Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4

Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4 Esther Rosales Sanchez, R. Jordan Price, Federico Marangelli, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4583627/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Plant Methods → Version 1 posted 9 You are reading this latest preprint version Abstract Background Plant breeding played a very important role in transforming strawberries from being a niche crop with a small geographical footprint into an economically important crop grown across the planet. But even modern marker assisted breeding takes a considerable amount of time, over multiple plant generations, to produce a plant with desirable traits. As a quicker alternative, plants with desirable traits can be raised through tissue culture by doing precise genetic manipulations. Overexpression of morphogenic regulators previously known for meristem development provides an efficient strategy for easier regeneration and transformation in multiple crops. In this study, we show the results for overexpression of chimeric GRF4-GIF1 in diploid strawberry Fragaria vesca Hawaii 4 (strawberry) where Vitis GRF4-GIF1 chimera provides significantly higher regeneration efficiency. Results We present here a comprehensive protocol for strawberry regeneration and transformation under control condition as compared to ectopic expression of GRF4-GIF1 chimeras from different plants. We report that ectopic expression of Vitis vinifera VvGRF4-GIF1 provide significantly higher regeneration efficiency during retransformation over wild-type plants. On the other hand, deregulated expression of miRNA resistant version of Vitis GRF4-GIF1 or TaGRF4-GIF (wheat) resulted in abnormalities. Transcriptomic analysis between the different chimeric GRF4-GIF1 lines indicate that differential expression of FvExpansin might be responsible for the pleiotropic effects. Similarly, cytokinin dehydrogenase/oxygenase and cytokinin responsive response regulators also showed differential expression indicating GRF4-GIF1 pathway playing important role in controlling cytokinin homeostasis. Conclusion Our data indicate that ectopic expression of Vitis vinifera VvGRF4-GIF1 chimera can provide significant advantage over wild-type plants during strawberry regeneration without producing any pleiotropic effects seen for the miRNA resistant VvGRF4-GIF1 . Strawberry regeneration transformation GRF4-GIF1 chimera leaf development cytokinin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND Somatic embryogenesis plays an essential role towards asexual propagation and regeneration of plants. Unlike organogenesis which requires a high cytokinin-to-auxin ratio [1,2], somatic embryogenesis is mostly dependent on auxin [2–4]. In the last decade, concerted effort has been made to understand the molecular mechanisms that drive the transition of a vegetative cell into an embryogenic competent cell under the influence of auxin and cytokinin signalling. Understanding of the regeneration pathways leading to embryogenesis under the influence of phytohormones resulted in the development of in vitro techniques for tissue culture of several plant species [5]. The Rosaceae is one such family where sexual hybridization, asexual propagation, and genetic improvements have been pivotal for developing better varieties for quite some time [6]. It is a large and diverse family that includes several economically relevant food crops such as apple ( Malus ), plum, peach, almond, cherry ( Prunus ), pear ( Pyrus ), raspberry ( Rubus ), strawberry ( Fragaria ) and other species with economic value. The cultivated strawberry, Fragaria x ananassa , is an octoploid species (2n =8x = 56) derived from the hybridization between F. chiloensis and F. virginiana [7]. Although breeding and genetic engineering tools are available in F. x ananassa [8], the polyploid genome makes crop improvement in this species difficult. For that reason, the diploid woodland strawberry ( Fragaria vesca ) that holds close kinship to commercial strawberry is widely used as a genetic model [6]. F. vesca offers favourable attributes including a ~240 Mb reference genome (versus 157 Mb in Arabidopsis thaliana ), short generation time, small plant size and a wide geographical distribution [7]. As a result, in the last few years, effective in vitro propagation, regeneration and transformation techniques have been developed for F. vesca to facilitate genetic engineering [9–11]. However, despite the establishment of regeneration and transformation techniques in woodland strawberry, the use of developmental regulators has never been tested, which might lead to the development of a better strategy that could not only induce better and faster regeneration but also facilitate more effective transformation in strawberry. Research in model organisms such as A. thaliana facilitated the identification of certain transcription factors that can integrate the signals leading to cellular reprogramming resulting in embryogenesis or meristematic fate [12]. These transcription factors are called developmental regulators as they coordinate spatial cellular distribution resulting in organ formation. For example, Somatic Embryogenesis Receptor Kinase ( SERK ), Leafy Cotyledon 1 ( LEC1 ), Leafy Cotyledon 2 ( LEC2 ), NiR, Baby Boom ( BBM ), Wound Induced Dedifferentiation 1 ( WIND1 ), Wuschel ( WUS ) and WOX5 have all been identified to be essential during somatic development [13–19]. Ectopic expression of these genes not only allowed regeneration of transformation-recalcitrant plant species but also increased regeneration efficiencies. However, overexpression of developmental genes like WUS and BBM induced pleiotropic effects, including callus necrosis, compromised differentiation of shoots and roots, reduced fertility of transgenic plants, and a variety of other aberrant phenotypes [20]. This necessitates the need for an alternative strategy to enhance regeneration without compromising the morphology of the plant. This search culminated with the finding that ectopic expression of a chimeric GRF-GIF protein complex could induce better regeneration of fertile cultivars [21]. The Growth-Regulating Factors (GRFs) are a small group of transcription factors that play an important role in plant development and are highly conserved in angiosperm, gymnosperm, and moss (bryophyte) lineages [22]. They encode proteins with conserved QLQ and WRC domains responsible for protein–protein and protein–DNA interactions, respectively. Many angiosperms and gymnosperm GRF genes carry the target site for micro-RNA 396 ( miR396 ), which attenuates its activity [23]. The GRF proteins form complexes with their transcription cofactor GRF-Interacting Factors (GIFs) and forms a transcription activation complex [24]. In these GRF-GIF complexes, GIFs recruit chromatin remodelling complexes and GRFs remove the nucleosomes from chromatin by virtue of the QLQ motifs to activate expression of target genes [25]. In general, callus formation and subsequent plant regeneration are accompanied by epigenetic changes on the packaging of DNA involving formation of an open-chromatin state facilitating gene expression [26]. Hence, GRF-GIF complexes are thought to confer meristematic potential to proliferative and formative cells during organogenesis by inducing the open-chromatin state [27]. Here, we report that the ectopic expression of chimeric GRF4-GIF1 from Citrus , Triticum and Vitis have differential effects in boosting regeneration and genetic transformation of diploid strawberry F. vesca Hawaii 4. We have also explored how the mutation of miR396 site in the Vitis GRF4 affect the activity of the Vitis miRGRF4-GIF1 chimera during the regeneration of F. vesca . Henceforth, in the paper we will call the GRF4-GIF1 chimera as CcGRF-GIF , VvGRF-GIF , Vv miRGRF-GIF and TaGRF-GIF . Transcriptomic analyses reveal several factors related to development and maturation differentially expressed by virtue of the transformation. We also report the increased potential of regeneration in VvGRF-GIF lines following re-transformation in comparison to wild-type plants. METHODS Seed germination and meristem propagation F. vesca Hawaii 4 seeds were harvested from the matured fruits and dried on filter paper at 37ºC. Dried seeds were labelled and packed in envelops and stored at 4ºC. Seeds were scarified with 70% ethanol followed by 1M sulphuric acid (H 2 SO 4 ) solution before they were thoroughly washed with water. To initiate germination, scarified seeds were plated on water agar and kept at 22ºC. The germinated seedlings were propagated on nutrient rich soil in a glasshouse at 22-24ºC under long day (16 hours days – 8 hours night) conditions to initiate runners. Fresh runners were harvested in water and transferred to the lab where, using a Leica stereomicroscope M165, the meristems were harvested using a scalpel. This tissue was immediately transferred to tubes containing strawberry propagation medium (SPM), taking special care to prevent desiccation. SPM is a MS-based medium (2.2 g/L) supplemented with 0.1 mg/L of BAP and 0.1 mg/L indole-3-butyric acid (IBA) and solidified with Daishin agar (Duchefa D1004, 9 g/L); the pH was adjusted to 5.8 before autoclaving. The tubes were maintained in a growth room at 20°C under long day conditions until they regenerated into plantlets. Following shoot maturation, the plantlets were transferred to honey jars (HS French Flint Ltd, London, UK) containing SPM, where they were maintained for regular work. Plant material and in vitro micropropagation In vitro shoot cultures of F. vesca Hawaii 4 were sub-cultured at 4–6-week intervals, 5 per honey jar containing 50 ml medium. Strawberry multiplication medium (SMM) and SPM were alternated in each round of subculturing. Both basal culture media were composed of Murashige and Skoog (MS) macro and micro elements and vitamins, supplemented with sucrose (30 g/L) and 0.5mg/L of 6-benzylaminopurine (BAP), solidified with Daishin agar (Duchefa D1004, 9 g/L) and the pH was adjusted to 5.8 before autoclaving. Construct assembly and transformation into A grobacterium tumefaciens The binary plasmid vector constructs pL2B-pNOS-Kan-tNOS-p35S-mCherry-t35S-54122 and pL2B-pNOS-Hyg-tmas-p35S-mCherry-t35S-5433 were assembled using Golden Gate cloning. The domesticated Level 0 constructs were synthesized by Thermo GeneArt and assembled into the Level 1 backbone using the Bsa1 restriction enzyme. The different Level 1 constructs were assembled into the respective binary vector backbones using Bbs1 restriction enzyme. The GRF4-GRF1 chimera constructs were obtained from Addgene in pDONR-zeo backbone [21]. The individual GRF4-GIF1 entry vectors: TaGRF4-GIF1 , VvGRF4-GIF1 , VvmiRGRF4-GIF1 and CcGRF4-GIF1 were recombined using LR clonase Gateway cloning kit (Invitrogen) into the pK7WG2D binary vector obtained from VIB Ghent [28]. Electrocompetent Agrobacterium tumefaciens strain EHA105 were mixed with 500 ng of binary vector constructs. The mixture was pipetted into an electroporation cuvette and loaded into the electroporator and pulsed for 2.5 sec at resistance (200 ohm), capacitance (25 µFD) with pre-set voltage (Gene Pulser, Biorad). 500 µl of LB media (L1704, Duchefa) was added to the mixture of cells and plasmid after the shock and then transferred to a microfuge tube. Tubes were incubated in a shaker for 3h at 200 rpm and 28 o C. Cells were spread to LB + appropriate antibiotics and grown for 2 days at 28 o C. Colonies were verified by PCR (Supplementary Table S1). Transformation and regeneration of transgenic plants Preparation of plant material for transformation: Petioles were harvested the day before the transformation from the youngest (most apical) leaves. The plant cultures used were four-six weeks old after the last subculture. Transformation and regeneration: A. tumefaciens strain EHA105 with the binary vector were grown overnight (200 rpm, 28 o C) in LB media with appropriate antibiotics. The culture was pelleted at 2,000 x g for 10 minutes and re-suspended in filter-sterilised liquid MS-based medium supplemented with glucose (30 g/L) and acetosyringone (100 µM), pH 5.2, to give OD 600 nm = 0.2 – 0.3. Petioles were cut into 4-5 mm pieces, submerged in the inoculum, and blotted on sterile filter paper to remove excess inoculum. The petiole pieces were then transferred to Strawberry Regeneration Medium (SRM) petri dishes (MS-based medium supplemented with 0.2 mg/L of α-naphthaleneacetic acid, 1 mg/L of thidiazuron (TDZ), 5 g/L of Agargel and 30 g/L of glucose and adjusted to pH 5.8). Petioles were co-cultivated in the dark for four days at 20°C. After the incubation, explants were washed in a solution of filter-sterilised ticarcillin disodium/clavulanate potassium (TCA, Duchefa) (400 mg/L) in water for 4 hours (60 rpm, 20 o C), then blotted and transfered to T25 Cell Culture Flasks (Nunc) containing 15 ml of liquid SRM with antibiotic selection. Flasks were placed in a shaker at 60 rpm, 20 o C, under low light intensity for 4 weeks, and then blotted and transferred to SRM selection petri dishes. Petioles were sub-cultured every 4 weeks until regeneration. Control (WT) shoots were regenerated using the same method, except that the explants were not co-cultivated with A. tumefaciens, and selection antibiotics were omitted from the culture media. The transformed shoots were transferred to 30 ml universal tubes (Fisher Scientific) containing 15 ml of rooting medium (Frag R) with selection. Frag R is MS-based medium (2.2 g/L) supplemented with 0.1 mg/L of BAP and 0.1 mg/L IBA and 20 g/L of glucose, solidified with 9 g/L of Daishin agar (Duchefa) and adjusted to pH 5.8. After 4 weeks, shoots were moved to tubes containing SMT medium (MS-based medium supplemented with 0.225 mg/L of BAP, 0.2 mg/L IBA, 0.1 mg/L gibberellic acid (GA3) and 30 g/L of glucose, solidified with 7.5 g/L of Sigma-Aldrich agar and adjusted to pH 5.6 before autoclaving). In the next subculturing step, plants were changed to SMM tubes. Mature transgenic plant propagation: After 4 weeks in SMM tubes, plants were mature enough to be moved to honey jars (5 plants per jar). Honey jars with SMM medium or Strawberry Medium for Rooting (SMR) were alternated at 4–6-week intervals. SMR medium is a MS-based medium supplemented with 0.4 mg/L of IBA, 0.1 mg/L gibberellic acid (GA3) and 30 g/L of glucose, solidified with 7.5 g/L of Sigma-Aldrich agar A1296 and adjusted to pH 5.6 before autoclaving. Genotyping of transgenic lines DNA were extracted from 50-100 mg of leaf tissue using an in-house protocol described subsequently. The frozen leaf tissue was ground with metal balls (IG100_5/32_PK1000; Simply Bearings Ltd., Leigh, UK) using a mechanical pulveriser (MiniG from Spex) at 1200 rpm for 30 seconds. 500µl of the extraction buffer (1.25% sodium dodecyl sulphate (SDS); 100 mM Tris HCl pH 8.0; 50 mM EDTA pH 8.0 and 25 mg PVP) were added to the disrupted tissue. The samples were mixed and incubated at 65°C for 30 min, inverting the tubes each 5 min. Samples were cooled placing them in ice for around 5 min and then 250 µl of chilled 5M NaCl, mix and incubate in ice for 15 more min. The samples were centrifuged for 10 min at 20,000 g. Supernatant was transferred into a new tube containing 360 µl of isopropanol. Samples were vortexed and incubated for 30 minutes or overnight at -20°C to allow DNA to precipitate. The samples were centrifuged for 20 min at 15,700 g. Supernatant was discarded and pellet was washed in 500 µl of 70% ethanol. The samples were centrifuged for 20 min at 15,700 g and supernatant was discarded. Washing step was repeated once more and supernatant was discarded. Each pellet was resuspended in 50 µl TE buffer (10 mM Tris HCl pH 8.0; 1 mM EDTA pH 8.0). PCR amplification was performed using gene specific primers and PCR-BIO Taq Mix Red (PCR Biosystems) following the manufacturer’s guidelines. RNA extraction RNA was extracted from 100-200 mg of leave tissue using an in-house protocol [29]. RNA integrity was assessed using the Agilent TapeStation system using RNA screen tape. Library preparation and paired-end RNA sequencing was performed by Novogene (Cambridge, UK) on an Illumina NovaSeq 6000 platform. Sequencing data were deposited at the NCBI under the Bioproject ID PRJNA986313. RNA sequencing analysis Raw reads were quality controlled using FastQC v0.11.9 [30], and adapters and low-quality regions were trimmed using Trimmomatic v0.39 using a sliding window of 4 and minimum PHRED score of 20 [31]. The first 10 nucleotides were trimmed and reads less than 100 nucleotides and unpaired reads were discarded. GRF-GIF transgene sequences (Supplementary File S1) were concatenated with the F. vesca v4.0.a1 genome. Assemblies were indexed and reads were aligned using HISAT2 v2.2.1 using the default settings for paired end reads [32]. Annotations for the GRF-GIF transgenes were generated using StringTie 2.1.7 and these were merged with the F. vesca v4.0.a2 gene annotations [33]. Quantification was performed using featureCounts v2.0.1 [34] and differential expression analysis was performed with the R package DESeq2 v3.17 [35]. Comparisons were made between the empty vector control ( pK7WG2D ) and each GRF-GIF construct. The Benjamin and Hochberg approach for control of the false discovery rate was used and an adjusted p-value below 0.05 was used to identify differentially expressed genes (DEGs). For visual inspection of samples distances, variance stabilizing transformation (VST) was used to normalise the raw read counts and a principal component analysis (PCA) was performed using R. KEGG and InterProScan functional annotations from the Genome Database for Rosaceae (GDR) [36] were used to annotate DEGs. A Venn diagram of shared DEGs was plotted using the R package ggvenn v0.1.10 (https://cran.r-project.org/web/packages/ggvenn) and heatmaps of DEG log2 (fold change) (log2FC) were produced using the python library seaborn v0.12.0 [37]. Heatmap clustering was performed using hierarchical clustering based on Euclidean distance. To visualise the expression of individual DEGs, raw read counts were TPM normalised using bioinfokit v2.1.0 and plotted using seaborn v0.12.0 [37]. Imaging Pictures of the plates were taken with a Canon DSLR camera EOS4000D. Visualization of mCherry fluorescence and eGFP-ER in plant tissue and pictures of the calli, shoots and plants were performed using a Leica Stereomicroscope M165. The leaves were scanned using an EPSON flatbed scanner. The images were assembled using Inkscape and Adobe Photoshop. Statistical analysis Histograms and statistical analyses were performed with R (2023.03.0 Build 386 © 2009-2023 Posit Software, PBC). Statistical differences were tested by performing a non-parametric Kruskal-Wallis test and the differences among samples were determined using pairwise comparisons with Wilcoxon rank sum test with continuity correction. Sequence alignments and phylogenetic tree Gene and protein sequences were obtained using NCBI (https://www.ncbi.nlm.nih.gov/) and OrthoDB (https://www.orthodb.org/?ncbi=18049678). Accession numbers and protein names used for the phylogenetic tree are available in the phylogenetic tree in Supplementary Figure S1 and Supplementary Table S2. Protein FASTA sequences were aligned using MUSCLE method with MEGA11 (11.03.13 Built 11220624 © 2013-2023). The phylogenetic tree was built in MEGA11 using the Maximum Likelihood method and JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates. All positions with less than 95% site coverage were eliminated. RESULTS Establishing F. vesca stock plants and a regeneration protocol. To establish a uniform population of F. vesca Hawaii 4 plants, surface sterilized and scarified seeds were germinated on water agar plates. Following their germination, the plantlets were propagated on soil mix in glasshouses. After 4-5 weeks growth in the glasshouse, the plants started to produce runners (Fig. 1a). The runners allow vegetative propagation of strawberry by producing a ‘clone-plant’ with each runner tip containing an apical meristem that can develop into a new plant (Fig. 1b). Apical meristem tissue was collected from the growing runner and propagated in Shoot Propagation Media (SPM) in tubes (Fig. 1c). Meristem culture is the most prevalent mode of vegetative propagation for strawberry as it allows selection of disease-free plants [38]. Subsequently, the plants growing from the meristem were moved into jars for shoot multiplication (Fig. 1d). 4-6 weeks post propagation into SPM, the plants produced enough petioles for the establishment of the regeneration experiment. Young petioles were harvested from the jars and sliced into 4-5 mm pieces under sterile condition to initiate regeneration (Fig. 1e). The petiole pieces were transferred to liquid shoot regeneration media (SRM) in flasks and maintained at 22°C with regular shaking under long day conditions (Fig. 1f). 2 weeks into the SRM, the petioles started to form callus at both the cut ends when they were transferred to SRM plates (Fig. 1g). Regeneration efficiency of plants from the callus were assessed at 4-, 8- and 12-weeks following incubation in SRM for 50 petiole edges (Fig. 1h). After 4 weeks, all petioles had calli on both edges, that started to produce shoots by 8 and 12 weeks with an efficiency of 71% and 86%, respectively. Thus, we could produce a running stock of Hawaii 4 plants and establish an efficient platform for regeneration that could be further exploited to produce transgenic plants. Selection of stable transgenics using antibiotic and fluorescent cassettes To successfully raise transgenic plants, it is important to select the positive lines from the wild type revertant. Antibiotics such as kanamycin and hygromycin previously allowed efficient selection of transgenic strawberry plants [39]. To compare the transformation efficiency of different antibiotic selection cassettes, 50 petiole pieces from 4-week-old Hawaii 4 crowns grown on SPM medium were infected with A. tumefaciens strain EHA105 containing plasmids carrying hygromycin or kanamycin selection cassettes (Fig. 2a). Negative control and non-transformed petioles did not survive the treatment with either antibiotic, turning brown by 4 weeks indicating senescence (Fig. 2b). Transformation efficiency was assessed at 4-, 8- and 12-weeks post transformation (WPT) and was estimated as frequency of petiole edges with regenerating shoots (Fig 2b-c). At all 3 time points, transformation efficiency of the hygromycin and kanamycin selection cassettes were found to be comparable (Fig. 2c). As a transformation marker, the plasmids were carrying mCherry fluorescent protein driven downstream to promoter 35S. Regenerating fluorescent shoots on the selection media were subsequently transferred to shooting and rooting media over the course of 12 weeks to facilitate development of stable plantlets with root systems (Fig. 2d). The uniform mCherry expression in all plant tissues throughout development indicated the lack of chimeric transformants. Moreover, the healthy physiology of the plants suggested the absence of any pleiotropic effects from the antibiotic selection cassettes (Fig 2d). Thus, the antibiotic and visual fluorescent markers cassette provides a dual selection for positive transformants all through the regeneration process. GRF4-GIF1 chimeras from different species increase regeneration efficiency Introduction of developmental genes has resulted in faster regeneration of callus for several plant species including certain recalcitrant plants [13–19]. To improve strawberry transformation efficiency, GRF4-GIF1 chimeras from different species were tested for their effect on strawberry transformation. Petiole pieces from 4-week-old Hawaii 4 crowns were infected with A. tumefaciens strain EHA105 carrying GRF4-GIF1 chimeras from Vitis vinifera (constitutive, VvGRF-GIF and miR396 -resistant version, Vv miR GRF-GIF ), Citrus clementina ( CcGRF-GIF ) and Triticum aestivum ( TaGRF-GIF ) (Supp Fig. S1-S2). Transformation efficiencies were assessed at 4-, 8- and 12- WPT and were estimated as frequency of petiole edges with shoots (Fig 3b-c). At 4 WPT, regeneration efficiency of petioles transformed with VvGRF-GIF , CcGRF-GIF and TaGRF-GIF chimeras were comparable to the empty vector transformed petioles (Fig 3c). At the same time point, Vv miR GRF-GIF showed regeneration efficiency comparable to 8 WPT for the empty vector control indicating a 4-week faster regeneration of shoot from callus which is evident in having much mature plantlets by 12 weeks (Fig. 3c). This is concomitant to the previous report where the miRNA resistant variety of GRF resulted in an increase in cell number and leaf size in Arabidopsis [40]. The Vv miR GRF-GIF construct contained four synonymous mutations to prevent the binding of miRNA396 , which regulates GRF4 expression (Supp Fig. S3). miRNA396 is known to target GRF1-4 family transcripts, controlling the activity of AtGRF3 during leaf development [40,41]. At week 8- and 12- WPT, all GRF4-GIF1 chimeras showed significantly higher shoot regeneration compared to the empty vector controls (Fig. 3b-c). While the introduction of the GRF4-GIF1 chimeras resulted in efficient regeneration, their constitutive expression also induced several pleiotropic effects on the plants [42]. To study the effect of GRF4-GIF1 chimeras on regenerating plant physiology, shoots were taken at 12 weeks and grown until rooted plantlets were established (Fig. 3d). As all the constructs were carrying eGFP transformation reporter, its expression was monitored throughout the experiment, from callus to regenerating plantlets, to ensure no chimeric plants were selected (Fig. 3d). Severe pleiotropic effects were observed for Vv miR GRF-GIF plants where, despite being more vigorous in regeneration, the plants failed to show the canonical leaf expansion and elongation that are hallmarks for proper development in strawberry (Fig. 3d-e). Similar observations were made for rice where the miRNA396 resistant variety of Vv miR GRF-GIF resulted in formation of large calli without proper regeneration [21]. As compared to the miRNA resistant version, VvGRF-GIF and CcGRF-GIF showed proper regeneration with healthy adult plants established under lab condition (Fig 3d). One of the reasons behind the better health of the VvGRF-GIF and CcGRF-GIF could be the presence of the miRNA396 target site where F. vesca miRNA396 could bind to regulate the expression of the GRF4 gene expression (Supp Fig. S3). TaGRF-GIF plants also exhibited aberrant leaf development in the regenerated plants. 3 out of 5 lines showed leaves with more lobes that were more serrated with sharp edges compared to empty vector transformed plants (Fig. 3e). Multiple protein alignment showed that TaGRF4 shares much less homology compared to the dicot GRFs (40% vs ~85%), but like its dicot counterparts still retains the miRNA396 binding site (Supp Fig. S4-S5). The presence of the miRNA target site suggests canonical transcriptional regulation by miRNA396 , but divergent protein structure of TaGRF4 might be activating phytohormone responses resulting in the aberrant leaf morphology. In a previous observation, OsbZIP48 from rice could complement an Arabidopsis Athy5 mutant but caused pleiotropic effect like semi-dwarfism [43]. Thus, our observation indicates that cross species activation of GRF4-GIF1 chimera can induce pleiotropic effects due to possibly mis-regulation at the transcriptional or translational level. Transcriptomic analysis of the GRF4-GIF1 chimeras shows differential gene activation All the chimeric GRF4-GIF1 produced a positive effect on regeneration efficiency irrespective of their source. But the pleiotropic effects in Vv miR GRF-GIF and TaGRF-GIF in strawberry indicates the presence of complex transcriptional landscape under different chimeric conditions. To investigate the issue, transcriptomic analysis was performed for each condition with leaf extracted RNA. Significantly high expression of the GRF4-GIF1 chimeras were observed for each of the transgenic lines assayed (Fig. 5a). Concomitant to the aberrant phenotypes, both Vv miR GRF-GIF and TaGRF-GIF lines shows 178 and 116 DEGs that were not represented in the other data sets (Fig. 5b-c). A particular group of DEGs showed very high expression in both Vv miR GRF-GIF and TaGRF-GIF as compared to the CcGRF-GIF and VvGRF-GIF identified as Unique cluster A. FvH4_3g44360, encoding a peroxidase from this cluster showed ~3.5-fold higher expression in Vv miR GRF-GIF1 compared to control plants (Fig. 5c-d; Supp Table S3). A previous report in Nicotiana benthamiana showed that overexpression of peroxidase leads to developmental abnormalities with retarded root development [44]. Another gene that is significantly upregulated in VvGRFmiR-GIF is FvH4_4g10610 encoding a EP3-like endochitinase (Fig. 5c-d; Supp Table S3). Endochitinase is an extracellular protein secreted by the non-embryogenic cells in the medium inducing somatic embryogenesis [45,46]. It could be possible that the higher expression of EP3-like endochitinase induced more somatic embryos in Vv miR GRF-GIF1 lines, but the sustained expression of the gene resulted in lack of regeneration. Interestingly, this gene is also upregulated in TaGRF-GIF lines indicating that the possible phenotypic effect of EP3-like endochitinase in plant development ranges from somatic embryogenesis to proper plant development (Supp Table S3). TaGRF-GIF lines showed an exclusive upregulation of FvH4_7g27130 encoding an expansin gene from the Unique cluster B where a cluster of DEGs show significantly higher expression in TaGRF-GIF as compared to the others (Fig. 5c-d). A recent report in Poplar showed that overexpression of GRF5 resulted in increased leaf size, and transcriptomic analysis assisted with DAP-seq showed significant representation of cell cycle and expansin gene families [47]. This paper also reported that poplar PpnGRF5 binds to promoter of Cytokinin oxidase/dehydrogenase ( pPpnCKX ) and negatively regulate its expression resulting in elevated cytokinin levels in the cells. Conversely, our transcriptomic data indicated ~3-fold increase of FvH4_2g39230 encoding Cytokinin oxidase/dehydrogenase (Supp Table S3). While an increase in the expression of CKX should ideally result in decrease of the cytokinin level, the transcriptome of TaGRF-GIF shows ~2-fold increase in FvH4_5g16240 expression encoding a type-A two-component response regulator (RRs) (Supp Table S3). Type-A response regulators act downstream to cytokinin signalling where upon activation, negatively regulate the pathway [48]. It could be possible that the deformed leaflet formation observed in TaGRF-GIF lines is due to abnormal cell division caused by deregulated levels of cytokinin. Differential activation of the cytokinin pathway is further evident by the fact that different sets of two-component RRs are activated in CcGRF-GIF and Vv miR GRF-GIF lines (Supp Table S3). Thus, our data indicates that GRF4-GIF1 chimera could differentially activates the cytokinin and expansin genes to control the developmental processes in strawberry. VvGRF-GIF plants show better regeneration efficiency following re-transformation Morphogenic regulators not only facilitate recalcitrant plants to undergo somatic embryogenesis but also allow better regeneration efficiency for plants that are already known to undergo somatic embryogenesis [20,21,42]. By virtue of their faster regeneration efficiency, we investigated whether GRF4-GIF1 stable lines in strawberry perform better during retransformation when compared to the empty-vector transformed lines and the wild-type plants. Due to the pleiotropic effects in Vv miR GRF-GIF and TaGRF-GIF lines, only VvGRF-GIF and CcGRF-GIF were considered for re-transformation with a vector carrying hygromycin resistance gene and mCherry fluorescent marker (Fig. 5a). As the transformed plants already had the GRF4-GIF1 chimeric cassette with kanamycin selection and eGFP marker, the vector with hygromycin selection cassette was chosen that was previously used in Fig. 2. 50 pieces of petioles from each line were infected and transformation efficiency were assessed at 4-, 8- and 12- WPT as before (Fig. 5b-c). At week 4, transformation efficiency was 0% in all the samples. By week 8- and 12-, the average transformation efficiency of two of the VvGRF-GIF lines were ~40% higher compared to the empty vector transformed petioles and wild-type plants (Fig. 5c). It is important to note that petiole regeneration following re-transformation is usually slower than single transformation possibly due to presence of multiple antibiotic selection. The shoots of these two VvGRF-GIF lines looked bigger and healthier by 12 weeks compared to the empty vector transformed lines (Fig. 5b). At week 8- and 12-, no significant difference in regeneration efficiency was noted for CcGRF-GIF lines (Fig. 5b-c). The calli and the regenerating plants were checked for fluorescence where the eGFP fluorescence indicated the consistent expression of the GRF4-GIF1 cassette and the mCherry indicated double transformation events (Fig. 5d). The double fluorescent calli were transferred to selection media for the propagation of transformed plants. Although, both VvGRF-GIF and CcGRF-GIF lines looked healthy at their rooted plantlet stages (Fig. 3), the differences in their regeneration efficiency after re-transformation is difficult to explain. One interesting difference is the upregulation of 3 cytokinin responsive RR genes FvH4_2g27180 , FvH4_5g16240 and FvH4_6g25290 in CcGRF-GIF as compared to VvGRF-GIF lines (Supp Table S3). Along with this difference, there are several other genes that were differentially regulated in the CcGRF-GIF lines which could contribute to their lack of regeneration phenotype (Fig. 4c). Thus, ectopic expression of VvGRF-GIF chimera could be a useful tool for expediting strawberry transformation without incurring unwanted pleiotropic effect. DISCUSSION Strawberry is a commercially important crop where several desirable agronomic traits define its value in the market. But fundamentally, the presence of physiological or genetically linked trade-offs limits the possibility for certain combinations of phenotypes to occur [ 49 ]​​. As often these viable traits are diametrically opposed, genetic engineering over normal breeding provides an opportunity to overcome the genetically linked traits ​[ 50 ]. In this study, we present an efficient strategy to expedite transformation in strawberry with the introduction of GRF4-GIF1 chimeras (Fig. 1 – 3 ). Using different types of GRF4-GIF1 chimeras coming from multiple plant species allowed us to explore the best possible chimera as complicated regulation of GRFs resulted in pleiotropic effects for Vv miR GRF-GIF1 and TaGRF-GIF (Fig. 3 ). Transcriptomic analysis of the different lines provided necessary insight into the possible causes of the pleiotropic effects and the complex regulon for GRF4-GIF1s (Fig. 4 ). We also found VvGRF-GIF lines has much better efficiency after re-transformation as compared to the empty vector or wild-type plants (Fig. 5 ). Diploid strawberry is an attractive system to study functional genomics in Rosaceae due to its small genome size, short life cycle and facile vegetative and seed propagation ​[ 10 ]. But the biggest bottle neck for doing any forward or reverse genetics is an efficient protocol to raise stable transgenics. In the last couple of decades there has been a considerable effort to establish various protocols for raising successful strawberry transgenics with various level of efficiencies ranging from 63–68% [ 10 , 51 ]​. Here, we have presented a comprehensive protocol for preparing a uniform line of clean stock plants using meristem culture which can be used for raising stable transgenics (Fig. 1 ). Strawberry is generally propagated using stolon which runs the risk of getting infected material into tissue culture spreading through vascular tissues [ 52 ]​. Meristem culture on the other hand allows an alternative strategy to obtain large quantities of virus free material due to active cell division and lack of differentiation [ 53 ]​​. Moreover, tissue culture of strawberry leads to somaclonal variation which can be avoided by meristeming and thus allowing true-to-type plants [ 54 ]​. The stability of the background is revealed by ~ 90% regeneration efficiency with very little variability (Fig. 1 d). Growth regulating factors (GRFs) are a small family of transcription factors that play important role in plant development by controlling various aspects of leaf developmental [ 40 , 47 , 55 ]​​, stem development, apical meristem development [ 56 , 57 ]​ and root development [ 58 ]​​. GRFs form complexes with GRF interacting proteins (GIFs) which act as transcriptional coactivators [ 59 ]​. From an evolutionary perspective, all land plants encode for GRF proteins except green algae, whereas GIFs are universally present ​[ 22 ]​. Several studies have shown that GRFs are active at the sites of active growth and differentiation, with expression gradually decreasing in the matured tissues [ 23 ]​. In A. thaliana , this expression regulation is primarily carried out by the miR396a and miR396b which shows near perfect sequence alignment with the transcripts of several GRFs [ 23 , 60 ]​. F. vesca encodes for miR396 gene which also shows sequence conservation with AtmiR396 indicating that miRNA mediated control of GRFs is highly conserved across the plant kingdom (Supp Fig. S3 ). This is in consonance to a previous report where ectopic expression of AtmiR396 resulted in reduction in gene expression of GRFs in N. benthamiana ​[ 61 ]. Vitis belonging to the order Vitales is phylogenetically closest to the Fragaria sharing maximum homology to FvGRF4 followed by Citrus from Sapindales and Triticum from Poales (Supp Fig. S1 ). Using the GRF4-GIF1 chimeras from both eudicot and monocot species allowed us to dissect the functional diversity within the GRF4 family in a heterologous system. miR396 expression and GRF4 expression work reciprocally, whereby GRF4 expression decreases in mature leaves with an increase in miR396 expression [ 40 ]. The importance of the transcriptional control of GRF4 by miR396 is revealed in the pleiotropic phenotype of the Vv miR GRF4-GIF1 lines (Fig. 3 ). While facilitating more regeneration events, the sustained expression of the GRF4-GIF1 chimera in the Vv miR GRF4-GIF1 lines prevented most of these plants to reach proper tissue differentiation (Fig. 3 b). Transcriptome analysis showed that there were many genes that were differentially up-regulated in the Vv miR GRF4-GIF1 lines as compared to the VvGRF4-GIF1 and CcGRF4-GIF1 (Fig. 4 ; Supp Table S3 ). Upregulation of certain vital genes required for the transition from cell division to differentiation like endochitinase and peroxidases might have played a significant role (Fig. 4 ; Supp Table S3 ). This agrees with a previous observation in Citrus where inactivation of peroxidase activity was shown to be important for in vitro plant differentiation [ 62 ]​. The expansion of the GRF family transcription factors happened due to large scale genome duplication [ 22 ]​. In case of the eudicots, a whole genome triplication event in the ancestor led to the formation of several GRF genes. Like eudicots, a similar duplication event led to formation of the monocot GRFs ​. The TaGRF4-GIF1 showed only 40% protein sequence homology to the eudicot GRF4-GIF1s compared to ~ 85% within the eudicots (Supp Fig. S4 -5). As genome duplication events are directly related to neofunctionalization [ 63 ], it could be possible that during evolution, GRFs gained functions in monocots that are different from eudicots. This is evident from our observation of the leaf phenotype in strawberry lines where ectopic expression of TaGRF4-GIF1 caused leaf deformations (Fig. 3 e). Transcriptome analysis showed that in these leaf tissue there was a significant increase in the expression of an expansin gene FvH4_7g27130 (Fig. 4 b). Previously in N. benthamiana , local expression of expansin recapitulated leaf formation from a meristem and could also alter the shape of the leaf lamina [ 64 ]​. GRF transcriptional activity tightly controls the cytokinin concentration in plants, which in turn is responsible for cell division and expansion ​[ 47 ]​. TaGRF-GIF lines showed higher expression of cytokinin responsive RRs in the abnormal leaves (Fig. 4 ; Supp Table S3 ). During the leaf expansion phase, an increase in cytokinin concentration can lead to abnormal leaf development ​​[ 65 ]​. Thus, the abnormality in the strawberry leaves in TaGRF-GIF lines could be due to misexpression of cytokinin responsive and expansin genes. The benefits of the developmental genes during transformation first came into prominence for their ability to jump start somatic embryogenesis [ 42 ]. Re-transformation of multiple genes in a desirable background, or ‘stacking’ of genes, has always been challenging for multiple reasons [ 66 ]. Here, we show that the introduction of the VvGRF-GIF in strawberry gives the plants certain advantages during re-transformation where the transformation efficiency increases by ~ 40% as compared to the empty vector transformed plants (Fig. 5 ). Moreover, the expression of both visual fluorescent transformation markers ensured that both the cassettes were properly transformed. But surprisingly, the CcGRF-GIF lines did not show any significant improvement during the re-transformation experiment (Fig. 5 ). Transcriptome analysis indicated significant differences between VvGRF-GIF and CcGRF-GIF lines despite the lack of any phenotypic discrepancies (Fig. 4 c, Supp Table S3 ). Cytokinin is intrinsically linked to regeneration of plants and GRF lines were shown to behave very differently during regeneration experiments depending upon its availability [ 21 ]​. As several cytokinin responsive RRs genes are activated in the CcGRF-GIF lines, it could be possible that disproportionate cytokinin levels affected its regeneration. CONCLUSIONS A comprehensive protocol for strawberry transformation will turn out to be extremely beneficial for understanding genetics within the Rosaceae family, which includes several economically important horticultural crops. The re-transformation protocol that we present here can be utilized in the future to raise stable transgenics of mutant backgrounds where faster screening strategies, such as virus-induced gene silencing (VIGS) can be introduced to study viable traits. Overall, not only have we presented VvGRF-GIF to be an effective GRF4-GIF1 chimera for enhancing regeneration in a strawberry transformation system, but also highlighted the pitfalls of using the wrong chimeras. This study provides an overarching scope for bringing more such important horticultural Rosaceae crops under tissue culture following the strawberry footsteps. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This work is supported by Bill and Melinda Gates Foundation as OPP1028264. Author’s contribution A.K. and R.H. conceived the idea. E.R.S and A.K. designed the experiments. K.M. provided her expertise to micro-propagate the strawberry cultivar in the tissue culture. E.R.S. and F.M carried out the experiments. E.R.S analysed the regeneration efficiency data and J.P. carried out the transcriptome analysis. A.K. supervised the project. A.K. wrote the manuscript with support from E.R.S and R.J.P. Acknowledgements We thank Fiona Wilson for optimizing the strawberry transformation protocol. 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Supplementary Files SupplementaryTableS1S2.xlsx SupplementaryTableS3a.xlsx SupplementaryTableS3b.xlsx Supplementaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 22 Aug, 2024 Reviews received at journal 22 Aug, 2024 Reviews received at journal 08 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers agreed at journal 19 Jun, 2024 Reviewers invited by journal 17 Jun, 2024 Editor assigned by journal 15 Jun, 2024 Submission checks completed at journal 15 Jun, 2024 First submitted to journal 14 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Jordan Price","email":"","orcid":"","institution":"NIAB","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"Jordan","lastName":"Price","suffix":""},{"id":321335153,"identity":"090666ec-3c78-4c30-a20e-a7ab0009bee9","order_by":2,"name":"Federico Marangelli","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"","lastName":"Marangelli","suffix":""},{"id":321335157,"identity":"7f8dfa6f-cb65-4e6a-a021-24934c8781f2","order_by":3,"name":"Kirsty McLeary","email":"","orcid":"","institution":"NIAB","correspondingAuthor":false,"prefix":"","firstName":"Kirsty","middleName":"","lastName":"McLeary","suffix":""},{"id":321335158,"identity":"7447bdb5-c023-4933-b874-58162306eb94","order_by":4,"name":"Richard J. Harrison","email":"","orcid":"","institution":"Wageningen University and Research","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"J.","lastName":"Harrison","suffix":""},{"id":321335160,"identity":"126fd5a6-a83f-4468-a2ca-256398e287ee","order_by":5,"name":"Anindya Kundu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFACxgYGHiAlAcYVQLYEaVrOEKUFCOBaGNugLHyAf3Zz24O3bXYMkjNyD97mnXdHhn92A+OHjzm4tUjcOdhuOLctmUFaIi/ZmnfbMx6JOweYJWduw2PNjcQ2ad42ZgY5iRwzad5th3kYbiSwMfPi0SIP0VIP1TLnMI88IS0GEC2HgQ4DaWk4zGNASIshUIvknHPHeSR73hhbzjl2mMfwzsFmvH6Ru5H+TOJNWbWcxPEcwxtvag7by91uPvjhIz7vgwAjGzhq4NwGAupB4A8RakbBKBgFo2DkAgA/g0wYn0uGCAAAAABJRU5ErkJggg==","orcid":"","institution":"NIAB","correspondingAuthor":true,"prefix":"","firstName":"Anindya","middleName":"","lastName":"Kundu","suffix":""}],"badges":[],"createdAt":"2024-06-14 18:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4583627/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4583627/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-024-01270-8","type":"published","date":"2024-10-18T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59488711,"identity":"5b7f4640-4da7-4b99-8d33-4c145fd017d2","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":697596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of a tissue culture stock of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHawaii 4 to use as a starting material in transformations with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. tumefaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e(a) Schematic representation of a F.\u003cem\u003evesca\u003c/em\u003e Hawaii 4 runner and (b) stereo-micrograph of a meristem, (c-d) Whole plant regeneration going through shoot (c) and root (d) development. (e-g) Schematic representation of \u003cem\u003eF. vesca\u003c/em\u003eHawaii 4 regeneration process using petioles as an explant going from petiole harvest (e), callusing (f) and shoot induction (g). (h) Boxplot represents the regeneration efficiency of \u003cem\u003eF. vesca\u003c/em\u003e petioles at 4-, 8- and 12- weeks post transfer to the regeneration media (SRM) where the box extends from the 25th to 75th percentiles; whiskers, 1.5 interquartile range points out of the whiskers. Statistical analysis is performed using Kruskal-Wallis test where n = 50 and p \u0026lt; 0.05. Scale bar = 500µm\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/5c75cd8e0e8d280bebc3f66d.png"},{"id":59488716,"identity":"0bb84b51-2815-420f-b46f-153a1aed357f","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":918355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of kanamycin and hygromycin selection cassette on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHawaii 4 regeneration at 4-, 8- and 12- weeks after transformation with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. tumefaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e EHA105.\u003c/strong\u003e (a) Schematic representation of Kan\u003csup\u003e+\u003c/sup\u003e (kanamycin selection) and Hyg\u003csup\u003e+\u003c/sup\u003e (Hygromycin selection) constructs. Regeneration of petiole with shoots studied at 4-, 8- and 12-weeks post transfer to the regeneration media where (b) micrograph represents regenerating petiole with shoots. Scale bar = 1cm and (c) Boxplot representing the regeneration efficiency where the box extends from the 25th to 75th percentiles; whiskers, 1.5 interquartile range; points out of the whiskers. Statistical analysis is performed using Kruskal-Wallis test where n = 100 and p \u0026lt; 0.05. (d) Representative images of Kan\u003csup\u003e+\u003c/sup\u003e and Hyg\u003csup\u003e+\u003c/sup\u003e calli at 12- weeks and regenerating plantlets under bright field and corresponding mCherry filter (red). Scale bar = 1mm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/2c2aa5336d80722827dcd57d.png"},{"id":59488713,"identity":"3e5a32e6-d538-421c-b22d-b4e58b86e76f","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2320787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGRF-GIF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e chimeras in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Hawaii 4 regeneration at 4-, 8- and 12- weeks after transformation with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. tumefaciens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eEHA105.\u003c/strong\u003e (a) Schematic representation of \u003cem\u003eGRF-GIF\u003c/em\u003e chimeras constructs. Regeneration of petiole with shoots studied at 4-, 8- and 12- weeks post transfer to the regeneration media where (b) micrograph represents regenerating petiole with shoots. Scale bar = 1cm and (c) Boxplot representing the regeneration efficiency where the box extends from the 25th to 75th percentiles; whiskers, 1.5 interquartile range; points out of the whiskers. Statistical analysis is performed using Kruskal-Wallis test where n = 100 and p \u0026lt; 0.05. Representative images of control (Hawaii4) and \u003cem\u003eGRF-GIF\u003c/em\u003e chimeras (d) calli at 12- weeks and regenerating plantlets under bright field and corresponding eGFP filter (green). Scale bar = 1mm. and (e) leaf margins under stereomicroscope. Scale bar = 1mm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/7cc08de2c639f13bcb13ee23.png"},{"id":59488718,"identity":"5f542197-7c11-4c26-a1f3-44ec18d56939","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":692463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGRF-GIF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003echimeras in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Hawaii 4. \u003c/strong\u003e(a) Relative expression of the \u003cem\u003eGRF-GIF\u003c/em\u003etransgenes in the leaves of three independent lines for each construct. (b) Heatmap showing the log2 transformed fold change (log2FC) of all differentially expressed genes (DEGs) shared across two or more \u003cem\u003eGRF-GIF\u003c/em\u003e chimeras. Two unique clusters of expression patterns are highlighted by hatched boxes. (c) Venn diagram representation of the total number of DEGs shared between the different \u003cem\u003eGRF-GIF\u003c/em\u003e chimeras. (d) Plots showing the log2FC of the unique cluster DEGs identified in b.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/acbc618c11e0a4837478dc84.png"},{"id":59489346,"identity":"9811152a-bc97-47b7-9f66-a2718e5f7430","added_by":"auto","created_at":"2024-07-02 11:46:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1215655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of re-transformating on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca GRF-GIF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003echimeras at 4-, 8- and 12- weeks after transformation with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. tumefaciens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eEHA105.\u003c/strong\u003e(a) Schematic representation of Hyg+ constructs transformed. (b) Regeneration of petiole with shoots studied at 4-, 8- and 12- weeks post transfer to the regeneration media where (b) micrograph represents regenerating petiole with shoots. Scale bar = 1cm and (c) Boxplot representing the regeneration efficiency where the box extends from the 25th to 75th percentiles; whiskers, 1.5 interquartile range; points out of the whiskers. Statistical analysis is performed using Kruskal-Wallis test where n = 100 and p \u0026lt; 0.05. (d) Representative images of control (Hawaii4), empty vector, \u003cem\u003eVvGRF-GIF\u003c/em\u003eand \u003cem\u003eCcGRF-GIF\u003c/em\u003e chimera callus at 12- weeks and regenerating plantlets under bright field mCherry (red) and GFP (green) filter. Scale bar = 1mm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/3f3d1a8140e034faf851f40a.png"},{"id":67149780,"identity":"18f92f3a-2ac6-44f4-90da-784cf864e50b","added_by":"auto","created_at":"2024-10-21 16:14:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8310070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/693670d8-e5bd-4ffe-8ab3-5aad33c974cc.pdf"},{"id":59488712,"identity":"fae37cbc-5a14-455a-aa35-9a2806874d1a","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19800,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/1651cb5bca0b73c233a16bf0.xlsx"},{"id":59488714,"identity":"af82abf7-492d-46ae-8a1c-1a72608a0637","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":120225,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3a.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/f4d7765e372e93a676dbba6e.xlsx"},{"id":59488719,"identity":"6aedab32-61ac-408d-9494-994cab02084d","added_by":"auto","created_at":"2024-07-02 11:38:33","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7171521,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3b.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/09c0ebbeac4d96ab228b9f35.xlsx"},{"id":59488720,"identity":"93e6dab2-c3ae-4af8-85e9-c69a4f8015b8","added_by":"auto","created_at":"2024-07-02 11:38:34","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4971959,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4583627/v1/61b7ad8d2b5cafd5b5200dd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eSomatic embryogenesis plays an essential role towards asexual propagation and regeneration of plants. Unlike organogenesis which requires a high cytokinin-to-auxin ratio [1,2], somatic embryogenesis is mostly dependent on auxin [2\u0026ndash;4].\u0026nbsp;In the last decade, concerted effort has been made to understand the molecular mechanisms that drive the transition of a vegetative cell into an embryogenic competent cell under the influence of auxin and cytokinin signalling. Understanding of the regeneration pathways leading to embryogenesis under the influence of phytohormones resulted in the development of \u003cem\u003ein vitro\u003c/em\u003e techniques for tissue culture of several plant species [5].\u0026nbsp;The Rosaceae is one such family where sexual hybridization, asexual propagation, and genetic improvements have been pivotal for developing better varieties for quite some time [6].\u0026nbsp;It is a large and diverse family that includes several economically relevant food crops such as apple (\u003cem\u003eMalus\u003c/em\u003e), plum, peach, almond, cherry (\u003cem\u003ePrunus\u003c/em\u003e),\u0026nbsp;pear (\u003cem\u003ePyrus\u003c/em\u003e), raspberry (\u003cem\u003eRubus\u003c/em\u003e), strawberry (\u003cem\u003eFragaria\u003c/em\u003e) and other species with economic value.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cultivated strawberry, \u003cem\u003eFragaria x ananassa\u003c/em\u003e, is an octoploid species (2n =8x = 56) derived from the hybridization between \u003cem\u003eF. chiloensis\u003c/em\u003e and\u003cem\u003e\u0026nbsp;F. virginiana\u003c/em\u003e [7].\u003cem\u003e\u0026nbsp;\u003c/em\u003eAlthough breeding and genetic engineering tools are available in \u003cem\u003eF. x ananassa\u0026nbsp;\u003c/em\u003e[8],\u0026nbsp;the polyploid genome makes crop improvement in this species difficult. For that reason, the diploid woodland strawberry (\u003cem\u003eFragaria vesca\u003c/em\u003e) that holds\u0026nbsp;close kinship to commercial strawberry\u0026nbsp;is widely used as a genetic model [6].\u0026nbsp;\u003cem\u003eF. vesca\u003c/em\u003e offers favourable attributes including a ~240 Mb reference genome (versus 157 Mb in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e), short generation time, small plant size and a wide geographical distribution\u0026nbsp;[7]. As a result, in the last few years, effective \u003cem\u003ein vitro\u003c/em\u003e propagation, regeneration and transformation techniques have been developed for \u003cem\u003eF. vesca\u0026nbsp;\u003c/em\u003eto facilitate genetic engineering\u0026nbsp;[9\u0026ndash;11].\u0026nbsp;However, despite\u0026nbsp;the establishment of regeneration and transformation techniques in woodland strawberry, the use of developmental regulators has never been tested, which might lead to the\u0026nbsp;development of a better strategy that could not only induce better and faster regeneration but also facilitate more effective transformation in strawberry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch in model organisms such as \u003cem\u003eA. thaliana\u003c/em\u003e facilitated the identification of certain transcription factors that can integrate the signals leading to cellular reprogramming resulting in embryogenesis or meristematic fate [12].\u0026nbsp;These transcription factors are called developmental regulators as they coordinate spatial cellular distribution resulting in organ formation. For example, Somatic Embryogenesis Receptor Kinase\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eSERK\u003c/em\u003e), Leafy Cotyledon 1 (\u003cem\u003eLEC1\u003c/em\u003e), Leafy Cotyledon 2 (\u003cem\u003eLEC2\u003c/em\u003e), NiR, Baby Boom (\u003cem\u003eBBM\u003c/em\u003e), Wound Induced Dedifferentiation 1 (\u003cem\u003eWIND1\u003c/em\u003e), Wuschel (\u003cem\u003eWUS\u003c/em\u003e) and \u003cem\u003eWOX5\u003c/em\u003e have all been identified to be essential during somatic development\u0026nbsp;[13\u0026ndash;19].\u0026nbsp;Ectopic expression of these genes not only allowed regeneration of transformation-recalcitrant plant species but also increased regeneration efficiencies. However, overexpression of developmental genes like \u003cem\u003eWUS\u003c/em\u003e and \u003cem\u003eBBM\u003c/em\u003e induced pleiotropic effects, including callus necrosis, compromised differentiation of shoots and roots, reduced fertility of transgenic plants, and a variety of other aberrant phenotypes [20].\u0026nbsp;This necessitates the need for an alternative strategy to enhance regeneration without compromising the morphology of the plant. This search culminated with the finding that ectopic expression of a chimeric GRF-GIF protein complex could induce better regeneration of fertile cultivars [21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Growth-Regulating Factors (GRFs) are a small group of transcription factors that play an important role in plant development and are highly conserved in angiosperm, gymnosperm, and moss (bryophyte) lineages [22].\u0026nbsp;They encode proteins with conserved QLQ and WRC domains responsible for protein\u0026ndash;protein and protein\u0026ndash;DNA interactions, respectively. Many angiosperms and gymnosperm GRF genes carry the target site for micro-RNA 396 (\u003cem\u003emiR396\u003c/em\u003e), which attenuates its activity [23].\u0026nbsp;The GRF proteins form complexes with their transcription cofactor GRF-Interacting Factors (GIFs) and forms a transcription activation complex [24]. In these GRF-GIF complexes, GIFs recruit chromatin remodelling complexes and GRFs remove the nucleosomes from chromatin by virtue of the QLQ motifs to activate expression of target genes [25].\u0026nbsp;In general, callus formation and subsequent plant regeneration are accompanied by epigenetic changes on the packaging of DNA involving formation of an open-chromatin state facilitating gene expression [26].\u0026nbsp;Hence, GRF-GIF complexes are thought to confer meristematic potential to proliferative and formative cells during organogenesis by inducing the open-chromatin state [27].\u003c/p\u003e\n\u003cp\u003eHere, we report that the ectopic expression of chimeric \u003cem\u003eGRF4-GIF1\u003c/em\u003e from \u003cem\u003eCitrus\u003c/em\u003e, \u003cem\u003eTriticum\u003c/em\u003e and \u003cem\u003eVitis\u003c/em\u003e have differential effects in boosting regeneration and genetic transformation of diploid strawberry \u003cem\u003eF. vesca\u003c/em\u003e Hawaii 4. We have also explored how the mutation of \u003cem\u003emiR396\u003c/em\u003e site in the \u003cem\u003eVitis GRF4\u003c/em\u003e affect the activity of the \u003cem\u003eVitis miRGRF4-GIF1\u003c/em\u003e chimera during the regeneration of \u003cem\u003eF. vesca\u003c/em\u003e. \u0026nbsp;Henceforth, in the paper we will call the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimera as \u003cem\u003eCcGRF-GIF\u003c/em\u003e, \u003cem\u003eVvGRF-GIF\u003c/em\u003e, \u003cem\u003eVv miRGRF-GIF\u003c/em\u003e and \u003cem\u003eTaGRF-GIF\u003c/em\u003e. Transcriptomic analyses reveal several factors related to development and maturation differentially expressed by virtue of the transformation. We also report the increased potential of regeneration in \u003cem\u003eVvGRF-GIF\u003c/em\u003e lines following re-transformation in comparison to wild-type plants.\u003c/p\u003e"},{"header":"METHODS","content":"\u003ch2\u003eSeed germination and meristem propagation\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eF. vesca\u003c/em\u003e Hawaii 4\u0026nbsp;seeds\u0026nbsp;were\u0026nbsp;harvested from the matured fruits and dried on filter paper at 37\u0026ordm;C. Dried seeds\u0026nbsp;were\u0026nbsp;labelled and packed in envelops and stored at 4\u0026ordm;C. Seeds\u0026nbsp;were\u0026nbsp;scarified with 70% ethanol followed by 1M\u0026nbsp;sulphuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e)\u0026nbsp;solution before they\u0026nbsp;were\u0026nbsp;thoroughly washed with water. To initiate germination, scarified seeds\u0026nbsp;were\u0026nbsp;plated on water agar and kept at 22\u0026ordm;C. The germinated seedlings\u0026nbsp;were\u0026nbsp;propagated on nutrient rich soil in a\u0026nbsp;glasshouse at 22-24\u0026ordm;C under long day (16 hours\u0026nbsp;days \u0026ndash; 8 hours\u0026nbsp;night)\u0026nbsp;conditions to initiate runners. Fresh runners\u0026nbsp;were\u0026nbsp;harvested in water and\u0026nbsp;transferred\u0026nbsp;to the lab where, using a\u0026nbsp;Leica stereomicroscope M165,\u0026nbsp;the\u0026nbsp;meristems were\u0026nbsp;harvested using\u0026nbsp;a\u0026nbsp;scalpel.\u0026nbsp;This tissue was immediately transferred to tubes containing strawberry propagation medium (SPM), taking special care to prevent desiccation. SPM is a\u0026nbsp;MS-based medium (2.2 g/L) supplemented with 0.1 mg/L of BAP and 0.1 mg/L indole-3-butyric acid (IBA) and solidified with Daishin agar (Duchefa D1004, 9 g/L); the pH was adjusted to 5.8 before autoclaving.\u0026nbsp;The tubes\u0026nbsp;were\u0026nbsp;maintained in a\u0026nbsp;growth room at 20\u0026deg;C\u0026nbsp;under long day conditions until they regenerated into plantlets. Following shoot maturation, the\u0026nbsp;plantlets were\u0026nbsp;transferred to honey jars (HS French Flint Ltd, London, UK) containing\u0026nbsp;SPM,\u0026nbsp;where they\u0026nbsp;were\u0026nbsp;maintained for regular work.\u003c/p\u003e\n\u003ch2\u003ePlant material and \u003cem\u003ein vitro\u003c/em\u003e micropropagation\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e shoot cultures of \u003cem\u003eF. vesca\u003c/em\u003e Hawaii 4 were sub-cultured at 4\u0026ndash;6-week intervals, 5 per honey jar containing 50 ml medium. Strawberry multiplication medium (SMM) and SPM were alternated in each round of subculturing. Both basal culture media were composed of Murashige and Skoog (MS) macro and micro elements and vitamins, supplemented with sucrose (30 g/L) and 0.5mg/L of 6-benzylaminopurine (BAP), solidified with Daishin agar (Duchefa D1004, 9 g/L) and the pH was adjusted to 5.8 before autoclaving.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConstruct assembly and transformation into A\u003cem\u003egrobacterium tumefaciens\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe binary plasmid vector constructs \u003cem\u003epL2B-pNOS-Kan-tNOS-p35S-mCherry-t35S-54122\u003c/em\u003e and \u003cem\u003epL2B-pNOS-Hyg-tmas-p35S-mCherry-t35S-5433\u003c/em\u003e were assembled using Golden Gate cloning. The domesticated Level 0 constructs were synthesized by Thermo GeneArt and assembled into the Level 1 backbone using the \u003cem\u003eBsa1\u003c/em\u003e restriction enzyme. The different Level 1 constructs were assembled into the respective binary vector backbones using \u003cem\u003eBbs1\u003c/em\u003e restriction enzyme. The \u003cem\u003eGRF4-GRF1\u003c/em\u003e chimera constructs were obtained from Addgene in \u003cem\u003epDONR-zeo\u003c/em\u003e backbone [21]. The individual \u003cem\u003eGRF4-GIF1\u003c/em\u003e entry vectors: \u003cem\u003eTaGRF4-GIF1\u003c/em\u003e, \u003cem\u003eVvGRF4-GIF1\u003c/em\u003e, \u003cem\u003eVvmiRGRF4-GIF1\u003c/em\u003e and \u003cem\u003eCcGRF4-GIF1\u003c/em\u003e were recombined using LR clonase Gateway cloning kit (Invitrogen) into the \u003cem\u003epK7WG2D\u003c/em\u003e binary vector obtained from VIB Ghent [28].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectrocompetent\u003cem\u003e\u0026nbsp;Agrobacterium tumefaciens\u003c/em\u003e strain EHA105 were mixed with 500 ng of binary vector constructs. The mixture was pipetted into an electroporation cuvette and loaded into the electroporator and pulsed for 2.5 sec at resistance (200 ohm), capacitance (25 \u0026micro;FD) with pre-set voltage (Gene Pulser, Biorad). 500 \u0026micro;l of LB media (L1704, Duchefa) was added to the mixture of cells and plasmid after the shock and then transferred to a microfuge tube. Tubes were incubated in a shaker for 3h at 200 rpm and 28\u003csup\u003eo\u003c/sup\u003eC. Cells were spread to LB + appropriate antibiotics and grown for 2 days at 28\u003csup\u003eo\u003c/sup\u003eC. Colonies were verified by PCR (Supplementary Table S1).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eTransformation and regeneration of transgenic plants\u003c/h2\u003e\n\u003ch3\u003ePreparation of plant material for transformation:\u003c/h3\u003e\n\u003cp\u003ePetioles were harvested the day before the transformation from the youngest (most apical) leaves. \u0026nbsp;The plant cultures used were four-six weeks old after the last subculture.\u003c/p\u003e\n\u003ch3\u003eTransformation and regeneration:\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105 with the binary vector were grown overnight (200 rpm, 28\u003csup\u003eo\u003c/sup\u003eC) in LB media with appropriate antibiotics. \u0026nbsp;The culture was pelleted at 2,000 x g for 10 minutes and re-suspended in filter-sterilised liquid MS-based medium supplemented with glucose (30 g/L) and acetosyringone (100 \u0026micro;M), pH 5.2, to give OD 600 nm = 0.2 \u0026ndash; 0.3. Petioles were cut into 4-5 mm pieces, submerged in the inoculum, and blotted on sterile filter paper to remove excess inoculum. The petiole pieces were then transferred to Strawberry Regeneration Medium (SRM) petri dishes (MS-based medium supplemented with 0.2 mg/L of \u0026alpha;-naphthaleneacetic acid, 1 mg/L of thidiazuron (TDZ), 5 g/L of Agargel and 30 g/L of glucose and adjusted to pH 5.8). Petioles were co-cultivated in the dark for four days at 20\u0026deg;C. After the incubation, explants were washed in a solution of filter-sterilised ticarcillin disodium/clavulanate potassium (TCA, Duchefa) (400 mg/L) in water for 4 hours (60 rpm, 20\u003csup\u003eo\u003c/sup\u003eC), then blotted and transfered to T25 Cell Culture Flasks (Nunc) containing 15 ml of liquid SRM with antibiotic selection. Flasks were placed in a shaker at 60 rpm, 20\u003csup\u003eo\u003c/sup\u003eC, under low light intensity for 4 weeks, and then blotted and transferred to SRM selection petri dishes. Petioles were sub-cultured every 4 weeks until regeneration. Control (WT) shoots were regenerated using the same method, except that the explants were not co-cultivated with \u003cem\u003eA. tumefaciens,\u003c/em\u003e and selection antibiotics were omitted from the culture media. The transformed shoots were transferred to 30 ml universal tubes (Fisher Scientific) containing 15 ml of rooting medium (Frag R) with selection. Frag R is MS-based medium (2.2 g/L) supplemented with 0.1 mg/L of BAP and 0.1 mg/L IBA and 20 g/L of glucose, solidified with 9 g/L of Daishin agar (Duchefa) and adjusted to pH 5.8. After 4 weeks, shoots were moved to tubes containing SMT medium (MS-based medium supplemented with 0.225 mg/L of BAP, 0.2 mg/L IBA, 0.1 mg/L gibberellic acid (GA3) and 30 g/L of glucose, solidified with 7.5 g/L of Sigma-Aldrich agar and adjusted to pH 5.6 before autoclaving). In the next subculturing step, plants were changed to SMM tubes.\u003c/p\u003e\n\u003ch3\u003eMature transgenic plant propagation:\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eAfter 4 weeks in SMM tubes, plants were mature enough to be moved to honey jars (5 plants per jar). Honey jars with SMM medium or Strawberry Medium for Rooting (SMR) were alternated at 4\u0026ndash;6-week intervals. SMR medium is a MS-based medium supplemented with 0.4 mg/L of IBA, 0.1 mg/L gibberellic acid (GA3) and 30 g/L of glucose, solidified with 7.5 g/L of Sigma-Aldrich agar A1296 and adjusted to pH 5.6 before autoclaving.\u003c/p\u003e\n\u003ch2\u003eGenotyping of transgenic lines\u003c/h2\u003e\n\u003cp\u003eDNA were extracted from 50-100 mg of leaf tissue using an in-house protocol described subsequently. The frozen leaf tissue was ground with metal balls (IG100_5/32_PK1000; Simply Bearings Ltd., Leigh, UK) using a mechanical pulveriser (MiniG from Spex) at 1200 rpm for 30 seconds. 500\u0026micro;l of the extraction buffer (1.25% sodium dodecyl sulphate (SDS); 100 mM Tris HCl pH 8.0; 50 mM EDTA pH 8.0 and 25 mg PVP) were added to the disrupted tissue. The samples were mixed and incubated at 65\u0026deg;C for 30 min, inverting the tubes each 5 min. Samples were cooled placing them in ice for around 5 min and then 250 \u0026micro;l of chilled 5M NaCl, mix and incubate in ice for 15 more min. The samples were centrifuged for 10 min at 20,000 g. Supernatant was transferred into a new tube containing 360 \u0026micro;l of isopropanol. Samples were vortexed and incubated for 30 minutes or overnight at -20\u0026deg;C to allow DNA to precipitate. The samples were centrifuged for 20 min at 15,700 g. Supernatant was discarded and pellet was washed in 500 \u0026micro;l of 70% ethanol. The samples were centrifuged for 20 min at 15,700 g and supernatant was discarded. Washing step was repeated once more and supernatant was discarded. Each pellet was resuspended in 50 \u0026micro;l TE buffer (10 mM Tris HCl pH 8.0; 1 mM EDTA pH 8.0). PCR amplification was performed using gene specific primers and PCR-BIO Taq Mix Red (PCR Biosystems) following the manufacturer\u0026rsquo;s guidelines.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eRNA extraction\u003c/h2\u003e\n\u003cp\u003eRNA was extracted from 100-200 mg of leave tissue using an in-house protocol [29]. RNA integrity was assessed using the Agilent TapeStation system using RNA screen tape. Library preparation and paired-end RNA sequencing was performed by Novogene (Cambridge, UK) on an Illumina NovaSeq 6000 platform. Sequencing data were deposited at the NCBI under the Bioproject ID PRJNA986313.\u003c/p\u003e\n\u003ch2\u003eRNA sequencing analysis\u003c/h2\u003e\n\u003cp\u003eRaw reads were quality controlled using FastQC v0.11.9 [30], and adapters and low-quality regions were trimmed using Trimmomatic v0.39 using a sliding window of 4 and minimum PHRED score of 20 [31]. The first 10 nucleotides were trimmed and reads less than 100 nucleotides and unpaired reads were discarded. \u003cem\u003eGRF-GIF\u003c/em\u003e transgene sequences (Supplementary File S1) were concatenated with the \u003cem\u003eF. vesca\u003c/em\u003e v4.0.a1 genome. Assemblies were indexed and reads were aligned using HISAT2 v2.2.1 using the default settings for paired end reads [32]. Annotations for the GRF-GIF transgenes were generated using StringTie 2.1.7 and these were merged with the \u003cem\u003eF. vesca\u003c/em\u003e v4.0.a2 gene annotations [33]. Quantification was performed using featureCounts v2.0.1 [34] and differential expression analysis was performed with the R package DESeq2 v3.17 [35]. Comparisons were made between the empty vector control (\u003cem\u003epK7WG2D\u003c/em\u003e) and each GRF-GIF construct. The Benjamin and Hochberg approach for control of the false discovery rate was used and an adjusted p-value below 0.05 was used to identify differentially expressed genes (DEGs). For visual inspection of samples distances, variance stabilizing transformation (VST) was used to normalise the raw read counts and a principal component analysis (PCA) was performed using R. KEGG and InterProScan functional annotations from the Genome Database for Rosaceae (GDR) [36] were used to annotate DEGs. A Venn diagram of shared DEGs was plotted using the R package ggvenn v0.1.10 (https://cran.r-project.org/web/packages/ggvenn) and heatmaps of DEG log2 (fold change) (log2FC) were produced using the python library seaborn v0.12.0 [37]. Heatmap clustering was performed using hierarchical clustering based on Euclidean distance. To visualise the expression of individual DEGs, raw read counts were TPM normalised using bioinfokit v2.1.0 and plotted using seaborn v0.12.0 [37].\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eImaging\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003ePictures of the plates were taken with a Canon DSLR camera EOS4000D. Visualization of mCherry fluorescence and eGFP-ER in plant tissue and pictures of the calli, shoots and plants were performed using a Leica Stereomicroscope M165. The leaves were scanned using an EPSON flatbed scanner. The images were assembled using Inkscape and Adobe Photoshop.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eHistograms and statistical analyses were performed with R (2023.03.0 Build 386 \u0026copy; 2009-2023 Posit Software, PBC). Statistical differences were tested by performing a non-parametric Kruskal-Wallis test and the differences among samples were determined using pairwise comparisons with Wilcoxon rank sum test with continuity correction.\u003c/p\u003e\n\u003ch2\u003eSequence alignments and phylogenetic tree\u003c/h2\u003e\n\u003cp\u003eGene and protein sequences were obtained using NCBI (https://www.ncbi.nlm.nih.gov/) and OrthoDB (https://www.orthodb.org/?ncbi=18049678). Accession numbers and protein names used for the phylogenetic tree are available in the phylogenetic tree in Supplementary Figure S1 and Supplementary Table S2. Protein FASTA sequences were aligned using MUSCLE method with MEGA11 (11.03.13 Built 11220624 \u0026copy; 2013-2023). The phylogenetic tree was built in MEGA11 using the Maximum Likelihood method and JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates. All positions with less than 95% site coverage were eliminated.\u003c/p\u003e"},{"header":"RESULTS ","content":"\u003ch2\u003eEstablishing \u003cem\u003eF. vesca\u003c/em\u003e stock plants and a regeneration protocol.\u003c/h2\u003e\n\u003cp\u003eTo establish a uniform population of \u003cem\u003eF. vesca\u003c/em\u003e Hawaii 4 plants, surface sterilized and scarified seeds were germinated on water agar plates. Following their germination, the plantlets were propagated on soil mix in glasshouses. After 4-5 weeks growth in the glasshouse, the plants started to produce runners (Fig. 1a). The runners allow vegetative propagation of strawberry by producing a \u0026lsquo;clone-plant\u0026rsquo; with each runner tip containing an apical meristem that can develop into a new plant (Fig. 1b). Apical meristem tissue was collected from the growing runner and propagated in Shoot Propagation Media (SPM) in tubes (Fig. 1c). Meristem culture is the most prevalent mode of vegetative propagation for strawberry as it allows selection of disease-free plants [38]. \u0026nbsp;Subsequently, the plants growing from the meristem were moved into jars for shoot multiplication (Fig. 1d). 4-6 weeks post propagation into SPM, the plants produced enough petioles for the establishment of the regeneration experiment. Young petioles were harvested from the jars and sliced into 4-5 mm pieces under sterile condition to initiate regeneration (Fig. 1e). The petiole pieces were transferred to liquid shoot regeneration media (SRM) in flasks and maintained at 22\u0026deg;C with regular shaking under long day conditions (Fig. 1f). 2 weeks into the SRM, the petioles started to form callus at both the cut ends when they were transferred to SRM plates (Fig. 1g). Regeneration efficiency of plants from the callus were assessed at 4-, 8- and 12-weeks following incubation in SRM for 50 petiole edges (Fig. 1h). After 4 weeks, all petioles had calli on both edges, that started to produce shoots by 8 and 12 weeks with an efficiency of 71% and 86%, respectively. Thus, we could produce a running stock of Hawaii 4 plants and establish an efficient platform for regeneration that could be further exploited to produce transgenic plants.\u003c/p\u003e\n\u003ch2\u003eSelection of stable transgenics using antibiotic and fluorescent cassettes\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo successfully raise transgenic plants, it is important to select the positive lines from the wild type revertant. Antibiotics such as kanamycin and hygromycin previously allowed efficient selection of transgenic strawberry plants [39]. To compare the transformation efficiency of different antibiotic selection cassettes, 50 petiole pieces from 4-week-old Hawaii 4 crowns grown on SPM medium were infected with \u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105 containing plasmids carrying hygromycin or kanamycin selection cassettes (Fig. 2a). Negative control and non-transformed petioles did not survive the treatment with either antibiotic, turning brown by 4 weeks indicating senescence (Fig. 2b). Transformation efficiency was assessed at 4-, 8- and 12-weeks post transformation (WPT) and was estimated as frequency of petiole edges with regenerating shoots (Fig 2b-c). At all 3 time points, transformation efficiency of the hygromycin and kanamycin selection cassettes were found to be comparable (Fig. 2c). As a transformation marker, the plasmids were carrying mCherry fluorescent protein driven downstream to promoter 35S. Regenerating fluorescent shoots on the selection media were subsequently transferred to shooting and rooting media over the course of 12 weeks to facilitate development of stable plantlets with root systems (Fig. 2d). The uniform mCherry expression in all plant tissues throughout development indicated the lack of chimeric transformants. Moreover, the healthy physiology of the plants suggested the absence of any pleiotropic effects from the antibiotic selection cassettes (Fig 2d). Thus, the antibiotic and visual fluorescent markers cassette provides a dual selection for positive transformants all through the regeneration process.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras from different species increase regeneration efficiency\u003c/h2\u003e\n\u003cp\u003eIntroduction of developmental genes has resulted in faster regeneration of callus for several plant species including certain recalcitrant plants [13\u0026ndash;19]. To improve strawberry transformation efficiency, \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras from different species were tested for their effect on strawberry transformation. Petiole pieces from 4-week-old Hawaii 4 crowns were infected with \u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105 carrying \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras from \u003cem\u003eVitis vinifera\u003c/em\u003e (constitutive, \u003cem\u003eVvGRF-GIF\u003c/em\u003e and \u003cem\u003emiR396\u003c/em\u003e-resistant version, \u003cem\u003eVv miR GRF-GIF\u003c/em\u003e), \u003cem\u003eCitrus clementina\u0026nbsp;\u003c/em\u003e(\u003cem\u003eCcGRF-GIF\u003c/em\u003e) and \u003cem\u003eTriticum aestivum\u0026nbsp;\u003c/em\u003e(\u003cem\u003eTaGRF-GIF\u003c/em\u003e) (Supp Fig. S1-S2). Transformation efficiencies were assessed at 4-, 8- and 12- WPT and were estimated as frequency of petiole edges with shoots (Fig 3b-c). At 4 WPT, regeneration efficiency of petioles transformed with \u003cem\u003eVvGRF-GIF\u003c/em\u003e, \u003cem\u003eCcGRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGRF-GIF\u003c/em\u003e chimeras were comparable to the empty vector transformed petioles (Fig 3c). \u0026nbsp;At the same time point, \u003cem\u003eVv miR GRF-GIF\u003c/em\u003e showed regeneration efficiency comparable to 8 WPT for the empty vector control indicating a 4-week faster regeneration of shoot from callus which is evident in having much mature plantlets by 12 weeks (Fig. 3c). This is concomitant to the previous report where the \u003cem\u003emiRNA\u003c/em\u003e resistant variety of GRF resulted in an increase in cell number and leaf size in \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003e[40]. The \u003cem\u003eVv miR GRF-GIF\u003c/em\u003e construct contained four synonymous mutations to prevent the binding of \u003cem\u003emiRNA396\u003c/em\u003e, which regulates \u003cem\u003eGRF4\u003c/em\u003e expression (Supp Fig. S3). \u003cem\u003emiRNA396\u003c/em\u003e is known to target \u003cem\u003eGRF1-4\u003c/em\u003e family transcripts, controlling the activity of \u003cem\u003eAtGRF3\u003c/em\u003e during leaf development [40,41]. At week 8- and 12- WPT, all \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras showed significantly higher shoot regeneration compared to the empty vector controls (Fig. 3b-c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile the introduction of the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras resulted in efficient regeneration, their constitutive expression also induced several pleiotropic effects on the plants [42]. To study the effect of \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras on regenerating plant physiology, shoots were taken at 12 weeks and grown until rooted plantlets were established (Fig. 3d). As all the constructs were carrying eGFP transformation reporter, its expression was monitored throughout the experiment, from callus to regenerating plantlets, to ensure no chimeric plants were selected (Fig. 3d). Severe pleiotropic effects were observed for \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eplants where, despite being more vigorous in regeneration, the plants failed to show the canonical leaf expansion and elongation that are hallmarks for proper development in strawberry (Fig. 3d-e). Similar observations were made for rice where the \u003cem\u003emiRNA396\u003c/em\u003e resistant variety of \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eresulted in formation of large calli without proper regeneration [21]. As compared to the \u003cem\u003emiRNA\u003c/em\u003e resistant version, \u003cem\u003eVvGRF-GIF\u003c/em\u003e and \u003cem\u003eCcGRF-GIF\u003c/em\u003e showed proper regeneration with healthy adult plants established under lab condition (Fig 3d). One of the reasons behind the better health of the \u003cem\u003eVvGRF-GIF\u003c/em\u003e and \u003cem\u003eCcGRF-GIF\u003c/em\u003e could be the presence of the \u003cem\u003emiRNA396\u003c/em\u003e target site where \u003cem\u003eF. vesca miRNA396\u003c/em\u003e could bind to regulate the expression of the \u003cem\u003eGRF4\u003c/em\u003e gene expression (Supp Fig. S3). \u003cem\u003eTaGRF-GIF\u003c/em\u003e plants also exhibited aberrant leaf development in the regenerated plants. 3 out of 5 lines showed leaves with more lobes that were more serrated with sharp edges compared to empty vector transformed plants (Fig. 3e). Multiple protein alignment showed that \u003cem\u003eTaGRF4\u003c/em\u003e shares much less homology compared to the dicot GRFs (40% vs ~85%), but like its dicot counterparts still retains the \u003cem\u003emiRNA396\u003c/em\u003e binding site (Supp Fig. S4-S5). The presence of the \u003cem\u003emiRNA\u003c/em\u003e target site suggests canonical transcriptional regulation by \u003cem\u003emiRNA396\u003c/em\u003e, but divergent protein structure of\u003cem\u003e\u0026nbsp;TaGRF4\u003c/em\u003e might be activating phytohormone responses resulting in the aberrant leaf morphology. In a previous observation, \u003cem\u003eOsbZIP48\u003c/em\u003e from rice could complement an \u003cem\u003eArabidopsis Athy5\u003c/em\u003e mutant but caused pleiotropic effect like semi-dwarfism [43]. Thus, our observation indicates that cross species activation of \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimera can induce pleiotropic effects due to possibly mis-regulation at the transcriptional or translational level.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eTranscriptomic analysis of the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras shows differential gene activation\u003c/h2\u003e\n\u003cp\u003eAll the chimeric \u003cem\u003eGRF4-GIF1\u003c/em\u003e produced a positive effect on regeneration efficiency irrespective of their source. But the pleiotropic effects in \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGRF-GIF\u003c/em\u003e in strawberry indicates the presence of complex transcriptional landscape under different chimeric conditions. \u0026nbsp;To investigate the issue, transcriptomic analysis was performed for each condition with leaf extracted RNA. Significantly high expression of the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras were observed for each of the transgenic lines assayed (Fig. 5a). Concomitant to the aberrant phenotypes, both \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGRF-GIF\u003c/em\u003e lines shows 178 and 116 DEGs that were not represented in the other data sets (Fig. 5b-c). A particular group of DEGs showed very high expression in both \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGRF-GIF\u0026nbsp;\u003c/em\u003eas compared to the \u003cem\u003eCcGRF-GIF\u003c/em\u003e and \u003cem\u003eVvGRF-GIF\u0026nbsp;\u003c/em\u003eidentified as Unique cluster A. \u003cem\u003eFvH4_3g44360,\u003c/em\u003e encoding a peroxidase from this cluster showed ~3.5-fold higher expression in \u003cem\u003eVv miR GRF-GIF1\u0026nbsp;\u003c/em\u003ecompared to control plants (Fig. 5c-d; Supp Table S3). A previous report in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e showed that overexpression of peroxidase leads to developmental abnormalities with retarded root development [44]. Another gene that is significantly upregulated in \u003cem\u003eVvGRFmiR-GIF\u0026nbsp;\u003c/em\u003eis \u003cem\u003eFvH4_4g10610\u003c/em\u003e encoding a EP3-like endochitinase (Fig. 5c-d; Supp Table S3). Endochitinase is an extracellular protein secreted by the non-embryogenic cells in the medium inducing somatic embryogenesis [45,46]. It could be possible that the higher expression of EP3-like endochitinase induced more somatic embryos in \u003cem\u003eVv miR GRF-GIF1\u003c/em\u003e lines, but the sustained expression of the gene resulted in lack of regeneration. Interestingly, this gene is also upregulated in \u003cem\u003eTaGRF-GIF\u003c/em\u003e lines indicating that the possible phenotypic effect of EP3-like endochitinase in plant development ranges from somatic embryogenesis to proper plant development (Supp Table S3). \u003cem\u003eTaGRF-GIF\u0026nbsp;\u003c/em\u003elines showed an exclusive upregulation of \u003cem\u003eFvH4_7g27130\u0026nbsp;\u003c/em\u003eencoding an expansin gene from the Unique cluster B where a cluster of DEGs show significantly higher expression in \u003cem\u003eTaGRF-GIF\u003c/em\u003e as compared to the others (Fig. 5c-d). A recent report in Poplar showed that overexpression of \u003cem\u003eGRF5\u003c/em\u003e resulted in increased leaf size, and transcriptomic analysis assisted with DAP-seq showed significant representation of cell cycle and expansin gene families [47]. This paper also reported that poplar \u003cem\u003ePpnGRF5\u003c/em\u003e binds to promoter of Cytokinin oxidase/dehydrogenase (\u003cem\u003epPpnCKX\u003c/em\u003e) and negatively regulate its expression resulting in elevated cytokinin levels in the cells. Conversely, our transcriptomic data indicated ~3-fold increase of \u003cem\u003eFvH4_2g39230\u003c/em\u003e encoding Cytokinin oxidase/dehydrogenase (Supp Table S3). While an increase in the expression of CKX should ideally result in decrease of the cytokinin level, the transcriptome of \u003cem\u003eTaGRF-GIF\u003c/em\u003e shows ~2-fold increase in \u003cem\u003eFvH4_5g16240\u003c/em\u003e expression encoding a type-A two-component response regulator (RRs) (Supp Table S3). Type-A response regulators act downstream to cytokinin signalling where upon activation, negatively regulate the pathway [48]. It could be possible that the deformed leaflet formation observed in \u003cem\u003eTaGRF-GIF\u003c/em\u003e lines is due to abnormal cell division caused by deregulated levels of cytokinin. Differential activation of the cytokinin pathway is further evident by the fact that different sets of two-component RRs are activated in \u003cem\u003eCcGRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eVv miR GRF-GIF\u003c/em\u003e lines (Supp Table S3). Thus, our data indicates that \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimera could differentially activates the cytokinin and expansin genes to control the developmental processes in strawberry.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eVvGRF-GIF\u0026nbsp;\u003c/em\u003eplants show better regeneration efficiency following re-transformation\u003c/h2\u003e\n\u003cp\u003eMorphogenic regulators not only facilitate recalcitrant plants to undergo somatic embryogenesis but also allow better regeneration efficiency for plants that are already known to undergo somatic embryogenesis [20,21,42]. By virtue of their faster regeneration efficiency, we investigated whether \u003cem\u003eGRF4-GIF1\u003c/em\u003e stable lines in strawberry perform better during retransformation when compared to the empty-vector transformed lines and the wild-type plants. Due to the pleiotropic effects in \u003cem\u003eVv miR GRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTaGRF-GIF\u0026nbsp;\u003c/em\u003elines, only \u003cem\u003eVvGRF-GIF\u003c/em\u003e and \u003cem\u003eCcGRF-GIF\u003c/em\u003e were considered for re-transformation with a vector carrying hygromycin resistance gene and mCherry fluorescent marker (Fig. 5a). As the transformed plants already had the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeric cassette with kanamycin selection and eGFP marker, the vector with hygromycin selection cassette was chosen that was previously used in Fig. 2. 50 pieces of petioles from each line were infected and transformation efficiency were assessed at 4-, 8- and 12- WPT as before (Fig. 5b-c). At week 4, transformation efficiency was 0% in all the samples. By week 8- and 12-, the average transformation efficiency of two of the \u003cem\u003eVvGRF-GIF\u003c/em\u003e lines were ~40% higher compared to the empty vector transformed petioles and wild-type plants (Fig. 5c). It is important to note that petiole regeneration following re-transformation is usually slower than single transformation possibly due to presence of multiple antibiotic selection. The shoots of these two \u003cem\u003eVvGRF-GIF\u003c/em\u003e lines looked bigger and healthier by 12 weeks compared to the empty vector transformed lines (Fig. 5b). At week 8- and 12-, no significant difference in regeneration efficiency was noted for \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines (Fig. 5b-c). The calli and the regenerating plants were checked for fluorescence where the eGFP fluorescence indicated the consistent expression of the \u003cem\u003eGRF4-GIF1\u003c/em\u003e cassette and the mCherry indicated double transformation events (Fig. 5d). The double fluorescent calli were transferred to selection media for the propagation of transformed plants. Although, both \u003cem\u003eVvGRF-GIF\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines looked healthy at their rooted plantlet stages (Fig. 3), the differences in their regeneration efficiency after re-transformation is difficult to explain. One interesting difference is the upregulation of 3 cytokinin responsive RR genes \u003cem\u003eFvH4_2g27180\u003c/em\u003e, \u003cem\u003eFvH4_5g16240\u003c/em\u003e and \u003cem\u003eFvH4_6g25290\u003c/em\u003e in \u003cem\u003eCcGRF-GIF\u003c/em\u003e as compared to \u003cem\u003eVvGRF-GIF\u0026nbsp;\u003c/em\u003elines (Supp Table S3). Along with this difference, there are several other genes that were differentially regulated in the \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines which could contribute to their lack of regeneration phenotype (Fig. 4c). Thus, ectopic expression of \u003cem\u003eVvGRF-GIF\u003c/em\u003e chimera could be a useful tool for expediting strawberry transformation without incurring unwanted pleiotropic effect.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eStrawberry is a commercially important crop where several desirable agronomic traits define its value in the market. But fundamentally, the presence of physiological or genetically linked trade-offs limits the possibility for certain combinations of phenotypes to occur [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]​​. As often these viable traits are diametrically opposed, genetic engineering over normal breeding provides an opportunity to overcome the genetically linked traits ​[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In this study, we present an efficient strategy to expedite transformation in strawberry with the introduction of \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Using different types of \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras coming from multiple plant species allowed us to explore the best possible chimera as complicated regulation of GRFs resulted in pleiotropic effects for \u003cem\u003eVv miR GRF-GIF1\u003c/em\u003e and \u003cem\u003eTaGRF-GIF\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Transcriptomic analysis of the different lines provided necessary insight into the possible causes of the pleiotropic effects and the complex regulon for \u003cem\u003eGRF4-GIF1s\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We also found \u003cem\u003eVvGRF-GIF\u003c/em\u003e lines has much better efficiency after re-transformation as compared to the empty vector or wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiploid strawberry is an attractive system to study functional genomics in Rosaceae due to its small genome size, short life cycle and facile vegetative and seed propagation ​[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. But the biggest bottle neck for doing any forward or reverse genetics is an efficient protocol to raise stable transgenics. In the last couple of decades there has been a considerable effort to establish various protocols for raising successful strawberry transgenics with various level of efficiencies ranging from 63\u0026ndash;68% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]​. Here, we have presented a comprehensive protocol for preparing a uniform line of clean stock plants using meristem culture which can be used for raising stable transgenics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Strawberry is generally propagated using stolon which runs the risk of getting infected material into tissue culture spreading through vascular tissues [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]​. Meristem culture on the other hand allows an alternative strategy to obtain large quantities of virus free material due to active cell division and lack of differentiation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]​​. Moreover, tissue culture of strawberry leads to somaclonal variation which can be avoided by meristeming and thus allowing true-to-type plants [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]​. The stability of the background is revealed by ~\u0026thinsp;90% regeneration efficiency with very little variability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eGrowth regulating factors (GRFs) are a small family of transcription factors that play important role in plant development by controlling various aspects of leaf developmental [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]​​, stem development, apical meristem development [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]​ and root development [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]​​. GRFs form complexes with GRF interacting proteins (GIFs) which act as transcriptional coactivators [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]​. From an evolutionary perspective, all land plants encode for GRF proteins except green algae, whereas GIFs are universally present ​[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]​. Several studies have shown that GRFs are active at the sites of active growth and differentiation, with expression gradually decreasing in the matured tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]​. In \u003cem\u003eA. thaliana\u003c/em\u003e, this expression regulation is primarily carried out by the \u003cem\u003emiR396a\u003c/em\u003e and \u003cem\u003emiR396b\u003c/em\u003e which shows near perfect sequence alignment with the transcripts of several \u003cem\u003eGRFs\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]​. \u003cem\u003eF. vesca\u003c/em\u003e encodes for \u003cem\u003emiR396\u003c/em\u003e gene which also shows sequence conservation with \u003cem\u003eAtmiR396\u003c/em\u003e indicating that \u003cem\u003emiRNA\u003c/em\u003e mediated control of GRFs is highly conserved across the plant kingdom (Supp Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This is in consonance to a previous report where ectopic expression of \u003cem\u003eAtmiR396\u003c/em\u003e resulted in reduction in gene expression of \u003cem\u003eGRFs\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e ​[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u003cem\u003eVitis\u003c/em\u003e belonging to the order Vitales is phylogenetically closest to the \u003cem\u003eFragaria\u003c/em\u003e sharing maximum homology to \u003cem\u003eFvGRF4\u003c/em\u003e followed by \u003cem\u003eCitrus\u003c/em\u003e from Sapindales and \u003cem\u003eTriticum\u003c/em\u003e from Poales (Supp Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras from both eudicot and monocot species allowed us to dissect the functional diversity within the GRF4 family in a heterologous system. \u003cem\u003emiR396\u003c/em\u003e expression and \u003cem\u003eGRF4\u003c/em\u003e expression work reciprocally, whereby \u003cem\u003eGRF4\u003c/em\u003e expression decreases in mature leaves with an increase in \u003cem\u003emiR396\u003c/em\u003e expression [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The importance of the transcriptional control of \u003cem\u003eGRF4\u003c/em\u003e by \u003cem\u003emiR396\u003c/em\u003e is revealed in the pleiotropic phenotype of the \u003cem\u003eVv miR GRF4-GIF1\u003c/em\u003e lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While facilitating more regeneration events, the sustained expression of the \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimera in the \u003cem\u003eVv miR GRF4-GIF1\u003c/em\u003e lines prevented most of these plants to reach proper tissue differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Transcriptome analysis showed that there were many genes that were differentially up-regulated in the \u003cem\u003eVv miR GRF4-GIF1\u003c/em\u003e lines as compared to the \u003cem\u003eVvGRF4-GIF1\u003c/em\u003e and \u003cem\u003eCcGRF4-GIF1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supp Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Upregulation of certain vital genes required for the transition from cell division to differentiation like endochitinase and peroxidases might have played a significant role (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supp Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This agrees with a previous observation in \u003cem\u003eCitrus\u003c/em\u003e where inactivation of peroxidase activity was shown to be important for \u003cem\u003ein vitro\u003c/em\u003e plant differentiation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]​.\u003c/p\u003e \u003cp\u003eThe expansion of the GRF family transcription factors happened due to large scale genome duplication [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]​. In case of the eudicots, a whole genome triplication event in the ancestor led to the formation of several \u003cem\u003eGRF\u003c/em\u003e genes. Like eudicots, a similar duplication event led to formation of the monocot \u003cem\u003eGRFs\u003c/em\u003e​. The \u003cem\u003eTaGRF4-GIF1\u003c/em\u003e showed only 40% protein sequence homology to the eudicot GRF4-GIF1s compared to ~\u0026thinsp;85% within the eudicots (Supp Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e-5). As genome duplication events are directly related to neofunctionalization [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], it could be possible that during evolution, GRFs gained functions in monocots that are different from eudicots. This is evident from our observation of the leaf phenotype in strawberry lines where ectopic expression of \u003cem\u003eTaGRF4-GIF1\u003c/em\u003e caused leaf deformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Transcriptome analysis showed that in these leaf tissue there was a significant increase in the expression of an expansin gene \u003cem\u003eFvH4_7g27130\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Previously in \u003cem\u003eN. benthamiana\u003c/em\u003e, local expression of expansin recapitulated leaf formation from a meristem and could also alter the shape of the leaf lamina [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]​. GRF transcriptional activity tightly controls the cytokinin concentration in plants, which in turn is responsible for cell division and expansion ​[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]​. \u003cem\u003eTaGRF-GIF\u003c/em\u003e lines showed higher expression of cytokinin responsive RRs in the abnormal leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supp Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). During the leaf expansion phase, an increase in cytokinin concentration can lead to abnormal leaf development ​​[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]​. Thus, the abnormality in the strawberry leaves in \u003cem\u003eTaGRF-GIF\u003c/em\u003e lines could be due to misexpression of cytokinin responsive and expansin genes.\u003c/p\u003e \u003cp\u003eThe benefits of the developmental genes during transformation first came into prominence for their ability to jump start somatic embryogenesis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Re-transformation of multiple genes in a desirable background, or \u0026lsquo;stacking\u0026rsquo; of genes, has always been challenging for multiple reasons [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Here, we show that the introduction of the \u003cem\u003eVvGRF-GIF\u003c/em\u003e in strawberry gives the plants certain advantages during re-transformation where the transformation efficiency increases by ~\u0026thinsp;40% as compared to the empty vector transformed plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, the expression of both visual fluorescent transformation markers ensured that both the cassettes were properly transformed. But surprisingly, the \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines did not show any significant improvement during the re-transformation experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Transcriptome analysis indicated significant differences between \u003cem\u003eVvGRF-GIF\u003c/em\u003e and \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines despite the lack of any phenotypic discrepancies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, Supp Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Cytokinin is intrinsically linked to regeneration of plants and GRF lines were shown to behave very differently during regeneration experiments depending upon its availability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]​. As several cytokinin responsive RRs genes are activated in the \u003cem\u003eCcGRF-GIF\u003c/em\u003e lines, it could be possible that disproportionate cytokinin levels affected its regeneration.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eA comprehensive protocol for strawberry transformation will turn out to be extremely beneficial for understanding genetics within the Rosaceae family, which includes several economically important horticultural crops. The re-transformation protocol that we present here can be utilized in the future to raise stable transgenics of mutant backgrounds where faster screening strategies, such as virus-induced gene silencing (VIGS) can be introduced to study viable traits. Overall, not only have we presented \u003cem\u003eVvGRF-GIF\u003c/em\u003e to be an effective \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimera for enhancing regeneration in a strawberry transformation system, but also highlighted the pitfalls of using the wrong chimeras. This study provides an overarching scope for bringing more such important horticultural Rosaceae crops under tissue culture following the strawberry footsteps.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by Bill and Melinda Gates Foundation as OPP1028264.\u003c/p\u003e\n\u003cp\u003eAuthor\u0026rsquo;s contribution\u003c/p\u003e\n\u003cp\u003eA.K. and R.H. conceived the idea. E.R.S and A.K. designed the experiments. K.M. provided her expertise to micro-propagate the strawberry cultivar in the tissue culture. E.R.S. and F.M carried out the experiments. E.R.S analysed the regeneration efficiency data and J.P. carried out the transcriptome analysis. A.K. supervised the project. A.K. wrote the manuscript with support from E.R.S and R.J.P.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Fiona Wilson for optimizing the strawberry transformation protocol. We thank Dr. Helen Bates for providing the RNA extraction protocol. We thank Dr. Emma Wallington and Crop Transformation team for their inputs. We would also like to acknowledge the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the \u0026ldquo;UK\u0026rsquo;s Crop Diversity Bioinformatics HPC\u0026rdquo; (BBSRC grant BB/S019669/1), used for analysis of results reported within this paper. We also want to acknowledge Bill and Melinda Gates Foundation for providing fellowship to A.K, E.R.S, F.M, R. J. P and supporting the project (ENSA).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSkoog F, Miller CO. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol. 1957;11:118\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eSugiyama M. Organogenesis in vitro. Curr Opin Plant Biol [Internet]. 1999;2:61\u0026ndash;4. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1369526699800120\u003c/li\u003e\n\u003cli\u003eZimmerman JL. Somatic Embryogenesis: A Model for Early Development in Higher Plants. Plant Cell. 1993. \u003c/li\u003e\n\u003cli\u003eMordhorst AP, Toonen MAJ, De Vries SC. Plant Embryogenesis. CRC Crit Rev Plant Sci. 1997;16:535\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eIsah T. Induction of somatic embryogenesis in woody plants. Acta Physiol Plant. 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Meristem culture for the elimination of the strawberry crown rot pathogen Phytophthora cactorum. J Berry Res. 2011;1:129\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eHanhineva KJ, K\u0026auml;renlampi SO. Production of transgenic strawberries by temporary immersion bioreactor system and verification by TAIL-PCR. BMC Biotechnol. 2007;7. \u003c/li\u003e\n\u003cli\u003eDebernardi JM, Mecchia MA, Vercruyssen L, Smaczniak C, Kaufmann K, Inze D, et al. Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity. Plant Journal. 2014;79:413\u0026ndash;26. \u003c/li\u003e\n\u003cli\u003eJones-Rhoades MW, Bartel DP. Computational Identification of Plant MicroRNAs and Their Targets, Including a Stress-Induced miRNA The primary method of identifying miRNA genes has been to isolate, reverse transcribe, clone, and sequence small cellular RNAs (Lagos-Quintana et al., 2001; Lau et [Internet]. Mol Cell. Lee and Ambros; 2004. 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Plant Biotechnol J. 2005. p. 141\u0026ndash;55. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Strawberry, regeneration, transformation, GRF4-GIF1 chimera, leaf development, cytokinin","lastPublishedDoi":"10.21203/rs.3.rs-4583627/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4583627/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePlant breeding played a very important role in transforming strawberries from being a niche crop with a small geographical footprint into an economically important crop grown across the planet. But even modern marker assisted breeding takes a considerable amount of time, over multiple plant generations, to produce a plant with desirable traits. As a quicker alternative, plants with desirable traits can be raised through tissue culture by doing precise genetic manipulations. Overexpression of morphogenic regulators previously known for meristem development provides an efficient strategy for easier regeneration and transformation in multiple crops. In this study, we show the results for overexpression of chimeric GRF4-GIF1 in diploid strawberry \u003cem\u003eFragaria vesca\u003c/em\u003e Hawaii 4 (strawberry) where \u003cem\u003eVitis GRF4-GIF1\u003c/em\u003e chimera provides significantly higher regeneration efficiency.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe present here a comprehensive protocol for strawberry regeneration and transformation under control condition as compared to ectopic expression of \u003cem\u003eGRF4-GIF1\u003c/em\u003e chimeras from different plants. We report that ectopic expression of \u003cem\u003eVitis vinifera VvGRF4-GIF1\u003c/em\u003e provide significantly higher regeneration efficiency during retransformation over wild-type plants. On the other hand, deregulated expression of \u003cem\u003emiRNA\u003c/em\u003e resistant version of \u003cem\u003eVitis GRF4-GIF1\u003c/em\u003e or \u003cem\u003eTaGRF4-GIF\u003c/em\u003e (wheat) resulted in abnormalities. Transcriptomic analysis between the different chimeric \u003cem\u003eGRF4-GIF1\u003c/em\u003e lines indicate that differential expression of \u003cem\u003eFvExpansin\u003c/em\u003e might be responsible for the pleiotropic effects. Similarly, cytokinin dehydrogenase/oxygenase and cytokinin responsive response regulators also showed differential expression indicating GRF4-GIF1 pathway playing important role in controlling cytokinin homeostasis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data indicate that ectopic expression of \u003cem\u003eVitis vinifera VvGRF4-GIF1\u003c/em\u003e chimera can provide significant advantage over wild-type plants during strawberry regeneration without producing any pleiotropic effects seen for the \u003cem\u003emiRNA\u003c/em\u003e resistant \u003cem\u003eVvGRF4-GIF1\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 11:38:28","doi":"10.21203/rs.3.rs-4583627/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-23T02:17:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-22T16:13:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T16:56:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112512258396643230813069220685237068694","date":"2024-07-01T16:37:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42458792067124686964800542822624110438","date":"2024-06-19T06:30:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-17T17:16:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-15T06:15:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-15T06:13:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2024-06-14T18:04:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8827b203-22ba-42be-b3ce-b5900818d0a3","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:11:56+00:00","versionOfRecord":{"articleIdentity":"rs-4583627","link":"https://doi.org/10.1186/s13007-024-01270-8","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2024-10-18 15:57:02","publishedOnDateReadable":"October 18th, 2024"},"versionCreatedAt":"2024-07-02 11:38:28","video":"","vorDoi":"10.1186/s13007-024-01270-8","vorDoiUrl":"https://doi.org/10.1186/s13007-024-01270-8","workflowStages":[]},"version":"v1","identity":"rs-4583627","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4583627","identity":"rs-4583627","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}