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Anthranilate synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis. more...

The effects of resveratrol and pterostilbene on in vitro growth of three soybean pathogens were tested to determine whether these stilbenic compounds could potentially be targets to increase innate resistance in transgenic soybean plants. Growth of Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum was measured on solid and in liquid media amended with resveratrol and pterostilbene (concentration in the media of resveratrol at 100 μg/ml and pterostilbene at 25 μg/ml). All three fungi were very sensitive to pterostilbene in potato dextrose agar (PDA), which reduced colony area of each of the three pathogens to less than half of the control 3 days after incubation. The three fungal pathogens were less sensitive to resveratrol compared with pterostilbene; however, area under the curve (AUC) calculated from colony areas measured over 3 days was significantly (P < 0.05) less than the control for S. sclerotiorum and R. solani on PDA with resveratrol or pterostilbene. AUC for M. phaseolina on PDA with pterostilbene was significantly (P < 0.05) lower than the control whereas, on PDA with resveratrol, AUC for M. phaseolina was lower than the control but the difference was nonsignificant (P > 0.05). AUC for all three fungi was significantly lower (P < 0.05) on PDA with pterostilbene than with resveratrol. In potato dextrose broth (PDB) shake cultures, AUC for all three fungi was significantly (P < 0.01) lower in pterostilbene than in the control. AUC for R. solani and S. sclerotiorum was significantly lower (P < 0.01) in resveratrol than the control, whereas AUC for M. phaseolina in resveratrol was lower, but not significantly (P > 0.05) different from the control. AUC in pterostilbene was highly significantly (P < 0.01) lower than in resveratrol for M. phaseolina and significantly (P < 0.05) lower for R. solani but the difference for S. sclerotiorum was nonsignificant (P > 0.05). There was a trend for lower mass accumulation of all three fungi in either pterostilbene or resveratrol compared with the control during the course of the experiment; however, S. sclerotiorum appeared to recover from the effects of pterostilbene between days 2 and 4. Results of biochemical analyses of the PDB over time indicated that the three fungi degraded resveratrol, with nearly 75% reduction in concentration in M. phaseolina, 80% in S. sclerotiorum, and 60% in R. solani PDB cultures by day 4 of fungal growth. M. phaseolina and S. sclerotiorum were able to resume growth after early inhibition by resveratrol after its concentration was reduced in the cultures through degradation, whereas R. solani was less efficient in resveratrol degradation and was not able to overcome its inhibitory effects on growth. The capacity to degrade pterostilbene was lowest in M. phaseolina compared with S. sclerotiorum and R. solani and the recovery of M. phaseolina cultures after initial growth inhibition by pterostilbene was minimal. The potential products of resveratrol and pterostilbene degradation by fungi were identified to be dimers and various oxidation products. more...

Soybean hairy roots transformed with the resveratrol synthase and resveratrol oxymethyl transferase genes driven by constitutive Arabidopsis actin and CsVMV promoters were characterized. Transformed hairy roots accumulated glycoside conjugates of the stilbenic compound resveratrol and the related compound pterostilbene, which are normally not synthesized by soybean plants. Expression of the non-native stilbenic phytoalexin synthesis in soybean hairy roots increased their resistance to the soybean pathogen Rhizoctonia solani. The expression of the AhRS3 gene resulted in 20% to 50% decreased root necrosis compared to that of untransformed hairy roots. The expression of two genes, the AhRS3 and ROMT, required for pterostilbene synthesis in soybean, resulted in significantly lower root necrosis (ranging from 0% to 7%) in transgenic roots than in untransformed hairy roots that had about 84% necrosis. Overexpression of the soybean prenyltransferase (dimethylallyltransferase) G4DT gene in soybean hairy roots increased accumulation of the native phytoalexin glyceollin resulting in decreased root necrosis. more...

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester-bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester-bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester-bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether-bound hydroxycinnamic acids varied greatly (about two-three-fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester-bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5-5-coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether-bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis. more...

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin. more...

Type II maize callus of the HiII genotype can be separated into regenerable and non-regenerable types based on the visible morphology of the callus. When the non-regenerable morphotype of callus initiated 6 mo or a yr earlier was treated with from 2% to 20% polyethylene glycol (PEG; 3,350 molecular weight) for three subculture periods of 21 d each, the morphotype changed to regenerable, and the callus did become highly regenerable. The PEG treatments did not improve the plant regeneration ability of the regenerable morphotype or of an old culture initiated 4 yr earlier. more...

The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 muM and 300 muM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T(1) progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 muM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography-mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Delta(1)-pyrroline-5-carboxylate (P5C). However, H(2)O(2) treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress. more...

The perennial grass, Miscanthus x giganteus is a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor-intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M x giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome-doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2-day treatments of 15μM oryzalin (78%) or 939μM colchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0 ± 0.2 μm) compared with regenerated triploid M. x giganteus plants (24.3 ± 1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M. x giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop. more...

Understanding the metabolic responses of the plant to a devastating foliar disease, soybean rust, caused by Phakopsora pachyrhizi, will assist in development of cultivars resistant to soybean rust. In this study, differences in phenolic metabolism were analyzed between inoculated and noninoculated plants using two susceptible and three resistant soybean genotypes with known resistance genes. Rust infection resulted in increased accumulation of isoflavonoids and flavonoids in leaves of all soybean genotypes tested. Although the soybean phytoalexin glyceollin was not detected in leaves of uninfected plants, accumulation of this compound at marked levels occurred in rust-infected leaves, being substantially higher in genotypes with a red-brown resistant reaction. In addition, there was inhibition of P. pachyrhizi spore germination by glyceollin, formononetin, quercetin, and kaempferol. However, there was no correlation between concentrations of flavonoids quercetin and kaempferol and rust-induced isoflavonoid formononetin in soybean leaves and rust resistance. Lignin synthesis also increased in all inoculated soybean genotypes whereas there was no significant difference in all noninoculated soybean genotypes. Cell wall lignification was markedly higher in inoculated resistant lines compared with inoculated susceptible lines, indicating a possible protective role of lignin in rust infection development. more...

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes. Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons. more...

Metabolic profiling using GC-MS and LC-MS analyses of soluble metabolites and cell wall bound phenolic compounds from maize calluses of different morphogenic competence revealed a number of biochemical characteristics that distinguish tissues with high plant regeneration ability from tissues that cannot efficiently regenerate plants in vitro. Maize cultures of different ages from H99 (compact type I callus) and HiII (friable type II callus) were divided into two different samples: regenerable (R) and non-regenerable (NR) based on known morphologies. Tissues from both genotypes with high morphogenic potential had higher asparagine and aspartate and indole-3-butenol concentrations, decreased sugar and DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) concentrations, low levels of 4-aminobutyric acid (GABA) and chlorogenic acid and lower levels of feruloyl- and sinapoyl glucosides compared to NR tissues. The ether bound cell wall phenolics of tissues with high regeneration potential had higher levels of the predominant G (guaiacyl) units and lower levels of H (p-hydroxyphenyl) and S (syringyl) units and higher ferulic acid/coumaric acid and ferulic acid/diferulic acid ratios. The same trends were found with the ester-bound phenolics of HiII, however, there were only small differences between the H99 R and NR tissues. Concentrations of the major sugars, organic acids, amino acids and soluble aromatic compounds tended to increase as the time after culture initiation increased. The results show that there are differences in general metabolism, phenolic secondary compounds and cell wall composition between R and NR cell types. more...

Common bean effects on health have been related to its dietary fiber content and other active compounds. This study assessed the content of flavonoids, coumestrol, phenolic acids, galactooligosaccharides, and phytic acid in wild and cultivated Mexican common bean seeds (raw and cooked) and that of flavonoids, coumestrol, and phenolic acids in germinated bean seeds. The presence of isoflavones in raw bean seeds was not confirmed by the UV spectra. Quercetin, kaempferol, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid mean contents were 10.9, 52.3, 10.1, 9.6, 5.4, and 18.2 microg/g, respectively; raffinose, stachyose, verbascose, and phytic acid mean contents were 8.5, 56.3, 5.5, and 11.5 mg/g, respectively, in raw seeds. All compounds were affected by autoclaving, and germination resulted in a de novo synthesis of flavonols, phytoestrogens, and phenolic acids. The impact on health of common bean seed is affected by dietary burden, specific compounds content, and processing. On the other hand, germinated bean seed or beans sprouts may be sources of antioxidants and phytoestrogens. more...

Sudden death syndrome (SDS), caused by the soilborne fungal pathogen Fusarium solani f. sp. glycines, is one of the most important diseases of soybean. Lignin degradation may play a role in the infection, colonization, and survival of the fungus in root tissue. Lignin degradation by F. solani f. sp. glycines was shown by the catalyzed release of 14CO2 from purified 14C-labeled Klason lignin, the degradation of polymeric aromatic dyes in culture (a method commonly used to test the ligninolytic capacity of microorganisms), and the production of laccase and lignin peroxidase (the major fungal lignin degrading enzymes). The laccase and lignin peroxidase activities and the amount of decolorization of aromatic polymeric dyes (Poly R-478 and Remazol Brilliant Blue R) by F. solani f. sp. glycines were intermediate or greater than that found with two known lignin-degrading fungi, Polyporus tulipifera and Schizophyllum commune. Studies of lignin synthesis from [14C]phenylalanine with soybean hairy root cultures showed that F. solani f. sp. glycines treatment stimulated lignin synthesis in 2 h, and by 24 h, some lignin degradation had occurred. These results indicate that F. solani f. sp. glycines was capable of degrading lignin which may be important in infection, colonization, and survival of the fungus. more...

Soybean [Glycine max (L.) Merr.] seed isoflavone concentration has been shown to be highly dependent on environmental conditions, but isoflavone concentrations have not been studied under controlled conditions to quantify the effects of specific factors. To determine the effect of air temperature and soil moisture status during soybean seed development on seed isoflavone concentration and composition, soybean plants were grown in the greenhouse under intermediate (18/28 degrees C), 9.5 h night/14.5 h daytime temperatures with high soil moisture conditions. Beginning at the R6 growth stage plants were subjected to either intermediate (18/28 degrees C), low (13/23 degrees C), or high (23/33 degrees C) 9.5 h night/14.5 h daytime temperatures with either low or high soil moisture conditions. Two French cultivars, Imari and Queen, and three U.S. cultivars, Dwight, Jack and Loda, all in maturity group II were studied. The overall results show that low temperatures and high soil moisture conditions produced the highest seed isoflavone concentrations with changes in temperature having the larger effect. The changes in daidzein and genistein concentrations were similar to changes in total isoflavones but the glycitein concentration was much less affected. The three U.S. cultivars were much less responsive to soil moisture than the two French cultivars. All five cultivars showed a two- to threefold increase in total isoflavone concentrations at the low temperature regime compared to the high temperature regime. Plant height was greatest under the intermediate temperatures; whereas, low temperatures and low soil moisture hastened maturity. Seed size was not significantly affected by any treatment. Soil moisture and air temperature have clear effects on the isoflavone concentrations in mature soybean seeds, but the ranking of all the cultivars based on average isoflavone concentration remained the same with all treatments. Environmental factors can have a large effect on isoflavone concentration, but the potential for isoflavone production is largely under genetic control. more...

The soil-borne fungus Fusarium solani (Mart.) Sacc. f. sp. glycines (FSG) infects soybean [Glycine max (L.) Merr.] roots and causes the disease sudden death syndrome (SDS). The biochemical response of soybean roots to FSG infection, which has not been studied before, was investigated by comparing FSG-inoculated and noninoculated roots of two partially resistant ('PI 520733' and 'PI 567374') and susceptible ('Spencer') genotypes. Activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid biosynthetic pathway, was increased in inoculated roots of all three genotypes. The phytoalexin glyceollin increased to much higher levels in inoculated roots of the partially resistant cultivars PI 520733 and PI 567374 than in the susceptible Spencer. The changes in phenolic metabolism were localized in lesion-containing areas of roots rather than in the new portion growing under the FSG inoculum. No clear correlation was found between the glyceollin precursor daidzein (4',7-dihydroxyisoflavone) and its conjugates and glyceollin levels in root tissues; however, isoflavone levels increased only in roots of inoculated plants of partially resistant lines, even though constitutive isoflavone levels were higher in the susceptible control. The FSG growth on potato dextrose agar medium was inhibited by increasing concentrations of glyceollin. Induction of lignin synthesis was found in the inoculated roots of all three lines, with the highest rate of lignification observed in roots of the partially resistant genotypes, especially PI 567374. These studies show for the first time that FSG inoculation of soybean roots in soil induces the phenylpropanoid pathway to synthesize isoflavones, the phytoalexin glyceollin, and lignin, indicating that these compounds may be involved in the partial resistance response. more...

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. more...

Summary The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration. more...

Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA3, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 μM ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 μM GA3-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 μM GA3-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 μM ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 μM ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA3, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 μM ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development. more...

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus byAgrobacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 μm 5MT, whereas the controls were completely inhibited by 15 μm 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 α-subunit can interact with the native A. sinicus β-subunit to form an active enzyme. TheASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance. more...

Experiments were carried out to estimate the amounts and nature of bonding of ferulic acid to cell walls of various dicotyledonous plant materials including soybean heterotrophic and mixotrophic cell suspension cultures, soybean leaves, buckwheat callus, flax phloem and xylem. Isolated cell walls were oxidized with CuSO4-NaOH, with phenol aldehyde products and ferulic acid produced estimated by GC. Ferulic acid content was also analyzed in the cell wall fraction extracted by 1 M alkali at room temperature, which cleaves ester linkages, and in the fraction extracted by hot concentrated alkali, which cleaves ether linkages. Overall, the bulk of the cell wall ferulic acid (60–80%) was found to be ether linked to cell-wall components. Room temperature alkali treatment may release from the cell wall a portion of the ferulic acid that is esterified to cell wall components via saponification. This treatment, however, also extracts a portion of the etherified ferulic acid that is bound to some cell wall components like proteins or glycoproteins that are acid precipitable. Our results demonstrate that hydroxycinnamic acids can form a significant part (0.01–0.19% of cell wall dry weight) of primary cell wall phenolics of dicots and the nature of linkages between ferulic acid and polymers of the primary cell wall varies in different plant materials. more...

Studies of the activity levels of the fungal cell wall degradative enzyme, beta-1,3-glucanase, were carried out with Zea mays L. embryogenic callus cultures and kernels of genotypes with different abilities to resist aflatoxin formation on the ears. Dual-culture experiments of the aflatoxin forming fungus, Aspergillus flavus Link: Fr. with maize callus, demonstrated that the growth of A. flavus on plates was inhibited more by callus of a resistant genotype (Tex 6 x Mo17 denoted TXM) than by a sensitive genotype (Pa91). The inhibition correlated with the activity levels of beta-1,3-glucanase in the callus and in the culture medium. The presence of the fungus caused an increase in enzyme activity in TXM but not in Pa91 callus. Direct treatment of callus of both genotypes with A. flavus spores caused the amount of callose to increase within 30 min. An elevated beta-1,3-glucanase activity in maize kernels was also correlated with lower A. flavus infection observed in the resistant genotype (Tex 6) compared with a susceptible one (B73). Fungal inoculation of the ears increased the enzyme activities both in kernels in contact with and not in contact with the fungus. These studies indicate that beta-1,3-glucanase activity may have a role in the inhibition of growth of the A. flavus fungus, that maize embryogenic callus and kernels do respond to the presence of this nonpathogenic fungus, and that these systems might be useful in further plant-fungal interaction studies. more...

In a study of the 5'-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the -272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, -1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between -1391 and -805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100-300 μM). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5' regulatory region is responsible for the positive ABA regulation of gene expression. more...

Somatic and zygotic embryos of soybean cv. Jack were analyzed for soluble carbohydrate, total lipids, and protein during development. Zygotic embryos accumulated trace amounts of fructose, galactose, and galactinol., whereas somatic embryos contained only trace amounts of galactose. Somatic embryos accumulated much higher glucose levels than zygotic embryos. Both somatic and zygotic embryos contain low levels of sucrose, myoinositol, and pinitol. Raffinose and stachyose accumulated in the late developmental stages of zygotic embryos, but only stachyose was found to accumulate in the late stage somatic embryos. Zygotic embryos contained low total lipid levels up to 50 d after flowering (DAF) and then the levels increased to 16% by 55 DAF and 21% at 65 DAF. Somatic embryos had low levels of total lipids throughout development with the maximum of only 4.7%. Soybean zygotic embryos contained about 40% protein throughout development, while the protein concentration of somatic embryos decreased from 44% to 25% as maturation approached. These studies demonstrate that the composition of Jack zygotic embryos is similar to that described for other cultivars during development while the somatic embryo composition and size is markedly different. The low somatic embryo germination often noted might be due to the abnormal development as shown by a composition different from that of mature zygotic embryos. The low concentration of the raffinose series sugars might be especially important factors. more...

Summary The stability of resistance to the herbicide glyphosate caused by amplification of the 5-enolpyruvylshiki-mate-3-phosphate synthase (EPSPS) gene was followed in six cloned Daucus carota (carrot) cell lines and other carrot lines grown in the absence of the selection agent for periods up to about four years. The lines originated from three different carrot suspension cultures with high levels of glyphosate resistance. The cloned lines lost resistance with a half-time of about two years. The resistance was correlated with decreased levels of EPSPS enzyme activity and EPSPS gene copy number. In some, but not all cases, glyphosate resistance was correlated with increased DNA content as measured by flow cytometry. The C1 wild type cell line, which had 2.3-times as much nuclear DNA as carrot plants, also had a correspondingly higher chromosome number, about 42 versus 18. The medium containing high glyphosate (70 mmol · L −1 ) was much less toxic if the K + level was decreased, indicating that growth inhibition of the most resistant lines was caused in part by the high K + concentration of the medium. These studies show that the selected glyphosate resistance was unstable and was lost slowly when grown away from the selection agent and that high K + concentrations limited growth of resistant lines at high glyphosate concentrations. more...

The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen. more...

The response of soybean transgenic plants, with suppressed synthesis of isoflavones, and nontransgenic plants to two common soybean pathogens, Macrophomina phaseolina and Phytophthora sojae, was studied. Transgenic soybean plants of one line used in this study were previously generated via bombardment of embryogenic cultures with the phenylalanine ammonia lyase, chalcone synthase, and isoflavone synthase (IFS2) genes in sense orientation driven by the cotyledon-preferable lectin promoter (to turn genes on in cotyledons), while plants of another line were newly produced using the IFS2 gene in sense orientation driven by the Cassava vein mosaic virus constitutive promoter (to turn genes on in all plant parts). Nearly complete inhibition of isoflavone synthesis was found in the cotyledons of young seedlings of transgenic plants transformed with the IFS2 transgene driven by the cotyledon-preferable lectin promoter compared with the untransformed control during the 10-day observation period, with the precursors of isoflavone synthesis being accumulated in the cotyledons of transgenic plants. These results indicated that the lectin promoter could be active not only during seed development but also during seed germination. Downregulation of isoflavone synthesis only in the seed or in the whole soybean plant caused a strong inhibition of the pathogen-inducible glyceollin in cotyledons after inoculation with P. sojae, which resulted in increased susceptibility of the cotyledons of both transgenic lines to this pathogen compared with inoculated cotyledons of untransformed plants. When stems were inoculated with M. phaseolina, suppression of glyceollin synthesis was found only in stems of transgenic plants expressing the transgene driven by a constitutive promoter, which developed more severe infection. These results provide further evidence that rapid glyceollin accumulation during infection contributes to the innate soybean defense response. more...

Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes. more...

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process. more...

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 (50μM) 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. more...

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement. more...

A photoautotrophic soybean suspension culture (SB-P) was used to study CO2 assimilation while exposed to elevated or ambient CO2 levels. These studies showed that under elevated CO2 (5% v/v) malate is the dominant fixation product, strongly suggesting that phosphoenolpyruvate carboxylase (PEPCase) is the primary enzyme involved in carbon fixation in these cells under their normal growth conditions. Citrate and [aspartate + glutamate] were also significant fixation products during fifteen minutes of exposure to 14CO2. During the ten minute unlabeled CO2 chase however, 14C-malate continued to increase while citrate and [aspartate + glutamate] declined. Fixation of 14CO2 under ambient CO2 levels (0.037%) showed a very different product pattern as 3-phosphoglycerate was very high in the first one to two minutes followed by increases in [serine + glycine] and [aspartate + glutamate]. Hexose phosphates were also quite high initially but then declined relatively rapidly. Thus, the carbon fixation pattern at ambient CO2 levels resembles somewhat that seen in C3 leaf cells while that seen at elevated CO2 levels more closely resembles that of a C4 plant. The initial fixation product of C3 plants, 3-PGA, was never detectable under high CO2 conditions. These data suggest that an in vitro photoautotrophic system would be suitable for studying carbon fixation physiology during photosynthetic and non-photosynthetic growth. more...

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5' flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells. more...

When Daucus carota L. (carrot) suspension cultures, which had been under gradual selection for increased glyphosate resistance, whre plated in glyphosate-containing medium, the populations became gradually more resistant with I 50 values about equal to the final selection concentration used in the liquid medium. Wild type cells did not grow on plates with 2 mM glyphosate, but some colonies formed after 90 days. Cells in these colonies, after growth away for the inhibitor to obtain sufficient cells, had increased glyphosate resistance and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity and usually had amplified EPSPS genes. One step selection in liquid or solidified medium with 10 mM glyphosate was not successful. However, in liquid medium with 2 mM glyphosate, the cell viability decreased to about 30% by 30 days and then increased as growth became evident. Kinetic analysis indicates that at least 0.8% of the cells grow. The growth rate seen when the cells are subjected to a gradual selection scheme, where glyphosate was about doubled at each step, also indicates that a high proportion of the original population survives to form the new population. These results indicate that the gene amplification which causes the glyphosate tolerance in carrot cell cultures is a gradual and relatively stable process that occurs in a relatively high proportion of the cell population. more...

When Zea mays callus cultures of two different genotypes were treated with the osmoticum mannitol (0.53M) for 24h their ability to reduce the tetrazolium derivative 2,3,5-triphenyltetrazolium chloride (TTC) to form the insoluble red compound formazan is stimulated. The formazan can be extracted with 95% ethanol for quantitation and this reaction has been used as a measure of viability since only live cells can carry out this reduction. In order to determine the cause of the increased TTC reduction caused by mannitol we carried out metabolic profiling analysis using GC-MS to identify 80 compounds. There were increases in sugar alcohols, hexoses except fructose and in total sugars. The total organic acid pools did not change and nitrogen containing compounds decreased slightly. Principle component analysis showed a large treatment effect due to changes in carbohydrate and nitrogen metabolism. These results indicate that the increased carbohydrate available for the citric acid cycle may be the cause of the increased TTC reduction observed after the mannitol treatment. more...

Metabolic changes were studied, which accompanied the conversion of 6 month old HiII maize non-regenerable (NR) calli into regenerable (R) calli when cultured for 63 days with 10% polyethylene glycol (PEG) (3350 MW) in culture medium. The conversion of 6 month old NR to R callus morphotype caused by PEG application decreased cell wall contents in callus dry mass and changed cell wall phenolics making their profile similar to that of R callus by reduction of lignin and ester- and ether-bound phenolic concentrations, including p -coumaric acid and ester- and ether-bound diferulates and by increase of the ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid in cell walls of NR callus. Some similar changes of cell wall phenolics caused by PEG application were also found in 48 month old NR callus, that changed the morphology, but did not regenerate plants. However, there were no changes in the old callus in levels of total ester and ether-bound cell wall phenolics and substantially smaller decreases were found in ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid, as well as in diferulate concentrations compared to young NR callus cultured with PEG. Remarkably, application of PEG also changed the primary metabolism of young NR callus tissues, so that they acquired metabolic features of highly regenerable callus. These data clearly suggest that PEG alters metabolism of NR calli, so they acquire biochemical characteristics of R calli, and that adaptive osmotic adjustments vary in different types of callus tissues. more...

A cell line ofDatura innoxia was selected in suspension culture to be resistant to 1% (vol/vol) ethanol (EtOHR). EtOHR cells were cross-resistant to 1% (vol/vol) methanol and 1% (vol/vol) 2-propanol but not 1% (vol/vol)n-propanol orn-butanol, whereas wild type (WT) cells were resistant only to methanol. Resistance in EtOHR cells is probably a result of a very low level of alcohol dehydrogenase (ADH) activity which was only 9 to 10% of that in WT cells and was undetectable during much of the EtOHR growth cycle. In the absence of ethanol, EtOHR cells have a I50 for the toxic ethanol analog allyl alcohol, which is nearly 3 times higher than that in WT cells. In the presence of ethanol, EtOHR cells have an I50 for allyl alcohol which is 12 times more than WT cells. This difference correlated well with the decrease in ADH activity found in EtOHR cells grown on ethanol. When ethanol was removed from the suspension medium, ADH activity in EtOHR cells gradually increased to WT levels. When re-exposed to ethanol after 200 cell generations, ADH activity quickly decreased and growth resumed after a 4- to 6-day lag period. Lipid analysis showed a 37% increase in total lipid in EtOHR cells, mostly in polar lipids, di- and triglycerides. The fatty acid composition of these lipid classes was shifted toward the more polyunsaturated. These lipid changes were probably a reflection of the increased plastid number in the EtOHR cells and may be a result of growth in ethanol rather than a reason for resistance. EtOHR cells seem to be regulatory mutants able to quickly lower ADH activity in the presence of ethanol. more...

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level. more...

The evaluation of soybean genotypes for resistance to iron deficiency through field experiments is complicated by variation in symptom expression. The objective of this study was to develop a tissue culture technique that could distinguish between Fe‐efficient and Fe‐inefficient genotypes. Ten soybean genotypes varying in sensitivity to iron deficiency were planted at 5 locations and rated based on chlorosis expression. For the lab evaluation, friable callus of the ten genotypes was placed on a 4MSII medium amended with 10 mM NaHCO3. Callus growth reduction relative to a control medium without NaHCO3 was recorded. Callus weight of all cultivars was affected by the addition of NaHCO3 however, significant differences in growth reduction among genotypes was observed. The high correlation between callus growth reduction and field chlorosis ratings observed (r2=0.92) and the fact that the expiant source plant can be grown to produce seed indicates that this technique would be useful in a soybean breed... more...

Photoautotrophic (PA) tissue cultures that rely entirely upon light for energy and CO2 for carbon have been initiated from a total of 29 species of higher plants. The species include C4 and Crassulacean acid metabolism species, legumes, and a number of important crop plants. However, no grasses have been grown successfully. PA culture initiation is time-consuming and appears to be associated with long-term adaptation physiologically and apparently genetically, since once initiated the cultures can be bleached and then rapidly become photosynthetically competent again. In addition, a series of unusual mutations have also been found in the psbA gene leading to triazine herbicide resistance in PA cultures in contrast to those found with triazine-resistant weeds. A large number of physiological, biochemical, and molecular biological studies have been carried out especially with the Chenopodium rubrum and Glycine max cell lines. The cultures can be grown in fermenters, hence, seem ideal for compound and macromolecule production, although the overall record of valuable secondary compound production has been disappointing. PA cultures are useful for many studies since they are axenic, grow relatively rapidly, are easy to view microscopically, can be heavily 14C labeled using 14CO2, are in easily changed medium for compound addition and removal and other conditions are readily altered, and the cells are easily extracted. Keywords: chlorophyll; phosphoenolpyruvate carboxylase; photoautotrophic; photomixotrophic; photosynthesis; plant biotechnology; plant cell culture; ribulose-bisphosphate carboxylase more...

Four antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 μ M APM or 10 μ M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 μ M), however, at a higher concentration (5 μM) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations. more...

Since the presence of glyphosate in maize tissue cultures of proprietary line L2 was very detrimental to plant regeneration, we determined metabolic changes associated with the glyphosate effects on plant regeneration in maize cultures. The polar fraction composition and soluble and cell-wall-bound phenolics were analyzed in the regenerable (R) and non-regenerable (NR) calluses of maize line L2. The tissues with high regeneration capacity had low sugar and 4-aminobutyric acid (GABA) concentrations and increased concentrations of most amino acids, polyamines and indole-3-butenol in the soluble polar fraction and higher ferulic acid/coumaric acid and ferulic acid/diferulic acid ratios and higher levels of the predominant G (guaiacyl) units in the cell wall fraction compared with NR calluses as was found before with H99 and HiII maize R and NR tissues, indicating an association of these metabolites with the capacity of maize cultured tissue to regenerate plants. We also found that di-coumaroyl spermidine and coumaroyl-feruoyl spermidine are present in the soluble fraction of L2 R tissues and are practically absent in NR tissues. However, we did not see such differences in HiII and H99 samples, which indicate that these are genotypic features not related to the capacity to regenerate plants in maize tissue cultures. Glyphosate treatment caused the accumulation of shikimic and quinic acids (not detected in untreated samples) in R and NR calluses (with higher levels found in R tissues) and also decreased the FA/diFA ratio in cell wall phenolics, polyamine and amino acid levels, and increased sugar concentrations in the R L2 tissues, indicating a metabolic shift of R callus to NR tissues. more...

Metabolite profiling of untransformed and cyanamide hydratase- (Cah) transformed (denoted 1C) soybean (Glycine max [L.] Merrill) leaves revealed only small differences in plants grown in the greenhouse or in the dark for 24 h, indicating that the Cah enzyme that converts cyanamide to urea has no substrates in soybean leaves and does not affect metabolism. Untransformed leaves sprayed with 0.5% cyanamide developed necrotic lesions within 2 h in the light but not in the dark. The sprayed 1C leaves showed little visible damage and accumulated high concentrations of urea, amino acids, and some sugars, but sucrose decreased over a 24 h period. The untransformed necrotic leaves also accumulated some urea and amino acids apparently due to cyanamide degradation, while sucrose and some organic acids decreased. Sprayed 1C leaves in the dark for 24 h contained very little urea and lower sugar levels. The untransformed sprayed leaves accumulated some organic acids, some sugars including sucrose, and urea and total amino acids. Unsprayed plants of both lines placed in the dark for 24 h showed increases in some amino acids and phosphate, and decreases in other amino acids, sugars, and organic acids. Thus the Cah enzyme can detoxify cyanamide by conversion to urea that is converted to amino acids. Other metabolic changes associated with leaf necrosis and darkness are also described. Principal component analysis confirmed the similarities and differences observed. Comparison of the GC-MS metabolic profiling analysis of amino acids with a dedicated system shows large differences, indicating a limitation of the former system. more...

Photosynthetic properties of photoautotrophic suspensions cultured in a minimal growth medium have been evaluated to determine whether changes have occurred in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, phosphoenol-pyruvate (PEP) carboxylase activity, chlorophyll content, or culture growth. Five photoautotrophic lines Amaranthus powellii, Datura innoxia, Glycine max, Gossypium hirsutum, and a Nicotiana tabacum-Nicotiana glutinosa fusion hybrid were grown in a medium without organic carbon other than phytohormones, and without vitamins. These photoautotrophic lines had total Rubisco activities ranging from 85 to 266 micromoles CO2 fixed per milligram chlorophyll hour−1, with percent activation of Rubisco ranging from 16 to 53%. Inclusion of protease inhibitors in the homogenization buffer did not result in higher Rubisco activity. PEP carboxylase activity for cells cultured in minimal medium was found to range from 16 to 146 micromoles CO2 per milligram chlorophyll hour−1, with no higher activity in the C4Amaranthus cells compared with PEP carboxylase activity in the C3 species assayed. Rubisco-to-PEP carboxylase ratios ranged from 2.2 to 1 up to 9.4 to 1. Chlorophyll contents increased in all but the Nicotiana cell line, and all of the photoautotrophic culture lines were capable of growth in vitamin-free medium with the exception of SB-P, which requires thiamine. more...

Regenerable callus cultures of Zea mays L. (maize) and organogenic callus cultures of Giycine max (L.) Merr. (soybean) were grown on media with decreased levels of N, P, K or Fe for three successive transfers on the same concentration. The soybean callus was generally more sensitive than maize to decreased levels of the elements. After three transfers soybean callus growth was completely inhibited with 1/3 of the normal Fe level and was reduced to 6 to 19% of the control growth with 1/30 of the normal medium levels of N, P or K. The maize callus growth was most sensitive to decreases in N with complete growth inhibition with 1/30 of the normal N concentration. When the P, K or Fe concentrations were 1/30 of normal, the maize callus growth was from 18 to 36% of the control. In most cases the growth decreased progressively from the first to the third transfer on the deficient media. These studies define the N, P, K and Fe concentrations which can be used for screening the maize and soybean callus s... more...

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. more...

Summary To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M 2 seedlings was screened on agar containing 0.2 mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms. more...

The polyphenolic composition of 62 wild and weedy Mexican bean collections from diverse origins, grouped by their seed coat color, was assessed. According to spectrophotometric analysis, the range of total phenols, condensed tannins, and total anthocyanins presented wide differences. Furthermore, the phenolic acid, flavonoid, and anthocyanin profiles were analyzed using HPLC. Ferulic was the main phenolic acid. Kaempferol and quercetin were the main flavonoids, and the isoflavones daidzein and coumestrol were found in only low levels in few collections. Delphinidin was the main anthocyanidin found, followed by petunidin, cyanidin, malvidin, pelargonidin, and peonidin. The wide variation observed in polyphenolic contents was more related to their genotype than to the color factor. These results show that some wild and weedy beans are good sources of phenolic compounds for use in breeding programs focused on nutrition and health. more...

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. more...

The biosynthesis of seed oil and starch both depend on the supply of carbon from the maternal plant. The biochemical interactions between these two pathways are not fully understood. In the Arabidopsis mutant shrunken seed 1 (sse1)/pex16, a reduced rate of fatty acid synthesis leads to starch accumulation. To further understand the metabolic impact of the decrease in oil synthesis, we compared soluble metabolites in sse1 and wild type (WT) seeds. Sugars, sugar phosphates, alcohols, pyruvate, and many other organic acids accumulated in sse1 seeds as a likely consequence of the reduced carbon demand for lipid synthesis. The enlarged pool size of hexose-P, the metabolites at the crossroad of sugar metabolism, glycolysis, and starch synthesis, was likely a direct cause of the increased flow into starch. Downstream of glycolysis, more carbon entered the TCA cycle as an alternative to the fatty acid pathway, causing the total amount of TCA cycle intermediates to rise while moving the steady state of the cycle away from fumarate. To convert the excess carbon metabolites into starch, we introduced the Escherichia coli starch synthetic enzyme ADP-glucose pyrophosphorylase (AGPase) into sse1 seeds. Expression of AGPase enhanced net starch biosynthesis in the mutant, resulting in starch levels that reached 37% of seed weight. However, further increases above this level were not achieved and most of the carbon intermediates remained high in comparison with the WT, indicating that additional mechanisms limit starch deposition in Arabidopsis seeds. more...

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin. more...