Analysis of organogenic competence of cotyledons of Jatropha curcas and their in vitro histological behavior

Using cut pieces cotyledons from germinating zygotic embryos of Jatropha curcas, we monitored the series of anatomical events leading to the generation of shoot through organogenesis by histological analysis. 14 days old cotyledons that were pre-cultured in a half strength Murashige and Skoog (MS) media supplemented with 100 mg/L myoinositol and 10 mg/L thiamine HCl, were cultured in organogenic competence induction media (CIM) comprising of MS salts with 1.5 mg/L benzyl adenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) and incubated for the induction of organogenic competence. Following their sequential transfer to shoot induction media (containing MS + 1.5 mg/L BA, 0.05 mg/L IBA and 0.5 mg/L GA3); shoot elongation media (containing MS + 0.3 mg/L BA) and rooting media (containing half strength MS + IBA at different concentrations), we selected individual explants and subjected them to histological analysis in order to study the morphological changes occurring during organogenesis. Our findings show that to induce organogenesis using cut pieces of cotyledons, these tissues must be harvested at least by the 14th day following germination. Optimal organogenic competence was attained after 21 days incubation period in the dark where most of the explants showed evidence of protruding shoots surrounded by calli with various morphological features. Up to 53.91% of the total explants cultured in this study produced well defined shoots with each explant producing an average number of 1.5 shoots bearing two to eight leaves. We recorded the highest percentage of root formation, which stood at 27.50% when the shoots were cultured in a rooting media containing 0.3 mg/L IBA. Histological analysis of the different events occurring during the process of organogenesis suggest that the protuberances arising from parenchymatous cells and perhaps bundle sheath forming meristematic centres acquiring organogenic competence have a multicellular origin, indicating that the regeneration process takes place through direct organogenesis.Key words: Jatropha curcas, organogenesis, auxins, histological analysis

Digestion of Starch Granules from Maize, Potato and Wheat by Larvae of the the Yellow Mealworm, Tenebrio molitor and the Mexican Bean Weevil, Zabrotes subfasciatus

Scanning electron microscopy images were taken of starch granules from different sources following exposure in vivo and in vitro to gut α-amylases isolated from Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae). One α-amylase was isolated from whole larval midguts of T. molitor using non-denaturing SDS-PAGE, while two other α-amylase fractions were isolated from whole larval midguts of Z. subfasciatus using hydrophobic interaction chromatography., Digested starch granules from larvae fed on maize, potato or wheat were isolated from midgut contents. Combinations of starch granules with isolated α-amylases from both species showed similar patterns of granule degradation. In vitro enzymatic degradation of maize starch granules by the three different α-amylase fractions began by creating small holes and crater-like areas on the surface of the granules. Over time, these holes increased in number and area resulting in extensive degradation of the granule structure. Granules from potato did not show formation of pits and craters on their surface, but presented extensive erosion in their interior. For all types of starch, as soon as the interior of the starch granule was reached, the inner layers of amylose and amylopectin were differentially hydrolyzed, resulting in a striated pattern. These data support the hypothesis that the pattern of starch degradation depends more on the granule type than on the α-amylase involved

Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.)

Recombinants encoding members of the trypsin/-amylase inhibitors family (also designated CM-proteins) were selected from a cDNA library prepared from developing barley endosperm. Inserts in two of the clones, pUP-13 and pUP-38, were sequenced and found to encode proteins which clearly belong to this family, as judged from the extensive homology of the deduced sequences with that of the barley trypsin inhibitor CMe, the only member of the group for which a complete amino acid sequence has been obtained by direct protein sequencing. These results, together with previously obtained N-terminal sequences of purified CM-proteins, imply that there are at least six different members of this dispersed gene family in barley. The relationship of this protein family to the B-3 hordein and to reserve prolamins from related species is discussed in terms of their genome structure and evolution

Cloning of cDNA and chromosomal location of genes encoding the three types of subunits of the wheat tetrameric inhibitor of insect a-amylase

We have characterized three cDNA clones corresponding to proteins CM1, CM3 and CM16, which represent the three types of subunits of the wheat tetrameric inhibitor of insect -amylases. The deduced amino acid sequences of the mature polypeptides are homologous to those of the dimeric and monomeric -amylase inhibitors and of the trypsin inhibitors. The mature polypeptides are preceded by typical signal peptides. Southern blot analysis of appropriate aneuploids, using the cloned cDNAs as probes, has revealed the location of genes for subunits of the CM3 and of the CM16 type within a few kb of each other in chromosomes 4A, 4B and 4D, and those for the CM1 type of subunit in chromosomes 7A, 7B and 7D. Known subunits of the tetrameric inhibitor corresponding to genes from the B and D genomes have been previously characterized. No proteins of this class have been found to be encoded by the A genome in hexaploid wheat (genomes AA, BB, DD) or in diploid wheats (AA) and no anti -amylase activity has been detected in the latter, so that the A-genome genes must be either silent (pseudogenes) or expressed at a much lower level

Characterization of Digestive Enzymes of Bruchid Parasitoids–Initial Steps for Environmental Risk Assessment of Genetically Modified Legumes

Genetically modified (GM) legumes expressing the α-amylase inhibitor 1 (αAI-1) from Phaseolus vulgaris L. or cysteine protease inhibitors are resistant to several bruchid pests (Coleoptera: Chrysomelidae). In addition, the combination of plant resistance factors together with hymenopteran parasitoids can substantially increase the bruchid control provided by the resistance alone. If the strategy of combining a bruchid-resistant GM legume and biological control is to be effective, the insecticidal trait must not adversely affect bruchid antagonists. The environmental risk assessment of such GM legumes includes the characterization of the targeted enzymes in the beneficial species and the assessment of the in vitro susceptibility to the resistance factor. The digestive physiology of bruchid parasitoids remain relatively unknown, and their susceptibility to αAI-1 has never been investigated. We have detected α-amylase and serine protease activities in all five bruchid parasitoid species tested. Thus, the deployment of GM legumes expressing cysteine protease inhibitors to control bruchids should be compatible with the use of parasitoids. In vitro inhibition studies showed that sensitivity of α-amylase activity to αAI-1 in the parasitoids was comparable to that in the target species. Direct feeding assays revealed that harmful effects of α-amylase inhibitors on bruchid parasitoids cannot be discounted and need further evaluation

Optimization of somatic embryogenesis and selection regimes for particle bombardment of friable embryogenic callus and somatic cotyledons of cassava (Manihot esculenta Crantz)

Although a number of transformation systems and selection regimes have been developed for cassava (Manihot esculenta Crantz), they have only been applied in a limited number of genotypes. This limitation of the applicability of the systems is due largely to variation in morphological responses of different genotypes to the regeneration and transformation procedures employed, which underscoresthe need to study all possible parameters for any given cultivar. Using two cultivars (“Rosinha” and “Bujá Preta”) we made an attempt to improve the frequency of somatic embryogenesis (SE), establishfriable embryogenic callus (FEC) lines from developing somatic embryos and evaluate the effect of different concentrations of the antibiotics kanamycin, on secondary somatic embryogenesis (SSE) andof paromomycin, on FEC in proliferation and histodifferentiation media. Further, we report on transient expression of the visual marker gene uidA following particle bombardment of FEC from ‘Bujá Preta’ andof cut pieces of green somatic cotyledons from ‘Rosinha’, using the plasmid pBI426. Higher number of embryos, which emerged as early as 8 days after culture on MS medium (CIM), was obtained whenabaxial part of the explant was in direct contact with the medium as against the adaxial side, and this did not depend on the explant size (0.5 cm² or 2.5 cm²). Highly friable and embryogenic callus was obtained on GD medium supplemented with 2% (w/v) sucrose and 12 mg/L picloram. While paromomycin, at concentration of 60 mg/L arrested the proliferation and histodifferentiation of FEC, kanamycin in CIM containing explants undergoing SSE led to a decrease in their embryogenic potential resulting in tissue death at 50 mg/L. The highest transient expression of uidA gene in FEC was observed combiningplasmolysis, M5 particle and a helium pressure of 1,200 psi, while in somatic cotyledons, the highest expression was observed when M5 particle was used along with the helium gas pressure of 900 psi

Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Acidithiobacillus ferrooxidans is a Gram negative, acidophilic, chemolithoautotrophic bacterium that plays an important role in metal bioleaching. During bioleaching, the cells are subjected to changes in the growth temperature and nutrients starvation. The aim of this study was to gather information about the response of the A. ferrooxidans Brazilian strain LR to K(2)HPO(4) starvation and heat stress through investigation of cellular morphology, chemical composition and differential proteome. The scanning electron microscopic results showed that under the tested stress conditions, A. ferrooxidans cells became elongated while the Fourier transform infrared spectroscopy (FT-IR) analysis showed alterations in the wavenumbers between 850 and 1,275 cm(-1), which are related to carbohydrates, phospholipids and phosphoproteins. These findings indicate that the bacterial cell surface is affected by the tested stress conditions. A proteomic analysis, using 2-DE and tandem mass spectrometry, enabled the identification of 44 differentially expressed protein spots, being 30 due to heat stress (40A degrees C) and 14 due to K(2)HPO(4) starvation. The identified proteins belonged to 11 different functional categories, including protein fate, energy metabolism and cellular processes. The upregulated proteins were mainly from protein fate and energy metabolism categories. The obtained results provide evidences that A. ferrooxidans LR responds to heat stress and K(2)HPO(4) starvation by inducing alterations in cellular morphology and chemical composition of the cell surface. Also, the identification of several proteins involved in protein fate suggests that the bacteria cellular homesostasis was affected. In addition, the identification of proteins from different functional categories indicates that the A. ferrooxidans response to higher than optimal temperatures and phosphate starvation involves global changes in its physiology.27614691479Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [02/07642-3