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.
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.
Authors:M. Özgen, M. Yildiz, N. Koyuncu, and S. Önde
., Chowdhury, S.H., Harada, S. 1991. Effect of culture condition on plant regeneration capacity of matureembryo derived callus in wheat (
L.). Wheat Information Service
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.
Authors:Feruzan Dane, Göksel Olgun, and Nuran Ekici
In this study ultrastructural differences between endothelial cells of different location in
have been examined with electron microscope at mature embryo sac phase. Embryo sac is of the
type and surrounded by endothelium except the micropylar region. The cuticle is located primarily around the chalazal three-fourths of the embryo sac. Endothelium cells around the chalaza and toward the micropylar region are rich in cytoplasmic organelles. The cytoplasm of endothelial cells near the central cell has large vacuoles and few organelles. There are also plasmodesmas on the anticlinal walls of endothelial cells. The endothelium and the micropylar integumentary cells play a role in transport of metabolites into the embryo sac.
Changes in relative water content (RWC), lipid peroxidation, proline and antioxidant system in relation to the tolerance to oxidative stress enzymes mediated high pressure were investigated in
L. cv. Tokak. For this purpose, mature embryos cultured on MS media were treated in a hyperbaric oxygenation chamber (approx. 59.06 feets, 2 kp/cm
) with pure oxygen for 60 minutes/day for a growth period of ten days in a plant growth chamber. Constitutive activities of SOD, APOX, GR and POX were higher in hyperbaric oxygenated (HBO) explants, being 96.07%, 28.57%, 77.77% and 54.14% for the 5th days; 95.78%, 40%, 37.5%, and 94.98% for the 10th days of culture, respectively, than in the control plants. Increase in SOD activity was also shown on polyacrilamide gel electrophoresis on the 10th day of application. Proline accumulation was increased in HBO-treated explants both on the 5th days (85.71%) and 10th days (37.14%) of treatment. MDA content was found to be higher in HBO treated explants both on the 5th (53.84%) and 10th (59.83%) days of culture.
plant regeneration in finger millet —
(L.) Gaertn. In Vitro Cell Dev Biol (in press)
Lee K, Jeon H, Kim M (2002) Optimization of a matureembryo based