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Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos.

ABSTRACT: In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize 'Qi319' IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25 min. We used A. tumefaciens to inoculate IEs derived from maize ears 9-15 d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~?3 to 4 times the %GFP compared with MC-treated IEs at 8 d after inoculation (3 d co-culture and 5 d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs.

SUBMITTER: Duan X

PROVIDER: S-EPMC6419711 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos.

Duan Xueqing X Zheng Liru L Sun Jinhao J Liu Wenbo W Wang Wenqian W An Hailong H

Physiology and molecular biology of plants : an international journal of functional plant biology 20190209 2

In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize 'Qi319' IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL ...[more]

PMID: 30956435

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Project description:The transformation efficiency (TE) was improved by a series of special chemical and physical methods using immature embryos from the cultivar Fielder, with the PureWheat technique. To analyze the reaction of immature embryos infected, which seemed to provide the necessary by Agrobacterium tumefaciens in PureWheat, a combination of scanning electron microscopy (SEM), complete transcriptome analysis, and metabolome analysis was conducted to understand the progress. The results of the SEM analysis revealed that Agrobacterium tumefaciens were deposited under the damaged cortex of immature embryos as a result of pretreatment and contacted the receptor cells to improve the TE. Transcriptome analysis indicated that the differentially expressed genes were mainly enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, plant-pathogen interaction, plant hormone signal transduction, and the MAPK (Mitogen-activated protein kinase) signaling pathway. By analyzing the correlation between differentially expressed genes and metabolites, the expression of many genes and the accumulation of metabolites were changed in glucose metabolism and the TCA cycle (Citrate cycle), as well as the amino acid metabolism; this suggests that the infection of wheat embryos with Agrobacterium is an energy-demanding process. The shikimate pathway may act as a hub between glucose metabolism and phenylpropanoid metabolism during Agrobacterium infection. The downregulation of the F5H gene and upregulation of the CCR gene led to the accumulation of lignin precursors through phenylpropanoid metabolism. In addition, several metabolic pathways and oxidases were found to be involved in the infection treatment, including melatonin biosynthesis, benzoxazinoid biosynthesis, betaine biosynthesis, superoxide dismutase, and peroxidase, suggesting that wheat embryos may be under the stress of Agrobacterium and, thus, undergo an oxidative stress response. These findings explore the physiological and molecular changes of immature embryos during the co-culture stage of the PureWheat technique and provide insights for Agrobacterium-mediated transgenic wheat experiments.

Project description:The insect resistant maize YieldGard MON810 was studied to assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes by comparison of various GM lines vs. their non-transgenic counterparts. To assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes, GM lines of the insect resistant maize YieldGard MON810 were compared with non-transgenic counterparts. For a more in-depth study, high-throughput deep sequencing together with microarrays were used to compare the transcriptomes of immature embryos of the MON810 variety DKC6575, with a cryIA(b) transgene, and its near-isogenic variety Tietar, grown under controlled environmental conditions. This technique also allows characterisation of the transgenic mRNAs produced. 3'UTR-anchored mRNA-seq produced 1,802,571 sequences from DKC6575 and 1,170,973 from Tietar, which mapped to 14,712 and 14,854 unigenes, respectively. Up to 32 reads from the transgenic embryos matched to the synthetic cry1A(b) sequence, similar to medium-abundant mRNAs. Gene expression analysis, using the R-bioconductor packages EdgeR and DEseq, revealed 140 differentially expressed genes mainly involved in carbohydrate metabolism, protein metabolism and chromatin organisation. Comparison of the expression of 30 selected genes in two additional MON810 and near-isogenic variety pairs showed that most of them were differentially expressed in the three pairs of varieties analysed. Analysis of functional annotation and the precise moment of expression of the differentially expressed genes and physiological data obtained suggest a slight but significant delay in seed and plant maturation of MON810 plants. However, these transcriptomic changes were not associated to undesirable changes in the global phenotype or plant behaviour. Moreover, while most expression changes in MON810 immature embryos were maintained in other transgenic varieties, some gene expression was found to be modulated by the genetic background in which the transgene was introduced through conventional breeding programs.

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Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos.

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Co-culturing on dry filter paper significantly increased the efficiency of <i>Agrobacterium</i>-mediated transformations of maize immature embryos.

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