Genotyping-by-Sequencing Analysis Reveals Associations between Agronomic and Oil Traits in Gamma Ray-Derived Mutant Rapeseed (Brassica napus L.).

Rapeseed (Brassica napus L.) holds significant commercial value as one of the leading oil crops, with its agronomic features and oil quality being crucial determinants. In this investigation, 73,226 single nucleotide polymorphisms (SNPs) across 95 rapeseed mutant lines induced by gamma rays, alongside the original cultivar ('Tamra'), using genotyping-by-sequencing (GBS) analysis were examined. This study encompassed gene ontology (GO) analysis and a genomewide association study (GWAS), thereby concentrating on agronomic traits (e.g., plant height, ear length, thousand-seed weight, and seed yield) and oil traits (including fatty acid composition and crude fat content). The GO analysis unveiled a multitude of genes with SNP variations associated with cellular processes, intracellular anatomical structures, and organic cyclic compound binding. Through GWAS, we detected 320 significant SNPs linked to both agronomic (104 SNPs) and oil traits (216 SNPs). Notably, two novel candidate genes, Bna.A05p02350D (SFGH) and Bna.C02p22490D (MDN1), are implicated in thousand-seed weight regulation. Additionally, Bna.C03p14350D (EXO70) and Bna.A09p05630D (PI4Kα1) emerged as novel candidate genes associated with erucic acid and crude fat content, respectively. These findings carry implications for identifying superior genotypes for the development of new cultivars. Association studies offer a cost-effective means of screening mutants and selecting elite rapeseed breeding lines, thereby enhancing the commercial viability of this pivotal oil crop.

Identification of Loci Governing Agronomic Traits and Mutation Hotspots via a GBS-Based Genome-Wide Association Study in a Soybean Mutant Diversity Pool.

In this study, we performed a genotyping-by-sequencing analysis and a genome-wide association study of a soybean mutant diversity pool previously constructed by gamma irradiation. A GWAS was conducted to detect significant associations between 37,249 SNPs, 11 agronomic traits, and 6 phytochemical traits. In the merged data set, 66 SNPs on 13 chromosomes were highly associated (FDR p < 0.05) with the following 4 agronomic traits: days of flowering (33 SNPs), flower color (16 SNPs), node number (6 SNPs), and seed coat color (11 SNPs). These results are consistent with the findings of earlier studies on other genetic features (e.g., natural accessions and recombinant inbred lines). Therefore, our observations suggest that the genomic changes in the mutants generated by gamma irradiation occurred at the same loci as the mutations in the natural soybean population. These findings are indicative of the existence of mutation hotspots, or the acceleration of genome evolution in response to high doses of radiation. Moreover, this study demonstrated that the integration of GBS and GWAS to investigate a mutant population derived from gamma irradiation is suitable for dissecting the molecular basis of complex traits in soybeans.

Stimuli-Responsive Drug Delivery of Doxorubicin Using Magnetic Nanoparticle Conjugated Poly(ethylene glycol)-g-Chitosan Copolymer.

Stimuli-responsive nanoparticles are regarded as an ideal candidate for anticancer drug targeting. We synthesized glutathione (GSH) and magnetic-sensitive nanocomposites for a dual-targeting strategy. To achieve this goal, methoxy poly (ethylene glycol) (MePEG) was grafted to water-soluble chitosan (abbreviated as ChitoPEG). Then doxorubicin (DOX) was conjugated to the backbone of chitosan via disulfide linkage. Iron oxide (IO) magnetic nanoparticles were also conjugated to the backbone of chitosan to provide magnetic sensitivity. In morphological observation, images from a transmission electron microscope (TEM) showed that IO nanoparticles were embedded in the ChitoPEG/DOX/IO nanocomposites. In a drug release study, GSH addition accelerated DOX release rate from nanocomposites, indicating that nanocomposites have redox-responsiveness. Furthermore, external magnetic stimulus concentrated nanocomposites in the magnetic field and then provided efficient internalization of nanocomposites into cancer cells in cell culture experiments. In an animal study with CT26 cell-bearing mice, nanocomposites showed superior magnetic sensitivity and then preferentially targeted tumor tissues in the field of external magnetic stimulus. Nanocomposites composed of ChitoPEG/DOX/IO nanoparticle conjugates have excellent anticancer drug targeting properties.

Differential Gene Expression Associated with Altered Isoflavone and Fatty Acid Contents in Soybean Mutant Diversity Pool.

Soybean seeds are consumed worldwide owing to their nutritional value and health benefits. In this study we investigated the metabolic properties of 208 soybean mutant diversity pool (MDP) lines by measuring the isoflavone and fatty acid contents of the seed. The total isoflavone content (TIC) ranged from 0.88 mg/g to 7.12 mg/g and averaged 3.08 mg/g. The proportion of oleic acid among total fatty acids (TFA) ranged from 0.38% to 24.66% and averaged 11.02%. Based on the TIC and TFA among the 208 MDP lines, we selected six lines with altered isoflavone content and six lines with altered oleic acid content compared with those of the corresponding wild-types for measuring gene expression. Each of twelve genes from the isoflavone and fatty acid biosynthesis pathways were analyzed at three different seed developmental stages. Isoflavone biosynthetic genes, including CHI1A, IFS1, and IFS2, showed differences in stages and expression patterns among individuals and wild-types, whereas MaT7 showed consistently higher expression levels in three mutants with increased isoflavone content at stage 1. Expression patterns of the 12 fatty acid biosynthetic genes were classifiable into two groups that reflected the developmental stages of the seeds. The results will be useful for functional analysis of the regulatory genes involved in the isoflavone and fatty acid biosynthetic pathways in soybean.

Development of vitrification protocol in Rubia akane (nakai) hairy roots using a systematic approach.

BACKGROUND: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions. OBJECTIVE: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions. METHODS: Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35%and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions. RESULTS: Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20 degree C and 40 degree C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress. CONCLUSION: R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.

Improved cryopreservation of chrysanthemum (Chrysanthemum morifolium) using droplet-vitrification.

A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5 percent glycerol + 17.5 percent sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50 percent glycerol + 50 percent sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9 percent and 84.9 percernt, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum.

Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation.

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.