A method of genetic transformation and gene editing of succulents without tissue culture

Abstract

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.

Keywords: cut–dip–budding (CDB) delivery system; gene editing; genetic transformation; succulents.

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.

Figure 2

Gene‐edited buds of K. blossfeldiana were obtained using the modified CDB method. (a) Transformation and gene editing efficiencies for K. blossfeldiana. (b) Fully and partially albino buds of K. blossfeldiana generated by editing of the PDS gene. The adventitious buds obtained from transformation with an empty vector were used as control at left (WT). (c) Mutation types in gene‐edited buds. The gRNA region is labelled with green colour, the PAM region is shown in blue colour letters and the number indicates deleted (−) or inserted (+) bases marked by red colour. WT, wild type. Scale bar: 1 cm.

Figure 3

Transformation of C. arborescens using the modified CBD method. (a) Transgenic bud of C. arborescens expressing the RUBY reporter gene. (b) The RUBY‐positive adventitious bud grew normally. (c) PCR test verifying that red buds from C. arborescens were RUBY transgene‐positive. (d) The transformation efficiency of C. arborescens using the modified CDB method. In Figure a,b, the tissue on the left is untransformed control. Red arrow indicates RUBY expression in transformed bud. Scale bar: 1 cm.

Figure 4

Transformation of S. trifasciata ‘Jinbianlan’ using the CDB system. (a) Diagram of the CDB workflow. (b) Hypertrophic leaves were cut and used as explants. White arrow indicates the site of Agrobacterium infection. (c) Explants inoculated with A. rhizogenes K599 were cultured in soil. (d and e) Formation of EGFP‐positive buds. (f and g) The EGFP‐positive buds were cultured to give rise to individual transgene‐positive plants. (h) PCR results showing that EGFP‐positive buds were transgene‐positive. (i) The results of Southern bot analysis. (j) Bar strip test of transgenic S. trifasciata. In (d), (e), (f) and (g), the sample on the left is untransformed control. In (e) and (g), samples were illuminated with a UV lamp. Red arrow indicates EGFP fluorescence in transformed tissue. Scale bar: 1 cm.

Figure 5

The CDB method of genetic transformation is genotype‐independent in S. trifasciata. (a and b) EGFP was transformed into S. trifasciata ‘Heijingang’. (c and d) EGFP was transformed into S. trifasciata ‘Hani’. (e and f) EGFP was transformed into S. trifasciata ‘Zuanshilan’. (g and h) EGFP was transformed into S. trifasciata ‘Yinmailan’. (i) PCR results showing that the EGFP‐positive buds of the five varieties of S. trifasciata were transgenic. (j) Transformation efficiencies for the four S. trifasciata varieties. Red arrow indicates EGFP fluorescence in transformed tissue. The sample on the left is untransformed control. Scale bar: 1 cm.