Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

Nishizawa, Keito; Takagi, Kyoko; Teraishi, Masayoshi; Kita, Akiko; Ishimoto, Masao

doi:10.5511/plantbiotechnology.10.0823a

Cited by 10 publications

(7 citation statements)

References 74 publications

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“…Down-regulation of the gene encoding Gly m Bd 30 K greatly suppresses the accumulation of the targeted protein in seeds of transgenic soybean [26]. The accumulation of α-, α'-, and β-subunits of 7S globulin in soybean seeds can be greatly decreased through RNA interference or artificial microRNA systems [27,28]. Recently, the transcription activator-like effector nucleases (TALE Ns) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) systems have become the main platforms for site-directed mutagenesis in higher plants [29][30][31][32].…”

Section: Introductionmentioning

confidence: 99%

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

et al. 2020

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Background Soybean (Glycine max) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods. Recently, the CRISPR/Cas9 system has been adopted as a powerful tool for the site-directed mutagenesis in higher plants. This system is expected to generate hypoallergenic soybean varieties. Results We used two guide RNAs (gRNAs) and Agrobacterium-mediated transformation for simultaneous site-directed mutagenesis of two genes encoding the major allergens Gly m Bd 28 K and Gly m Bd 30 K in two Japanese soybean varieties, Enrei and Kariyutaka. We obtained two independent T0 Enrei plants and nine T0 Kariyutaka plants. Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations were induced in both targeted loci of both soybean varieties. Sequencing analysis showed that deletions were the predominant mutation type in the targeted loci. The Cas9-free plants carrying the mutant alleles of the targeted loci with the transgenes excluded by genetic segregation were obtained in the T2 and T3 generations. Variable mutational spectra were observed in the targeted loci even in T2 and T3 progenies of the same T0 plant. Induction of multiple mutant alleles resulted in six haplotypes in the Cas9-free mutants derived from one T0 plant. Immunoblot analysis revealed that no Gly m Bd 28 K or Gly m Bd 30 K protein accumulated in the seeds of the Cas9-free plants. Whole-genome sequencing confirmed that a Cas9-free mutant had also no the other foreign DNA from the binary vector. Our results demonstrate the applicability of the CRISPR/Cas9 system for the production of hypoallergenic soybean plants. Conclusions Simultaneous site-directed mutagenesis by the CRISPR/Cas9 system removed two major allergenic proteins from mature soybean seeds. This system enables rapid and efficient modification of seed components in soybean varieties.

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Section: Introductionmentioning

confidence: 99%

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

et al. 2020

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“…Metabolic pathways for soybean seed components and their regulation at the molecular level should provide important information for improvement of seed quality by genetic engineering (Nishizawa et al , 2010). However, due to the long life cycle of soybeans, using developing seed (zygotic embryos) as an experimental system is neither an efficient nor a practical approach for screening the large number of genes (by knockout, overexpression, or underexpression) implicated in seed quality.…”

Section: Introductionmentioning

confidence: 99%

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition

Truong

Koch

Yoon

et al. 2013

Journal of Experimental Botany

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Soybean [Glycine max (L.) Merr.] seed are valued for their protein and oil content. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. This report describes the growth dynamics and compositional changes of SHaM embryos in response to change in the carbon to nitrogen ratio of the medium. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates, embryo protein content was strongly correlated with decreasing C:N ratios and increasing glutamine consumption rates. However, oil content remained relatively unchanged across the C:N ratio range tested, and resources were instead directed towards the starch and residual biomass (estimated by mass balance) pools in response to increasing C:N ratios. Protein and oil were inversely related only at concentrations of sucrose in the medium <88mM, where carbon limited growth and no starch was found to accumulate in the tissues. These observations and the high reproducibility in the data indicate that SHaM embryos are an ideal model system for the application of metabolic flux analysis studies designed to test hypotheses regarding assimilate partitioning in developing soybean seeds.

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“…Efforts have been made to improve the quality and quantity of macronutrients such as protein/amino acids, oils/fats and carbohydrates as well as to increase the bioavailability of micronutrients, vitamins and antioxidants through transgenics and breeding. In soya bean, most of the work was carried out to improve seed protein quality (Cunha et al ., ; Nishizawa et al ., ; Qi et al ., ), oil content (Kajikawa et al ., ; Li et al ., ), biotic resistance (Cunha et al ., ; Dang and Wei, ; McLean et al ., ; Tougou et al ., ) and abiotic resistance (DeRonde et al ., ; Valente et al ., ). However, on the other hand, there have been limited efforts in the management of antinutrients and/or toxins, which are the by‐products of plant metabolism and considered as health hazards to humans and animals.…”

Section: Discussionmentioning

confidence: 99%

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

Kumar

Chattopadhyay

Ghosh

et al. 2016

Plant Biotechnology Journal

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SummarySoya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of b-N-oxalyl-L-a,b-diaminopropionic acid (b-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, b-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDCexpressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.

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Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

Cited by 10 publications

References 74 publications

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

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