Abstract
Plant genome editing has been mainly mediated by T-DNA transformation to study plant biology and improve crop quality and quantity. The T-DNA must eventually be removed (called T-DNA-free) in siblings via genetic segregation after completing editing using the CRISPR/cas9 mechanism. Here, we developed an easy and efficient method to select T-DNA-free segregants with kanamycin treatments at 10 days after germination (DAG) onto seedlings of T1 siblings propagated from transgenic plants in Solanum nigrum harboring NPTII on the CRISPR/cas9 vector. To quantify the responses in root and shoot growth and leaf color, three types of kanamycin treatments were applied as follows: (1) submerging germinated seeds in kanamycin liquid (Submerged), (2) growing seeds on agar MS media containing kanamycin (MS agar media), and (3) spraying kanamycin onto seedlings at 10 DAG (Sprayed). Optimal kanamycin concentrations were determined as 200 mg/L for “Submerged,” 50 mg/L for “MS agar media,” and 400 mg/L for “Sprayed” to distinguish wild type (WT) seedlings treated with kanamycin from untreated seedlings. Furthermore, T-DNA-free plants were selected from T1 siblings of genome-edited S. nigrum plants using the same methods of distinguishing untreated WT plants, followed by confirmation of T-DNA-free status using polymerase chain reaction. Notably, the efficiency of T-DNA-free selection was 100% with the “Sprayed” method, and after 2 weeks, all the selected plants recovered from the strong stress. However, although “Submerged” and “MS agar media” methods were useful for removing mutants harboring T-DNA from the population, the methods showed relatively low accuracy in selection of T-DNA-free mutants with efficiencies of 5% and 17%, respectively. This indicates that in addition to stress from kanamycin treatment, plant growth is likely controlled by many factors owing to which mutations often occur during the regeneration process; however, photosynthesis is often not affected by such mutations. Therefore, we suggest that the “Sprayed” method is an easy, simple, and accurate method for isolating genome-edited S. nigrum plants that do not contain T-DNA in the genome and could be generally applicable to breed T-DNA-free variants in other crops such as tomato.
References
Ahangarzadeh S et al (2012) Cloning, transformation and expression of human interferon α2b gene in tobacco plant (Nicotiana tabacum cv. Xanthi). Jundishapur J Nat Pharmaceut Prod. https://doi.org/10.5812/jjnpp.3678
Barton KA et al (1983) Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell.
Belhaj K et al (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:1–10. https://doi.org/10.1186/1746-4811-9-39
Bell CC et al (2014) A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. BMC Genom. https://doi.org/10.1186/1471-2164-15-1002
Bhatta BP, Malla S (2020) Improving horticultural crops via crispr/cas9: current successes and prospects. Plants. https://doi.org/10.3390/plants9101360
Brooks C et al (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166(3):1292–1297. https://doi.org/10.1104/pp.114.247577
Chakraborty M et al (2016) Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker. Physiol Mol Biol Plants. https://doi.org/10.1007/s12298-015-0334-y
Danilo B et al (2019) Efficient and transgene-free gene targeting using Agrobacterium-mediated delivery of the CRISPR/Cas9 system in tomato. Plant Cell Rep. https://doi.org/10.1007/s00299-019-02373-6
Datukishvili N et al (2015) New multiplex PCR methods for rapid screening of genetically modified organisms in foods. Front Microbiol. https://doi.org/10.3389/fmicb.2015.00757
Falk J et al (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves. FEBS Lett. https://doi.org/10.1016/S0014-5793(03)00166-2
Jeong DH et al (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 130(4):1636–1644. https://doi.org/10.1104/pp.014357
Kapildev G et al (2016) High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper). Acta Physiol Plant. https://doi.org/10.1007/s11738-016-2215-6
Kwon CT et al (2020) Rapid customization of Solanaceae fruit crops for urban agriculture. Nat Biotechnol 38(2):182–188. https://doi.org/10.1038/s41587-019-0361-2
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Park S et al (2020) Rapid generation of transgenic and gene-edited Solanum nigrum plants using Agrobacterium-mediated transformation. Plant Biotechnol Rep. https://doi.org/10.1007/s11816-020-00616-7
Sakamoto T et al (2003) Genetic manipulation of gibberellin metabolism in transgenic rice. Nat Biotechnol 21(8):909. https://doi.org/10.1038/nbt847
Vu TV et al (2020) Precision genome engineering for the breeding of tomatoes: recent progress and future perspectives. Front Genome Editing. https://doi.org/10.3389/fgeed.2020.612137
Weber E et al (2011) Assembly of designer tal effectors by golden gate cloning. PLoS One. https://doi.org/10.1371/journal.pone.0019722
Weide R, Koornneef M, Zabel P (1989) A simple, nondestructive spraying assay for the detection of an active kanamycin resistance gene in transgenic tomato plants. Theor Appl Genet 78(2):169–172. https://doi.org/10.1007/BF00288794
Acknowledgements
We would like to thank the members of Park’s laboratory for discussions, A. Cho, Y. Chae, and Y.S. La for technical assistance. This research was supported by the National Research Foundation of Korea (NRF-2020R1A2C1101915) funded by the Korean Ministry of Science, ICT and Future Planning to S.J.P. and supported by a grant from the New Breeding Technologies Development Program (Project No. PJ01652302) funded by the Rural Development Administration, Republic of Korea, to C.M.K.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kim, M.J., Beak, H.K., Choi, J.E. et al. Simple methods for selection of T-DNA-free segregants from offspring of gene-edited Solanum nigrum. Plant Biotechnol Rep 16, 257–264 (2022). https://doi.org/10.1007/s11816-022-00754-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11816-022-00754-0