Colchicine Mutagenesis from Long-term Cultured Adventitious Roots Increases Biomass and Ginsenoside Production in Wild Ginseng (Panax ginseng Mayer)

Panax ginseng Mayer is a perennial herb that has been used as a medicinal plant in Eastern Asia for thousands of years. The aim of this study was to enhance root biomass and ginsenoside content in cultured adventitious roots by colchicine mutagenesis. Adventitious P. ginseng roots were treated with colchicine at different concentrations (100, 200, and 300 mg·L−1) and for different durations (1, 2, and 3 days). Genetic variability of mutant lines was assessed using random amplification of polymorphic DNA (RAPD) analysis. Ginsenoside biosynthesis gene expression, ginsenoside content, enzyme activities, and performance in bioreactor culture were assessed in four mutant lines (100–1-2, 100–1-18, 300–1-16, and 300–2-8). The results showed that ginsenoside productivity was enhanced in all mutant lines, with mutant 100–1-18 exhibiting the most pronounced increase (4.8-fold higher than the control). Expression of some ginsenoside biosynthetic enzymes was elevated in mutant lines. Enzyme activities varied among lines, and lipid peroxidation activity correlated with root biomass. All four lines were suitable for bioreactor cultivation, with mutant 100–1-18 exhibiting the highest biomass after culture scale-up. The results indicated that colchicine mutagenesis of P. ginseng roots increased biomass and ginsenosides production. This technique, and the root lines produced in this study, may be used to increase industrial yields of P. ginseng biomass and ginsenosides.


Introduction
Cultivation of plant cell tissues and organs is one of the most powerful tools available for research into plant propagation, crop development, production of valuable phytocompounds, and preservation of endangered plant species. However, in vitro plant materials accumulate genetic and epigenetic changes at high frequencies during long-term culture. Long periods of continuous subculture also enhance somaclonal variation [1], which leads to changes in phenotypes such as plant size, yield, Root tips, 1.5 cm long, were washed three times with MS medium and then transferred to fresh solid medium supplement with 5.0 mg·L −1 IBA, 3.0% sucrose and cultured in the dark at 24 ± 1 • C. Lateral root induction, the number of lateral roots per explant and lateral root length were recorded after eight weeks. The lateral roots of treated roots from each treatment were detached and cultured on the same medium describe above. The putative mutant roots were firstly screened for morphology and biomass after six weeks of culture. The four putative lines with the highest biomass were selected and screened again for ginsenoside content.
Mutant roots were transferred to a flask containing 100 mL of MS liquid medium containing IBA and 3% sucrose and assessed in a scaled-up 3 L bioreactor containing 2 L of MS liquid medium. The inoculation density of root in flask and bioreactor culture was 5.0 mg·L −1 . Lateral root number, root length, and biomass (fresh and dry weight) were measured in the flask and bioreactor cultures. Mutant root morphology was also examined.

DNA Content Analysis by Flow Cytometry
DNA content of colchicine-treated adventitious roots was analyzed by flow cytometry (Partec PA, Münster, Germany) after six weeks of culture. Three to four lateral roots were chopped using a razor blade in 0.4 mL nuclei extraction buffer (CyStain UV Precise P, Partec, Münster, Germany) for 30 s. The sample was incubated for 5 min and filtered through a Partec 50 µm celltrics ® disposable filter. Staining buffer (1.6 mL) was added to the sample tube, incubated for 30 s and analyzed in a flow cytometer. At least 3000-6000 nuclei were measured per sample and processed using FloMax software (Partec, Münster, Germany). The peak of nuclei insulated from a tetraploid (control) lateral root of P. ginseng was adjusted at channel 100.

DNA Isolation
Fresh 6-week-old adventitious roots (0.1 g) from selected putative mutant lines and from three tetraploid control lines were homogenized using a Tissuelyser II (Qiagen, Hilden Germany). Genomic DNA was isolated following the cetyl trimethyl ammonium bromide (CTAB) extraction method [26]. CTAB buffer solution and 0.2% β-mercaptoethanol were added to the homogenized adventitious roots samples. Samples were incubated at 65 • C for 30 min, chloroform:isoamyl alcohol extraction solution (24:1) was added, and DNA was precipitated with isopropanol. Pellets were washed with 70% ethanol and resuspended in 50 µL of sterilized water. DNA concentration was measured using a DS-11 + spectrophotometer (Denovix, Inc., Wilmington, DE, USA).

Preparation and Extraction of Mutant Lines
The 6-week-old root samples (0.5 g dry weight) of control and selected mutant roots were placed in 100 mL flasks with 50 mL of 80% (v/v) ethanol. Flasks were sonicated in an ultra-sonication bath (SD-D250H by Mujigae Co., Seoul, Korea) for 1 h at room temperature. Thereafter, extracts were filtered through Whatman filter paper (No. 1002 110) and collected in a round-bottom flask. The solvent was evaporated to dryness using a rotary evaporator (N-1000, Eyela, Tokyo, Japan) at 40 • C, and the residue was dissolved in 50 mL of distilled water. The aqueous solution was washed twice with 50 mL of ethyl ether, and the aqueous layer was then extracted twice with 50 mL of water-saturated n-butanol. The n-butanol fraction was evaporated using a rotary evaporator at 50 • C. The sample solution was dissolved in 2 mL of methanol and filtered through a 0.2 µm Whatman syringe filter before analysis by High Performance Liquid Chromatography (HPLC).

Lipid Peroxidation
Lipid peroxidation contents were measured using a modified malondialdehyde (MDA) method [27]. The 6-week-old root tissue (0.4 g) of control and selected mutant roots were disrupted in liquid nitrogen with a mortar and pestle and homogenized in 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 5000 rpm for 5 min. The supernatant was then blended with 0.6% TBA and placed in a water bath at 90 • C for 30 min. Absorbance was measured at 532 and 600 nm using a spectrophotometer (Optizen POP, Mecasys Co., Ltd, Daejeon, Korea).
Peroxidase, POD (EC 1.11.1.7) activity was measured using a modified method described by Bisht et al. [28]. The reaction mixture contained 1.5 mL of phosphate buffer (pH 7.0), 1% guaiacol, and 1% H 2 O 2 . POD activity was assessed as the change in absorption at 470 nm as a result of guaiacol oxidation. The results were expressed as unit min -1 ·mg -1 protein.

Statistics
RAPD data were scored for the absence "0" or presence "1" of electrophoresis band. Scoring data were entered into a binary matrix and analyzed using NTSYSpc 2.1. Similarities were calculated using Jaccard's coefficient, and a dendrogram was established using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Data were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Where a significant difference (p < 0.05) was observed for a measured parameter, means were separated using Duncan's multiple range test at the 5% level.

Effect of Colchicine Treatment on Lateral Root Induction and Growth
The induction of secondary roots after exposure to different concentrations of colchicine (100, 200, and 300 mg·L −1 ) for different durations (1, 2, and 3 days) was estimated eight weeks after treatment (Table 2; Figure 1). All the colchicine-treated adventitious root explants survived until eight weeks after treatment (data not shown). Colchicine treatment appreciably affected the number of explants with induced lateral roots ( Table 2). Treatment slowed the induction and branching of lateral roots during the initial 2-4 weeks of culture (data not shown). However, there was no significant difference in lateral root formation between control and treated roots after eight weeks of cultivation (Table 2). It was possible that the colchicine-treated explants exhibited slowed root induction as a result of a physiological disturbance that reduced the cell division rate and caused initial growth retardation [29]. Colchicine-treated adventitious roots produced fewer, shorter lateral roots than untreated roots (Table 2). This may have been due to the loss of microtubules and the occurrence of sticky supercoiled chromosomes and c-mitoses. Loss of cortical microtubules likely resulted in cell expansion rather than cell elongation, producing shorter and thicker lateral roots after colchicine treatment (Figure 1C,D,G,H) [29,30]. Obute et al. [23], and Hewawasam et al. [31] observed similar suppression of growth in Vigna unguiculata, Cucumeropsis mannii, and Crossandra infundibuiformis. The presence of IBA in culture medium might have counteracted the initial harmful effects of colchicine [29]. New adventitious roots were detached, cultured, and screened by biomass. Four biomass-enhanced root lines were selected, and the histogram of nuclei (Figure. S1) and DNA index (Table 3) were assessed using flow cytometry, which showed that all four lines and controls were tetraploid. The capacity of colchicine to infiltrate inside cell of living organisms to interact with the DNA produces the typical toxic effects related to colchicine properties. Thereby, the mutagenic effects are principally caused by the direct interaction between the mutagen and DNA molecules [10,17].  (Table 2). This may have been due to the loss of microtubules and the occurrence of sticky supercoiled chromosomes and c-mitoses. Loss of cortical microtubules likely resulted in cell expansion rather than cell elongation, producing shorter and thicker lateral roots after colchicine treatment (Figure 1C,D,G,H) [29,30]. Obute et al. [23], and Hewawasam et al. [31] observed similar suppression of growth in Vigna unguiculata, Cucumeropsis mannii, and Crossandra infundibuiformis. The presence of IBA in culture medium might have counteracted the initial harmful effects of colchicine [29]. New adventitious roots were detached, cultured, and screened by biomass. Four biomassenhanced root lines were selected, and the histogram of nuclei (Figure. S1) and DNA index (Table 3) were assessed using flow cytometry, which showed that all four lines and controls were tetraploid. The capacity of colchicine to infiltrate inside cell of living organisms to interact with the DNA produces the typical toxic effects related to colchicine properties. Thereby, the mutagenic effects are principally caused by the direct interaction between the mutagen and DNA molecules [10,17].  The four putative lines with the highest biomass among the total of 254 lines after screened by morphology and biomass (data not showed) were selected and screened again for ginsenoside content. Putative mutant roots were propagated in 250 mL flasks containing 100 mL of MS medium The four putative lines with the highest biomass among the total of 254 lines after screened by morphology and biomass (data not showed) were selected and screened again for ginsenoside content. Putative mutant roots were propagated in 250 mL flasks containing 100 mL of MS medium supplemented with 5 mg·L −1 IBA and 3% sucrose, in the dark, on a rotary shaker (100 rpm) ( Table 3).
The putative mutant roots were initially screened for high root biomass after six weeks of culture. Further experiments using DNA marker techniques were used to elucidate the genetic backgrounds of the four selected lines.
Four mutant root lines (100-1-2, 100-1-18, 300-1-16, and 300-2-8) exhibited higher growth ratios and more rapid growth than the control. Lateral root number, root length, and fresh and dry biomass were also recorded in the four lines after six weeks of culture ( Table 3). The mutant roots (Figure 1E,F,G,H) were thicker and formed more abundant and longer lateral roots than the control roots ( Figure 1A,B,C,D). The biomass obtained after six weeks of culture was approximately 1.5-fold higher for mutant roots than for control roots (Table 3).

RAPD Analysis of Mutant Roots
RAPD analysis was used to assess genetic differences between the control and four putative mutant lines because it is a rapid detect method that does not require prior information regarding the nucleotide sequence, is inexpensive, and is suitable for detecting DNA alternations after exposure with mutagenic agents [32,33]. Six of eighteen RAPD primers (Table 1) produced different band patterns between control and mutant lines (data not shown). The principal differences seen in the RAPD profiles were the presence or absence of varying bands. Amplification data from the differential primers were scored and used to assess genetic distance (Figure 2A). Genetic distances between the control and the four mutant lines were 0.46-1.0, as assessed using Jaccard's coefficient matrix ( Figure 2B). UPGMA analysis of RAPD data assigned the control to one cluster and the four mutant lines to a separate cluster. Of the four mutant lines, 100-1-18 differed substantially from the other three lines. Although RAPD analysis is most commonly used for phytogenetic, taxonomic, and genetic mapping studies, RAPD can also be used for detection of DNA damage and mutation [34]. In the present study, RAPD markers were effective for analysis of P. ginseng mutant adventitious root lines.

Expression Analysis of Ginsenoside Biosynthetic Genes
The expression levels of four important genes involved in ginsenoside synthesis (PgSS, PgSE2, PPDS, and PPTS) were analyzed to better understand the molecular characteristics of the four mutant lines (Figure 3). Triterpene ginsenosides are principally biosynthesized through the mevalonic acid pathway in the cytoplasm and the methylerythriol phosphate pathway in the chloroplast [35,36]. Squalene synthase (PgSS) catalyzes the biosynthesis of triterpenes [37], and squalene epoxidase (PgSE2) is involved in the production of 2,3-oxidosqualene [38]. Cytochrome P450 enzymes are involved in a further stage of ginsenoside synthesis. PPDS (CYP716A47) catalyzes the formation of protopanaxadiol (PPD) from drammarenediol-II, and PPTS (CYP716A53v2) catalyzes the formation of protopanaxatriol (PPT) from PPD. Differences in PgSE2, PPDS, and PPTS expression were observed among mutant and control lines. PgSS transcription was elevated in the four mutants compared with the control. PgSS, PgSE2, PPDS, and PPTS were highly expressed in mutant 100-1-18 ( Figure 3). The PgSE2 gene was also highly expressed in mutant 300-2-8, whereas the PgSE2, PPDS, and PPTS genes were minimally expressed in mutants 100-1-2 and 300-1-16 ( Figure 3). The results clearly demonstrated that ginsenoside biosynthesis genes were expressed at higher levels in the mutant 100-1-18 than in the other mutant lines.

Expression Analysis of Ginsenoside Biosynthetic Genes
The expression levels of four important genes involved in ginsenoside synthesis (PgSS, PgSE 2 , PPDS, and PPTS) were analyzed to better understand the molecular characteristics of the four mutant lines (Figure 3). Triterpene ginsenosides are principally biosynthesized through the mevalonic acid pathway in the cytoplasm and the methylerythriol phosphate pathway in the chloroplast [35,36]. Squalene synthase (PgSS) catalyzes the biosynthesis of triterpenes [37], and squalene epoxidase (PgSE 2 ) is involved in the production of 2,3-oxidosqualene [38]. Cytochrome P450 enzymes are involved in a further stage of ginsenoside synthesis. PPDS (CYP716A47) catalyzes the formation of protopanaxadiol (PPD) from drammarenediol-II, and PPTS (CYP716A53v2) catalyzes the formation of protopanaxatriol (PPT) from PPD. Differences in PgSE 2 , PPDS, and PPTS expression were observed among mutant and control lines. PgSS transcription was elevated in the four mutants compared with the control. PgSS, PgSE 2 , PPDS, and PPTS were highly expressed in mutant 100-1-18 ( Figure 3). The PgSE 2 gene was also highly expressed in mutant 300-2-8, whereas the PgSE 2 , PPDS, and PPTS genes were minimally expressed in mutants 100-1-2 and 300-1-16 ( Figure 3). The results clearly demonstrated that ginsenoside biosynthesis genes were expressed at higher levels in the mutant 100-1-18 than in the other mutant lines.

Ginsenoside Content Analysis by HPLC
Ginsenoside content was determined in the four mutant lines after six weeks of culture. The HPLC chromatogram with the retention time values of the determined ginsenosides and their standards are shown at Table S2 and Figure S2. The total contents of 11 ginsenosides increased in all mutant lines compared with the control (Table 4). Mutant 100-1-18 produced the highest amounts of total PPD and PPT ginsenosides (2.5-, 2.12-, and 2.68-fold higher than the control, respectively) ( Table  4). In the PPD group, the Rb1, Rb2, and Rc contents of 100-1-18 were 3.7-, 4.1-, and 2.3-fold higher than the control, respectively (Table 4). In the PPT group, the Re, Rf, Rg1, and Rg2 contents of 100-1-18 were 2.87-, 4.7-, 1.7-, and 3.2-fold higher than the control, respectively ( Table 4). The PPD/PPT ratio differed among mutant and control roots: the 100-1-2 roots exhibited the highest PPD/PPT ratio, and the 300-2-8 roots had the lowest ratio. Overall, mutant 100-1-18 had the highest ginsenoside productivity (186.6 mg·L −1 ), which was 4.85-fold higher than that of the control. Ginsenoside contents differed among the mutant and control lines as a result of disparate expression of ginsenoside

Ginsenoside Content Analysis by HPLC
Ginsenoside content was determined in the four mutant lines after six weeks of culture. The HPLC chromatogram with the retention time values of the determined ginsenosides and their standards are shown at Table S2 and Figure S2. The total contents of 11 ginsenosides increased in all mutant lines compared with the control (Table 4). Mutant 100-1-18 produced the highest amounts of total PPD and PPT ginsenosides (2.5-, 2.12-, and 2.68-fold higher than the control, respectively) ( Table 4). In the PPD group, the Rb1, Rb2, and Rc contents of 100-1-18 were 3.7-, 4.1-, and 2.3-fold higher than the control, respectively (Table 4). In the PPT group, the Re, Rf, Rg1, and Rg2 contents of 100-1-18 were 2.87-, 4.7-, 1.7-, and 3.2-fold higher than the control, respectively ( Table 4). The PPD/PPT ratio differed among mutant and control roots: the 100-1-2 roots exhibited the highest PPD/PPT ratio, and the 300-2-8 roots had the lowest ratio. Overall, mutant 100-1-18 had the highest ginsenoside productivity (186.6 mg·L −1 ), which was 4.85-fold higher than that of the control. Ginsenoside contents differed among the mutant and control lines as a result of disparate expression of ginsenoside synthesis genes. The high accumulation of ginsenosides in mutant 100-1-18 corresponded with elevated expression of PgSE 2 , PPDS, and PPTS ( Figure 3; Table 4). The increased accumulation of ginsenoside content in mutant lines indicated that colchicine had a pronounced effect on ginsenoside synthesis. Mutants with elevated biomass and increased production of effective compounds are particularly valuable for medicinal plants [20,39]. Kharde et al. [20] reported that bacoside production increased 4-fold in colchicine-treated in vitro Brahmi plants. In this study, mutant roots exhibited higher ginsenoside contents than control roots, demonstrating that this phytochemical characteristic was strongly affected by mutagenesis (Table 4). Mutant roots exhibited thicker and longer growth than control roots, which may have contributed to the elevated ginsenoside levels. Further studies are needed to fully understand the molecular mechanisms governing high ginsenoside content and productivity in P. ginseng. The ginsenoside content of mutant line 100-1-18 produced the highest PPD (Rb1, Rb2, Rg3, and Rh2) and PPT (Re, Rg1, and Rg2) levels. The 100-1-18 line is therefore of possible commercial pharmacological value.

MDA, CAT, and POD Activity of Mutant Roots
Antioxidase (POD and CAT) activities and MDA content were analyzed for clarify the distinctive characteristic in tetraploid and mutant roots. This allows a more detailed understanding of the molecular mechanism through which the mutagenesis effects on bioactive compounds stay obscure. MDA content was higher in mutant root lines than in the control line, and MDA contents were directly proportional to biomass which showed through the linear regression coefficients (r2) in the four mutant root lines (Figure 4). Mutant root lines 100-1-2 and 300-2-8 exhibited the lowest POD activities, whereas 100-1-18 and 300-1-16 activities were similar to those of the control. Mutant 100-1-2 also exhibited the lowest CAT value, whereas the other mutants resembled the control ( Figure 5). No regression relationship among the POD and CAT actives and biomass was observed (data not shown). The colchicine-induced genetic alterations in the mutant lines may explain the differences in concentration and activities of some enzymes [40,41]. The difference in antioxidant enzyme activities and MDA content may be due to the upregulated expression of the corresponding genes.

MDA, CAT, and POD Activity of Mutant Roots
Antioxidase (POD and CAT) activities and MDA content were analyzed for clarify the distinctive characteristic in tetraploid and mutant roots. This allows a more detailed understanding of the molecular mechanism through which the mutagenesis effects on bioactive compounds stay obscure. MDA content was higher in mutant root lines than in the control line, and MDA contents were directly proportional to biomass which showed through the linear regression coefficients (r2) in the four mutant root lines (Figure 4). Mutant root lines 100-1-2 and 300-2-8 exhibited the lowest POD activities, whereas 100-1-18 and 300-1-16 activities were similar to those of the control. Mutant 100-1-2 also exhibited the lowest CAT value, whereas the other mutants resembled the control ( Figure 5). No regression relationship among the POD and CAT actives and biomass was observed (data not shown). The colchicine-induced genetic alterations in the mutant lines may explain the differences in concentration and activities of some enzymes [40,41]. The difference in antioxidant enzyme activities and MDA content may be due to the upregulated expression of the corresponding genes.

Bioreactor Culture of Mutant Lines
The selected mutant roots were scaled-up in a bioreactor system. All four mutant lines grew successfully in the bioreactor system during the six-week culture duration (Table 3; Figure 6). Mutant 300-2-8, which showed the highest fresh biomass, exhibited particularly rapid adaptation capacity in the bioreactor system. After six weeks of culture, lateral root number and length were highest in mutant 100-1-18 (Table 3). Overall, all four mutant lines were suitable for scaled-up cultivation in the bioreactor system, suggesting their amenability to commercial production at industrial levels. Mutagenesis led to increased vegetative and reproductive organ production in P. ginseng. Similarly, Nura et al. [21] showed that leaf and seed yield were improved in sesame mutants. Colchicine induction of mutagenesis in sesame caused genomic alterations that enhanced cell division and meristematic expansion [21].

Bioreactor Culture of Mutant Lines
The selected mutant roots were scaled-up in a bioreactor system. All four mutant lines grew successfully in the bioreactor system during the six-week culture duration (Table 3; Figure 6). Mutant 300-2-8, which showed the highest fresh biomass, exhibited particularly rapid adaptation capacity in the bioreactor system. After six weeks of culture, lateral root number and length were highest in mutant 100-1-18 (Table 3). Overall, all four mutant lines were suitable for scaled-up cultivation in the bioreactor system, suggesting their amenability to commercial production at industrial levels. Mutagenesis led to increased vegetative and reproductive organ production in P. ginseng. Similarly, Nura et al. [21] showed that leaf and seed yield were improved in sesame mutants. Colchicine induction of mutagenesis in sesame caused genomic alterations that enhanced cell division and meristematic expansion [21].