Dominant expression of an amino-acid uptake deficiency in carrot somatic fusion hybrids

Somatic hybrids were selected previously by their ability to grow in medium containing normally inhibitory levels of the two amino acid analogs aminoethylcysteine (AEC) and 5-methyltryptophan (5MT) following fusion of protoplasts from a cell strain resistant to AEC with protoplasts resistant to 5MT. The hybrid nature of the selected clones was shown by several criteria including the presence of another resistance, azetidine-2-carboxylate (A2C), carried by one of the parental strains which was not selected for in the initial hybrid selection scheme. The characterization presented here shows that the AEC resistance in the parental strain, as well as the two somatic hybrids, was due to decreased AEC uptake. Also the 5MT resistance in the hybrids, as in the parent was caused by a feedback altered form of the tryptophan biosynthetic control enzyme, anthranilate synthase which leads to increases in free tryptophan. The A2C resistance was caused by the accumulation of free proline by a mechanism which has not been studied. These studies confirm that AEC resistance caused by decreased uptake can be expressed dominantly in protoplast fusion hybrids.Abbreviations A2C Azetidine-2-carboxylate - AEC Aminoethylcysteine - 5MT 5-methyltryptophan     

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO₂ (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO₂ levels (5%). Photoautotrophy was indicated by the requirement for CO₂ and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C₄Amaranthus species. The cells showed high dark respiration rates and had lower net CO₂ fixation under high O₂ conditions. Dark CO₂ fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO₂ conditions showed the unique properties of a high RuBPcase activation level in ambient CO₂ and a stable ability to show net CO₂ fixation in 21% O₂ conditions.     

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.     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I₅₀ values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [¹⁴C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.     

Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [¹⁴C]mevalonate ([¹⁴C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [¹⁴C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [¹⁴C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.     

Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Comparison of RAPD and RFLP markers for mapping F2 generations in maize (Zea mays L.)

The F₂ generations from two maize crosses were used to compare the ability of RAPD and RFLP marker systems to create a genetic linkage map. Both RFLPs and RAPDs were shown to provide Mendelian-type markers. Most of the RFLPs (80%) could be placed with a good level of certainty (LOD>4) on the genetic linkage map. However, because of their dominant nature, only between 37% and 59% of the RAPDs could be placed with such a LOD score. The use of combined data from RFLPs and RAPDs increases the level of information provided by RAPDs and allows the creation of a combined RFLP/RAPD genetic linkage map. Thus, the RAPD technique was found to be a powerful method to provide improved probes coverage on a previously created RFLP map and to locate markers linked to chromosomal regions of interest.     

A simple procedure for the expression of genes in transgenic soybean callus tissue

Cotyledons from germinating seeds of the soybean cultivar Peking were inoculated with virulent Agrobacterium tumefaciens strain A281:pZA-7 which carries a wild type Ti plasmid pTiBo542 and a disarmed Ti plasmid (a binary vector)pZA-7 which contains the glucuronidase (uidA) and neomycin phosphotransferase (nptII) genes. Tumors were produced on all inoculated explants and 82% of these tumor lines were cotransformed by the nptII gene from the binary vector pZA-7 as shown by PCR analysis (18 of 22 lines tested). Eleven of these 18 lines were also resistant to kanamycin. Eleven lines expressed -glucuronidase activity (GUS), six of which were also kanamycin resistant. Since there is a high rate of coexpression of genes carried by the binary vector, this system provides a simple and rapid method for the expression of genes of interest in transformed soybean tissue which has been used successfully to test constructs designed for soybean transformation.