Somatic Embryogenesis and Genetic Transformation of Caragana intermedia
Abstract
:1. Introduction
2. Results
2.1. Effects of Cotyledon Inoculation Modes on SE
2.2. Effects of Cotyledon Development Stages on SE
2.3. Effects of PGRs on SE
2.4. Development Progress of Embryos
2.5. Germination of Cotyledon-Stage Embryos
2.6. Genetic Transformation of C. intermedia
2.7. Identification of Transgenic Materials
2.8. TRV-VIGS System Verification
3. Discussion
4. Materials and Methods
4.1. Seed Developmental Progression
4.2. Somatic Embryo Induction
4.3. Paraffin Section
4.4. Genetic Transformation
4.4.1. Reporter Genes
4.4.2. Activation of Agrobacterium
4.4.3. Preparation of Infective Bacterial Suspension
4.4.4. Cotyledons Agroinfection
4.4.5. Co-Culture and Somatic Embryos Screening
4.5. Identification of Transgenic Material
4.5.1. RNA Extraction, cDNA Synthesis, and PCR Analysis
4.5.2. Real-Time Quantitative Polymerase Chain Reaction
4.6. Image Collection and Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Weeks After Flowering (WAF) | Seed Length (mm) | Seed Width (mm) | Cotyledon-Stage Embryo Induction Rate (%) | Number of Cotyledon-Stage Embryos |
---|---|---|---|---|
1 | 1.52 ± 0.05 h | 0.72 ± 0.01 i | 0.00 g | 0.00 e |
2 | 1.78 ± 0.02 g | 1.09 ± 0.03 h | 0.00 g | 0.00 e |
3 | 4.98 ± 0.04 f | 1.99 ± 0.06 g | 0.00 g | 0.00 e |
4 | 5.57 ± 0.11 e | 2.85 ± 0.05 f | 5.66 ± 0.22 e | 0.87 ± 0.09 d |
5 | 7.05 ± 0.11 d | 4.16 ± 0.03 d | 37.67 ± 0.46 c | 7.51 ± 0.30 c |
6 | 7.55 ± 0.01 c | 4.33 ± 0.02 c | 55.00 ± 0.48 b | 9.58 ± 0.09 b |
7 | 8.37 ± 0.02 b | 4.58 ± 0.05 b | 57.14 ± 0.40 a | 11.80 ± 0.42 a |
8 | 8.89 ± 0.05 a | 4.86 ± 0.07 a | 12.31 ± 0.17 d | 1.25 ± 0.05 d |
9 | 9.02 ± 0.05 a | 4.96 ± 0.01 a | 1.48 ± 0.18 f | 0.35 ± 0.03 e |
10 | 7.02 ± 0.13 d | 3.41 ± 0.05 e | 0.00 g | 0.00 e |
Treatment Number | NAA (mg/L) | 6-BA (mg/L) | Cotyledon-Stage Embryo Induction Rate (%) | Number of Cotyledon-Stage Embryos | ||||||
---|---|---|---|---|---|---|---|---|---|---|
5 WAF | 6 WAF | 7 WAF | Means | 5 WAF | 6 WAF | 7 WAF | Means | |||
1 | 3 | 3 | 79.17 ± 0.31 a | 53.89 ± 0.79 b | 34.72 ± 1.30 e | 55.93 ± 6.45 b | 11.98 ± 0.95 bc | 8.38 ± 0.23 bc | 11.56 ± 0.39 bc | 10.64 ± 0.64 b |
2 | 3 | 5 | 58.33 ± 1.08 c | 65.00 ± 1.46 a | 41.67 ± 1.03 cd | 55.00 ± 3.52 bc | 10.04 ± 0.10 d | 10.20 ± 0.46 a | 6.88 ± 0.97 e | 9.04 ± 0.62 cd |
3 | 3 | 7 | 54.17 ± 0.60 cd | 40.74 ± 1.60 c | 30.56 ± 1.24 e | 41.82 ± 3.47 f | 10.61 ± 0.47 cd | 7.55 ± 0.65 c | 5.35 ± 0.44 e | 7.84 ± 0.81 ef |
4 | 5 | 3 | 64.88 ± 1.43 b | 55.56 ± 1.78 b | 54.76 ± 1.85 b | 58.40 ± 1.83 a | 15.23 ± 0.78 a | 9.82 ± 0.55 ab | 11.35 ± 0.48 bc | 12.13 ± 0.86 a |
5 | 5 | 5 | 37.67 ± 2.50 e | 55.00 ± 0.90 b | 57.14 ± 0.73 b | 49.94 ± 3.18 e | 7.51 ± 0.24 e | 9.58 ± 0.54 ab | 11.80 ± 0.49 bc | 9.63 ± 0.66 c |
6 | 5 | 7 | 51.33 ± 0.72 d | 25.56 ± 1.93 d | 40.21 ± 0.44 d | 39.03 ± 3.78 g | 8.33 ± 0.07 e | 5.89 ± 0.55 de | 10.30 ± 0.39 cd | 8.17 ± 0.67 de |
7 | 7 | 3 | 54.53 ± 1.23 cd | 43.06 ± 1.05 c | 55.83 ± 2.56 b | 51.14 ± 2.21 de | 12.74 ± 0.94 b | 6.85 ± 0.75 cd | 12.34 ± 0.92 b | 10.64 ± 1.05 b |
8 | 7 | 5 | 67.50 ± 1.80 b | 28.61 ± 0.96 d | 61.90 ± 1.72 a | 52.67 ± 6.12 cd | 14.83 ± 0.41 a | 5.05 ± 0.08 e | 15.63 ± 0.75 a | 11.84 ± 1.72 a |
9 | 7 | 7 | 40.00 ± 1.75 e | 25.28 ± 0.26 d | 46.16 ± 2.13 c | 37.15 ± 3.20 g | 4.87 ± 0.20 f | 7.01 ± 0.20 cd | 9.28 ± 0.23 d | 7.05 ± 0.65 f |
Number | Research Purpose | Medium Components |
---|---|---|
1# | Effects of cotyledon inoculation modes on SE | MS + 5 mg/L NAA + 5 mg/L 6-BA + 30 g/L sucrose + 7 g/L agar + 500 mg/L hydrolyzed casein, pH 5.8 |
2# | Effects of different PGR combinations on SE | MS + (3, 5, 7) mg/L NAA + (3, 5, 7) mg/L 6-BA + 30 g/L sucrose + 7 g/L agar + 500 mg/L hydrolyzed casein, pH 5.8 |
3# | Germination of cotyledon-stage embryos | 1/2 MS + (0, 0.005, 0.05, 0.01) mg/L NAA + 30 g/L sucrose + 7 g/L agar, pH 5.8; |
MS, HS-MS, 1/4 MS + 30 g/L sucrose + 7 g/L agar, pH 5.8 | ||
4# | Co-culture | MS + 5 mg/L NAA + 3 mg/L 6-BA + 8 g/L agar + 30 g/L sucrose + 200 µM AS, pH 5.4 |
5# | Somatic embryo selection | MS + 5 mg/L NAA + 3 mg/L 6-BA + 8 g/L agar + 30 g/L sucrose + 500 mg/L hydrolyzed casein + 400 mg/L Cef + 13 mg/L Hyg, pH 5.8 |
6# | Cotyledon-stage embryo differentiation | HS-MS + 30 g/L sucrose + 7 g/L agar + 250 mg/L Cef + 50 mg/L Kan, pH 5.8 |
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Tian, J.; Zhu, J.; Deng, X.; Zhu, X.; Wang, R.; Li, G. Somatic Embryogenesis and Genetic Transformation of Caragana intermedia. Plants 2025, 14, 1545. https://doi.org/10.3390/plants14101545
Tian J, Zhu J, Deng X, Zhu X, Wang R, Li G. Somatic Embryogenesis and Genetic Transformation of Caragana intermedia. Plants. 2025; 14(10):1545. https://doi.org/10.3390/plants14101545
Chicago/Turabian StyleTian, Ju, Jialei Zhu, Xiaohan Deng, Xu Zhu, Ruigang Wang, and Guojing Li. 2025. "Somatic Embryogenesis and Genetic Transformation of Caragana intermedia" Plants 14, no. 10: 1545. https://doi.org/10.3390/plants14101545
APA StyleTian, J., Zhu, J., Deng, X., Zhu, X., Wang, R., & Li, G. (2025). Somatic Embryogenesis and Genetic Transformation of Caragana intermedia. Plants, 14(10), 1545. https://doi.org/10.3390/plants14101545