Identification of elicitors enhances the polyphenolic compounds and pharmacological potential in hairy root cultures of <i>Aster scaber</i>

Ghimire, B.K.,Thiruvengadam, M.,Chung, I.-M. Elsevier 2019 South African journal of botany : official journal Vol.125 No.-

<P><B>Abstract</B></P> <P> <I>Aster scaber</I> Thunb is an important Korean mountainous vegetable and also a medicinal herb, which produces valuable bioactive compounds. This study showed that hairy root cultures can be established to improve the accumulation of biomass and phenolic compounds in <I>A. scaber.</I> Hairy roots were molecularly confirmed by polymerase chain reaction (PCR). We examined the influences of methyl jasmonate (MeJA) and yeast extract (YE) elicitors for the enhancement of phenolic compounds and pharmaceutical activities on hairy root cultures of <I>A. scaber</I>. Application of 100 mg/L of YE resulted in significantly higher fresh and dry root biomass (97.91 and 9.82 g/L) than MeJA and non-elicited control root cultures. MeJA (100 μM) and YE (100 mg/L) elicitation dramatically induced the accumulation of total phenolics (244.5 ± 2.5; 215.10 ± 2.0 GAE) and flavonoids (6.7 ± 0.3; 6.0 ± 0.2 QE) than non-elicited root cultures (181.65 ± 1.5 mg/g GAE and 4.65 ± 0.2 mg/g QE). Phenolic compounds including six hydroxycinnamic acids, seven flavonols, seven hydroxybenzoic acids, vanillin, homogentisic acid, and resveratrol were identified via UHPLC analysis in elicited (MeJA 100 μM and YE 100 mg/L) and control root cultures. Because of these phenolic compounds enhancement, the antioxidant, antimicrobial, antidiabetic, anti-inflammatory and anticancer activities were high in the in the MeJA- and YE-elicited hairy roots than in the control cultures. MeJA was more effective in the elicitation of phenolic compounds and pharmaceutical potential than YE. The identification of elicitors can lead the large quantity of phenolic compounds to encounter extensive range demand through marketable production without endangering of <I>A. scaber</I>. This elicitation approach could be large scale production of the commercially possible amount of these valuable bioactive compounds by hairy root cultures in <I>A. scaber.</I> </P> <P><B>Highlights</B></P> <P> <UL> <LI> Hairy root biomass was enhanced in methyl jasmonate (MeJA) or yeast extract (YE) elicitation. </LI> <LI> Phenolic compounds were significantly raised in MeJA or YE elicited hairy roots. </LI> <LI> Biological activities were exhibited higher in MeJA or YE than non-elicited hairy roots. </LI> </UL> </P>

Establishment of Tripterygium wilfordii Hook. f. Hairy Root Culture and Optimization of Its Culture Conditions for the Production of Triptolide and Wilforine

( Chuan Shu Zhu ),( Guo Qeng Miao ),( Jia Guo ),( Yan Bo Huo ),( Xing Zhang ),( Jia Hua Xie ),( Jun Tao Feng ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.6

In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes. Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 μM methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 μM salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 μM had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.

Production of Red Ginseng Specific Ginsenosides $(Rg_2, Rg_3, Rh_1 and Rh_2)$ from Agrobacterium-transformed hairy Roots of Panax ginseng by Heat Treatment

Yang, Deok-Chun,Yang, Kye-Jin,Park, Yong-Eui Korean Society of Photoscience 2001 Journal of Photosciences Vol.8 No.1

It was reported that Red ginseng contains specific ginsenoside-Rg$_2$,-Rg$_3$,-Rh$_1$and -Rh$_2$, which show various pharmacological effects. However, production of these specific ginsenosides from Red ginseng is not commercially applicable because of high cost of the raw material, roots. This work was carried out to examine the production of Red ginseng specific ginsenosides from Agrobacterium-transformed hairy roots. Hairy roots were induced from 3 year-old root segment of Korean ginseng (Panax ginseng C.A. Meyer) after infection with Agrobacterium rhizogenes A4. Among many lines of hairybroots, KGHR-8A was selected. Steam heat treatment of hairy roots was resulted in the changes of ginsenoside composition. Eleven ginsenosides were detected in heat-treated hairy roots but eight in freeze dried hairy roots. In heat treated hairy root, content of ginsenoside-Rb$_1$,Rb$_2$,Rc, Rd, Re, Rf, and Rg$_1$were decreased compared to those of freeze dried hairy roots. However, heat treatment strongly enhanced the amount of Red ginseng specific ginsenogides (ginsenoside-Rg$_2$,-Rg$_3$,-Rh$_1$and -Rh$_2$). Amounts of ginsenoside-Rg$_3$,-Rh$_1$and -Rh$_2$ in heat-treated hairy roots were 2.58, 3.62 and 1.08 mg/g dry wt, respectively, but these were detected as trace amount in hairy roots without heat treatment. Optimum condition of heat treatment for the production of Red ginseng specific ginsenoside was 2 h at 105$^{\circ}C$. This result represents that Red ginseng specific ginsenoside can be producted from hairy roots by steam heat treatment.

Induction of Ginseng Hairy Roots And Their Possible Application To Large Scale Culture

Yang,Deok-Chun 한국자원식물학회 2003 Plant Resources Vol.6 No.1

Ginseng(Panax ginseng C. A. Meyer) is important medicinal plant but requires 4-year cultivation for root harvest because of slow growth. In contrast, ginseng hairy roots induced by introducing Ri-plasmid of Agrobacterium rhizogenes into genomic DNA of plant cells show vigorous growth, and the hairy roots produce the same or more saponins than natural ginseng roots. Therefore, hairy roots can be used for commercial purposes. The present study was carried out to induce hairy roots with both active growth and high saponin contents. Numerous hairy roots of Panax ginseng were obtained after root disks of three-year old roots were infected with Agrobacterium rhizogenes R1000 A4T in dark condition after one month of culture. About 3 hundred lines of hairy roots were selected according as morphological characters on medium with carbenicillin. After pre-selection of fifteen lines of hairy roots with active growth, KGHR-l and KGHR-8 lines were finally selected which had characters of high content of ginsenoside-Rd and ginsenoside-Re, respectively. The optimum growth of hairy roots was achieved in the culture of 1/2 MS liquid medium in dark (22℃) under 60 rpm gyratory shaking. Hairy roots grew well in 5L Erlenmeyer flasks, lL roller drums, 10L jar-fermenters, and especially in 20L air-lift culture vessels.

갈퀴꼭두선이의 Hairy Root 배양에 의한 Anthraquinone계 색소생산 연구(II)

김유선,신승원,Kim, You-Sun,Shin, Seung-Won 한국생약학회 1996 생약학회지 Vol.27 No.4

Hairy roots induced from stems of Rubia cordifolia var. pratensis were cultured in the liquid medium under a variety of auxins to find the optimal condition for the growth and production of pigments. Culture of the hairy roots on NN liquid medium containing NAA 0.5 mg/l was best for growth of hairy roots. Production of yellow anthraquinone derivatives and purpurin in hairy roots was enhanced by the culture on NN liquid medium without auxins. Effects of L-phenylalanine, L-tyrosine and juglone, synthesized via the shikimic acid pathway, on growth and production of pigments in hairy roots were studied in the present study. Concentration of exogeneous L-phenylalanine. L-tyrosine and juglone in liquid culture system of hairy root containing NAA 0.1 mg/l was decreased quickly in its early stages of the culture period. Addition of juglone to NN liquid medium containing NAA 0.1 mg/l enhanced the productivity of pigments in hairy roots.

인삼 뿌리 부위별 및 모상근 세포주간 ginsenoside 양상 및 함량

양덕춘,양계진 한국식물생명공학회 2000 식물생명공학회지 Vol.27 No.6

The patterns and contents of ginsenosides were examined in normal root parts and hairy root lines of Panax ginseng C. A. Meyer. Ginsenoside-Rb$_1$, -Rb$_2$, -Rc, -Rd, -Re, -Rf, -Rg$_1$, -Rg$_2$ were detected in normal roots and hairy roots of ginseng. The patterns and contents of ginsenosides in that were very difference each other. The contents of total ginsenoside of hairy root (KGHR-1) was 17.42 mg/g dry wt, it's highest compared to others. Ginsenoside contents of hairy root (KGHR-1) was higher on ginsenoside-Rd, Rg$_1$, KGHR-5 was higher on ginsenoside-Rb$_1$, Rg$_1$, and KGHR-8 was higher on ginsenoside-Rd, Re than others. The contents of total ginsenosides on 6 years old ginseng cultured in the field were high in the order of main root, lateral root and fine roots, and content of ginsenosides in fine roots was 3.2 times higher than that in main root. The ratio of ginsenoside-Rg$_1$to total ginsenosides were about 3.43%, 8.68% and 14.18% respectively on fine root, lateral root and main root, it's very lower than that in hairy roots. It is suggested that specific ginsenosides can be produce in cultures of ginseng hairy roots. 생장이 우수한 인삼모상근 세포주 (KGHR-1, KGHR-5, KGHR-8) 및 6년생 인삼근의 부위별로 ginsenoside 양상 및 생성특성을 조사하였다. 인삼모상근 및 6년생 인상근에서 ginsenoslde-Rb$_1$, Rb$_2$, Rc, Rd, Re, Rf, Rg$_1$, Rg$_2$을 확인하였으며, 인삼모상근 세포주간 및 인삼근 부위별로 ginsenoside의 함량은 큰 차이를 나타내었다. 8종류의 ginsenoside함량이 가장 높은 인삼모상근은 KGHR-1 세포주로 17.42 mg/g dry wt와 함량을 나타내었다. 모상근세포주 KGHR-1은 ginsenoside-Rd, Rg$_1$을, KGHR-5는 ginsenoside-Rb$_1$, Rg$_1$을, 그리고 KGHR-8은 ginsenoside-Rd, Re을 상대적으로 많이 생성하는 특징을 지니고 있으며, ginsenoside-Rf의 생성은 매우 낮았다. 6년생 인삼근의 부위별 ginsenoside의 함량은 주근, 지근, 세근순으로 많았으며, 주근에서 ginsenoside-Rc의 생성은 ginsenoside의 50.99%로써 모상근 세포주의 4.90~6.89%보다 매우 높았다. 6년생 인삼근의 총 ginsenoside에 대한 ginsenoside-Rg$_1$의 비율은 3.43~14.18% 수준으로 주근, 지근, 세근순으로 급격히 감소하였으며, 모상관의 17.14~24.43%와 비교할 때 매우 낮은 수준을 나타내었다. 따라서 인삼모상근 배양을 통하여 특정 ginsenosides생산이 가능하리라 생각된다.

Induction of Hairy Roots and Characterization of Peroxidase Expression as a Potential Root Growth Marker in Sesame

Chun, J. -A.,Lee, J. -W.,Yi, Y. -B.,Park, G. -Y.,Chung, C. -H. Taylor Francis 2009 PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY Vol.39 No.4

<P> Using hypocotyl and cotyledon of sesame seedlings, hairy root cultures were established and cDNA coding for a peroxidase was cloned from the roots. The frequency of sesame hairy root formation was higher in hypocotyl (33.4%) than cotyledon (9.3%). Applicable levels of kanamycin and hygromycin as a selectable marker were 100 µg/mL and 30 µg/mL, respectively. The peroxidase cDNA showed relatively high sequence identity with and similarity to plant class III peroxidase family. The cDNA encoded polypeptide was identified with the presence of three sequence features: 1) the putative 4 disulfide bridges, 2) an ER-targeted signal sequence in the N-terminus, and 3) two triplets, NXS for glycosylation. A real-time RT-PCR exhibited an abrupt increase in the peroxidase transcription activity after 4-week cultures of the sesame hairy roots and its highest level in 6-week cultured hairy roots. In contrast, the growth pattern of sesame hairy roots showed a typical sigmoidal curve. The active hairy root growth began after 2-week culture and their stationary growth phase occurred after 5-week culture. These results suggested that the peroxidase expression patterns at its transcription level could be used a potential indicator signaling a message that there will be no longer active growth in hairy root cultures. The sesame peroxidase gene was differentially expressed in different tissues.</P>

Establishment of hairy root lines of several economic plant species by inoculation with agrobacterium rhizogenes

Chang Soon Ahn(安獎,淳) 한국육종학회 1993 한국육종학회지 Vol.25 No.3

Seedlings of 5 crop species ; Lactuca sativa L., Capsicum annuum L., Cucumis sativus L., Lycopersicon esculentum Mill. and Sesamum indicum L. were inoculated with 6 wild strains of Agrobacterium rhizogenes, A4, A13, C8, M123, 1724 and 15834. Hairy roots were induced from almost all the combinations, though the plant species showed marked differences in the per cent hairy root formation. The root cultures derived from the initial hairy root tips displayed the typical hairy root phenotypes such as vigorous growth, diminished geotropism and abundant lateral roots. Hairy roots of cucumber, tomato and lettuce kept growing vigorously in subcultures, while the growth of those of hot pepper and sesame suspended in a few rounds of subcultures. Addition of GA₃ to the culture medium effectively accelerated the growth of hot pepper hairy roots. The formation of green and compact calli from the hairy roots cultured on the media containing NAA and zeatin indicated that plant regeneration would be readily induced by modification of culture medium or environmental parameters.

인삼 모상근 유도를 위한 최적 조건

양덕춘,김용해,양덕조,신성련,최광태 한국자원식물학회 1999 한국자원식물학회지 Vol.12 No.1

인삼 뿌리 절편으로 부터 모상근 유기를 위한 최적 조건을 확립하고자 Agrobacterium rhirogenes와 인삼 뿌리 절편의 항생제 내성 조사 및 최적의 모상근 유도 배지를 조사하기 위하여 수행하였다. NaOCl로 인삼 뿌리를 멸균하였을 때, 오염 정도가 감소하면서 조직의 손상이 일어나지 않는 NaOCl의 농도는 7% NaOCl에서 15-20분, 9% NaOCl에서 5분으로 나타났다. 인삼근은 년수가 증가할수록 오염 정도가 심하였으며, 특히 6년근중 표피가 있는 처리구는 오염 정도가 매우 높았다. Agrobacterium의 성장억제를 위한 항생제는 tetracycline이 가장 효과적이었으며, 30mg/L 이상의 농도에서 균의 성장이 억제되었다. 하지만 30mg/L tetracycline에서 인삼 조직이 고사하였으며, cefotaxime(500mg/L), carbenicillin(500mg/L)에서 균의 성장을 완전히 억제하였으며, 조직의 손상이 일어나지 않았다. 3년근 인삼에서 모상근 유도을 위한 배지로는 1/2MS 배지에 500mg/L의 cefotaxime이 첨가된 배지가 가장 좋았으며, 인삼 뿌리 절편에 Agrobacterium을 발라주는 것 보다는 균과 공동배양할때가 절편이 좋았다. Agrobacterium접종 2주 후부터 callus가 유기되기 시작한 후, 다시 2주 후에 모상근이 유도되었다. 유도된 hairy roots는 PCR에 의하여 rol C유전자를 조사함으로서 형질전환체임을 확인하였다. The experiments were carried out to determine the optimum conditions for the induction of hairy roots in ginseng(Panax ginseng C.A. Meyer) by Agrobacterium spp. We were examined the antibiotics resistance of Agrobacterium spp and various ginseng parts, and the media for induction of hairy roots. The optimum concentration of NaOCl for sterilization of ginseng root segments without tissue damage with reduce of contamination was 7% NaOCl for 15-20 min and 9% NaOCl for 5 min, respectively. The more ginseng ages, the more contamination of ginseng root segment by sterilized in 7% NaOCl for 20 min, and especially in ginseng root segments with epidermis in six-year old roots. The growth of Agrobacterium spp were inhibited, but ginseng root segments was death in 30mg/L tetracycline. In 500mg/L cefotaxime or 500mg/L carbenicillin, the growth of Agrobacterium sup were inhibited, and root segments was grown normally. The optimum conditions for induction of hairy roots were using the root segments of three-year old ginseng cultured in 1/2MS medium supplemented with 500mg/L cefotaxime, and inoculation of Agrobacterium to root segments were better co-culture than smear method. After 2 weeks co-culture, the callus induced in cambium of root segments cultured in 1/2MS solid medium with 500mg/L cefotaxime. And then after 2 weeks, ginseng hairy roots were induced in callus of root segments. PCR analysis of rot C gene fragment confirmed that hairy roots were transgenic tissues.

Effect of Plant Growth Regulators on Growth and Biosynthesis of Phenolic Compounds in Genetically Transformed Hairy Roots of Panax ginseng C. A. Meyer

Jeong, Gwi-Taek,Woo, Je-Chang,Park, Don-Hee Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.2

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots of Panax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. ${\alpha}-Naphthaleneacetic$ acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.