Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02836.x

Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

World consumption of soybean ( Glycine max ) in 2008 was over 221 million metric tons, with approximately 50% of this supply coming from U.S. production, where soybean plantings on an annual basis are over 77 million ha. Soybeans are desired on the marketplace as a valuable source of protein and oil

https://www.jstor.org/stable/pdfplus/40537933.pdf

Soybean Oil: Genetic Approaches for Modification of Functionality and Total Content

Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12201

Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family

Soybean (Glycine max [L.] Merr.) is one of the most important oil and protein crops. Ever-increasing soybean consumption necessitates the improvement of varieties for more efficient production. However, both correlations among different traits and genetic interactions among genes that affect a single trait pose a challenge to soybean breeding. To understand the genetic networks underlying phenotypic correlations, we collected 809 soybean accessions worldwide and phenotyped them for two years at three locations for 84 agronomic traits. Genome-wide association studies identified 245 significant genetic loci, among which 95 genetically interacted with other loci. We determined that 14 oil synthesis-related genes are responsible for fatty acid accumulation in soybean and function in line with an additive model. Network analyses demonstrated that 51 traits could be linked through the linkage disequilibrium of 115 associated loci and these links reflect phenotypic correlations. We revealed that 23 loci, including the known Dt1, E2, E1, Ln, Dt2, Fan, and Fap loci, as well as 16 undefined associated loci, have pleiotropic effects on different traits. This study provides insights into the genetic correlation among complex traits and will facilitate future soybean functional studies and breeding through molecular design.

/pdf/genome-wide-association-studies-dissect-the-genetic-networks-1p4qp4vxpx.pdf

Genome-wide association studies dissect the genetic networks underlying agronomical traits in soybean

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.

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

Dry seeds of soybean [Glycine max (L.) Merr. var. Bay] were irradiated by X-rays (21.4 kR) and the M2 generations were examined for the oleic acid content in their oil. The genetic variability of oleic acid content in the oil of the Bay variety was significantly increased by the X-ray treatment when compared with the Bay control. A mutant, designated as M23, was selected from 2747 M2 plants, having an oleic acid content of 46.1%, twice as much as that in the normal Bay variety. A distinct inverse relationship was observed between the oleic and linoleic acid contents in M23. Mutant M23 isolated from the M2 generations was confirmed to be always associated with a high oleic acid content under different environmental conditions in the M3 generations.

High Oleic Acid Mutant in Soybean Induced by X-Ray Irradiation

Identification of corresponding genes for three low-α-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed

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

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

The generation of useful mutant alleles of specific genes would accelerate conventional breeding programs in various commercially important crops. Common soybean oil is easily oxidized because it is rich in polyunsaturated fatty acids (PUFAs). Microsomal omega-6 fatty acid desaturase (FAD2), which introduces a second unsaturated bond into oleic acid, is a primary target for elevating oleic acid levels and reducing PUFA levels. The paleopolyploid soybean genome contains five FAD2 gene homologues, at least three of which (GmFAD2-1a, 2-1b, and 2-2a) are functional. In spite of their importance, very little genetic variation has been identified in these genes except in GmFAD2-1a, because fatty acid content is easily affected by environmental conditions such as temperature. Here we isolated novel mutant alleles of GmFAD2-1b from ethyl methanesulfonate-treated soybean mutant populations through Targeting Induced Local Lesions In Genomes (TILLING), a reverse genetic method. Evaluation of enzyme activity in a yeast heterologous expression system suggested that two mutant lines, ‘B12’ and ‘E11’, contain near-null and null alleles, respectively, of GmFAD2-1b. Furthermore, by combining GmFAD2-1a and GmFAD2-1b mutant alleles, we successfully generated soybean lines with >80% oleic acid content. TILLING could provide a practical method for expanding the genetic diversity of polyploid crops.

https://www.jstage.jst.go.jp/article/jsbbs/60/4/60_4_419/_pdf

Novel GmFAD2-1b mutant alleles created by reverse genetics induce marked elevation of oleic acid content in soybean seeds in combination with GmFAD2-1a mutant alleles

Yutaka Takagi

Papers

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

High Oleic Acid Mutant in Soybean Induced by X-Ray Irradiation

Identification of corresponding genes for three low-α-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed

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

Novel GmFAD2-1b mutant alleles created by reverse genetics induce marked elevation of oleic acid content in soybean seeds in combination with GmFAD2-1a mutant alleles