The Development of a Transformation System for Four Local Rice Varieties and CRISPR/Cas9-Mediated Editing of the OsCCD7 Gene

Abstract

Agrobacterium-mediated transformation systems are extensively applied in japonica rice varieties. However, the adaptability of local rice varieties to existing transformation systems remains limited, owing to their complex genotypes, posing a substantial challenge to transformation. In this study, four local rice varieties were selected to optimize the effects of different culture media on callus induction, browning resistance, contamination resistance, callus tolerance, differentiation, regeneration, and root development, and then two varieties were selected to improve plant architecture and tiller development by CRISPR/Cas9-mediated gene editing, based on constructive transformation systems. The goal was to enhance the transformation efficiency of local varieties and innovate germplasms. The results demonstrated that japonica rice varieties XG293 and WD68 exhibited higher induction rates under the treatment of 2 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) + 1 mg/L NAA (Naphthaleneacetic acid), whereas indica rice varieties H128 and E33 performed the best under 3 mg/L 2,4-D + 1 mg/L NAA. Severe browning in H128 was effectively mitigated by a carbon source of 20 g/L maltose supplemented with 40 mg/L ascorbic acid. Contamination after Agrobacterium infection was controlled by 300 mg/L Tmt (Timentin). Under a treatment of 200 µM/L acetosyringone +10 min infection duration, XG293 and WD68 exhibited higher callus tolerance, differentiation rates, and GUS staining rates, achieving transformation efficiencies of 43.24% and 52.38%, respectively. In contrast, H128 and E33 performed better under the treatment of 200 µM/L Acetosyringone + 5 min, with transformation efficiencies of 40.00% and 40.74%, respectively. The mutants after OsCCD7 gene editing in WD68 and H128 displayed a dwarfness of plant height, a significant increase in tiller numbers, and compact architecture. These findings demonstrate that an optimized combination of plant growth regulators and infection durations effectively improves transformation efficiency for local varieties, and the OsCCD7 gene regulates plant architecture and tiller development with variable effects, depending on the rice complex genotypes. This study provides a theoretical basis for the efficient transformation of local rice varieties and germplasm innovation.

1. Introduction

Rice is one of China’s primary staple crops and significantly contributes to national grain production [1]. Advances in biotechnology provide the theoretical foundation for precise improvements during rice breeding. Through extensive biotechnology applications, numerous rice varieties with desirable traits have been successfully developed. These advances not only enhance rice productivity but also reduce chemical pesticide usage, thus effectively contributing to environmental protection [2]. Genetic transformation is a fundamental technique in biotechnology, employing transformation methods such as biolistics, electroporation, pollen tube pathway, and Agrobacterium mediation [3]. Agrobacterium-mediated transformation is widely preferred owing to its high transformation frequency, low copy insertion, and broad host range [4].

Efficient tissue culture systems are essential for successful Agrobacterium-mediated genetic transformation of rice, requiring effective callus induction, differentiation, and plant regeneration. Callus induction and plant regeneration are affected by multiple factors, including genotype, explant type, medium composition, phytohormone regulators, and culture conditions [5]. Although highly efficient tissue culture systems are widely applied in model rice varieties, such as Nipponbare and Kasalath, local rice varieties exhibit complex genotypes, encountering problems such as low callus induction rates, severe browning, poor differentiation, low transformation efficiency, and poor seedling survival [6,7]. Establishing common and efficient transformation systems for local japonica and indica rice is thus essential for enhancing genetic transformation efficiency and innovating rice germplasm resources. With the development of the Asian economy, rice breeding faces the challenge of regionalized demand for high-yielding, high-quality varieties, which traditional breeding methods struggle to meet [8]. CRISPR-Cas9 genome editing technology, with its high efficiency and precision, has become a key tool for improving rice quality and agronomic traits, such as stress tolerance and yield [9]. This technology has successfully edited multiple quality-related genes and advanced functional genomics research, providing a new approach for developing high-quality rice varieties that adapt to environmental and market demands [10]. CCD7 (Carotenoid Cleavage Dioxygenase 7), a key enzyme in the biosynthesis of strigolactones (SLs), is responsible for catalyzing the carotenoids’ cleavage to generate SL precursor molecules. SLs negatively regulate rice tiller number by inhibiting the growth and development of lateral buds (axillary buds) [11]. Mutation of the CCD7 gene disrupts SL synthesis, thereby releasing the suppression of tillering and increasing the tiller numbers [12]. SL deficiency can indirectly affect the biosynthesis of gibberellin and auxin, leading to reduced plant height [13].

This study aimed to establish an efficient genetic transformation system for local rice varieties and utilized the CRISPR/Cas9-mediated gene editing tool to create novel germplasms with improved traits. Four local rice varieties were selected as materials to optimize hormone types and concentrations in induction media, reduce callus browning during subculture, and minimize contamination after Agrobacterium infection, thereby establishing an optimized induction culture system. This study also adjusted callus infection durations and Acetosyringone (AS) concentrations of infection buffer, transformed the GUS gene as a marker, assessed GUS-expressing calli activity, and optimized the rooting medium for regenerated seedlings. Positive rates of regenerated seedlings were detected to confirm the transgenic lines and establish a highly efficient transformation system. The OsCCD7 gene, regulating plant architecture in rice, was used to create novel germplasms using the CRISPR/Cas9 tool, based on the same efficient transformation system as above.

2. Materials and Methods

2.1. Experimental Materials

Two japonica rice varieties (XG293 and WD68) and two indica rice varieties (H128 and E33) were provided by Anhui Youxin Agricultural Technology Co., Ltd., Hefei, Anhui, China. as transformation recipients. The four varieties have relatively high plant height, low tiller numbers, stable yield, strong stress resistance, and a short growth period, making them suitable for crop rotation in the main grain-producing transitional zone in Anhui province (

Table 1. Source and type of four rice varieties.

Varieties Name	Variety Type	Plant Height/cm	Seed Setting Rate/%	Total Growth Period/d
XG293	japonica rice	95.4	89.4	147.1
WD68	japonica rice	90.0	80.0	149.0
H128	indica rice	118.5	89.9	112.7
E33	indica rice	124.0	85.0	135.0

).

2.2. Construction of CRISPR/Cas9 Vector for OsCCD7 Gene

The cDNA sequence of the OsCCD7 gene in Nipponbare rice was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 18 June 2023) and submitted to the CRISPR-P v2.0 database (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/SCORE, accessed on 18 June 2023) for target site prediction. The target sites proximal to the start codon (ATG) were selected to verify the location within exonic regions, referring to the alignment between the gDNA and cDNA sequences in the OsCCD7 gene. The target regions were amplified from varieties WD68 and H128 to confirm identity sequences with the Nipponbare genome, or else the target sites were redesigned. Based on the GT bon scaffold (gRNA scaffold), two optimal targets were isolated and cloned into the pBK1-Cas9-U3 vector by Golden Gate assembly (Eco31 I restriction sites) to obtain the fusion vector CRISPR/Cas9-ΔOsCCD7. The fusion vector was transformed into E. coli DH5α to prepare plasmid stocks. The pBK1-Cas9-U3 vector was ordered from the Wuhan Boyuan Biotechnology Co., Ltd. (#REC42-I, Wuhan, Hubei, China), with the vector map and construction procedures following the manufacturer’s protocol. The gRNA sequences and target regions are listed in Table S1 and Figure S1, respectively.

2.3. Establishment of Regeneration System of Callus

2.3.1. Callus Induction

Mature seeds without glume were selected, rinsed three times with sterile water, sterilized with 75% ethanol for 3 min, and then disinfected using a 3% NaClO solution for 40 min under shaking conditions (180 rpm). Seeds were rinsed five times with sterile water and dried on sterile filter paper. Sterilized seeds were cultured on induction medium for 15 days to obtain primary callus, followed by subculture on fresh induction medium for more 15 days. Callus size, fresh weight, dry weight, and induction rate were recorded. Dark cultivation was performed in a controlled environment incubator equipped with a humidity control system at 28 ± 2 °C, and the humidity was maintained with 60–70%. The induction media were prepared with the different combinations of 2,4-D and NAA (Table S2).

2.3.2. Anti-Browning Treatment of Callus

To prevent browning during subculture, different carbon sources (sucrose and maltose) were tested to identify the optimal type and concentration (Table S3). After determining the optimal carbon source, various concentrations of anti-browning agents were tested, such as vitamin C (Vc) and polyvinylpyrrolidone (PVP). The browning rate, browning index, size, and fresh and dry weight of the callus were evaluated to determine resistance to browning. The degree of browning was calculated based on the proportion of browned area to the total callus area and categorized into five levels (Figure S2) [14].

2.3.3. Pre-Infection and Recovery Culture of Callus

Agrobacterium strain EHA105 (Sangon Biotech, Shanghai, China), without plasmids, was cultured and centrifuged, and the bacterial suspension was diluted with infection buffer to the optimal density and measured at a wavelength of 600 nm to obtain an optical density value of 0.6–0.8 (OD₆₀₀ = 0.6–0.8, 1 × 10⁸ CFU/mL). The callus induced for 30 days was immersed in Agrobacterium suspension for 30 min, dried on sterile filter paper, and transferred onto co-cultivation medium at 28 °C for 3 days. The callus was subsequently rinsed 6–7 times with sterile water containing 400 mg/L cefotaxime (Cef), dried on sterile filter paper for 30 min, and transferred to recovery medium for 7 days. Contamination and initial differentiation rates were recorded. Recovery media with different antibiotics and concentrations were tested (Table S4). Culture conditions were maintained at 28 ± 2 °C, 60–70% humidity, a 16 h light/8 h dark photoperiod, and a light intensity of 1000–2500 lux.

2.3.4. Callus Differentiation Culture

After 45 days of recovery culture, the calli were transferred to differentiation media for 30 days, and differentiation rates were recorded. Differentiation media containing various types and concentrations of plant growth regulators (PGRs) were tested (Table S5). Culture conditions were the same as for the recovery culture.

2.4. The Establishment of the Transformation System

2.4.1. Preparation of Infection Suspension

The expression vector pCAMBIA2301-GUS (CaMV35S promoter) was obtained from our laboratory. The plasmid was extracted and transformed into Agrobacterium tumefaciens strain EHA105 using heat shock treatment at 42 °C. The transformed Agrobacterium was streaked on LB solid medium containing 50 mg/L kanamycin (Kan⁺) and 100 mg/L rifampicin (Rif⁺) and incubated at 28 °C for 3 days. A single bacterial colony was cultured in 50 mL LB liquid medium (containing Kan⁺) at 28 °C, with shaking for 24 h. The bacterial suspension was centrifuged, and the pellet was resuspended in infection buffer to an OD₆₀₀ of 0.6–0.8 for subsequent use.

2.4.2. Callus Infection and Recovery Culture

Using the optimized callus regeneration system, 30-day-old calli were immersed in Agrobacterium infection suspension containing the pCAMBIA2301-GUS plasmid and shaken for different durations at 28 °C with 180 rpm. The co-cultivation and rinse of the calli were performed according to the pre-infection steps as described above, and transferred to a recovery medium. The medium was refreshed every 15 days to promote calli regrowth, and the calli resistance rates were recorded after 45 days. Different combinations of AS concentrations and infection durations were evaluated (Table S6).

Healthy, compact, and uncontaminated calli were selected, and a subset was used for GUS staining. Calli were immersed in GUS staining solution at 37 °C for 12 h and sequentially decolorized in ethanol concentrations of 30%, 40%, 50%, 60%, and 70% (v/v) for 1 h (180 rpm). Stained tissues were observed and photographed under a microscope (YSZ710T, YUESHI, Suzhou, China). The GUS staining solution was prepared according to the manual (PH1727, PHYGENE, Fuzhou, China). The remaining calli were transferred to the differentiation medium (300 mg/L Tmt) for shoot formation over 15 days, and the differentiation rates were recorded.

2.4.3. Rooting Culture of Regenerated Seedlings

Regenerated seedlings with uniform growth and good morphology were transferred to a rooting medium. After 15 days, plant height and stem diameter were measured. Root parameters, including total root length, average root diameter, root surface area, and root volume, were analyzed using a root scanner analysis system (Regent instruments, Quebec, QC, Canada). Rooting media included N (N6 medium), NA (N6 medium + 0.2 mg/L NAA), M (1/2 MS medium), and MA (1/2 MS medium + 0.2 mg/L NAA).

2.4.4. Transformation of CRISPR/Cas9-ΔOsCCD7 into Rice

The plasmid of the CRISPR/Cas9-ΔOsCCD7 fusion vector was transformed into Agrobacterium tumefaciens strain EHA105, according to the established procedures above, and identified with the primers U3cef and CCD7cer for a positive test (Table S1). The calli of japonica rice WD68 and indica rice H128 were immersed in Agrobacterium infection suspension containing the CRISPR/Cas9-ΔOsCCD7 plasmid. The transformed calli were regenerated into transgenic seedlings.

2.4.5. Medium Components

(1)

LB solid medium: 5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 15 g/L agar (pH = 7).

(2)

Induction medium: N6 macronutrients, B5 micronutrients, B5 organics, iron salts, 0.2 g/L Glu, 0.1 g/L L-Pro, 0.1 g/L CH, X mg/L 2,4-D, X mg/L NAA, 30 g/L sucrose, and 8 g/L agar (pH = 5.8).

(3)

Co-cultivation medium: N6 macronutrients, B5 micronutrients, B5 organics, iron salts, 2 g/L myo-inositol, 3.9 g/L MES, 0.5 g/L CH, 30 g/L sucrose, X µM AS, and 8 g/L agar (pH = 5.5).

(4)

Recovery medium: N6 macronutrients, B5 micronutrients, B5 organics, iron salts, 0.2 g/L Glu, 0.1 g/L L-Pro, 0.1 g/L CH, 2.0 mg/L 2,4-D, 30 g/L sucrose, 8 g/L agar, X mg/L Tmt, and X mg/L Cef (pH = 5.8).

(5)

Differentiation medium: N6 macronutrients, B5 micronutrients, B5 organics, iron salts, 0.2 g/L Glu, 0.1 g/L L-Pro, 0.1 g/L CH, 30 g/L sucrose, 8 g/L agar, X mg/L KT, X mg/L NAA, and X mg/L 6-BA (pH = 5.8).

(6)

Infection buffer: AA macronutrients, B5 micronutrients, B5 organics, iron salts, 3.9 g/L MES, and 0.5 g/L CH (pH = 5.5).

(7)

The composition of the N6 and 1/2 MS media was based on Ali et al. [15] and Murashige et al. [16].

2.5. Positive Detection of Transgenic Materials

2.5.1. Histochemical Staining

Leaves and root tips of 120-day-old seedlings were immersed in GUS staining solution (PH1727, PHYGENE, China) at 37 °C for 12 h, followed by decolorization. Decolorized tissues were examined microscopically to confirm GUS expression activity.

2.5.2. PCR-Based Detection

After 120 days, transgenic seedlings were individually labeled, and T₁-generation leaf samples were ground into powder. Genomic DNA was extracted using the CTAB method [17], followed by PCR analysis to assess transformation efficiency. Positive detection primers for the GUS gene were GUS-PTF1 and GUS-PTR1, and for the ΔOsCCD7 gene were the primers of cas9-339f and cas9-339r. PCR products for positive detection were analyzed by agarose gel electrophoresis. The target sites were isolated using the primers Cas9-Os1081f and Cas9-Os1081r from transgenic mutant plants, and sent for sequencing for mutation detection. The PCR system and reaction conditions followed the manual for the PCR Taq Mix (PC106, Wuhan Sanying Biotechnology, Wuhan, Hubei, China).

2.6. Measurement of Agronomic Traits in Transgenic Rice

The transgenic lines were planted in a net greenhouse, with the individual plants labeled. At the end of the grain-filling stage, the plant height, tiller numbers, effective tiller numbers, main stem diameter, and the length and width of the main stem sword leaves were determined. After harvest, the spike length, spike width, number of primary and secondary branches, the length, width, and thickness of the grains, and the 1000-grain weight were measured. All traits were measured in 15 replicates.

2.7. Data Analysis

All experiments were conducted using a completely randomized design (CRD), with each replicate containing 15 technical replicates (n = 15). The data were analyzed using SPSS 18.0, and significant differences were determined by Duncan’s multiple comparisons and the probability value (p-value) of the data from the Student’s t-test. The optimal tissue culture formulation was comprehensively evaluated by calculating the D value using the membership function method [18]. The raw data were verified using GraphPad Prism 8 to generate bar charts/line charts, with the error bars representing the mean ± standard error (SEM). R software 4.5.0 (ggplot2 package) was used to draw complex graphics, such as heat maps. Calculation formulas for callus-related parameters were listed as follows:

(1)

Induction rate (%) = (Number of induced calli/Number of cultured mature seeds) × 100.

(2)

Browning rate (%) = (Number of browned calli/Number of subcultured calli) × 100.

(3)

Browning index (%) = (Each browning level × Number of calli at the browning level)/(Total number of calli × Highest browning level) × 100.

(4)

Contamination rate (%) = (Number of contaminated calli/Number of Agrobacterium-infected calli) × 100.

(5)

Initial differentiation rate (%) = (Number of calli showing differentiation/Total number of calli) × 100.

(6)

Differentiation rate (%) = (Number of shoot-forming calli/Total number of calli) × 100.

(7)

Resistance rate (%) = (Number of resistant calli/Number of Agrobacterium-infected calli) × 100.

(8)

GUS staining rate (%) = (Number of successfully GUS-stained calli/Number of Agrobacterium-infected calli) × 100.

(9)

Transformation efficiency (%) = (Number of positive plants/Number of T0 plants) × 100.

3. Results

3.1. Effects of Different Combinations of 2,4-D and NAA on Callus Formation

Numerous calli growths were seen after 7 days of induction culture, and by day 21, compact, pale-yellow embryogenic calli had developed (Figure 1A). The average induction rates for the 2,4-D-only treatments (A1–A4) were 53.17–57.44% for all types, according to the results (Figure 1B). On the other hand, induction rates rose to 57.35–63.48% for the combined 2,4-D and NAA treatments (A5–A20), indicating that this combination encourages callus formation. Under 2,4-D alone, callus measurements showed average callus sizes of 10.72–10.97 mm², fresh weights of 32.70–40.35 mg, and dry weights of 5.94–7.36 mg (Figure 1C). However, combined treatments produced denser, more robust calli, increasing callus diameters to 21.99–27.29 mm², fresh weights to 34.84–72.93 mg, and dry weights to 5.98–9.95 mg.

The analysis of membership functions determined that A9 is the optimal treatment for japonica rice varieties XG293 and WD68, followed by A7, A8, A10, and A6. For indica rice varieties H128 and E33, A13 emerged as the best treatment option, followed by A17, A3, A9, and A6. A9 achieved the highest induction rates in japonica rice varieties XG293 (79.34%) and WD68 (83.89%), resulting in callus sizes of 27.40 mm² and 38.53 mm², respectively. The fresh weights recorded were 53 mg for XG293 and 81.5 mg for WD68, while the dry weights measured were 12.52 mg and 23.68 mg, respectively. In contrast, for indica rice varieties H128 and E33, treatment A13 yielded the highest induction rates of 75.87% and 86.61%, with callus areas measuring 40.28 mm² and 43.53 mm², respectively; fresh weights were noted at 59.4 mg for H128 and an impressive 229 mg for E33; and dry weights were recorded as 9.68 mg and 12.6 mg, accordingly. Further, we observed increases in callus size (36.52 mm²), fresh weight (230.20 mg), and dry weight (9.56 mg) in the E33 variety following A17 treatment. These results indicate a significant difference in optimal response concentrations between japonica rice versus indica rice when subjected to combinations of 2,4-D and NAA.

3.2. Effects of Different Additives on Callus Browning

During subculture, H128 exhibited significantly higher callus browning rates compared to other varieties, with a browning rate of 59.55% (Figure S3). To address this issue, various carbon sources were evaluated (Figure 2). In comparison to the control (S3), treatments S1 and S4 resulted in a significant reduction in browning rates, while treatment S5 led to an even more pronounced decrease. The browning index showed significant reductions under S1, S4, and S5 treatments when compared to the control group, with the lowest index recorded at 29.66% for treatment S4. Although the fresh weight was significantly reduced in treatment S1 relative to the control, both the callus size and dry weight remained unaffected.

Treatment S5 significantly decreased browning rates, although there were only modest improvements in callus quality. In order to further enhance callus quality, the anti-browning agents Vc and PVP were evaluated after S5 (20 g/L maltose) was determined to be the best carbon source (Figure 3). The Vc treatments significantly reduced browning compared to the control. Browning was considerably decreased by the PVP treatments P1 and P2, while P3 showed only a slight improvement. When compared to controls, treatments V1, V2, and P1 considerably increased callus size, while treatment V4 showed a moderate improvement. The dry weights did not significantly differ between the treatments, but the V2 treatment had the highest fresh weight. Both Vc and PVP significantly reduced the browning index, with V2 and P2 achieving ideal indices of 2.08% and 7.14%, respectively. The anti-browning treatments that ranked highest were V2 > V1 > V4 > P1 > V5. As a result, it was shown that S5 (20 g/L maltose) and 40 mg/L Vc (V2) substantially reduced browning in H128 calli.

3.3. Effects of Different Antibiotics and Their Concentrations on Callus Contamination Rate and Differentiation

According to the results, contamination in all four varieties was considerably decreased by M3 and M4 treatments (Figure 4A). In M3, fresh callus formation was visible, indicating satisfactory callus growth, despite low contamination and mild browning. On the other hand, calli turned white and lost their ability to differentiate in M4 due to increased antibiotic concentrations.

According to a statistical examination of contamination rates, the contamination rates decreased as antibiotic concentration rose (Figure 4B). The contamination rates in japonica types XG293 and WD68 showed efficient suppression with increased Tmt concentration, with a substantial decrease in M3 and M4 treatments, but no significant difference between M1 and M2. M2 considerably decreased contamination in indica cultivars H128 and E33 as compared to M1, and H128 showed additional reductions under M3 and M4, underscoring the efficacy of greater Cef and Tmt concentrations. XG293, WD68, H128, and E33 showed the highest differentiation rates after M3 treatment, with respective rates of 9.44%, 10.42%, 10.53%, and 5.97% (Figure 4C). These results suggest that M3 (300 mg/L Tmt) is the best recovery medium for all four rice types in terms of preventing contamination and encouraging differentiation.

3.4. Effects of Different PGR Combinations on Callus Differentiation

Regeneration capability is essential for improving rice transformation efficiency in addition to embryogenic callus formation. The calli gave rise to shoot primordia after 15 days on differentiation media. The calli were highly sensitive to plant growth regulators (PGRs) at this point, and several combinations successfully encouraged the production of shoots. When branch buds became apparent after 30 days, they were moved to rooting media to continue growing (Figure S4).

The findings showed that the rates of callus differentiation varied significantly between treatments (

Table 2. Effects of different PGR combinations on callus differentiation rates.

Treatments	Different Varieties
	XG293	WD68	H128	E33
G1	12.50 ± 0.03 bc	23.02 ± 3.45 ab	30.00 ± 0.01 b	17.50 ± 2.50 c
G2	16.67 ± 0.03 b	13.89 ± 2.78 ab	22.50 ± 4.79 bc	12.50 ± 2.50 c
G3	19.44 ± 0.03 b	22.22 ± 10.14 ab	12.50 ± 2.50 cd	25.00 ± 8.66 bc
G4	5.56 ± 0.03 c	11.11 ± 0.01 b	5.00 ± 2.89 d	12.50 ± 2.50 c
G5	11.11 ± 0.05 bc	16.67 ± 3.21 ab	15.00 ± 2.89 bcd	15.00 ± 2.89 c
G6	11.11 ± 0.01 bc	10.00 ± 0.01 b	12.50 ± 4.79 cd	17.50 ± 2.50 c
G7	32.50 ± 0.03 a	25.00 ± 6.45 ab	30.00 ± 9.13 b	60.00 ± 5.77 a
G8	20.00 ± 0.04 b	29.17 ± 6.94 a	22.50 ± 4.79 bc	52.50 ± 6.29 a
G9	13.89 ± 0.03 bc	19.44 ± 2.78 ab	72.50 ± 6.29 a	45.00 ± 17.56 ab
Cv	48.51%	34.45%	80.02%	65.26%

Note: PGR: plant growth regulator, and Cv: coefficient of variation. Values with different letters for each variety are significantly different at p < 0.05. Values were represented by means ± standard error (n = 15).

). The differentiation rates for XG293 had a coefficient of variation of 48.51% and varied from 5.56% (G4) to 32.50% (G7). With a 34.45% coefficient of variation, WD68 showed differentiation rates that ranged from 10.00% (G6) to 29.17% (G8). With differentiation rates ranging from 5.00% (G4) to 72.50% (G9), and a coefficient of variation of 80.02%, H128 demonstrated significant variability and showed a high degree of responsiveness to various PGR combinations. Likewise, E33 showed differentiation rates with a coefficient of variation of 65.26% that ranged from 12.50% (G2 and G4) to 60.00% (G7).

Comprehensive analysis demonstrated that indica varieties H128 and E33 exhibited higher differentiation potential. H128 responded particularly well to G9 treatment, highlighting its sensitivity to high concentrations of KT and 6-BA. Overall, among the tested rice varieties, treatments G7, G8, and G9, all containing high KT concentrations (3 mg/L), were most effective in promoting shoot formation.

3.5. Effects of Different AS Concentration and Infection Duration on Rice Transformation

For all varieties, callus resistance rates decreased as infection time increased (Figure 5A). The greatest callus resistance happened during a 5 min infection (W1) under 100 µM AS treatments (W1–W5). While indica varieties H128 and E33 produced the highest resistance rates at 5 min (W6), japonica types XG293 and WD68 had optimal resistance rates at 10 min (W7) with 200 µM AS (W6–W10). At 40 min infection durations (W5 and W10), resistance rates were lowest across all types, suggesting that extended infections resulted in toxicity and decreased transformation effectiveness. According to callus morphological observations, the majority of the calli that underwent W6 and W7 treatments looked compact and yellow with no browning or callus death, suggesting a successful recovery. The small amount of callus death that was seen was probably caused by genotypic variations (Figure 5B). Furthermore, during induction and differentiation, japonica variants XG293 and WD68 showed higher rates of shoot production and faster development than indica varieties H128 and E33 (Figure 5C).

Significant variations between treatments were found in the differentiation rates (

Table 3. Effects of AS concentrations and infection durations on callus differentiation.

Treatments	Callus Differentiation Rate (%)
	XG293	WD68	H128	E33
W1	9.56% ± 2.54 c	6.96% ± 1.26 c	8.77% ± 2.74 d	1.76% ± 1.12 c
W2	6.92% ± 3.07 c	14.43% ± 2.53 b	6.70% ± 2.56 d	7.72% ± 3.18 bc
W3	9.67% ± 2.05 c	19.60% ± 1.89 b	5.09% ± 1.36 d	5.92% ± 2.42 bc
W4	5.09% ± 2.24 c	7.74% ± 3.21 c	7.12% ± 1.96 d	10.82% ± 4.79 bc
W5	8.79% ± 2.32 c	4.23% ± 0.86 c	6.86% ± 2.13 d	6.67% ± 2.34 bc
W6	21.39% ± 3.52 b	3.56% ± 1.83 c	28.80% ± 2.43 a	23.36% ± 3.05 a
W7	41.71% ± 3.79 a	31.15% ± 3.17 a	19.18% ± 1.36 b	13.57% ± 2.12 b
W8	28.07% ± 3.33 b	20.37% ± 1.65 b	17.09% ± 3.73 bc	8.10% ± 2.35 bc
W9	9.14% ± 1.46 c	18.45% ± 2.96 b	10.32% ± 3.69 cd	8.92% ± 3.77 bc
W10	6.01% ± 2.41 c	15.36% ± 2.16 b	8.07% ± 1.14 d	7.97% ± 2.50 bc

Note: Values with different letters for each variety are significantly different at p < 0.05. Values were represented by means ± standard error (n = 15).

). The differentiation rates of XG293 and WD68 under the W7 treatment were significantly greater than those of the other treatments, at 41.71% and 31.15%, respectively. With differentiation rates of 28.80% and 23.36%, respectively, H128 and E33 had the greatest rates under the W6 treatment; these rates were also noticeably greater than those of the other treatments. These results suggest that japonica rice is more suited for transformation since it showed higher differentiation rates than indica rice cultivars after Agrobacterium infection.

Transgenic calli were screened via Agrobacterium-mediated GUS gene transformation after 45 days post-transformation. GUS staining was employed to conduct a preliminary evaluation of transformation efficiency. The results indicated that the XG293 and WD68 strains in the W7 treatment group exhibited the highest staining rates of 13.75% and 17.5%, respectively. In contrast, the H128 and E33 strains in the W6 treatment group demonstrated the highest staining rates of 60% and 27.5%, respectively (Figure 6). The results aligned with the data on healing tissue resistance, indicating that the W7 treatment is optimal for Japanese rice varieties, whereas the W6 treatment is more appropriate for indica rice varieties.

3.6. Effects of Different Rooting Media on Root Growth in Regenerated Seedlings

After shoot regeneration, seedlings placed in various rooting media demonstrated notable differences in root development. Data analysis indicated that seedlings cultured on the MA medium exhibited greater stem diameter, increased plant height, and overall enhanced development relative to other media (Figure 7). Seedlings cultivated on the M medium frequently demonstrated the lowest values. Significant differences in stem diameter were observed between the MA and M treatments across all four varieties, while significant differences in plant height were noted only in XG293 and H128. The MA medium significantly increased total root length in three varieties, with the exception of E33. The root surface area and root volume were maximized under the MA treatment, significantly surpassing those observed in the M treatment. The root tip count in E33 was reduced under the MA treatment, while the other three varieties showed significantly higher counts compared to the M treatment. In comparison to the NA medium, the MA medium markedly enhanced stem diameter, total root length, root surface area, and root volume, reaching a significant level in root surface area, except in H128, indicating superior rooting effects. The analysis of membership functions ranked the treatments in the following order: MA > N > M > NA, thereby confirming MA as the optimal rooting medium. MA significantly enhanced root branching, increased root volume, and improved root–medium contact compared to the M treatment, thereby facilitating water and nutrient uptake (Figure S5). Differences between the N and NA treatments were minimal, suggesting that the addition of NAA to the N6 medium (NA) had a negligible impact on root development, while NAA significantly improved rooting in the 1/2 MS medium (MA).

3.7. Positive Detection of Transgenic Seedlings

At 120 days post-transformation, japonica varieties XG293 and WD68 attained the heading stage, while indica varieties H128 and E33 were still in the booting stage (Figure 8). PCR analysis of individually labeled plants confirmed the presence of a distinct 488-bp band corresponding to the GUS gene, thereby verifying successful transformation. The GUS staining of the PCR-positive plants confirmed the gene expression, revealing blue-stained mesophyll cells near leaf veins and root tip cells in transgenic plants, while wild-type plants exhibited no staining. The transformation efficiencies were observed to be 43.24% and 52.38% for XG293 and WD68, respectively, under optimal conditions (W7 for japonica, W6 for indica), while H128 and E33 exhibited efficiencies of 40.74% and 40.00%, respectively. The results demonstrate that the optimized transformation protocol attained high transformation efficiency and stable gene expression, establishing a dependable basis for genetic transformation in local japonica and indica rice varieties.

3.8. Identification of Agronomic Traits in OsCCD7 Mutants

The two editing sites in both varieties were sequenced. Target I showed no mutation in the WD68 genome, whereas there was a base mutation and a base insertion upstream of the PAM site in H128. Target II showed two base deletions upstream of the PAM site in both H128 and WD68 (Figure S6). These changes led to mutations in the OsCCD7 gene, inducing phenotypic variations in two varieties.

(I)

Plant architecture-related traits between wild type (WT) and OsCCD7 mutants

The growth of seedlings in the mutant lines exhibited reduced plant height and a significant increase in tillers (Figure 9). In comparison to WT, the results for WD68 indicated a significant reduction in the plant height of the mutant lines by 45.22% and in the stem diameter by 60.05%. Conversely, tiller numbers and effective tiller numbers increased by 4.21 and 3.94 times, respectively. Additionally, the leaf length and width of the sword leaves in the mutant lines decreased by 40.57% and 45.12%, respectively. In H128, the dimensions of the sword leaves in the mutants measured 16.23 cm in length and 0.91 cm in width, reflecting decreases of 42.99% and 52.60%, respectively. The plant height and stem diameter decreased significantly by 32.70% and 62.37%, respectively, while the tiller numbers and effective tiller numbers increased significantly by 10.26 and 9.83 times, respectively. All the differences attained highly significant levels. The results confirm the stability of the new genetic transformation system for local rice and indicate that OsCCD7 plays a significant role in regulating plant height and tiller, as well as influencing stem diameter and leaf development in rice.

(II)

Spike-related traits in WT vs. OsCCD7 mutants

The investigation of spike-related traits in the mutant lines revealed a significant decrease in spike length, width, and the number of primary and secondary branches (Figure 10). In WD68, the spike length and width of the mutants decreased significantly by 31.43% and 72.88%, respectively, compared to each variety of WT. Additionally, the number of primary and secondary branches decreased significantly by 60.13% and 83.71%, respectively. In H128, the mutants exhibited a spike length of 12.6 cm and a width of 0.45 cm, along with primary and secondary branch counts of 5.5 and 2.7, respectively. These measurements represent significant reductions of 38.27%, 81.56%, 56.69%, and 93.56% compared to each trait of the wild type (WT). The mutants exhibited a significant reduction in grain width and thickness by 76.27% and 86.74%, respectively, while grain length showed no significant changes. Consequently, the 1000-grain weight was 60.13% lower than that of the wild type (WT). The results demonstrate that the grain yield of the mutation lines was significantly diminished following OsCCD7 gene editing, indicating that OsCCD7 not only enhances tiller numbers and decreases plant height but also may exert a negative regulatory influence on rice yield.

4. Discussion

4.1. Effects of 2,4-D and NAA Combinations on Callus Induction

Embryogenic calli that can undergo cell division are crucial for effective Agrobacterium-mediated rice transformation. Prior research has identified 2,4-D as the principal plant growth regulator that facilitates embryogenic callus induction and proliferation in rice [19]. In japonica varieties like Nipponbare, a concentration of 2 mg/L 2,4-D is generally effective for callus induction, resulting in induction rates as high as 82.9% [20]. Previous studies have indicated that a combination of 3 mg/L 2,4-D and 2 mg/L NAA increased induction rates to 90%, enhancing callus quality and development [21]. This study evaluated combinations of 2,4-D and NAA, confirming their synergistic effects on callus induction. Japonica rice XG293 and WD68 exhibited optimal callus induction at 2 mg/L 2,4-D and 1 mg/L NAA, while indica rice H128 and E33 reached optimal responses at 3 mg/L 2,4-D and 1 mg/L NAA. 2,4-D, a synthetic auxin, effectively promotes cell dedifferentiation and callus initiation [22]. However, its sole application may result in tissue browning or regeneration issues. NAA exhibits reduced induction activity; however, it effectively sustains cell division and enhances tissue quality [23]. The synergistic effect of both hormones may be that 2,4-D dominates the formation of initial callus tissue, while NAA maintains tissue activity and differentiation potential by regulating the balance of endogenous hormones [24].

4.2. Effects of Different Additives on Anti-Browning in Calli

Callus browning adversely impacts callus quality and frequently leads to cell death [25]. The influence of various carbon sources on callus browning during rice transformation is significant. This study observed severe browning in indica rice H128, which maltose significantly mitigated. Maltose effectively mitigates browning; however, excessively high concentrations may enhance polyphenol oxidase activity, thereby exacerbating browning. While 20 g/L maltose significantly decreased callus browning, it did not substantially improve callus quality on its own. Consequently, the efficacy of anti-browning agents, specifically Vc and PVP, was assessed to enhance callus quality. Vc inhibits browning mediated by polyphenol oxidase, while PVP mitigates browning through metal–ion chelation [26,27]. Among the treatments evaluated, 20 mg/L Vc demonstrated the most substantial reduction in browning and significantly enhanced callus quality.

4.3. Effects of Different Antibiotics and Their Concentrations on Contamination and Differentiation in Calli

Cef and Tmt are commonly employed antibiotics in Agrobacterium-mediated transformation to minimize bacterial contamination. These antibiotics interfere with bacterial cell wall synthesis or protein synthesis, typically without affecting plant callus viability [28]. This study found that higher concentrations of Cef and Tmt significantly decreased callus contamination rates. In the presence of Cef, differentiation capacity was limited, while Tmt promoted differentiation. At elevated Tmt concentrations of 500 mg/L, callus differentiation was inhibited, suggesting toxic effects on callus growth. Under the tested conditions, a recovery medium with 300 mg/L Tmt significantly decreased contamination while enhancing differentiation across all four rice varieties.

4.4. Effects of Different PGR Combinations on Rice Callus Differentiation

Prior research has demonstrated that the capacity for callus regeneration is contingent upon particular hormonal responses. Calli accumulate elevated levels of auxins and demonstrate significant sensitivity to cytokinins. This characteristic enables calli to regenerate roots at low auxin concentrations, while elevated cytokinin concentrations stimulate shoot-related gene expression, facilitating shoot development [29]. Auxins and cytokinins typically exhibit inhibitory interactions in differentiated somatic cells; however, both classes of hormones, including cytokinins such as KT and 6-BA, and auxins like NAA, are essential for rice transformation [30]. NAA primarily facilitates cell division, elongation, and shoot induction, with varying responses observed among different rice genotypes to its concentrations. For example, the Moroberekan rice genotype “CBMH” demonstrated a high differentiation rate in a medium with 2.0 mg/L KT and 0.1 mg/L NAA, while the genotype “JDJ” reached only a 10% differentiation rate [31]. This study demonstrated that KT significantly enhances cell division and callus proliferation, while 6-BA predominantly stimulates lateral shoot formation. XG293 and E33 exhibited optimal responses to low NAA concentrations (1 mg/L, G7), while H128 necessitated a higher NAA concentration (2 mg/L, G9) for optimal differentiation. WD68 demonstrated optimal differentiation at an intermediate NAA concentration of 1.5 mg/L (G8), significantly enhancing callus differentiation and shoot formation. NAA concentration exerts a dual influence on plant cell differentiation and regeneration. NAA at a concentration of 0.1 mg/L facilitates callus formation and lateral bud differentiation; however, elevated concentrations hinder differentiation or induce abnormal proliferation [22]. The effects are regulated synergistically by 6-BA and KT, potentially operating through mechanisms including metabolic pathways and redox status [32,33]. The optimal concentration of NAA differs across various varieties, necessitating further research to clarify its specific regulatory mechanisms.

4.5. Effects of Different AS Concentrations and Infection Durations on Rice Transformation

Prior research indicates that AS is crucial for the effective transformation of monocotyledonous plants, as it activates the Vir genes required for T-DNA transfer into plant cells. The optimal concentration of AS is essential for preventing necrosis and enhancing the efficiency of rice transformation [34]. Appropriate duration of Agrobacterium infection is essential for achieving effective transient gene expression and high rates of selection differentiation. Prolonged infection duration can lead to hypoxic toxicity and necrosis of calli, while inadequate duration restricts bacterial adhesion and the efficiency of gene transfer [35]. Prior studies have determined that 200 µM AS is optimal for rice transformation, resulting in transformation efficiencies ranging from 10% to 30% [6]. Furthermore, elevating AS concentration enhances transformation rates. Specifically, at 200 µM AS, the IET-4786 rice attained a transformation efficiency of 33.33% [36]. This study found that 200 µM AS generated high-quality calli; however, extended infection durations reduced callus resistance rates. The optimal transformation efficiency was observed at infection durations of 10 min for japonica rice XD293 (43.24%) and WD68 (52.38%), and 5 min for indica rice H128 (40.74%) and E33 (40.00%).

4.6. Effects of Different Rooting Media on Root Growth in Regenerated Seedlings

Two basal media were compared for their effects on root growth. The N6 medium, which is abundant in mineral elements, is commonly utilized for monocot tissue culture, particularly in the induction and regeneration of calli in rice and maize [37]. Conversely, the 1/2 MS medium has elevated levels of inorganic salts, which facilitate rapid root growth in seedlings by preserving ionic balance [38]. NAA, an auxin, facilitates the formation of root primordia and is crucial for rooting in cultured seedlings and cuttings [39]. Significant differences in root growth and branching were observed among the four varieties when NAA was added to each medium. The MA medium (1/2 MS + 0.2 mg/L NAA) resulted in enhanced root length, surface area, volume, and root tip quantity. The enhanced performance is due to the micronutrient-rich 1/2 MS medium (including iron, manganese, zinc, copper, boron, and molybdenum) and the optimal concentration of NAA, which improves lateral root formation, root volume, and the uptake of water and nutrients, thereby promoting healthy seedling growth

4.7. Agronomic Characteristics Analysis of OsCCD7 Mutants

HTD3 gene editing using CRISPR/Cas9 resulted in a single base insertion or a 2 bp deletion in Exon 1, which increased the tiller numbers of the htd3 mutant by 2–3 times and reduced plant height by 35% [40]. After editing the OsCCD7 gene in japonica rice WD68 and indica rice H128, researchers observed a 2 bp base deletion. This alteration resulted in reduced plant height in the mutants of both varieties, a notable increase in tiller numbers, and a more compact plant morphology. Editing effects varied: the grain size of WD68 mutants remained largely unchanged relative to the WT, whereas the grain width, thickness, and 1000-grain weight of H128 mutants were significantly diminished compared to the WT, resulting in a reduced grain yield. This decrease may be attributed to the high tiller numbers in H128 mutants, leading to a reduced allocation of photosynthetic products to the grain. The regulatory effects of OsCCD7 on plant height and tiller development vary among different rice varieties. The effective utilization of OsCCD7 to enhance major cultivated varieties and innovate high-quality rice germplasm necessitates further experimental verification. The germplasm materials of WD 68 and H128 developed in this study can serve as parental resources and can undergo backcrossing with the corresponding wild type to produce superior offspring lines.

5. Conclusions

Japonica rice varieties XG293 and WD68 exhibited optimal callus induction under A9 (2 mg/L 2,4-D + 1 mg/L NAA), whereas indica rice varieties H128 and E33 responded best to A13 (3 mg/L 2,4-D + 1 mg/L NAA), achieving high induction rates and favorable growth traits. To mitigate callus browning during subculture, 20 g/L maltose combined with 20 mg/L Vc effectively improved callus quality. Agrobacterium contamination was effectively suppressed using 300 mg/L Tmt. The optimal transformation conditions consisted of infection suspension with 200 µM AS for 10 min infection durations in japonica varieties and 5 min durations in indica varieties. Under these optimized conditions, the stable expression of the Gus gene and ΔOsCCD7 was achieved, with transformation efficiencies of 43.24% and 52.38% for japonica varieties XD293 and WD68, respectively, and 40.74% and 40.00% for indica varieties H128 and E33, respectively. The rice mutants after OsCCD7 gene editing by CRISPR/Cas9 displayed a dwarfness of plant height and a significant increase in tiller numbers. These findings provide a robust theoretical basis to improve transformation efficiency for germplasm innovation in local rice varieties.