Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota.

We have co-transformed carrot (Daucus carota) and Arabidopsis thaliana with an Agrobacterium tumefaciens non-tumorigenic T-DNA carrying the maize transposable element Activator (Ac) and an Agrobacterium rhizogenes Ri T-DNA. We present evidence that the Ac element transposes in transformed root or root-derived callus cultures of both species. We show that fertile plants can be regenerated from transformed, root-derived callus cultures of Arabidopsis, demonstrating the utility of the Ri plasmid for introducing the maize Ac element into plants. We also present evidence that Ac elements that excise from the transforming T-DNA early after transformation continue to be mobile in carrot root cultures.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

Transgenic maize plants by tissue electroporation.

In this paper, we describe the transformation of regenerable maize tissues by electroporation. In many maize lines, immature zygotic embryos can give rise to embryogenic callus cultures from which plants can be regenerated. Immature zygotic embryos or embryogenic type I calli were wounded either enzymatically or mechanically and subsequently electroporated with a chimeric gene encoding neomycin phosphotransferase (neo). Transformed embryogenic calli were selected from electroporated tissues on kanamycin-containing media and fertile transgenic maize plants were regenerated. The neo gene was transmitted to the progeny of kanamycin-resistant transformants in a Mendelian fashion. This showed that all transformants were nonchimeric, suggesting that transformation and regeneration are a single-cell event. The maize transformation procedure presented here does not require the establishment of genotype-dependent embryogenic type II callus or cell suspension cultures and facilitates the engineering of new traits into agronomically relevant maize inbred lines.     

A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.     

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°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°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°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.     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Callus induction and maintenance of Zea mays kernels

Summary Totipotent tissue cultures of maize (Zea mays L.) have previously been initiated from various explant tissues. In this paper, we present an alternative source of callus induction.A callus of maize (G 204 hybrid) was obtained from intact kernels grown on Linsmair and Skoog RM medium supplemented with 20 mg 2,4-dichlorphenoxyacetic acid (2,4-D) per litre. The callus growth was greatest from the first node of the seedling shoot. Occasionally, callus growth was observed from the radicle and coleopticle regions. The callus was easily transferred and maintained on a medium with 2 mg/L 2,4-D. This callus formed numerous roots and leaf-like structures when grown on a medium containing 800 mg/L yeast extract, 30 g/L sucrose and 10 g/L agar.     

Tissue culture of maize: Selection of friable callus lines

Subnodal callus of the F 71 maize /Zea mays L./ inbred was subjected to selection for friability and intensive proliferation. This procedure resulted in three callus lines characterized by uniform structure and improved growth rate. Some characteristics of these lines as well as attempts to establish maize suspension cultures are described.     

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.     

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the antidifferentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.     

Diamine oxidase is important in assessment of polyamine effects on hemopoietic cell proliferation in vitro

Summary A difference was observed in the effect of difluoromethlyornithine (DFMO), a specific inhibitor of ornithine decarboxylase, on human and murine granulocyte-macrophage precursor cell (CFU-C) proliferation in vitro, in the presence of fetal bovine serum (FBS) and horse serum (HS). A dose of DFMO which almost totally abolished CFU-C colonies in cultures containing FBS had no effect or very little effect on CFU-C in cultures supplemented with HS. This effect could be reversed by aminoguanidine reacting with diamine oxidase (DAO), which is present in FBS but not in HS. The importance of DAO in the assessment of polyamine effects is also suggested by decreased colony formation in cultures containing HS and DFMO only after the addition of this enzyme. Additionally, Mo T cell line cultures containing DFMO demonstrated a substantially lower intracellular concentration of putrescine in the presence of FBS rather than HS. Supported in part by National Institutes of Health grant RO1-AM27423 and American Cancer Society grant CH-334.     

Synergistic effect of DFMO and 2,4-D on regeneration of Tabernaemontana persicariaefolia jacq in vitro

Callus cultures of Tabernaemontana persicariaefolia, (Apocynaceae), an endangered species endemic to the Mascarene Islands, were established from leaf explants on MS medium containing either 5 mg·l⁻¹ 2,4-D and 0.5 mg·l⁻¹ BA or 5 mg·l⁻¹ 2,4-D, 0.5 mg·l⁻¹ BA and 200 mg·l⁻¹ DFMO. Histological studies showed regenerating nodules resembling globular embryos in calli after 4 weeks on the DFMO medium. Green shoot formation was achieved by sequential subculture of the induced calli on media with gradually decreasing 2,4-D concentrations (5→1→0 mg·l⁻¹). Regeneration was greatly stimulated in the presence of DFMO. The first emergence of shoots occured 3 weeks earlier than in untreated callus cultures.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

Globulin-1 gene expression in regenerable<Emphasis Type="Italic"> Zea mays</Emphasis> (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.     

Studies on the morphogenetic response of maize tissue cultures of different origin

The participation of the genotype and of organ specifity effect in the quality of morphogenetic response (callogenesis, bud and root formation) of primary maize explants has been investigated. The presence of synthetic auxins — especially 2,4-D at 1 to 5 mg 1^?1 conc. - in cultivation medium was essential for both callus formation and continuous growth of tissue and suspension cultures. Anatomic structure of callus cultures is permanently heterogeneous, their growth is ensured by the action of meristems of the type found in root tips, and by repeated callogenesis from malformed roots. Adventive buds and plants could be regenerated only from cultures of embryonal origin (of one line). The presence or absence of the endosperm gene “opaque” did not influence callogenesis intensity in cultures of isolated embryos; however the morphogenetic response was clearly “line specific”.     

Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.