Project description:Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In order to investigate the related genes affecting the transformation efficiency of embryogenic calli of different rice cultivars, we used Affymetrix GeneChipM-BM-. Rice Genome Array to measure the global gene expression profiling just before transformation and at four different time points after transformation (1 h, 6 h, 12 h, 24 h) in both japonica rice cultivar Nipponbare and indica rice cultivar Zhenshan 97. The mature embryo-derived embryogenic calli of Nipponbare (Nip) and Zhenshan 97 (ZS) were infected by Agrobacterium. Calli of Nip and ZS were sampled just before infection (0 h) and 1h, 6h, 12 h and 24h after infection, respectively. Three independent biological replications for each time point of the two varieties were used. To avoid the influence of polymorphisms between the probe sequence on the array and the genomes of the varieties used, we used a genomic DNA (gDNA)-based probe-selection strategy based on the hybridization efficiency of gDNA from Nip and ZS with the PM oligonucleotide probes on the rice array. The genomic DNA of Nip and ZS were extracted and hybridized to the Affymetrix Rice Genome Arrays. Three biological replications per cultivar were performed.
Project description:Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In order to investigate the related genes affecting the transformation efficiency of embryogenic calli of different rice cultivars, we used Affymetrix GeneChip® Rice Genome Array to measure the global gene expression profiling just before transformation and at four different time points after transformation (1 h, 6 h, 12 h, 24 h) in both japonica rice cultivar Nipponbare and indica rice cultivar Zhenshan 97.
Project description:Carotenoids have been demonstrated to be indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we utilized genomic expression investigation to gain a deep insight into the carotenoid-related biological processes in the citrus calli overexpressing CrtB. Abortive ovule embryogenic calli from four citrus genotypes were used in this study. They were derived from Star Ruby grapefruit (C. paradise Macf.), Marsh grapefruit (C. paradise Macf.), and Sunburst mandarin [Citrus reticulata Blanco M-CM-^W (C. paradisi Macf. M-CM-^W C. reticulata)], designated as RB, M, and SBT, respectively. Engineered cell models (ECMs) were established by over-expressing 35S::CrtB (tpM-bM-^@M-^SrbcSM-bM-^@M-^SCrtB) [CrtB protein, phytoene synthase from Erwinia herbicola (now known as Pantoea agglomerans), containing a Pea rbcS transit peptide] in citrus embryogenic calli. Twenty-day-old calli were harvested and used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR. Examination of 2 different small RNA expression profilings in 2 developmental stages of meristems.
Project description:Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR.
Project description:Four days old rice calli were selected to grow 324h under spaceflight controls, 1g-flight controls and 1g-ground controls. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Rice calli were selected at different treatment for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the diff-genes that caused by the microgravity.
Project description:Four days old rice calli were selected to grow 324h under spaceflight controls, 1g-flight controls and 1g-ground controls. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process.
Project description:Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (ECs) and nonembryogenic calli (NECs) of C. papaya using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. ECs and NECs presented remarkable differences in anatomical and histochemical characteristics. ECs showed greater reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate the role of mitochondrial metabolism in the embryogenic competence of C. papaya calli. Greater participation of alternative oxidase (AOX) enzymes in respiration, as well as stronger accumulation of mitochondrial stress response proteins, was observed in ECs. In addition, ECs showed a greater abundance of proteins related to oxidative phosphorylation and higher total respiration (TR). Auxin-responsive Gretchen Hagen 3 (GH3) family proteins may play an important role in decreasing the contents of free 2,4-dichlorophenoxyacetic acid (2,4-D) in ECs. The accumulation of stress response proteins among total proteins was observed in ECs. ECs also showed higher endogenous hydrogen peroxide (H2O2) contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos.
Project description:Somatic embryogenesis (SE) is the development of embryo-like structures from somatic plant tissues. Recently, weve shown that transcription factor MtWOX9-1 belonging to WOX family is able to stimulate SE in the callus culture in Medicago truncatula. In this research, transcriptomic analysis of highly embryogenic calli with MtWOX9-1 overexpression was performed in comparison with wildtype calli. It was shown that MtWOX9-1 overexpression leads to the activation of several groups of genes, including genes related with cell division and tissue differentiation, and also with seed development. Enriched GO pathways included several groups related with histone methyltransferase activity as well as DNA methylation and chromatin binding, suggesting major epigenetic changes occuring in MtWOX9-1 overexpressing calli.