The effects of low levels of chloramphenicol and methotrexate on somatic embryogenesis inCitrus

Summary The effects of the antibiotics methotrexate and chloramphenicol on somatic embryogenesis inCitrus were evaluated. Relatively low levels (0.1 to 1.0 μg/ml) of these antibiotics did not inhibit embryo production from undeveloped ovules of ‘Key’ lime [C. aurantifolia) (Christm.) Swing.]. Surprisingly, both antibiotics induced the formation of embryogenic callus in these cultures. This is usually a rare event in cultures of undevelopedCitrus ovules, and ‘Key’ lime is especially recalcitrant. The effects of these antibiotics on embryogenic callus appeared to be limited to the induction stage, because there was no consistent effect, either stimulatory or inhibitory, on established, lines of embryogenic callus. Florida Agricultural Experiment Station Journal Series No. 8958. This research was supported in part by a grant to Moore and Cline from the Competitive Grants Office of the SEA, USDA (85-CRCR-1-1623).     

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices

Summary Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells. This work was supported by an operating grant from the Medical Research Council of Canada.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Hydrogen cyanide and embryonal dormancy in apple seeds

Embryos of apple ( Malus domestica Borh. cv. Antonówka) were treated with 1 m M gaseous HCN for 6 h and cultured under a 12 h photoperiod. HCN pretreatment stimulated germination, increased the length of hypocotyls, shortened the main root and decreased the percentages of seedlings with asymmetrically grown as well as with asymmetrically greened cotyledons. High activity of β-cyanoalanine synthase (EC 4.4.1.9) and a sharp increase in cyanogen content during embryo culture suggested very low levels of endogenous HCN. despite the activity of HCN releasing enzymes. The obtained data allow us to postulate an important role for cyanide in the regulatory complex controlling dormancy in apple seeds. Experiments with respiratory inhibitors indicated, however, that HCN pretreatment affected neither the alternative electron transport pathway nor residual respiration.     

Cyanide controls enzymes involved in lipid and sugar catabolism in dormant apple embryos during culture

The activities of alkaline lipase (EC 3.1.1.3, AlkL), isocitrate lyase (EC 4.1.3.1. ICL), pyruvate kinase (EC 2.7.1.40. PK) and glucose–6–phosphate dehydrogenase (EC 1.1.1.49, G6PDH) were determined in cultured, dormant embryos of apple (Malus domestica Borb. cv. Antonówka), pretreated with gaseous HCN. The C₆/C₁ , ratio was estimated in the same material. The activities of AlkL and ICL were not stimulated by HCN pretreatment until the period of maximum stimulation of germination. The activity of G6PDH was inhibited by cyanide only late during the culture of embryos. Therefore, the changes in these activities are considered to be the result and not the cause of enhanced germination. On the other hand, also PK, active very early during the culture of embryos, was modified as a result of the treatment. The cyanide-induced changes in activity of this enzyme in cotyledons (inhibition followed by stimulation) were similar to those in the whole embryo, whereas its changes in the embryonal axis (stimulation followed by inhibition) resembled CN-induced changes in PK in axes of apple seeds submitted to cold stratification (Bogatek and Lewak 1988). The estimation of C₆/C₁ ratios partly confirmed these observations. A role of HCN-induced modifications of PK activity in embryonal dormancy is proposed.     

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum

Summary This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the -glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of -rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for -glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.     

Increasing in vitro germination of Musa balbisiana seed

Seeds of Musa balbisiana were soaked in water for five days prior to excision of embryos. Embryos with their longitudinal axis laid flat and half-way embedded on agar-solidified medium produced the highest germination and the most desirable plantlet characteristics. Germination in vitro was 94% within 7 days compared to 50% after 54 days for greenhouse-sown seeds.     

A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.