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.
Keywords: cut–dip–budding (CDB) delivery system, gene editing, genetic transformation, succulents
Introduction
Gene editing technology, particularly the CRISPR‐Cas system, has brought about a breakthrough in plant genetic improvements. An effective delivery system of gene editing tools is often the bottleneck that limits the application of gene editing technologies. Agrobacterium tumefaciens‐mediated transformation and particle bombardment are the main methods for plant genetic transformation. However, many plants cannot be transformed using these methods. Besides requiring the tedious process of tissue culture, the transformation efficiencies of these methods are dependent on plant species and genotype. Although plant regeneration factors such as the BABY BOOM (BBM8), WUSCHEL (WUS), LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2) and GRF‐GIF genes have been used to enhance plant genetic transformation efficiencies and to expand the range of transformable genotypes (Debernardi et al., 2020; Horstman et al., 2017; Liu et al., 2014; Stone et al., 2001; Wang et al., 2022), most plants remain difficult to be transformed using these methods.
It is highly desirable to transform plants without the requirement for tissue culture. One such system is the widely used floral dip method of genetic transformation in Arabidopsis (Clough and Bent, 1998). Another is the recently developed ‘cut‐dip‐budding’ (CDB) method (Cao et al., 2023). Using this latter innovative technique, successful transformation has been achieved in multiple plant families, including two herbaceous plants (Taraxacum kok‐saghyz and Coronilla varia), a tuberous root plant (sweet potato) and three woody plant species (Ailanthus altissima, Aralia elata and Clerodendrum chinense). These plants possess root suckering capacity. When infected with Agrobacterium rhizogenes, genetically transformed hairy roots generated from their explants can be induced to develop transformed shoots.
Root suckering represents a method of vegetative reproduction found in certain plants, wherein fresh shoots or stems sprout from the roots of an established plant (Akkerman, 2003; Del Tredici, 1995). This process generally entails the activation of dormant buds or meristematic cells within root tissues, ultimately giving rise to new shoots. It is conceivable that, upon Agrobacterium infection, dormant meristematic cells in shoot tissues may undergo transformation and develop transformed buds. If this hypothesis holds true, it may open up the possibility of extending the CDB gene delivery system to plant species lacking root suckering capabilities but instead harboring dormant meristematic cells in other plant organs. Succulent plants form a notable category within this group. Numerous succulent species possess the capacity to generate shoots from their leaves, particularly when these leaves are cut or torn.
In this study, we demonstrated that the CDB system can be adapted to transform succulent plants with a shoot regeneration ability from cut leaves. Three succulent species, including both monocotyledonous and dicotyledonous plants, were successfully transformed and transgenic or gene edited plants obtained. Our work greatly expands the application of the CDB system for plant transformation and gene editing.
Materials and methods
Plant materials
We chose three succulent species as research materials, including Kalanchoe blossfeldiana, Crassula arborescens and Sansevieria trifasciata (Figure S1A–C). All three species have strong ability to generate shoots from cut leaves.
Vector construction and Agrobacterium rhizogenes preparation
The binary plasmid pCAMBIA1300 was used for constructing the transformation vector. The EGFP (Figure S2A) or RUBY (Figure S2B) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, was used as a reporter gene. Cas9, sgRNA and AtU6 expression boxes were inserted in front of the EGFP reporter cassette for simultaneous delivery into plant cells (Figure S2A). The phytoene desaturase (PDS) gene of K. blossfeldiana was targeted by CRISPR/Cas9 for easy visualization of gene edited plants. The plasmids were introduced into A. rhizogenes K599 cells via heat‐shock transformation. The transformed Agrobacteria were cultured (28 °C, 220 rpm) to reach OD600 of 0.8–1 in TY medium (5 g/L TRYPTONE and 3 g/L YEAST EXTRACT) containing 50 mg/L streptomycin, 50 mg/L kanamycin and 10 mM CaCl2. Three hundred microlitres of this culture was spread and cultured overnight on TY solid agar medium (5 g/L TRYPTONE, 3 g/L YEAST EXTRACT and 15 g/L AGAR) until the agar medium was covered with a uniform layer of bacteria. The bacterial suspension in liquid TY medium and the slush bacterial layer from the solid agar medium were then ready for plant infection. PCR primers are listed in Table S1.
Plant transformation
In this study, we modified the original CDB method in previously published article (Cao et al., 2023). In the original CDB method, cut stems were used as explants and inoculated with A. rhizogenes K599 bacteria to generate hairy roots. Transformed hairy roots are grown in soil before being cut into segments; later, the root segments generate buds due to their root suckering ability. In the current study, we used the leaves of succulents as explants, which were inoculated with bacteria and then generated buds directly from the infected explants, without the need to produce hairy roots. Briefly, hypertrophic leaves from approximately two‐year‐old K. blossfeldiana plants were cut into leaf segments using a scalpel. These cut leaf segments were used as explants and the cut end at the base of the leaf segments were immersed for 20–30 min in A. rhizogenes K599 suspension from liquid culture that were sedimented and resuspended in the infection solution [10 mm MgCl2, 10 mm 2‐(N‐morpholino) ethanesulfonic acid (MES), 100 μm acetosyringone (AS), pH 6.0]. The cut ends of these explants were then coated with bacterial layers from the TY solid agar medium. Next, these inoculated leaf segments were placed in a culture box containing moist soil (nutrient soil: vermiculite = 1:2). The inoculated explants were cultured at 26 °C with a 16‐h light/8‐h dark cycle until buds developed from the leaf cuttings (approximately 2 months). Putative transgenic buds were then transferred to potted soil and grown under normal growth conditions. The transformation method for C. arborescens was identical to that used for K. blossfeldiana.
For S. trifasciata, the cut leaf segments required vacuum infiltration instead of soaking in A. rhizogenes K599 suspension before being further infected by coating the cut sites with A. rhizogenes K599 layers from agar media. For vacuum infiltration, the incisions near the base of the leaf segments were soaked in the A. rhizogenes K599 suspension and were then subjected to vacuum pressure (0.08 MPa, 5s) in a tightly sealed vessel. After the degassing process to facilitate Agrobacterium entry into the leaf segments, the cut sites of leaf segments were coated with bacterial layers and then cultured in soil until buds emerged.
PCR test
We isolated DNA from the young leaves of succulents using the cetyl‐trimethylammonium bromide (CTAB) method and dissolved it in H2O containing 100 ng/mL of RNase. This genomic DNA served as a template for subsequent PCR and Southern blotting or gene editing analysis. To verify the presence of transgenes, we amplified the EGFP or RUBY gene fragment using 1 μL of diluted DNA (100–150 ng/μL) and the corresponding primers.
Bar protein test
For tests of the phosphinothricin acetyltransferase (bar) herbicide resistance gene, we detected the presence of the bar protein in genetically modified plants using the QuickStixTM Kit (ENVIROLOGIX, Cat. NO. AS013LS). Plant leaf tissue was placed into the tapered bottom of a 1.5 mL centrifuge tube and ground with a pestle by rotating it against the sides of the tube with twisting motions for 20–30 s. Five hundred microlitres of extraction buffer (at room temperature) was added and the grinding step was repeated to ensure thorough mixing of the tissue with the buffer. The test strip was placed into the extraction tube right away and the result read within 10–15 min. If two lines appeared, the sample was considered positive.
Southern blot analysis
Southern blot analysis was carried out to determine the copy number in the PCR‐positive transgenic plants. Approximately 40 μg of genomic DNA from transformed or non‐transformed control plants was digested overnight with Hind III. Seven micrograms of the digested DNA were separated on a 0.8% agarose gel. After electrophoresis, the digested DNA was transferred to a Hybond‐N+ nylon membrane (Amersham Pharmacia) and hybridized with a DIG‐labelled Bar gene probe prepared by PCR. The blots were washed at 65 °C under stringent conditions and analysed using a Typhoon‐8600 scanner (Zhou et al., 2008). All PCR primers are listed in Table S1.
Statistical analysis
The statistical tests, sample sizes and replication are reported in the figure legends and tables.
Results
Transformation of Kalanchoe blossfeldiana with the CDB gene delivery system
The succulents, especially those of the Crassulaceae family, can develop new roots and shoots from leaf cuttings. We hypothesized that the shoot regeneration ability of cut leaves could be used to deliver transgenes or gene editing constructs. We tested our hypothesis first in K. blossfeldiana, a popular ornamental marketed worldwide both as indoor potted plant and as a garden plant. Although K. blossfeldiana can be transformed using conventional transformation methods (Favero et al., 2021; Lutken et al., 2010), the skills required for these methods and genotype dependency have limited the genetic engineering of this plant.
We adapted the CDB method to transform K. blossfeldiana as depicted (Figure 1a). Hypertrophic leaves from mother plants were cut into leaf segments with a surgical blade (Figure 1b). The leaf segments were infected with Agrobacteria carrying the EGFP reporter and then cultured in a box (Figure 1c). About 2 months later, we observed EGFP positive adventitious buds (Figure 1d,e). These EGFP‐positive buds were transferred to nutrient‐rich soil for further cultivation. The plants developed from these buds showed strong EGFP fluorescence (Figures 1f,g). PCR analysis (Figure 1h), Southern blotting (Figure 1i) and Bar test (Figure 1j) confirmed that these plants were transgenic.
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.
The young leaves of the plants from EGFP positive buds were fully fluorescent under UV light. Older leaves did not exhibit green fluorescence or only parts of them were fluorescent (Figure S3A,B). PCR and bar strip analysis showed that both young and old leaves were positive for the transgenes (Figure S3C,D). It appeared that EGFP expression was influenced greatly by the developmental stage of leaves in the succulent plants.
The usefulness of the CDB gene delivery system for K. blossfeldiana transformation was also evaluated using RUBY, a visual reporter known to perform well in all tested plants (He et al., 2020). The results show successful generation of transgenic buds expressing RUBY in a stable manner in K. blossfeldiana (Figure S4A). These RUBY‐positive buds exhibited normal growth (Figure S4B) and distinctive red leaf phenotype owing to RUBY expression. PCR results further confirmed the transgenic nature of these individuals with red leaves (Figure S4C). Collectively, these findings indicate the reproducibility of utilizing the CDB gene delivery system for generating transgenic K. blossfeldiana plants.
Efficient delivery of gene editing tools using the CDB system
To determine whether the modified CDB system could be employed for delivering gene‐editing tools, we selected the phytoene desaturase (PDS) gene as a target for gene editing experiments in K. blossfeldiana. Successful editing of the PDS gene would result in an albino phenotype, simplifying the identification of gene‐edited plants. The CDB experiments were conducted three times and gene‐edited albino plants were obtained in all three experiments.
In experiment I, out of the 50 explants infected with Agrobacteria, 35 produced transgene‐positive buds, 34 of which were successfully edited at the PDS locus (Figure 2a). The results of the subsequent two experiments (experiment II and III) closely mirrored those of experiment I. Taken together, the transformation efficiency of K. blossfeldiana across all three experiments averaged approximately 74%, with an editing efficiency of approximately 70%. Despite the presence of 20 chimeric plants, all of the edited plants exhibited the albino phenotype at least in some parts of the plants (Figure 2b), suggesting that homozygous edits had been achieved in all edited plants. Sequence analysis confirmed that all albino and chimeric plants had undergone editing at the target site (Figures 2c). These data suggest that the CDB system is a highly reliable method for delivering gene editing tools to generate gene‐edited plants.
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.
Transformation of Crassula arborescens using the CDB delivery system
To determine whether the modified CDB method can be used to transform other succulent plants, we applied it to Crassula arborescens, another species from the Crassulaceae family. C. arborescens is cultivated as an ornamental plant for use in succulent gardens and as a houseplant for indoor growing. Since the modified CDB method worked well in K. blossfeldiana, we chose to use RUBY as the visual marker to evaluate the effectiveness of the method in C. arborescens.
Using the modified CDB protocol that was employed for K. blossfeldiana, we obtained red adventitious buds at the cut site of the leaf base of C. arborescens (Figure 3a). These red buds grew normally and were considered as putative transgenic buds expressing the RUBY gene (Figure 3b). These putative transgenic buds were confirmed through PCR and sequence analysis (Figure 3c). Out of 120 explants infected with Agrobacteria in three separate experiments, 6 produced transgene‐positive buds, indicating a 5% transformation efficiency (Figure 3d). Although this efficiency is lower than that of K. blossfeldiana, the modified CDB method provides a viable means for genetic improvement of C. arborescens through gene editing or transgenic approaches.
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.
Transformation of Sansevieria trifasciata with the CDB delivery system
After successfully applying the CDB gene delivery system to the two succulents as described above, we tested whether the modified CDB method could be used transform Sansevieria trifasciata ‘Jinbianlan,’ a monocotyledonous species for which no genetic transformation method has been reported to date (Figure S5A). S. trifasciata ‘Jinbianlan’ belongs to the genus Sansevieria within the lily family. It is an evergreen perennial succulent plant with hypertrophic leaves and can propagate by regenerating adventitious buds from cut and scarred leaves. Additionally, it holds significant ornamental value and has potential applications in traditional Chinese medicine, air purification and soil repair (Guo, 2007; Li and Yang, 2020).
We conducted the CDB experiment on S. trifasciata using the workflow shown in the Figure 4a. Leaves were removed from the base of the donor plant and were meticulously cut into leaf segments using surgical blades (Figure 4b). These leaf segments served as explants and they were inoculated with Agrobacterium K599 carrying EGFP through vacuum infiltration and bacterial layer application. The inoculated explants were then placed into a culture box filled with moist soil for further cultivation (Figure 4c). Approximately 3–4 months later, adventitious buds began to form (Figure 4d). EGFP‐positive buds were identified using a UV lamp and were transplanted into nutrient‐rich soil (Figure 4e) to grow into adult plants (Figures 4f,g). All of the EGFP‐positive plants were subsequently confirmed as transgene‐positive through PCR detection, Bar protein testing and Southern blotting (Figures 4h–j). The results showed that the CDB gene delivery system was effective for genetic transformation of this monocotyledonous succulent plant.
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.
CDB delivery system is genotype‐independent in S. trifasciata
To evaluate whether the effectiveness of the CDB method may be plant genotype‐dependent in S. trifasciata, we assessed its efficiency in five distinct cultivars. In addition to S. trifasciata ‘Jinbianlan’, we applied the CDB method to S. trifasciata ‘Heijingang’, S. trifasciata ‘Hani’, S. trifasciata ‘Zuanshilan’ and S. trifasciata ‘Yinmailan’, cultivars exhibiting different plant heights and varying leaf coloration patterns (Figure S5A–E).
The EGFP gene, under the control of the CaMV 35S promoter, was introduced into these four cultivars using the CDB delivery system. The results indicated successful transformation in all four cultivars (Figures 5a–h), as confirmed by PCR analysis of EGFP gene fragments (Figure 5i). To assess the transformation efficiency of different S. trifasciata species using the CDB system, we subjected 180 explants to infiltration for each of the five varieties tested. Ultimately, we obtained 13, 14, 13, 8 and 6 transgene‐positive buds from S. trifasciata ‘Jinbianlan’, S. trifasciata ‘Heijingang’, S. trifasciata ‘Hani’, S. trifasciata ‘Zuanshilan’ and S. trifasciata ‘Yinmailan’, respectively (Figure 5j). The results suggest that genetic transformation through the CDB gene delivery system is genotype independent.
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.
We also introduced the RUBY gene into S. trifasciata ‘Heijingang’ using the aforementioned CDB protocol and obtained transgenic buds stably expressing RUBY (Figure S6A–C). The results confirmed the reproducibility of the CDB method for S. trifasciata transformation.
Discussion
The efficient delivery of transgenes or editing tools to plants continues to be an important challenge to date (Mao et al., 2019). Fewer than 0.1% of the more than 370 000 species of higher plants can be genetically modified by Agrobacterium tumefaciens‐mediated transformation or particle bombardment, which require laborious and time‐consuming tissue culture work. Recently, we reported tissue culture‐free CDB method for genetic transformation and gene editing in several plant species with root suckering ability (Cao et al., 2023). In this study, we modified the CDB method and used it to transform succulent plants with shoot regeneration ability from cut leaves. Our work broadens the application of the CDB gene delivery system and make more plant species amenable to improvement through genetic engineering.
The CDB method holds great promise for enhancing economically significant fruit trees and succulent plant varieties and any other plant species capable of root suckering or regenerating shoots from cut leaves. Breeding fruit trees and succulent plants has long been a challenging endeavour due to their unusually protracted juvenile stages (Hewitt, 1997). The transition from seed to flowering in these plants can span from several years to a decade or more. Fortunately, most trees and succulents can be reproduced vegetatively, offering a workaround to this obstacle. The combination of gene editing technology, the CDB gene delivery system and asexual propagation presents a solution. Utilizing the CDB system, gene editing tools can be effectively introduced into these traditionally difficult‐to‐transform species, allowing for the incorporation of desired traits or the modification of existing traits. These enhanced traits can then be rapidly propagated asexually (e.g. cut leaves), streamlining the process and eliminating the necessity of waiting for multiple generations and many years to achieve the desired results. This would revolutionize plant breeding in fruit trees, succulent plants and other recalcitrant plants.
Expanding the application of the CDB delivery method to succulents, plant species lacking the ability for root suckering, opens up the possibility of utilizing this technique for a wider range of plant species. A key similarity between plants with root suckering capacity and succulents seems to lie in the presence of dormant buds or dormant meristematic tissues in their roots and leaves, respectively. For the successful application of the CDB method, it is imperative to not only create wounds for Agrobacterium infection but also to stimulate these dormant buds. Various external disturbances, such as the cutting of plant tissues, can serve as triggers for the activation of these dormant buds.
Since dormant meristematic cells or buds are present in a wide variety of plant species (Anderson et al., 2001; Lang et al., 1987), subjecting these dormant meristematic cells or buds to Agrobacterium infection under suitable conditions could potentially lead to successful CDB‐mediated transformation, especially if we can identify explants where dormant buds can be activated when detached from their parent plants. Our results here indicate that the CDB gene delivery system holds great potential to extend genetic transformation and gene editing to a diverse array of plant species.
Author contributions
J.L. and J.‐K.Z. designed and supervised the research. J.L., S.L., Y.W., S.L. and C.S. carried out the experiments. J.L., S.D., M.W., J.D., M.L., G.L., Z.L. and J.‐K.Z. discussed and analysed the data. J.L., S.L., G.L. and J.‐K.Z. wrote the paper.
Declaration of interests
The authors declare no competing interests.
Supporting information
Figure S1 Shoot regeneration from cut leaves of three succulents. (A) K. blossfeldiana. (B) S. trifasciata. (C) C. arborescens. Scale bar: 1 cm.
Figure S2 Schematic of the gene constructs used in this study. (A) The gene‐editing vector carrying EGFP and Bar. (B) The vector carrying RUBY and Bar.
Figure S3 EGFP‐positive buds of K. blossfeldiana produced leaves that were all transgene‐positive. Scale bar: 1 cm.
Figure S4 RUBY‐positive buds were obtained in K. blossfeldiana using the CDB method.
Figure S5 The five cultivars of S. trifasciat used in this study.
Figure S6 RUBY‐positive adventive buds obtained in S. trifasciata.
Table S1 Primers used in this study.
Acknowledgements
This research was supported by Shandong Shunfeng Biotechnology Co. Ltd., Jinan, China and by grants from the National Natural Science Foundation of China (32188102 to J.‐K.Z.), the Project of Sanya Yazhou Bay Science and Technology City (Grant No. SCKJ‐JYRC‐2023‐72), the Innovational Fund for Scientific and Technological Personnel of Hainan Province (Grant No. KJRC2023D18), and the Hainan Provincial Natural Science Foundation of China (Grant No.324MS088).
Data availability
All the data supporting the findings of this study are available in the paper and supplementary data.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1 Shoot regeneration from cut leaves of three succulents. (A) K. blossfeldiana. (B) S. trifasciata. (C) C. arborescens. Scale bar: 1 cm.
Figure S2 Schematic of the gene constructs used in this study. (A) The gene‐editing vector carrying EGFP and Bar. (B) The vector carrying RUBY and Bar.
Figure S3 EGFP‐positive buds of K. blossfeldiana produced leaves that were all transgene‐positive. Scale bar: 1 cm.
Figure S4 RUBY‐positive buds were obtained in K. blossfeldiana using the CDB method.
Figure S5 The five cultivars of S. trifasciat used in this study.
Figure S6 RUBY‐positive adventive buds obtained in S. trifasciata.
Table S1 Primers used in this study.
Data Availability Statement
All the data supporting the findings of this study are available in the paper and supplementary data.