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New aspects of soybean somatic embryogenesis 总被引：4，自引：0，他引：4
Somatic embryo formation from immature cotyledons was improved in the following ways: by cutting into sections, supplementing culture media with spermine and using solid/liquid/solid type of culture. Cut cotyledons of the eight genotypes examined expressed a higher ability for somatic embryogenesis than whole cotyledons. Of the three polyamines tested, spermine considerably stimulated and putrescine slightly inhibited induction of somatic embryos. The ability of embryoid formation on medium with spermidine depended on the genotype. The solid/liquid/solid type of culture was better than the continuous solid culture. The best nitrogen ion content for the subculture of somatic embryos was 10 mM NH4NO3 and 30 mM KNO3. The possibility of using these modifications in Agrobacterium transformation is discussed. 相似文献
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Sebastian Gasparis Maciej Kała Mateusz Przyborowski Leszek A. Łyżnik Wacław Orczyk Anna Nadolska-Orczyk 《Plant methods》2018,14(1):111
Background
Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.Results
We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants.Conclusions
We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.3.
The goal of this work was to establish which enzymes – peroxidases or NADPH oxidases – play the most important role in the resistance‐related oxidative burst response of wheat to infection by brown rust (Puccinia triticina). The expression of four peroxidases and two NADPH oxidases was analysed in the susceptible wheat cv. Thatcher and isogenic lines with different Lr resistance genes after pathogen inoculation. Of the peroxidases, TaPrx118 and TaPrx112 were induced several times more strongly than TaPrx103 and TaPrx107. The induction of peroxidases was more pronounced than that of NADPH oxidases. The patterns of peroxidase expression clearly differentiated moderately resistant from highly resistant lines and corresponded to oxidative response profiles. The possible involvement of peroxidases or NADPH oxidases was verified with enzyme‐specific inhibitors. The oxidative burst in the susceptible cv. Thatcher and in the lines TcLr24, TcLr25, TcLr9 was peroxidase‐dependent, while the response in line TcLr26 was NADPH‐oxidase‐dependent. It is postulated that class III peroxidases play a leading role in the formation of reactive oxygen species molecules during the response of wheat to pathogen infection. The results suggest a high level of redundancy of some peroxidase genes induced in biotic stress. The role of both enzyme systems in wheat response/resistance to brown rust is discussed in relation to the oxidative response, the efficiency of resistance, and the presence and origin of particular Lr resistance genes. 相似文献
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The accumulation of H2O2 (oxidative burst) and the progress of pathogen development were studied in compatible and incompatible wheat‐brown rust interactions. The accumulation of H2O2 was detected in 98·7% of guard cells with appressoria 8 h post inoculation (hpi). The reaction in both susceptible and resistant plants declined 2–3 days post inoculation (dpi). The second phase of the oxidative burst was observed in the mesophyll and/or epidermis. In susceptible plants it began 4–5 dpi and was detected only in the epidermis. In resistant plants the response was observed in the mesophyll. In moderately resistant plants it was induced 1–3 dpi, and the percentage of infection units reached 80–90% 8 dpi. This corresponded with severe necrotic symptoms. In highly resistant plants, the oxidative burst was short and transient. The percentage of infection units with H2O2 accumulation reached its highest level (60–70%) 2 dpi, and decreased thereafter. Four days later, the low percentage and weak DAB staining indicated very low H2O2 accumulation. The localization and the time‐course changes of the oxidative burst correlated with the profiles of the micronecrotic response, haustorium mother cell formation and pathogen development termination. An early and localized induction of oxidative burst followed by its rapid quenching correlated with high resistance and almost no disease symptoms. The possible correlation of the oxidative burst and pathogen development patterns with the level and durability of resistance conferred by Lr genes are discussed. 相似文献
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