledons and hypocotyls resulted valid in terms of regeneration of green fluorescent calli. In fact, the highest average number of shoots per explant was recorded when both flower-derived SEs explants of Thompson Seedless were cultured on M2 (more than five shoots/explant from cotyledons and four shoots/explant from hypocotyls). (2019b). Sci. SS, AR, and IP contributed to the regeneration and transformation experiments and plant acclimatization. In total, nine putative transgenic Thompson Seedless lines and one Ancellotta line, derived from cotyledons and hypocotyls, were successfully acclimatized in plastic pots, and grown in a dedicated greenhouse. The presence of the transgene in the selected regenerated lines was confirmed by PCR amplification of a portion of the 35S promoter present in the T-DNA sequence used in this study. (A, B) refer to Thompson Seedless cotyledons cultured on M1; (C, D) refer to Thompson Seedless cotyledons cultured on M2; (E, F) refer to Lambrusco Salamino non-organogenic calli regenerated on M2 from cotyledons; (G, H) refer to Ancellotta cotyledons cultured on M2; (IJ) refer to Thompson Seedless hypocotyls cultured on M1; (KL) refer to Thompson Seedless hypocotyls cultured on M2 (bar = 2mm). The frequency of eGFP fluorescent calli and the transformation efficiency for each type of explant were reported and calculated as described above, at 9 weeks after infection. At the third subculture, explants cultured on M1 were moved to elongation M1 supplemented with 1.1 M BAP and 0.49 M IBA as PGRs, while explants cultured on M2 were transferred to elongation M2 supplemented with 4.4 M BAP. From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks. (2022). LC, SS, and BMe conceived and designed the study. Although MB slices had on average a significantly higher number of calli showing eGFP fluorescence than hypocotyls, the number of regenerated shoots showing eGFP fluorescence was higher in hypocotyls. Biol. Finally, only elongated shoots expressing eGFP were cultured on a kanamycin-enriched PGR-free MS medium to stimulate in vitro root emergence in vitro. In vitro regeneration of two grapevine (Vitis vinifera l.) varieties from leaf explants. doi:10.1016/J.PLAPHY.2022.10.027, Ricci, A., Capriotti, L., Mezzetti, B., Navacchi, O., Sabbadini, S. (2020). 4. doi:10.1016/S0176-1617(89)80004-5, Wang, Q., Li, P., Hanania, U., Sahar, N., Mawassi, M., Gafny, R., et al. The response to genetic transformation was first evaluated after 9 weeks of tissues cultivation on the two selection media, each supplemented with 146 M kanamycin, 420 M cefotaxime, and 475 M carbenicillin, by the identification of the number of cotyledons and hypocotyls that developed green fluorescent calli lines and expressed as the percentage of the total explants treated (Figure5). in vitro: formation of adventitious buds on hypocotyls and cotyledons of somatic embryos. doi:10.1007/S11240-008-9378-9/FIGURES/5, Li, Z. T., Dhekney, S., Dutt, M., Van Aman, M., Tattersall, J., Kelley, K. T., et al. Methods mol. A. H., Jones, A., Godin, C., Traas, J. A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars. Front. 78, 2937. In vitro shoot regeneration from an intact and a sectioned embryo-axis of apple seeds. High efficiency somatic embryogenesis and plant germination in grapevine cultivars Chardonnay and brachetto a grappolo lungo. With the aim to identify the most efficient genetic transformation protocol, the MB protocol was compared with the new protocol based on the use of cotyledons and hypocotyls obtained from flowerderived SEs. These major drawbacks could be bypassed by the application of this innovative regeneration strategy via organogenesis, avoiding plant losses, and increasing the regeneration potential in all the three grapevine cultivars, which showed comparable high competence to regenerate adventitious shoots. Trends Food Sci. J. Mol. Plant Sci. (2016). Bioz Stars score: 86/100, based on 1 PubMed citations. 24, 977. doi:10.3390/IJMS24020977, Nuzzo, F., Gambino, G., Perrone, I. All the explants (MBs, cotyledons, and hypocotyls) were inoculated with the same gene construct used in the previous transformation experiment (35S::eGFP::nptII). J. Int. Plant Sci. Cotyledons showed the highest significant transformation efficiency in terms of explants displaying eGFP fluorescent shoots, compared to MB slices and hypocotyls explants (Figure7A). The results achieved showed the overcoming of the embryo-plantlet conversion stage, leading to an higher regeneration capacity of these explants compared to intact somatic embryos (Vilaplana and Mullins, 1989). Approximately ~ 15 g of genomic DNA was digested with KpnI enzyme, separated on a 0.7% agarose gel at 4.5V cm-1 and transferred on Hybond-N+ nylon membrane (GE Healthcare). Dev. GoldBio's DH5-alpha Chemically Competent E. coli cells are suitable for high efficiency transformation in a wide variety of applications such as cloning and sub-cloning. During the process of organogenesis, cells acquire the competence to give rise to a specific meristem (i.e., the shoot apical meristem), and then later regenerate the missing portion (i.e., the root system); while somatic embryogenesis allows the simultaneous development of both the caulinar and radical poles in the new regenerated individual. Incubate plates overnight at 37C. 90, 973979. (2002). 160, 877887. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Somatic embryogenesis {{/amp]]mdash; stress-induced remodeling of plant cell fate. J. Enology Viticulture 53, 183190. Transformation efficiency (TE) was calculated in vitro for each case study as the [(number of independent eGFP fluorescent shoots/total number of explants treated)100]. Figure2 De novo adventitious shoot organogenesis from cotyledons (AD) and hypocotyls (EH) of Ancellotta (A, E), Lambrusco Salamino (B, F), and Thompson Seedless (C, G) after three weeks of culture. Development of highly efficient genetic transformation protocols for table grape sugraone and crimson seedless at low Agrobacterium density. These explants derive from the development of embryogenic calli induced from whole flowers, stamens, and pistils of the respective Vitis vinifera cultivars, following the protocols optimized by Capriotti etal., 2022. The original contributions presented in the study are included in the article/supplementary material. Such explants, having regained juvenile characteristics, retain a high propensity for regeneration as well as good transformation efficiency. The two almost-united cotyledons (Figure1B) and the hypocotyls (Figure1C) were placed on 90 mm-Petri dishes, filled with two different novel shoot regeneration media, which consisted of Murashige and Skoog (MS) (Murashige and Skoog, 1962) basal salts and vitamins supplemented with 15g L1 sucrose, 7g L1 plant agar (Duchefa Biochemie, Haarlem, The Netherlands). The analysis confirmed that the Thompson Seedless transgenic lines originated from independent transformation events. Morphogenic competence of Vitis rupestris s. secondary somatic embryos with a long culture history. Callus, dedifferentiation, totipotency, somatic embryogenesis: what these terms mean in the era of molecular plant biology? Explants were blotted dry and cotyledons were placed with the abaxial face in contact with the culture media, while hypocotyls were cultured horizontally in microboxes (Micropoli, IT) filled with two different regeneration and selective media containing MS medium (Murashige and Skoog, 1962), and including 30g L1 sucrose, 7g L1 plant agar, 146 M kanamycin, 420 M cefotaxime, and 475 M carbenicillin. Am. The lanes labelled Negative control show the PCR result using deionized sterile water. In addition, these small explants easily undergo necrosis and it is generally necessary to adapt the transformation protocol to the genotype used (Torregrosa etal., 2002; Lpez-Prez etal., 2008). As reported by Vilaplana and Mullins (1989), we observed the continuous induction of adventitious shoots from the explants, which appeared during the first stage of development as round green buds producing the first plantlets after only 40 days from culture initiation (Figures2D, G). Add 900 L Luria-Bertani (LB) media w/o Ampicillin. At 12 weeks, the initial explants (cotyledons, and hypocotyls) were no longer distinguishable, as they developed callus-like structures characterized by continuous shoot regeneration (Figures4AD). 10. 204, 123127. Explants were cultured on two different MS-based culture media, one having a combination of 4.4 M BAP and 0.49 M IBA (M1), and the other only supplemented with 13.2 M BAP (M2). Improvement of Agrobacterium-mediated transformation efficiency and transgenic plant regeneration of Vitis vinifera l. by optimizing selection regimes and utilizing cryopreserved cell suspensions. In previous experimental trials carried out on these Italian grapevine cultivars by our research group, the direct transformation of somatic embryos or somatic embryogenic calli, by following the protocol described by Dhekney etal. All the data were recorded after 9 weeks of culture under the same culture conditions. These explants were cultured on regeneration/selection media M1 and M2 supplemented with 146 M kanamycin, 420 M cefotaxime, and 475 M carbenicillin. This confirms that chimerism remains a major problem in establishing genetic transformation protocols for different grapevine cultivars (Franks etal., 2002; Dalla Costa etal., 2019). The state-of-the-art of grapevine biotechnology and new breeding technologies (NBTS) OENO one. 11. doi:10.5897/AJB2018.16700, Larrouy, J., Jaksons, P., Bicknell, R. (2017). Optimizing the concentrations of plant growth regulators for in vitro shoot cultures, callus induction and shoot regeneration from calluses of grapes. 1864, Kumar, S., Barone, P., Smith, M. (New York, NY: Humana Press), 191202. Then, the filter was washed twice for 5 minutes in 2X SSC/0.1% SDS at 42C and twice for 15 minutes in 0.1XSSC/0.1%SDS at 42C and exposed to Carestream Kodak BioMax XAR films. Electrophoresis, Western Blotting and ELISA, Chromatography and Mass Spectrometry Reagents, Laboratory Syringe Needles and Accessories, Lab Coats, Aprons, and Other Safety Apparel, Sharps Disposal Containers and Accessories, Classroom Laboratory Supplies and Consumables, Applied Biosystems TaqMan Assay and Arrays Search Tool, Applied Biosystems TaqMan Custom Assay Design Tools, Applied Biosystems Custom qPCR Primers and TaqMan Probes Tool, Chemical Storage and Management Resource Center, The 80dlacZM15 marker provides -complementation of the -galactosidase gene from pUC or similar vectors to allow blue/white colony screening on bacterial agar plates containing Bluo-Gal or X-Gal, RecA1 and endA1 mutations in DH5 cells improve insert stability and quality of the extracted plasmid DNA, High transformation efficiency: >1x109 cfu/g pUC19 DNA, Suitable for routine and high-throughput cloning applications, Genetic markers that allow for blue/white colony screening, Convenient two reactions per vial packaging. DNA extraction from the putative transgenic lines was performed by using the Thermo Scientific Phire Plant Direct PCR Kit (Fisher Scientific), following the manufacturers instructions. All analyses were performed with the Statistica 7 software (Statsoft Tulsa, CA, USA). Fisher Scientific is always working to improve our content for you. Using DH5 as a Transient Host To use the DH5 strain as a transient host, follow the transformation protocol provided on the previous page with the following changes: NEB 5-alpha Competent E. coli (High Efficiency) | DH5 | NEB A total of 1, 7, and 26 regenerating fluorescent shoots were obtained from MBs (Figures7B, C), hypocotyls (Figures7D, E), and cotyledons (Figures7F, G), respectively. Factors affecting somatic embryogenesis in eight Italian grapevine cultivars and the genetic stability of embryo-derived regenerants as assessed by molecular markers. Regeneration of grapevines (Vitis spp.) 11. Frontiers | Efficient protocol of de novo shoot organogenesis from For hypocotyls, M1 was also slightly more efficient than M2, although there was not statistical difference in terms of eGFP fluorescent calli, settlings on values between 20% and 24% (Figure5B). Additional cells may be plated out the next day, if desired. However, similar experiments on other woody plants are scarce, probably due to the high level of heterozygosity and the need to cultivate clones identical to those typical of the selected crop variety (Capriotti etal., 2020). Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Roots emitting fluorescence, directly regenerated from the explants, were also observed in Thompson Seedless cotyledons cultured on M1 (highlighted with arrows in Figures6A, B). 134, 413419. The regeneration protocols fine-tuned in this study were then applied to obtain transgenic grapevine plants via Agrobacterium-mediated transformation.
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