Find in Library

Transformation of cereals is one of the emerging areas for plant genomic and biotechnology research. Wheat was among the last major crops to be transformed by particle bombardment about 10 years ago. However, Agrobacterium-mediated transformation has several advantages over bombardment, including a reduction in copy number, fewer rearrangements and preferential integration into transcriptionally active chromosome regions. As a first step, we started to adapt an immature embryo-based transformation method for the model variety ‘Cadenza’. The regeneration of this variety was low and especially the cost of generating donor plants was high. Therefore, we decided (i) to test regeneration capacity of winter and spring wheats using four different explants, (ii) to determine the optimal genotype-regeneration system combinations, and (iii) to work out the details of mature embryo transformation with Agrobacterium. The experiment was carried out with 16 cultivated winter wheat and 2 model spring wheat varieties. Four different explants: anther, immature embryo, mature embryo and dry seed were tested for callus induction and plant regeneration. The regeneration capacity was the lowest in the case of anther culture and ranged from 20% (‘Mv Béres’) to 0.1% (‘Mv Magvas’) with four varieties exerting significantly higher regeneration than ‘Cadenza’. Plant regeneration from immature embryos ranged between 59% (‘Mv Regiment’) and 0.1% (‘Mv Toborzó’). Again, four varieties produced significantly more plants than the control ‘Cadenza’. We tested two systems for the plant regeneration from mature embryos. First, mature embryos were isolated from seeds, which resulted in an average of 17% plant regeneration (from 63% in ‘Fatima’ to zero in ‘Mv Palotás’). ‘Cadenza’ was one of the worse regenerating genotype (7%). The highest plant regeneration (average 54%) was in the case of seed explants. There were no significant differences between the varieties tested. Comparing the regeneration reaction in the different regeneration systems we can conclude that each variety has good regeneration capacity in at least one or more regeneration systems. Based on these results we started optimizing the Agrobacterium-mediated transformation of mature and immature embryos of three cultivars for each method. Induction of vir genes was confirmed in the presence of embryos as well as induced calli. Transient expression of the GUS reporter gene was demonstrated after 3 and 5 days of cocultivation and selected shoots are currently being regenerated.