Maize Callus Cultures Research Articles

Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45°C (optimum 40°C), pH5-7 (optimum pH6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

Breakage-fusion-bridge cycles and de novo telomere formation on broken chromosomes in maize callus cultures.

Breakpoints involved in chromosome alterations associated with heterochromatin have been detected in maize plants regenerated from callus culture. A cytogenetic analysis of plants regenerated from a maize callus was performed aiming to analyze the stability of a chromosome 7 bearing a deficiency-duplication (Df-Dp), which was interpreted as derived from a chromatid type breakage-fusion-bridge (BFB) cycle. The Df-Dp chromosome 7 was stable in mitotic and meiotic cells of the regenerated plants. Fluorescence in situ hybridization showed signals of telomeric sequences on the broken chromosome arm and provided evidence of de novo telomere formation. The stability of two types of altered chromosome 7 was investigated in C-banded metaphases from samples of the original callus that were collected during a period of 30-42 months after culture initiation. New alterations involving heterochromatic knobs of chromosomes 7 and 9 were observed. The aberrant chromosomes were stable in the subcultures, thus providing evidence of broken chromosome healing. The examination of anaphases showed the presence of bridges, which was consistent with the occurrence of BFB cycles. De novo telomere formation occurred in euchromatic and heterochromatic chromosome termini. The results point to events of chromosomal evolution that might occur in plants.

Use of maize callus cultures for assessing chilling stress resistance

The production of maize callus lines and their use for assessing chilling injury are described. The substantial influence of physiological factors (type of explant, age of plant and culture) on the stress resistance of cells is shown. It is concluded that maize callus cultures can be used as a model object when studying stress resistance at the cellular level.

Геномна мінливість у калюсних культурах кукурудзи лінії Р346 і отриманих від неї сомаклональних ліній

Геномна мінливість у калюсних культурах кукурудзи лінії Р346 і отриманих від неї сомаклональних ліній

Globulin-1 gene expression in regenerable Zea mays (maize) callus.

Since maize callus cultures regenerate plants via somatic embryogenesis, one might expect to find similar proteins in both zygotic embryos and tissue cultures. The 63-kD globulin protein designated GLB1, the expression of which is regulated by abscisic acid (ABA), is one such protein. When maize Type I regenerable callus was exposed for 24 h to 0.1 m M ABA or a water stress induced by 0.53 M mannitol, GLB1 was produced as determined by Western analysis. This protein was not detected in ABA or mannitol-treated regenerable cultured tissue of a null genotype or in tissues not exposed to ABA or water stress. Exposure to ABA in the culture medium increased the callus ABA levels greatly but a mannitol-induced water stress had only a small effect on ABA levels. Regenerable callus exposed to 0.1 m M ABA also produced mRNA that hybridized on a Northern blot with a globulin- 1 gene ( Glb1) probe. When both Type I and Type II regenerable cultured tissues were exposed to regeneration medium without ABA or mannitol, several GLB1 antibody immunoreactive proteins were produced. These proteins were not detected in regenerated plants nor in non-regenerable callus treated with ABA. These results suggest that: (1) at least for expression of Glb1, somatic embryogenesis is similar to zygotic embryogenesis, (2) there may be a regulatory role for auxin in the processing of Glb1-encoded polypeptides since fewer are seen when dicamba is present in the medium, (3) ABA has a role in somatic embryogenesis, and (4) regenerability of a maize callus culture may be assessed by treating the cultured tissue with 0.1 m M ABA to determine if GLB1 proteins are induced.

A mannose selection system for production of fertile transgenic maize plants from protoplasts.

Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T1 and T2 plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation.

Behaviour of chromosomes in anaphase cells in embryogenic callus cultures of maize (Zea mays L.)

Mitotic anaphase cells of highly friable and embryogenic calluses which had been induced from immature embryos of two inbred lines of maize that have contrasting levels of heterochromatic knobs were analysed for the presence of abnormalities 3, 6, 9 and 12 months after the initiation of culture. A total of 500 typical anaphases was scored at each time point, and various aberrations, such as delay in the separation of sister chromatides, chromosome bridges (single, double and multiple) and chromosome fragments, were revealed to occur extensively in the cultures of both genotypes. Preparations after C-banding revealed that primary breakages often occurred inside knobs or at junction regions between the euchromatin and the heterochromatin of the knobs. Figures characterized by the delayed separation of sister chromatids, which originated preferentially at the knob level and was considered to be an initial event in the development of breakages, were observed at constant frequencies throughout the experiment. Increasing numbers of aberrant cells were detected with time, mainly due to the accumulation of cells with chromosome bridges and fragments. Several mitotic figures suggested the occurrence of breakagefusion-bridge cycles that were initiated by broken chromosomes. The overall frequencies of aberrant cells were similar for both genotypes, despite the differences in knob composition. However, callus cultures induced from the genotype having the higher level of knobs had more aberrant cells with abnormalities that involved several chromosomes, such as multiple bridges and multiple fragments.

Behaviour of chromosomes in anaphase cells in embryogenic callus cultures of maize (Zea mays L.)

Behaviour of chromosomes in anaphase cells in embryogenic callus cultures of maize (Zea mays L.)

Influence of carbon source and CO 2-enrichment on biochemical parameters associated with photomixotrophia in maize callus cultures

Summary To learn about the biochemical processes underlying the induction of photomixotrophia in maize cell culture, maize calli were cultured in medium containing either glucose or starch as the carbon source. The effect of a CO 2 -enriched atmosphere on different parameters was tested. Levels of chlorophyll and CO 2 -fixing enzymes were measured to assess the greening process concomitant to histological observations of chloroplast development. Both starch and glucose promoted higher chlorophyll accumulation in callus cultured under light than sucrose. Histological analysis of green callus grown on glucose-containing medium revealed the formation of poorly developed chloroplasts containing starch grains, whereas in starch medium a large number of elongated chloroplasts containing thylakoids were observed. Exposure of these calli to a CO 2 -enriched atmosphere enhanced the plastid differentiation process up to mature chloroplasts with grana and intergranal thylakoids. Western-blot analysis demonstrated the presence of CO 2 -fixing enzymes, Rubisco (EC 4.1.1.39) and PEP carboxylase (EC 4.1.1.31), as well as Rubisco activase in greening callus. Rubisco and PEP carboxylase activities showed large values when starch was the carbon source in the medium. Results of histological analysis and a/b chlorophyll ratios indicated that the chloroplasts formed were of the C 3 -type. PEP carboxylase kinetic properties were also consistent with a C 3 -type enzyme involved in anaplerotic functions. It is concluded that under the experimental conditions tested, starch plus CO 2 -enriched atmosphere are the best carbon source for inducing and supporting photomixotrophia in maize cultures, as indicated by several biochemical parameters.

Endogenous gibberellins from callus cultures of maize

This paper reports the identification of gibberellin A 1(GA 1), GA 17, GA 19, GA 20, GA 29, and GA 53 from extracts of normal (wild-type) maize callus using full-scan GC-Mass Spectrometry and Kovats retention indices (KRIs). In addition, evidence is presented for the presence of GA 8 based on its molecular ion at the appropriate KRI. In a preliminary experiment, the presence of GA 3, GA 5, and GA 44 was suggested, based on GC-selected ion monitoring (SIM); however, the presence of these GAs was not confirmed by full-scan GC-Mass Spectrometry. The information represents the first identification of GAs from maize callus. All of the identified GAs are members of the early-13-hydroxylation pathway, a biosynthetic pathway that leads to bioactive GA 1. GA 1, GA 8, GA 19, GA 20, and GA 29 were quantified from GC-SIM isotopic dilution data. GA 17 and GA 53 were quantified from GC-SIM data by comparing the ion intensity from each gibberellin to a known amount of a GA 19 standard. The levels of the identified GAs ranged from 6 pg g −1 fr. wt to 210 pg g −1 fr. wt.

Morphotypes of friable embryogenic maize callus

Friable, embryogenic callus cultures of maize are normally maintained as a heterogeneous mixture of various morphotypes in different stages of development (Fransz and Schel 1991). With stringent selection during subculture, three Type II callus morphotypes have been enriched and maintained. Cultured tissue segments from the three morphotypes referred to as "pre-embryogenic", "early embryogenic", and "late embryogenic" were analyzed using light and scanning electron microscopy. The results suggest a developmental relationship between the three morphotypes. Regeneration studies substantiated that all morphotypes were capable of regenerating through somatic embryogenesis.

Influence of pretreatments with inhibitors of putrescine synthesis on polyamine metabolism and differentiation processes of maize calluses

The effect of short treatments with 10 mM DL-α-difluoromethylornithine (DFMO) and DL-α-difluoromethylarginine (DFMA), irreversible inhibitors of putrescine synthesis on growth, plant regeneration and endogenous polyamine content of maize ( Zea mays L.) callus cultures was investigated. The effect of exogenous putrescine and the enzymatic activities of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were also studied. Of all the assayed treatments, only DFMA significantly decreased callus growth versus control. Enzymatic activity was markedly decreased by the addition of the respective inhibitors in all the treatments. Furthermore, a decrease in ODC and ADC activities was observed when DFMA and DFMO were added respectively to the medium. Whereas a predominance of ODC activity was detected in maintenance medium, ADC activity was the prevalent in differentiation medium. Moreover, DFMO pretreated calluses led to a marked increase in the number of buds with respect to all the other treatments and maintained the capacity of the calluses to regenerate plants. By contrast, although DFMA pretreated calluses raied the number of regenerated plants versus control, the number of buds were decreased and the calluses tended to be eliminated at the end of the differentiation period. The combined addition of putrescine and inhibitors reduced the regeneration rate and the enzymatic activities of the calluses. Nevertheless, the addition of putrescine to the control medium increased the regeneration rate and the number of plants. Differences in endogenous polyamine content (especially putrescine), depending on the treatments, were also observed.

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.

Effect of DL-Alpha-Difluoromethylornithine Pretreatments in Maize Callus Differentiation

The effect of pretreatments with DL-alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of the ornithine decarboxylase (ODC) activity, in plant differentiation, polyamine (PA) and amino acid contents of maize callus cultures was investigated. This study indicates that DFMO pretreatments can be used to improve regenerative response from maize callus cultures. These findings may also be useful in other recalcitrant cultures.

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.

Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants.

Embryogenic maize (Zea mays L.) callus cultures were transformed by microprojectile bombardment with a chimeric hygromycin phosphotransferase (HPT) gene and three transformed lines were obtained by selecting for hygromycin resistance. All lines contained one or a few copies of the intact HPT coding sequence. Fertile, transgenic plants were regenerated and the transmission of the chimeric gene was demonstrated through two complete generations. One line inherited the gene in the manner expected for a single, dominant locus, whereas two did not.

Improved plant regeneration in maize callus cultures after pretreatment with dl-alpha-difluoromethylarginine

The influence of a three-month-long pretreatment with dl-alpha-difluoromethylarginine (DFMA), an irreversible suicide inhibitor of arginine decarboxylase activity (ADC; EC 4.1.1.19), on plant regeneration, protein and polyamine (PA) composition of Zea mays callus cultures has been investigated. A four-fold increase in the number of regenerated plants is obtained after pretreatment with 0.5 mM DFMA. In addition, the regeneration frequency increases 3-fold in the treated calluses and the plants regenerated from such cultures are more developed than the untreated controls. The data obtained on protein and PA contents suggest that a senescence effect is exerted on the calluses grown in the presence of DFMA. However, after DFMA removal a rejuvenation effect occurs on the calluses that may explain the improvement of morphogenic capacity. This study indicates that DFMA pretreatment can be used to increase regeneration efficiency from maize callus cultures.

Proline Is Not the Primary Determinant of Chilling Tolerance Induced by Mannitol or Abscisic Acid in Regenerable Maize Callus Cultures

Chilling sensitive regenerable maize (Zea mays L.) callus cultures can be induced to survive prolonged exposure to 4 degrees C by treatments with mannitol, abscisic acid (ABA), and/or high levels of proline. Maize callus with a free proline content of about 122 micromoles/grain fresh weight survived longer exposures to 4 degrees C than did callus with a free proline content of about 68 micromoles/grain fresh weight. The addition of 0.53 molar mannitol or 0.1 millimolar ABA to culture medium produced a free proline content in maize callus of about 136 and 145 micromoles/grain fresh weight, respectively, if the medium contained 12 millimolar proline or about 36 and 1 micromoles/grain fresh weight, respectively, if no proline was in the medium. Although these mannitol and ABA treatments produced drastically different free proline levels in maize callus, callus grown on these media survived longer exposures to 4 degrees C than did maize callus grown on any proline treatment alone. Thus, the internal free proline level of treated callus is not the primary factor conferring chilling tolerance on these tissues.

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.

The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I50) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I50 for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO4 - to MnO2 (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus.