RESUMO
Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.
Assuntos
Hordeum/genética , Plantas Geneticamente Modificadas/genética , Plântula/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Vetores Genéticos , Genoma de Planta , Genômica , Germinação/genética , Hordeum/embriologia , Hordeum/crescimento & desenvolvimento , Canamicina Quinase/genética , Meristema/embriologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/embriologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plântula/embriologia , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , TransgenesRESUMO
Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.
Assuntos
Ácido Abscísico/metabolismo , Hordeum/metabolismo , Oxigênio/metabolismo , Sementes/metabolismo , Germinação/fisiologia , Hordeum/embriologia , TemperaturaRESUMO
The 5-(n)-alkylresorcinol fraction of the epicuticular waxes of Hordeum vulgare seeds appeared to be responsible for their in-born resistance to pathogenic fungi such as Aspergillus niger and Penicillium crysogenum. The antifungal properties of this fraction were evaluated qualitatively and quantitatively with a novel bioassay where the extreme lipophilicity of these compounds was taken into account. The minimum inhibitory concentration in the fungi tested ranged from 5.6 to 10 micrograms cm-2 for the alkyresorcinols. The behaviour of the different cultivars against these fungi could be predicted by measuring the natural amount of resorcinols of each variety by TLC-scanning densitometry. The ranking of cultivars thus established correlated well with the field behaviour of each cultivar, providing a useful and rapid method for predicting the behaviour against fungi of new varieties being developed.