Jane Elizabeth Kraus

Astra Blue and Basic Fuchsin Double Staining of Plant Materials

Methods for double staining plant materials using astra blue and basic fuchsin are described here. These methods can be applied to free hand and microtome sections embedded in paraffin, paraplast or historesin. Also, they can be used to study isolated epidermal peels and pollen preparations. Temporary, semipermanent and permanent preparations were studied. Astra blue stained polysaccharides of the cell wall such as cellulose and pectins. Basic fuchsin showed an affinity for lignified, suberized or cutinized walls. The easy preparation of the reagents, excellent color contrast of the histological preparations, and brief staining times of some methods makes them useful for both routine research and didactic purposes. Also, excellent color or black and white photomicrography can be obtained after the double staining described here.

Métodos de coloração de Roeser (1972): modificado - e Kropp (1972) visando a substituição do azul de astra por azul de alcião 8GS ou 8GX

foram testados metodos de coloracao baseados em Roeser (1972) modificado e Kropp (1972), visando a substituicao do corante azul de astra por azul de alciao 8GS ou 8GX. As amostras foram fixadas em FAA, desidratadas em serie butilica terciaria e incluidas em parafina. Os cortes histologicos transversais foram corados segundo diferentes baterias de coloracao, modificadas quanto ao tipo de corante usado, diferenciador e serie de desidratacao. As lâminas permanentes foram preparadas com balsamo-do-canada sintetico. Os resultados obtidos evidenciaram que o metodo de Roeser (1972) modificado e melhor que o de Kropp (1972), nas condicoes deste experimento. O azul de astra pode ser substituido por azul de alciao 8GX e a desidratacao pode ser em serie isopropilica ou etilica, sem grande diferenca entre elas. Sao discutidos os resultados provenientes das di ferentes coloracoes. Palavras-chave: metodo de coloracao, azul de astra. azul de alciao, fucsina basica, safranina, histologia vegetal ABSTRACT - (Staining methods of modified Roeser (1972) and Kropp (1972), aiming at substituing the astra blue by aleian blue 8GS or 8GX). Staining methods based on modified Roeser (1972) as well as that of Kropp (l972) were done with leaves of

Shoot regeneration capacity from roots and transgenic hairy roots of tomato cultivars and wild related species

The organogenetic competence of roots and Agrobacterium rhizogenes-induced hairy roots of twelve Lycopersicon genotypes was investigated. Both roots and hairy roots of L. peruvianum, L. chilense, L. hirsutum and two L. peruvianum-derived genotypes regenerated shoots after 2–4 weeks of incubation on zeatin-contained medium. Anatomical analysis showed that shoot regeneration in roots could be direct or indirect, depending on the genotype considered. Hairy roots showed considerable differences in their morphogenetic responses, when compared to the corresponding non-transgenic roots. The differences observed may reflect the influence of the introduced rol genes on hormonal metabolism/sensitivity. Hairy root-derived T0 plants had shortened internodes, wrinkled leaves and abundant root initiation, and most produced flowers and fruits with viable seeds. The hairy root syndrome was detected early in germinating T1 seedlings as a strong reduction in the hypocotyl length. Our data point to the possibility of the use of A. rhizogenes, combined with regenerating Lycopersicon genotypes, in a very simple protocol, based on genetic capacity instead of special procedures for regeneration, to produce transgenic tomato plants expressing rol genes, as well as, genes present in binary vectors. Furthermore, the regeneration differences observed in each Lycopersicon genotype and in transgenic materials expressing rol genes open the possibility for their use in the analysis of both the biochemical and the genetic background of organogenetic competence.

Endogenous auxin and cytokinin contents associated with shoot formation in leaves of pineapple cultured in vitro

The in vitro culture of pineapple leaves on a shoot induction medium (SIM) results in the formation of protuberances and further development in shoots, and plantlets. The contents of endogenous indoleacetic acid (IAA) and five cytokinins (Cks), N6(2-isopentenyl)adenine (iP), N6(2-isopentenyl)adenosine (iPR), zeatin (Z), zeatin riboside (ZR) and N6-benzyladenine (BA), present in the basal portion of those leaves, were correlated to the organogenic response that occurs over 15 days of culture. The endogenous auxin/cytokinins ratio was lowest on the 3rd day, mainly due to a strong increase in the iP level. It seems that endogenous iP concentration triggered the induction signal for an organogenic response in pineapple leaf bases. The rise in iP content required the presence of BA and a-naphthaleneacetic acid (NAA) in the medium, suggesting that endogenous iP production is regulated in response to these growth regulator uptakes.

TEMPERATURE DETERMINES THE OCCURRENCE OF CAM OR C3 PHOTOSYNTHESIS IN PINEAPPLE PLANTLETS GROWN IN VITRO

SummaryAnanas comosus (L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperature regimes developed as CAM or as C3 plants. The plants used in this study were developed from the lateral buds of the nodal etiolated stem explants cultured on Murashige and Skoog medium for 3 mo. The cultures were maintained under a 16-h photoperiod for different thermoperiods. With 28°C light/15°C dark thermoperiod, as compared with constant 28°C light and dark, pineapple plants had a succulence index two times greater, and also a greater nocturnal titratable acidity and phosphoenolpyruvate carboxylase (PEPCase) activity, indicating CAM-type photosynthesis. The highest abscisic acid (ABA) level occurred during the light period, 8 h prior to maximum PEPCase activity, while the indole-3-acetic acid (IAA) peak was found during the dark period, coinciding with the time of highest PEPCase activity. These plants were also smaller with thicker leaves and fewer roots, but had greater dry weight. Their leaves showed histological characteristics of CAM plants, such as the presence of greater quantities of chlorenchyma and hypoderm. In addition, their vascular system was more conspicuous. In contrast, under constant temperature (28°C light/dark) plants showed little succulence in the leaves. There was no significant acid oscillation and diurnal variation in PEPCase activity in these plants, suggesting the occurrence of C3 photosynthesis. Also, no diurnal variation in ABA and IAA contents was observed. The results of this study clearly indicate a role for temperature in determining the type of carbon fixation pathway in in vitro grown pineapple. Evidence that ABA and IAA participate in CAM signaling is provided.

Relationships between endogenous hormonal levels and axillary bud development of Ananas comosus nodal segments

The effects of some endogenous hormones on the control of axillary bud development of pineapple Ananas comosus (L.) Merr. nodal segments cultivated in vitro were verified. Nodal segments with the apex (control) and decapitated nodal segments were used as explants and were cultured on hormone-free medium. Histological modifications occurring during the developmental process were also observed. Endogenous levels of indole-3-acetic acid (IAA) and four cytokinins (Cks) (isopentenyladenine (iP), isopentenyladenine 9-riboside (iPR), zeatin (Z) and zeatin riboside (ZR)) were quantified by means of high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). During the first 4 h of culture, a marked reduction in the level of IAA (309%) of the decapitated nodal segment was detected and this coincided with the beginning of cell division in the leaf axil. Cks levels decreased during the first 12 h of culture in both explants. However, this reduction was 150% higher in the control, and occurred mainly due to a decrease in the level of ZR. The reduction in the level of IAA probably favored Cks biosynthesis and/or inhibited its degradation in the nodal tissue of the decapitated segment. After 16 h of culture, there was an increase in the level of IAA probably as a consequence of the establishment of the new auxin-producing shoot apex. After 1 d, a progressive increase in ZR occurred, suggesting that Z-type Cks have an important role in the leaf development of the new plant. Comparatively, auxin/total Cks ratio was always lower in the decapitated nodal segment throughout the process of the axillary bud development.

Anatomical and ultrastructural aspects of leaf galls in Ficus microcarpa L.f. (Moraceae) induced by Gynaikothrips ficorum Marchal (Thysanoptera)

Several species of Ficus present leaf galls and the goal of this research is to study the structural alterations involved in the formation of leaf galls caused by Gynaikothrips ficorum on F. microcarpa, an ornamental plant. The galls of young and mature leaves were separated into two developmental stages based on the presence of lesions on leaf lamina and the degree of leaf folding. Swellings of the lamina were observed in young and mature leaves during gall development which coincided with the areas of cellular hypertrophy and tissue hyperplasia. Swellings were detected in a greater amount and more precociously on young leaves when compared to mature ones. In young leaves, the cecidogenetic responses were quicker and led to further structural differences because younger cells are not completely differentiated. Cell hypertrophy and tissue hyperplasia were striking processes involved in the ontogenesis of the studied gall, similar to other galls induced by thrips. Nevertheless, in spite of the numerous sites of feeding and the wide area of attack, F microcarpa galls can be considered rudimentary, since no new tissue differentiation was observed.

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.

Anatomy and ontogenesis of hymenopteran leaf galls of Struthanthus vulgaris Mart. (Loranthaceae)

Em folhas de Struthanthus vulgaris Mart. (Loranthaceae) foram observadas galhas induzidas por um Hymenoptera. Essas galhas apresentam cinco estagios de desenvolvimento. No primeiro estagio de desenvolvimento a galha e evidenciada como uma pequena protuberância de cor castanha na superficie da folha. Internamente ao redor dos ovos notase a presenca de celulas do clorenquima divididas. No segundo estagio, a galha aumenta em tamanho e sua superficie mostra-se ondulada, com uma depressao na regiao central. Internamente, na regiao da depressao observa-se uma câmara larval incompletamente dividida, onde estao os embrioes. Ao redor da câmara esta presente um parenquima neoformado e as fibras e esclereides perdem as paredes secundarias. No parenquima vascular tambem ocorre hiperplasia. No terceiro estagio, a galha aumenta em tamanho, assumindo um formato elipsoidal. A epiderme apresenta-se com fissuras e o parenquima neoformado e mais evidente, com uma regiao cortical e outra medular. Nesta, cada câmara contem um indutor em fase larval e e revestida por uma ou duas camadas de tecido nutritivo. No quarto estagio, a galha e ainda maior, sendo revestida em sua maior parte por periderme. Novos feixes vasculares, esclereides e fibras sao formados. Os indutores estao em fase larval avancada e o tecido nutritivo nao e mais observado. No quinto estagio de desenvolvimento, a galha atinge o tamanho definitivo e os indutores estao em fase pupal. As celulas da regiao cortical apresentam-se ligeiramente hipertrofiadas. As galhas senescentes mostram os orificios resultantes dos canais de emergencia, feitos pelos indutores adultos. Os estudos anatomicos da galha induzida por himenoptero possibilitaram uma analise comparativa do desenvolvimento desta com outra causada por um diptero previamente descrita na mesma planta hospedeira. Sugere-se que durante a maturacao da galha, processos chaves especificos sao desencadeados possibilitando uma cecidogenese especifica.

Anatomia de galhas de ambrosia em folhas de Baccharis concinna e Baccharis dracunculifolia (Asteraceae)

Ambrosia galls are induced by dipteran (Cecidomyiidae) and no nutritive tissue is present: fungi hyphae are the source of food to the inducer larva. Ambrosia galls in Baccharis concinna and B. dracunculifolia have only one chamber with one inducer. The fungi hyphae are observed. In B. dracunculifolia galls the hyphae are restricted to the larval chamber. The parenchyma palisade cells are elongated. In B. concinna galls hyphae spread also among chlorenchyma cells around of larval chamber. The chlorenchyma cells near to the chamber elongate slightly. In both galls, pericyclic fibers of vascular system lose their secondary walls. When the inducer is in pupal phase, the amount of hyphae increases and they fill several parts of the larval chamber. Hyphae of B. concinna galls present lipophilic globules which are not observed in the hyphae of B. dracunculifolia galls. Picnids are found only in the senescent galls of B. dracunculifolia. This paper is the first contribution to the knowledge of the ambrosia galls in the Brazilian flora.