Matriconditioning integrated with gibberellic acid to hasten seed germination and improve stand establishment of pepper and tomato

Emergence and stand establishment of tomato (Lycopersicon lycopersicum (L.) (Karsten ex Farw) and pepper (Capsicum annus L.) seeds are often slow and erratic, particularly under stress conditions. Field emergence trials sometimes have not responded to priming in pepper. This study examined the combined effects of matriconditioning and gibberellin application on the germination and stand establishment of pepper and tomato seeds. Pepper and tomato seeds were conditioned with a solid carrier, Micro Cel E, in the presence of gibberellic acid (GA) for 1, 2 , 3 and 4 days at 15 and 25ºC. The results showed that, in all cases, even under stress conditions, the combination of matriconditioning with GA was effective in improving germination and emergence of pepper and tomato. The germination time was, in average, reduced by 2 to 3 days by primed seeds. Thus, matriconditioning, during which germination is suspended, provides an unique means to rapidly and efficiently digest the endosperm by GA-induced enzymes and reduce the mechanical restraints of endosperm thus providing energy to start and sustain embryo growth.

Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Molecular genetic framework for protophloem formation.

The phloem performs essential systemic functions in tracheophytes, yet little is known about its molecular genetic specification. Here we show that application of the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) specifically inhibits specification of protophloem in Arabidopsis roots by locking the sieve element precursor cell in its preceding developmental state. CLE45 treatment, as well as viable transgenic expression of a weak CLE45(G6T) variant, interferes not only with commitment to sieve element fate but also with the formative sieve element precursor cell division that creates protophloem and metaphloem cell files. However, the absence of this division appears to be a secondary effect of discontinuous sieve element files and subsequent systemically reduced auxin signaling in the root meristem. In the absence of the formative sieve element precursor cell division, metaphloem identity is seemingly adopted by the normally procambial cell file instead, pointing to possibly independent positional cues for metaphloem formation. The protophloem formation and differentiation defects in brevis radix (brx) and octopus (ops) mutants are similar to those observed in transgenic seedlings with increased CLE45 activity and can be rescued by loss of function of a putative CLE45 receptor, BARELY ANY MERISTEM 3 (BAM3). Conversely, a dominant gain-of-function ops allele or mild OPS dosage increase suppresses brx defects and confers CLE45 resistance. Thus, our data suggest that delicate quantitative interplay between the opposing activities of BAM3-mediated CLE45 signals and OPS-dependent signals determines cellular commitment to protophloem sieve element fate, with OPS acting as a positive, quantitative master regulator of phloem fate.

Precision and scaling in morphogen gradient read-out.

Morphogen gradients infer cell fate as a function of cellular position. Experiments in Drosophila embryos have shown that the Bicoid (Bcd) gradient is precise and exhibits some degree of scaling. We present experimental results on the precision of Bcd target genes for embryos with a single, double or quadruple dose of bicoid demonstrating that precision is highest at mid-embryo and position dependent, rather than gene dependent. This confirms that the major contribution to precision is achieved already at the Bcd gradient formation. Modeling this dynamic process, we investigate precision for inter-embryo fluctuations in different parameters affecting gradient formation. Within our modeling framework, the observed precision can only be achieved by a transient Bcd profile. Studying different extensions of our modeling framework reveals that scaling is generally position dependent and decreases toward the posterior pole. Our measurements confirm this trend, indicating almost perfect scaling except for anterior most expression domains, which overcompensate fluctuations in embryo length.

Influence of environmental factors on the expression of phenotypic characters by chicken dorsal root ganglion cells in culture.

Primary sensory neurons were grown under four conditions of culture. The influence of nonneuronal cells, horse serum or both was studied on the phenotypic expression of certain neuronal subpopulations. The number of neurons expressing acetylcholinesterase, alpha-bungarotoxin-binding sites or a high uptake capacity for glutamine was enhanced by nonneuronal cells. The horse serum increases the neuronal subpopulation exhibiting a carbonic anhydrase activity. Certain phenotypic changes fit conditions consistent with an epigenetic induction rather than a cell selection.

Developmental changes in cardiac recovery from anoxia-reoxygenation.

The developing cardiovascular system is known to operate normally in a hypoxic environment. However, the functional and ultrastructural recovery of embryonic/fetal hearts subjected to anoxia lasting as long as hypoxia/ischemia performed in adult animal models remains to be investigated. Isolated spontaneously beating hearts from Hamburger-Hamilton developmental stages 14 (14HH), 20HH, 24HH, and 27HH chick embryos were subjected in vitro to 30 or 60 min of anoxia followed by 60 min of reoxygenation. Morphological alterations and apoptosis were assessed histologically and by transmission electron microscopy. Anoxia provoked an initial tachycardia followed by bradycardia leading to complete cardiac arrest, except for in the youngest heart, which kept beating. Complete atrioventricular block appeared after 9.4 +/- 1.1, 1.7 +/- 0.2, and 1.6 +/- 0.3 min at stages 20HH, 24HH, and 27HH, respectively. At reoxygenation, sinoatrial activity resumed first in the form of irregular bursts, and one-to-one atrioventricular conduction resumed after 8, 17, and 35 min at stages 20HH, 24HH, and 27HH, respectively. Ventricular shortening recovered within 30 min except at stage 27HH. After 60 min of anoxia, stage 27HH hearts did not retrieve their baseline activity. Whatever the stage and anoxia duration, nuclear and mitochondrial swelling observed at the end of anoxia were reversible with no apoptosis. Thus the embryonic heart is able to fully recover from anoxia/reoxygenation although its anoxic tolerance declines with age. Changes in cellular homeostatic mechanisms rather than in energy metabolism may account for these developmental variations.

Mouse fetal trisomy 13 and hypotrophy of the spinal cord: effect on calbindin-D28k and calretinin expressed by neurons of the spinal cord and dorsal root ganglia.

Trisomy 13 was detected in 10% of mouse embryos obtained from pregnant females which were doubly heterozygous for Robertsonian chromosomes involving chromosome 13. The developing dorsal root ganglia and spinal cords were examined in trisomy 13 and littermate control mice between days 12 and 18 of gestation (E12-18). The overall size of the dorsal root ganglia and number of ganglion cells within a given ganglion were not altered, but the number of neurons immunoreactive for calbindin and calretinin was reduced. The trisomic spinal cord was reduced in size with neurons lying in a tightly compact distribution in the gray matter. In trisomic fetuses, the extent of the neuropil of the spinal cord was reduced, and may represent a diminished field of interneuronal connectivity, due to reduced arborization of dendritic processes of the neurons present, particularly of calbindin-immunostained neurons. Furthermore, the subpopulation of calretinin-immunoreactive neurons and axons was also reduced in developing trisomic gray and white matter, respectively. Thus, overexpression of genes on mouse chromosome 13 exerts a deleterious effect on the development of neuropil, affecting both dendritic and axonal arborization in the trisomy 13 mouse. The defect of calbindin or calretinin expression by subsets of dorsal root ganglion or spinal cord neurons may result from deficient cell-to-cell interactions with targets which are hypoplastic.

Concentrations sériques de protéines placentaires au 1er trimestre dans des grossesses monofoetales et multiples après FIV : implications pour le dépistage de la trisomie 21 [First trimester serum concentrations of placental proteins in singleton and multiple IVF pregnancies: implications for Down syndrome screening]

First trimester biochemical trisomy screening is based on serum concentrations of pregnancy-associated plasma protein A (PAPP-A) and human chorionic gonadotrophin (hCG). Our aim was to confirm previously suggested modifications in serum marker concentrations after in vitro fertilisation (IVF) and embryo transfer (ET), and to assess the need of establishing normal medians for trisomy screening in these. We compared 56 singleton pregnancies obtained after ET (of which 40 in gonadotrophin stimulation cycles) with 120 gestation-matched spontaneous controls. For multiple pregnancies, 17 treated cycles were compared with 25 controls. The levels of PAPP-A, hCG, and pregnancy-specific β1-glycoprotein were determined and compared between treated and spontaneous pregnancies. Serum PAPP-A levels were reduced in pregnancies achieved after gonadotrophin-stimulated IVF and ET, and this was more pronounced in earlier gestational stages. SP1 followed the same trend, while hCG tended to be increased, and this not only in pregnancies obtained from gonadotrophin-stimulated but also from oestrogen supported cycles, and with a more pronounced effect in the later gestational ages examined here. Decreased PAPP-A together with increased hCG concentrations produce falsely elevated results in first trimester Down syndrome screening, but we do not recommend the establishment of normal medians for IVF pregnancies due to the variations in stimulation protocols.

Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L.

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways

Postmortem angiography after vascular perfusion with diesel oil and a lipophilic contrast agent.

OBJECTIVE: The objective of our study was to establish optimal perfusion conditions for high-resolution postmortem angiography that would permit dynamic visualization of the arterial and venous systems. MATERIALS AND METHODS: Cadavers of two dogs and one cat were perfused with diesel oil through a peristaltic pump. The lipophilic contrast agent Lipiodol Ultra Fluide was then injected, and angiography was performed. The efficiency of perfusion was evaluated in the chick chorioallantoic membrane. RESULTS: Vessels could be seen up to the level of the smaller supplying and draining vessels. Hence, both the arterial and the venous sides of the vascular system could be distinguished. The chorioallantoic membrane assay revealed that diesel oil enters microvessels up to 50 microm in diameter and that it does not penetrate the capillary network. CONCLUSION: After establishing a postmortem circulation by diesel oil perfusion, angiography can be performed by injection of Lipiodol Ultra Fluide. The resolution of the images obtained up to 3 days after death is comparable to that achieved in clinical angiography.

Rescue of a non-viable accession and rapd analysis of recovered plants of Arachis retusa

In vitro regeneration of Arachis retusa was examined for the purpose of germplasm renewal and conservation. Random amplified polymorphic DNA (RAPD) fingerprinting was used to evaluate the genetic stability of plants derived from embryo axes and apical segments. Ten arbitrary decamer primers were screened and five of them were selected. Ninety genomic regions were evaluated, with an average of 18 loci per clone. All amplified segments were monomorphic. The results indicate that recovered plants are genetically stable at the assessed genomic regions and that both regeneration processes are suitable for in vitro germplasm preservation of Arachis species.

Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD

The objectives of this work were to evaluate the frequency of polyembryony, and to identify zygotic and nucellar seedlings of Citrus volkameriana using RAPD. Twenty-five polyembryonic and eight monoembryonic seeds were cultivated in vitrofor six months. DNA from seedlings was extracted and used in combination with five RAPD primers to identify zygotic or nucellar origin of the seedlings. Environmental conditions of the year affected significantly (P<0.05) the morphological characteristics of fruitsand the number ofembryos per seed. Polyembryonic seeds ranged from 30.9%, 44.8% to 54.4% over three years. Morphological characteristic was not correlated with polyembryony. In vitro culture enable all embryos of each seed to grow, favoring the percentage of seedlings identified as zygotic. In polyembryonic and monoembryonic seeds, 25.9% and 87.5% of the seedlings, respectively, were sexually originated. In polyembryonic seeds, not all zygotic seedlings were produced by small embryos located at the micropyle.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

Estudio de la expresión de Stxbp3, Stxbp6 y Claudin F en la formación del lumen en el oído interno del Pez Cebra, Danio rerio

Durante los últimos años se ha fomentado la investigación mediante el pez cebra como modelo biológico gracias a las considerables ventajas que ofrece respecto a modelos utilizados habitualmente. Una de las aplicaciones más destacadas de este modelo es en el estudio de las células sensoriales en el oído interno, ya que tienen un gran parecido con las células sensoriales de los humanos. Gracias a la facilidad de visualización y estudio de estas células en el pez cebra, se han podido llevar a cabo numerosas investigaciones sobre enfermedades que afectan a este órgano sensorial, así como la sordera. No obstante, para poder analizar todas las estructuras y células que forman parte del oído interno, es importante entender la morfogénesis de este órgano. Este proyecto se basa en el estudio de la morfogénesis del oído interno, concretamente, en la formación de lumen, una estructura que se forma en el oído interno en estadios tempranos del embrión, y que a partir de la cual se forman las demás estructuras que constituirán el oído interno. Para poder entender la formación del lumen en estadios tempranos del embrión, es necesario la caracterización de proteínas que participen en este proceso. Por lo que el objetivo principal de este proyecto es el estudio de la de la expresión de los genes Stxbp3, Stxbp6 y Claudin F en la apertura del lumen en el oído interno del pez cebra.