Yutaka Takagi

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.

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.

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.

Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean

Palmitic acid is the most abundant (approx. 11% of total fatty acids) saturated fatty acid in conventional soybean seed oil. Increasing the saturated acid content of soybean oil improves its oxidative stability and plasticity. We have developed three soybean mutants with high palmitic acid content by X-ray irradiation. In this study, we successfully identified the mutated sites of two of these high-palmitic-acid mutants, J10 and M22. PCR-based mutant analysis revealed that J10 has a 206,203-bp-long deletion that includes the GmKASIIA gene and 16 other predicted genes, and M22 has a 26-bp-long deletion in the sixth intron of GmKASIIB. The small deletion in M22 causes mis-splicing of GmKASIIB transcripts, which should result in nonfunctional products. In addition, we designed co-dominant marker sets for these mutant alleles and confirmed the association of genotypes and palmitic acid contents in F2 seeds of J10 X M22. This information will be useful in breeding programs to develop novel soybean cultivars with improved palmitic acid content. However, in the third mutant, KK7, we found no polymorphism in either GmKASIIA or GmKASIIB, which suggests that several unknown genes in addition to GmKASIIA and GmKASIIB may be involved in elevating the palmitic acid content of soybean seed oil.

A novel GmFAD3-2a mutant allele developed through TILLING reduces α-linolenic acid content in soybean seed oil

Soybean (Glycine max (L.) Merr.) oil typically contains 8% α-linolenic acid that is highly unstable and easily oxidized. This property is undesirable in many food and industrial applications. Genetic strategies for reducing α-linolenic acid content would enhance the commercial value. However, genetic resources for low α-linolenic acid content are limited among natural soybean variations. Microsomal omega-3-fatty acid desaturase (FAD3) is responsible for the synthesis of α-linolenic acid in the polyunsaturated fatty acid pathway. There are four FAD3 homologs (Glyma02g39230, Glyma11g27190, Glyma14g37350 and Glyma18g06950) in the soybean genome. While non-functional alleles have been reported for Glyma02g39230 (GmFAD3-1a) and Glyma14g37350 (GmFAD3-1b), little variation is seen in Glyma18g06950 (GmFAD3-2a). We isolated seven mutant GmFAD3-2a alleles, each containing a single-nucleotide substitution, from 39,100 independent mutant lines by using targeting induced local lesions in genomes (TILLING). Analysis of GmFAD3-2a transcripts and enzyme activities revealed that one missense mutant, ‘P1-A9’, contains a non-functional allele of GmFAD3-2a. By combining three non-functional alleles (GmFAD3-1a, GmFAD3-1b, and GmFAD3-2a), we generated soybean lines containing <2% α-linolenic acid in their seeds. The reverse-genetics-based development of novel mutant alleles in the fatty acid metabolic pathway will allow the improvement of soybean with better oil quality through conventional breeding.

Effect of some structurally altered B chromosomes on the pairing of standard B chromosomes of rye in wheat

SUMMARYMeiotic pairing of the B chromosomes of rye in the genetic environment of wheat was studied in order to investigate the effect of some structurally altered derivatives of B chromosomes on the pairing of the standard B chromosomes in the plant series containing 42 wheat chromosomes plus 2stBs + 1defB, 3stBs + 1defB, 3stB+ lsB, 3stBs+ 1siB, 3stBs+11iB, 3stBs + 2mBs, 4stBs + 1siB, 4stBs+11iB, 5stBs + 1siB, and 6stBs + 1siB chromosomes, and a comparison was made with control plants bearing only an equal number of standard B chromosomes. In the presence of deficient B, small B and small iso B chromosomes in the above plant series together with standard Bs, frequencies of pairing of the standard B chromosomes were markedly lower than the control plants containing only the corresponding number of standard Bs whereas the frequencies of standard B univalents were remarkably higher in the presence of the aforesaid derivatives of B chromosomes. This indicates that the dificient B, small B and small iso B chro...