Microgametophyte development in Cannabis sativa L. and first androgenesis induction through microspore embryogenesis

[EN] Development of double haploids is an elusive current breeding objective in Cannabis sativa L. We have studied the whole process of anther and pollen grain formation during meiosis, microsporogenesis, and microgametogenesis and correlated the different microgametophyte developmental stages with bud length in plants from varieties USO31 and Finola. We also studied microspore and pollen amyloplast content and studied the effect of a cold pretreatment to excised buds prior to microspore in vitro culture. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% were found. A high uniformity in the developmental stage of microspores and pollen grains contained in anthers was observed, and this allowed the identification of bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains. The starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis. Although at a low frequency, cold-shock pretreatment applied on buds can deviate the naturally occurring gametophytic pathway toward an embryogenic development. This represents the first report concerning androgenesis induction in C. sativa, which lays the foundations for double haploid research in this species.Galán-Ávila, A.; García-Fortea, E.; Prohens Tomás, J.; Herraiz García, FJ. (2021). Microgametophyte development in Cannabis sativa L. and first androgenesis induction through microspore embryogenesis. Frontiers in Plant Science. 12:1-22. https://doi.org/10.3389/fpls.2021.669424S1221

Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus embryogenic microspores

[EN] The change in developmental fate of microspores reprogrammed toward embryogenesis is a complex but fascinating experimental system where microspores undergo dramatic changes derived from the developmental switch. After 40 years of study of the ultrastructural changes undergone by the induced microspores, many questions are still open. In this work, we analyzed the architecture of DNA-containing organelles such as plastids and mitochondria in samples of B. napus isolated microspore cultures covering the different stages before, during, and after the developmental switch. Mitochondria presented a conventional oval or sausage-like morphology for all cell types studied, similar to that found in vivo in other cell types from vegetative parts. Similarly, plastids of microspores before induction and of non-induced cells showed conventional architectures. However, approximately 40% of the plastids of embryogenic microspores presented atypical features such as curved profiles, protrusions, and internal compartments filled with cytoplasm. Three-dimensional reconstructions confirmed that these plastids actually engulf cytoplasm regions, isolating them from the rest of the cell. Acid phosphatase activity was found in them, confirming the lytic activity of these organelles. In addition, digested plastid-like structures were found excreted to the apoplast. All these phenomena seemed transient, since microspore-derived embryos (MDEs) showed conventional plastids. Together, these results strongly suggested that under special circumstances, such as those of the androgenic switch, plastids of embryogenic microspores behave as autophagic plastids (plastolysomes), engulfing cytoplasm for digestion, and then are excreted out of the cytoplasm as part of a cleaning program necessary for microspores to become embryos.We especially thank Professor L. Andrew Staehelin for his help and advice during the stay of JMSS at his lab at UC Boulder, where part of the samples used in this work was processed. We also want to express our thanks to the staff of the COMAV greenhouses and to the staff of the Electron Microscopy Service of Universitat Politecnica de Valencia for their excellent technical help. This work was supported by the following grants to JMSS: AGL2010-17895 from Spanish MICINN and ACOMP/2012/168 from Generalitat Valenciana.Parra Vega, V.; Corral Martínez, P.; Rivas-Sendra, A.; Seguí-Simarro, JM. (2015). Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus embryogenic microspores. Frontiers in Plant Science. 6(94). https://doi.org/10.3389/fpls.2015.00094S694Aubert, S., Gout, E., Bligny, R., Marty-Mazars, D., Barrieu, F., Alabouvette, J., … Douce, R. (1996). 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<i>In vitro</i> imaginal disc development and moulting hormone

ABSTRACT imaginal leg and wing discs obtained from late-third-instar Drosophila larvae were cultured in vitro in various concentrations of ecdysterone ranging from 10−10 to 10−5 M in order to test the effect of hormone concentration on evagination and cell differentiation. At the optimal concentration of 8 × 10−8 M discs evaginated normally, secreted the pupal cuticle, underwent apolysis, differentiated imaginal structures and secreted the imaginal cuticle. At suboptimal concentrations (10−8 M and less), evagination was incomplete in a variable proportion of appendages. Morphogenetic movements were limited to the earlier ones; so that appendages did not emerge from the peripodial sac. Subsequent development, whenever it occurred, took place inside the peripodial sac. This particular type of ‘endoevagination’ was only obtained with sub-optimal hormone concentration. At supra-optimal concentrations (10−6 M and more), evagination was always complete but further differentiation was inhibited. These results show that endoevagination is strictly related to insufficient supply of hormone and that morphogenesis and cell differentiation in imaginal discs are two independent phenomena, which respond to different levels of hormone stimulation.</jats:p

RGD-dependent growth of maize calluses and immunodetection of an integrin-like protein

AbstractWhen maize calluses are grown in the presence of the RGD peptide, important morphological changes are observed indicating the presence of a likely RGD-binding receptor. Polyclonal antibodies generated against the human β1 integrin subunit, the platelet integrin αIIbβ3 (P23) and antibodies specific for either the β3 platelet chain or the αIIb polypeptide cross-react with glycoproteins in Western blot analyses. Immunoprecipitation assays indicate that this maize integrin-like protein shares structural similarities with the animal αIIbβ3 complex. We also show that AcAt2, a polyclonal antibody raised against Arabidopsis proteins purified on an RGD column, interacts with a maize protein