Enhanced production of saikosaponins by root culture of Bupleurum falcatum L. using two-step control of sugar concentration

 The effect of sugar concentration on the production of saikosaponins was investigated using a root culture of Bupleurum falcatum L. The formation of the lateral roots, which were induced in the presence of indolebutyric acid, was suppressed as the sugar concentration was increased. After the lateral root tips had emerged from the inoculated roots, however, high concentrations of sugar showed no inhibitory effect on the development of the lateral roots. A two-step culture, with 1% sucrose at the beginning of the culture and addition of 6% sucrose at 14 days, when lateral roots have emerged, greatly improved the productivity, affording 0.8 g/l of saikosaponin-a and -d. Received: 28 October 1999 / Revision received: 19 April 2000 / Accepted: 27 April 2000     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

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.     

Stimulation of in vitro root and shoot growth of potato by increasing sucrose concentration in the presence of fluridone,an inhibitor of abscisic acid synthesis

Fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, strongly stimulated rooting of nodal stem segments of potato (Solanum tuberosum L.) cultivar Arran Banner cultured in darkness on tuberisation medium. Inclusion of 10^-6 M ABA in the culture medium prevented this rooting response, indicating that root proliferation in the presence of fluridone could be due to inhibition of ABA synthesis. The rooting response to fluridone (increased total root number and root fresh weight) was obtained only at high sucrose concentrations (0.175 and 0.234 M) and was demonstrated with two potato cultivars and two culture media; one which favoured tuberisation and one which did not. Shoot numbers were also increased, but to a lesser extent than root numbers, and total fresh weight of plant material per culture was greatly increased by inclusion of both fluridone (10^-6 or 10^-5 M) and 0.234 M sucrose in the culture medium. The role of sucrose was not simply osmotic because when the osmolarity of fluridone medium was increased using mixtures of mannitol and sucrose, no root proliferation occurred unless sucrose predominated in the mixture.     

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.     

Propagation,growth, and carbohydrates of <Emphasis Type="Italic">Dendrobium</Emphasis> Second Love (Orchidaceae) <Emphasis Type="Italic">in vitro</Emphasis> as affected by sucrose,light, and dark

In general, plant material grown in vitro has low photosynthetic ability to achieve positive carbon balances. Therefore, a continuous supply of carbohydrates from the culture medium is required, and sucrose has been the most commonly used carbon source. In this paper, we investigate the effects of different sucrose concentrations and the presence and absence of light on the endogenous levels of soluble carbohydrates and starch as well as on the proliferation and growth of Dendrobium Second Love (Orchidaceae) in vitro. The possibility of using etiolated stem segments as a means for micropropagating this hybrid was also verified. The results obtained indicated that the presence and absence of light and the sucrose concentrations used influenced the amounts of soluble carbohydrates and starch and the proliferation of D. Second Love shoots and roots. An increase in sucrose concentration caused a progressive increase in the amounts of total carbohydrates and starch. Under both light conditions, sucrose was the main sugar found in the shoots followed by glucose and fructose. The addition of sucrose to the culture medium up to 2% and 4% was advantageous to the number of shoots produced per explant and the root longitudinal growth in the presence and absence of light, respectively. Shoot and root dry matter and the number of roots formed per explant increased as sucrose concentration was raised up to 6% in both light treatments. The use of dark-grown shoot segments proved to be a useful and reliable alternative for the micropropagation of this hybrid.     

蔗糖浓度对三裂叶野葛毛状根生长及其次生物质产生的影响

本文研究了蔗糖浓度对发根农杆菌ATCC15834诱导产生的三裂叶野葛毛状根生长及其葛根素和异黄酮类化合物产生的影响以及液体培养基中蔗糖的消耗变化。结果表明：毛状根在含5％、4％、3％和2％蔗糖的MS培养基中培养16天后的干重增殖倍数分别为11．7、11．9、10．1和5．9;其中尤以3％的蔗糖浓度最有利于毛状根中异黄酮类化合物及葛根素的积累;培养12天后,毛状根的葛根素含量达到最高,约5．147mg／gDW;而其异黄酮类化合物的含量则在培养16天后达到最高,约27．76mg／gDW。在毛状根液体培养过程中培养基的蔗糖浓度随着毛状根的生长而降低,其消耗速率与毛状根的生长速度及其可溶性总糖含量成正比。毛状根的可溶性总糖含量在培养12天时达到最高,而培养16天后培养基中的蔗糖消耗完毕。     

Alteration of biomass and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots by media sterilization method and sugars

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.     

An analysis of seed development in Pisum sativum. VII. Embryo development and precocious germination in vitro

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.     

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)     

Production of ginkgolides and bilobalide from optimized the<Emphasis Type="Italic">Ginkgo biloba</Emphasis> cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1–8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.     

Induction,characterization, and NMR-based metabolic profiling of adventitious root cultures from leaf explants of <Emphasis Type="Italic">Gynura procumbens</Emphasis>

Gynura procumbens is a medicinal plant used in South East Asia to treat various ailments such as rash, hemorrhoids, inflammation, and diabetes. In order to develop a large-scale culture system for G. procumbens biomass containing bioactive compounds, adventitious root cultures were initiated from leaf explants. Murashige and Skoog (MS) media containing different compositions of indole-3-butyric acid (IBA), 1-naphthalene-acetic acid (NAA), and combinations of both plant growth regulators (PGRs) were evaluated for root induction. A combination of 3 mg/l NAA + 1 mg/l IBA gave the highest root induction (48%) as compared to other PGRs treatments after 9 weeks of incubation period. Subsequently, the adventitious roots were established in liquid culture containing MS medium and the combination of 3 mg/l NAA + 1 mg/l IBA. A study on the medium strength, sucrose concentration, pH, and light versus dark was conducted to optimize the in vitro culture conditions. The results showed that differences in MS medium strength from half to double strength, and light or dark condition did not significantly affect the biomass production, while the initial medium pH of 5.5 and 2% w/v sucrose concentration were most suitable for the root culture growth. Nuclear magnetic resonance (NMR) spectroscopy was performed to characterize the metabolite content in the root cultures of G. procumbens. Among the elucidated metabolites were some phenylpropanoids identified as caffeic acid, chlorogenic acid, and 3,5-di-O-caffeoylquinic acid which might be the bioactive compounds associated to the folk use of this plant.     

Production of scopolamine by normal root culture of Hyoscyamus niger

Summary Normal root of Hyoscyamus niger was cultivated in various nutrient conditions to optimize root growth and scopolamine production. Nitrate or phosphate concentration had little effect on root growth, while the low levels of nitrate or phosphate enhanced specific scopolamine content by 44% and 39%, respectively. Glucose or fructose was not adequate for the root culture as a carbon source, while the root growth was markedly enhanced at high sucrose concentration (up to 9%). The growth rate (0.59g dw./L.d.) and scopolamine productivity (1.98 mg/L.d.) was increased greater in roots with 9% initial sucrose concentration as compared to 3% sucrose (0.28 g dw./L.d., 0.98 mg/L.d.)     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Hinokitiol Production in a Suspension Culture of Calocedrus formosana Florin

A suspension culture of Calocedrus formosana Florin was studied as a material for efficient production of hinokitiol. Murashige-Skoog’s medium containing 3% sucrose and 1 mg/l 1-naphthylacetic acid was most desirable for cell growth. Cell growth, expressed as fresh cell weight, showed a 20-fold increase after 4 weeks of culture in this medium. Adding potassium acetate or chitosan to the medium increased hinokitiol production. The highest hinokitiol yield, 1700 μg/g fresh cells, was obtained when cells were cultured in the growth medium with chitosan.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.