Medium optimization of transformed root cultures of Stizolobium hassjoo producing L-DOPA with response surface methodology

Medium optimization of B5 medium for hairy root cultures producing secondary metabolites was studied through statistical experimental design. Transformed root cultures of Stizolobium hassjoo producing L-DOPA were used as a model system. The serial dilution experiments facilitated logical choice of the upper and lower bounds on executing 2(11)(-)(6) fractional factorial design. Steepest ascent method as well as central composite design were sequentially employed to optimize the media of shake flask cultures. The modified B5 media of GM, PM, and GPM were obtained, indicating the optimum medium compositions for enhancing hairy root dry weight, L-DOPA content in hairy roots, and L-DOPA production, respectively. When cultivating S. hassjoo hairy roots in GM, PM and GPM for 16 days, the dry wt of hairy roots, L-DOPA content, and L-DOPA production obtained were ca. 530 mg per flask (10.6 g/L), 10.8% dry wt, and 806 mg/L, which were 1.8-, 2-, and 2.8-fold of basal B5 medium control runs, respectively.     

Enhanced accumulation of secondary metabolites in hairy root cultures of Scutellaria lateriflora following elicitation

Hairy root cultures of Scutellaria lateriflora were established using Agrobacterium rhizogenes A4 and produced acteoside (18.5?mg?g(-1) dry wt), baicalin (14.5?mg?g(-1) dry wt) and wogonoside (12?mg?g(-1) dry wt). Yeast extract (50?μg?ml(-1)) increased acteoside production 1.4-fold and flavone production 1.7-fold after 7 and 14?days of elicitation. Addition of Pectobacterium carotovorum lysate in the stationary phase of the hairy root culture stimulated only the accumulation of wogonin to 30?mg?g(-1) dry wt. The production of wogonin in hairy roots could be associated with its role as a phytolaexin.     

ENHANCEMENT OF RUTIN PRODUCTION IN Fagopyrum tataricum HAIRY ROOT CULTURES WITH ITS ENDOPHYTIC FUNGAL ELICITORS

Tartary buckwheat (Fagopyrum tataricum) is a potentially important source of rutin, a natural bioactive flavonoid with antihyperglycemic, antioxidative, antihypertensive, and anti-inflammatory properties. This study examines the effects of endophytic fungi on rutin production in the hairy root cultures of F. tataricum. Without obvious changes in the appearance of the hairy roots, the exogenous fungal mycelia elicitors efficiently stimulated the hairy root growth and rutin biosynthesis, and the stimulation effect was mainly dependent on the mycelia elicitor species, as well as its treatment dose. Two endophytic fungal isolates Fat9 (Fusarium oxysporum) and Fat15 (Alternaria sp.) were screened as promising candidates for promoting F. tataricum hairy root growth and rutin production. With application of polysaccharide (PS) of endophyte Fat9 (200 mg/L), and PS of endophyte Fat15 (100 mg/L) to the hairy root cultures on day 25, the rutin yield was increased to 45.9 mg/L and 47.2 mg/L, respectively. That was about 3.1- to 3.2-fold in comparison with the control level of 14.6 mg/L. Moreover, the present study revealed that the accumulation of rutin resulted from the stimulation of the phenylpropanoid pathway by mycelia PS treatments. This may be an efficient strategy for enhancing rutin production in F. tataricum hairy root culture provided with its endophytic mycelia elicitors.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Study on Hairy-Root Cultures of Fagopyrum cymosum

Transformed hairy root cultures of Fagopyrum cymosum (Trey.) Meisn. have been established by infecting petioles of shoots cultured in vitro with Agrobacterium rhizogenes. These hairy root cultures grew vigorously in MS hormone-free medium. They showed a 1861-fold increase in fresh weight with in 25 days, and grew faster than cell cultures of F. cymosum (only increased 26.7-fold with in the same days). The cultures were shon to synthesis dimeric procyanidin up to 4.5% (dry wt.) approximating the level of original plant. The growth rates of hairy root cultures up to 149.3 mg dry w-t/L/day in large culture vessels (3 L) and dimeric procyanidin (3.58%, dry wt.) were detected.     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

不同理化因子对雪莲毛状根生长和总黄酮生物合成的影响

在 1 2MS液体培养基上研究了不同理化因子对水母雪莲毛状根生长和总黄酮生物合成的影响。实验结果表明 :氮源总浓度 (包括NH+4和NO-3)为 30mmol L ;NH⁺₄ NO^-₃比例为 5∶2 5 ;2 %蔗糖和 3%葡萄糖组合 ;0.5mg LGA₃和 0.5mg LIBA ;pH5.8;18h d的光照 (光强为 35.0.0lx) ;2.4℃ ;摇床转速为 100rmin有利于毛状根生长及总黄酮的生物合成。在此培养条件下 ,经过21d的培养毛状根生长量达到 12.8g L(DW) ,总黄酮合成量为 192.2mg L ,即总黄酮含量占毛状根干重的 15 % ,约为干重野生水母雪莲植株总黄酮含量的 2.5倍。     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

Growth and Pigment Production by Hairy Root Cultures of Beta Vulgaris L. in a Bubble Column Reactor

Beet hairy root cultures established from red and yellow varieties were grown in a 2 L bubble column reactor. The yellow clone showed profuse root hairs and a predominance of betaxanthin pigment with the red clone showed fewer root hairs and both betaxanthin and betacyanin pigments. The cultures displayed different ionic and sugar yields: 2.1 mg dry wt / mS.mL and 0.361 g dry wt / g sugar for the yellow clone and 2.3 mg dry wt / mS.mL and 0.375 g dry wt / g sugar for the red one. Both cultures grew at the same specific growth rate of 0.22 d^-1in the bubble column, as compared to 0.32 d^-1in shake flasks, indicating mass transfer limitations for growth in reactors.     

Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze

Rosmarinic acid, an important phenolic active compound, is one of the main active constituents of Agastache rugosa Kuntze and has astringent properties, antioxidant capacity, anti-inflammatory activity, antimutagenic ability, antimicrobial capacity, and antiviral properties. To investigate in vitro production of rosmarinic acid, we established a hairy root culture of A. rugosa by infecting leaf and stem explants with Agrobacterium rhizogenes R1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (14.1 g dry wt/l) was attained after 14 days of culture, at which time the content of rosmarinic acid was 116 mg/g dry wt. The present results demonstrate that hairy root culture of A. rugosa is a valuable alternative approach for the production of rosmarinic acid.     

蔗糖和光对三裂叶野葛毛状根生长及次生物质产生的影响

研究了蔗糖浓度和光对固体培养的三裂叶野葛毛状根生长及其总异黄酮和葛根素产生的影响。结果表明：在供试的分别添加1%、3%、5%、7%和9%蔗糖的MS固体培养基中,3%蔗糖能促进三裂叶野葛毛状根的生长及其异黄酮类化合物和葛根素的积累;培养20d后,其生物量达到0.48g(DW,干重)/瓶,总异黄酮和葛根素含量分别为25.44mg/g(DW)和11.64mg/g(DW)。与添加3%蔗糖的MS培养基培养的三裂叶野葛毛状根相比,含5%蔗糖的培养基培养的毛状根干重增殖倍数提高了7.0％,而含1％、7％和9％蔗糖的培养基培养的毛状根干重增殖倍数分别下降62.4％、42.8％和65.3％;其总异黄酮含量分别降低574％、13％和33.4％,葛根素含量分别下降47.9％、15.8％和35.1％,但其毛状根培养物的可溶性糖含量则分别增加了0.52、1.45和1.54倍。暗培养30d的毛状根的生物量达到0.83g(DW)/瓶,分别比蓝光和白光培养的毛状根提高37.1%和23.3%。在蓝光和白光下培养的部分毛状根的表面呈淡绿色;但白光处理的毛状根中总异黄酮含量比蓝光和暗培养处理的分别提高了14.7%和19.2%;蓝光抑制毛状根中葛根素含量的积累,白光和暗培养的毛状根培养物中的葛根素含量分别是蓝光处理的1.61倍和1.52倍。     

Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid

Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g(-1) of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45-50 days). The level was significantly higher than that found in untransformed root culture (19 mg g(-10 of dry wt).     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Diterpenoid production in hairy root culture of Salvia sclarea L

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L(-1) sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 +/- 0.6) mg g(-1) dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 +/- 0.15) mg g(-1) dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate