Jinghua Lu, Shanshan Li, Shuai Deng, Mugui Wang, Yinghuang Wu, Ming Li, Jinsong Dong, Suhui Lu, Chunli Su, Guofu Li, Zhaobo Lang, Jian-Kang Zhu

Plant Biotechnology Journal; First published: 29 February 2024; https://doi.org/10.1111/pbi.14318

Kalanchoe blossfeldiana

Summary

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties – the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%–7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14318

Figure 1: The CDB gene delivery system is effective for the succulent plant K. blossfeldiana. (a) CDB delivery system workflow. (b) Hypertrophic leaves were cut and used as explants. White arrow indicates the site of infection. (c) Explants inoculated with K599 were cultured in soil. (d and e) The formation of EGFP-positive buds showing green fluorescence. (f and g) Plants grown from transformed buds. (h) PCR test showed that all tested eight buds were transgene-positive. (i) Determination of transgene copy number in the transgenic lines using Southern blot analysis. (j) Bar protein test of transgene-positive buds. In (d), (e), (f) and (g), the sample on the left is untransformed control. In (e) and (g), the sample was illuminated with UV light. Red arrow indicates EGFP fluorescence in transformed tissues. Scale bar: 1 cm.