Production of Adventitious Ginseng Roots Using Biorectors

Yu, Kee-Won,Hahn, Eun-Joo,Paek, Kee-Yoeup The Korean Society of Plant Biotechnology 2000 식물생명공학회지 Vol.27 No.4

Panax ginseng is an important medicinal plant that has been used worldwide for geriatric, tonic, stomachic, and aphrodisiac treatments. Ginsenosides contained in the ginseng root are the main substances having active functions for human body. The price of ginseng is very expensive due to a complex process of cultivation, and the yield of ginseng is limited, which cannot meet the demand of the increasing market. Researchers have applied plant biotechnology to solve the problems but there are still things to be determined towards ginsenoside production by large-scale adventitious root culture. In this experiment, 5 to 20 liter bioreactors were employed to determine optimal conditions for adventitious root culture and ginsenoside production of Panax gineng. Callus was induced from the ginseng root on MS agar medium containing 1.0 mg. $L^{-1}$ 2,4-D and 0.1 mg. $L^{-1}$ kinetin. Then the callus was cultured on MS agar medium supplemented with 2.0 mg. $L^{-1}$ IBA, 0.1 mg. $L^{-1}$ kinetin, and 30 g. $L^{-1}$ to induce adventitious roots. The maximum root growth and ginsenoside production were obtained in 1/2 MS medium. 2.0 mg. $L^{-1}$ naphthalene acetic acid resulted in greater root growth than 2.0 mg $L^{-1}$ indole-3-butyric acid. Ginsenoside content increased with 2.0 mg. $L^{-1}$ benzyl adenin or kinetin. High concentrations of benzyl adenin (above 3.0 mg. $L^{-1}$ ) decreased the adventitious root growth and ginsenoside productivity. N $H_{4}$$^{+}$ inhibited the ginsenoside accumulation, while high concentrations of $K^{+}$, $Mg_{2}$$^{+}$, and $Ca_{2}$$^{+}$ increased it. N $H_{4}$$^{+}$ at 0.5 and 1.0 times of the normal amount in 3/4 SH medium resulted in the greatest biomass increase, but the highest ginsenoside productivity was obtained when N $O_{3}$$^{-}$ was used as the sole nitrogen source in the medium. Most microelements at high concentrations in the medium inhibited the root growth, but high concentrations of MnS $O_4$enhanced the root growth. Root dry weight increased with increasing sucrose concentrations up to 50 g. $L^{-1}$ , but decreased from 70 g $L^{-1}$ Ginsenoside productivity was maximized at the range of 20 to 30 g. $L^{-1}$ sucrose. In the experiment on bioreactor types, cone and balloon types were determined to be favorable for both adventitious root growth and ginsenoside production. Jasmonic acid was effective for increasing ginsenoside contents and Rb group ginsenosides mainly increased. These results could be employed in commercial scale bioreactor cultures of Panax ginseng.x ginseng.

Adventitious root cultures of Clitoria ternatea L. and its potential as a memory enhancer alternative

Lee Rui Xuan,Hassan Zurina,Subramaniam Sreeramanan,Chew Bee Lynn 한국식물생명공학회 2021 Plant biotechnology reports Vol.15 No.2

Clitoria ternatea L., known as the butterfy pea plant, is from the Fabaceae family and is commonly used in traditional medicine or as a brain tonic in Asia. The root extracts of C. ternatea have been previously studied for its neuroprotective properties linked to memory enhancement of the mammalian brain, indirectly delaying the occurrence of neurodegenerative diseases. The current study aims to establish in vitro adventitious root cultures of C. ternatea via cotyledon explants and to identify the phytoconstituents of the induced adventitious roots. In this study, the highest percentage of induction (95.24%) was achieved in half-strength MS medium supplemented with 2.50 mg/L NAA (1-naphthaleneacetic acid) after 6 weeks of culture. Results also indicated that the supplementation of 1.50 mg/L NAA induced the highest number of adventitious roots (12.86±2.14), while 2.50 mg/L 4-Cl-IAA (4-chloroindole-3-acetic acid) yielded higher adventitious root length (0.75±0.13 cm) in comparison with the other auxin treatments. Microscopic analysis indicated thicker in vitro root morphology for all NAA treatments in comparison with the roots of in vitro seedling. Ethanolic extract of in vitro-generated adventitious roots showed potential anti-acetylcholinesterase activities (IC50=1519.89±0.18 μg/mL) albeit being lower than the control (IC50=70.37±0.01 μg/mL), while gas chromatography–mass spectrometry analysis identifed the presence of pentacyclic triterpenes, phytosterols and fatty acids in the adventitious root culture that are linked to the mammalian neuroprotective properties of C. ternatea. The current investigation reports on the potential of C. ternatea root cultures being further utilized to produce valuable metabolites linked to the mammalian memory enhancement.

Inhibition of Adventitious Root Growth in Boron-Deficient or Aluminum-Stressed Sunflower Cuttings

Hong, Jung-Hee,Go, Eun-Jung,Kim, Tae-Yun 한국환경과학회 2003 한국환경과학회지 Vol.12 No.11

The effect of boron and aluminum on the development of adventitious roots was studied in sunflower cuttings. Three-day-old seedlings were de-rooted and grown in nutrient solutions with or without boron and supplemented with different concentrations (from 50 to 700 μM) of aluminum. The number and length of the adventitious roots and proline content in adventitious roots in response to insufficient boron and aluminum stress were determined periodically. The micronutrient boron caused the development of numerous roots in the lower parts of the hypocotyl. A dose-response of boron-induced rooting yielded an optimum concentration of 0.1 mM boron. In the absence of boron, in the majority of the adventitious roots, a significant inhibition was observed with or without aluminum, indicating that the most apparent symptom of boron deficiency is the cessation of root growth. Increasing concentrations of aluminum caused progressive inhibition of growth and rooting of the hypocotyls, and a parallel increase in proline levels of adventitious roots. Supplemental boron ameliorated the inhibitory effect of aluminum, suggesting that aluminum could inhibit root growth by inducing boron deficiency. Ascorbate added to medium in the absence of boron improved root growth and induced a significant decrease in proline levels. These findings suggest that adventitious root growth inhibition resulting from either boron deficiency or aluminum toxicity may be a result of impaired ascorbate metabolism.

대두 (Glycine max L.)의 자엽 부정근 형성시 특이단백질의 변화 양상

한태진 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.2

The potency of adventitious root initiation was investigated in relation with germination days and ike end of culture days healed with root induction medium on the initiation of adventitious roots from explants of soybean (Glycine max L.) cotyledon. Also, the changes in the pattern of protein profiles related with adventitious mot initiation have been examined by two-dimensional polyauyl-amide gel elecoophoresis during the germination and the initiation of adventitious roots. The potency of adventitious root initiation from germinated cotyledons was high after 4 days, and adventitious roots were initiated from after 4 days and very high after 6 days healed with mot induction medium There was a reproducible quantitative change in 34 specific protein spots during germination and adventitious root initiation. A necessary basic protein for adventitious root initiation with rotative molecular weight of 27 kD was induced during the germination. And three basic protein groups with relative molecular weight 22 kD, 23 kD and 33 kD, and two acidic protein groups with relative molecular weight 27kD and 29 kD were induced during the initiation of adventitious roots. 대두(Glycine max L) 자엽의 발아 정도에 따른 부정근 형성능과 자엽 절편의 부정근 형성 배지 처리 시간에 따른 부정근 형성 정도를 조사하였으며 부정근 형성과 관계 있는 발아 및 부정근 유기시의 단백질 변화 양상을 2차원 전기영동법으로 조사하였다. 발아된 자엽의 부정근 형성능은 발아 4일 이후에 높았으며 부정근 형성 배지에서의 부정근 형성은 처리 4일 이후부터 형성되어 6일 이상 처리한 것이 가장 부정근 형성이 높았다. 또한 부정근 형성과 관계 있는 단백질 34개의 단백질 spot를 얻었는데 부정근 형성을 위한 발아 자엽의 주요 단백질은 염기성 단백질 27d이었다. 그리고 부정근 유기시 생성되는 단백질은 염기성의 길 kD, 23 kD 및 33 kD였으며 산성의 27 kD와 29 kD이었다.

품종별 대두 〔Glycine max L.〕 자엽에서의 부정근 형성

하건수,한태진,Ha, Keon-Soo,Han, Tae-Jin 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.1

대두의 자엽 절편을 이용한 부정근 형성과 품종 반응을 비교하였다. 품종별 부정근 형성 양상은 직접적인 부정근 형성 품종과 callus형성이 동시에 이루어지는 품종들로 구분되었으며, callus 형성이 많은 품종에서는 부정근 형성수가 적었다. 자엽 절편에서의 부정근 형성은 절편의 향축면 치상시에만 부정근이 형성되었고, 배축면과 향축면이 동시에 치상될 경우에도 향축면에서만 부정근이 형성되며, 절편의 배축이 존재하였던 방향에서만 부정근이 형성되는 방향성이 존재하였다. 부정근 형성시 자엽 절편 내 탄수화물은 형성초기보다 형성이 가장 많은 유기 4일에 가장 많이 관찰되었으며 ,형성된 부정근의 생장이 이루어지는 7일 이후에는 관찰되지 않았다. The patterns of adventitious root formation from cotyledons for each cultivar of soybeans were compared. The results of adventitious root formation in cultivars are classified as two groups; the first group showed the direct adventitious root formation, and the second group resulted in the callus and adventitious root formation. The cultivars that have much callus formation had less the adventitious root formation. The adventitious root formation in the cotyledonary explants was occured only at the inoculation of adaxial side. When adaxial and abaxial side was inoculated simultaneously, the adventitious roots were formed at the adaxial side. Thus, it suggests that there must be direction to some extent. Starch in the cotyledonary explants were more abundant at the 4 days after induction than at the early stage of the adventitious root formation, but the starch was not observed after 7 days, that the growth stage of adventitious roots.

Cryopreservation of Panax ginseng Adventitious Roots

Suk Young Oh,Chun Hua Wu,Elena Popova,Eun Joo Hahn,백기엽 한국식물학회 2009 Journal of Plant Biology Vol.52 No.4

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L−1 was recorded for post-freezeregenerated cultures versus 9.1 g dw L−1 for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.

킬레이트제가 액체배양 중 인삼 부정근의 게르마늄 축적에 미치는 영향

장은정(Eun Jung Chang),오훈일(Hoon Il Oh) 고려인삼학회 2007 Journal of Ginseng Research Vol.31 No.3

식물조직배양기술을 이용한 Ge 함유 인삼 부정근 생산 시 Ge의 생산성을 향상시키고자, 다양한 킬레이트제가 인삼 부정근의 Ge 축적과 사포닌 형성 및 생육에 미치는 영향을 조사하였다. GeO₂ 50ppm과 함께 citric acid, oxalic acid, phosphoric acid, EDTA 및 EGTA와 같은 킬레이트제를 인삼 부정근 배양시 첨가하여 Ge 흡수에 미치는 영향을 조사한 결과, phosphoric acid, EDTA 및 EGTA가 Ge 흡수를 촉진시킨다는 사실을 알 수 있었다. 그러나, EDTA와 EGTA는 인삼 부정근의 생육을 억제시킬 뿐만 아니라 Ge 흡수 촉진 효과도 phosphoric acid에 비해 낮으므로 인삼 부정근의 Ge 흡수를 촉진하는데 가장 효과적인 킬레이트제는 phosphoric acid인 것으로 확인되었다. 인삼 부정근의 Ge 흡수 촉진을 위한 phosphoric acid의 최적 농도는 1.0 mM이었고, 이때의 Ge 함량은22.7±0.3 ㎎%로 대조구(16.8±0.7 ㎎%)의 1.4배에 해당하는 양이다. 한편, 인삼 부정근의 생장률과 총 사포닌 함량은 phosphoric acid의 농도가 증가할수록 감소하는 경향을 나타내었다. In order to increase the content of germanium in ginseng adventitious roots, the effects of chelating agents on germanium content and root growth were investigated in the submerged cultures of ginseng adventitious roots. Chelating agents such as citric acid, oxalic acid, phosphoric acid, EDTA (Ethylenediamine tetraacetic acid) or EGTA (Ethylene glycol-bis (β-aminoethylether)-tetraacetic acid) were administrated in the submerged culture of ginseng root containing 50 ppm GeO₂. After 6 weeks of cultivation, fresh weight, germanium and saponin contents in the roots were analyzed. Among chelating agents, addition of 1.0mM phosphoric acid was found to be best for germanium accumulation. Under this condition, germanium content increased 1.4 times as compared to that of the control. The germanium content in the adventitious roots also increased with addition of EDTA or EGTA, while they inhibited the growth of ginseng adventitious root. Citric and oxalic acids were not effective for increasing germanium content in adventitious roots. As the results, it suggests that the phosphoric acid can be proved as the optimal agent for the enhancement of germanium accumulation in ginseng adventitious roots. These results can be served as a guideline for the mass production of ginseng adventitious roots containing germanium by large-scale production.

Comparative study of essential oil in wild and in vitro cultures of Valeriana jatamansi Jones in Nepal

Pokharel Basanta Raj,Pandey Sushma,Manandhar Mangala Devi,Pant Bijaya 한국식물생명공학회 2023 Plant biotechnology reports Vol.17 No.3

Comparative analysis was carried out to determine the variation in the composition of essential oils of hairy roots produced in vitro and in vivo rhizomes of Valeriana jatamansi Jones collected from three different districts in Nepal i.e., Pyuthan (1200 m), Gulmi (3000 m), Godawari (1400 m) was carried out. In vitro adventitious root proliferation was established in MS media supplemented with Naphthalene acetic acid (NAA). GC–MS analysis were performed on the essential oils extracted from the sample. The findings showed that the yield of the essential oil obtained from the hydrodistillation of in vitro adventitious roots and dried rhizomes ranged between 0.52 and 0.80%. The highest percentage of essential oil (0.80%) were produced in the in vitro adventitious roots and minimum in the in vivo rhizomes collected from Pyuthan district, i.e., (0.52%). GC–MS analysis revealed a total of 17 volatile compounds in the essential oil from the dried rhizomes of V. jatamansi Jones from different locations and in vitro adventitious roots. It was found that the compound carotol was present only in sample from Pyuthan district, but absent in other samples. The compound matricarin was found in samples from Gulmi and Godawari district but absent in samples from Pyuthan district and in vitro adventitious roots. Similarly, the compound jatamansone was found in samples from Gulmi, Godawari, and adventitious roots but absent in sample from Pyuthan district. The compounds patchoulinone, 1, 2-butyl octyl ester of benzene dicarboxylic acid, and bis (2-ethyl hexyl) phthalate, one of the major compounds were present only in sample from adventitious roots The findings of the present study showed that the essential oil composition in both in vitro and in vivo roots, at different altitudes significantly affect the essential oil content of V. jatamansi Jones.

Effect of Boron on the Development of Adventitious Roots in Sunflower Seedlings

Go, Eun-Jung,Hong, Jung-Hee Korean Society of Life Science 2002 생명과학회지 Vol.12 No.6

암하에서 3일간 발아시킨 해바라기 유식물의 유근을 절단한 후 붕소 첨가구와 비첨가구에서 온도와 pH 처리에 의한 부정근 발생을 조사하였다. 붕소 미첨가구에서는 부정근이 발생하지 않았으나, 붕소 첨가구에서는 배축의 하위부에서 부정근 발생이 유도되었다. 낮은 pH 처리에 의해 부정근이 손상되었으나 $Ca^{2+}$을 함께 처리한 붕소 첨가구에서는 현저하게 회복되었는데, 이것은 proton 독성을 극복하는데 calcium이 필요하며 proton 독성이 뿌리에서 중요한 기능을 하는 펙틴 다당류의 안정성을 방해하는 현상과 관련이 있음을 시사해준다. 부정근 생장에 최적온도는 $25^{\circ}C$이었다. 붕소 결핍의 가장 뚜렷한 현상은 부정근 생장의 정지이었으나 붕소 첨가구에서 부정근 생장은 향상되었다. 붕소 비첨가구에 ascorbate를 처리하였을 때 부정근의 생장은 촉진되었다. 이러한 사실들로 보아 붕소 결핍, 낮은 pH 및 온도저해로부터 야기된 부정근 생장의 억제는 ascorbate대사가 교란된 결과에 기인되는 것으로 사료된다. Three-day-old sunfower(Helianthus annuus L.) seedlings were do-rooted and incubated in the nutrient solutions that contained either deficient or sufficient boron supply, and exposure to various pH and temperatures. In the absence of boron, no adventitious roots were formed in the majority of the seedlings. Boron caused the development of numerous adventitious roots in the lower part of the hypocotyl. The low pH damage was ameliorated by the simultaneous application of calcium, implying the involvement of calcium-requiring process in overcoming proton toxicity. Borate showed a strong ameliorative effect with $Ca^{2+}$. These results suggest that the primary target of proton toxicity may be linked to a disturbance of the stability in the pectic polysaccharide network, where calcium plays a key role in plant roots. Maximum temperature for the growth of adventitious roots was $25^{\circ}C$. Although cessation of growth is the most apparent symptom of boron deficiency, exogenous ascorbate improved adventitious root growth in plants in the absence of boron. From the results it is suggested that the inhibition of adventitious root resulting from boron deficiency, low pH and temperature damage may be a consequence of disrupted ascorbate metabolism.

Chitosan과 Jasmonic acid 처리에 의한 인삼 부정근의 Ginsenosides의 생산성 증대

이범수,인준교,송원섭,양덕춘 한국자원식물학회 2004 한국자원식물학회지 Vol.17 No.1

인삼 부정근의 생장과 ginsenosides 함량에 미치는 elicitor의 영향을 조사한 결과 chitosan처리구의 생장량은 10 mg/L에서 가장 양호하였으며, ginsenosides의 함량은 5 mg/L처리농도에서 14.48 mg/g $.$ DW로 대조구에 비해 약9%의 함량증대를 가져왔다. 세포내에서 이차대사계를 활성화시키는 중간 신호전달 물질(signal transducer)로 알려진 jasmonic acid를 인삼 부정근 배양에 처리한 결과 부정근의 생장량은 10 uM 처리구에서 ginsenosides함량이 가장 양호하였으나, 인삼 부정근의 생장은 다소 억제되었다. In order to investigate the effects of elicitors on the growth and ginsenosides biosynthesis of ginseng adventitious roots, chitosan and jasmonic acid were treated with various concentrations. The growth rate of adventitious roots was increased with the addition of chitosan at higher concentrations (10 mg/L), but the best accumulation of ginsenosides was observed at the lower concentration (5 mg/L). Jasmonic acid was an effective elicitor for ginsenosides biosynthesis in ginseng adventitious roots. The maximum accumulation of ginsenosides was observed at the treatment of 10 uM jasmonic acid. But the jasmonic acid was found to decrease the growth rate of adventitious roots.