Optimasi  Optical Density  (OD) Dan Dosis Kanamisin Dalam Transformasi Gen  Sucrose Phosphate Synthase  (SoSPS1) Pada Anggrek  Dendrobium  sp.

Arsy Chairiyah; Bambang Sugiharto; Purnama Okviandari; Parawita Dewanti; Khiar Ayatina Hasbi

doi:10.22146/veg.106229

Optimasi Optical Density (OD) Dan Dosis Kanamisin Dalam Transformasi Gen Sucrose Phosphate Synthase (SoSPS1) Pada Anggrek Dendrobium sp.

https://doi.org/10.22146/veg.106229

Arsy Chairiyah⁽¹⁾, Bambang Sugiharto⁽²⁾, Purnama Okviandari⁽³⁾, Parawita Dewanti^(4*), Khiar Ayatina Hasbi⁽⁵⁾

(1) Universitas Jember
(2) Universitas Jember
(3) Universitas Jember
(4) Universitas Jember
(5) Universitas Jember
(*) Corresponding Author

Abstract

Genetic transformation in plants involves the insertion of isolated foreign genes into the plant genome. Sucrose Phosphate Synthase (SPS) is a key enzyme in sucrose biosynthesis, the primary sugar serving as an energy source and carbon transport molecule in most plants. Dendrobium orchids, which utilize Crassulacean Acid Metabolism (CAM), often exhibit a slow growth rate, thereby limiting their commercial potential. The insertion of the SPS gene into the orchid genome is expected to optimize the sucrose metabolic pathway, potentially influencing the rate of photosynthesis, carbon allocation, and growth characteristics of the orchid. Optimizing the optical density (OD) of the Agrobacterium suspension and the dosage of kanamycin is crucial for enhancing the success rate of the gene transformation process. This study aimed to determine the optimal concentrations of OD and kanamycin to achieve efficient SoSPS1 gene transformation in orchids. Three-month-old Protocorm-Like Bodies (PLBs) of Dendrobium Glory White were used as the explant source. The transformed explants were subsequently evaluated for the effectiveness of the kanamycin dosage, the percentage of explant shoots, the number of shoots per explant, the transformation efficiency, and phenotypic changes in the transformed plants. The results showed that a transformation efficiency of up to 40% was achieved using an Agrobacterium suspension at an OD600nm of 0.4. A lethal dose (LD50) of 100 mgL-1 kanamycin resulted in 34% of plantlets surviving post-selection. Transformation with the SoSPS1 gene in Dendrobium sp. induced distinct phenotypic plants including split leaves, swollen stems (pseudobulb), and increased shoot multiplication.

Keywords

gene insertion; orchid PLB; SoSPS1 gene; sucrose

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References

Ambros, P. F., Matzke, A. J. M., & Matzke, M. A. 1986. Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. The EMBO Journal, 5(9): 2073–2077. DOI: 10.1002/j.1460-2075.1986.tb04468.x

Andayani, R., Rahmatika, W., dan Fitriyah, N. 2023. Transfomasi genetik : Peluang dan tantangan. Jurnal Ilmiah Agrineca, 23(1): 80–91. DOI: org/10.36728/afp.v23i1.2350

Anur, R. M., Mufithah, N., Sawitri, W. D., Sakakibara, H., & Sugiharto, B. 2020. Overexpression of sucrose phosphate synthase enhanced sucrose content and biomass production in transgenic sugarcane. Plants, 9(2): 200. DOI: 10.3390/plants9020200

Bilska-Kos, A., Mytych, J., Suski, S., Magoń, J., Ochodzki, P., & Zebrowski, J. 2020. Sucrose phosphate synthase (SPS), sucrose synthase (SUS) and their products in the leaves of Miscanthus × giganteus and Zea mays at low temperature. Planta, 252(2): 23. DOI: 10.1007/s00425-020-03421-2

Chen, C., Fu, X., Peng, R., Tian, Y., & Yao, Q. 2020. Detoxifying processes during kanamycin-induced stress to Arabidopsis thaliana seedling growth. Biotechnology & Biotechnological Equipment, 34(1): 673–679. DOI: 10.1080/13102818.2020.1798811

Gelvin, S. B. 2021. Plant DNA repair and Agrobacterium T−DNA Integration. International Journal of Molecular Sciences, 22(16): 8458. DOI: 10.3390/ijms22168458

Huang, H., Ullah, F., Zhou, D.-X., Yi, M., & Zhao, Y. 2019. Mechanisms of ROS regulation of plant development and stress responses. Frontiers in Plant Science, 10. DOI: 10.3389/fpls.2019.00800

Indraloka, A. B., Dewanti, P., & Restanto, D. P. 2019. Morphological Characteristics and Pollinia Observation of 10 Indonesia Native Dendrobium Orchids. In BIOVALENTIA: BIOLOGICAL RESEARCH JOURNAL, 5(2).

Kirchhoff, J., Schiermeyer, A., Schneider, K., Fischer, R., Ainley, W. M., Webb, S. R., Schinkel, H., & Schillberg, S. 2020. Gene expression variability between randomly and targeted transgene integration events in tobacco suspension cell lines. Plant Biotechnology Reports, 14(4): 451–458. DOI: 10.1007/s11816-020-00624-7

Krispil, R., Tannenbaum, M., Sarusi-Portuguez, A., Loza, O., Raskina, O., & Hakim, O. 2020. The position and complex genomic architecture of plant T-DNA insertions revealed by 4SEE. International Journal of Molecular Sciences, 21(7): 2373. DOI: 10.3390/ijms21072373

Lian, Z., Nguyen, C. D., Wilson, S., Chen, J., Gong, H., & Huo, H. 2020. An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus. Plant Cell, Tissue and Organ Culture (PCTOC), 142(3): 527–536. DOI: 10.1007/s11240-020-01877-4

Ma, P., Zhang, X., Chen, L., Zhao, Q., Zhang, Q., Hua, X., Wang, Z., Tang, H., Yu, Q., Zhang, M., Ming, R., & Zhang, J. 2020. Comparative analysis of sucrose phosphate synthase (SPS) gene family between Saccharum officinarum and Saccharum spontaneum. BMC Plant Biology, 20(1): 422. DOI: 10.1186/s12870-020-02599-7

Martha, K. M., Dewanti, P., Restanto, D. P., Ratnasari, T., & Alfian, F. N. 2023. In Vitro Mutagenesis of Dendrobium Gabriella Suryajaya Using Ethyl Methane Sulfonate (EMS) and Plantlet Regeneration. Biosaintifika, 15(1): 87–96. DOI: 10.15294/biosaintifika.v15i1.40997

Mulyatama, R. A., Neliana, I. R., Sawitri, W. D., Sakakibara, H., Kim, K.-M., & Sugiharto, B. 2022. Increasing the Activity of Sugarcane Sucrose Phosphate Synthase Enhanced Growth and Grain Yields in Transgenic Indica Rice. Agronomy, 12(12): 2949. DOI: 10.3390/agronomy12122949

Peach, C., and Velten, J. 1991. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Molecular Biology, 17(1): 49–60. DOI: 10.1007/BF00036805

Satria, D. B. R., Sugiharto, B., dan Restanto, D. P. 2015. Transformasi gen SoSPS1 menggunakan vektor Agrobacterium tumefaciens dan eksplan tunas apikal padi Indica cv. Inpari 14 SS. Universitas Jember, Jember

Singh, R. K., and Prasad, M. 2016. Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops. Protoplasma, 253(3): 691–707. DOI: 10.1007/s00709-015-0905-3

Utami, E. S. W., Hariyanto, S., & Manuhara, Y. S. W. 2018. Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid. Journal of Genetic Engineering and Biotechnology, 16(2): 703–709. DOI: 10.1016/j.jgeb.2018.02.002

Verma, S., Kumar, A., & Modgil, M. 2023. Impact of cefotaxime and kanamycin on in vitro regeneration via Agrobacterium mediated transformation in apple cv. Red Chief. Plant Physiology Reports, 28(1): 34–42. DOI: 10.1007/s40502-023-00708-w

Yang, C., Liu, J., Qin, X., Liu, Y., Sui, M., Zhang, Y., Hu, Y., Mao, Y., & Shen, X. 2023. Effect of nitric oxide on browning of stem tip explants of malus sieversii. Horticulturae, 9(11): 1246. DOI: 10.3390/horticulturae9111246

Yang, Z.-H., Huang, W., Yang, Q.-Y., Chang, W., & Zhang, S.-B. 2018. Anatomical and diffusional determinants inside leaves explain the difference in photosynthetic capacity between Cypripedium and Paphiopedilum, Orchidaceae. Photosynthesis Research, 136(3): 315–328. DOI: 10.1007/s11120-017-0466-8

Zhang, W., Dong, X.-M., Zhang, Y.-W., Fan, Z.-X., & Zhang, S.-B. 2023. Age-related differences in physiological and metabolic responses of Pleione aurita (Orchidaceae) pseudobulbs to drought stress and recovery. Plant Physiology and Biochemistry, 197: 107655. DOI: 10.1016/j.plaphy.2023.107655

Zhang, Y., Zeng, D., Liu, Y., & Zhu, W. 2022. SlSPS, a Sucrose Phosphate Synthase gene, mediates plant growth and thermotolerance in tomato. Horticulturae, 8(6): 491. DOI: 10.3390/horticulturae8060491

DOI: https://doi.org/10.22146/veg.106229