Shaikh M. Rahman

Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene.

Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid α-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, α-linolenic acid content of the T1 seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T2 and T3 progenies. These results demonstrate that the α-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.

Inheritance of high oleic acid content in the seed oil of soybean mutant M23.

A mutant line, M23, of soybean [Glycine max (L.) Merr.] was found to have two fold increases in oleic acid content in the seed oil compared with the original variety, Bay. Our objective was to determine the inheritance of the high oleic acid content in this mutant. Reciprocal crosses were made between M23 and Bay. There were no maternal and cytoplasmic effects for oleic acid content. The F1 seeds and F1 plants were significantly different from either parents or the midparent value, indicating partial dominance of oleic acid content in these crosses. The oleic acid content segregated in the F2 seeds and F2 plants in a trimodal pattern with normal, intermediate and high classes, satisfactorily fitting a 1∶2∶1 ratio. The seeds of a backcross between M23 and F1 segregated into intermediate and high classes in a ratio of 1∶1. These results indicated that oleic acid content was controlled by two alleles at a single locus with a partial dominant effect. Thus, the allele in M23 was designated ol and the genotypes of M23 and Bay were determined to be olol and 0l0l, respectively. The oleic acid contents of the F2 seeds and F2 plants were inversely related with the linoleic acid content which segregated in a trimodal pattern with normal, intermediate and low classes in a 1∶2∶1 ratio. Thus, it was assumed that the low linoleic acid content in M23 was also controlled by the ol alleles. Because a diet with high oleic acid content reduces the content of low density lipoprotein cholesterol in blood plasma, the mutant allele, ol, would be useful in improving soybean cultivars for high oleic acid content.

Genetic control of high stearic acid content in seed oil of two soybean mutants

Abstract Stearic acid is one of the two saturated fatty acids found in soybean [Glycine max (L.) Merr.] oil, with its content in the seed oil of commercial cultivars averaging 4.0%. Two mutants, KK-2 and M25 with two- and six-fold higher stearic acid contents in the seed oil than cv ‘Bay’, were identified after X-ray seed irradiation. Our objective was to determine the genetic control of high stearic acid content in these mutants. Reciprocal crosses were made between each mutant and ‘Bay’, and between the two mutants. No maternal effect for stearic acid content was observed from the analysis of F1 seeds in any of the crosses. Low stearic acid content in ‘Bay’ was partially dominant to high stearic acid content in KK-2 and M25, and high stearic acid content in KK-2 was partially dominant to high stearic acid content in M25. Cytoplasmic effects were not observed, as demonstrated by the lack of reciprocal cross differences for stearic acid content in our analysis of F2 seeds from F1 plants. The stearic acid content in F2 seeds of KK-2בBay’ and M25בBay’ crosses segregated into three phenotypic classes which satisfactorily fit a 1:2:1 ratio, indicating that high stearic acid content in KK-2 and M25 was controlled by recessive alleles at a single locus. The data for stearic acid content in F2 seeds of the KK-2×M25 cross satisfactorily fit a 3:9:1:3 phenotypic ratio. The F2 segregation ratio and the segregation of F3 seeds from individual F2 plants indicated that KK-2 and M25 have different alleles at different loci for stearic acid content. The alleles in KK-2 and M25 have been designated as st1 and st2, respectively. The stearic acid content (>30.0%) found in the st1st1st2st2 genotype is the highest known to date in soybean, but it was not possible to develop the line with this genotype because the irregular seeds failed to grow into plants after germination. Therefore, tissue culture methods must be developed to perpetuate this genotype.

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.

Inheritance of reduced linolenic acid content in soybean seed oil

Abstract Linolenic acid is the unstable component of soybean [Glycine max (L.) Merr.] oil that is responsible for the undesirable odors and flavors commonly associated with poor oil quality. Two mutants, M-5 and KL-8, have been identified that have lower linolenic acid levels in the seed oil than the ‘Bay’ cultivar. Our objective was to determine the relationships between the genetic systems controlling linolenic acid in these mutants. Reciprocal crosses were made between the mutants and ‘Bay’, and between the two mutants. No maternal effect for linolenic acid content was observed from the analysis of F1 seeds in any of the crosses. The data for linolenic acid content in F2 seeds of M-5בBay’ and KL-8בBay’ crosses satisfactorily fit a 1 : 2 : 1 and 3 : 1 ratio, respectively. For the M-5×KL-8 cross, segregation observed from the analysis of F2 seeds for linolenic acid content satisfactorily fit a ratio of 3 more than either mutant: 12 within the range of the two mutants: 1 less than either mutant. The segregation ratio of F2 seeds and the segregation of F3 seeds from F2 plants indicated that M-5 and KL-8 have alleles at different loci that control linolenic acid content. The allele in KL-8 has been designated as fanx (KL-8) to distinguish it from fan (M-5). The low linolenic acid segregates with the genotype fanfanfanxfanx provide additional germplasm to reduce the linolenic acid content from the seed oil of soybean.

Differential response of methionine metabolism in two grain legumes, soybean and azuki bean, expressing a mutated form of Arabidopsis cystathionine γ-synthase.

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

NK-104, A Potent New 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitor, Enhances Posttranslational Catabolism of Apolipoprotein B-100 and Inhibits Secretion of Apolipoprotein B-100 and Triacylglycerols from HepG2 Cells

Abstract The effects of NK-104, a new, potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on the secretion and intracellular catabolism of apolipoprotein B-100 (apo) and on lipid metabolism in HepG2 cells were investigated. The cells were treated with NK-104 1 or 10 uM for 3 or 24 hours in 1% bovine serum albumin-containing medium. To examine intracellular apo B catabolism, the cells were labeled with 85 S-methionine for 10 minutes and followed for up to 90 minutes. NK-104 reduced the secretion of apo B and 35 S-labeled apo B. The degradation rate of intracellular 35 S-labeled apo B was faster in cells treated with NK-104 than in controls, suggesting that apo B stability was decreased by treatment with NK-104. NK-104 reduced significantly the intracellular levels and synthesis of cholesterol and cholesteryl ester in HepG2 cells. The agent reduced the secretion of labeled triacylglycerols without affecting intracellular triacylglycerol synthesis. Results of this study suggest that NK-104 reduced the secretion of apo B by enhancing degradation of the protein. This effect may be mediated through changes in the intracellular metabolism of cholesterol or cholesteryl ester.

Efficient in vitro germination and shoot proliferation of chilling-treated water chestnut (Trapa japonica Flerov) embryonal explants

SummaryEmbryonal explants from water chestnut (Trapa japonica Flerov) seeds germinated with high efficiency following a 40-d cold treatment at 5°C on half-strength MS (Murashige and Skoog) medium supplemented with 2.7 μM N6-benzyladenine (BA), 0.5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM gibberellic acid (GA3). Control and chill-treated (different durations) embryonal explants were cultured onto media which contained half-strength MS medium supplemented with different concentrations and combinations of cytokinins [BA, thidiazuron (TDZ), kinetin, zeatin], auxin (NAA) and GA3. A liquid half-strength MS medium with 1.1 μM BA and 0.5 μM NAA resulted in the best shoot proliferation of control or chill-treated explants, and the addition of 0.5 μM GA3 stimulated axillary shoot elongation. Germination and shoot proliferation were always greater for chill-treated explants compared with control explants under the same culture conditions. Shoots produced in vitro rooted 100% of the time in a liquid half-strength MS medium with 1.1 μM BA, 0.5 μM NAA and 1.1 μM indole-3-butyric acid, and the regenerated plantlets were established successfully in a water chestnut paddy field.