RESUMEN
Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.
Asunto(s)
Abies/genética , Agrobacterium tumefaciens/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente , Semillas/genética , Abies/embriología , Toxinas de Bacillus thuringiensis , Vectores Genéticos , Técnicas de Embriogénesis Somática de Plantas , Regiones Promotoras Genéticas , Semillas/crecimiento & desarrollo , Transcripción Genética , Transformación GenéticaRESUMEN
In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.
Asunto(s)
Abies/embriología , Criopreservación/métodos , Semillas , Abies/genética , Marcadores GenéticosRESUMEN
The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial K (ATP)(+) channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the K (ATP)(+) channel activity could induce an entry of K(+) into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.
Asunto(s)
Abies/citología , Abies/embriología , Apoptosis , Desarrollo Embrionario , Metabolismo Energético , Mitocondrias/metabolismo , Abies/efectos de los fármacos , Abies/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Citocromos c/metabolismo , Fragmentación del ADN/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/farmacología , Glucosa-6-Fosfato/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Proteínas Mitocondriales , NADP/metabolismo , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Proteínas de Plantas , Canales de Potasio/metabolismoRESUMEN
Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network (ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba x Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing beta-(1-->6)-galactotetraosyl group as a first molecular component of ECMSN covering embryogenic cells in gymnosperms.