Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid × kinetin combinations in callus cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue ofCereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22°C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32°C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22°C and was indicated as a marker of stress conditions that affect the growth ofC. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies ofC. peruvianus callus cultures.This research was supported by the CNPq.     

Isozymes in nutrient medium of suspension-cultured apple cells (Malus sylvestris Mill.)

The time course of the activities of esterase, -galactosidase, and -glucosidase in cell sap and nutrient medium in in vitro cultured apple cells (Malus sylvestris Mill.) was studied. The corresponding isozyme patterns and the intracellular and extracellular isozyme patterns of acid phosphatase and polyphenol oxidase were compared using isozyme visualization methods adapted to ultra-thin-layer isoelectric focusing. Neither quantitative (total activity) nor qualitative (isozyme pattern) data were congruent for cell saps and nutrient media. Malate dehydrogenase, malic enzyme, and glutamate dehydrogenase occurred in cell sap only. The extracellular activities probably originate to a great part from a programmed release by intact cells. Nutrient media of plant cell cultures constitute a rich source of active plant isozymes.     

Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.)

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.     

DNA RNA and protein content of tissue during growth and embryogenesis in wild-carrot suspension cultures

Summary A highly selected population of cells (clumps from 63 to 125 μm in diameter), obtained by screening 14-day-old stock suspension cultures of wild carrot (Daucus carota L.), was used to initiate cultures in this study. Time-course changes in DNA, RNA and protein were followed when these cultures were grown in the presence or absence of 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The data show that growth of these cultures, particularly in the early part of the growth curve, is different from that in most other studies reported on suspension cultures initiated without screening. The gross compositional analysis shows that this difference stems from the very high RNA:DNA and protein:DNA ratios of the cellular material used as the inoculum in this study. The presence of 2,4-D in the medium promoted total RNA and protein levels. Correlations were sought between the appearance of embryos in the absence of exogenous 2,4-D and gross compositional differences developing in cultures grown in the presence and absence of 2,4-D. The handling of cultures during inoculation appeared to have led to a substantial loss of DNA. This had, however, little effect on dry weight or protein content of the tissue. This research was supported by the W. Alton Jones Foundation.     

Ethanol production and toxicity in suspension-cultured carrot cells and embryos

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH alcohol dehydrogenase - 6-BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - FW fresh weight     

Isoperoxidase Activity and Induction in Cultured Tissues of Wild Carrot: a Comparison of Proembryos and Embryos

Several anodic isoperoxidases were found in embryonic tissues of cultured wild carrot, Daucus carota L., which were not present in the proembryo masses from which they originate. This difference is further reflected in the higher specific activity of peroxidase in embryo extracts as compared to proembryonic tissues. The absence of anodic isoperoxidases and depressed peroxidase activity in carrot tissue cultures in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) suggests a regulatory role for this plant growth regulator in controlling peroxidase activity.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

A comparison of 2,4-dichlorophenoxyacetic Acid metabolism in cultured soybean cells and in embryogenic carrot cells

The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D-free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4-D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D-amino acid conjugates.     

Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.This research was supported by the CNPq     

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Somatic hybridization between selected Lycopersicon and Solanum species

Summary Mesophyll protoplasts of an interspecific Lycopersicon esculentum Mill, (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 10⁵/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium.Abbreviations ABA abscisic acid - BAP 6 benzylaminopurine - 2,4-D 2,4 dichlorophenoxy acetic acid - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - GOT glutamate oxaloacetate - IAA indoleacetic acid - IBA indolebutyric acid - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - MES morpholinoethane-sulfonic acid - PEG polyethylene glycol - 6-PGDH 6 phosphogluconate dehydrogenase - PGI phosphoglucoisomerase     

Embryogenesis and ribosomal DNA in carrot cell suspensions cultured in vitro

The contents of nucleic acids and rDNA were estimated during the development of carrot cell suspensions cultured under two different conditions. The cells transferred from stock culture to the medium without 2,4-dichlorophenoxyacetic acid (2,4-D) induced the embryogenesis (embryogenic culture), while the cells inoculated to the medium with 0.2 mg/l 2,4-D did not form any embryos (non-embryogenic culture). The ratio of RNA to DNA of both cultures increased in the early stage of the culture. The rise of the ratio in embryogenic culture was much higher than that in non-embryogenic culture, which showed that embryogenic culture accumulated RNA prior to the formation of embryos. The rDNA amount of non-embryogenic culture remained constant throughout the culture period. Although embryogenic culture showed a slight change in rDNA amount, the differences were at most 12% and the quantitative stability of the rDNA was demonstrated during the development of carrot cell suspension cultures.     

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.)

Summary The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 M 2,4-dichlorophenoxyacetic acid for 6–8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 M. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.Abbreviations Acp acid phosphatase - BAP 6-benzylaminopurine - cv cultivar - df degree of freedom - 2,4-D 2,4-dichlorophenoxyacetic acid - Est esterase - Got glutamate oxaloacetate transaminase - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - Prx peroxidase - Tris tris(hydroxymethyl)aminomethane     

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - SEM scanning electron microscopy     

Developmental genetics of teleosts: a biochemical analysis of lake chubsucker ontogeny

Developing embryos of the lake chubsucker, Erimyzon sucetta, were analyzed with regard to both gross morphological changes and specific enzymatic changes from the unfertilized egg stage until some 3 weeks posthatching. Total activities of three enzymes—lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase—were determined throughout the course of development. Each of these different enzymes exhibited a different pattern of change during ontogeny. Electrophoretic analysis of qualitative changes in isozyme patterns was accomplished for these three enzymes and for α-amylase, glucosephosphate isomerase, mannosephosphate isomerase, creatine kinase, esterase, glutamate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, malate dehydrogenase, hexose diphosphatase, phosphoglucomutase, and phosphogluconate dehydrogenase. Many of the enzyme systems investigated exhibited rich patterns of ontogenetic change, while a few remained relatively unchanged throughout the interval studied. Several of the enzymes in particular metabolic pathways exhibited coincident changes suggestive of coordinate control. The appearance of several rather “tissue-specific” isozymes was closely correlated with the morphological and functional differentiation of these particular tissues or organs.