Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture

We investigated the nutrient requirements of rice in liquidculture and developed a revised medium of mineral salts, sucrose,thiamine and 2,4-dichlorophenoxyacetic acid. The following majornutrients at the indicated concentrations were beneficial: NO₃-N(40 mM), NH₄-N (5.0 mM), P (2.0 mM) and K (40 mM). Cobalt, iodine,pyridoxine, nicotinic acid and m-inositol were not essentialand were excluded from the revised R-2 medium. Growth was betterwith ammonium and nitrate together than with nitrate as thesole nitrogen source. Cell growth in the R-2 medium was superiorto that obtained in B5, Heller, Murashige-Skoog and White media. (Received March 31, 1973; )     

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.     

Importance of myo-inositol,calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l^-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.Abbreviations BA benzylaminopurine - CaCl₂ calcium chloride, 2,4,-D-2,4-dichlorophenoxyacetic acid - EDTA ethylene diamine tetraacetic acid - KCl potassium chloride - MES-2-N morpholino ethane sulfonic acid - MgCl₂ magnesium chloride - NH₄NO₃ ammonium nitrate - NAA naphthalene acetic acid, p-protoplasts     

Uptake of phosphate and its effect on phenylalanine ammonia-lyase activity and cinnamoyl putrescine accumulation in cell suspension cultures of Nicotiana tabacum

The influence of phosphate on the medium-induced formation of cinnamoyl putrescines in cell cultures of Nicotiana tabcum was investigated. Phosphate added to a phosphate-free production medium was completely accumulated in the cells within 24h after inoculation at initial concentrations up to 2 mM. At higher concentrations phosphate was partly accumulated with an intracellular saturation at approx. 0.65 mmol/g dr. wt. equivalent to approx. 45 mM intracellular concentration. Enhanced activities of phenylalanine ammonialyase and increased product levels of cinnamoyl putrescines, induced by cell transfer into phosphatefree medium were suppressed similarly at initial phosphate concentrations of 0.02–0.5 mM. At the same time growth was stimulated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - fr. wt. fresh weight - dr. wt. dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Effects of auxins and cytokinins on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis

Cell suspension cultures of Anchusa officinalis required exogenous phytohormones for their normal growth. Cell lysis was observed at the third passage in a hormone-free medium. Using hormone — depleted cells, the effects of auxins (2,4-D, NAA, IAA and CFP) and cytokinins (BA, kinetin, and zeatin) on cell growth and RA production were investigated. All auxins tested could maintain growth and integrity of the cells whereas cytokinins alone could not, suggesting that this culture is auxindependent. Among the auxins tested, NAA had a pronounced effect on RA production. The total RA content obtained at optimum NAA concentration (0.25 mg/l) reached 1.7 g/l (12% of dry weight). The kinetics of growth and RA production suggested that the increase in final RA content was due to both an increase in the rate of RA synthesis and initiation of the period of synthesis in the exponential rather than the linear growth phase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - CFP 2-chloro-4-fluorophenoxyacetic acid - BA 6-benzyladenine - RA rosmarinic acid     

Decolorization of a kraft mill effluent with fungal mycelium immobilized in calcium alginate gel

A white-rot fungus Coriolus versicolor was immobilized by entrapment in calcium alginate beads. Treatment of a kraft mill effluent with the immobilized fungus in the presence of sucrose resulted in 80% loss of color of the effluent within 3 days. The minimal concentration of sucrose required for the decolorization was 10 mM. Other carbon sources (xylose, glucose, glycerol, and ethanol) could also be used.     

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L^–1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L^–1 NAA and 1 mg·L^–1 K (N₂K₁MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d^–1 and 0.08 d^–1 were obtained in MS medium supplemented with 1 mg·L^–1 NAA, 0.1 mg·L^–1 K and 30 g·L^–1 sucrose (N₁K_0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L^–1 sucrose (N₁K_0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N₁K_0.1MS and N₁K_0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L^–1 and 7.9 g·L^–1, and yields on carbohydrate were 0.39 and 0.45 in N₁K_0.1MS and N₁K_0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.Abbreviations B5 Gamborg mineral salt medium - d.w. Dry weight - K Kinetin - MS Murashige and Skoog mineral salt medium - N or NAA Naphthaleneacetic acid - N_iK_jMS i and j are the respective concentrations (mg·L^–1) of NAA and K - n Number of experimental points - r Linear regression correlation coefficient     

Release and crystallization of berberine in the liquid medium of Thalictrum minus cell suspension cultures

Cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. were found to produce a large amount of berberine (400–800 mg/l) when 5–10 M 6-benzyladenine was added to Linsmaier and Skoog's medium containing 100 M 1-naphthaleneacetic acid. Most of the berberine produced was released continuously from the cells into the liquid medium, and an excess of berberine crystallized as its nitrate in the medium. When the cells were cultured in a modified LS medium containing 20 mM KNO₃ and 40 mM NH₄Cl in place of 20.6 mM NH₄NO₃ as nitrogen source, most of the alkaloid crystallized to form berberine chloride instead of nitrate. Minor alkaloids, thalifendine and magnoflorine, were also isolated from the medium and identified.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine     

Factors influencing induction,propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Sugars and Amino Acids on Androgenesis of Cucumis sativus

The effects of sugars (sucrose, maltose, glucose and fructose) and amino acids (glutamine, glycine, arginine, asparagine and cysteine) on embryogenesis and plantlet regeneration from cultured anthers of Cucumis sativus L. cv. Calypso and Green Long were studied. Type and concentration of sugar and amino acid influenced embryogenesis. Among the different sugars tested, sucrose was the best for embryo induction with an optimal concentration of 0.25 M. Maximum of 72 and 80 embryos per 60 anthers of Calypso and Green Long, respectively, were induced on embryo induction medium [B5 (Gamborg, Miller and Ojima (1968) Exp. Cell Res. 50: 151–158) supplemented with 2.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 μM 6-benzyladenine (BA)] containing 0.25 M sucrose. The addition of amino acids to the embryo induction medium improved embryo yield with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) giving the best response. Embryo differentiation was achieved on B5 medium supplemented with 0.25 μM of α-naphthaleneacetic acid (NAA), 0.25 μM kinetin (KN) and 0.09 M sucrose. Embryos were converted on B5 medium supplemented with abscisic acid (ABA) (10 μM) and 0.09 M sucrose. Embryos that developed on B5 medium supplemented with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) exhibited the highest plantlet regeneration frequency.     

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.     

A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus

Protoplasts of several spring and winter varieties of Brassica napus were isolated from hypocotyl tissue. Protoplasts divided and formed cell colonies at high frequency, without browning when cultured in modified Shepards' medium. This high efficiency of proliferation was sustained through to plant regeneration with all varieties cultured. This has been attributed to the incorporation of a reservoir medium, the presence of 2,4-D in the proliferation medium, and the presence of kinetin in conjunction with lowering of the sucrose concentration in the regeneration medium.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension

In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.     

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8–4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid.The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53–39 mg/L per cycle during five recycling cycles.     

Micropropagation of Uniola paniculata L.

Uniola paniculata L. is a major sand-dune stabilizing grass which is being utilized to prevent shoreline erosion. In vitro cultured caryopses of U. paniculata produced callus on MS medium supplemented with 22.5 M 2,4-D, 4.4 M BA and 87.6 mM sucrose. Shoot induction occurred after these calli were inoculated onto the same medium without 2,4-D. Rooting of in vitro-derived shoots occurred when transferred to a one-half strength MS medium containing 43.8 mM sucrose and 14.7 M IBA. Plantlets were planted after the roots reached a length greater than 20 mm.Abbreviations BA N-(phenyl-methyl)-lH-purine-6-amine - IBA lH-indole-3-butanoic acid - MS Murashige and Skoog (1962) - 2,4-D (2,4-dichlorophenoxy) acetic acid - TC agar tissue culture agar, (K.C. Biologicals, Lenexa, KS) - Subdue methaxyl:N-(2,6-dimethylphenyl)-N-(methyoxyacetyl) alanine methyl ester (CIBA- GEIGY)     

Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459

The nutritional requirements of Xanthomonas campestris NRRL B1459 for optimal xanthan production were studied in a chemically defined medium. Of the carbon sources tested, a 4% sucrose or glucose medium yielded the highest xanthan titers. The further addition of certain organic acids, such as succinate, pyruvate, and α-ketoglutarate, stimulated xanthan production; excess concentrations of these organic acids inhibited xanthan formation. Certain amino acids (e.g., glutamate) and nitrate salts were superior to ammonium salts for xanthan production. Concentrations of these nitrogen sources higher than the optimal levels inhibited xanthan production while stimulating growth. Xanthan production was also sensitive to high concentrations of inorganic phosphate. High xanthan potencies, up to 30 g/kg of broth, were achieved in these shake-flask studies, in which completely defined media were used.     

Isolation and in vitro fusion of egg and sperm cells in Oryza sativa.

Protocols for isolation of gametes of Oryza sativa were developed and initial results on in vitro fusion of sperm and egg cells are reported. The best yield of viable sperm cells was obtained when pollen grains were cultured in a medium containing of 1.3 mM boric acid, 3.6 mM calcium chloride, 0.74 mM potassium phosphate, and 438 mM sucrose. Embryo sacs were isolated using cell wall degrading enzyme treatments for 2-5 h followed by mechanical manipulation. The maximum yield (38.2%) of egg cell was achieved when 2% cellulase and 0.55% pectinase were used in the medium. However, the optimum concentration of cellulase and pectinase was found to be 1% and 0.85%, respectively. Fluorescein diacetate (FDA) stain was used to determine the viable sperm and egg cells. The optimal procedures (fusion conditions) for gametes fusion occurred in a medium containing calcium chloride at a concentration of 7 mM (pH 7.5) and the best result obtained (55.5%) in terms of fused gametes, is reported.     

Effect of sugars,amino acids,and culture technique on maturation of somatic embryos of Pinus strobus on medium with two gellan gum concentrations

Maturation of five embryogenic lines of Pinus strobus L. was tested on media with various sugars and sources of organic nitrogen, and solidified with two gellan gum concentrations (0.6 and 1.0%). Mature somatic embryo production was more abundant at 1.0% gellan gum than at 0.6%. Complex combinations of amino acids had little effect on mature embryo production of most tested embryogenic lines. Increasing glutamine concentration of the maturation medium from 1.7 to 7.3 g l⁻¹ was beneficial to one embryogenic line. Increasing sucrose concentration or substituting part of the sucrose with mannitol or sorbitol had variable effects on somatic embryo maturation depending on the embryogenic line. A medium with 88 mM sucrose plus 175 mM sorbitol solidified with 1.0% gellan gum produced high numbers of somatic embryos in four out of five embryogenic lines tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Regeneration systems from immature embryos of Bulgarian pea genotypes

Ten genotypes from Pisum sativum and Pisum arvense were screened for their regeneration abilities. Most of them were created through experimental mutagenesis from Bulgarian varieties and have various valuable agronomic traits. Embryonic axes from immature embryos were plated on modified Murashige and Skoog medium, containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d), α-naphthaleneacetic acid (NAA) and benzyladenine (BA). Two schemes for direct and indirect organogenesis were established. Callus and shoot formation were induced on media containing 0.2 mM 2,4-d or 5 mM BA, respectively. Embryonic axes formed buds directly when plated on medium with 10 mM BA and 1 mM NAA. Organogenesis and adventitious bud formation were maintained on medium supplemented with BA and NAA. Rhizogenesis was induced on Gamborgs' B5 medium. All screened genotypes were able to regenerate plants with a high efficiency (50–100%) although some differences in their organogenetic response were observed. This revised version was published online in June 2006 with corrections to the Cover Date.