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2023 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Plant materials
Protein extraction
Trypsin digestion
Affinity enrichment of ubiquitinated peptides
Lc-ms/ms and data analysis
Bioinformatics methods
Results
Overview of the ubiquitomic analysis
Properties of ubiquitinated proteins and motifs
A temperature-regulated rice endosperm ubiquitome
Functions for differentially expressed ubiquitinated proteins
Discussion
Ubiquitination of heat shock proteins and transcription factors under high-temperature stress
Sucrose and starch metabolism are modified by ubiquitination under high-temperature stress
Ubiquitination affects sucrose hydrolysis
Ubiquitination of adp-glucose pyrophosphorylase (agpase)
Ubiquitination regulates amylose biosynthesis
Ubiquitination regulates amylopectin biosynthesis
Ubiquitination affects ribosomal systems under high-temperature stress
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
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References
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ARTICLE   Open Access    

Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis

  • College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China

More Information
  • Fragaria nilgerrensis Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for F. nilgerrensis has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for F. nilgerrensis leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4−5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of an efficient regeneration system provided a feasible platform for genetic transformation, which provided the foundation for further gene functional studies in F. nilgerrensis.

  • Introduction

    Rice (Oryza sativa L.) is a world staple crop that feeds over half of the world's population[1,2]. In recent years, high-temperature events are becoming more frequent and intensive as a result of global warming, which can severely affect rice grain yield and quality[3,4]. During flowering and grain filling stages, high-temperature stress can result in a significant reduction in seed setting rate and influence amylose content, starch fine structure, functional properties and chalkiness degree of rice[5−7]. Transcriptome and proteome analysis in rice endosperm have also been used to demonstrate the differences in high-temperature environments at gene and protein expression levels[8−10]. In addition, as an important post-translational modification, protein phosphorylation has proven to be involved in the regulation of starch metabolism in response to high-temperature stress[11]. However, little is known about whether protein ubiquitination regulates seed development under high-temperature stress.

    Ubiquitination is another form of post-translational modification that plays key roles in diverse cellular processes[12]. Several reports have described the functions of ubiquitination in rice defense responses based on ubiquitome analysis. Liu et al.[13] investigated relationships between ubiquitination and salt-stress responses in rice seedlings using a gel-based shotgun proteomic analysis and revealed the potential important role of protein ubiquitination in salt tolerance in rice. Xie et al.[14] identified 861 peptides with ubiquitinated lysines in 464 proteins in rice leaf cells by combining highly sensitive immune affinity purification and high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These ubiquitinated proteins regulated a wide range of processes, including response to stress stimuli. A later study revealed the relationships between ubiquitination status and activation of rice defense responses, and generated an in-depth quantitative proteomic catalog of ubiquitination in rice seedlings after chitin and flg22 treatments, providing useful information for understanding how the ubiquitination system regulates the defense responses upon pathogen attack[15]. Although many studies have shown that ubiquitination plays improtant roles in the heat response of plant[16,17], there has been little systematic discussion on the ubiquitome of rice endosperm in the context of global climate change.

    In this study, we examine the high-temperature induced ubiquitination change in two rice varieties with different starch qualities, through a label-free quantitative ubiquitome analysis. This study provides a comprehensive view of the function of ubiquitination in high-temperature response of rice developing seed, which will shed new light on the improvement of rice grain quality under heat stress.

    Materials and methods

    Plant materials

    Two indica rice varieties with different starch quality, 9311 and Guangluai4 (GLA4), were used as materials. 9311 is a heat-sensitive variety, which displays low amylose content with good starch quality; while GLA4 is known to be the parental variety of HT54, an indica breeding line with heat tolerance, and thus GLA4 is possibly heat tolerant, which shows high amylose content with poor starch quality[18,19]. Rice growth conditions, sample treatment and collection were conducted as previously described[11].

    Protein extraction

    Husk, pericarp and embryo were detached from immature rice grains on ice[20]. Rice endosperm was then ground with liquid nitrogen, and the cell powder was sonicated 10 rounds of 10 s sonication and 15 s off-sonication on ice in lysis buffer (6 M Guanidine hydrochloride, pH 7.8−8.0, 0.1% Protease Inhibitor Cocktail) using a high intensity ultrasonic processor. Following lysis, the suspension was centrifuged at 14,000 g for 40 min at 4 °C to remove the debris. The supernatant was collected, and the protein concentration was estimated using BCA assay (Pierce BCA Protein assay kit, Thermo Fisher Scientific, Waltham, MA, USA) before further analysis.

    Trypsin digestion

    The protein mixture was reduced by DTT with the final concentration of 10 mM at 37 °C for 1 h, alkylated by iodoacetamide with a final concentration of 50 mM at room temperature in the dark for 0.5 h, and digested by trypsin (1:50) at 37 °C for 16 h. Then the sample was diluted by adding trifluoroacetic acid (TFA) to the final concentration of 0.1%. The enzymatic peptides were desalted on a Sep-Pak C18 cartridge (Waters, Milford, MA, USA), concentrated by lyophilization and reconstituted in precooled IAP buffer (50 mM MOPS-NaOH PH 7.2, 10 mM Na2HPO4, 50 mM NaCl) for further analysis.

    Affinity enrichment of ubiquitinated peptides

    The peptides solution was incubated with prewashed K-ε-GG antibody beads (PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit), and gently shaken at 4 °C for 1.5 h. The suspension was centrifuged at 2,000 g for 30 s, and the supernatant was removed. The Anti-K-ε-GG antibody beads were washed with IAP Buffer three times and with ddH2O three times. The peptides were eluted from the beads with 0.15% trifluoroacetic acid (TFA). Finally, the eluted fractions were combined and desalted with C18 Stage Tips.

    LC-MS/MS and data analysis

    LC-MS/MS analysis were performed using the methods of Pang et al.[11]. Raw mass spectrometric data were analyzed with MaxQuant software (version 1.3.0.5) and were compared with the indica rice protein sequence database (Oryza sativa subsp. indica-ASM465v1). Parameters were set according to Pang et al.[11]. All measurements were obtained from three separate biological replicates.

    Bioinformatics methods

    Quantification of the modified peptides was performed using the label-free quantification (LFQ) algorithm[11]. Differentially ubiquitinated sites (proteins) in response to high-temperature were identified by Student's t-test (p < 0.05, log2(fold-change) > 1) with at least two valid values in any condition or the ubiquitination sites that exhibited valid values in one condition (at least two of three replicates) and none in the other.

    Subcellular localization was performed using CELLO database (http://cello.life.nctu.edu.tw). Gene Ontology (GO) annotation proteome was derived from the AgriGO (http://bioinfo.cau.edu.cn/agriGO/). The differential metabolic profiles were visualized with MapMan software (version 3.6.0RC1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed by using KEGG Automatic Annotation Server (KAAS) software. A p-value of < 0.05 was used as the threshold of significant enrichment. SWISS-MODEL was used to generate the tertiary structure of GBSSI (SWISS-MODEL, http://swissmodel.expasy.org/). The figures were annotated with Adobe Illustrator (Adobe Systems, San Jose, CA, USA).

    Results

    Overview of the ubiquitomic analysis

    To elucidate how high-temperature stress influences rice developing endosperm at the ubiquitination level, a label-free analysis was performed to quantify ubiquitome from two indica rice varieties under normal (9311-C and GLA4-C) and high-temperature conditions (9311-H and GLA4-H). The distribution of mass error of all the identified peptides was near zero and most of them (71.5%) were between -1 and 1 ppm, suggesting that the mass accuracy of the MS data fits the requirement (Fig. 1a). Meanwhile, the length of most peptides distributed between 8 and 42, ensuring that sample preparation reached standard conditions (Fig. 1b).

    Figure 1.  Characteristics of the ubiquitinated proteome of rice endosperm and QC validation of MS data. (a) Mass error distribution of all identified ubiquitinated peptides. (b) Peptide length distribution. (c) Frequency distribution of ubiquitinated proteins according to the number of ubiquitination sites identified.
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    In all endosperm samples, a total of 437 ubiquitinated peptides were identified from 246 ubiquitinated proteins, covering 488 quantifiable ubiquitinated sites (Supplemental Table S1). Among the ubiquitinated proteins, 60.6% had only one ubiquitinated lysine site, and 18.7%, 8.1%, 5.3%, or 7.3% had two, three, four, or five and more ubiquitinated sites, respectively. In addition, four proteins (1.6%, BGIOSGA004052, BGIOSGA006533, BGIOSGA006780, BGIOSGA022241) were ubiquitinated at 10 or more lysine sites (Fig. 1c, Supplemental Table S1). The proteins BGIOSGA008317 had the most ubiquitination sites with the number of 16. It was noted that besides ubiquitin, two related ubiquitin-like proteins NEDD8 and ISG15 also contain C-terminal di-Gly motifs generated by trypsin cleavage, and the modifications of these three proteins cannot be distinguished by MS[21]. Here, the di-Gly-modified proteome therefore represents a composite of proteins modified by these three proteins. However, the sites from NEDD8 or ISG15 modifications were limited because they mediate only a few reactions in cells[21].

    Properties of ubiquitinated proteins and motifs

    To better understand the lysine ubiquitome changes in rice endosperm induced by high-temperature, we performed a Gene Ontology (GO) functional annotation analysis on all identified ubiquitinated proteins (Fig. 2a). In the biological process GO category, 'metabolic process' and 'cellular process' were mainly enriched, accounting for 75.1% and 74.1% of ubiquitinated proteins, respectively. In addition, 34.6% proteins were associated with 'response to stimulu', emphasizing the regulatory role of ubiquitination modification in response to high-temperature stress. From the cellular component perspective, ubiquitinated proteins were mainly associated with 'cellular anatomical entity' (99.4%), 'intracellular' (84.4%) and 'protein-containing complex' (29.9%). The molecular function category result suggested that these proteins were largely involved in 'binding' (62.7%), 'catalytic activity' (43.4%) and 'structural molecule activity' (16.5%). Furthermore, subcellular location annotation information indicated that 34.7%−39.4% proteins were located in the cytoplasm, and other were mostly located in the nucleus (23.5%−27.7%), plasma membrane (9.4%−11.4%), and chloroplast (9.6%−12.8%) (Fig. 2b). It is noteworthy that the ubiquitinated proteins located in the cytoplasm were decreased in high-temperature environments in both varieties.

    Figure 2.  Analysis of ubiquitinated proteins and motifs. (a) Gene ontology (GO) functional characterization of ubiquitinated proteins. (b) Subcellular localization of ubiquitinated proteins. From the inside out, the ring represents 9311-C, 9311-H, GLA4-C and GLA4-H, respectively. (c) Motif enrichment analysis of ubiquitinated proteins.
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    The following two significantly enriched motifs from all of the identified ubiquitinated sites were identified using MoDL analysis: [A/S]xKub and Kubxx[E/Q/R/V]x[E/G/L/P/Q/R/Y], which covered 84 and 100 sequences, respectively (Fig. 2c). Further analysis showed that the conserved alanine (A) and glutamic acid (E) were included in upstream and downstream of the ubiquitinated lysine sites in rice endosperm. A similar phenomenon also occurred in rice leaf[14], wheat leaf[22], and petunia[23], indicating that alanine (A) and glutamic acid (E) were likely to be the specific amino acids in conserved ubiquitination motifs in plants. Additionally, serine (S) was enriched at the position -2 (upstream) of the ubiquitinated lysine, while various amino acids such as arginine (R), glutamic acid (E), glutamine (Q), valine (V) were found at positions +3 and +5 (downstream).

    A temperature-regulated rice endosperm ubiquitome

    To detect possible changes in rice endosperm ubiquitome attributable to high-temperature stress, we then performed LFQ analysis on all quantifiable ubiquitination sites within our dataset. As shown in Fig. 3a, more ubiquitinated proteins, peptides and sites were detected in the treatment groups (9311-H and GLA4-H), suggesting that exposure to high-temperature stress may increase the ubiquitination events in rice endosperm. Only 282 common ubiquitinated sites in 158 proteins were quantifiable for all sample groups due to reversible ubiquitination induced by high-temperature (Fig. 3b). Principal component analysis (PCA) showed that three repeats of each sample clustered together, and four groups were clearly separated (Fig. 3c). Furthermore, the differentially expression profiles of ubiquitination sites (proteins) in 9311 and GLA4 under high-temperature stress were depicted to further understand the possible changes (Fig. 3d). Where LFQ values were missing, the data were filtered to identify those ubiquitination sites with a consistent presence/absence expression pattern. These analyses yielded 89 ubiquitination sites that were only present in 9311-H and six that were only present in 9311-C (Fig. 3d, Supplemental Table S2). Similarly, 51 differentially expressed ubiquitination sites were present in GLA4-H and 13 ubiquitination sites only occurred in GLA4-C (Fig. 3d & Supplemental Table S3). Beyond that, a total of 113 and 50 significantly changed ubiquitination sites (p < 0.05, log2(fold-change) >1) were screened out in 9311 and GLA4, respectively (Fig. 3d, Supplemental Tables S4 & S5). For subsequent comparative analysis, the ubiquitination expression profiles with consistent presence/absence and ubiquitination sites with significant differences in statistical testing were combined and named as 9311-Up, 9311-Down, GLA4-Up, and GLA4-Down, respectively (Fig. 3d). The number of significantly up-regulated ubiquitination sites was far greater than down-regulated ubiquitination sites in both 9311 and GLA4 varieties. These findings indicated that high temperature not only induced the occurrence of ubiquitination sites, but also significantly upregulated the intensity of ubiquitination. Beyond that, the magnitude of the up-regulation in 9311 was higher than that in GLA4 (Fig. 3b & d), indicating that the ubiquitination modification of heat-sensitive varieties was more active than heat-resistant varieties in response to high-temperature stress.

    Figure 3.  A temperature regulated rice endosperm ubiquitome. (a) The number of ubiquitinated proteins, peptides and sites detected in four group samples. (b) Venn diagram of ubiquitination sites (proteins) detected in four group samples. (c) PCA based on ubiquitination intensity across all four sample groups with three biological repetitions. (d) Differentially expression profiles of ubiquitination sites (proteins) in 9311 and GLA4 under high-temperature stress. The expression profiles of selected ubiquitination sites (p < 0.05, log2(fold-change) >1) were normalized using the Z-score and presented in a heatmap.
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    To further investigate the ubiquitination regulatory pattern under high temperature stress in two varieties, four groups with significantly regulated sites were analyzed. There were 37 ubiquitination sites showed the same regulatory trend in 9311 and GLA4, accounting for 17.8% and 32.5% of the total differentially expressed sites in 9311 and GLA4, respectively. Among them, 36 ubiquitination sites were upregulated and one site was downregulated (Fig. 4a). In addition, 159 upregulated ubiquitination sites and three downregulated sites were only present in 9311, while 53 upregulated sites and 15 downregulated sites were only present in GLA4. Moreover, nine ubiquitination sites showed opposite regulatory trends in 9311 and GLA4. A similar regulatory trend of ubiquitination proteins is shown in Fig. 4b. It is noted that some proteins had both upregulated and downregulated ubiquitination sites (Supplemental Tables S6 & S7), indicating that significant differences in ubiquitination were, to some extent, independent of protein abundance.

    Figure 4.  Comparison of differentially ubiquitinated sites and proteins in 9311 and GLA4 under high-temperature stress.
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    Functions for differentially expressed ubiquitinated proteins

    To understand the function of ubiquitination in response to the high-temperature stress of rice endosperm, we conducted GO enrichment-based clustering analysis of the differentially ubiquitinated proteins in 9311 and GLA4 at high temperature, respectively (Fig. 5). In the biological process category of 9311, proteins were relatively enriched in the carbohydrate metabolic process, polysaccharide metabolic process, starch biosynthetic process, cellular macromolecule localization, protein localization, intracellular transport, and phosphorylation (Fig. 5). For the molecular function analysis, we found that the proteins related to kinase activity, nucleotidyltransferase activity, phosphotransferase activity, and nutrient reservoir activity were enriched (Fig. 5). The two principal cellular components were intrinsic component of membrane and integral component of membrane (Fig. 5). There was no significantly enriched GO term in the GLA4 group due to the dataset containing relatively few proteins, and thus, further enrichment analysis was conducted on the proteins that were common to both varieties. The results showed that proteins were over-represented in carbon metabolism, including starch biosynthesis and metabolism, glucan biosynthesis and metabolism, and polysaccharide biosynthesis and metabolism (Fig. 5), indicating the importance of carbohydrate synthesis and metabolism in the ubiquitination regulatory network.

    Figure 5.  Enrichment analysis of differentially expressed ubiquitinated proteins based on Gene Ontology (GO) terms.
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    To identify pathways which were differentially ubiquitinated under high-temperature stress, the KEGG pathway-based clustering analysis was conducted. The results showed that the differentially ubiquitinated proteins in both 9311 and GLA4 were mostly abundant in the pathways of carbohydrate metabolism, starch and sucrose metabolism, folding, sorting and degradation, translation, ribosome, and protein processing in endoplasmic reticulum (Fig. 6a). In the 9311 group, the pathways of carbohydrate metabolism, starch and sucrose metabolism, glycosyltransferases, glycolysis, and energy metabolism were enriched in the differentially ubiquitinated proteins (Fig. 6b); while there was no significantly enriched KEGG pathway in the GLA4 group. We further found the proteins that were common to both varieties were only significantly enriched in the starch and sucrose metabolism pathways (p = 0.04). The ubiquitination proteins involved in the starch and sucrose metabolism mainly include: sucrose hydrolysis (SUS, FK, UGPase), and starch synthesis (AGPase, GBSSI, BEI, BEIIb, PUL, PHO1), which are discussed below.

    Figure 6.  KEGG classification and enrichment analysis of differentially ubiquitinated proteins. (a) Number of differentially ubiquitinated proteins based on KEGG classification in 9311 and GLA4. (b) KEGG enrichment analysis of differentially ubiquitinated proteins in 9311.
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    Discussion

    Although many reports have described specific examples of ubiquitination in rice defense responses[13,15,16], our knowledge on global changes in the developing endosperm ubiquitome under high-temperature stress is still lacking. In this study, a label-free quantitative proteomic analysis of ubiquitination was applied to examine the high-temperature induced ubiquitination change of two indica rice varieties (9311 and GLA4) with distinct starch quality. We identified many new lysine modification sites on proteins involved in various pathways, highlighting the complexity of the ubiquitination-mediated regulatory system in high-temperature stress responses in rice.

    Ubiquitination of heat shock proteins and transcription factors under high-temperature stress

    Heat shock proteins accumulate under various stresses and play important roles in plant defenses against abiotic stresses[24,25]. Research has shown that a number of heat shock proteins were prominent in the rice ubiquitome network, of which OsHSP71.1, and OsHSP82A showed increased ubiquitination levels under chitin and flg22 treatment[15]. Here, seven lysine residues on five heat shock proteins possessed ubiquitination modification in rice endosperm. Three sites (BGIOSGA011420-K78, BGIOSGA026764-K99, BGIOSGA029594-K106) showed significant up-regulation in 9311 under high-temperature stress, while in GLA4, the ubiquitination level of BGIOSGA011420-K78 was down-regulated. This differential ubiquitination of heat-tolerant and heat-sensitive varieties provided a basis for studying the regulation of post-translational modification of heat shock proteins under high-temperature stress, despite the regulatory role of those heat shock proteins being still unclear.

    Transcription factors (TFs) play an essential role in the regulation of gene expression. A total of three transcription factors were identified in the ubiquitination dataset, of which two were NAC family members. As one of the largest plant-specific TF families, NAC is involved in the responses to abiotic and biotic stresses[26]. The ubiquitination modification of K173 in BGIOSGA018048 was specifically expressed in the 9311-H group, which may affect the stress resistance level in high-temperature environments. In addition, two sites K148 and K149 of ERF TF family member BGIOSGA024035 was downregulated in GLA4 under high-temperature stress. This differential ubiquitination was likely to affect the expression of related genes regulated by this transcription factor.

    Sucrose and starch metabolism are modified by ubiquitination under high-temperature stress

    Ubiquitination affects sucrose hydrolysis

    The results of GO and KEGG enrichment analysis of differential ubiquitination proteins indicated that the sucrose and starch metabolic pathway was largely affected by ubiquitination regulation under high-temperature stress (Figs 5 & 6). The ubiquitination sites involved in sucrose and starch metabolism are listed in Table 1. To assess how high-temperature stress affects the crucial pathway, the significantly differential ubiquitination sites in 9311 and GLA4 were displayed in the heatmap of specific proteins (Fig. 7).

    Table 1.  Ubiquitination sites related to sucrose and starch metabolism in rice endosperm.
    Gene nameAnnotationProtein entryModification site(s)
    SUS1Sucrose synthase 1BGIOSGA010570K172, K177
    SUS2Sucrose synthase 2BGIOSGA021739K160, K165, K176, K804
    SUS3Sucrose synthase 3BGIOSGA026140K172, K177, K541, K544, K588
    FKFructokinaseBGIOSGA027875K143
    UGPaseUDP-glucose pyrophosphorylaseBGIOSGA031231K27, K150, K303, K306
    AGPS1ADP-glucose pyrophosphorylase small subunit 1BGIOSGA030039K94, K464, K484
    AGPS2ADP-glucose pyrophosphorylase small subunit 2BGIOSGA027135K106, K132, K385, K403, K406, K476, K496
    AGPL2ADP-glucose pyrophosphorylase large subunit 2BGIOSGA004052K41, K78, K134, K191, K227, K254, K316, K338, K394, K396, K463, K508, K513
    AGPL3ADP-glucose pyrophosphorylase large subunit 3BGIOSGA017490K509
    GBSSIGranule bound starch synthase IBGIOSGA022241K130, K173, K177, K181, K192, K258, K371, K381, K385, K399, K462, K517, K530, K549, K571, K575
    BEIStarch branching enzyme IBGIOSGA020506K103, K108, K122
    BEIIbStarch branching enzyme IIbBGIOSGA006344K134
    PULStarch debranching enzyme:PullulanaseBGIOSGA015875K230, K330, K431, K736, K884
    PHO1Plastidial phosphorylaseBGIOSGA009780K277, K445, K941
     | Show Table
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    Figure 7.  Sucrose and starch pathway at the ubiquitination levels in rice endosperm under high-temperature stress.
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    In cereal endosperm, sucrose is the substrate for the biosynthesis of starch. The formation of glucose 1-phosphate (G1P, used in starch synthesis, see below) from sucrose requires a series of enzymes[27]. Here, we found that sucrose synthase 1 (SUS1), SUS2, SUS3, fructokinase (FK), and UDP-glucose pyrophosphorylase (UGPase) were ubiquitinated among all sample groups (Table 1, Fig. 7). In the ubiquitome of seedling and leaf in japonica rice, ubiquitination sites have been found in SUS1, SUS2, UGPase, and FK, which were related to sucrose hydrolysis[14,15]. SUS catalyzed the process of cleaving sucrose into UDP-glucose (UDPG) and fructose. Two ubiquitination sites, K172 and K177, were identified in SUS1 in rice endosperm, which were also found in rice leaves[14]. A total of four ubiquitination sites were identified in SUS2, two of which were also reported in rice seedling and leaf, indicating the conservation of the lysine residues in different rice tissues. It was noted that all four ubiquitination sites in SUS2 were upregulated in high-temperature environments, although the regulated sites of 9311 and GLA4 were different. In 9311, the ubiquitination levels of K160, K174, and K804 were increased, while GLA4 was only upregulated in K176. The ubiquitination sites K541, K544, and K588 in SUS3 were screened from developing rice seeds for the first time. In addition, SUS3 had two completely overlapping sites K172 and K177 with SUS1, and it was difficult to determine which enzymes the two sites belonged to. The ubiquitination levels of SUS3-K541 and SUS3-K544 in 9311 significantly increased in high-temperature environments, while there was no significant difference in the ubiquitination level of SUS3 in GLA4. Overall, the ubiquitination sites of SUS in rice endosperm were located in the functional domain except for SUS2-K804, reflecting the importance of ubiquitination regulation in SUS.

    UGPase catalyses the conversion of glucose 1-phosphate and UTP into UDPG[28]. Research has shown that the mutation of UGPase gene lead to chalky endosperm[29]. As shown in Table 1, four ubiquitination sites K27, K150, K303, and K306 were identified in rice endosperm, which were completely inconsistent with the seven ubiquitination sites in rice seedlings and two in leaves[14,15], reflecting the tissue specificity. We speculated that the UGPase with different modification sites may play different regulatory roles in metabolic pathways in different tissues. Under high-temperature stress, the ubiquitination level of UGPase-K27 was 8.1-fold up-regulated. Liao et al.[10] demonstrated that the expression of UDPase was down-regulated in both heat-tolerant and heat-sensitive rice lines under high temperature conditions, which could reasonably explain the significant up-regulation of UGPase-K27 ubiquitination level. The ubiquitination site K143 of FK was also reported in seedling tissues[15].

    Ubiquitination of ADP-glucose pyrophosphorylase (AGPase)

    The AGPase reaction represents the first committed step of starch biosynthesis[27]. A total of 22 lysine ubiquitination sites were identified in four AGPase subunits (AGPL2, AGPL3, AGPS1, AGPS2). AGPL2 had 13 ubiquitination sites, of which six were located in NTP_transferase domain, including K254, K338, K191, K134, K227, and K316. High-temperature stress resulted in an increase in the ubiquitination level of K254 in both 9311 and GLA4, and significant upregulation of K508 and K513 in 9311, as well as K191, K227, and K316 in GLA4. In contrast, AGPL2-K394 were significantly downregulated in GLA4. AGPL3 contained one ubiquitination site K509, and the modification level of AGPL3-K509 was up-regulated in high-temperature environments in 9311. AGPS1 had one specific ubiquitination site K464 and another two sites K94 and K484 that completely overlapped with AGPS2-K106 and AGPS2-K496, respectively. The modification levels of K464 and K484 significantly increased in high-temperature environments in 9311, and K94 was significantly up-regulated in both varieties. There were seven ubiquitination sites in AGPS2 in rice endosperm, which were different with the sites found in rice leaves[14]. In addition to the two sites that overlapped with AGPS1, AGPS2 had another two ubiquitination sites (K406 and K132) that upregulated in high-temperature environments.

    Ubiquitination regulates amylose biosynthesis

    Amylose content is one of the key determinants that strongly influence rice grain quality[30]. The biosynthesis of amylose requires the catalytic effect of granule-bound starch synthase I (GBSSI)[30,31]. Here, a total of 16 ubiquitination sites were identified in GBSSI (Table 1, Fig. 8a). Among these ubiquitination sites, six lysine residues (K130, K173, K177, K181, K192, K258) were located in glycosyltransferase 5 (GT5) domain, and three sites (K399, K462, K517) were located in GT1 domain (Fig. 6), indicating the important role of ubiquitination regulation of GBSSI. Under high-temperature stress, the ubiquitination levels of six sites (K130, K177, K399, K381, K385, K549) increased in two indica rice varieties, while one sites (K258) showed downregulation in 9311 (Fig. 8a). Numerous studies had described that the amylose content was reduced under high-temperature stress in rice[5,7], which might be due to the degradation of GBSSI proteins caused by the increased significantly up-regulated ubiquitination sites. These ubiquitination sites identified in rice GBSSI with significant differences under high-temperature stress were expected to become a new breakthrough point for the improvement of starch quality.

    Figure 8.  Structure of GBSSI. (a) Domain structure of GBSSI and ubiquitination sites with significant differences in response to high-temperature stress. (b) 3D model of GBSSI and the relationship between ubiquitination sites K462 and ADP, SO4 (salt bridge or hydrogen bond).
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    To further determine the regulatory role of the ubiquitination sites in GBSSI, SWISS-MODEL was used to predict 3D structural model. As shown in Fig. 8b, GBSSI had three SO4 (sulfate ions) and one ADP ligand. These ligands interact with GBSSI through hydrogen bonds and salt bridges. Three sites, K447, R458, and K462, were associated with SO4 through salt bridges, while G100, N265, Q412, K462, and Q493 interact with the hydrogen bonds of ADP in GBSSI[32,33]. Based on this finding, it can be reasonably inferred that the K462 site with ubiquitination modification located in the GT1 domain played an important role in the interaction between GBSSI, SO4, and ADP. An in-depth investigation was necessary to gain a more comprehensive understanding of the regulatory function of ubiquitination modification at GBSSI-K462, although there was no significant difference in the ubiquitination level under high-temperature stress.

    Ubiquitination regulates amylopectin biosynthesis

    Amylopectin, the major component of starch, is synthesized by the coordinated action of multiple enzymes including soluble starch synthase (SSs), starch branching enzyme (BEs), starch debranching enzyme (DBEs), and phosphorylases (PHOs or Phos) with ADPG as a substrate. In this study, ubiquitination sites were detected in BEs, DBEs, and Phos.

    BEs, covering two isoforms, BEI and BEII, are responsible for catalyzing the formation of α-1,6-glucosidic linkages of amylopectin[34]. There were three ubiquitination sites (K103, K108, and K122) identified in BEI (Fig. 9a). K122 was the first amino acid in the carbohydrate-binding module 48 (CBM48) domain. Sequence alignment analyses of BEs from eight plants revealed K122 was conserved among all plants' BEI (Fig. 9a), suggesting a high probability of the functional effects of ubiquitination modification at this site. In high-temperature environments, ubiquitination levels of K108 and K122 were significantly up-regulated in 9311, while no significantly regulated ubiquitination sites of BEI were observed in GLA4. Only one ubiquitination site, K134, was found in BEIIb (Fig. 9a). The ubiquitination levels showed a slightly upward trend with no significant differences in high-temperature environments in both varieties. These changes could be one of the reasons for increased gelatinization temperature and relative crystallinity of rice starch in response to high-temperature[5].

    Figure 9.  Domain structure of (a) BEs, (b) PUL and (c) Pho1 as well as their ubiquitination sites with significant differences in response to heat stress. Residues in red indicate the ubiquitination site. Non-ubiquitinated residues are shown in dark grey.
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    DBEs consists of isoamylase (ISA) and pullulanase (PUL) with catalytic function for hydrolyzing α-1,6-glucosic linkages[35]. In the present study, we found that only PUL was ubiquitinated in rice endosperm (Fig. 9b). Among five ubiquitination sites (K230, K330, K432, K736, and K884) identified in PUL, K230 was located in the PULN2 domain, while K330 was in the CBM48 domain. Under high-temperature stress, K330 showed completely opposite regulatory trends in two cultivars. In addition, the ubiquitination level of K884, located in the DUF3372 domain, was significantly up-regulated in 9311. Previous study has reported that the expression of PUL was significantly up-regulated in 9311 under high-temperature stress, while GLA4 showed down-regulation in PUL abundance[11]. Consequently, there might be two possible functions of these ubiquitination sites. One possibility is that ubiquitination sites were unrelated to protein degradation; instead, they regulated the biosynthesis of amylopectin by affecting other functions of the protein. Secondly, ubiquitination sites were associated with protein degradation, and the levels of ubiquitination modification were based on protein abundance, resulting in a completely consistent regulation of ubiquitination modification and protein abundance under high-temperature stress.

    PHOs, including two types, Pho1/PHO1 and Pho2/PHO2, are responsible for the transfer of glucosyl units from Glc-1-P to the non-reducing end of a-1,4-linked glucan chains[36]. Pho1 is a temperature-dependent enzyme and considered crucial not only during the maturation of amylopectin but also in the initiation process of starch synthesis[37,38]. The three ubiquitination sites (K277, K445, K941) identified in Pho1 were located in two phosphorylase domains. We found that two sites, Pho1-K277 and Pho1-K445, were only ubiquitinated in high-temperature environments in 9311 and GLA4, respectively. Pang et al.[11] has demonstrated that the protein abundance of Pho1 decreased under high-temperature stress, especially in GLA4. Satoh et al.[38] reported that the functional activity of Pho1 was weakened under conditions of high temperature and its function might be complement by one or more other factors. Hence, these ubiquitination modifications that specifically occurred in high-temperature environments might be related to the degradation of Pho1 proteins.

    Ubiquitination affects ribosomal systems under high-temperature stress

    As a factory for protein synthesis in cells, the ribosome is an extremely crucial structure in the cell[39]. It has been proven that multiple ribosomal subunits were abundantly ubiquitinated in Arabidopsis and wheat[22]. In the present study, 57 ubiquitination sites involving 33 ribosome subunits were identified in 40S and 60S ribosome complexes in rice. Under high-temperature stress, the ubiquitination levels of some sites were significantly upregulated or downregulated, implying that ubiquitination of ribosomal proteins is likely to be an important regulatory mechanism in high-temperature response in rice endosperm. The results of GO and KEGG enrichment analysis indicated that the ribosome system was one of the most active systems for ubiquitination regulation under high-temperature stress. We speculated that the ubiquitin-proteasome system might be involved in the removal of subunits or entire ribosomes that were improperly folded in high-temperature environments. As shown in Fig. 10, the S10e, L18Ae, S27Ae, L9e, S3e, S28e, S20e, and S2e subunits were significantly up-regulated in 9311, while L13e subunits showed a completely opposite regulatory trend at the ubiquitination sites K81 and K88. In GLA4, the ubiquitination levels of S10e, S27Ae, L10Ae, L9e, S3e, S2e, and L4e showed a significant increase, while the ubiquitination level of L17e was significantly down-regulated under high-temperature stress. A total of seven ubiquitination sites involving S10e, S27Ae, L9e, S3e, and S2e subunits were jointly up-regulated in both two varieties. These sites might be related to the degradation of improperly folded ribosome subunits under high-temperature stress, while other ubiquitination sites with variety specificity might be associated with ribosomal function.

    Figure 10.  Ribosome system at the ubiquitination levels in rice endosperm under high-temperature stress. Grey shadings represent ubiquitinated proteins with no significant differences under heat stress. Red and blue shadings indicate up-regulated and down-regulated ubiquitinated proteins, respectively. Orange shading displays a combination of up- and down-regulated ubiquitinated sites in the same ubiquitinated protein.
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    In conclusion, this study provides the first comprehensive view of the ubiquitome in rice developing endosperm, and demonstrated that ubiquitination has diverse functions in the high-temperature response of rice endosperm by modulating various cellular processes, especially the sucrose and starch metabolism. Comparative analysis of the temperature-induced ubiquitination status revealed some similarities and more interesting differences between 9311 and GLA4. These differences might be the reason for the different qualities formation of the two indica rice varieties, which could provide potential genetic resources for the improvement of the heat resistance in rice. Considering the diversity of ubiquitination modification, it is worthwhile to further validate and explore the function and regulatory mechanism of the key targets and key pathways. The findings provide valuable insights into the role of ubiquitination in response to high-temperature stress and lay a foundation for further functional analysis of lysine ubiquitination in rice.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Bao J, Pang Y; data collection: Pang Y; analysis and interpretation of results: Pang Y; draft manuscript preparation: Ying Y; Revised manuscript preparation: Ying Y, Pang Y, Bao J. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    Acknowledgments

    This work was financially supported by the AgroST Project (NK2022050102) and Zhejiang Provincial Natural Science Foundation (LZ21C130003).

  • The authors declare that they have no conflict of interest.

  • Supplementary Fig. S1 Schematic of pRI201 vector.
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  • Cite this article

    Jiang S, Ji Y, Wang M, Xue L, Zhao J, et al. 2023. Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis. Fruit Research 3:9 doi: 10.48130/FruRes-2023-0009
    Jiang S, Ji Y, Wang M, Xue L, Zhao J, et al. 2023. Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis. Fruit Research 3:9 doi: 10.48130/FruRes-2023-0009
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Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis

  • College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China

  • Received: 27 November 2022
  • Accepted: 01 March 2023
  • Published online: 14 April 2023
Fruit Research  3,  Article number: 9  (2023)  |  Cite this article

Abstract: Fragaria nilgerrensis Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for F. nilgerrensis has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for F. nilgerrensis leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4−5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of an efficient regeneration system provided a feasible platform for genetic transformation, which provided the foundation for further gene functional studies in F. nilgerrensis.

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    INTRODUCTION

    • Establishing a high-frequency regeneration system is a prerequisite for genetic transformation, which is one of the most effective biotechnological approaches in crops[1,2]. Strawberry (Fragaria spp.), an economically important berry crop, has become a model crop for fundamental research in recent years[3], and numerous studies have been undertaken to develop regeneration systems of strawberry cultivars or species, including the octoploid genotype LF9[4], Chandler[5] and the diploid wild species Fragaria vesca[6]. The diploid wild Chinese strawberry species Fragaria nilgerrensis Schlechtendal ex J. Gay is mainly distributed in Sichuan, Yunnan, Guizhou, Shaanxi, Hubei, Chongqing, and Hunan Provinces (China) with many excellent characteristics, such as fruit with white color, peach fragrance, and strong disease resistance[7]. Thus, the regeneration of F. nilgerrensis has attracted wide research attention[8]. To date, a few studies on regeneration in F. nilgerrensis have been reported. For example, the shoot regeneration from detached leaves was established, and the regeneration frequency did not reach 80%[9]. Although the callus was induced from F. nilgerrensis leaves with an induction rate of 95%, the differentiation of callus into plantlets did not continue to be studied[10]. Despite reports of regeneration in F. nilgerrensis, the regeneration efficiency remains low, thereby preventing its application to genetic transformation. Therefore, there is an urgent need to establish a high-efficiency regeneration system for F. nilgerrensis.

      With the development of genomics of strawberries, the understanding of gene function is gradually deepened. Both gene manipulation technologies including overexpression and knockout/down and gene editing technology (CRISPR/Cas9) depend on a high-efficiency transformation system[11−13]. A large number of studies on gene manipulation and trait improvement through Agrobacterium-mediated transformation have been carried out in different strawberry species[14−17]. Pi et al. generated diploid strawberry F. vesca variety 'H4' transgenic lines with parthenocarpic fruit growth and delayed fruit ripening by mutating gene FveSEP3 generated by CRISPR/Cas9[18]. The RAP gene was successfully transformed into the F. vesca variety 'Yellow Wonder' and octoploid cultivated strawberry respectively by overexpressing and knockout technology. The results showed that RAP is one important gene in the fruit color breeding of strawberry[19]. It is reported that strawberry cultivars with a high regeneration rate are more likely to regenerate into plantlets during the Agrobacterium-mediated genetic transformation[16]. For example, a high-efficiency regeneration method was developed with 6.67 shoots per explant in the strawberry cultivar 'Chandler'. Based on this, the genetic transformation efficiency was up to 31.25%[5,20]. Therefore, establishing a stable and high-efficiency transformation system that relies on efficient regeneration is essential for strawberry. F. nilgerrensis, an important diploid strawberry, could be used as a model plant species for various fundamental research due to its relatively small genome size (250 Mbp). The completion of sequencing and assembling of F. nilgerrensis has accelerated the identification and mining of its candidate genes controlling some traits[21]. However, the lack of an efficient genetic transformation system for this species has hindered the verification of these gene functions.

      Various factors could influence strawberry regeneration and genetic transformation, including genotype, explant type, hormone concentration, Agrobacterium strain, infection time, antibiotic concentration, and so on. It has been reported that regeneration and genetic transformation efficiency are closely related to genetic background and the genotype is a major factor[13]. For example, the octoploid strawberry cultivars 'Festival' and 'Fortuna' have a higher percentage of callus induction than 'Sweet Charlie'[22]. The transformation percentage achieved in 'Rapella' was only 0.02%[14], while that in 'Hecker' was 68%[15]. These indicated wide differences in the regeneration and transformation rates among strawberry cultivars. The hormone is another crucial factor involved in the regulation of regeneration. The cytokinin thidiazuron (TDZ) and 6-benzylaminopurine (6-BA), and the auxins indole-3-acetic acid (IAA), 1-naphthlcetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) are generally used for strawberry regeneration[10,23,24]. In particular, TDZ has been frequently utilized in many strawberry species for rapid plantlet regeneration, probably due to its function as a regulator of endogenous auxin levels and its cytokinin-like activity[5,25]. Other regeneration and transformation factors have also been studied in strawberries including exogenous nutrient casein hydrolysate, Fe-EDTA or KNO3[26−27], dark culture time[28], explant (leaf disc)[26], Agrobacterium strain, infection time, antibiotic types and concentrations[5]. The above factors were mainly studied in octoploid strawberry cultivars, however, the high ploidy and heterozygosity of this species pose problems and difficulties for further genetic and molecular studies[5,29]. Studies have shown that the diploid species with a relatively small genome size is a valuable resource for various fundamental research as model plant species[21,30]. However, only a few factors influencing regeneration and transformation have been studied in diploid F. nilgerrensis.

      In the present study, several factors affecting in vitro regeneration of F. nilgerrensis leaf discs were optimized, including dark culture time, CH, TDZ/6-BA, and IBA concentrations to establish the efficient regeneration system that could guarantee the generation of transgenic shoots. Meanwhile, we analyzed callus morphology using paraffin sections to determine the regeneration pathways. Subsequently, the transgenic plants were obtained in Agrobacterium-mediated genetic transformation. PCR-positive transformants were confirmed by PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. This study may be useful in gene functional research and trait improvement for F. nilgerrensis.

    RESULTS

      Optimization of regeneration conditions in F. nilgerrensis leaf discs

    • The effects of plant growth regulators (PGRs) on shoot differentiation were analyzed using different concentrations of TDZ (1, 2, 3, and 4 mg·L−1) and 6-BA (1, 2, 3, and 4 mg·L−1) combined with IBA (0.1, 0.2, and 0.3 mg·L−1). The analysis demonstrated that the rates of callus induction and regeneration and the number of regenerated shoots per explant varied significantly between the treatments at 45 d of inoculation (Table 1). At constant IBA concentration, the rate of shoot regeneration first increased and then decreased as TDZ concentration increased, which is consistent with the 6-BA treatment. The higher rates of callus induction and regeneration and more regenerated shoots per explant were obtained with 2 mg·L−1 TDZ. Similarly, when the concentration of TDZ or 6-BA was maintained constant, the three indexes reached the maximum at 0.2 mg·L−1 IBA. Simultaneously, lower callus formation and shoot regeneration rates were obtained when 6-BA was used instead of TDZ (Table 1). At 25 d of inoculation, most leaves curled, but the callus did not cover the entire leaf in the presence of 6-BA in the medium. In contrast, in the medium containing TDZ, thick, loose, yellow, and granular callus covered the whole leaves (Fig. 1a). The effect of TDZ on regeneration and callus induction was more obvious than that of 6-BA. In summary, the A2C2 medium containing 0.2 mg·L−1 IBA and 2 mg·L−1 TDZ exhibited the highest rates of callus induction (97.8%) and shoot regeneration (97.3%) and maximum regenerated shoots per explant (2.52) at 45 d of culture and was further used for genetic transformation.

      Table 1.  Effect of different PGRs on regeneration of F. nilgerrensis leaf discs at 45 d of culture.

      Media codeConcentration (mg·L−1)Callus induction rate (%)Shoot regeneration rate (%)Shoot no. per explant
      TDZBAIBA
      A1C110.121.1 ± 1.91 i9.1 ± 2.08 j0.11 ± 0.04 gh
      A1C210.294.2 ± 2.14 ab37.8 ± 1.91 e0.40 ± 0.09 e
      A1C310.361.1 ± 1.91 e0.0 ± 0.00 l0.0 ± 0.00 i
      A2C120.180.0 ± 2.00 c79.0 ± 3.60 c1.47 ± 0.08 c
      A2C220.297.8 ± 0.57 a97.3 ± 0.58 a2.52 ± 0.09 a
      A2C320.364.5 ± 3.87 de4.4 ± 1.93 k0.07 ± 0.03 hi
      A3C130.125.6 ± 1.96 h17.8 ± 1.91 h0.19 ± 0.02 fg
      A3C230.292.1 ± 1.82b89.5 ± 0.50 b1.96 ± 0.07 b
      A3C330.362.2 ± 3.89 e20.3 ± 0.58 g0.25 ± 0.09 f
      A4C140.167.8 ± 1.91 d0.0 ± 0.00 l0.0 ± 0.00 i
      A4C240.245.6 ± 1.96 g0.0 ± 0.00 l0.0 ± 0.00 i
      A4C340.37.1 ± 2.54 j0.0 ± 0.00 l0.0 ± 0.00 i
      B1C110.15.6 ± 1.93 j0.0 ± 0.00 l0.0 ± 0.00 i
      B1C210.25.6 ± 1.93 j0.0 ± 0.00 l0.0 ± 0.00 i
      B1C310.327.8 ± 1.91 h0.0 ± 0.00 l0.0 ± 0.00 i
      B2C120.127.8 ± 1.91 h0.0 ± 0.00 l0.0 ± 0.00 i
      B2C220.247.7 ± 1.31 fg14.3 ± 2.31 i0.13 ± 0.05 gh
      B2C320.367.8 ± 1.91 d66.7 ± 3.51 d0.6 ± 0.10 d
      B3C130.125.2 ± 2.54 h0.0 ± 0.00 l0.0 ± 0.00 i
      B3C230.24.5 ± 1.91 j0.0 ± 0.00 l0.0 ± 0.00 i
      B3C330.350 ± 3.30 f34.4 ± 1.96 f0.35 ± 0.08 e
      B4C140.15.6 ± 1.91 j0.0 ± 0.00 l0.0 ± 0.00 i
      B4C240.28.9 ± 5.06 j0.0 ± 0.00 l0.0 ± 0.00 i
      B4C340.38.3 ± 1.67 j0.0 ± 0.00 l0.0 ± 0.00 i
      Values represent mean ± SE; Different lowercase letters indicate significant differences among treatments according to the LSD test (P < 0.05).

      Figure 1. 

      Effect of PGRs on regeneration of F. nilgerrensis leaf discs. (a) Effects of TDZ and 6-BA on callus formation at 25 d of culture. (b) Shoot regeneration curve cultured on A2C2 medium for different days. (c) Shoot regeneration process cultured on A2C2 medium for different days.

      The shoot regeneration rate was measured every 5 d on A2C2 medium (Fig. 1b). It can be seen from Fig. 1b that shoot regeneration rate was 2.6% at about 25 d of culture and the regeneration rate rapidly increased from 35 to 45 d of culture, reached the peak of 97.3% at 45 d of culture and became stable. Moreover, the process of callus induction and shoot regeneration were continuously observed on A2C2 media for 60 d (Fig. 1c). A large amount of callus was formed around 15 d of culture, and began to differentiate into shoots at 25 d of culture. The height of shoots reached 1.0−2.0 cm at 45−60 d of culture.

      To analyze the influence of dark culture on regeneration efficiency, five treatments of dark culture with 0, 7, 14, 21, and 28 d respectively were used in the experiment. The callus weight and shoot regeneration rate first increased and then decreased as the dark culture duration increased (Fig. 2a), and at 45 d of inoculation, the shoot regeneration rate of 14 d dark culture was the highest (98.5%), which was similar to that of 21 d dark culture. Furthermore, the rate of callus induction and shoot regeneration rate were very low in the treatment of 0 d dark culture (Fig. 2c), which is significantly lower than that of 7 d dark culture. While dark culture for over 28 d led to browning and blackening of callus and decreased the callus induction and regeneration rates (Fig. 2c). These results indicated that dark culture duration affected the rate of callus induction and quality of callus, and then affected the regeneration rate.

      Figure 2. 

      Effects of CH concentrations and dark culture duration on the regeneration of F. nilgerrensis leaf discs at 45 d of culture. (a) The callus weight and shoot regeneration rates under different CH concentrations and dark culture duration. (b) The callus growth curve on the media containing 0.0 and 0.5 g·L−1 CH. (c) Effect of dark culture for 0, 7, 14, 21, and 28 d on regeneration. (d) The effect of 0.0, 0.2, 0.5 , 0.7, and 1.0 g·L−1 CH on regeneration. Bars indicate the S.E. of the means; different lowercase letters indicate significant differences among the treatments according to the LSD test (P < 0.05).

      The effects of different concentrations of CH on regeneration were evaluated in our study. After 45 d of culture, CH at five concentrations significantly affected the rates of shoot regeneration. The shoot regeneration rate and callus weight first increased and then decreased as CH concentration increased, and the highest regeneration rate (96.6%) and callus weight (7.2 g) was attained at 0.5 g·L−1 CH, similar to at 0.2 g·L−1 CH (Fig. 2a). The percentage of callus with thick, yellow, granular, and tight texture was higher at 0.2 or 0.5 g·L−1 CH, while the percentage of callus with brown color increased on the medium with 0.0, 0.7, or 1.0 g·L−1 CH (Fig. 2d).

      In the current experiment, two types of callus appeared: Type I callus, the callus was light yellow and compact, with a higher regeneration rate; Type II callus, the callus was brown, massive, or spongy, with a lower regeneration rate. The effect of different concentrations of CH on the percentages of Type I and Type II callus were compared (Table 2). The induction rate of Type I callus was only 6.11% on the medium without CH. However, the highest induction rate of Type I callus (87.23%), and the lowest induction rate of Type II callus (9.44%) were obtained when the 0.5 g·L−1 CH was added to the medium. When CH concentration continued to increase, the induction rate of Type I callus began to decrease. Therefore, the addition of CH with optimal concentration in the medium was beneficial to the formation of Type I callus and the optimal concentration of CH was 0.5 g·L−1.

      Table 2.  The rates of Type I and II callus on the medium with different CH concentrations of F. nilgerrensis leaf discs at 45 d of culture.

      CH concentration (g·L−1)No. of leaf discsRate of callus induction (%)Rate of Type I callus (%)Rate of Type II callus (%)
      0.06075.56 ± 0.96 c6.11 ± 0.96 e69.44 ± 0.96 a
      0.26091.11 ± 0.96 b81.11 ± 0.96 b10.00 ± 1.67 d
      0.56096.67 ± 1.67 a87.23 ± 1.93 a9.44 ± 1.93 d
      0.76071.11 ± 1.92 d21.67 ± 1.67 c49.45 ± 2.54 b
      1.06062.78 ± 0.96 e17.22 ± 0.96 d45.56 ± 1.93 c
      Values represent mean ± SE; Different lowercase letters indicate significant differences among treatments according to the LSD test ( P < 0.05).

      Furthermore, the effects of 0.0 and 0.5 g·L−1 CH on the callus growth were compared every 5 d for 45 d. The growth curve presented a typical 'S' shape on both concentrations (Fig. 2b), but the callus growth was significantly higher at 0.5 g·L−1 CH than at 0.0 g·L−1 from 25 d to 45 d of culture. The biomass of the callus steadily rose with prolonged culture. The callus growth rate was the highest at 25 d to 35 d and became stable after 35 d.

      Observation of callus paraffin sections

    • The process of callus growth in our experiment was as follows: first, the leaves began to curl, and the callus appeared within 7 d of culture when the leaf discs were cultured on the regeneration medium in the dark. A large amount of white callus was formed around 10−20 d of culture and covered with the whole leaves. Then some white callus turned into Type I callus at 20−35 d of culture on the A2C2 medium with 2.0 mg·L−1 TDZ, 0.2 mg·L−1 IBA, and 0.2 or 0.5 g·L−1 CH under the dark culture for 14−21 d of culture, and began to differentiate into shoots at 25 d of culture, which proved the regenerative pathway of F. nilgerrensis leaf discs was indirect organogenesis. However, the white callus turned brown (Type II) with a lower differentiation rate.

      Light yellow and brown callus was observed at 25 d of culture through paraffin sections. Yellow callus was formed by smaller cells with regular shape, close arrangement, and obvious nuclei, which showed the morphological characteristics of embryogenic callus (Fig. 3a). However, the cells of brown non-embryonic callus were bigger, irregular, more loosely arranged, and highly vacuolated ones with larger intercellular spaces, and non-obvious nucleus (Fig. 3b). These results indicated that the regeneration pathway of indirect organogenesis for F. nilgerrensis leaf discs was yellow embryogenic callus as the intermediate process.

      Figure 3. 

      Paraffin section observation of callus obtained from F. nilgerrensis leaf discs. (a) Type I callus (left) and its cells (right, black arrow), bar = 100 μm. (b) Type II callus (left) and its cells (right, black arrow), bar = 100 μm.

      Optimization of Kan concentrations and Agrobacterium-mediated method for the transformation of F. nilgerrensis leaf discs

    • The non-transformed explants were cultured in the shoot regeneration medium supplemented with different Kan concentrations (0, 5, 10, and 15 mg·L−1) for screening of threshold concentrations and to inhibit non-transgenic shoots. The results showed that shoot differentiation was inhibited at 5 mg·L−1 Kan concentration and all explants died at 10 and 15 mg·L−1 Kan concentrations (Table 3). Therefore, 10 mg·L−1 Kan was selected as the threshold for non-transformed explants.

      Table 3.  The regeneration rates in different Kan concentrations of F. nilgerrensis leaf discs at 45 d of culture.

      Kan concentration (mg·L−1)No. of leaf discsRate of regeneration (%)
      06098.32 ± 2.01a
      56051.36 ± 1.62b
      10600.08 ± 0.01c
      15600.00c
      Values represent mean ± SE; Different lowercase letters indicate significant differences among treatments according to the LSD test (P < 0.05).

      Kan concentrations were set to 10, 20, 30, 40 and 50 mg·L−1 to determine the effects on transformation efficiency. The results showed that significant differences were detected among the different concentrations and the percentages of resistant shoot regeneration and transformation decreased with increasing Kan concentrations (Table 4). At 10 mg·L−1 Kan concentrations, the number of PCR-positive transformants was less than half that of resistant shoots, indicating some false positive plantlets. At 50 mg·L−1 Kan, the percentage of the resistant shoot regeneration and transformation efficiency was 0.00%, demonstrating a substantial impairment in the ability of shoot regeneration from leaf discs. Furthermore, we found that compared with 30 mg·L−1 Kan, although 90% resistant shoots were identified as PCR-positive transgenic lines using 20 mg·L−1 Kan, the resistant shoot regeneration rate and transformation percentage were greater, and a relatively high transformation efficiency was obtained. Therefore, 20 mg·L−1 Kan was used as the optimal concentration for efficient genetic transformation.

      Table 4.  Effect of Kan concentrations on Agrobacterium-mediated transformation of F. nilgerrensis leaf discs.

      Kan concentration
      (mg·L−1)      		No. of infected explantsRate of resistant shoot regeneration (%)
      at 65 d post-infection      		Percentage of transformation (%)
      at 130 d post-infection
      106024.74 ± 0.62a10.98 ± 0.13a
      206010.25 ± 0.18b9.22 ± 0.20b
      30603.33 ± 0.14c3.22 ± 0.25c
      40601.67 ± 0.01d1.67 ± 0.01d
      50600.00e0.00e
      Values represent mean ± SE; Different lowercase letters indicate significant differences among treatments according to the LSD test (P < 0.05).

      The result of Agrobacterium-mediated transformation for 600 leaf discs of F. nilgerrensis showed the formation of callus at 15 d and the regeneration of resistant shoots at 65 d post-infection (Fig. 4). The putative transgenic plantlets were obtained through 60−90 d of elongation culture of resistant shoots. Meanwhile, the experiment showed the regeneration rates of resistant shoots (%) at 65 d post-infection for the three replicates were 10.12%, 9.56%, and 9.11% respectively, and the average value was 9.60%, which indicated that a relatively stable transformation system was established. PCR analysis of resistant transgenic lines will be performed to identify positive transgenic lines at 130 d post-infection.

      Figure 4. 

      Agrobacterium-mediated genetic transformation of F. nilgerrensis leaf discs.

      Identification of transgenic lines

    • A total of 58 putative transgenic lines were recovered by Agrobacterium-mediated transformation of F. nilgerrensis leaf discs, of which 52 (89.66%) were confirmed as transgenic lines after PCR analysis. It can be seen from the PCR results that the 1,800 bp GUS gene was amplified to varying degrees from 52 positive transgenic lines (Fig. 5), while the negative controls showed no amplification. From the above, the transformation percentage for F. nilgerrensis leaf discs was 8.67% (52 out of 600). Meanwhile, using this system, it took approximately 4−5 months to form PCR-positive transgenic lines.

      Figure 5. 

      PCR analysis of GUS gene in transgenic lines. Lane M, DNA marker; Lanes 1–58, 58 putative transgenic lines; Lane WT, wild-type plant; Lane P, plasmid control; Lane H, ddH2O.

      GUS staining showed that the positive lines' whole or part of the leaves showed a blue reaction, which confirmed the expression of the GUS gene. Meanwhile, the control leaves displayed no blue GUS staining (Fig. 6).

      Figure 6. 

      Histochemical GUS staining of transgenic lines. (a) Wild-type plant. (b)–(d) PCR-positive transgenic plants.

      The regeneration rates of leaf discs from transgenic lines were assessed to further segregate Kan resistance transformants and identify the transgene integration. The results showed that the regeneration rates of leaf discs for three repetitions were 90%, 80% and 90%, respectively, with an average regeneration rate of 86.67%, which indicated the transgenic lines were resistant to Kan and the transgene integration of PCR-positive transformants (Fig. 7). Meanwhile, all leaf discs of the non-transgenic plants were brown within 45 d of culture.

      Figure 7. 

      Transformants regeneration on medium with Kan. (a) Leaf regeneration of PCR-positive transgenic lines under Kan selective pressure. (b) Non-transformed plants (control).

    DISCUSSION

    • The construction of a high-efficiency regeneration system is essential for the successful genetic transformation of strawberries. There are two ways to establish a strawberry regeneration system: organogenesis and somatic embryogenesis, both of which include direct and indirect regeneration. The indirect regeneration pathway is the formation of the callus as the intermediate process. Despite reports of somatic embryogenesis regeneration from various strawberry explants, this pathway was still much less efficient and generally occurred directly, without the formation of callus[5,32−34], which hinders genetic transformation. Therefore, the main regeneration pathway for strawberry is organogenesis. Numerous studies have demonstrated that the regeneration of various strawberry cultivars is based on the callus stage[9] and the formation of embryogenic callus with a crisp texture, compact structure, and nodular or tumor-like protrusions can significantly increase the regeneration rate[35]. The most effective induction rate (71.4%) of embryogenic callus was obtained for 'Jukhyan' strawberry anther after optimizing the factors including AgNO3, Fe-EDTA and types of media, and the regeneration rate was highest (92.7%) on 1/2 MS medium[36]. It is reported that adding yeast extract[37], CH[38], dicamba[39], AgNO3[40], carbon sources[41], and activated charcoal[35] into the medium increased the induction rate of embryogenic callus and affected the maintenance and differentiation rate of the embryogenic callus. For example, nutrients including CH and potassium nitrate that were added to the medium increased the rate of the callus and shoot formation for 'Allstar' strawberry leaf[42]. Some researchers showed that different CH concentrations affected the callus induction and status and identified 0.2−1.0 g·L−1 as the suitable concentration for the regeneration of different crops[37,43]. In our experiment, in the presence of CH at 0.2 or 0.5 g·L−1, embryogenic callus was formed at 20−35 d of culture and directly differentiated into shoots at 25 d of culture, and the regeneration rate increased and reached 97.3% at 45 d of culture. Therefore, the regenerative pathway for F. nilgerrensis leaf discs was indirect organogenesis and embryogenic callus induction is an effective way to build an efficient regeneration system; here, CH plays a key role. The formation of embryogenic callus was the key to the substantial increase in the regeneration rate of F. nilgerrensis leaf discs, which guaranteed the regeneration of putative transgenic shoots.

      Efficient shoot regeneration and rigorous antibiotic screening will considerably reduce the possibility of chimeric shoots in the genetic transformation of strawberries and facilitate the recovery of transgenic plants[20,44]. In our study, based on efficient shoot regeneration (97.3%), the transformation percentage for F. nilgerrensis leaf discs reached 8.67%, and PCR-positive transgenic lines were obtained in 16–20 weeks, which significantly improved over 20−24 weeks[15]. This may be due to the high-efficiency regeneration system based on embryonic callus development, which might boost the efficiency of selecting and proliferating single transformed cells, thereby reducing fewer chimeras and increasing the transformation efficiency[45]. Simultaneously, non-transgenic plants and false positive plants were eliminated by using different Kan concentrations; at Kan concentrations greater than 10 mg·L−1, non-transgenic leaf disc regeneration was severely hampered, which was in line with the findings of other studies[13]. However, a concentration of 20 mg·L−1 achieved efficient genetic transformation while avoiding the risk of chimerism or false positive plants in our experiment, which was lower than the value (25%−50%) reported in 'Chandler' by Husaini[20]. These differences are most likely due to the cultivars with varying levels of antibiotic tolerance.

    CONCLUSIONS

    • In this study, the high-efficiency regeneration system for F. nilgerrensis leaf discs with a 97.3% regeneration rate was established on the MS medium with 2.0 mg·L−1 TDZ, 0.2 mg·L−1 IBA, and 0.2 or 0.5 g·L−1 CH in the dark culture for 14 to 21 d after optimizing several regeneration factors, which guaranteed the regeneration of putative transgenic shoots. By paraffin section observation of the callus from the leaf disc regeneration, the light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Furthermore, an average 8.67% transformation percentage was achieved by screening Kan concentration and referring to transformation procedures described by predecessors and PCR-positive transformants were obtained within 4−5 months. These findings will speed up studies on the gene functions and trait improvement of this species.

    MATERIALS AND METHODS

      In vitro apical meristem culture and tube plantlet formation in F. nilgerrensis

    • The plants of F. nilgerrensis (accession code SN11-6) were collected from Yiliang County (25°4'16" N, 103°13'16" E), Kunming City, Yunnan Province (China), in 2019 and grown in the open field of Shenyang Agricultural University (41°49'48" N, 123°34'12" E). Then 2−3 cm long runner-tips of F. nilgerrensis were collected, disinfected with 75% (v/v) alcohol for 10 s and 0.1% (v/v) mercuric chloride for 15 min in turn, and then rinsed three times with sterile and distilled water. The 0.5−1.0 mm apex meristems of the runners were then inoculated on MS media to grow into plantlets. The tube plantlets used for leaf regeneration were allowed to proliferate on the subculture medium MS + 0.5 mg·L−1 6-BA + 0.2 mg·L−1 GA3 (gibberellic acid). These plants on the subculture media were maintained in a growth chamber at 25 ± 2 °C under a 16 h photoperiod with 250 mol·m−2·s−1 illumination intensity and sub-cultured every four weeks.

      Optimization of leaf regeneration conditions in F. nilgerrensis

    • Regeneration trials were performed using young expanded leaves detached from the tube plantlets. The leaf was cut transversely along the mid-vein to obtain the leaf discs of 0.8−1.2 cm as explants; these leaf discs were placed with the downward abaxial surface in contact with the regeneration media. A total of 34 assays with various combinations of treatments were designed, including different plant growth regulators (PGRs) (Table 1), dark culture durations (0, 7, 14, 21 and 28 d) and CH concentrations (0.0, 0.2, 0.5, 0.7 and 1.0 g·L−1), to optimize the regeneration system.

      Thirty explants were maintained as three replicates for each treatment. After 45 d of culture, the percentages of callus induction and shoot regeneration were calculated as follows, and the number of shoots per leaf explant was measured. Callus induction rate (%) = (No. of leaf discs induced callus/ No. of inoculated leaf discs) × 100; Shoot regeneration rate (%) = (No. of explants regenerated shoots/ No. of cultured explants) × 100.

      Observation of callus paraffin section

    • Paraffin section of the callus cultured for 25 d was used for further observation. The callus was fixed in formaldehyde–acetic acid–ethanol (FAA) solution for 2−4 d, dehydrated, embedded and stained[31]. The stained sections were observed, and images were captured using an Olympus microscope (SZX10, Olympus Corporation, Japan).

      Agrobacterium strain and plasmid used for genetic transformation

    • A binary vector pRI201-AN-GUS (TaKaRa, Dalian, China) (Supplemental Fig. S1), carrying the kanamycin (Kan) resistance gene NPTII (Neomycin phosphotransferase II) as a selection marker and the CaMV 35S promoter-driven GUS reporter gene, was used for genetic transformation. The Agrobacterium tumefaciens strain GV3101 was used for this process [46].

      Optimization of kanamycin concentrations and Agrobacterium-mediated transformation of F. nilgerrensis leaf discs

    • The method of Agrobacterium-mediated genetic transformation for F. nilgerrensis leaf discs was referred to the previously described procedure with a slight modification[20].

      The GV3101 cells transformed with the pRI201-AN-GUS plasmid were cultured in a YEP solid medium containing 50 mg·L−1 Kan and 20 mg·L−1 rifampicin (Rif) overnight at 28 °C on a rotary shaker (180 rpm·min−1). A single colony was confirmed by PCR using F: AGTCACGACGTTGTA and R: CAGGAAACAGCTATGAC primers, chosen, injected into 2 mL YEP liquid medium with the same antibiotics and cultured for 12−16 h at 28−30 °C on a rotary shaker (180 rpm·min−1). Then, 2 mL of the bacterial culture was added to 50 mL YEP liquid medium and cultured for 6−8 h to an OD600 value of 0.5−0.6 under the same antibiotics, temperature and speed. Then, the bacterial culture was centrifuged at 5000 r·min−1 for 5 min, and the obtained bacterial pellet was diluted in MS liquid medium (Table 5) to an OD600 value of 0.5. Finally, the bacterial suspension was mixed with 100 μmol·L−1 acetosyringone (AS) for explant infection. The whole processes of genetic transformation were co-culture, delayed-selection culture, selection and regeneration culture, shoot elongation culture and rooting culture, respectively.

      Table 5.  Composition of media used for genetic transformation of F. nilgerrensis leaf discs.

      ProcedureMedia compositionDuration
      Agrobacterium infectionMS liquid medium, 100 μmol·L−1 AS, 5 g·L−1 glucose and 15 g·L−1 sucrose10 min
      Co-cultureMS, 2.0 mg·L−1 TDZ, 0.2 mg·L−1 IBA, 0.5 g·L−1 CH, 7 g·L−1 agar, 5 g·L−1 glucose and 30 g·L−1 sucrose3 d
      Delayed-selection cultureMS, 2 mg·L−1 TDZ, 0.2 mg·L−1 IBA, 0.5 g·L−1 CH, 7 g·L−1 agar, 30 g·L−1 sucrose, 250 mg·L−1 Tim and 250 mg·L−1 Cef4 d
      Selection and regeneration cultureMS, 2.0 mg·L−1 TDZ, 0.2 mg·L−1 IBA, 0.5 g·L−1 CH, 7 g·L−1 agar, 30 g·L−1 sucrose, 250 mg·L−1 Tim, 250 mg·L−1 Cef and 10/20 mg·L−1 Kan60 d
      Shoot elongation cultureMS, 2.0 mg·L−1 TDZ, 0.2 mg·L−1 IBA, 0.5 g·L−1 CH, 7 g·L−1 agar, and 30 g·L−1 sucrose, 250 mg·L−1 Cef and 10 mg·L−1 Kan60−90 d
      Rooting culture1/2 MS, 0.1 mg·L−1 IBA, 15 g·L−1 sucrose and 5 g·L−1 agar30 d

      For co-culture, green leaves from 20-day-old plantlets were cut into leaf discs. The explants immersed in bacterial solution directly were gently shaken every 2 min for 10 min, dried on sterile filter paper, and then cultured in Petri dishes (100 mm × 25 mm) containing a co-culture media with the abaxial side up (Table 5). The plate was sealed with parafilm and incubated at 25 ± 2 °C in the dark for 3 d.

      After 3 d of co-culture, explants were washed in a liquid co-culture medium supplemented with 250 mg·L−1 cefotaxime (Cef) and 250 mg·L−1 timentin (Tim), rinsed with vigorous shaking to remove the Agrobacterium cells, and then blotted dry on sterile filter paper. After washing, the explants were transferred to a delayed-selection media for 4 d (Table 5) and cultured under the same conditions as mentioned earlier.

      The different concentrations of Kan (0, 5, 10, and 15 mg·L−1) were screened to determine the threshold concentration for eliminating non-transformed explants. Three replicates with 60 non-transformed explants each were used. The regeneration rate was calculated at 45 d of culture. After 4 d of delayed-selection culture, infected explants were transferred to a fresh selection and regeneration media with 10 mg·L−1 Kan for the selection of transformed cells and to inhibit further Agrobacterium growth for 14 d (Table 5). Further, to evaluate the effects on transformation efficiency, treatments with different Kan concentrations were used in this assay. After 14 d selective regeneration culture, the explants were subcultured on the selective media with different concentrations of Kan (10, 20, 30, 40 and 50 mg·L−1) for eliminating false positive transformed shoots for more than 4 weeks and the regeneration rate of the resistant shoot was calculated at 65 d post-infection. Sixty infected explants were cultured with three replicates per treatment.

      The regeneration shoots were then subcultured on a selective elongation media with 10 mg·L−1 Kan and 250 mg·L−1 Cef for 8−12 weeks, and the transformation percentage was determined through PCR analysis at 130 d post-infection. After elongation culture, 4−5 cm long shoots were transferred to root induction medium for rooting (Table 5), and then transferred into 72-hole plug trays, followed by 10 cm × 10 cm pots, and finally planted in the open field. All the cultures were kept in the culture room at 25 ± 2 °C. Regeneration rate of resistant shoots (%) = No. of explants regenerated / No. of infected explants × 100; Transformation percentage (%) = No. of PCR-positive transgenic lines / No. of infected explants × 100.

      To test the system's stability, 600 leaf discs with three replicates were used for genetic transformation. The regeneration rate of resistant shoots at 65 d post-infection and transformation percentage at 130 d post-infection was calculated.

      Confirmation of transgenic lines

    • Polymerase chain reaction (PCR) analysis was performed using putative-resistant transgenic lines to detect the GUS marker gene and confirm the positive transgenic lines. One plantlet was randomly selected from each line for the experiment. Genomic DNA was extracted from leaves using a DNA extraction kit (Aidlab, Beijing, China). DNA samples from non-transgenic wild-type plants and ddH2O were used as negative controls, while the overexpression binary vector pRI201 plasmid acted as a positive control. The PCR to amplify the GUS gene (1800 bp) was performed in a 20 μL reaction mixture consisting of 1 μL of each primer (GUS-F: 5'-ATGTTACGTCCTGTAGAAAC-3' and GUS-R: 5'-TCATTGTTTGCCTCCC TGCT-3'), 10 μL 2×Taq Master Mix (Vazyme, Nanjing, China), 7 μL distilled sterile water, and 1 μL DNA (100 ng) on a PCR system with the conditions of denaturation (95 °C for 1 min), amplification with 28 cycles (95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s) and extension (72 °C for 7 min) in turn, following the method by Li et al.[13].

      Histochemical GUS assay was performed using a GUS staining kit (Biosharp, Anhui, China) with PCR-positive transgenic lines to confirm the transgenic lines following the method by Li et al.[13]. Wild-type plants were used as control.

      To further segregate Kan resistance PCR-positive transgenic lines and identify the transgene integration, the regeneration rates of leaf discs from the progeny of primary transgenic lines were assessed. Leaves of transgenic lines were randomly selected and cut into leaf discs to be inoculated on the regeneration medium with 10 mg·L−1 Kan for 2−3 weeks in the dark. Thirty leaf discs with three replicates were used. The leaves of the wild-type plants were used as the control. The regeneration rate was determined after 45 d of culture.

      Statistical analysis

    • Data significant differences were calculated using the IBM SPSS Statistics 20 test and significant differences were set at P < 0.05.

      Acknowledgments

      • This work was supported by Liaoning Provincial Science and Technology Project of 'Jiebangguashuai' (No. 2022JH1/10400016) and Shenyang Academician and Expert Workstation Project (2022-15).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Supplementary information
      Rights and permissions
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (7)  Table (5) References (46)
  • About this article
    Cite this article
    Jiang S, Ji Y, Wang M, Xue L, Zhao J, et al. 2023. Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis. Fruit Research 3:9 doi: 10.48130/FruRes-2023-0009
    Jiang S, Ji Y, Wang M, Xue L, Zhao J, et al. 2023. Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transformation in Fragaria nilgerrensis. Fruit Research 3:9 doi: 10.48130/FruRes-2023-0009
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Catalog

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  • Introduction
  • Results

  • Optimization of regeneration conditions in f. nilgerrensis leaf discs

  • Observation of callus paraffin sections

  • Optimization of kan concentrations and agrobacterium-mediated method for the transformation of f. nilgerrensis leaf discs

  • Identification of transgenic lines

  • Discussion
  • Conclusions
  • Materials and methods

  • in vitro apical meristem culture and tube plantlet formation in f. nilgerrensis

  • Optimization of leaf regeneration conditions in f. nilgerrensis

  • Observation of callus paraffin section

  • agrobacterium strain and plasmid used for genetic transformation

  • Optimization of kanamycin concentrations and agrobacterium-mediated transformation of f. nilgerrensis leaf discs

  • Confirmation of transgenic lines

  • Statistical analysis

  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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