Extracellular accumulation of high specific-activity peroxidase by cell suspension cultures of cowpea

Cell suspension cultures of cowpea (Vigna sp.) were able to produce extracellular peroxidase. Different growth regulator concentrations induced different peroxidase activity in callus. The crude extracellular medium after four weeks of culture showed higher (6 times) specific peroxidase activity and higher thermo stability than commercial horse-radish peroxidase. The commercial production of peroxidase enzyme from cowpea by utilizing plant cell cultures is discussed.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with α-difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.     

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.     

丹参的冠瘿组织培养和丹参酮的产生

用根癌农杆菌感染丹参无菌苗获得冠瘿组织,除菌后的冠瘿组织在无激素的Ms培养基上生长良好。经高压纸电泳检查,冠瘿组织中含有冠痿碱,证实根癌农杆菌的Ti质粒转化成功。冠瘿组织的生长和丹参酮的积累与基本培养基有关,B5和Ms培养基有利于生长．月增殖倍数分别达到102倍和90倍,而67-V和WP培养基则有利于丹参酮的合成,在培养过程中丹参酮能分泌到培养液中。研究表明用冠瘿组织作为培养系统,生产药用植物有效成分具有良好的开发前景。     

Acid phosphatase activity of normal and tumor tissue of tobacco grown in vitro and in vivo

Tissue cultures of Nicotiana labacum consisting of green, albino and habituated (normal origin) and teratoma (tomorous origin) were grown under asceptic conditions for 6 to 8 weeks and their extracts were analyzed for phosphatase activity. Comparative enzyme analyses were also made on crude stem extracts of greenhouse-grown normal and tumor tissues of Nicotiana tabacum (var. Wisconsin) and a hybrid (N. glauca × N. langsdorffii).

All the crude extracts showed acid phosphatase activity with a pH optimum at 5.8 to 6.0. The total protein content and enzyme acivity of teratoma tissue (tumor) was higher than that of green, albino or habituated tissue (normal). Similar increased levels were seen in tumor tissue grown in greenhouse in comparison with greenhouse-grown normal tissues. The crude extracts of each of the tissues did not exhibit any qualitative difference in specificity with the 5 different substrates tested; however, differences in the level of activity was observed.

The effect of 4 different culture media was tested on the growth, protein content and acid phosphatase activity of habituated tobacco in tissue culture. Tissues growing in medium containing high salt concentrations showed higher activities than tissues grown in a basal control medium. From the results, it is suggested that although many factors like auxin and other growth factors can influence growth of habituated tobacco tissue, they need not necessarily affect this specific enzyme activity.

     

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

Summary The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.     

The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cotton cultivars (Gossypium hirsutum L.)

Summary To determine NaCl effects on callus growth and antioxidant activity, callus of a salt-tolerant and a salt-sensitive cultivar of cotton was grown on media amended with 0, 75, and 150 mM NaCl. Callus of the salt-tolerant cultivar, Acala 1517-8 8, grown at 150 mM NaCl, showed significant increases in superoxide dismutase, catalase, ascorbate peroxidase, peroxidase and glutathione reductase activities compared to callus tissue grown at 0 mM NaCl. In contrast, callus tissue of the salt-sensitive cultivar, Deltapine 50, grown at 0, 75, and 150 mM NaCl, showed no difference in the activities of these enzymes. At the 150 mM NaCl treatment, peroxidase was the only antioxidant enzyme from Deltapine 50 with an activity as high as that observed in Acala 1517-88. The NaCl-induced increase in the activity of these enzymes in Acala 1517-88 indicates that callus tissue from the more salt-tolerant cultivar has a higher capacity for scavenging and dismutating superoxide, an increased ability to decompose H₂O₂, and a more active ascorbate-glutathione cycle when grown on media amended with NaCl.     

Ethylene production in habituated and auxin-requiring tobacco callus cultures. Does ethylene play a role in the habituation?

Ethylene production of habituated and auxin-requiring tobacco ( Nicotiana tabacum L. cv. Xanthi) callus cultures were compared. More ethylene was produced by auxinrequiring i.e. auxin-heterotrophic cultures than by habituated ones. Treatment with 2,4-dichlorophenoxyacetic acid increased the ethylene evolution of habituated cultures over the range 10⁻⁷ to 10⁻⁴ M , which suggests that the higher ethylene production of auxin-dependent callus is caused by the 2,4-D in the medium. The IAA levels depended on the age of both types of callus cultures.     

Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated.Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C).The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred.The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

Effect of pyridoxal phosphate and cysteine on δ-aminolaevulinate synthetase and dehydratase in soya

The specific activity of ALA-S extracts prepared from lyophilized soya callus growing in light or dark showed a striking increase when tissue cultures were grown in the presence of pyridoxal phosphate (PyP) in order to complex aminothiols. By contrast ALA-D specific activity in the light grown callus was reduced on incorporation of PyP into the growth medium. Cysteine (Cy) added to the culture medium did not influence the specific activities of either enzyme.     

Relationships among iron deficit-induced potato callus growth inhibition,Fe distribution,chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H₂O₂) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.     

Somatic embryogenesis in Asparagus officinalis can be an in vitro selection process leading to habituated and 2,4-D dependent embryogenic lines

Long-term embryogenic lines were repeatedly obtained from nine asparagus (Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Isoenzymes of Acid Phosphatase and Non-specific Esterases in Cultures of Neoplastic and Normal Tobacco Tissues

Axenic cultures of normal, habituated and crown gall teratoma tissues were grown under varying conditions to examine the effects of environment on the expression of neoplastic character. Acid phosphatase patterns on polyacrylamide gels did not vary greatly among tissues although there were differences in acid phosphatase activity between various strains of Agrobacterium tumefaciens , the bacteria which cause crown gall. Certain esterase isoenzymes were found only in tissues grown on specific media, while others were tissue-specific but independent of the nature of the medium. Comparisons of liquid and solid grown cultures revealed that culture conditions also influence esterase expression. Both sunflower and tobacco crown gall tissue contained an esterase not found in habituated or normal tissues, and similar in electrophoretic mobility to an esterase found in extracts of the bacteria that had induced the tumors. The basic difference between the three tissue types studied is the manner in which they respond to a given environment.     

Peroxidase activity in calluses and cell suspension cultures of radishRaphanus sativus var. Cherry Bell

Calluses ofRaphanus sativus var. Cherry Bell were induced in a medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine. The biomass and peroxidase activities were determined, both in agar cultures and cell suspension cultures. Different growth-regulator concentrations induced different responses measured as peroxidase activity in callus. The suspended-cell cultures showed the importance of selecting the cell line, in order to obtain an optimal response in extracellular peroxidase activity. The commercial production of this enzyme by utilizing plant cell tissue cultures is discussed.     

Biochemical comparisons of habituated and non-habituated callus lines of Glycine max (L.) cv. Acme

A cytokinin habituated non-organogenic callus line of Glycine max L. cv. Acme, was isolated. Cytokinins were detected in both non- and habituated calli. HPLC analysis indicated that the highest biological activity was associated with compounds that cochromatographed with zeatin and derivatives thereof. Cytokinin-like activity in the habituated callus was higher than in the non-habituated callus. No difference was observed in the ability of the two callus types to metabolise trans-zeatin. The habituated callus however, metabolised N⁶(²-isopentenyl)adenine (iP)at a slower rate than the non-habituated line. The regulation of proline and putrescine levels during the growth cycle was similar in both callus types, although less proline was present in the habituated than the non-habituated callus. In both callus types a decrease in putrescine levels was accompanied by an increase in the proline levels on day 21 of the growth cycle. Neither spermine nor spermidine were detected in either the habituated or non-habituated lines. A comparison of the total RNA levels of the habituated and non-habituated material revealed higher RNA levels in the non-habituated than the habituated line. Two-dimensional gel electrophoresis indicated the presence of 3 polypeptides in the habituated callus on day 13 of the growth cycle. These were not detected in the non-habituated callus, of similar age.Abbreviations Ade Adenine - DTT 1,4-dithiothreitol - IEF isoelectrofocussing - iP N⁶(²-isopentenyl)adenine - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulphate     

Selection of salt tolerant plants of Nicotiana tabacum L. through in vitro and its biochemical characterization

Sodium chloride tolerant organogenic callus lines of Nicotiana tabacum were developed in vitro on Murashige and Skoog [16] medium supplemented with BA, IAA and different concentration of NaCl. The maximum shoot bud regeneration was achieved from both tolerant and non-tolerant calluses on MS medium supplemented with 1.0 mg/l BA, 0.1 mg/l IAA with or without NaCl within 4 weeks of culture. Standard growth parameters such as fresh weight and dry weight of organogenic callus, growth tolerant index and enzyme activity (peroxidase and catalase) were used as indicators of salt tolerance. The growth tolerance index in the 4-week after the beginning of treatments yielded significant differences among the non-tolerant and tolerant organogenic callus lines. The regenerated shoots were rooted on half-strength MS basal salts supplemented with 2% sucrose but devoid of growth regulator. The regenerated plants from tolerant callus lines were capable of growing in vitro in presence of 175 mM NaCl. SDS-PAGE profile showed that the progenies derived from tolerant sources were tolerant to salt. This investigation may help in the selection and characterization of salt tolerance in plant improvement programme.