Alterações celulares em ápices caulinares e estabilidade genética de plantas de Eucaliptos submetidos à criopreservação / Cellular alterations in kaolinitic apexes and genetic stability of Eucalyptus plants submitted to cryopreservation

O eucalipto é amplamente explorado, principalmente para produção de papel e na construção civil , o que gera uma alta demanda por mudas de eucalipto, tendo-se a necessidade de buscar técnicas biotecnológicas para aumentar a produtividade e conservar o germoplasma de híbridos elite. Dentre essas técnicas a criopreservação apresenta alto potencial para conservação em longo prazo de genótipos de interesse. Os objetivos deste trabalho foram desenvolver um protocolo de criopreservação por meio da técnica droplet-vitrification para o híbrido comercial Eucalyptus grandis X Eucalyptus urophylla, identificar alterações celulares nos ápices caulinares cripreservados, e verificar estabilidade genética das plantas. Ápices caulinares foram pré-cultivados em meio MS com sacarose a 0,2 M e 0,5 M, por 24 horas. Posteriormente, os ápices foram dispostos em papel alumínio contendo uma gota de PVS2 à 0 ºC, antes da imersão em nitrogênio líquido (NL), em  diferentes tempos (20, 40, 60, 80, 100 e 120 minutos). Os ápices foram reaquecidos por 10 minutos em solução de descarregamento a 25ºC e transferidos para o meio de regeneração MS suplementado com 0,14 µM de BAP. Após 60 dias, avaliaram-se a sobrevivência, brotações e a estabilidade genética das plantas por citometria de fluxo. Os ápices caulinares nos tempos: recém excisados, submetidos à criopreservação, e após uma semana de recuperação foram coletados para análise de histologia e Microscopia Eletrônica de Varredura. O tempo de 60 minutos de PVS2 seguido de imersão em nitrogênio líquido proporcionou as maiores taxas de sobrevivência  (66%), e número de brotações (6 brotos/ápice). O conteúdo estimado de DNA nuclear das plantas de eucalipto tratadas com PVS2 e criopreservadas não apresentaram diferenças estatísticas, quando comparado com as plantas do controle. As observações histológicas e ultraestruturais revelaram que o protocolo de Droplet-vitrification estabelecido com 60 minutos de exposição ao PVS2 causou menores danos celulares dos ápices caulinares.Alterações celulares em ápices caulinares e estabilidade genética de plantas de Eucaliptos submetidos à criopreservação / Cellular alterations in kaolinitic apexes and genetic stability of Eucalyptus plants submitted to cryopreservatio

Regeneração de pitaya por organogênese indireta avaliada por microscopia eletrônica de varredura e citometria de fluxo

The objective of this work was to evaluate the induction of indirect organogenesis by concentrations of dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) in pitaya (Hylocereus undatus) explants, using scanning electron microscopy and the flow cytometry technique. The treatments consisted of the concentrations of 0, 2.0, and 4.0 mg L-1 2,4-D and TDZ and of the combinations of these regulators. Percentages of callus coverage at 45 and 60 days were evaluated. The explants subjected to the treatments were analized by flow cytometry and scanning electron microscopy. All treatments induced endoreduplication, and there was no somaclonal variation. Under the combination of 2.0 mg L-1 TDZ and 4.0 mg L-1 2,4-D, calluses were formed in 95% of the explants, but were smaller than those produced with 2,4-D separately. The concentration of 2.0 mg L-1 TDZ induces the indirect organogenesis in pitaya explants, confirmed by the presence of conducting vessels through scanning electron microscopy.O objetivo deste trabalho foi avaliar a indução de organogênese indireta por concentrações de ácido diclorofenoxiacético (2,4-D) e tidiazurom (TDZ) em explantes de pitaia (Hylocereus undatus), por meio de microscopia eletrônica de varredura e da técnica de citometria de fluxo. Os tratamentos consistiram das concentrações de 0, 2,0 e 4,0 mg L-1 de 2,4-D e TDZ e das combinações desses reguladores. Avaliaram-se as percentagens de cobertura de calos aos 45 e 60 dias. Os explantes submetidos aos tratamentos foram analizados por citometria de fluxo e microscopia eletrônica de varredura. Todos os tratamentos induziram endorreduplicação, e não houve variação somaclonal. Na combinação de 2,0 mg L-1 TDZ e 4,0 mg L-1 2,4-D, calos foram formados em 95% dos explantes, mas foram menores do que os produzidos com 2,4-D separadamente. A concentração de 2,0 mg L-1 de TDZ induz organogênese indireta em explantes de pitaia, comprovada pela presença de vasos condutores por meio da microscopia eletrônica de varredura

Leaf anatomy of Cordiera sessilis (Vell.) Kuntze (Rubiaceae)

A study on the leaf anatomy of Cordiera sessilis (Rubiaceae), a native medicinal shrub from Brazilian Cerrado was carried out to contribute for species identification and provide support for future biochemical studies and medicinal application. Leaves were collected, fixed and processed by usual techniques, and studied by light and electron microscopy. Quantitative analyzes of stomata and trichomes were performed. The leaves showed typical characteristics of Rubiaceae family, like hypostomatic leaves, paracytic stomata, dorsiventral mesophyll and collateral bundles. Two types of vascular patterns were identified in the petiole: in distal part, the vascular system is arranged cylindrically surrounded by sclerenchyma sheath and in proximal part, the vascular system is arranged in U-shape coupled to sclerified cells. The micromorphological organization of leaf surface, epicuticular wax types, the petiole pattern and histochemical characteristics as the presence of druses, sand crystals and alkaloids and absence of raphids in the mesophyll, midrib and petiole are considerate representative characteristics of C. sessilis and may be useful in the species identification.A study on the leaf anatomy of Cordiera sessilis (Rubiaceae), a native medicinal shrub from Brazilian Cerrado was carried out to identify features that may be useful in species recognition. Leaves were collected, fixed and processed by usual techniques, and studied by light and electron microscopy. Quantitative analyzes of stomata and trichomes were performed. In addition to the typical anatomical characteristics of Rubiaceae leaves, two types of vascular patterns were identified in the petiole: in distal part, the vascular system is arranged cylindrically surrounded by sclerenchyma sheath and in proximal part the vascular system is arranged in U-shape coupled to sclerified cells. The micromorphological organization of leaf surface, epicuticular wax types, the petiole pattern and histochemical characteristics as the presence of druses, crystal sand and alkaloids and absence of raphides in the mesophyll, midrib and petiole are considerate representative characteristics of C. sessilis and may be useful in the species recognition.

In vitro ROOTING OF TENERA HYBRID OIL PALM (Elaeis guineensis Jacq.) PLANTS1

ABSTRACT Oil palm is a woody monocot of economic importance due to high oil production from its fruits. Currently, the conventional method most used to propagate oil palm is seed germination, but success is limited by long time requirements and low germination percentage. An alternative for large-scale propagation of oil palm is the biotechnological technique of somatic embryogenesis. The rooting of plants germinated from somatic embryos is a difficult step, yet it is of great importance for later acclimatization and success in propagation. The aim of this study was to evaluate the effect of the auxins indole acetic acid (IAA) and indole butyric acid (IBA) on the rooting of somatic embryos of Tenera hybrid oil palm. Plants obtained by somatic embryogenesis were inoculated in modified MS medium with 10% sucrose and 0.6% agar and supplemented with IAA or IBA at concentrations of 5 µM, 10 µM, and 15 µM, and the absence of growth regulators. After 120 days, the presence of roots, root type, length of the longest root, number of roots, number of leaves, and shoot length were analyzed. Growth regulators were favorable to rooting; plants cultivated with IBA growth regulator at 15 µM showed higher rooting percentage (87%) and better results for the parameters of number of roots (1.33) and shoot length (9.83)

In vitro development and acclimatization of dendezeiro (Elaeis guineensis)

Fruits and almond from the dendezeiro, oil palmbelonging to the Elaeis genus,are widely used for the production of cookingoils or for the pharmaceutical and cosmetic industries.In the last decade, this oil palm also emerged as a promising source for commercialbiofuel production. This study evaluated the effect of different culture media, MS (MURASHIGUE AND SKOOG) and Y3 (EEUWENS)and carbohydrates duringin vitro germination of zygotic embryos, the effect of growth regulators GA3, NAA and BA Ponin vitro seedling development, and the survival rate of acclimatized seedlingsof Manicoré hybrid (Elaeis oleifera x E. guineensis). Zygotic embryos were inoculated on MS and modified Y3 media, supplemented with different sucrose concentrations (30, 45, and 60 gL-1) or sorbitol (36 gL-1), and the germination rate was evaluated after 30 days. Subsequently, seedlings were transferred to modified Y3 culture medium supplemented with differentGA3 concentrations (3.5 and 7 mgL-1) or without it, combined or not with 1 mgL-1 of NAA, 5 mgL-1 of BAP.The highest germinationpercentage of germinated embryos (92%) was observed in MS medium supplemented with 36 gL-1 sorbitol. Culture media supplemented with growth regulatorsGA3, NAA and BAP promoted greater shoot lengththan control media. Rooted seedlings showed high survival percentage (85%) during acclimatization

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

ABSTRACT The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages

Morphological and ultrastructural analysis of various types of banana callus, cv. Prata anã

This work was carried out to characterise morphologically and ultrastructurally the banana callus, obtained from the scalp method. Genotypes of banana, cv. Prata anã, cultivated in vitro were used to induce meristems at the leaf base; subsequently, structures known as scalps were formed. For ultrastructural analysis, five samples of callus were collected, fixed in Karnovsky solution and analysed by scanning electron microscope (SEM) and transmission electron microscope (TEM). The formation of three types of callus was observed: Type 1 - transparent watery callus, Type 2 - yellow callus with small clusters, Type 3 - yellow callus with large clusters. SEM analysis showed that Type 1 callus cells were elongated and that Type 2 and Type 3 callus cells were isodiametric, which is a characteristic of embryogenic cells. The TEM analysis showed that Type 1 callus cells had thin walls, a large number of small vacuoles and dispersed cytoplasm. The Type 2 callus cells showed dense cytoplasm, large vacuoles and a large amount of mitochondria. The Type 3 callus cells had thick and intercellular spaces. Thus, the Type 2 callus cells had characteristics consistent with embryogenic callus cells

Morphological characteristics and cell viability of coffee plants calli

The aim of this research was to characterize and compare two types of calli from leaf explants of Coffea arabica (cultivar Catiguá). Cells of different types of callus were successfully characterized regarding viability and internal and external morphological characteristics. It was obtained two morphologically distinct types of callus: (i) yellow friable and (ii) transparent watery. The yellow friable calli showed higher cell viability and embryogenic characteristics. Scanning and transmission electron microscopy showed embryogenic characteristics in cells of the yellow friable calli evidenced by the presence of small and isodiametric cells, while transparent watery calli showed elongated cells and large cytoplasm vacuolization