Protocol for efficient ginseng transformation

Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher rate compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the selection media until identification of newly growing cells. In β-glucuronidase (GUS) assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased than non-treated control. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. We re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.


Introduction
Panax ginseng is a major medicinal plant (Sharma and Pandit. 2009). Ginseng synthesizes unique triterpene saponin called ginsenosides, which are beneficial to human health (Kim et al. 2015). Therefore, investigating the genes involved in the ginsenoside biosynthesis pathway by generating transgenic ginseng plants is important. However, functional studies using efficient and stable ginseng transformation are lacking, and limited due to the low success rate of stable transformation of P. ginseng.
Agrobacterium tumefaciens has been used for plant transformation because of its Ti-plasmid, and the insertion of bacterial Ti-plasmid into plant genomes results in the genetic modification of plants (Hwang et al. 2017). In P. ginseng, Agrobacterium-mediated transformation has been used to infect cotyledons (Lee et al. 1995), which are from zygotic embryos. Zygotic embryos are obtained from mature ginseng seeds. After sterilization of ginseng seeds, they are cut to take out embryos, and each embryo provides two cotyledons that are used for transformation. After transformation, transgenic tissues can be induced by plant hormones: 2, 4-dichlorophenoxyacetic acid (2, 4-D), one of auxin type hormones (Phillips et al. 2019), is used for the induction of embryogenic callus in ginseng (Wang et al. 1990). 6-Benzylaminopurine (BAP) is an artificial cytokinin that used to induce callus with 2, 4-D in P. ginseng (Lee et al. 2014). Callus has totipotency, which makes differentiation and regeneration possible if there are proper plant hormones. As plant genetic engineering and biotechnology advanced, especially for medicinal plants, callus cultures can be used to synthesize secondary metabolites and generate new breeds that have desirable features like stress resistance (Efferth et al. 2019). Moreover, callus can be material for stable transformation (Švubová and Blehová. 2013). Therefore, the generation of callus from plant material is important. In P. ginseng, application of indole-3-butyric acid (IBA), which is one of the auxin hormones, is used to differentiate adventitious roots from embryogenic callus (Lee et al. 2014).
MgSO 4 pre-treatment of plant material before infection with Agrobacterium can increase the efficiency of ginseng transformation. Addition of MgSO 4 to plant medium is reported to improve in vitro plant culture and transformation efficiency (Dalton et al. 2020). MgSO 4 pretreatment before Agrobacterium infection increased efficiency of transformation using ginseng cotyledons (Choi et al. 2003). In β-glucuronidase (GUS) assay, the expression of the GUS gene in transgenic explants were stronger than non-treated control. However, the success rate of ginseng transformation is low, thereby indicating the inability of cotyledons for transformation. In preparing the ginseng embryo for the plant material, seed sterilization and germination of the zygotic embryo must be performed (Lee et al. 2021). Comparing with the preparation of embryos, which involves complicated procedures, using calli for transformation can reduce the preparation of plant material, and it is easier to maintain and proliferate ginseng calli than embryos. Calli can be utilized for transformation by just cutting them into small pieces to facilitate infection mediated by Agrobacterium to calli before the experiment (Bevitori et al. 2014), and we suggest our efficient protocol for ginseng transformation (Fig. 1).
Introduction of CRISPR/Cas9 through plant tissue culture is actively performed in various plants (Montecillo et al. 2020). Gene editing by Cas9 expression in transformed cell requires to check mutation through DNA and RNA examination. Therefore, the efficiency of gene insertion and maintenance of transgenic explants are important to ensure deletion. However, certification of transgenic plant tissues at the DNA level includes many procedures, from DNA extraction to verifying the gene insertion with PCR using specific primers that are designed, resulting in a quantitative loss of transgenic lines. To increase the convenient and efficiency of selection for transgenic plants, we applied the new visible reporter RUBY system to ginseng, which changes the color of transgenic cells to red due to the accumulation of betalain (He et al. 2020). Through this new reporter, it became possible to identify transformed cells by their red-color phenotype without any other treatments for explants. Comparing with analysis methods such as GUS assay or DNA qualification, the application of the RUBY system was more convenient for distinguishing transformants.

Materials and methods
Ginseng callus induction and proliferation using medium culture 1. Ginseng calli induced on D1Ba0.5 medium containing 2.2 g/L Murashige and Skoog (MS) powder, 3% sucrose and 0.8% plant agar with 1 mg/L 2, 4-D and 0.5 mg/L BAP were transferred and maintained in a flask containing the same medium without plant agar. 2. They were cultured in a flask for 1 month (16 h light period, 60% relative humidity at 22 °C, 5000 lx). 3. After 1 month, calli cultured in liquid medium were used for the experiment or transferred into a new flask of liquid medium.
Ginseng stable transformation 1. Agrobacterium seed culture 1. Agrobacterium tumefaciens (strain LBA4404) was streaked on the yeast extract peptone (YEP) solid medium containing 10 g/L bacro-peptone, 10 g/L yeast extract, 5 g/L NaCl and added with 50 mg/L kanamycin or 5 mg/L tetracycline (depending on the antibiotic resistance gene of introduced vectors) and 200 mg/L rifampicin. 2. The plate was cultured in a dark 28 °C incubator (Republic of Korea, JSR, JSBI-050 C) for 48 h. 3. Five milliliters of YEP liquid medium (added with antibiotics) was prepared in a test tube, and a single colony was selected from the solid medium. 4. The test tube was cultured overnight in a 28 °C shaking incubator (Republic of Korea, DOOREE Science, DF-94 F), and the incubator was maintained in the dark.

Agrobacterium mass culture
1. Twenty milliliters of antibiotic-free YEP medium was prepared for one construct in a flask.
2. All seed culture liquid was added to the as-prepared 20 mL YEP medium for mass culture. 3. The flask containing medium and Agrobacterium was cultured until OD 600 reaches 0.6 in the 28 °C shaking incubator in the dark. 4. When OD 600 reaches 0.6, the flask was placed on ice or refrigerated to stop the growth of Agrobacterium, keeping it fresh.

Callus preparation
1. During the Agrobacterium mass culture, liquid-cultured ginseng calli were removed from the flask. 2. The compact calli were cut into small pieces, approximately 0.5 cm 3 .

MgSO 4 pre-treatment
1. The cut calli were dipped into 25ml of 0.05 M MgSO 4 for 30 min.

Resuspend
1. The calli pre-treated with MgSO 4 were moved to 1/2MS liquid medium and resuspended for 5 min.

Infection
1. The Agrobacterium mass culture liquid was centrifuged at 4,500 RPM for 5 min in 4 °C, and the supernatant was removed. 2. Acetosyringone was added to MS liquid medium at a concentration of 50 µM. 3. After removing supernatant, we added same volume of infection solution into cells. The same volume of infection solution was added to the total volume of mass culture liquid used to maintain the concentration of Agrobacterium. 4. The centrifuge bottle added with the infection solution was carefully shaken to resuspend Agrobacterium. 5. The ginseng calli pre-treated with MgSO 4 were dipped into the infection solution. 6. All calli must be dipped into the solution, and infection was performed for 5 min. 7. After 5 min, the calli were removed from the infection solution and transferred onto filter paper. 8. The infection solution on the surfaces of the calli was completely removed.

Co-culture
1. The dried calli were transferred into D1 solid medium containing 2.2 g/L MS powder, 3% sucrose and 0.8% plant agar with 1 g/L 2, 4-D. 2. The plate was cultured in a dark chamber for 48 h.

Selection culture
1. D1Ba0.5 CH medium, D1Ba0.5 solid medium containing cefotaxime and hygromycin antibiotic, were prepared. The concentrations of the antibiotics are 200 mg/L for cefotaxime and 50 mg/L for hygromycin. 2. The co-cultured calli were transferred from D1 medium to D1Ba0.5 CH medium. 3. They were cultured in a light chamber and moved into a new medium every 2 weeks. When moving them into a new medium, dead calli were removed, and only surviving calli were transferred into the new medium. 4. After 6 weeks, the surviving calli were transferred into the same medium with a low concentration of antibiotics (100 mg/L of cefotaxime and 25 mg/L of hygromycin). 5. They were moved into new medium that contain lower antibiotics every 2 weeks. 6. After 6 weeks, selection was completed. The surviving calli were transferred into an antibiotic-free medium.

Cloning of p35S:RUBY
RUBY vector without a promoter was provided by the Crop Biotech Institute, Kyung Hee University. We digested the vector with HindIII and HpaI at 37 °C for 2 h, and cloned the p35S promoter in front of the RUBY gene. The modified vector was named p35S:RUBY.

Results and discussion
In this study, we updated the protocol for P. ginseng transformation using callus in an efficient and convenient way. Previous studies on ginseng transformation have used the cotyledon from zygotic embryos for stable transformation. However, plant callus has also been used for Agrobacteriummediated transformation (Hossain et al. 2007). Similarly, P. ginseng callus can be used for transformation (Choi et al. 2006). Therefore, the transformation efficiency of the ginseng callus was tested and compared with that of the cotyledon. We used cotyledons and calli for transformation (Fig. 2a). Although we could obtain calli from cotyledons and calli (Fig. 3), the survival rate of callus 3 months after transformation was 13.28% (Table 1). By contrast, about 1% could be obtained from the cotyledon, indicating that the survival rate during selection culture is higher when using the callus rather than the cotyledon. The generation of callus tissue was also faster in transgenic calli than in transgenic cotyledons (Fig. 3). Comparing convenience of preparation of plant materials, we tested ginseng calli for the plant materials, which were proliferated with liquid medium with 2, 4-D and BAP, and calli were just cut into 0.5cm 3 before transformation. In contrast, cotyledons require seed maturation, seed sterilization, and dissection of embryos for every transformation. Therefore, compared with the cotyledon, using the callus is much more convenient to generate callus. An Agrobacterium strain can affect the success rate of transformation (Zhi et al. 2015); thus, we performed ginseng transformation by using two Agrobacterium strains, namely, LBA4404 and C58C1, using p35S:GUS for constructs under same conditions. Calli infected with C58C1 were easily contaminated by Agrobacterium in all explants (Fig. 2f), despite decrease of infection time. Therefore, we updated our system using the LBA4404 strain.
In previous study, plant hormones contained in the medium affected the efficiency of transformation (Li et al. 2017). We introduced p35S:GUS into ginseng calli and conducted the GUS assay after 12 weeks of selection culture using selection medium containing hormones and without hormones. The expression of the GUS gene was shown in transgenic calli that were selected with hormones, but the expression was rarely shown in transgenic calli that were selected in hormone-free medium during the same selection ginseng. a-d The materials used for transformation: embryogenic callus (callus) and cotyledon of germinated zygotic embryo (cotyledon). After co-culture with Agrobacterium, cotyledon (b) and callus (d) were grown on selection medium. However, after 2 months of transformation, survival lines using callus as starting materials were 20-40%, whereas (c) cotyledons survive about 1%. e The effect of hormone on selection medium. GUS-reacted callus is shown in blue on hormone-treated medium, but less reaction occurs in hormonefree medium. f The calli after 1 month of transformation using Agrobacterium tumefaciens LBA4404 (left side) and C58C1 (right side) with the same construct. Survival rate of callus after 1 month is about 30% and 0%, respectively. g The effect of MgSO4 pretreatment before Agrobacterium co-cultivation. GUS-reacted callus is shown in blue during treatment. Scale bar = 10 mm Fig. 3 Callus generation after selection from antibiotic medium. Generation of callus cells in the explants was accelerated on antibioticfree medium. Calli (a) could generate the cells faster than cotyledons (b). During the same period, cells generated from calli (a3 ~ a6) were bigger then cotyledons (b1 ~ b6). Dedifferentiation of cells consisting cotyledons must occur to generate callus, so it takes more time to obtain callus cells than using calli for transformation. Red circles indicate newly formed callus cells, which were transferred to induce adventitious roots. Scale bar = 4.0 mm procedures (Fig. 2e). The survival rate after 3 months after transformation was highest in calli that were cultured on hormone-free medium (Table 1), however, we failed to obtain transgenic explant lines due to inefficient gene delivery. Therefore, we applied D1Ba0.5CH medium, which contains plant hormones for selection culture, to our system.
As callus is a hard tissue, we hypothesized that all cells in explants could not be transformed. Similar with the previous report (Abdallat et al. 2011), newly growing antibiotic resistant cells appeared on the surface of the degenerated explants. In the GUS assay of p35S:GUS transgenic lines, the expression of the GUS gene was shown in antibiotic We conducted MgSO 4 pretreatment to increase the efficiency of transformation. Transformant calli that were pretreated with MgSO4 showed larger area of GUS expression compared with control in newly growing cells, as observed by the GUS assay (Figs. 2g and 3a). The success rate of the transformation from survival lines during culture selection was 68% and 77.27% in control and MgSO 4 pre-treatment, respectively (Table 1). We hypothesized MgSO4 promotes recovery of calli after infection to Agrobacterium, resulting higher efficiency of transformation. Although it is not clear for how MgSO 4 increases transformation efficiency, it was reported the effect of MgSO 4 on plant growth especially against stress condition (Ren et al. 2022), and further role needs to be investigated.
Following our protocol, the highest survival rate of explants after 3 months was 25.38% in hormone-free medium (Table 1), but none of them were transformants. The highest rate for the generation of transgenic lines was 77.27% (Table 1). We successfully generated transgenic adventitious roots from calli on B5I3 media under dark condition to obtain stable transgenic tissues ( Fig. 4c and d). We certified p35S:GUS transgenic callus lines with GUS reaction and DNA qualification with PCR using specific primers (Table 2) of the inserted gene (Fig. 4). During DNA qualification, the result of the GUS assay and DNA qualification was inconsistent, particularly for callus (Fig. 4e). As shown in Fig. 4a and d, adventitious roots induced from a callus showed accurate and uniform expression, which was induced from one cell of the callus.
However, p35S:GUS transgenic lines have a limitation that requires the GUS assay, indicating that cells are not examined in vivo. Therefore, we introduced the visual reporter RUBY system in P. ginseng for the first time (Fig. 5). We cloned the p35S promoter into the promoterless RUBY vector (Salazar-González et al. 2022), which was digested with two different restriction enzymes, HindIII and HpaI (p35S:RUBY, Fig. 5a). Using our protocol, we could obtain calli and observe with the naked eye that red cells We inserted a p35S promoter in front of RUBY to induce its strong expression in ginseng. HindIII and HpaI were treated and cut in front of RUBY, and a p35S promoter was inserted to induce strong expression of RUBY. Scale bar = 10 mm started to grow on the surface of explants without any chemical treatments (Fig. 5b). In p35S:RUBY transgenic lines, cells can be easily separated for proliferation. By using the RUBY system, we could certify our hypothesis that transformant cells are newly growing cells, which are shown in red on the surface of RUBY transgenic callus. Therefore, genetic modification likely occurred in newly generating cells around the cut surface of callus explants. Using this new reporter system in ginseng, the efficiency of gene insertion could be significantly increased by selecting red cells, which are transgenic.

Conclusion
• By a liquid medium containing 2, 4-D and BAP, calli could be easily propagated. • Agrobacterium LBA4404 was appropriate for ginseng transformation than C58C1. • The transformation efficiency of growing cells was increased through MgSO 4 pretreatment. • The rate of ginseng transformation was increased up to 77.27% in surviving lines during culture selection. • Selecting transgenic cells is important. Thus, a visible reporter RUBY system was introduced to identify transgenic cells through a red color with the naked eye and to easily generate transgenic plants, particularly CRISPR/ Cas9-mediated knockout lines.