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| C. Ciências Biológicas - 11. Morfologia - 5. Histologia | ||
| HISTOLOGY OF SOMATIC EMBRYOGENESIS IN THE CACTUS PRICKLY-PEAR. | ||
| Fabíola Fernandes Heredia 1 (fabiolafh@yahoo.com.br), Priscilla Barbetta e Silva 2, Francisco de Assis Paiva Campos 1 e Francisco Linhares Arruda Ferreira Gomes 2 | ||
| (1. Depto. de Bioquímica e Biologia Molecular, Universidade Federal do Ceará - UFC.; 2. Curso de Ciências Biológicas, Centro de Ciências da Saúde, Universidade Estadual do Ceará - UECE.) | ||
| INTRODUÇÃO:
The techniques of plant tissue culture and genetic engineering could provide an optional tool to improve Opuntia genus and develop novel varieties. Successful application of tissue culture and genetic transformation techniques in breeding programs depends on the development of in vitro plant regeneration systems. Although regeneration through somatic embryogenesis (SE), has been documented for a wide range of higher plants, for Opuntia ficus-indica there is no complete SE protocol available. In vitro regeneration via somatic embryogenesis or organogenesis is important for clonal propagation and is usually an integral part of any plant transformation studies. The most suitable regeneration systems for transformation are direct or repetitive production of somatic embryos or de novo shoot organogenesis, which is originated from single cells of the epidermal layer. The aim of this study was to demonstrate the developmental stages, the origin and the type of the SE induced in isolated shoot apices of prickly-pear. |
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| METODOLOGIA:
Direct somatic embryogenesis was induced, on shoot apical meristem of prickly-pear devoid of its primordial leafs on semisolid MS basal medium supplemented with 4 mg.L-1 picloram, 20 g.L -1 sucrose and solidified with 7 g.L-1 agar in absence of light, for up to six weeks. Every other day and up to 30 days after the beginning of the cultivation of isolated shoot apices on IM (induction medium), samples were collected for histological analysis. These samples were fixed in Karnovsky’s solution overnight. After fixation, they were dehydrated with a graded ethanol series (40 -100%), and infiltrated with glycol methacrylate resin. Sections (4 - 5 μm thick) were transferred to glass slides and doubled-stained with 0.12 % toluidine blue 0 with 5.0% borax, for 15 minutes and 0,05% fuchsine for 2 seconds. Excess stain was washed away and sections were secured to glass slides using gentle heat. Macroscopic features were photographed using a stereoscope. Histological photographs were taken on a microscope using Kodak Ultra film at ASA 400. |
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| RESULTADOS:
The first somatic embryos appeared on the surface of the explant without previous callus proliferation, within 15 -18 days. The first stage of embryogenesis was reached within the first week of culture, and was characterized by the presence of small and isodiametric cells with a prominent deeply-stained nucleus and nucleolus, a dense nonvacuolated cytoplasm and a high nucleus/cytoplasm ratio. The next step in ontogenesis was the formation of proembryos which contained about 6 to 10 vigorously dividing cells undergoing periclinal and anticlinal divisions. Subsequently globular-shaped embryo appeared possessing a spherical to oblong shape and a distinct protoderm. Ahead in the development, procambium strands initiated to form at the middle part of the embryo towards the cotyledonal primordia. At the next stage torpedo-shaped embryos were seen presenting a cylindrical shape as a result of lateral expansion, and the formation of tracheary elements was evident at the apical part of the structure. By the time of cotyledon formation, somatic embryos had passed through all the steps of histogenesis. The root meristem was located in the basal part of somatic embryo, owing to active proliferation of embryonal cortex cells adjacent to the procambium; somatic embryos had well-developed pith, shoot apex, a discrete radicular end and narrow suspensor. |
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| CONCLUSÕES:
In the present study we demonstrated that Opuntia ficus-indica like many other angiosperm species can readily produce somatic embryos from isolated shoot apices cultured in vitro. Somatic embryos development progressed through typical embryogenic stages, namely: globular, heart, torpedo, and cotyledon-shaped, similar to those of its zygotic counterparts. Histological examination of early events during embryogenesis indicated that the normal morphogenetic pathway was initiated from subepidermal cells and was mainly unicellular in origin. The direct and unicellular origin of the somatic embryos attained here opens the possibility of producing unique transgenic prickly-pear genotypes in subsequent transformation experiments. To the best of our knowledge, this study presents the first detailed histological description of the ontogeny of directed-derived somatic embryo in the Cactaceae family. |
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| Instituição de fomento: Funcap - Fundação Cearense de Amparo a Pesquisa | ||
| Palavras-chave: somatic embryogenesis; histology; cactaceae. | ||
| Anais da 57ª Reunião Anual da SBPC - Fortaleza, CE - Julho/2005 |