Elicitation of Glycyrrhiza glabra (L.) hairy roots by methyl jasmonate enhanced glycyrrhizin production

doi:10.21203/rs.3.rs-4820225/v1

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Elicitation of Glycyrrhiza glabra (L.) hairy roots by methyl jasmonate enhanced glycyrrhizin production

https://doi.org/10.21203/rs.3.rs-4820225/v1

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Glycyrrhizin is a valuable secondary metabolite with high medicinal properties, which is known as the most important compound in the licorice (Glycyrrhiza glabra L.). Considering the problems in growing plants in the field, the use of tissue culture techniques, especially hairy root culture, makes the production of glycyrrhizin simpler and more affordable. The influence of Agrobacterium rhizogenes strains on the efficiency of hairy root induction showed that the highest hairy roots were observed using MSU strain (80%). Also, the effect of explant type (leaf, stem and petiole) and age (two-week-old, three-week-old and four-week-old), light conditions (24 hours of darkness and 16 hours of light-8 hours of darkness) and carbohydrate source (3% glucose and 3% sucrose) showed that the production of hairy roots from this medicinal plant depends on the type and age of explant, light treatment and the composition of the culture medium and the highest hairy roots induction was observed in leaf explant (80%), three-week-old leaf (76.66%) and MS medium containing 3% sucrose (86.66%) and treatment of 24 hours of darkness (86.66%). The amount of glycyrrhizin in hairy root lines treated by 100 µM methyl jasmonate (MJA) after 24 hours increased in the no cutting treated line and decreased in the cutting treated lines compared to the control line. In conclusion, elicitation of G. glabra hairy root culture is a suitable platform for achieving high glycyrrhizin yields.

glycyrrhizin

Agrobacterium rhizogenes

methyl jasmonate

hairy roots culture

Medicinal plants synthesize a wide range of natural products known as secondary metabolites, which are used for various purposes such as food ingredients, pharmaceutical drugs, and raw materials for the chemical and other industries (Omidbaigi 2005).

One of the most valuable medicinal and industrial plants is Glycyrrhiza glabra L., commonly known as licorice. Various forms of its products are used in different industries including tobacco, confectionery, and pharmaceuticals (Jatav et al. 2011; Omidbaigi 2005). The root of this plant is rich in glycyrrhizin, which exhibits various medicinal activities, including antiviral activity against different DNA and RNA viruses (Nassiri Asl and Hosseinzadeh 2007), including the coronavirus. Glycyrrhizin is recognized as a prominent and the most important active compound in licorice, functioning as a glycoside saponin (Mousa et al. 2006). Glycyrrhizin and its hydrolyzed products possess important medicinal activities, including anti-inflammatory (Shibata 2000), antibacterial, antioxidant (Hong et al. 2009), antifungal (Fatima et al. 2009), antitumor (Zhang and Ye 2009), and antiviral (Nassiri Asl and Hosseinzadeh 2007) activities. They are used in the food industry and also for masking the bitter taste of medications (Hui-yan et al. 2020). Due to these properties, it has high demand in the market (Wahab et al. 2021).

Considering the slow biosynthesis rate of secondary metabolites production in nature and the significant amount of time required, their production is not economically feasible. Therefore, it seems necessary to utilize plant tissue culture techniques to achieve faster and higher production of its secondary metabolites and medicinal compounds (Wongwichaa et al. 2008).

Plant tissue culture is one of the important techniques for industrial production of secondary metabolites in medicinal plants (Loyola-Vargas and Ochoa-Alejo 2018). In addition, plant tissue culture can also be used for the biosynthesis of novel compounds through biotransformation. Hairy roots culture is one of the plant tissue culture methods employed for secondary metabolite production (Teoh 2015). The main advantage of hairy roots induced by Agrobacterium rhizogenes compared to callus and cell suspension cultures is their high growth rate and their independence from external growth regulators (Sharafi et al. 2014). Although the type of culture medium and the concentration of its components have an impact on the growth of hairy roots, they have shown sensitivity to the source of carbohydrates in the culture medium, bacterial strain, and the type and age of the explant (tissue sample) (Sharafi et al. 2014; Shirazi et al. 2012).

One of the most effective and widely used biotechnological tools for inducing new secondary metabolites or advanced biosynthesis and accumulation of secondary metabolites in plant tissue culture is elicitation (Krstić-Milošević et al. 2017). Methyl jasmonate plays a key role as an elicitor in increasing the production of high-value pharmaceutical secondary metabolites, in such a way that it reduces the time required to achieve a high amount of these metabolites by enhancing the rate of secondary metabolite biosynthesis (Eder and Cosio 1994; Singh and Dwivedi 2018). Cultivation of hairy roots in medicinal plants is a highly promising strategy for increasing the yield of secondary metabolites due to the high demand for bioactive compounds and their limited availability in natural sources (Veena and Taylor 2007). The study conducted by Khan et al. (2015) evaluated the induction of callus and plantlet regeneration of G. glabra using three different culture media (MS, W, B) containing various growth regulators. The results indicated that the basal medium and plant growth regulators play an important role in tissue culture initiation, differentiation, and production of secondary metabolites. Shirazi et al. (2012) subjected the extract of hairy roots induced by A. rhizogenes to High-Performance Liquid Chromatography (HPLC) for high-efficiency analysis. The results of the study demonstrated the production of glycyrrhizin through this method in hairy roots of G. glabra. In another research, Wongwichan et al. (2011) investigated the optimization of cell growth conditions and glycyrrhizin production, as well as the effect of yeast extract, methyl jasmonate, and chitosan as elicitors on the accumulation of glycyrrhizin in hairy root cultures of G. inflata

Today, due to the increased demand for glycyrrhizin, overharvesting of the G. glabra plant occurs, leading to unregulated harvesting of medicinal plants and a decline in resources in their natural habitats (Hayashi et al. 2003). The present study aims to optimize the production conditions of hairy roots in G. glabra to achieve a higher yield of glycyrrhizin. This is done through in vitro plant tissue culture, where various factors such as different strains of A. rhizogenes, light conditions, type of carbohydrate source in the growth medium, and the type and age of explants are evaluated for their effect on hairy root production. In addition, this research evaluates the effect of the methyl jasmonate elicitor on the production level of glycyrrhizin in the hairy roots of G. glabra.

Plant material

G. glabra seeds were purchased from Pakan Bazer Company (Isfahan, Iran). In order to disinfect the seeds, they were first soaked in sulfuric acid (H₂SO₄ 96%) for 20 min. In the next step, the seeds were washed three times with sterile distilled water, and then they were disinfected with a 5% sodium hypochlorite solution for 15 min. In the final step, the seeds were washed three times with sterile distilled water and then cultured in MS culture medium (Murashige and Skoog 1962) without any growth regulators. The cultures were incubated for germination in a growth chamber at a temperature of 24 ± 2^oC and a photoperiod of 16 h of light. The 21-day-old plantlets produced under in vitro conditions were used as a source to prepare explants for inoculation with bacteria and induction of hairy roots.

Bacterial strains

In this research, three strains of A. rhizogenes named as MSU, A7, and A4-N (obtained from the Institute of Secondary Metabolites Biotechnology, Isfahan, Iran) were used to induce hairy roots. To prepare a bacterial suspension for inoculation with plant explants, a single colony of each desired strain was cultured in 10 ml of Luria-Bertani (LB) liquid medium supplemented with 50 mg/L of rifampicin antibiotic (Sigma Chemical Co.) under sterile conditions. The bacterial cultures were placed in a shaking incubator at a temperature of 28^oC and a rotary shaker of 110 rpm for 24 h in dark conditions. The bacterial cultures were then centrifuged at 7000 g for 10 min using a centrifuge machine. After discarding the supernatant, 15 ml of liquid MS medium supplemented with 50 g/L sucrose were added. The bacterial cultures were placed again in a shaking incubator at a temperature of 28^oC for 2 to 5 h. The prepared suspension was used for inoculating the explants.

Hairy roots induction

In order to inoculate the explants with the bacterial suspension, leaf, stem, and petiole explants were first separated from the in vitro plant cultures. To investigate the effect of different bacterial strains, leaf explants from 21-day-old plantlets were used for inoculation with three strains (MSU, A7, and A4-N) of A. rhizogenes. Furthermore, to investigate the effect of different explant types on hairy roots induction, each type of leaf, stem, and petiole explants was individually immersed in the bacterial suspension of the MSU strain after creating a surface wound on the explants. After 15 min of inoculating the explants with the bacterial suspension, the explants were cultured in MS culture medium supplemented with 100 \(\:{\mu\:}\text{l}\) of acetosyringone and 0.1 g/L of L-glutamine for 48 h in dark conditions at a temperature of 24 ± 2^oC. Next, the explants were cultured in 1/2 MS culture medium supplemented with 0.5 g/L of cefotaxime antibiotic and 0.1 g/L of L-glutamine. They were then maintained in a growth chamber at a temperature of 24 ± 2^oC until hairy roots appeared. The explants were subcultured every two weeks to ensure their continued growth and development.

In order to investigate the effect of the age of leaf explants on the rooting percentage, leaf explants of two, three, and four-week-old plants were inoculated with the MSU strain of A. rhizogenes for hairy root induction. The leaf explants were individually immersed in the bacterial suspension for a duration of 15 min. Afterward, the leaf explants were cultured in 1/2 MS culture medium supplemented with 0.5 g/L of the cefotaxime antibiotic and 1.0 g/L of L-glutamine.

To investigate the effect of two different carbon sources, glucose and sucrose, on the rooting percentage, leaf explants of 21-day-old plantlets were used for inoculation with the MSU strain. After inoculating the leaf explants with the bacteria, the explants were individually cultured in two different growth media containing 3% glucose or sucrose.

In another experiment, the effect of light conditions on the rooting percentage of leaf explants from 21-day-old G. glabra plantlets inoculated with the MSU bacterial strain was investigated. The leaf explants were subjected to two different light treatments: 24 h of darkness or a 16 h light-8 h darkness cycle, for hairy root induction. Finally, the explant cultures were maintained in a growth chamber at a temperature of 24 ± 2^oC, and the process of subculturing the explants was performed every two weeks.

DNA isolation and PCR analysis

In order to molecularly confirm the nature of hairy roots in G. glabra, genomic DNA extraction was performed from putative hairy roots and non-transformed roots using the CTAB (cetyl trimethylammonium bromide) method (Murray and Thompson 1980). Additionally, plasmid extraction from three bacterial strains, MSU, A7, and A4-N, was performed using the alkaline lysis method with SDS (sodium dodecyl sulfate) (Sambrook and Russell 2006). The extracted DNA was evaluated using polymerase chain reaction (PCR) amplification with the specific primers for the rolC and virD genes. The forward primer 5'-TAACATGGCTGAAGACGACC-3' and reverse primer 5'-AAACTTGCACTCGCCATGCC-3' the gene rolC and also forward primer 5'-ATGTCGCAAGGCAGTAAGCCCA-3 and reverse primer 5'-GGAGTCTTTCAGCAGGAGCAA-3 the gene virD were used for the amplification of the target genes (Fattahi et al. 2013). The PCR reaction was carried out under the following conditions: denaturation at a temperature of 94^oC for a duration of 30 s, primer annealing at 58°C for 30 seconds, and extension at 72°C for 1 min for 37 cycles. The amplified products were analyzed using 1% agarose gel electrophoresis.

Determine the growth rate of hairy roots

Three samples containing hairy roots were selected to determine the growth rate of the hairy roots. From each selected small sample, 0.5 g of hairy roots were transferred to 250 mL flasks containing 5 mL of liquid 1/2 MS culture medium. The flasks were then placed in an incubator shaker at a temperature of 25^oC and maintained in darkness. To prepare a growth rate chart of hairy roots, the weight of the hairy roots was measured once every three days for a duration of 13 weeks.

Elicitor treatment

The effect of methyl jasmonate elicitor on the production of glycyrrhizin in the hairy roots of G. glabra, was investigated. A stock solution of methyl jasmonate (Sigma Co., Germany) was prepared using ethanol at a concentration of 100 \(\:{\mu\:}\text{M}\). To treat the hairy roots with methyl jasmonate, 1 to 2 cm segments of the root were separated from the leaf sample and transferred to 250 mL flasks containing 10 mL of liquid 1/2 MS culture medium. The flasks were then placed in a shaker incubator at a temperature of 25^oC and maintained in darkness. After the roots have grown sufficiently in the flasks, methyl jasmonate with a concentration of 100 \(\:{\mu\:}\text{M}\) was added to the flasks containing the hairy roots. In the following, to evaluate the effect of physical stress (cutting) on the hairy roots, one line was considered as the control among the five lines, and the cutting operation was performed on the other four lines of the hairy roots. Sampling was performed from the hairy roots of G. glabra after 48 h of adding the elicitor. Furthermore, a 1\(\:\:{\mu\:}\text{L}\) sample was taken from the liquid 1/2 MS culture medium in two of the flasks containing hairy roots to measure the level of released glycyrrhizin in the medium. All treatments were performed in triplicate.

High-performance liquid chromatography (HPLC) analysis

Standard glycyrrhizin (glycyrrhizic acid ammonium salt) was purchased from Sigma Co., Germany. The isolation, tracking, and quantification of glycyrrhizin from the hairy roots of G. glabra, as well as the normal roots of the plant, were performed using an HPLC instrument equipped with a 20 \(\:{\mu\:}\text{L}\) injection syringe and a detector set at a wavelength of 250 nm. For better separation of glycyrrhizin, a reverse-phase chromatography column was used, and the mobile phase was washed by changing the ratio of acetonitrile to acetic acid. The isoliquiritigenin separation was performed according to the Liu et al. (2005). In order to accurately calculate the concentration of glycyrrhizin, solutions with different concentrations of the standard glycyrrhizin were prepared, and 20 \(\:{\mu\:}\text{L}\) of each solution was injected into the injection port of the instrument. The concentration of glycyrrhizin was calculated in triplicate based on the standard curve.

Statistical analysis

Data analysis was performed using SPSS software, and Excel software was used for plotting the figures. The differences between the means of the samples were analyzed using the Least Significant Difference (LSD) test at a significance level of P < 0.01.

Hairy roots induction

Effect of A. rhizogenes strains on hairy roots transformation

The in vitro cultured plantlets of G. glabra were inoculated with strains MSU, A7, and A4-N of A. rhizogenes. The first hairy roots appeared at the site of the wounds on the explants 20 days after the inoculation. In the subsequent stages, the hairy roots obtained continued to grow in free-hormones liquid 1/2 MS cultures medium.

After one month, the percentage of explants that developed hairy roots was examined (Table 1). The results showed that the MSU strain had the highest effect on hairy root induction, with 80% of the three-week-old leaf explants showing root formation. Following that, the A7 and A4-N strains had effects on hairy root induction from the three-week-old leaf explants with percentages of 50% and 13%, respectively (Fig. 1, A).

Table 1

variance analysis of the effects of strain type and plant explant type on hairy roots induction.
Sources of changes	df	MS
Strain type	2	3344/44**
Error test	6	177/78
Sample type	2	4433/33**
Error test	6	222/22
In the variance analysis table above, the symbol ** indicates significance level of P < 0.01.

Table 2

Dry weight (D.W) produced glycyrrhizin contents (µg/g DW) by non- treatment hairy root and hairy root lines of *G. glabra.*
Line	DW (g)	Glycyrrhizin (µg/g)
L1	6/7	397
L1 T	5/1	191
L2	6	282
L2 T	5	177
L3	6/8	382
L3 T	5/3	197
L4	5/8	256
L4 T	4/7	174
L5	6/4	116
L5 T	7	382

Different strains of A. rhizogenes have varying potential in transferring T-DNA to host plant explants and subsequently inducing hairy roots (Porter and Flores 1991; Sharafi et al. 2013). That is because the number of T-DNA copies transferred from the Ri plasmid of A. rhizogenes, the integration site of T-DNA within the host genome, and the complete or partial transfer of T-DNA to the host cell genome all contribute to this variation. Therefore, it can be concluded that different strains of A. rhizogenes have varying abilities to induce hairy roots, and this is dependent on their pathogenicity. It is possible for a plant species to not be sensitive to one strain of bacteria and show a different response to another strain. Ionkova et al. (1997) demonstrated that different strains of A. rhizogenes had varying effects on the induction of hairy roots in Astragalus mongolicus. Furthermore, the results of this study are consistent with the findings of the study conducted by Dobigny et al. (1995), which investigated the induction of hairy roots by different strains of A. rhizogenes in two potato cultivars.

Effect of type and age of explants on hairy roots transformation

The comparison of means between the types of explants used, including leaf, stem, and petiole, indicated that leaf explants had the highest impact on the production of hairy roots (80% root induction) following inoculation with the MSU strain of A. rhizogenes. Following that, stem and petiole explants showed lower hairy root induction percentages, with 23% and 6%, respectively, in response to inoculation with the MSU strain of A. rhizogenes (Fig. 1, B).

Additionally, in some leaf explants inoculated with the MSU strain, both root induction and callus formation were observed. Most of the hairy roots obtained from the petiole explants also underwent necrosis and destroyed after five days. According to the observations, the site of hairy root initiation in the leaf explants of G. glabra was at the location of the wound created during the experiment.

According to the results of this study, the plant species and the type of plant explant are important factors that influence the success of transformation and induction of hairy roots. In studies conducted on the species Withania somnifera and Solanum surattense, leaves have been identified as the best plant explants for inducing hairy roots (Pawar and Maheshwari 2004).

A comparison was made between leaf explants of two, three, and four-week-old plants to assess the percentage of hairy root induction during the experiment. The results of the experiment showed that the age of the leaf explants had the greatest impact on hairy root induction, with the three-week-old leaf explants exhibiting the highest percentage of root induction (76.66%) (Fig. 1, C). The study conducted by Mehrotra et al. (2008) on the induction of hairy roots in G. glabra by Agrobacterium demonstrated that the age and type of explant, as well as the composition of the culture medium, have a significant influence on hairy root induction. This is because the age of the explant is the main factor in altering the physiological properties of the cells.

Effect of light conditions and carbohydrate source on hairy roots transformation

The results of the T-test showed that the application of 3% sucrose in 1/2 MS medium (86.66% root induction) and the maintenance of explants in complete darkness (86.66% root induction) had a more favorable response in terms of hairy root induction percentage (Fig. 1, D), compared to 1/2 MS medium containing 3% glucose (36.66% root induction) and 16 h light conditions (Fig. 1, E). Studies have shown that sucrose is the most suitable carbon source for cell cultures and the production of secondary metabolites. In addition, sucrose has been found to have stimulatory effects on the biomass accumulation of hairy roots (Hirasuna et al. 1996). The results of this research indicate that the explants placed in complete darkness exhibited better hairy root induction compared to the explants subjected to a 16 h light and 8 h dark photoperiod.

Molecular identification of hairy roots by PCR analysis

In order to provide definitive evidence of the transgenic nature of hairy roots in G. glabra, DNA extraction was initially performed from hairy roots, non-transgenic roots, and A. rhizogenes strains (MSU, A7, and A4-N). To confirm the presence of the rolC gene in the genomic DNA of hairy roots and verify the presence of the rolC and virD genes in the plasmid DNA of A. rhizogenes, as well as the absence of both genes in non-transgenic roots, a polymerase chain reaction (PCR) assay was conducted. The presence of the rolC gene was confirmed in the genomic DNA of hairy roots through the amplification of a fragment approximately 534 bp in length. The observation of bands indicates the transfer of the T-DNA fragment from the A. rhizogenes plasmid to the genome of hairy roots, thereby confirming their transgenic nature. The amplification of DNA fragments was observed in the extracted plasmid DNA, confirming the presence of a gene region called virD with an approximate length of 438 bp. However, the absence of any bands in both transgenic and non-transgenic hairy roots indicates the lack of bacterial gene remnants within the transgenic hairy roots. Furthermore, the absence of a band on the gel in non-transgenic roots (negative control sample) indicates their non-transgenic nature (Fig. 2).

The bacterial T-DNA region consists of two parts, TL-DNA and TR-DNA, which are transferred and integrated into the host plant genome separately. However, for inducing hairy root formation, the presence of TL-DNA containing the rolA, rolB, rolC, and rolD genes is essential (Georgiev et al. 2007).

After confirming the transgenic nature of the hairy roots, they were transferred to flasks containing liquid culture medium, and a growth curve of G. glabra hairy roots was obtained to monitor their growth pattern. This growth curve indicates that during the first week after transferring the hairy roots from solid medium to liquid medium, the hairy roots entered a lag phase. The average weight of the hairy roots at the end of the first week is 0.5 g. From the beginning of the second week until the end of the ninth week, the growth of the hairy roots followed a logarithmic pattern. By the start of the tenth week, the hairy roots entered a stationary phase, indicating a stable growth pattern. The late logarithmic phase (ninth week) was chosen as the appropriate time to add elicitor (methyl jasmonate) to the desired hairy roots samples (Fig. 3). The growth pattern of G. inflata hairy roots showed that the ninth week of the transfer hairy roots to the flask is the best time to add elicitor (Wongwichaa et al. 2011).

Methyl jasmonate treatment and HPLC analysis

The fresh weight of the hairy roots was measured at the appropriate time for adding methyl jasmonate elicitor, which showed that the L5 hairy root line had the highest weight (8/6 g). Following that, the L1, L2, L3 and L4 hairy roots lines have root weights of 8g, 7.8 g, 6.6 g, and 6.3 g, respectively.

Methyl jasmonate, as a biotic elicitor, induces a plant defense response (Eder and Cosio 1994). On the other hand, secondary metabolites are often produced in the plant's defense response to environmental stresses (Palazón et al. 2003). To determine the amount of glycyrrhizin in G. glabra hairy roots for four selected lines, HPLC was used after 48 h of adding methyl jasmonate. The results indicate that in line 5 (302 \(\:{\mu\:}\text{g}/\text{g}\)), compared to its own control line (116 \(\:{\mu\:}\text{g}/\text{g}\)), the addition of the elicitor methyl jasmonate led to an increase in glycyrrhizin production in the hairy roots. However, in contrast, in the other four lines where cutting was performed on the roots, the glycyrrhizin production in the hairy roots decreased compared to their respective control line. After 24 hours of cutting in the hairy a pink color with varying shades was observed in the medium (Fig. 4). Analysis of two culture medium samples from L5 and L3 showed the presence of glycyrrhizin in the culture medium of line 3 hairy roots and its absence in the culture medium of line 5. In addition, among the control treatments, L1 hairy roots line had the highest amount of glycyrrhizin (397 \(\:{\mu\:}\text{g}/\text{g}\)). Following that, the L3, L2, L4 and L5 hairy roots lines had glycyrrhizin contents of 382, 281, 256 and 116\(\:\:{\mu\:}\text{g}/\text{g}\), respectively (Table. 2). In continuation, with the addition of methyl jasmonate elicitor at a concentration of 100\(\:\:{\mu\:}\text{M}\), the results indicate a decrease in the produced amount of glycyrrhizin in the G. glabra hairy roots compared to the control treatment. Furthermore, among the cut roots and treated with the elicitor, the L3 hairy roots line had the highest amount of glycyrrhizin (197\(\:\:{\mu\:}\text{g}/\text{g}\)). Following that, the L1, L2, and L4 hairy roots lines had glycyrrhizin contents of 191, 177, and 174 \(\:{\mu\:}\text{g}/\text{g}\), respectively (Fig. 5).

The hairy root morphology is very sensitive as the roots respond to the little changes in the local environmental conditions like temperature and shear stress. Any change in the morphology of hairy root such as density, thickness and root length, significantly affect the production of secondary metabolite (Srivastava and Srivastava 2012). In a study, after producing hairy roots from the G. glabra plant, the highest amount of glycyrrhizin was measured 45.68 \(\:{\mu\:}\text{g}/\text{g}\) of dry weight, (6 times higher than the non-hairy root) (Shirazi et al. 2012). In the study by Wongwichaa et al. (2011), the use of methyl jasmonate elicitor at a concentration of 100 \(\:{\mu\:}\text{M}\) on G. inflata hairy roots after five days resulted in an increase in glycyrrhizin production to 109 \(\:{\mu\:}\text{g}/\text{g}\) (five times higher than the control sample). In the study by Shabani et al. (2008), the use of methyl jasmonate elicitor at a concentration of 100 \(\:{\mu\:}\text{M}\) did not significantly affect the level of glycyrrhizin present in the hairy root samples or the expression of the bAS gene compared to the control sample. However, when a concentration of 1\(\:\:\text{m}\text{M}\) of methyl jasmonate was applied, it resulted in an increase in the expression of the bAS gene. In this study, the amount of glycyrrhizin in L1, L2, L3, and L4 hairy roots lines decreased significantly compared to the control lines after cutting the hairy roots. This indicates that despite the addition of the elicitor, the production of glycyrrhizin in the cut lines decreased due to the change in the morphology of the hairy roots during shear stress. However, in line 5, where the roots were not cut, after 48 hours of adding methyl jasmonate elicitor at a concentration of 100 \(\:{\mu\:}\text{M}\) resulted in an increase in glycyrrhizin production in the hairy roots. Indeed, the use of G. glabra hairy root culture in this research has been effective in achieving a significant production of glycyrrhizin compared to other studies, considering the low production of glycyrrhizin in previous research. The hairy root culture system offers advantages such as genetic stability, rapid growth, and the potential for enhanced secondary metabolite production, making it a suitable platform for achieving high glycyrrhizin yields.

Authors Contribution Statement

ZA carried out the experiment and performed the data analyses and wrote the manuscript. MT was the project supervisor, designed the research, provided all the technical support during the laboratory work, and reviewed the manuscript. BEST was the project supervisor and helped in the design of the experiment.

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Elicitation of Glycyrrhiza glabra (L.) hairy roots by methyl jasmonate enhanced glycyrrhizin production

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Abstract

Figures

Introduction

Material and Methods

Plant material

Bacterial strains

Hairy roots induction

DNA isolation and PCR analysis

Determine the growth rate of hairy roots

Elicitor treatment

High-performance liquid chromatography (HPLC) analysis

Statistical analysis

Results and Discussion

Hairy roots induction

Effect of type and age of explants on hairy roots transformation

Effect of light conditions and carbohydrate source on hairy roots transformation

Molecular identification of hairy roots by PCR analysis

Methyl jasmonate treatment and HPLC analysis

Declarations

References

Status:

Version 1