Bioprocess engineering of <Emphasis Type="Italic">Echium italicum</Emphasis> L.: induction of shikonin and alkannin derivatives by two-liquid-phase suspension cultures

An in vitro cell suspension culture of Echium italicum was established and assayed for the production of shikonin and alkannin derivatives. Callus tissues were induced from cotyledon explants of the plant incubated onto the solidified B5 medium. A two-liquid-phase system suspension culture was then established to elicit pigments of shikonin and alkannin derivatives using liquid paraffin. The presence of liquid paraffin efficiently induced production of pigments in cultured cells. The production and/or accumulation of these compounds in the E. italicum cells was examined using fluorescence microscopy as the naphthoquinone molecules display autofluorescent properties. Phytochemical analysis of the n-hexane extract of the medium was also carried out using preparative HPLC. The chemical structure of shikonin and alkannin derivatives were characterized by UV, 1H-NMR, and 13C-NMR techniques. Based on our findings, this bioprocess engineering approach resulted in induction of shikonin and alkannin derivatives, whereupon it may be recruited for production of these important secondary metabolites.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant

Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l⁻¹ of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Influence of auxins,cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (<Emphasis Type="Italic">Morus</Emphasis><Emphasis Type="Italic">alba</Emphasis> L.)

Rutin is an economically valuable flavone compound with anticancer activity, dietary effects, and anti-aging activity. In this study, callus and adventitious roots were induced from three Morus (mulberry) species. Among the three mulberry species tested for rutin production, roots of the Sugye (M. alba L.) had the highest levels (242.2 μg/g fresh tissue) of rutin. In addition, the mature leaves of this type of tree promoted higher levels of rutin compared to those of young leaves or those undergoing senescence. Adding auxins such as indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) not only enhanced the development of callus and adventitious roots but also increased the protein and rutin contents. In contrast, adding cytokinins such as 6-benzyladenine (BA) and kinetin (KN) retarded callus and adventitious root development as well as the protein and rutin contents. Callus in suspension culture in the presence of IAA produced more rutin than that in the absence of IAA. However, rutin secretion into a medium was greater in the absence of IAA. Different ammonium/nitrate (AM/NI) ratios in a root suspension culture also greatly affected rutin production and its secretion into a liquid medium. As a result, the highest level of rutin was produced when adventitious roots were grown in a 34/66 AM/NI full-strength standard MS medium containing 5 mg/l IAA.     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Saponins in tissue culture of <Emphasis Type="Italic">Primula veris</Emphasis> L.

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.     

Naphthazarins from Arnebia hispidissima

Besides arnebin-7, alkannin acetate, alkannin isovalerate, alkannin and β-sitosterol, a new isohexenylnaphthazarin, alkannin β-hydroxyisovalerate, has been isolated from the roots of Arnebia hispidissima.     

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.     

太子参细胞悬浮培养及其皂苷含量分析

以太子参的幼叶为外植体,诱导培养获得太子参愈伤组织,并通过细胞悬浮培养获取皂苷.结果表明：用MS＋BA 0.2 mg L^-1＋2,4-D 1.0 mg L^-1＋KT 1.0 mgL^-1液体培养基可获得大量繁殖速度快、生长均匀一致的悬浮细胞.由细胞悬浮培养获得的太子参皂苷的HPLC色谱峰值与常规种植及组培苗的相同,但纯度较好.细胞悬浮培养约30 d时,每克干重细胞的培养液内可提取总皂苷量为2.13-2.92 mg,略低于大田常规种植所收获的每克干重太子参块根内的总皂苷含量（3.6-4.3 mg）,与组培苗收获的太子参块根内的总皂苷含量相近.     

Establishment of cell suspension cultures of yew (<Emphasis Type="Italic">Taxus × media</Emphasis> Rehd. and assessment of their genomic stability

Summary Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inibited growth while no effect on nuclear DNA content was noted.     

Miscanthus×giganteus plant regeneration: effect of callus types,ages and culture methods on regeneration competence

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large‐scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot‐forming calli having a yellow or white compact callus with light‐green shoot‐like structures showed the highest regeneration frequency. Percentage of shoot‐forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4‐d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic‐like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana‐Champaign, Illinois, were 78% and 56%, respectively.     

In vitro culture and fructan production by Vernonia herbacea (Asteraceae)

Vernonia herbacea is a native species from the Brazilian Cerrado that accumulates about 80 % of inulin-type fructans in the underground reserve organs, the rhizophores. This work aimed at establishing a protocol for in vitro culture of V. herbacea, using seeds (achenes) and leaf discs as explants. Following germination and seedling growth, stem nodes from 6-month-old in vitro germinated plants were isolated and incubated on culture medium free of growth regulators for plant propagation and rhizophore formation. Fructan content and composition were evaluated in leaves, stems, roots and rhizophores from plants grown in vitro and compared with those of greenhouse-grown plants, in order to evaluate inulin production in vitro. Fructan contents of aerial organs and roots from in vitro plants were higher, compared with greenhouse plants, while in rhizophores, the opposite was observed. High performance anion exchange chromatography/pulsed amperometric detection profiles revealed the presence of the inulin homologous series in the aerial organs exclusively for in vitro plants, while in roots and rhizophores, this series was detected in plants grown in both conditions. These results indicate a modification in the source/sink ratio, leading to changes in the distribution of carbohydrates in in vitro plants. The leaf disc cultures on medium supplemented with indole-3-butyric acid induced the formation of roots (0.24, 0.49 µM) and friable callus (2.46 µM), while 6-benzylaminopurine (from 1.1 through 4.43 µM) induced compact callus. However, no shoot formation was observed. The use of seeds allowed the establishment of a protocol for in vitro culture and provides a model system for a better understanding of fructan metabolism in V. herbacea.     

Cryopreservation of embryogenic cell suspensions of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don.

The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l⁻¹ Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l⁻¹ IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l⁻¹) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek^™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.     

Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

The effect of polyamines on the efficiency of multiplication and rooting of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal and content of some withanolides in obtained plants

An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5 mg L^−l], indole-3-acetic acid (IAA) [0.3 mg L^−l] and with the addition of polyamine, spermidine (20 mg L^−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L^−l) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l^?1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l^?1 each of Kin, BA and 0.1 mg l^?1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite^®, if their base was treated with 500 mg l^?1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M₅ medium having 0.5 mg l^?1 each of 2, 4-D and Kin, 100 mg l^?1 l-phenylalanine and 5 mg l^?1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.