Confocal microscopy is widely used in cell biology. Like other filter-based systems, traditional confocal microscopes are limited by the spectral bands established by each optical filter. As a result, emission spectra from labels and/or autofluorescence can be overlapped leading to spectral crosstalk and inability to quantify the amount of signal originating from each individual fluorescent species. The need for accurate quantification of in vivo cellular processes and in-depth knowledge of organelle development and microstructure led Monsanto to search for non-commercial microscopes that could achieve those goals. Through a cooperative research and development agreement (CRADA) established between Monsanto and Sandia Corporation in August 2006, we built a new 3D-hyperspectral confocal fluorescence imaging system, specifically designed to meet the analytical requirements of plant specimens.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.

The combination of hyperspectral confocal fluorescence microscopy and multivariate curve resolution (MCR) provides an ideal system for improved quantitative imaging when multiple fluorophores are present. However, the presence of multiple noise sources limits the ability of MCR to accurately extract pure‐component spectra when there is high spectral and/or spatial overlap between multiple fluorophores. Previously, MCR results were improved by weighting the spectral images for Poisson‐distributed noise, but additional noise sources are often present. We have identified and quantified all the major noise sources in hyperspectral fluorescence images. Two primary noise sources were found: Poisson‐distributed noise and detector‐read noise. We present methods to quantify detector‐read noise variance and to empirically determine the electron multiplying CCD (EMCCD) gain factor required to compute the Poisson noise variance. We have found that properly weighting spectral image data to account for both noise sources improved MCR accuracy. In this paper, we demonstrate three weighting schemes applied to a real hyperspectral corn leaf image and to simulated data based upon this same image. MCR applied to both real and simulated hyperspectral images weighted to compensate for the two major noise sources greatly improved the extracted pure emission spectra and their concentrations relative to MCR with either unweighted or Poisson‐only weighted data. Thus, properly identifying and accounting for the major noise sources in hyperspectral images can serve to improve the MCR results. These methods are very general and can be applied to the multivariate analysis of spectral images whenever CCD or EMCCD detectors are used. Copyright © 2008 John Wiley & Sons, Ltd.

Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

Plant somatic cells have the remarkable ability to regenerate an entire organism. Many species in the genus Kalanchoë, known as "mother of thousands," develop plantlets on the leaf margins. Using key regulators of organogenesis (STM) and embryogenesis (LEC1 and FUS3) processes, we analyzed asexual reproduction in Kalanchoë leaves. Suppression of STM abolished the ability to make plantlets. Here, we report that constitutive plantlet-forming species, like Kalanchoë daigremontiana, form plantlets by coopting both organogenesis and embryogenesis programs into leaves. These species have a defective LEC1 gene and produce nonviable seed, whereas species that produce plantlets only upon stress induction have an intact LEC1 gene and produce viable seed. The latter species are basal in the genus, suggesting that induced-plantlet formation and seed viability are ancestral traits. We provide evidence that asexual reproduction likely initiated as a process of organogenesis and then recruited an embryogenesis program into the leaves in response to loss of sexual reproduction within this genus.

We now know that nitric oxide (NO) bursts are early and transient characteristics of plant stress under many biotic and abiotic conditions. NO originates non-enzymatically from nitrite (NO2-), as a by-product from nitrate reductase (NR). It arises enzymatically from nitrite:nitric oxide reductase (NI-NOR), and putative nitric oxide synthase (NOS) activity. NOS substrates (L-arginine, NADPH, and oxygen), and products (NO and L-citrulline) are ubiquitous in plants, and pivotal components of intermediary N metabolism. NO has both beneficial and harmful effects, depending on its concentrations and milieu. At low concentrations, it is a chemical messenger that directly integrates and differentiates time-dependent responses to stress and defence against pathogens. Prime direct targets of NO are the haems that shuttle three gases key to plant life, viz. -NO, carbon dioxide, and oxygen. -NO and thiols are nitrosated to S-nitrosothiols, stored, and probably shuttled by transnitrosylation of proteins. At high levels, and in the presence of reactive oxygen species (ROS), -NO produces

The effect of mechanical stress (centrifugation) on the induction of nitric oxide (NO) formation and DNA fragmentation was investigated in leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifuged leaves from wild-type and nitrate reductase (NR)nia1, nia2 double mutant, defective in the assimilation of nitrate, were labelled with 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualize in vivo NO production. After these treatments, DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labelling (TUNEL) method. Exposure to an NO-releasing compound, sodium nitroprusside (SNP) mimicked the cell response to centrifugation (20 g). The involvement of endogenous NO as a signal in mechanical stress and in DNA fragmentation was confirmed by inhibition of NO production using a nitric oxide synthase (NOS) inhibitor viz. NG-monomethyl- L -arginine (L -NMMA). These results indicate that NOS-like activity was present in A. thaliana leaves and was increased by mechanical stress. The effect of leaf-wounding on nitric oxide production was identical to that of centrifugation. Experiments with A. thaliana NR mutant also showed that NO bursts were induced by mechanical and wounding stresses and that NO was not a by-product of NR activity. A positive and significant correlation between NO production and DNA fragmentation was recorded for both centrifuged and non-centrifuged cells. Our results suggest that factors other than NO contribute to DNA damage and cell death, and furthermore, that an inducible form of NOS is present in A. thaliana.

Different gravity environments have been shown to significantly affect leaf-plantlet formation and asexual reproduction in Kalanchoë daigremontiana Ham. and Perr. In the present work, we investigated the effect of gravity at tissue and cell levels. Leaves and leaf-plantlets were cultured for different periods of time (min to 15 d) in different levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 x 10(-4) g), 1 g (control) and hypergravity (centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity affected cell death (apoptosis) in this species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO) formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis, mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal differentiation. Exposures to acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with N(G)-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how chloroplasts, cells and leaves may respond (and adapt) to gravity changes.

Leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalanchoë and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.

Plant cells either die by "accident" (traumatic cell death) or by "design" (programmed cell death; PCD). There is clear evidence that cell death during plant development and interactions with the environment involves PCD (in Gray and Johal, 1998). K. daigremontiana reproduces asexually by forming plantlets from leaf indentations which fall to soil and convert into adult plants. In nature, its entire plant body except leaf-plantlets senesces as consequence of floral differentiation or stressful environmental conditions. At unit gravity, PCD precedes plantlet detachment from the mother-leaf, leading to an abscission scar after plantlet fall. Earlier experiments have shown that leaf-plantlet formation and asexual reproduction increased with short duration hypergravity treatments and decreased in simulated hypergravity (Pedroso and Durzan, 1998). The present experiments were designed to determine if and what type of cell death occurs following gravitational changes, and the sequence of events leading to it. Our study shows that changes in gravitational environment cause a burst in nitric oxide, followed by a sequence of events that may ultimately led to programmed cell death by apoptosis.

Camellia japonica L, commonly known as camellia, is an evergreen ornamental plant of the Theaceae family. Native from Eastern Asia, its origin is still controversial, being considered by some as a species indigenous from Japan and, by others, from China (Ta and Leng, 1983). It was introduced into Europe by the Portuguese in 1542 (Anderson, 1961) and soon spread to Spain, England, France and Italy. It was later introduced into the United States at the beginning of the 18th century, and in Australia during the mid 19th century.

Haploid plants have the gametophytic chromosome number. They are of great importance for the production of homozygous plants and for mutation studies. The use of anther and microspore cultures for the induction of pollen embryogenesis hastens the production of haploids and gametoclonal variants. These systems can be useful tools for cultivar improvement. Camellia japonica L. (Theaceae family) is one of the most important species in the genus Camellia. Economically, it is valuable as an ornamental woody species and has a potential to be used for oil and wood production. The available Camellia varieties are genetically highly heterozygous. In this genus, frost and pathogens cause serious damage that results in drastic annual economic loses. So far, in vitro culture of this species, and other Camellia species, has been mainly concerned with the establishment of protocols for micropropagation from juvenile and adult materials (Bennet, 1977, 1978; Bennet & Scheibert, 1982; Carlisi & Torres, 1986; Crezé, 1983; Kato, 1989a; Pedroso-Ubach, 1991; Samartin et al., 1984, 1986; Samartin, 1989; Vieitez et al., 1989a,b, 1992), and for mass propagation by somatic embryogenesis (Barciela & Vieitez, 1993; Kato, 1986, 1989b; Nakamura, 1988; Pedroso & Pais, 1993, 1994d,e; San-José & Vieitez, 1993; Vieitez & Barciela, 1990; Vieitez et al., 1991). Plant improvement has been obtained in some species by field selection and artificial crossing techniques (Yamaguchi et al., 1987). Anther culture, as a strategy for plant improvement, was reported for C. sinensis (Raina & Iyer, 1974; Chen & Liao, 1982, 1988) and C. japonica (Pedroso-Ubach, 1991). In C. sinensis, the production of haploid callus from anthers has been obtained in at least nine cultivars (Raina & Iyer, 1974; Chen & Liao, 1982, 1988) but haploid shoot regeneration was successful only in one of the cultivars (Chen & Liao, 1982, 1988). Embryo production has also been reported in this species by Shimokado et al.(1986) but apparently no plant regeneration was achieved. As far as we know, successful regeneration from anthers and microspore culture has been only reported in C. japonica (Pedroso-Ubach, 1991). Although pollen-derived plants have been obtained by anther culture in many species, plant regeneration from isolated microspores has only been successful in a few species (Bajaj, 1990). Microspore culture presents some potential advantages over anther culture, especially concerning in vitro selection strategies, genetic studies and genetic transformation. The culture of isolated microspores has become a valuable system for studying in vitro embryogenesis (Taylor et al., 1990). Complementing earlier work (Pedroso & Pais, 1992, 1993, 1994c,d), direct microspore embryogenesis in C. japonica would be valuable for studying gene expression during microspore embryogenesis and the factors that divert microspore development onto an embryogenic pathway.

Contents of endogenous free and conjugated polyamines were determined in embryogenic and non‐embryogenic leaf regions of Camellia japonica leaf explants, before (day 0), and 20 and 45 days after the induction of direct somatic embryogenesis, to clarify whether or not polyamines are linked to the specific morphogenic responses previously reported in the leaf regions. The analysis was carried out by high‐performance liquid chromatography. The results showed that there were no significant differences in the endogenous free and conjugated putrescine (Put), spermidine (Spd) and spermine (Spm) contents between the embryogenic and non‐embryogenic leaf regions of the same leaf. Thus, leaf region‐specific embryogenic response in C. japonica is not being determined/conditioned by the endogenous levels of Put, Spd and/or Spm. However, soluble and insoluble conjugated Put and soluble conjugated Spd seem to be related to the formation and development of globular embryos.

In Plant Kingdom, one of the strategies for plants to survive is the spontaneous production of direct somatic embryos [1]. Examples can be found in angiosperms, both in dicots and monocots. In artificial conditions, somatic embryogenesis is achieved using different types of initial explants and following different types of stress [for a review see, 1,8,9,11,12]. Although both direct and indirect somatic embryogenesis have been successfully achieved for many species [9], the knowledge: on the early events occurring during the induction of embryogenesis is still scarce. Here, we describe some of the molecular and elemental changes occurring during the early stages of direct somatic embryogenesis using, as model plant, the woody species Camellia japonica. Our previous results shown that direct embryogenesis from camellia leaves is region specific [5]. This specificity was suggested to be related to pre-established elemental gradients, namely, of calcium, sodium and sulphur [4,6]. After the induction treatment, two types of response were detected: a ''fast'' and a slower one [6]. The first one, common to all parenchyma cells, was suggested to be a stress response, due to leaf excision and to the induction treatment. The following morphogenic or non-morphogenic responses, that were cell or explant-region specific, were suggested to be dependent on the cell-state of each cell at the time of leaf isolation [6]. Some data on the possible effect of stress on embryogenic induction is presented. The role of Ca2(+), Mg2(+) and competing polyamines (free and conjugated) on cell division and morphogenesis is also discussed.

Embryogenic and non-embryogenic induced leaves of Camellia japonica, with the same shoot origin and submitted to the same culture conditions, were used to study protein changes during the induction of direct embryogenesis. The analysis of protein changes in these samples, based on two-dimensional polyacrylamide gel electrophoresis, revealed that 91% and 66.2% of the polypeptides detected, respectively, in embryogenic and non-embryogenic induced leaves, were already present in the non-induced control leaves. The results of the differential expression of eight selected polypeptides detected in induced and non-induced leaves are presented. A spotlist report was obtained for the 9% (43) embryogenesis-associated leaf polypeptides. From these polypeptides, two polypeptides were identified which seem to be specifically associated with somatic embryogenesis in C. japonica: E1 (pl 5.6; mol. wt. 43.5) and E2 (pl 6.0; mol. wt. 25.7).

Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.

The culture conditions for direct, indirect, and repetitive embryogenesis were established forCamellia japonica cv. Elegans and cv. Ville de Nantes. Direct embryo production from leaves averaged 15.3 embryos per responsive leaf on Murashige and Skoog medium (MS) with 1.0 mg·liter−1 N6-benzyladenine and 0.5 mg·liter−1 2,4-dichlorophenoxyacetic acid. Plantlet production was 7.1 (±1.5) plantlets per leaf. Direct embryo production from stems averaged 5.7 embryos per shoot, and 2.7 embryos per stem portion, on MS medium supplemented with 1.0 mg·liter−1 N6-benzyladenine and 0.1 mg·liter−1 indolbutyric acid (MS28). Conversion was only obtained after repetitive embryogenesis. Embryogenesis from leaf-derived callus occurred in all callus after transfer to MS/2–25 medium (half strength MS medium with 25 g·liter−1 D-glucose) (production stage). Plantlet production was 16.3 (±3.6) plantlets per callus. Repetitive embryogenesis increased embryo population by 2.3- to 3.6-fold every 4 wk. Conversion of secondary embryos was obtained on MS medium supplemented with 2.0 mg·liter−1 N6-benzyladenine, 0.2 mg·liter−1 indolbutyric acid, 5 mg·liter−1 gibberellic acid (MS56). Direct embryo formation from leaves, stems, and cotyledons, and embryogenic callus formation from leaves were restricted to specific regions of the explant.