Authors:A. Behpouri, A. Perochon, F.M. Doohan, and C.K.-Y. Ng
Brachypodium distachyon has emerged as the model species for important temperate grass crops such as wheat and barley and the genome of the B. distachyon community inbred line Bd21 has been sequenced. Methods for tissue culture and Agrobacterium-mediated transformation have been developed for this model grass as a resource for reverse genetics and functional genomic analyses. In order to obtain a high quantity and quality of compact embryogenic callus (CEC) in B. distachyon, it is important to examine and optimize the optimal concentration of the auxin 2,4-D (dichlorophenoxyacetic acid) to use in both callus induction and callus proliferation media. Here, we investigated the effects of different concentrations of 2,4-D on callus induction and callus proliferation of B. distachyon Bd21. Our results showed that 2.5 mg l–1 2,4-D is an optimal concentration to use for both callus induction and proliferation, although 5.0 mg l–1 may also be used for callus proliferation. Additionally, the suitability of hygromycin or bialaphos as selectable markers was examined and results indicated that hygromycin is significantly more efficient than bialaphos when using the Agrobacterium-mediated transformation system.
Authors:R. Murín, K. Mészáros, P. Nemeček, R. Kuna, and J. Faragó
The effect of explant type (immature vs. mature embryos) and two auxin types (2,4-dichlorophenoxyacetic acid vs. Dicamba) on the callogenesis and plant regeneration ability of 26 wheat cultivars was studied. In general, the callus induction, plant regeneration and shoot formation frequencies were higher in mature embryo-derived cultures as compared to immature ones on media originally developed for mature wheat embryo cultures. In both culture types, the auxin Dicamba was found to be more efficient, especially when mature embryos were cultured. The separation of means using Duncan’s multiple range test revealed the best in vitro response, in terms of the frequency of callus regeneration, in the cultivar Astella for both immature and mature embryo cultures. This cultivar gave very promising results, suggesting that it could be used in the future for further tissue culture investigations and as a donor material for genetic transformation experiments in wheat. Correlation analyses revealed significant similarities between the evaluated parameters within each group (immature and mature embryo-derived cultures). However, there were no significant correlations between these two groups for most of the parameters. This suggests that the mechanism of plant regeneration in the two in vitro regeneration systems (mature vs. immature embryo culture) may be different enough to hamper the development of an optimal plant regeneration protocol for use in both systems.
The first steps in studies on the female and male gametophytes of wheat involved the light microscope analysis of semi-thin sections of embryo sacs containing egg-cells developing in planta. The information thus obtained on the development of the egg-cell from its initial formation to maturity contributed to the successful isolation of egg-cells. The morphological and ultrastructural details of egg-cells isolated 3, 6, 9, 12, 15 and 18 days after emasculation were examined to determine the function of the female gametophyte and its suitability for micromanipulation. A sufficient number of gametoplasts in the right stage of development are required for the successful microinjection and in vitro fusion of egg-cells and male gametes. No data are yet available on the fusiogenicity of wheat egg-cells in various stages of development. Various in vitro fertilisation and microinjection techniques could be of service in gamete fusion experiments aimed at the creation of interspecific and intergeneric hybrids which do not occur in nature due to sporophytic incompatibility. The results acquired in investigations on immature embryos can be used for the study of embryos developing from egg-cells fertilised in vitro.
Authors:H. Aadel, R. Abdelwahd, S.M. Udupa, G. Diria, A. El Mouhtadi, K. Ahansal, F. Gaboun, A. Douira, and D. Iraqi
Although significant progress has been made on Agrobacterium-mediated wheat transformation, current methodologies using immature embryos as recipient tissues are labor intensive, time consuming and expensive. The use of mature embryos as explants is increasingly being recognized as an optimal method for developing regenerable cell lines during wheat transformation. Therefore, we have developed an Agrobacterium-based transformation protocol using mature embryos while adjusting several factors that influence genetic transformation efficiency. In this study, we focussed on acetosyringone concentrations, genotypes and different types of mature embryos (intact or longitudinally halved-embryos or fragmented into four latitudinal pieces) used as a source of explants for the genetic transformation. A. tumefaciens strain EHA101 harboring the plasmid vector pTF101.1 carrying the barley HVA1 gene and bar-selectable marker gene were used. Mature intact-embryos and longitudinally halved-embryos yielded the highest number of putative transgenic plantlets on the selection medium. However, no plantlets were obtained from latitudinal fragmented mature embryos. ‘Amal’ and ‘Rajae’ genotypes regenerated the highest number of putative transgenic plants and 200 μM acetosyringone was found to be the optimal concentration for their transformation. A total of 47 transgenic plants were selected with 11 plantlets showing resistance to leaf painting. Molecular analysis revealed that 1% and 0.66% of T0 regenerated plantlets were successfully transformed and carried the HVA1 gene for the ‘Amal’ and ‘Rajae’ genotypes, respectively. Additional analysis shows the transgene is stably inherited in the T1 generation. Based on the results, we conclude that among the influencing factors tested, genotypes, mature embryo explant types and acetosyringone concentration contribute significantly to the success of bread wheat transformation.