Twisting development, the birth of a potential new gene.

Evolution has long been considered to be a conservative process in which new genes arise from pre-existing genes through gene duplication, domain shuffling, horizontal transfer, overprinting, retrotransposition, etc. However, this view is changing as new genes originating from non-genic sequences are discovered in different organisms. Still, rather limited functional information is available. Here, we have identified TWISTED1 (TWT1), a possible de novo-originated protein-coding gene that modifies microtubule arrangement and causes helicoidal growth in Arabidopsis thaliana when its expression is increased. Interestingly, even though TWT1 is a likely recent gene, the lack of TWT1 function affects A. thaliana development. TWT1 seems to have originated from a non-genic sequence. If so, it would be one of the few examples to date of how during evolution de novo genes are integrated into developmental cellular and organismal processes.

Anther Culture of the Gametophytic Self-Incompatible Species Physalis ixocarpa Brot.

Here we present an optimized protocol for in vitro embryo formation and plant regeneration through anther culture of the Mexican husk tomato (Physalis ixocarpa Brot.). This protocol relies on the application of an anther thermal shock at a specific developmental stage prior to the in vitro culture, ensures embryo formation from anthers without callus formation, and allows spending less time to regenerate doubled haploid complete plants. This protocol has been used for different cultivars of Physalis ixocarpa (Chapingo, Rendidora, Puebla, Arandaz, Manzano, Tamazula, Salamanca, and Milpero), and also for two wild-type accessions, all of them cultivated in Mexico. Chapingo cultivar responded with the highest percentage of androgenesis on the embryo induction medium (EIM).

Whole-Mount Immunolocalization Procedure for Plant Female Meiocytes.

Here we present an optimized protocol for immunolocalization of meiotic proteins during female meiosis in whole mount tissues. It ensures ovule morphology integrity and homogeneous reagent penetration. The method relies on paraformaldehyde tissue fixation, polyacrylamide embedding, tissue permeabilization, antibody incubation, counterstaining, and confocal microscopy analysis. This protocol has been used in diverse Arabidopsis ecotypes and in the legume Vigna unguiculata.

Whole-mount immunolocalization to study female meiosis in Arabidopsis.

Here we describe a whole-mount immunolocalization protocol to follow the subcellular localization of proteins during female meiosis in Arabidopsis thaliana, a model species that is used to study sexual reproduction in flowering plants. By using confocal microscopy, the procedure allows one to follow megasporogenesis at all stages before differentiation of the functional megaspore. This in particular includes stages that occur during prophase I, such as the installation of the axial and central elements of the synaptonemal complex along the meiotic chromosomes. In contrast to procedures that require microtome sectioning or enzymatic isolation and smearing to separate female meiocytes from neighboring cells, this 3-day protocol preserves the constitution of the developing primordium and incorporates the architecture of the ovule to provide a temporal and spatial context to meiotic divisions. This opens up the possibility to systematically compare the dynamics of protein localization during female and male meiosis. Steps describe tissue collection and fixation, preparation of slides and polyacrylamide embedding, tissue permeabilization, antibody incubation, propidium iodide staining, and finally image acquisition by confocal microscopy. The procedure adds an essential technique to the toolkit of plant meiotic analysis, and it represents a framework for technical adaptations that could soon allow the analysis of plant reproductive alternatives to sexual reproduction.

An efficient method for miRNA detection and localization in crop plants.

microRNAs are a class of non-coding small RNAs (sRNAs) that are important regulators of gene expression at the post-transcriptional level by mRNA cleavage or translation inhibition. Another class of sRNAs are siRNAs, which also regulate gene expression but by causing DNA methylation. This epigenetic regulatory role has been observed for some miRNAs as well. The use of sRNAs allows the development of biotechnological applications in plants. To develop these types of applications, and to better understand the natural roles they play, it is important to be able to detect and to localize these sRNAs at the plant tissue level. Sometimes, in crop plants this can be challenging. Therefore, we developed a tissue printing hybridization protocol for easy and efficient detection of sRNAs and demonstrate this by the analysis of the spatio-temporal expression patterns of the miRNAs miR159 and miR164 in fruits of various crop plants. Moreover, we show the possibility to also detect the expression of miRNAs in fruit juice using a dot blot hybridization approach.

Polymorphism and methylation patterns in Agave tequilana Weber var. 'Azul' plants propagated asexually by three different methods.

Genetic variation in three forms of asexually propagated Agave tequilana Weber var. 'Azul' plants namely offsets, bulbils and in vitro cultured individuals was studied by AFLP analysis. Low levels of variation were observed between mother plants and offsets and a higher level between mother plant and bulbils. Families obtained from commercial plantations showed lower levels of variation in comparison to families grown as ornamentals. No variation was observed between the original explant and four generations of in vitro cultured plants. Epigenetic variation was also studied by analyzing changes in methylation patterns between mother plants and offspring in each form of asexual reproduction. Offsets and bulbils showed an overall decrease in methylation whereas in vitro cultured plants showed patterns specific to each generation: Generations 1 and 4 showed overall demethylation whereas Generations 2 and 3 showed increased methylation. Analysis of ESTs associated with transposable elements revealed higher proportions of ESTs from Ty1-copia-like, Gypsy and CACTA transposable elements in cDNA libraries obtained from pluripotent tissue suggesting a possible correlation between methylation patterns, expression of transposable element associated genes and somaclonal variation.