Enhancement of paclitaxel production by abscisic acid in cell suspension cultures of Taxus chinensis

Addition of 5 mg abscisic acid l^–1 after 12 days' growth of Taxus chinensis suspension culture gave the greatest paclitaxel accumulation at 11 mg l^–1, which was almost 5 times that of the control culture. The highest paclitaxel production, 18 mg l^–1, was obtained using 5 mg abscisic acid l^–1 and 20 mg methyl jasmonate l^–1.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

Initiation and growth characterization of some hop cell suspension cultures

Cell suspension cultures of some hop (Humulus lupulus L.) cultivars were initiated from corresponding callus cultures induced on different media. Dissimilation curves were determined to characterize the growth of the suspension cultures maintained in Gamborg's B5 and in Murashige and Skoog's medium. Both media proved to be suitable; a comparison of the curves obtained for suspension cultures of the hop cultivar Wye Northdown grown in both media did not reveal striking differences. For four hop cell lines, the concentration of nitrate and sugar in the culture medium was analysed by HPLC during the growth of their suspension cultures. The cell suspension cultures of the various hop cultivars were also screened for the presence of bitter acids by HPLC and of volatile compounds by capillary GC. However, neither bitter acids nor volatile compounds could be detected; the addition of a non-toxic lipophylic phase (XAD-2, XAD-1180 or Miglyol 812) to the culture media did not help to induce the formation of volatile compounds.This paper will also appear as a chapter of the Ph.D. thesis of the first author; this thesis will be distributed among colleagues only.     

Accumulation of chlorogenic acid in cell suspension cultures of Eucommia ulmoides

Suspension cultures of Eucommia ulmoides were developed and shown to accumulate chlorogenic acid. MS medium plus 2.0 mg l^–1 2,4-dichlorophenoxy acetic acid was used for the cell suspension cultures of Eucommia ulmoides. The chlorogenic acid content of suspension cells was analyzed by capillary electrophoresis, and the mean content was 2.15%, approximate to that of Eucommia ulmoides leaves.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Photosynthetic properties of two different soybean suspension cultures

The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration.     

Regeneration of plantlets from embryogenic suspension cultures of pickling cucumber (Cucumis Sativus L. CV. Endeavor)

Summary Procedures have been developed for the initiation and long-term maintenance of embryogenic suspension cultures of pickling cucumber (Cucumis sativus) cultivar Endeavor and for the regeneration of normal plantlets. Embryogenic calluses from petiole explants plated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), both at 5μM, were used to initiate the embryogenic suspension cultures. Among various growth regulator combinations evaluated for initiation and maintenance of these suspension cultures, only MS medium with 2,4-D and BA, both at 1μM, produced cultures that were yellow, friable, and still regenerable after repeated subculture (every two wk) over a 3- to 15-mo. period. The effects of various concentrations of auxin and cytokinin in the plating medium, the addition of AgNO₃, and various plating procedures were also evaluated. The highest frequency of regeneration of shoots and plantlets was achieved by plating aggregates onto filter paper overlaid on MS medium with naphthalene acetic acid (NAA)/BA at a concentration of 2:1 or 1:1μM. The addition of activated charcoal (0.5%) or AgNO₃ (30μM) in the plating medium did not enhance the frequency of plantlet regeneration. The highest frequency of normal-appearing plantlets recovered was 42 to 46% per petri dish. The procedures described in this study can be used to increase plantlet recovery from individual embryogenic calluses of pickling cucumber.     

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

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.     

Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens

From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90000±5000 dalton, and consisted of two subunits, each of 42000±1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5–6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono-and o-dihydroxysubstrates were determined.Abbreviations DEAE diethylaminoethyl - HPLC high-performance liquid chromatography - L-DOPA L-3,4-dihydroxyphenylalanine     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Establishment and characterization of American elm cell suspension cultures

Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A₇₀₀ and media conductivity. Combined cell growth data for all experiments within a genotype showed relatively low variances and between-genotype contrasts during repeated passages showed no significant differences. Subculturing exponentially growing cells at 8–14 day intervals, within readily measured parameters of media conductivity (4.95–4.2 mmhos) and cell concentration (≥ 1.4 A₇₀₀), consistently resulted in repeatable profiles of elm cell growth and minimized lag phase. Culture cells were essentially homogeneous after 5 subculture passages and their overall appearance was stable. We conclude that the described procedure resulted in consistent cultures suitable for elicitor treatment experiments. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l⁻¹, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l⁻¹ of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.     

Codominant autosomal inheritance of polymorphic red cell acid phosphatases of lemurs and some properties of the enzymes

Red cell acid phosphatase phenotypes of 207 captive animals of the genera Lemur, Hapalemur, and Propithecus were determined by starch gel electrophoresis and phosphatase-specific staining. In Lemur fulvus, three phenotypes, designated A, B, and AB, were observed. In each of the species L. catta, L. macaco, L. mongoz, and L. variegatus, a single phenotype was observed. In Hapalemur griseus, three phenotypes were found: A, B, and AB. In Propithecus verreauxi, a single phenotype was found. Examination of breeding records in conjunction with the results of the electrophoretic analyses supports the conclusion that the erythrocytic acid phosphatases in this group of nonhuman primates are the products of at least two codominant autosomal alleles. There is a wide range of specific activities of the acid phosphatases as determined by colorimetric assays. The values range from 60.6 moles of p-nitrophenol released per gram of hemoglobin per 30 min in Lemur catta to 429.1 moles in Propithecus verreauxi. The enzymes of L. fulvus and P. verreauxi were purified approximately 400-fold, and Michaelis-Menten constants were determined on the purified preparations. For L. fulvus phenotype A, K _m =0.8 mM; for L. fulvus phenotype B, K _m =0.8 mM; and for P. verreauxi, K _m =0.6 mM; the substrate in each case was p-nitrophenylphosphate.The work reported here was taken in part from a doctoral dissertation submitted by G.A.M. to the Graduate School of Duke University in partial fulfillment of the requirements for the Doctor of Philosophy degree. This work was supported by grants from the National Science Foundation (Nos. GS 39635X and BNS-74-02504), the USPHS (fellowship to G.A.M., No. GM-02007), and the Wenner-Gren Foundation (No. 2697).     

Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago Truncatula cv. Jemalong

Summary Plants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.