-Subcellular changes are relevant to understand plant organogenesis and embryogenesis in the early stages of cell development. The cytology during cell development in tissue culture is however still poorly characterized. This study aimed to characterize the ultrastructural differences related to callogenesis of anthers, ovaries, leaf and nodal segments of Inga vera Willd. subsp. Affinis (DC.) T.D. Penn. Flower buds, nodal segments and leaves were disinfected and inoculated in test tubes containing MS medium with 3% sucrose and 4.5µM 2.4-D, except for leaf callogenesis, where 9µM of this auxin was used, and for the callogenesis of anthers and ovaries, where the culture medium was enriched with 0.25% activated charcoal and 90µM PVP. After 45 days in culture medium, the anther, ovary, leaf and nodal segment calli were fixed in Karnovisky and prepared for visualization by scanning and transmission electron microscopy. Ultrastructural differences were observed among the callus cells of anthers, ovaries, segments and leaves. There was no evidence of somatic embryo formation in the anther, leaf and nodal segment calli, in spite of some embryogenic characteristics in the cells. The ovary calli, with indications of embryo formation, seem to be the most responsive explant source for embryogenesis. Keywords: Anthers, Ovaries e Nodal segments.
ANÁLISE ULTRAESTRUTURAL DE CALOS DE INGAZEIRO (
In vitro conservation techniques can be utilized for germplasm maintenance. However, few reports on the in vitro conservation of sugarcane species are present in the literature. The objective of this study was to subject sugarcane plants to in vitro under minimal growth conditions and to evaluate the survival, regeneration, and the monitoring of nuclear DNA content levels of the plants. Shoots from 10 sugarcane varieties (Saccharum spp.) were introduced into two media: MC1, consisting of half-strength Murashige and Skoog (MS) salts and 3% sorbitol, or MC2, similar to the first formulation, but additionally supplemented with 3.8 μM abscisic acid (ABA). The shoots were maintained for up to 12 mo at 18°C in the presence of light. At the end of the period, the explants were inoculated onto multiplication medium containing 0.9 μM 6-benzylaminopurine (BAP) and 0.47 μM kinetin (Kin) for growth recovery. Flow cytometry analysis of shoots was verified at every 6 mo of storage. As a result, we found distinct behaviors of the varieties studied over the storage time, but in general, MC1 provided the greatest explant survival rates, with an average of approximately 80% cultures being able to recover. Once in the recovery media, the explant regrowth was fast, and the ability to multiply shoots was reestablished from the second 30-d subculture. However, by flow cytometry analysis, we observed a decrease in the estimated relative amount of DNA at 12 mo storage for most varieties examined, which was not observed when the monitoring was done at 6 mo. From these results, we conclude that sugarcane plants survived the minimal growth condition; however, maintaining the genotypes for extended periods in vitro may lead to variations in the estimated amount of nuclear DNA and, thus, be at risk of somaclonal variation.
The current analysis describes an improved protocol for somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis) through liquid medium, and assesses the influence of successive subcultures during induction of calluses in three Brazilian oil palm varieties. Calluses were induced in a Murashige and Skoog (MS) medium with 450 Picloram, 0.5 g L −1 glutamine, 2.5 g L −1 activated charcoal, 30 g L −1 sucrose, and solidified with 2.5 g L −1 Phytagel. In a first experiment, the effect of continued subculture of explants every 30 days to fresh culture medium was determined. During a second experiment, part of the embryogenic calluses obtained were transferred to a liquid medium under agitation, consisting of MS with 5 µM picloram or 2,4-dichlorophenoxyacetic acid (2,4-D). After 210 days, the calluses were transferred to semi-solid media for differentiating somatic embryos. It was observed that continued subculture of explants monthly was a determinant in stimulating and improving the formation of embryogenic calluses. Embryogenic calluses in liquid medium with 2,4-D significantly improved the percentage of differentiated somatic embryos (up to 80.2%), with the largest amount of torpedo embryos (8.3 per callus). Regenerated plants with roots were individualised and transferred to a greenhouse, with close to 95% survival.
Stryphnodendron adstringens is a medicinal plant considered as endangered species due to the destructive methods used for tannins extraction. The objective of this study was to induce calli development from nodal segments of Stryphnodendron adstringens (Mart.) Coville and to evaluate the total phenol and tannin contents of the induced calli. Results indicated that calli induced in the presence of 0.5 and 2.0 mg L -1 picloran or picloran associated with 0.1 mg L -1 kinetin, showed higher fresh matter values (0.120 g and 0.116 g, respectively). The highest total phenol contents were obtained in calli grown in the absence of growth regulators (9.58%) and in the presence of 0.1 mg L -1 kinetin (9.23 %). Highest yields of total tannins was observed in calli induced in the absence of growth regulators (2.36%) and in the presence of 2.0 mg L -1 picloran (1.71%).
RES UMOStryphnodendron adstringens é uma planta medicinal considerada uma espécie ameaçada de extinção devido aos métodos destrutivos usados para a extração dos taninos. O objetivo deste estudo foi desenvolver calos a partir de segmentos nodais de Stryphnodendron adstringens (Mart.) Coville e avaliar os teores de fenol total e de taninos dos calos induzidos. Os resultados indicaram que calos obtidos na presença de 0,5 e 2,0 mg L -1 de picloram ou picloram associado com 0,1 mg L -1 de cinetina apresentaram os maiores valores de matéria fresca (0,120g e 0,116 g, respectivamente). Os maiores teores de fenol total foram verificados em calos cultivados na ausência de regulador de crescimento (9,58%) e na presença de 0,1mg L -1 de cinetina (9,23%). Maior produção de taninos totais foi observada em calos induzidos na ausência de reguladores de crescimento (2,36%) e na presença de 2,0mg L -1 de picloram (1,71%).
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