Simple method for transformation and gene editing in medicinal plants

A sample delivery method, modified from cut-dip-budding, uses explants with robust shoot regeneration ability, enabling transformation and gene editing in medicinal plants, bypassing tissue culture and hairy root formation. This method has potential for applications across a wide range of plant species. Gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding. However, a critical challenge hindering the widespread adoption of these technologies is the efficient delivery of gene-editing tools. The predominant methods for introducing these tools into plants typically involve Agrobacterium tumefaciens-mediated transformation or particle bombardment (Mao et al., 2019). Unfortunately, these traditional gene delivery methods require delicate and time-consuming tissue culture procedures and show limited success, especially in medicinal and other less studied plants. A recent breakthrough is the development of the Cut-Dip-Budding (CDB) gene delivery system. The CDB system is highly effective for plants with root-suckering capabilities. It allows the delivery of transgenes and gene-editing tools into plants through hairy root induction followed by shoot regeneration from transformed hairy roots, bypassing the need for tissue culture processes (Cao et al., 2023). Medicinal plants hold immense importance due to their medical, economic, ecological, and botanical values. These plants are rich sources of specialized secondary metabolites. There remains a notable bottleneck in exploring and utilizing the key genes responsible for producing bioactive compounds (Sun et al., 2022). Until recently, genetic transformation protocols were available for only a very small number of medicinal plant species. Therefore, there is an urgent need for innovative genetic transformation methods for medicinal plants. Fortunately, quite a few medicinal plant species possess root-suckering ability and can be transformed using the initial CDB method. Here, we present the development of a modified CDB method that achieves genetic transformation and gene editing in medicinal plants through direct Agrobacterium infection of various plant organs with a strong ability to generate shoots, bypassing not only tissue culture but also hairy root formation. One such organ we used is the root from medicinal plant species such as Pugongying (Taraxacum mongolicum) and Dihuang (Rehmannia glutinosa) (Figure 1A). Pugongying is widely used in treating inflammatory disorders (Zhang et al., 2022). Dihuang is a medicinal plant species from the Scrophulariaceae family and is always added into anti-diabetes compound recipes (Li et al., 2004). Detached roots of both medicinal plant species exhibit a robust ability to regenerate into a whole plant. We initiated the transformation process by cultivating Pugongying until their primary roots reached a thickness of 3–5 mm. These roots were then cut into 2–3 cm segments. We introduced a plasmid carrying Cas9 driven by the AtUBQ1 promoter, along with sgRNA targeting TmPDS driven by the AtU6 promoter, into the Agrobacterium rhizogenes K599 strain (Figure 1B). Both ends of the root segments were coated with solid agar-grown A. rhizogenes K599 bacteria. The infected root segments were then placed on moist vermiculite. Approximately 2–3 weeks later, we observed direct regeneration of albino shoots from the ends of Pugongying root segments (Figure 1C). PCR detection, TA cloning, and Sanger sequencing confirmed homozygous TmPDS mutations in the triploid Pugongying plants (Figure 1D). This experiment was repeated three times, with each experiment generating 6, 4, and 7 transgenic plants from 24, 26, and 27 root segments, and yielding 3, 2, and 3 edited mutants (Figure 1E), respectively. Using a procedure similar to that for Pugongying, Dihuang root segments were inoculated with A. rhizogenes K599. After 1–2 months of cultivation on moist vermiculite, we obtained a total of seven transgenic buds expressing the mCherry reporter gene among the regenerated buds at the ends of 47 root segments in three independent experiments (Figure 1F, G). Furthermore, we introduced the RUBY reporter gene into Dihuang and produced plants exhibiting betacyanin pigmentation (He et al., 2020) (Figure S1). Genetic transformation and gene editing of medicinal plants using a modified CDB protocol (A) Diagram of modified CDB protocol of Pugongying transformation. Red arrows indicate the cut site. The ends of root segments were infected with Agrobacterium and genetically modified shoots were regenerated from the root segments. (B) Structure of gene-editing construct for Pugongying. (C) Photographs of WT and TmPDS gene edited albino Pugongying mutant seedling. (D) Sequencing of homozygous TmPDS gene edited mutant line #1. (E) Statistics of Pugongying transformation and gene-editing efficiencies. (F) Photograph of bud regeneration from Dihuang root segments (upper) and photograph of the Dihuang bud under mCherry excitation light (lower). (G) Statistics of Dihuang transformation efficiency. (H) Diagram of the modified CDB protocol for Danshen transformation. (I) Structure of the gene-editing construct for Danshen. (J) Danshen explant inoculation with Agrobacterium and generation of the SmPDS gene edited albino mutant. Red arrow indicates the cut site. (K) Statistics for Danshen and Yuanzhi transformation efficiencies, and Danshen gene-editing efficiency. (L) Photograph of Yuanzhi regenerated bud (left) and photograph under mCherry excitation light (right). BF: bright field; Chi: chimeras; He: heterozygotes; Ho: homozygotes; PAM: protospacer adjacent motif; WT: wild type. Bars = 1 cm. Another such organ is the petiole of the medicinal plant species Danshen (Salvia miltiorrhiza), where shoots can easily regenerate (Figure 1H). We constructed a vector in which Cas9 was driven by the SlEF1α promoter, sgRNA targeting SmPDS was driven by the AtU6 promoter, and the GFP reporter was driven by the 35S promoter (Figure 1I). We inoculated the leaf petioles with A. rhizogenes K599 (Figure 1J), and then inserted the leaf petioles into moist vermiculite for cultivation. Approximately 2 months later, albino buds regenerated from the wounded sites on the leaf petioles (Figure 1J). Sanger sequencing confirmed homozygous editing of the SmPDS (Figure S2). We also obtained edited smlac15 mutant Danshen by targeting the SmLAC15 gene (Figure S3), which is involved in phenolic acid biosynthesis (Zhou et al., 2021). Out of 109 explants, we obtained seven transgenic plants, with four plants successfully edited (Figure 1K). The third such organ is the hypocotyl of Yuanzhi (Polygala tenuifolia), another important traditional Chinese medicinal plant. The hypocotyls of Yuanzhi can easily regenerate buds without tissue culture (Figure 1L). Yuanzhi, a species with no reported transformation success so far, possesses functions such as treating insomnia and forgetfulness, and enhancing cognitive function (Chen et al., 2022). Developing a genetic transformation system is crucial for understanding its medicinal mechanisms and drug development. To carry out transformation using CDB, we cut the hypocotyls of Yuanzhi and infected the cut sites with A. rhizogenes K599, resulting in regenerated transgenic plants expressing mCherry or RUBY (Figure 1K, L, S4). In summary, using the modified CDB method, we succeeded in genetic transformation or gene editing in four important medicinal plants. Our work demonstrated that the CDB gene delivery system can be used to transform medicinal plants with organs that, when detached, exhibit a strong ability to generate shoots. These organs include roots, petioles, and hypocotyls. The application of CDB methods to these explants with robust shoot regenerating ability not only increases the number of medicinal plants that can be conveniently transformed and genetically modified but also opens up the possibility of utilizing this very simple method across a wide range of plant species, including non-medicinal ones. This work was supported by the National Key R&D Program of China (2021YFA1300404 to J.-K.Z) and National Natural Science Foundation of China (32188102 to J.-K.Z), and by Bellagen Biotechnology Co. Ltd., Jinan, China. We are grateful to Zhenhua Cao from Shanxi Medical University for providing Taraxacum mongolicum and Polygala tenuifolia. The authors declare no conflict of interest. X.C. and J.-K.Z. designed the research. X.C., L.Z., and H.X. carried out the experiments. X.C., H.X., M.S., H.L., and J.-K.Z. analyzed the data. X.C., H.X., G.L., and J.-K.Z. wrote the paper. All authors read and approved of its content. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13593/suppinfo Figure S1. Photograph of Dihuang expressing RUBY Figure S2. Sequencing result of smpds homozygous mutant Figure S3. SmLAC15 gene editing result in a transgenic Danshen plant Figure S4. Photograph of Yuanzhi expressing RUBY Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.