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.
In this short note a protocol was summarized based on almost a decade of experience on the regeneration and transformation of pepper. The recipe presented could be used efficiently in the genetic engineering of pepper. The essence of the regeneration and transformation of pepper is derived from detailed descriptions previously published by the authors on how the method was developed.
Genetic engineering of cereal crops could be refined and made more publicly acceptable by the use of promoters that direct transgene expression to particular organs at defined stages of plant development. A fusion between the barley
promoter and green fluorescence protein was used to transform bread wheat. Expression analyses showed that the
promoter is active in the organs surrounding the wheat floret at anthesis. It is not active in other vegetative and seed tissues. The organ specificity and developmental regulation of the
promoter suggest that it would be useful for engineering Fusarium head blight resistance in wheat.
Authors:E. Kiss-Bába, S. Pánczél, K. Simonyi, and G. Bisztray
Shah, P., Singh, N. K., Khare, N., Rathore, M., Anandhan, S., Arif, M., Singh, R. K., Das, S. C., Ahmed, Z., Kumar, N. (2008):
mediated genetictransformation of summer squash (
L. cv. Australian green) with
Authors:A. Tiwari, Y. Bharti, S. Tripathi, N. Mishra, M. Lal, G. Rao, P. Sharma, and M. Sharma
Manickavasagam, M., Ganapathi, A., Anbazhagan, V. R., Sudhakar, B., Selvaraj, N., Vasudevan, A. and Kasthurirengan, S. (2004): Agrobacterium mediated genetictransformation and development of herbicide-resistant sugarcane (
species hybrids) using
Authors:D. Tinak Ekom, S. Udupa, F. Gaboun, M. Benchekroun, M. Ennaji, and D. Iraqi
The use of mature embryos as explants to initiate cultures is a best alternative to save time and costs, especially for producing somatic embryos for genetic transformation of durum wheat. However, plantlets regeneration from cultures derived from matured embryos is usually low. In this study, we tested matured embryos as explants from eight Moroccan durum wheat varieties (‘Irden’, ‘Marzak’, ‘Kyperounda’, ‘Isly’, ‘Amria’, ‘Karim’, ‘Marouane’ and ‘Tomouh’) to define suitable culture media for obtaining high frequencies of somatic embryogenesis and in vitro plantlets regeneration. For this purpose, we tested five induction and maintenance media (M1 to M5) based on MS media (macro and oligo-elements) which differed with respect to concentrations of plant hormones (2,4-D and BA), vitamins, sucrose, maltose, L-asparagine, and solidifying agents. All tested media induced embryogenic callus for the varieties and regenerate plantlets. However, a significant effect of variety, medium and variety × medium interaction were observed for callus induction and regeneration. Average callus growth as measured by relative fresh weight growth rate (RFWGR) across different media was the highest for ‘Amria’ (7215.4%) and the lowest for ‘Tomouh’ (2088.2%). M1 (2 mg/L 2,4-D) and M5 (3 mg/L 2,4-D) media gave highest RFWGR(6892.1% and 6332%, respectively) and M3 (1 mg/L 2,4-D) was the lowest (3708.8%), across different varieties. However, the embryogenic callus from M3 media regenerated the highest percentage of plantlet, upon transfer to regeneration medium, for most of the varieties. For the varieties ‘Marouane’, ‘Kyperounda’, ‘Marzak’, ‘Karim’, and ‘Tomouh’, the favourable medium was M3, whereas, for ‘Isly’, ‘Irden’ and ‘Amria’, both M2 (2.5 mg/L BA and 2.5 mg/L 2,4-D) and M3 were the favourable media for embryogenic callus induction. In this study, for the first time, favourable media for induction and regeneration from mature embryo of Moroccan durum wheat varieties were identified. These media will be used for callus induction and genetic transformation.