RESUMO
Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6-8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.
Assuntos
Agrobacterium tumefaciens/metabolismo , Retículo Endoplasmático/metabolismo , Linho/genética , Proteínas de Fluorescência Verde/metabolismo , Agrobacterium tumefaciens/genética , Meios de Cultura/metabolismo , Farmacorresistência Bacteriana , Retículo Endoplasmático/genética , Linho/citologia , Linho/crescimento & desenvolvimento , Linho/metabolismo , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos , Transformação Genética , TransgenesRESUMO
Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mesembryanthemum/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Rhizobium/genética , Transformação Genética/genética , Proteínas de Fluorescência Verde/genética , Mesembryanthemum/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.