Vanessa Cristina Stein

ULTRASTRUCTURAL CALLI ANALYSIS OF Inga vera WILLD. SUBSP. Affinis (DC.) T.D. PENN

A compreensao da organogenese e embriogenese de plantas, nos estagios iniciais de desenvolvimento das celulas, requer a observacao das mudancas subcelulares. No entanto, a caracterizacao citologica durante o desenvolvimento desses estagios nao tem sido realizada com frequencia na cultura de tecidos. Portanto, o objetivo deste trabalho foi caracterizar as diferencas ultraestruturais relacionadas a calogenese de anteras, ovarios, folha e segmentos nodais de Inga vera Willd. subsp. Affinis (DC.) T.D. Penn. Para tanto, botoes florais, segmentos nodais e folhas foram desinfestados e inoculados em tubos de ensaio contendo meio MS com 3% de sacarose e 4,5 µM de 2,4-D, exceto na calogenese de folhas, em que foram utilizados 9 µM dessa auxina, e na calogenese de anteras e ovarios, cujo meio de cultura foi acrescido de 0,25% de carvao ativado e 90 µM de PVP. Apos 45 dias em meio de cultura, os calos de anteras, ovarios, folhas e segmentos nodais foram fixados em Karnovisky e preparados para a visualizacao em microscopia eletronica de varredura e microscopia eletronica de transmissao. Assim, foram observadas diferencas ultraestruturais entre as celulas dos calos provenientes de anteras, ovarios, segmentos e folhas. Os calos de anteras, folhas e segmentos nodais nao apresentaram evidencias de formacao de embrioes somaticos, apesar de suas celulas possuirem algumas caracteristicas embriogenicas. No entanto, os calos de ovarios parecem ser a fonte de explante mais responsiva a embriogenese, mostrando indicios de formacao de embrioes.

Induction and characterization of oil palm (Elaeis guineensis Jacq.) pro-embryogenic masses

Oil palm is one of the most economically valuable oil seed plants, but the expansion of plantations has been limited by availability of seedlings, as the conventional propagation is through seeds, which have low germination rates. One possible solution for the large-scale production is the use of somatic embryogenesis. The aim of this study was evaluate the effects auxins 2,4-D and picloram on the induction of pro-embryogenic masses in E.guineenesis hybrid leaf explants and characterize, regarding embryogenic characteristics, with cytochemical and ultrastructural analysis. Specifically, in vitro plantlets leaves fragments were inoculated in Y3 culture medium supplemented by 2.4-D or picloram at different concentrations (0.0, 1.0, 3.0, 6.0 and 9.0 mg l⁻¹). After 90 days the presence/ absence of cell masses were evaluated. Both growth regulators efficiently induced cellular masses regardless of the concentrations applied. As the cell masses were not homogeneously formed, they were classified according to color and shape into four types: TYPE 1--elongated and translucent, TYPE 2--uneven and translucent, TYPE 3--globular and beige, TYPE 4--globular and white. Based on the anatomical and ultrastructural features, TYPE 2, 3 and 4 cell masses were considered to have the highest embryogenic potential and therefore may be most suited to large-scale vegetative propagation of oil palm.

GENETIC TRANSFORMATION OF Eucalyptus camaldulensis BY AGROBALISTIC METHOD

Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1) and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2). To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun), to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS), for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes). The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained.

Induction and Morpho-Ultrastructural Analysis of Organogenic Calli of a Wild Passionfruit

This work studied a new protocol for organogenic ca lli induction and characterization of the morpholog y and ultrastructure of callogenesis in leaf explants of Passiflora gibertii N. E. Brown , a native passion fruit species from Brazil. Calli induction was performed in different growth conditions (light and dark), different MS me dium salt concentrations (MS and MS half strength) and the pr esence or absence of coconut water. The leaf explan ts maintained in the dark were more responsive to bud formation. In order to reduce spending on in vitro culture, the most suitable induction medium for P. gibertii organogenesis could, therefore be the MS half stre ngth salt concentration medium maintained in the dark. The ad dition of coconut water to the culture medium was e ssential for both calli induction and bud formation. The mor phological and ultrastructural features of the orga nogenic calli were isodiametric cells, characterized by an organi zed cellular system, nucleus with prominent nucleol i, presence of starch grains and dense cytoplasm rich in endoplasm ic reticulum. The scanning electron microscopy demo nstrated that buds were present on these calli.

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 Manicore 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.

Growth Curve and Development of the Internal Calli Structure of Eucalyptus camaldulensis Dehn

The objective of this work was to elucidate the growth curve of Eucalyptus camaldulensis Dehn. calli analyzing their anatomical modifications. A sigmoid aspect of the growth curve of the calli fresh matter was observed, with five different phases (lag, exponential, linear, deceleration and decline). In the lag phase, the highest growth percentage 87%, was observed, which reduced during the evaluation period to 17% in the linear phase. As for the anatomical analyses, cellular multiplications was observed during the lag and exponential phases and increase in cell size during the linear phase, promoting the calli volume growth and the establishment of the globular conformation.