Strain R1601 Research Articles

Comparative Analysis of Glucosinolate and Phenolic Compounds in Green and Red Kimchi Cabbage (Brassica rapa L. ssp. pekinensis) Hairy Roots after Exposure to Light and Dark Conditions

Brassica rapa L. ssp. pekinensis (Lour.) Hanelt (kimchi cabbage) is a major vegetable cultivated in Korea, and its hairy roots (HRs) are rich in glucosinolates and phenolic compounds. This study aimed to induce HRs from cotyledon explants via the transformation of the Agroacterium rhizogenes strain R1000 and examine the glucosinolate and phenolic compounds present in the HRs of two kimchi cabbage (green and red) cultivars after exposure to 16 h light/8 h dark conditions (photosynthetic photon flux density of 54.6 µmol m−2 s−1) and continuous dark conditions. The highest HR production was achieved in the green kimchi cabbage grown under dark conditions (0.37 ± 0.01 DW g/30 mL). The highest glucosinolate and phenolic contents were neoglucobrassicin and catechin hydrate, which were highest in the green kimchi HRs grown under dark (GKHD) conditions (5268.29 ± 292.84 µg/g DW) and green HRs grown under light (GKHL) conditions (203.49 ± 4.70 µg/g DW), respectively. A heat map showed that the red kimchi HRs grown under dark conditions (RKHD) and the GKHL condition accumulated the highest glucosinolate and phenolic contents. Principal component (PCA) and partial least-squares discriminant (PLS-DA) analyses of the 13 identified metabolites showed a clear separation. According to a variable importance in projection (VIP) analysis, quercetin was the most important metabolite, leading to a clear separation. The most suitable conditions for enhancing the glucosinolate and phenolic contents were the GKHD and GKHL conditions, respectively, whereas both compounds were enhanced in the RKHD condition. HRs cultures cultivated under light and dark conditions are a promising method to enhance the production of specific health-promoting bioactive metabolites, which might be helpful in the pharmaceutical and nutraceutical industries.

Composite plants of cucumber and buckwheat as a tool to study auxin distribution and transport in the root system

Genetic transformation of most dicotyledonous plants by Rhizobium rhizogenes (also known as Agrobacterium rhizogenes) results in production of composite plants plants consisting of wild-type shoot and transgenic root system. Composite plants are the suitable model for investigation of hormonal mechanisms related to development of the root system as regulatory links between the root system and the shoot maintains in such plants.
 In most plants initiation of lateral root primordia occurs above the elongation zone [1]. However, in cucurbits and some other species, including important cereal crop buckwheat (Fagopyrum esculentum Moench), lateral root primordia initiation and development occurs in the apical meristem of the parental root [2, 3].
 The phytohormone auxin is a key regulator of lateral root development. Fusions of auxin-responsive promoters and reporter genes can be used to study the role of auxin in the development of root system of non-model plants such as cucumber (Cucumis sativus L.) and buckwheat [4].
 The agrobacterium mediated transformation technique of cucurbits [5] has been adapted for buckwheat. R. rhizogenes strain R1000 was used in all transformations. Set of binary vectors based on pKGW-RR-MGW or pKGW-MGW was developed to study auxin response maxima (DR5::mNeonGreen) or auxin transport (fusions of genes encoding auxin efflux proteins PIN and mNeonGreen).
 Pattern of auxin response maxima was similar in both species and included quiescent center and initial cells, columella, xylem cell files and lateral root primordia on all stages of development. Members of CsPIN1 (CsPINb and CsSoPIN1) group contributed unequally in generation of auxin maximum required for lateral root primordium initiation.
 The research was supported by the RFBR grant 20-016-00233-a.

Checking transformation efficiency for different Lagenaria siceraria genotypes by using seed germination pouches as a growth carrier

Bottle gourd [Lagenaria siceraria (Mol.) Standl.] is one of the most common horticultural and medicinal crops. Genetic transformation is a rate-limiting step in unraveling the function of genes and molecular breeding in this non-model crop. In this study, we established an optimized protocol for transformation using a seed germination pouch as a growth carrier and developed bottle gourd composite plants. Overground explants were inoculated with Agrobacterium rhizogenes strain R1000, which harbored a binary vector pBI121 carrying the GUS reporter gene. The integration of GUS into transgenic roots was confirmed via reverse transcription-polymerase chain reaction and histochemical staining. Furthermore, we screened for desirable genotypes from 14 bottle gourd germplasm collections based on the number of induced roots, transformation efficiency, and induction rate, and “Changguang” (transformation efficiency, approximately 36.8%) was selected as the genotype suitable for hairy root induction in bottle gourd. The optimized protocol presented here is a rapid, suitable, and efficient tool for studying the function of genes and root biology of bottle gourd in the future.

​​An Agrobacterium rhizogenes Strain R1000-mediated Efficient Hairy Root Transformation Protocol for Common Bean

Background: Common bean (Phaseolus vulgaris L.) is a globally important grain and vegetable legume crop, providing a substantial portion of the diet protein and minerals for many people in the developing world. However, the genetic studies and improvement on this crop has long been impeded by its recalcitrance to Agrobacterium-mediated whole plant genetic transformation. Established Agrobacterium rhizogenes-based hairy root transformation in common bean heavily relies on the strain K599. Methods: In order to develop an efficient alternative protocol for hair transformation in common bean, the efficiency of Agrobacterium rhizogenes strain R1000 in inducing hairy roots from 6-day-old seedlings with root below cotyledons excised by the soaking and smearing method were tested. The binary plasmid pBI121 with the reporter gene GUS (pBI121-GUS) or eGFP (pBI121-eGFP) driven by the constitutive promoter was used for transformation and rapid identification of the transgenic hairy roots. Result: We established a strain R1000-based system for the induction of hairy roots in common bean. The plant receptor genotypes and infection methods were optimized, which led to a high transformation rate of hairy roots up to 60%. This method therefore provides a useful alternative means for functional genomic studies in common bean.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Enhanced production of valtrate in hairy root cultures of Valeriana jatamansi Jones by methyl jasmonate, jasmonic acid and salicylic acid elicitors

Valtrate is a pharmacologically active epoxyiridoid ester found in the roots and rhizomes of Valeriana jatamansi Jones. The plant produces only small amounts of this metabolite naturally, and so induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, strain R1601 of Agrobacterium rhizogenes was used to induce hairy roots in V. jatamansi, and stable hairy root cultures of V. jatamansi were established successfully. The influence of three exogenous elicitors including methyl jasmonate (MJ), jasmonic acid (JA) and salicylic acid (SA) on valtrate production in the hairy root cultures of V. jatamansi was also investigated, and the 25-day-old hairy root cultures were treated with different concentrations of the elicitors at exposure time of 7 days. This present study showed that MJ (100 mg/L) highly promoted valtrate production at 7 days after elicitation, to a level of 3.63 times higher than that of non-elicited control. SA did not significantly increase the production of valtrate. This is the first-time study to assess the elicitation of hairy root cultures to promote valtrate biosynthesis in V. jatamansi and the resulting experiments demonstrated that MJ was indeed a potent inducer of valtrate biosynthesis.

Elicitation of withaferin-A in hairy root culture of Withania somnifera (L.) Dunal using natural polysaccharides

Withaferin-A (WA) is a major bioactive compound in the roots of Withania somnifera. It is reported to possess inhibitory effects against breast cancer, liver cancer, melanoma cancer and prostate cancer cells. We developed an elicitation based on the hairy root culture (HRC) protocol for the enhancement of the WA production. Agrobacterium rhizogenes strain R1000 was used for the establishment of hairy root culture. PCR analysis confirmed the integration of the root loci C (rol C) gene in transformed roots. Elicitation of the hairy roots with natural polysaccharides of sodium alginate (SA), k-carrageenan (kC) and chitosan (CH) at various concentrations in the most cases resulted in a higher amount of WA compound. Among the various elicitors, 100 mg L−1 CH resulted 4.03-fold increase in WA production than control. This study is the first report in W. somnifera hairy roots, where the natural polysaccharides are used as elicitors for the large-scale production of WA.

Comparison of Different Strains of Agrobacterium rhizogenes for Hairy Root Induction and Betulin and Betulinic Acid Production in Morus alba

Three Agrobacterium rhizogenes strains were tested for their ability to transform the plant Mois alba L. and to induce production of the secondary metabolites betulin and betulinic acid. All the tested strains of A. rhizogenes (R1601, LBA9402 and R1000) were able to induce hairy root formation in leaf tissue explants. Strain LBA9402 had the highest rate of infection (92.7% ± 8.8%), whereas strain R1601 had the lowest rate (87.4% ± 9.3%). The highest number of hairy roots per explant (5.6 ±0.5) and the greatest root length (2.4 ± 0.2 mm) were obtained with strain LBA9402. We also evaluated dry weight (a measure of growth) and betulin and betulinic acid production in hairy roots and found that the highest growth (167.8 ± 14.5 mg/flask) occurred after infection with strain LBA9402. Furthermore the highest production of betulin (5.4 ± 0.4 mg/g dry weight) and betulinic acid (2.3 ± 0.2 mg/g dry weight) was noted using strain LBA9402. Among three elicitors, yeast extract showed the highest induction of betulin production (8.7 ± 0.4 mg/g) and silver nitrate induced the highest yield of betulinic acid (4.1 ± 0.2 mg/g). Our study showed that A. rhizogenes strain LBA9402 was the most effective of the three tested strains for production-of transformed root cultures and betulin and betulinic acid.

Response of Different <i>Agrobacterium Rhizogenes</i> Strains for <i>in vitro</i> Hairy Root Induction and Accumulation of Rosmarinic Acid Production in <i>Agastache Rugosa</i>

In this study a total of seven different Agrobacterium rhizogenes strains were evaluated for their ability to transform the plant Agastache rugosa and to produce the secondary metabolite rosmarinic acid. All the strains of A. rhizogenes i.e., 13333, 15834, A4, LBA9402, R1000, R1200 and R1601 strains tested here in this study, were able to induce hairy root formation in leaf tissue explants. The strain A4had the highest rate of infection (94.1±8.3%) and the strain R1000 had the lowest rate (88.6±6.9%). The highest frequency of hairy roots per explant (13.6±1.4) was found for strain R1601 and the tallest root length (20.4±1.7 mm) was found for strain 13333. We also evaluated dry weight and level of rosmarinic acid in the hairy roots and found that the highest growth (310.1±14.6 mg/flask) was occurred after infection with strain R1200, while the highest production of rosmarinic acid (68.2±3.8 mg/g dry weight) was noted using strain 13333. Our study showed that A. rhizogenes strain 13333 was the most effective of the 7 tested strains for production of transformed root cultures as well as rosmarinic acid in the hairy roots.

Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein. Sucrose, galactose and fructose markedly influenced the production of major flavones and were therefore chosen for subsequent experiments. HRC growth and flavone accumulation were examined following culture with 30, 100 and 150 mM sucrose, galactose and fructose, respectively. From these data, 150 mM sucrose was found to be the optimal carbon source for the enhancement of baicalein production and growth of S. baicalensis HRCs. Fructose caused the greatest increase in baicalin accumulation. Additionally, galactose was the optimal carbon source for wogonin production. These results provide important insights into the optimal growth conditions, particularly the appropriate carbohydrate source, for S. baicalensis.

Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment.

An improved Agrobacterium rhizogenes-mediated genetic transformation of Withania somnifera (L.) Dunal was developed using the bacterial strain R1000 with leaf segment explants of in vitro raised plantlets. Out of the three strains used (R1000, MTCC 2364 and MTCC 532), the strain R1000 proved to be more efficient than others. Among the different conditions tested, the highest (93.3 %) transformation rate was observed after 3 weeks when the explants were subjected to sonication (15 s) and heat treatment (41 °C for 5 min). Transgenic status of the hairy roots was confirmed by PCR using rol B-specific primers. HPLC analysis showed the ability of hairy roots to synthesize withaferin A and withanolide A, both steroidal lactones of medicinal value. This protocol offers new avenue in A. rhizogenes-mediated hairy root induction and is useful for large-scale production of these bioactive compounds from W. somnifera.

Specific activity of thymidine nucleotide biosynthetic enzymes in hairy roots extracts of Sesamum indicum L. transformed by two strains of Agrobacteruim rhizogenes

The study concluded induction of transformed hairy roots from leaves and decapitated seedlings of Sesamum indicum L. using two strains of Agrobacterium rhizogenes considered as natural vector of transformation. The strain R1601 stimulated roots on leaves and seedlings during 20 days of inoculation placed on solidified Arnon and Hoagland medium. Whereas they involved 12 days when inoculated with the strain R15834. Generally strain R15834 was efficient in inducing these roots and their numbers than strain R1601 which approached 54.4% and 41.6% respectively. The results indicated an increase in the specific activity of enzymes Thymidlate synthase (TS), Dihydrofolate reductase (DHFR), Serine hydroxy methyl transfrase (SHMT) in extract of transformed hairy roots producing agropine by stain R15834 and approach 4.610, 1.057, 0.480 µmolmin mg of protein respectively compared with the activity of 1.256, 0.097, 0.125µ molmin mg in the control samples. This was coupled with an increase in amount of DNA and RNA that approached 105, 1020 µg per gram respectively compared to 44, 462 µg per gram in control samples. The transformation of these hairy roots was pointed out through the separation of agropine spots from their extracts when electrophoreted in the presence of standared agropine.

Hairy Root Cultures and Plant Regeneration in <i>Solidago nemoralis</i> Transformed with <i>Agrobacterium rhizogenes</i>

By screening a native plant extract library we identified Solidago nemoralis as a novel source of agonists for alpha7 nicotinic receptors for acetylcholine with therapeutic potential. The next phase of our drug discovery strategy is to increase the yields of active compounds in the plant species by gain of function mutations in hairy root cultures [1]. Here we report a protocol for Agrobacterium rhizogenes-mediated genetic transformation of hairy root cultures of Solidago nemoralis which will enable this. Leaf explants of this species were successfully transformed with a frequency of 30%-35% using A. rhizogenes strain R1000 harboring the binary vector pCambia 1301. Transformation was confirmed using the β-glucuronidase (GUS) histochemical assay. Transformed hairy roots showed spontaneous regeneration of adventitious shoots in media without the addition of cytokines, albeit at very low frequency. However, media supplementation with auxin (α-naphthaleneacetic acid, NAA) increased shoot regeneration frequency to 35% and resulted in viable adventitious shoots. Transformation was confirmed at all phases of plant regeneration by GUS staining. Hairy root transformation of Solidago altissima has been previously reported, but this is the first report of genetic transformation of S. nemoralis. The protocol will allow for a large population of activation tagged mutants of S. nemoralis to be generated which will be then screened for the presence of stable mutants which are over-producing metabolites with activity at alpha7 nicotinic receptors. These over-producing mutant cultures will then be regenerated into intact mutant plants.

An efficient protocol for genetic transformation of watercress (Nasturtium officinale) using Agrobacterium rhizogenes

Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01-0.02 μmol/g of DW) and 4-methoxyglucobrassicin (0.06-0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06-0.21 μmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).

An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes

The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes, and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this, the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant, and the concentration of antibiotics (75mg/l kanamycin) was elucidated for selection after transformation. Wild-type and transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such as phenolic compounds from P. grandiflorum hairy root cultures.

Establishment of Withania somnifera Hairy Root Cultures for the Production of Withanolide A

Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 microg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 microg/g dry weight).

Decursin Production from Hairy Root Culture of Angelica gigas

Angelica gigas Nakai, commonly known as Korean Angelica, is a perennial herb belonging to the Umbelliferae family and one of the most important medicinal plants in Korea. For thousands of years, the roots of A. gigas has been used as traditional Oriental herbal medicine to treat anemia, abdominal pain, injuries, migraine, arthritis, and female afflictions, and the material has also been prescribed for health-promoting effects [Chi and Kim, 1970; Choi et al., 2003; Sarker and Nahar, 2004]. Decursin (Fig. 1) and decursinol angelate (a decursin isomer), the major secondary metabolites of A. gigas, have significant neuroprotective, anticancer, and anti-androgen-receptor signaling activities [Kang et al., 2005; Yim et al., 2005; Guo et al., 2007]. Various species of bacteria can transfer genes to higher plants, among which Agrobacterium rhizogenes, a Gramnegative soil bacterium, is one of the most widely studied. A. rhizogenes infects the plant cell and leads to the formation of hairy roots [Signs and Flores, 1990]. In many plants, hairy root cultures have proven to be an efficient production system for secondary metabolites. Such cultures have genetic and biochemical stabilities, rapid growth rate, and the ability to synthesize natural compounds at levels comparable to those of intact plants [Giri and Narasu, 2000; Guillon et al., 2006]. To the best of our knowledge, this is the first report on decursin production by hairy root culture of A. gigas. Here, we describe the production of decursin by hairy root cultures of A. gigas transformed with A. rhizogenes R1000. A. rhizogenes strain R1000 cultures were grown to the log phase at 28oC on a gyratory shaker (180 rev/min) in liquid Luria-Bertani medium. The bacterial cells were collected by centrifugation for 10 min at 2000 rpm, and resuspended at a cell density of A600 = 0.5 in MS [Murashige and Skoog, 1962] liquid inoculation medium. Young leaf and stem of A. gigas were taken from plants grown in vitro and were cut at the ends into sections of 0.7×0.7 cm2 and 0.7 cm. Excised leaves and stems were dipped into A. rhizogenes culture in the liquid inoculation medium for 10 min, blotted dry on a sterile filter paper, and incubated in the dark at 25oC on the agar-solidified MS medium. After 2 days of co-cultivation, the explant tissues were transferred to a hormone-free solid MS medium containing 200 mg/L Timentin. Within 4 weeks numerous hairy roots were observed emerging from the wound sites. The hairy roots were separated from the explant tissue and subcultured in the dark at 25oC on the agar-solidified MS medium. After repeated transfers to fresh media, rapidly growing hairy root cultures were obtained. Wild type root cultures were established by inoculating MS liquid medium with excised roots from A. gigas seedlings grown in vitro. Isolated wild type and hairy roots (200 mg) were transferred to 30 mL MS liquid medium, containing 30 g/ L sucrose, in 100 mL flasks. Wild type and hairy root cultures were maintained at 25oC on a gyratory shaker (100 rev/min) in a growth chamber. After 24 days of culture, hairy roots were harvested. Dry weight and decursin contents were determined. Three flasks were used for each culture condition, and the experiments were performed in duplicates. Hairy roots were dried at −80oC in brown paper bags for at least 48 h using a freeze-dryer. Dried samples were ground into a fine powder using a mortar and pestle. Samples (0.5 g) were extracted with 30 mL of 70:30 (% v/v) ethanol-water at 50oC in a water bath for 1 h. After *Corresponding author Phone: +82-42-821-5730; Fax: +82-42-822-2631 E-mail: supark@cnu.ac.kr

Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate

Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A(4)GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B(5) medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (T +/L and T +/R) and the GM2 (T +/L and T -/R DNA) type clones in considerably higher amount whether as two T -/L but T +/R callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B(5) vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B(5) media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T(L)-DNA and T(R)-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.