Production of ferruginol by cell suspension cultures of Salvia miltiorrhiza

The time-course production of ferruginol, a diterpene, was examined during growth of Salvia miltiorrhiza in cell culture. Ferruginol was produced in the lag and stationary phases of growth and so production of this secondary metabolite was inversely related to active cell division. The effects of auxins and light on ferruginol production were also examined.     

Polysaccharide from cell walls of Chlamydomonas reinhardtii

The cell wall fraction of the green alga Chlamydomonas reinhardtii contained about 25% carbohydrate after prolonged treatment with salivary amylase     

Properties of hydroxycinnamate: CoA ligase from stems of Salix babylonica cultured in vitro

Hydroxycinnamate: CoA ligase was extracted from stems of in vitro willow cultures and characterized. One peak of activity was obtained after column chromatography on Sephadex G 100 or DEAE Sephacel. p-Coumaric acid gave the highest V_max among the cinnamates examined. The K_mvalues for p-coumaric, caffeic and ferulic acid were 31.0, 4.7 and 46 μM, respectively. The MW of the CoA ligase was 57 000 and the pH optimum was 7.0. The characteristics of the enzyme correspond to its physiological role in lignin biosynthesis.     

Effects of precursors on serially propagated Digitalis lanata leaf and root cultures

Serially propagated Digitalis lanata leaf and root cultures established from germinated seeds were studied for digoxin production. Leaf cultures were grown and maintained in a medium containing benzyl adenine, and root cultures in the same medium with indoleacetic acid. A consistently high digoxin content, as determined by radio-immunoassay, is present in the leaf culture (9.0 mg % dry wt) and in root culture (1.9 mg % dry wt) as compared to unorganized cells (0.06 mg % dry wt). Leaf liquid cultures grew very rapidly as compared to the root cultures and the unorganized cell suspension cultures. The concentration of digoxin increased in both leaf and root cultures by adding to the medium either sodium glycocholate, cholesteryl acetate, or progesterone. Smilageninacetate increased the digoxin content of root cultures but not that of leaf cultures. Lanosterol and 5β-androstan-3,17-dione did not significantly increase the concentration of digoxin. Deoxycholic acid was toxic to the tissues studied.     

Invertase in cultured Daucus carota cells

Invertase activity of cultured carrot cells was distributed between cell wall and supernatant fractions of the cell homogenate. The enzyme associated with the cell wall fraction was solubilized by alkaline NaCl solution and the proportions found in the cell wall and soluble fractions depended on the concentration of NaCl. Formation of protoplasts by the action of cellulase and pectinase was accompanied by release of 50–60% of the invertase activity from the cells.     

Effect of exogenous gibberellic acid,abscisic acid,and benzylaminopurine on epicotyl dormancy of cultured herbaceous peony embryos

Epicotyl dormancy was broken in cultured peony (Paeonia lactiflora Pall.) embryos after topical application of agarose gels containing gibberellic acid, with optimum growth at 1.5 mM gibberellic acid. Addition of 100 M abscisic acid to the medium resulted in complete inhibition of gibberellic acid-stimulated promotion of dormant epicotyls. Epicotyl dormancy was also broken in embryos by culture on media containing 1 or 10 M benzylaminopurine. A highly significant increase in leaf number occurred when embryos were both cultured on medium containing benzylaminopurine and treated topically with gibberellic acid. Anatomical and morphological studies indicated that the increase in shoot growth was due to the development and growth of 1) buds formed at the cotyledonary node, 2) axillary buds, and 3) adventitious meristems originating from subepidermal parenchymatous tissue.Abbreviations ABA abscisic acid - BA N⁶-benzylaminopurine - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - LS Linsmaier and Skoog     

Structure of hemicellulosic polysaccharides of Avena sativa coleoptile cell walls

The glycosidic linkage compositions of intact and, in some cases, enzyme-degraded polysaccharides extracted from the cell walls of oat coleoptiles and subsequently purified have been examined. A major component is shown to be a glucuronoarabinoxylan similar in structure to those described for a variety of other monocots. The noncellulosic glucan component is a β-linked polymer containing both 1,4- and 1,3-linked glucosyl residues in a ratio of 2 to 1. Analysis of the oligosaccharide produced by ‘lichenase’ digestion of this β-glucan suggests that the the 1,3- and 1,4-glucosyl linkages repeat in regular fashion. A small amount of xyloglucan polysaccharides like those described for cell walls of dicots was also detected.     

Alkaloid production by plants regenerated from cultured cells of Datura innoxia

Cellular aggregates in Datura innoxia suspension cultures give rise to large numbers of shoots when such aggregates are cultured in the light on an auxin-free agar medium supplemented with kinetin. These shoots form roots on a kinetin-free medium to develop into complete plants. Most of the regenerated plants are diploid, and the frequencies of ancuploid or polyploid plants are much lower than might be expected from the distribution of chromosome number in the cultured cells. During root ditterentiation and plant development, scopolamine synthesis is initiated and there is a progressive increase in the alkaloid content. Consequently, the general pattern of alkaloid composition is restored to a normal state in the majority of the regenerated plants including aneuploid or polyploids. Nevertheless, some of the plants show an abnormal expression in alkaloid metabolism, such as the complete hydrolysis of scopolamine in the dried leaves.     

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and kinetin (K) were added to the ASP 12‐NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4‐D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole‐3‐acetic acid (1.0–10.0 μmol), 2,4‐D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.     

Hormonal effects on diosgenin biosynthesis and growth in Dioscorea deltoidea tissue cultures

Analysis of Dioscorea deltoidea tissue cultures grown in the presence of 2,4-D, indole-3-butyric acid, isopentenyladenine, benzyladenine and GA singly and in combination showed that the medium with 2,4-D most consistently favored diosgenin production. GA and high benzyladenine concentrations were toxic.     

动物细胞培养技术在人造肉研究中的应用

人造肉作为2018年全球十大突破和新兴科技之一,因其来源可追溯、食品安全性和绿色可持续等优势得到广泛的关注。欧美等国家已经投入大量资源开展细胞培养人造肉研究,未来将对我国的肉制品及食品市场造成一定的冲击。现阶段,细胞培养人造肉生产的挑战在于如何高效模拟动物肌肉组织生长环境,并在生物反应器中实现大规模的生产。尽管动物细胞组织培养技术已经得到深入的研究,并取得了不同程度的成功应用,但由于现有动物细胞组织培养成本与技术要求较高,仍不能实现大规模的产业化培养。因此,对于人造肉的生产来说,开发高效、安全的大规模细胞培养技术是亟需解决的问题,可以有效降低生产成本,实现产业化应用。文中通过介绍基于人造肉生物制造的动物细胞组织培养技术研究现状,具体阐述了目前的挑战和关键技术问题,并初步探讨了其可能的解决策略和应用前景。     

Influence of plant growth regulators, polyamines and glycerol interaction on growth and morphogenesis of carposporelings of Grateloupia cultured in vitro

The influence of the plant growth regulators 2,4-D, GA₃, BA and kinetin, and the polyamines putrescine, spermidine and spermine were tested on axenic in vitro cultures of carposporelings of Grateloupia doryphora. The auxin 2,4-D (10^-3 M) and the polyamine spermine (10^-6 M and 10^-3 M) induced a callus (disorganised cell mass that arose from the organised tissue of the carposporeling, as demonstrated by microscopic monitoring of the tissue). Putrescine and spermidine (10^-3 M) transformed the carposporelings into cell masses that produced shoots. BA (10^-3 M) and kinetin (10^-6 M and 10^-3 M) were inhibitory. In 10^-1 M glycerol-containing culture medium, which is known to induce the formation of morphogenic cell masses, the addition of GA₃ M) resulted in the inhibition of the morphogenesis (i.e. shoot emission) in the cell mass. The kinetin at 10^-6 M inhibited morphogenesis, whilst at 10^-3 M inhibited even the formation of the cell masses. The combination of glycerol (10^-1 M) and the auxin 2,4-D (10^-6 and 10^-3 M) or the polyamines putrescine, spermidine and spermine (10^-6 and 10^-3 M) resulted in a bigger size of the cell masses that led to a higher amount of shoots per cell mass than in glycerol alone. This revised version was published online in June 2006 with corrections to the Cover Date.     

6 Lignin-protein complex in cell walls of Pinus elliottii: Amino acid constituents

Cell walls of Pinus elliottii callus contain ca 12 % protein. Klason lignin prepared from the walls contained 9 % protein and represented 4.5 % of the wall. The lignin fraction was increased to 22 % of the wall weight by reacting washed cell-wall tissue with coniferyl alcohol and H₂O₂, a reaction catalysed by peroxidase that remained bound to the wall. The augmented lignin preparation yielded 10 % protein. The acid hydrolysate of whole wall tissue included five amino acids at a concentration higher than hydroxyproline. The hydrolysates of both natural and augmented lignin preparations yielded distributions of amino acids in which the concentration of hydroxyproline was higher than that of all other amino acids. The results suggest that polymerizing lignin links covalently with cell-wall glycoprotein, and that the bonds may be formed preferentially with hydroxyproline.     

Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro

We succeeded in cultivating onion plants in vitro with a high potential for shoot regeneration. The apex must be destroyed or injured to obtain axillary buds. This capacity was restricted to the abaxial base of the youngest sheaths. It was shown necessary to restore plant individuality before further proliferation; this process constituted one cycle. For successive regeneration each cycle was composed of three steps: shoot proliferation in the presence of a cytokinin, shoot individualization and plant development in the absence of growth regulators. Effect of growth regulators on the physiological status of onion plants cultured in vitro is discussed.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid     

The charophycean green algae provide insights into the early origins of plant cell walls

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Characterization of a lignin-specific O-Methyltransferase in aspen wood

O-Methyltransferases were extracted from the differentiating xylem of 10-yr-old Populus euramericana. The enzymes were partially purified by ammonium sulfate precipitation, and column chromatography on DEAE-cellulose, Sephadex G200 and hydroxyapatite. The enzymes were resolved into two peaks by DEAE-cellulose chromatography, and the MWs of the respective enzymes were estimated to be 72 000 and 75 000 by gel filtration chromatography. The enzyme corresponding to the latter peak was unstable and thus only the former peak enzyme was characterized completely. Magnesium ions had no effect, EDTA moderately stimulated and heavy metals and SH group inhibitors strongly inhibited enzyme activity. K_mm values for caffeate and 5-hydroxyferulate were estimated to be 3.8 x 10⁻⁴ and 3.1 x 10⁻⁴ M, respectively. The ratio of V_max/K_m for 5-hydroxyferulate was 5.4 times greater than that for caffeate. The enzyme(s) catalysing the formation of ferulate from caffeate and of sinapate from 5-hydroxyferulate were not separated during the purification or by the disc electrophoresis using polyacrylamide gel. Quercetin, cyanin and catechin were not methylated by the enzyme preparation. The O-methyltransferase of aspen wood, where the phenolic metabolism is almost exclusively directed to lignin biosynthesis, catalyses the methylation of both guaiacyl and syringyl lignin precursors, with preferential utilization of the latter substrate. These findings lead to the conclusion that the enzyme is a typical angiosperm-type O-methyltransferase related to guaiacyl and syringyl lignin biosynthesis in aspen wood.     

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.     

Morphinane alkaloids in cultured tissues and redifferentiated organs of Papaver somniferum

The capacity of alkaloid synthesis was examined in cultured tissues of Papaver somniferum. Callus, derived meristemoids, redifferentiated roots and