Diego Carlos Zappacosta

Increased apomixis expression concurrent with genetic and epigenetic variation in a newly synthesized Eragrostis curvula polyploid

Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a “tetraploid-dihaploid-tetraploid” series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003–2007) to reach levels of 85–90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements.

Peroxidase and polyamine activity variation during the in vitro rooting of Berberis buxifolia

Abstract Berberis buxifolia is a Patagonian shrub with great economic potential for tinctorial, pharmacological, and food industries. Clonal propagation is possible through in vitro culture and is also useful for metabolite production. However, this species is difficult to root, and to improve this, more knowledge of rhizogenesis processes is needed. Polyamines and peroxidases are useful biochemical markers during analysis of rooting phases for correlation with tissue morphological changes. Therefore, endogenous polyamine (putrescine, spermidine, spermine) changes, peroxidase activity evolution, and morphological development were studied to characterise the in vitro rhizogenesis of microshoots of B. buxifolia and, thus, to define the rooting phases. Polyamine and peroxidase changed significantly during the rooting period, and had opposite behaviours which were directly related to the IBA media content. The lower polyamine concentration and the higher peroxidase activity were found in a treatment with a dark period during the first four days and with IBA in the culture medium. Putrescine was the most abundant polyamine found in B. buxifolia tissues, 14‐ to 18‐fold more than spermidine and spermine, respectively. Therefore, these compounds were used to define the rooting phases: an induction phase (0 to 4–7 days) followed by an expression phase (4–7 to 28 days). The observed changes in the biochemical markers could be correlated with microscopic and macroscopic tissue observations in the microshoots, and the time course of rooting percentage. Successive culture media can be developed including polyamines, or other compounds and environmental conditions, which positively modify the studied biochemical markers behaviour.

Successive media to improve the in vitro rhizogenesis of Nothofagus nervosa (Phil.) Dim. et Mil.

In vitro rooting constitutes a difficult step during the micropropagation process of forest species. The successive media culture technique represents one way to overcome this barrier and includes modifying physical (e.g. photoperiod) and chemical (e.g. flavonoids) factors during the rooting phases. The aim of this study was to obtain a successive media protocol based on the incorporation of flavonoids during the in vitro rooting of Nothofagus nervosa. The factors evaluated were the type, concentration, and combination of flavonoids in relation to the rooting phases, the presence of IBA in the culture medium, the photoperiod, and the effect of flavonoids on total tissue peroxidase activity. The photoperiod used included a darkness period during the rooting induction stage and the presence of 0.61 µMIBA in the culture medium. The results showed that flavonoid incorporation at a concentration of 20 µM accelerated the appearance of roots and improved the quality of the already formed ones. Each type and concentration of flavonoid produced different responses, with (±)naringenin giving the best results. The latter caused a peak in the peroxidase activity that was absent in the control treatments. This work allowed identifying an optimized rooting protocol through a successive media culture technique that improved the speed of appearance, as well as the quantity and quality of roots for a single N. nervosa clone.

Evaluation of different methods for assessing the reproductive mode of weeping lovegrass plants, Eragrostis curvula (Schrad.) Nees

Weeping lovegrass is a forage grass cultivated in semiarid regions of the world that reproduces mainly by apomixis (diplospory), a process that involves the formation of asexual seeds and bypasses the processes of meiosis and fertilisation. The aim of this work was to evaluate and compare different techniques (cytoembryology, callose deposition, flow cytometry and progeny tests) to determine the reproductive mode of weeping lovegrass. Typical sexual and apomictic processes were clearly differentiated using cytoembryology, and different callose deposition patterns were observed in sexual and apomictic genotypes. Previous studies indicated that presence of callose on the cell wall of the megaspore mother cell is associated only with sexual processes. Nevertheless, our results also found callose deposition in apomictic genotypes, although clearly different from the pattern found in sexual processes, allowing discrimination between sexual and apomictic plants. Flow cytometry seed screening using individual seeds did not differentiate between sexual and apomictic plants as the embryo : endosperm DNA content ratio was similar in sexual and apomictic plants. Progeny tests using molecular markers showed uniform patterns in offspring from apomictic plants and variable patterns among the progeny of sexual plants. The results obtained from cytological studies and progeny tests were similar, indicating that both methods provide useful tools for determination of reproductive mode. However, the callose test with aniline blue was faster and easier to use than other techniques.

Apomixis frequency under stress conditions in weeping lovegrass (Eragrostis curvula)

To overcome environmental stress, plants develop physiological responses that are triggered by genetic or epigenetic changes, some of which involve DNA methylation. It has been proposed that apomixis, the formation of asexual seeds without meiosis, occurs through the temporal or spatial deregulation of the sexual process mediated by genetic and epigenetic factors influenced by the environment. Here, we explored whether there was a link between the occurrence of apomixis and various factors that generate stress, including drought stress, in vitro culture, and intraspecific hybridization. For this purpose, we monitored the embryo sacs of different weeping lovegrass (Eragrostis curvula [Schrad.] Nees) genotypes after the plants were subjected to these stress conditions. Progeny tests based on molecular markers and genome methylation status were analyzed following the stress treatment. When grown in the greenhouse, the cultivar Tanganyika INTA generated less than 2% of its progeny by sexual reproduction. Plants of this cultivar subjected to different stresses showed an increase of sexual embryo sacs, demonstrating an increased expression of sexuality compared to control plants. Plants of the cv. Tanganyika USDA did not demonstrate the ability to generate sexual embryo sacs under any conditions and is therefore classified as a fully apomictic cultivar. We found that this change in the prevalence of sexuality was correlated with genetic and epigenetic changes analyzed by MSAP and AFLPs profiles. Our results demonstrate that different stress conditions can alter the expression of sexual reproduction in facultative tetraploid apomictic cultivars and when the stress stops the reproductive mode shift back to the apomixis original level. These data together with previous observations allow us to generate a hypothetical model of the regulation of apomixis in weeping lovegrass in which the genetic/s region/s that condition apomixis, is/are affected by ploidy, and is/are subjected to epigenetic control.

De novo transcriptome sequencing and assembly from apomictic and sexual Eragrostis curvula genotypes

A long-standing goal in plant breeding has been the ability to confer apomixis to agriculturally relevant species, which would require a deeper comprehension of the molecular basis of apomictic regulatory mechanisms. Eragrostis curvula (Schrad.) Nees is a perennial grass that includes both sexual and apomictic cytotypes. The availability of a reference transcriptome for this species would constitute a very important tool toward the identification of genes controlling key steps of the apomictic pathway. Here, we used Roche/454 sequencing technologies to generate reads from inflorescences of E. curvula apomictic and sexual genotypes that were de novo assembled into a reference transcriptome. Near 90% of the 49568 assembled isotigs showed sequence similarity to sequences deposited in the public databases. A gene ontology analysis categorized 27448 isotigs into at least one of the three main GO categories. We identified 11475 SSRs, and several of them were assayed in E curvula germoplasm using SSR-based primers, providing a valuable set of molecular markers that could allow direct allele selection. The differential contribution to each library of the spliced forms of several transcripts revealed the existence of several isotigs produced via alternative splicing of single genes. The reference transcriptome presented and validated in this work will be useful for the identification of a wide range of gene(s) related to agronomic traits of E. curvula, including those controlling key steps of the apomictic pathway in this species, allowing the extrapolation of the findings to other plant species.