Project description:Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several transgenic rice lines. We found that all tested transgenic plants had significant losses of methylation compared to untransformed plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. Whole genome methylation maps of rice were generated using BS-seq (Hume Stroud, Suhua Feng, Steve Jacobsen at UCLA). Whole genome expression maps of rice were generated using mRNA-seq and smRNA (Stacey Simon, Blake Meyers at Univ of Delaware).
Project description:Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of transgenic plants for most species. Our aims are to improve knowledge of gene regulatory circuits important to meristem organization, and to identify regulatory genes that might be useful for improving the efficiency of regeneration during transformation. We analyzed gene expression during poplar regeneration using an Affymetrix GeneChip® array representing over 56,000 poplar transcripts. Experiment Overall Design: We focused on callus induction and shoot formation, thus sample RNAs were collected from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. Experiment Overall Design: Two biological replicates were used for each of the five time points.
Project description:Major latex proteins (MLPs) play critical roles in defense and stress responses in plants. However, the functions of MLPs from the apple (Malus × domestica) have not been clearly characterized. Here, we focused on the biological function of MdMLP423, which was previously identified as a potential pathogenesis-related gene. Phylogenetic analysis and conserved domain analysis revealed that MdMLP423 was a protein with a `Gly-rich loop' (GXGGXG) domain and belonged to the Bet v_1 subclass. Gene expression profile revealed that MdMLP423 was predominantly expressed in the flower. Additionally, the expression of MdMLP423 was significantly inhibited by Macrophoma kawatsukai and Alternaria alternata apple pathotype (AAAP) infection. To verify the function of MdMLP423, we generated its overexpressing transgenic lines in apple callus. The MdMLP423-overexpressing callus exhibited lower resistance to Macrophoma kawatsukai and AAAP infection, as evidenced by the lower expression of resistance-related genes, higher degree and faster speed of the disease than those of non-transgenic callus. RNA-seq analysis for MdMLP423-overexpressing callus and non-transgenic callus was constructed, and the expression analysis indicated that MdMLP423 regulated the expression of a series of differential expression genes (DEGs) and transcription factors, including DEGs involved in phytohormone signaling pathways, cell wall reinforcement, defense-related proteins, AP2-EREBP, WRKY, MYB, NAC, Zinc finger protein and ABI3. Taken together, our results demonstrate that MdMLP423 plays negative regulation on Macrophoma kawatsukai and AAAP resistance through inhibiting the expression of cooperating with defense- and stress-related genes and transcription factor.
Project description:OsMKK4 is a rice MAPKK and immediately activated by treatment with chitin elicitor, a fungal MAMP. OsMKK4 phosphrylate and activate OsMPK6. We compared gene expression in osmpk6 and osmpk6 complemented cells expressing active OsMKK4 using rice 44K oligoarray. We used transgenic osmpk6 mutant and osmpk6 complemented callus expressing active OsMKK4 (OsMKK4DD) under the dexamethazone (DEX)-inducible promoter. Callus were treated with DEX or control ethanol. Samples were derived from 3 biological replicates and labeled by one-color method.
Project description:Auxin and cytokinin can regulate callus formation from developed plant organs and shoot regeneration from callus. The regulation of dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation was considered to be caused by the regulation of reprograming of callus cells, but the hypothesis had been argued still in now. Although elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus regulation. In this study, we identified the compound Fipexide (FPX) as a useful regulatory compound through chemical biology-based screening. Compared with the activity of auxin and cytokinin, FPX showed higher efficiency as a chemical inducer in callus formation, shoot regeneration, and Agrobacterium infection. In regards to morphology, the cellular organization of FPX-induced callus differed from that produced under auxin/cytokinin conditions. According to a microarray analysis, the expressions of approximately 971 genes were two-fold up-regulated by FPX treatment for 2 days. Among these genes, 598 genes were also induced by auxin/cytokinin, while 373 genes, including metabolic regulation-related genes, were specifically expressed only under FPX treatment. FPX can promote callus formations in rice, poplar, and several vegetables. FPX should be a useful tool to reveal unknown mechanisms of plant development and to increase the number of transgenic plant species.
Project description:Auxin and cytokinin can regulate callus formation from developed plant organs and shoot regeneration from callus. The regulation of dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation was considered to be caused by the regulation of reprograming of callus cells, but the hypothesis had been argued still in now. Although elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus regulation. In this study, we identified the compound Fipexide (FPX) as a useful regulatory compound through chemical biology-based screening. Compared with the activity of auxin and cytokinin, FPX showed higher efficiency as a chemical inducer in callus formation, shoot regeneration, and Agrobacterium infection. In regards to morphology, the cellular organization of FPX-induced callus differed from that produced under auxin/cytokinin conditions. According to a microarray analysis, the expressions of approximately 971 genes were two-fold up-regulated by FPX treatment for 2 days. Among these genes, 598 genes were also induced by auxin/cytokinin, while 373 genes, including metabolic regulation-related genes, were specifically expressed only under FPX treatment. FPX can promote callus formations in rice, poplar, and several vegetables. FPX should be a useful tool to reveal unknown mechanisms of plant development and to increase the number of transgenic plant species.
Project description:Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several transgenic rice lines. We found that all tested transgenic plants had significant losses of methylation compared to untransformed plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.
Project description:Background: Maize (Zea Mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. Results: We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. Conclusions: These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.
Project description:In the microarray experiment a comparison of different flower organs of transformed transgenic gerbera line to wild type gerbera was done. In the transgenic gerbera line the gene gmyb10 that regulates anthocyanin pathway is over-expressed. In this line anthocyanin pigmentation is altered compared to the wild type. In this experiment RNA from dark red callus, two stage of petal development and stamens of the transgenic lines were compared to RNA of wild type in order to identify target genes for gmyb10.