A Fruit-Pulp-Derived Callus-Level Agrobacterium-Mediated Transformation Platform for Ziziphus jujuba

TL;DR

A new transformation system for jujube using fruit pulp callus improves genetic studies.

Contribution

A novel fruit-pulp-derived callus transformation system for jujube with high efficiency.

Findings

Fruit-pulp-derived callus showed genotype-specific auxin and antibiotic sensitivity.

The pCY vector achieved 77.8% transformation efficiency in jujube.

ZjCBF3 transgene integration and expression were confirmed using PCR and fluorescence.

Abstract

The jujube (Ziziphus jujuba Mill.) is a significant economic fruit tree, valued for its nutritional and medicinal properties. However, advances in functional genomics are hindered by the lack of an efficient transformation system. To overcome the limitations of conventional explant, we established a fruit-pulp-derived, callus-based Agrobacterium-mediated transformation system using fruit-pulp harvested 50 days after pollination. Through orthogonal experimental design, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid were identified as key regulators for inducing high-quality, friable callus in two jujube genotypes, ‘JZ60’ and ‘LWCZ’. This system revealed significant genotype-specific variation in auxin requirements for callus proliferation and in differential antibiotic sensitivity. Transformation efficiency, as evaluated by fluorescence screening, was primarily determined by acetosyringone concentration and the binary vector architecture. The results revealed that the compact pCY (kanamycin resistance) vector achieved higher transformation efficiency (up to 77.8%) than pCAMBIA1301, whereas the pCAMBIA1301 (hygromycin resistance) vector enabled more uniform transgene expression. Integration and expression of the ZjCBF3 transgene were confirmed by polymerase chain reaction (PCR), reverse transcription quantitative PCR, and green fluorescent protein fluorescence assays. This study established a fruit-pulp-based callus transformation system for jujube, providing a rapid platform for its functional genomic studies.

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1. Introduction

The jujube (Ziziphus jujuba Mill.), a perennial fruit tree of the Rhamnaceae family, is one of China’s most economically, nutritionally, and medicinally valuable woody crops. Owing to its exceptional adaptability to arid and semi-arid environments and high content of sugars, vitamins, and bioactive compounds, the jujube has been widely cultivated in more than 40 countries worldwide [1,2]. In recent years, increasing demand for enhanced fruit quality, nutritional value, and stress resistance has intensified efforts to improve fruit quality and plant resistance through genomic strategies. However, the lack of an efficient transformation system continues to restrict progress in functional genomics of jujube.

Agrobacterium tumefaciens-mediated transformation remains the most widely used method for stable gene integration in plants due to its simplicity, low copy-number insertions, and high transgene stability [3,4]. This approach has been successfully applied to several fruit trees, providing efficient gene delivery and expression system [5,6,7,8]. Agrobacterium-mediated transformation of jujube shoot tips has been reported to achieve plantlet regeneration [9]. Recently, an Agrobacterium rhizogenes-mediated hairy root transformation system was established in sour jujube seedlings, providing a rapid root-based functional assay route [10]. However, the transformation systems specifically based on fruit pulp remain limited. Callus-based transformation from fruit tissues presents a strategic alternative for the functional study of fruit biology, whereas the callus-level transformation has proven to be a rapid platform for functional genomic studies. For instance, stable transgenic callus systems have been successfully employed to characterize key regulatory modules involved in anthocyanin biosynthesis and stress responses, such as the MdSIZ1-mediated low-temperature response [11], the MdMYB2-dependent cold tolerance pathway [12], and the MdNAC1 regulatory network for anthocyanin synthesis [13]. These studies demonstrate that stable callus transformation can reliably phenotype metabolic and stress-related traits without the prolonged cycle of whole-plant regeneration. Currently, reported methods, such as those applied to the cultivar ‘JZ39’ to investigate sugar metabolism-related genes [14] and to fruit pulp-derived callus of ‘Beiqi’ to study lignin biosynthesis genes [15], have achieved limited success. This is largely due to a combination of insufficient methodological details, genotype dependence, phenolic compound oxidation, and consequently, poor reproducibility.

Callus-mediated transformation can alleviate seasonal and physiological constraints in woody perennials by providing rapidly proliferating, developmentally plastic tissue for gene delivery and selection [6,16,17]. However, calli induced from conventional explants (e.g., leaf, cotyledon, anther) often suffer from browning and strong genotype dependence [18,19]. Fruit pulp is a low-lignified, metabolically active tissue that can generate stable callus cultures; in apples and related species, pulp-derived calli can be maintained long-term with robust phenolic and triterpene metabolism [20,21,22,23,24]. These observations suggest that fruit pulp may be a promising explant for transformation in Z. jujuba, but a transformation platform has not yet been established.

Transformation efficiency in woody plants is strongly influenced by genotype-specific hormonal responses and antibiotic sensitivity. Studies have demonstrated that the balance between auxin and cytokinin is closely associated with callus friability and regenerative capacity, and that these hormonal responses can differ substantially among genotypes [25]. In woody perennials, variations in tissue organization, phenolic metabolism, and antibiotic or selection-agent sensitivity among cultivars can further affect transformation and regeneration performance [26]. These genotype-dependent physiological traits necessitate cultivar-specific optimization of hormonal and selection regimes. Moreover, vector architecture, including T-DNA size, promoter-terminator configuration, and selectable marker genes, is decisive in balancing transformation efficiency and expression patterns [27,28]. Compact binary vectors such as pSiM24 and pCY often improve delivery due to reduced plasmid size and higher replication stability, but they can also increase mosaic or partial expression. In contrast, larger constructs (e.g., pCAMBIA1301) are more frequently associated with more uniform expression [28,29]. Understanding vector-genotype interactions is therefore critical for achieving both efficiency and stability in the transformation of recalcitrant woody species.

In this study, we developed a fruit-pulp-based Agrobacterium-mediated transformation platform for Z. jujuba using pulp tissues harvested 50 days after pollination (DAP) from two genotypes (‘JZ60’ and ‘LWCZ’). We used an L_18_ (3^7^) orthogonal design to optimize plant growth regulators, carbon sources, and selection conditions for callus induction and proliferation. We then evaluated the vector backbone and acetosyringone (AS) concentration to examine how transformation parameters interact with host tissue physiology. This callus-level workflow provides a robust route for functional studies in jujube and can be integrated with downstream regeneration protocols in future work.

2. Results

2.1. Orthogonal Screening Identifies 6-BA and 2,4-D as the Primary Drivers of Callus Induction

Based on the orthogonal screeding results, the test No. 10 produced the highest callus biomass in both genotypes, with the best values of 0.399 g for ‘JZ60’ and 0.351 g for ‘LWCZ’ (Table 1). Range analysis and Type II ANOVA consistently identified 6-BA as the dominant factor and 2,4-D as the second-most-influential factor for biomass accumulation in both genotypes (Tables S3 and S4). Post hoc LSD tests indicated optimal concentration ranges of 0.5–1.0 mg L^−1^ for 6-BA and 1.5–2.5 mg L^−1^ for 2,4-D (Table S5). Treatments within these ranges produced significantly higher biomass than the 0 mg L^−1^ controls, whereas differences among treatments within the ranges were not significant, indicating a plateau response. All high-performing treatments (Nos. 7, 10, and 16) fell within these empirically identified ranges (Table 1 and Table S5).

2.2. Callus Morphological Grading Correlates with Biomass

A four-tier morphological grading system (Grades I–IV) was established to classify calli based on key phenotypic traits: texture, color, and proliferation vigor (Figure 1; for detailed criteria, see Table S2). Grade I calli had a loose, friable structure, a creamy-white to pale-yellow appearance, and vigorous marginal proliferation with minimal browning. In contrast, Grade IV calli were dense and compact, with extensive browning and little or no proliferative activity. A strong positive correlation was observed between morphological grade and biomass accumulation. All Grade I calli, derived from the most effective media formulations (e.g., Test Nos. 7, 10, and 16), achieved the highest FWG. These Grade I calli were obtained under the high-performing media combinations identified in the orthogonal screening (Table 1; see Section 4.1) and consistently showed the highest FWG. Conversely, Grade IV calli showed negligible growth and were primarily induced under suboptimal conditions, frequently lacking 6-BA and/or 2,4-D or containing elevated levels of TDZ.

2.3. Proliferation Optimization for the Dominance of 6-BA and Genotype-Specific Response to 2,4-D

In proliferation assays, both genotypes showed strong dependence on 6-BA, with 1.0 mg L^−1^ generally supporting greater biomass than 0.5 mg L^−1^ (Figure 2; Tables S6 and S7). The optimal 2,4-D concentration differed between genotypes, peaking at 2.5 mg L^−1^ in ‘JZ60’ and 1.5 mg L^−1^ in ‘LWCZ’ (Tables S6 and S7). A significant 6-BA × 2,4-D interaction was detected in ‘LWCZ’, indicating genotype-specific auxin responsiveness across cytokinin levels (Table S7). Based on both biomass and morphology (Figure 2), 1.0 mg L^−1^ 6-BA plus 1.5 mg L^−1^ 2,4-D was selected as the standard proliferation medium for subsequent transformation experiments.

2.4. Determination of Genotype-Specific Antibiotic Selection Thresholds

The two jujube genotypes exhibited distinct responses to kanamycin and hygromycin (Figure 3). ‘LWCZ’ calli were highly sensitive to kanamycin, with visible browning at 10 mg L^−1^ and widespread necrosis at 30 mg L^−1^, indicating that 30 mg L^−1^ was an effective concentration for selection. In contrast, ‘JZ60’ was more tolerant, requiring 50 mg L^−1^ to achieve complete growth inhibition (Figure 3A). For hygromycin, the response pattern was reversed: ‘JZ60’ was highly sensitive, with growth nearly completely suppressed at 10 mg L^−1^, whereas ‘LWCZ’ required 20 mg L^−1^ for effective inhibition (Figure 3B). These results indicated that ‘LWCZ’ was more sensitive to kanamycin but more tolerant to hygromycin than ‘JZ60’. Based on these results, the following genotype-specific antibiotic regimes were used in subsequent transformations: 30 mg L^−1^ kanamycin and 20 mg L^−1^ hygromycin for ‘LWCZ’; 50 mg L^−1^ kanamycin and 10 mg L^−1^ hygromycin for ‘JZ60’.

2.5. Orthogonal Screening of Acetosyringone and Vector Backbone

Transformation efficiency varied widely across treatments, ranging from 1% to 74% (Table 2). Range analysis and Type II ANOVA identified acetosyringone (AS) concentration as the primary determinant of transformation efficiency in both genotypes (Tables S8 and S9). Omitting AS reduced efficiency to 1–4% (Table 2). The vector backbone made a secondary but significant contribution, with pCY outperforming pCAMBIA1301 (Table 2 and Table S9). The remaining factors (OD_600_, infection duration, and co-cultivation duration) had comparatively minor effects. A broadly effective parameter set was OD_600_ 0.8–1.0, 20 min of infection, and 3–4 days of co-cultivation. AS concentration remained the primary genotype-dependent variable for achieving high transformation efficiency.

2.6. EGFP Fluorescence Analysis for Transformation Efficiency

EGFP fluorescence analysis revealed distinct vector-dependent outcomes (Figure 4). In ‘JZ60’, the pCY vector yielded the highest transformation efficiency (77.8%), but exhibited greater phenotypic heterogeneity (Class I: 12/36; Class II: 16/36). The remaining 8 explants were classified as Class III (yellow-dominant, ambiguous) and excluded to avoid autofluorescence-related false positives (Figure 4B). In contrast, pCAMBIA1301 in ‘JZ60’ resulted in lower efficiency (47.2%), but superior uniformity, with the majority of positive explants classified as Class I (Figure 4A). In ‘LWCZ’, pCY provided a moderate efficiency advantage (50.0%) over pCAMBIA1301 (44.4%), while maintaining high expression homogeneity (Figure 4C,D).

2.7. Validation of ZjCBF3 Integration and Expression

Molecular analyses provided clear evidence of successful transformation. Genomic PCR confirmed the integration of the ZjCBF3 transgene in all EGFP-positive lines, with no amplification detected in wild-type controls (Figure 5A–D). RT-qPCR revealed strong transgene expression, with transcript levels several hundred-fold higher in transformed lines compared to wild-type calli (Figure 5E–H). The pCAMBIA1301 vector drove significantly higher ZjCBF3 expression than pCY in both genotypes, with a 2.3-fold increase in ‘JZ60’ and a 1.3-fold increase in ‘LWCZ’. Expression variability, however, differed among genotype-vector combinations. The most uniform expression was observed in ‘JZ60’ lines transformed with pCY (CV = 22.9%), while the greatest variability occurred in ‘LWCZ’ lines with pCAMBIA1301 (CV = 39.9%). These results confirmed the successful integration and expression of the transgene at the molecular level and highlighted the influence of vector choice and genotype on expression strength and homogeneity.

3. Discussion

A reproducible transformation workflow for recalcitrant woody species requires uniform, highly proliferative target tissues. In this study, immature fruit pulp collected approximately 50 days after pollination consistently provided friable callus suitable for Agrobacterium-mediated transformation and selection. Orthogonal screening followed by factorial refinement identified 6-BA and 2,4-D as the principal drivers of callus induction and proliferation, whereas higher TDZ tended to increase compactness/browning and reduce growth (Figure 2 and Figure 3; Table 1). Replacing sucrose with maltose further improved callus uniformity and reduced browning, consistent with reports that maltose can support in vitro competence and stabilize carbon/osmotic responses during tissue culture [30,31]. To minimize false positives in fluorescence-based screening, we applied conservative EGFP scoring criteria and calculated transformation efficiency using only clearly positive classes (Section 4.7; Figure 4). Collectively, these optimizations yielded a practical, fruit-pulp-derived callus platform for gene delivery at the callus level and downstream molecular assays in jujube.

Genotype strongly influenced tissue culture responses and selection stringency. Under identical induction conditions, ‘JZ60’ required a higher 2,4-D level for vigorous callus proliferation than ‘LWCZ’. This is consistent with genotype-dependent auxin perception and feedback regulation reported across species [30,31,32] and with AUX/IAA-mediated control of callus proliferation during 2,4-D-driven reprogramming [33,34]. Antibiotic responses were also genotype-specific: ‘LWCZ’ calli were more sensitive to kanamycin yet more tolerant of hygromycin than ‘JZ60’. Similar genotype-dependent selection responses have been documented in woody plants [6,35,36] and may reflect differences in antibiotic uptake, detoxification capacity, and stress physiology [37,38,39].

The vector system further shaped callus-level transformation outcomes and interacted with genotype-dependent selection stringency. Across both genotypes, pCY produced higher transformation efficiencies than pCAMBIA1301, whereas pCAMBIA1301 tended to yield fewer positives but a higher proportion of uniformly fluorescent calli (Figure 5 and Figure 6). This pattern is consistent with an efficiency–uniformity trade-off reported in woody plants and fruit trees [6,35,37]. Mechanistically, the compact pCY backbone may facilitate T-DNA delivery [27,40]. In contrast, differences in selectable markers, promoter–terminator combinations, T-DNA size, and cassette configuration may influence expression uniformity and variability through transcriptional stability and early epigenetic events associated with transgene silencing [41,42,43,44,45,46,47,48]. Importantly, the pCAMBIA1301-associated advantage is interpreted as improved expression uniformity among surviving calli rather than as evidence of more stable integration. Demonstrating integration stability will require additional molecular characterization (e.g., copy-number determination and insertion-site characterization) and validation in regenerated plants.

Although Agrobacterium-mediated transformation of Z. jujuba shoot tips has been achieved [9], shoot-tip explants may be less suitable for addressing questions closely linked to fruit tissues, such as aspects of fruit-associated secondary metabolism. Recently, a rapid Agrobacterium rhizogenes-mediated hairy root transformation system was successfully established in sour jujube using germinated seedlings [10]. While highly efficient for root-focused assays, transformation approaches relying on germinated seeds or seedlings introduce significant genetic heterogeneity because jujube is a highly heterozygous species. This genetic variation reduces reproducibility and poses challenges when targeting specific, cultivar-dependent fruit traits. In contrast, the primary advantage of using fruit pulp over germinated seeds is that pulp is a maternal somatic tissue, ensuring that the exact genetic background of the elite cultivar is perfectly preserved. Furthermore, fruit pulp provides a low-lignified, metabolically active tissue that yields abundant soft explants with reduced browning and strong callus competence. In this context, our fruit-pulp-derived callus platform offers a complementary option by enabling genotype-adapted selection and fluorescence-based screening directly in fruit-derived tissues, facilitating rapid, preliminary evaluation of candidate genes related to fruit traits before investing in whole-plant regeneration. Overall, efficient jujube transformation appears to require coordinated optimization across explant stage, culture regime, selection conditions, and vector system. While transformation was validated at the callus level, integrating this platform with an efficient regeneration system remains an important next step for broader application. Furthermore, this framework provides a practical basis for rapid functional assays not only in jujube but potentially in other recalcitrant woody plant species characterized by rich fruit pulp.

4. Materials and Methods

4.1. Plant Materials

Immature fruits of two jujube genotypes (Figure S1), ‘JZ60’ (Ziziphus jujuba Mill. cv. Jingzao60) and ‘LWCZ’ (Ziziphus jujuba Mill. cv. Lingwuchangzao), were harvested 50 days after pollination (DAP) at our experimental garden of Northwest A&F University in Qingjian, Shaanxi, China. Fruit pulps at 50 DAP were selected, rinsed with water for 5–10 min, and gently scrubbed in a detergent solution. They were then thoroughly rinsed with tap water for 30–60 min to ensure complete removal of detergent residues. Subsequently, the fruits were immersed in 75% (v/v) ethanol for 40 s and rinsed 1–2 times with sterile distilled water. Following this, they were treated with 2% (v/v) sodium hypochlorite (NaClO) for 8 min and subjected to 3–5 additional rinses with sterile distilled water to eliminate residual sterilants. Under strict aseptic conditions, fruit peels were removed, and the pulp tissues were sliced into explants measuring approximately 0.4–0.5 cm in thickness for subsequent experimental use.

4.2. Callus Induction: Medium and L18 (37) Design

The callus induction medium was prepared using MS basal medium (Duchefa Biochemie B.V., Haarlem, Netherlands) as the base, supplemented with 6 g L^−1^ agar (i.e., 4.44 g of MS powder dissolved in 1 L of purified water). The medium was further enriched with various carbon sources and combinations of plant growth regulators (PGRs). A five-factor, three-level orthogonal experimental design was conducted using an L_18_ (3^7^) orthogonal array to systematically evaluate the effects of sugar type (sucrose, maltose, or a mixture of sucrose and maltose) and four plant growth regulators, including thidiazuron (TDZ), 6-benzylaminopurine (6-BA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D), on callus induction from fruit pulp explants of two jujube genotypes, ‘JZ60’ and ‘LWCZ’. The factors and their respective levels were defined as follows: carbon source (20.00 g L^−1^ sucrose, 10.00 g L^−1^ sucrose and 10.00 g L^−1^ maltose, or 20.00 g L^−1^ maltose), TDZ (0.00, 1.00, or 2.00 mg L^−1^), 6-BA (0.00, 0.50, or 1.00 mg L^−1^), IBA (0.00, 0.20, or 0.50 mg L^−1^), and 2,4-D (0.00, 1.50, or 2.50 mg L^−1^). For each genotype-medium combination, 5–10 explants were inoculated per replicate, with five replicates per treatment. Cultures were maintained in complete darkness at 25–28 °C. After 35 days of culture, callus biomass was quantified as the increase in fresh weight. Morphological characteristics, including texture, color, proliferative ability, and overall growth patterns, were observed, recorded, and classified into four distinct grades (I–IV) based on color, texture/compactness, and growth vigor. Briefly, Grade I callus is pale/cream-colored, friable or granular, and shows vigorous proliferation; Grade II is light yellow with moderate friability and sustained growth; Grade III is yellow-brown, relatively compact, with reduced proliferation; and Grade IV is dark brown/necrotic with minimal growth, typically considered non-viable for downstream applications. Full grading descriptors and representative phenotypes are provided in Table S2.

4.3. Callus Proliferation

Following successful callus induction from fruit pulp explants, a 2 × 2 factorial experimental design was implemented to optimize proliferation for the two jujube genotypes, ‘JZ60’ and ‘LWCZ’. The experiment evaluated the effects of two key plant growth regulators: 6-BA at 0.50 and 1.00 mg L^−1^, and 2,4-D at 1.50 and 2.50 mg L^−1^. All proliferation media were based on MS salts supplemented with 20.00 g L^−1^ maltose as the carbon source and contained no TDZ or IBA. The four combinations were designated as T1 (0.5/1.5), T2 (0.5/2.5), T3 (1.0/1.5), and T4 (1.0/2.5), where the ratios represent the concentrations of 6-BA and 2,4-D (mg L^−1^), respectively. For each of the four treatment combinations, three biological replicates were established, with 36 callus pieces inoculated per replicate. Cultures were maintained in complete darkness at 25–28 °C, with subcultures performed every 21 days. At each subculture, fresh weight gain (FWG) was measured, and morphological characteristics (e.g., texture, color, proliferation vigor) were recorded. Calli demonstrating vigorous growth, friable texture, and minimal browning were selected for subsequent genetic transformation experiments, with the optimized medium designated as the standard proliferation medium.

4.4. Determination of Antibiotic Selection Thresholds for Transformation

To establish effective selection conditions for genetic transformation, an antibiotic sensitivity assay was conducted using untransformed calli of the jujube genotypes ‘JZ60’ and ‘LWCZ’. Calli were cultured on the optimized proliferation medium containing MS basal salts and maltose, supplemented with graded concentrations of kanamycin (10, 30, 50, or 70 mg L^−1^) or hygromycin (5, 10, 20, or 40 mg L^−1^). For each antibiotic concentration, 36 callus pieces were inoculated per Petri dish, with three independent biological replicates. Cultures were maintained in complete darkness at 25–28 °C for 21 days. Callus growth status, morphological development (including browning and necrosis), and survival rates were monitored and recorded weekly. The minimum antibiotic concentration that completely inhibited callus growth and induced severe tissue browning or necrosis was defined as the selection threshold for subsequent transformation experiments.

4.5. Binary Vector Construction

Two recombinant binary vectors for ZjCBF3 (GenBank accession No. CP157405.1) overexpression, pCAMBIA1301::35S::ZjCBF3 and pCY::35S::ZjCBF3, were constructed using established methods [49,50]. See Figure 6 for schematic diagrams and Table S1 for primer sequences. Both constructs, along with their corresponding empty vectors (pCAMBIA1301 and pCY), were verified by Sanger sequencing. The verified plasmids were introduced into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw transformation method. Positive transformants were selected on LB solid medium supplemented with 50 mg L^−1^ kanamycin and 25 mg L^−1^ rifampicin. The empty vectors pCAMBIA1301 (conferring hygromycin resistance) and pCY (conferring kanamycin resistance) were used as negative controls during parameter optimization (Section 4.6).

4.6. Optimization of Agrobacterium-Mediated Transformation Parameters

An L_18_ (2^1^·3^4^) orthogonal experimental design was employed to systematically optimize key parameters affecting Agrobacterium-mediated transformation efficiency in calli derived from fruit pulp of jujube genotypes ‘JZ60’ and ‘LWCZ’. The design incorporated one two-level factor, the vector backbone (empty pCAMBIA1301 vs. empty pCY), along with four three-level factors: bacterial optical density (OD_600_ = 0.6, 0.8, or 1.0), infection duration (10, 20, or 30 min), acetosyringone (AS) concentration (0, 100, or 200 μM), and co-cultivation duration (2, 3, or 4 days). Callus fragments (2–5 mm in diameter) were used as explants. Each treatment combination, consisting of 36 explants per Petri dish, was independently replicated three times. All infection and co-cultivation steps were conducted in the dark at 25–28 °C. After co-cultivation, explants were transferred to proliferation medium supplemented with genotype-specific selection antibiotics (determined in Section 4.4) and 400 mg L^−1^ carbenicillin to suppress Agrobacterium growth. Transformation efficiency, defined as the percentage of EGFP-positive calli, was quantified after 21 days of selection. The optimal parameters identified in this screening experiment were subsequently used for genetic transformation with the full-length recombinant vectors pCAMBIA1301::35S::ZjCBF3 and pCY::35S::ZjCBF3.

4.7. EGFP Fluorescence Observation and Transformation Efficiency Assessment

EGFP fluorescence in putative transgenic calli was assessed using a LUYOR fluorescence excitation system (LUYOR-3415RG; LUYOR, Newark, DE, USA) under blue light excitation (450 nm excitation, 500 nm emission filter). To ensure comparability across treatments, imaging was performed with the same optical setup, working distance, and acquisition settings for all samples within each experiment. Images were acquired with a Canon EOS 6D camera (Canon Inc., Tokyo, Japan) equipped with a 105-mm lens (manual mode; center-weighted metering; flash disabled; auto white balance). Automatic exposure was disabled, and fixed settings were applied across images: aperture was set to f/5.6, ISO to 100, and exposure time to 0.5 s. For reliable and objective evaluation, calli were systematically categorized into four distinct phenotypic classes: Class I (uniform and intense green fluorescence) and Class II (predominantly green fluorescence with minor yellow speckling or localized heterogeneity) were classified as EGFP-positive, indicating successful transgene expression. Class III (yellow-dominant signals with weak or discontinuous green fluorescence) was designated as ambiguous. Because Class III fluorescence signals may originate from endogenous autofluorescence or represent unstable, weak transgene expression, distinguishing true transformants from artifacts was unreliable without independent DNA-level validation. Consequently, to ensure rigorous data accuracy and avoid false positives, these calls were strictly treated as ambiguous and excluded from the efficiency calculation in this study. Class IV included non-fluorescent or necrotic tissues, representing non-viable or untransformed explants.

Transformation efficiency was evaluated under optimized experimental conditions using 36 explants per treatment. The metric was calculated as the percentage of EGFP-positive explants (sum of Class I and Class II) relative to the total number of explants (n = 36), according to the following equation: Transformation efficiency (%) = [(Number of Class I + Class II calli)/36] × 100.

5. Conclusions

We established a fruit-pulp-derived, callus-level Agrobacterium-mediated transformation workflow for Ziziphus jujuba by optimizing carbon sources (maltose), hormone regimes, and genotype-specific selection parameters. The results highlighted cultivar-dependent differences in auxin requirement and antibiotic sensitivity, supporting the need for tailored hormone and selection regimes. Vector architecture further influenced callus-level outcomes, with pCY yielding higher frequencies of EGFP-positive calli and pCAMBIA1301 showing more uniform fluorescence among positives. This framework provides a practical basis for callus-based transformation and rapid functional assays in jujube.