Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

The pathways of micro- and megagametophyte development in Agave fourcroydes (henequén) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequén is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.

No abstract

To induce somatic embryogenesis in habanero pepper (Capsicum chinense Jacq.), the cultivar BVll-03, belonging to the red type, was used. Different explants were evaluated, as were different culture media, the composition of which varied in the content of plant growth regulators. Results showed the formation of somatic embryos from cotyledons, zygotic embryos, germinated zygotic embryos, hypocotyls, and cotyledonary leaves. Explants were cultured on Murashige and Skoog medium supplemented with 2,4-D (9.05 μm). The somatic embryos always formed directly from the explant, without callus formation, and the greatest efficiency was obtained when segments of hypocotyls were cultured, obtaining 175 ± 20 somatic embryos per explant. Only the somatic embryos obtained on Murashige and Skoog medium containing 2,4-D (9.05 μm) and treated with abscisic acid (ABA) (1.89 μm) before their transfer to the germination media (Murashige and Skoog + 1.1 μm GA3) emitted their radicule and expanded their cotyledonary leaves (60%), whereas the remaining embryos did not achieve germination because of different causes (abnormalities, delayed development). Not only is this protocol of somatic embryogenesis the first to be reported for this species (C. chinense Jacq.), but it is also the most efficient reported so far, within the Capsicum genus.

Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from Carica papaya cv. Maradol. We isolated four genes (CpRap2.4a, CpRAap2.4b, CpRap2.1 and CpRap2.10), and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2) domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.

Spanish red cedar (Cedrela odorata L.) is a tropical timber tree native to the Americas from southern Mexico to northern Argentina. Commercial plantations are scarce and, consequently, natural populations are overexploited. Traditional propagation practices for the establishment of large-scale plantations have had limited success in this species due to the relative scarcity of seeds, its broad genetic diversity and the lack of domesticated varieties. In vitro clonal propagation provides an effective method to overcome this situation and increase the yield of commercial plantations through the rapid multiplication of elite materials. Somatic embryogenesis (SE) is one of the most promising strategies for tree propagation due to the possibility of producing artificial seeds, the ability to store and rapidly mobilize germplasm and the opportunity for genetic manipulation. We report here the induction of indirect SE in C. odorata from calli derived from immature zygotic embryos after 12 weeks of culture. Macroscopic, histological, and scanning electron microscopic analyses of the calli revealed the presence of embryogenic cell clusters that formed cotyledonary embryos with clear bipolar structures and no vascular connections with the mother tissue. Different media preparations containing combinations of diverse auxins and cytokinins are known to have different effects on the type and frequency of embryogenic structures. Embryo conversion was achieved using an MS-based medium [Murashige T, Skoog F (1962) Physiol Plant 15:473-497, 1962 supplemented with abscisic acid, and transfer to soil was successful at a rate of 75%. The method described here provides a basis for optimizing the clonal propagation and genetic manipulation of this valuable species.

In recent years, the massive influx of pelagic Sargassum spp. has generated great interest in the scientific community, highlighting the urgency of addressing the physiology and biochemical composition of these species. Until now, the presence of lignified cells in the tissue of Sargassum natans and Sargassum fluitans has not been reported. Although ‘‘lignin-like’’ compounds have been identified in green algae, the presence of true lignin in the Sargassum genus has not been confirmed. Our work is the first report of lignified cells forming the secondary cell wall in these Sargassum. This study used histological techniques applied to thick sections for identifying lignin-like tissues in Sargassum spp. The dyes as Safranin O and Toluidine have been used to differentiate lignin and cellulose in conducting tissue and to indicate the presence, absence, and distribution of these compounds in tissues. This work is the initial study of the cell wall heteropolymers structure and arrangement in Sargassum spp., providing insights into the unique cell wall architecture of these seaweeds.

The ontogenesis of direct high-frequency somatic embryogenesis of C. chinense induced from hypocotyl was characterized through a histological analysis of the different phases in the histodifferentiation process during the development of the somatic embryo. The anatomical analysis was carried out since the hypocotyl segments were placed in the culture medium until 45 days of culture. The somatic embryos were induced and maintained in Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (9.5 μm). Samples of tissues and organs were taken every 24 h, fixed in formalin acetic alcohol, and embedded in plastic resin. They were cut into serial sections (5 μm) and stained with toluidine blue. The analysis revealed that the proembryogenic cells originated just from provascular hypocotyl cells. Provascular cells acquired the embryogenic competence 48 h after induction and an intense mitotic division was observed and embryogenic structures were generated first along the vascular strands, which subsequently evolved into somatic embryos. After 2 weeks, there were observed embryos at different stages of development (preglobular, globular, heart-shaped, torpedo-shaped, and cotyledonary). This is the first report dealing with the ontogenesis of the direct somatic embryogenesis of C. chinense, and it is the most complete histological characterization carried out on somatic embryogenesis in the Capsicum genus to date.