Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus

[EN] Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.This work was supported by grant PID2020-115763RBI00 to JS-S from Spanish MICINN and by a Juan de la Cierva -Incorporacion Fellowship and a Marie Sklodowska-Curie Individual Fellowship (656579) to PC-M. RM holds a CDEIGENT (2018/023) fellowship from Generalitat Valenciana.Camacho-Fernández, C.; Seguí-Simarro, JM.; Mir Moreno, R.; Boutilier, K.; Corral-Martínez, P. (2021). Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus. Frontiers in Plant Science. 12:1-16. https://doi.org/10.3389/fpls.2021.7371391161

Obtención de doble haploides en especies de interés agronómico: análisis de agentes y mecanismos celulares implicados en la inducción androgénica en berenjena, colza y tomate

La androgénesis es un proceso experimental que permite la obtención de líneas puras doble haploides a través de embriogénesis o callogénesis inducida partiendo, en la mayoría de los casos, del gametófito masculino (polen bicelular joven) o en la mayoría de los casos, su precursor, la microspora. Desde una perspectiva biotecnológica, esta posibilidad adquiere gran relevancia, pues las líneas puras son la base del proceso de obtención de semilla híbrida, y este proceso permite reducir a una sola generación, las 8 o 9 generalmente necesarias en el caso de utilizar métodos de mejora genética clásica para obtener las líneas puras. En la presente Tesis Doctoral se aborda el estudio de la inducción de androgénesis aplicando en paralelo dos abordajes: uno básico, mediante el estudio de factores que influyen y cambios que tienen lugar en la microspora al ser inducida, y uno aplicado, orientado a mejorar la eficiencia de inducción en especies recalcitrantes. El estudio se lleva a cabo en tres especies: la colza (Brassica napus), utilizada como sistema modelo para el estudio básico, y dos recalcitrantes, tomate (Solanum Lycopersicum), y berenjena (Solanum melongena). En resumen, mediante los estudios planteados en esta Tesis, se pretende conocer mejor algunos de los procesos de la androgénesis mediante su estudio en especies modelo como la colza, y recalcitrantes como el tomate. Además se pretende mejorar la eficiencia de la obtención de doble haploides en berenjena, desarrollando un método eficiente de cultivo de microsporas aisladas de berenjena.Corral Martínez, P. (2013). Obtención de doble haploides en especies de interés agronómico: análisis de agentes y mecanismos celulares implicados en la inducción androgénica en berenjena, colza y tomate [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31643TESI

Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis

[EN] Doubled haploid (DH) technology allows for the production of pure lines, useful for plant breeding, through a one-generation procedure that reduces considerably the time and resources needed to produce them. Despite the advantages of microspore culture to obtain DHs, this technique is still insufficiently developed in eggplant, where DHs are produced from microsporederived calli through organogenesis. At present, very little is known on the best in vitro conditions to promote this process. This is why in this work we addressed the optimization of the process of regeneration of eggplant DH plants from microspore-derived calli. We evaluated the effect of different media compositions in the induction of organogenesis, in the promotion of shoot growth and elongation, and in root growth. According to our results, we propose the repeated subculture of the calli in MS medium with 0.2 mg/l IAA and 4 mg/l zeatin to produce shoots, and then the repeated subculture of the excised shoots in basal MS medium to promote their conversion into entire plantlets. This procedure yielded 7.6 plants per 100 cultured calli, which represents a *49 increase with respect to previous reports. We also evaluated by flow cytometry and SSR molecular markers the effect of these in vitro culture conditions in the rate of DH plant production, finding that*70 % of the regenerated plants were true DHs. These results substantially improve the efficiencies of DH recovery published to date in eggplant, and may be useful to those working in the field of eggplant doubled haploidy and breeding.We acknowledge Dr. Rosa Peiro for her statistical advice, and the staff of the COMAV greenhouses for their valuable help. This work was supported by the AGL2014-55177-R grant from Spanish MINECO to JMSS.Rivas Sendra, A.; Corral Martínez, P.; Camacho Fernández, C.; Seguí-Simarro, JM. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture. 122(3):759-765. https://doi.org/10.1007/s11240-015-0791-6S7597651223Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D (2014) Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat. Plant Cell Tissue Organ Cult 116:125–130Borgato L, Conicella C, Pisani F, Furini A (2007) Production and characterization of arboreous and fertile Solanum melongena plus Solanum marginatum somatic hybrid plants. Planta 226:961–969Castillo AM, Nielsen NH, Jensen A, Vallés MP (2014) Effects of n-butanol on barley microspore embryogenesis. Plant Cell Tissue Organ Cult 117:411–418Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tissue Organ Cult 104:359–373Dumas de Vaulx R, Chambonnet D (1982) Culture in vitro d’anthères d’aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements à 35°C associés à de faibles teneurs en substances de croissance. Agronomie 2:983–988Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8:377–424Eshaghi ZC, Abdollahi MR, Moosavi SS, Deljou A, Seguí-Simarro JM (2015) Induction of androgenesis and production of haploid embryos in anther cultures of borage (Borago officinalis L.). Plant Cell Tissue Organ Cult 1–9. doi: 10.1007/s11240-015-0768-5Franklin G, Sheeba CJ, Sita GL (2004) Regeneration of eggplant (Solanum melongena L.) from root explants. In Vitro Cell Dev Biol Plant 40:188–191Gisbert C, Prohens J, Nuez F (2006) Efficient regeneration in two potential new crops for subtropical climates, the scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants. New Zeal J Crop Hort Sci 34:55–62Kaur M, Dhatt AS, Sandhu JS, Gosal SS (2011) In vitro plant regeneration in brinjal from cultured seedling explants. Indian J Hortic 68:61–65Kim M, Park E-J, An D, Lee Y (2013) High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:191–201Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L). Plant Cell Rep 15:391–395Mohinuddin AKM, Chowdhury MKU, Abdullah Zaliha C, Napis S (1997) Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tissue Organ Cult 51:75–78Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant growth regulators III: gibberellins, ethylene, abscisic acid, their analogues and inhibitors; miscellaneous compounds. In George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture, 3 edn, vol 1. Springer, DordrechtParra-Vega V, Renau-Morata B, Sifres A, Seguí-Simarro JM (2013) Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:353–360Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 3. Kluwer, Dordrecht, pp 115–141Salas P, Prohens J, Seguí-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274Seguí-Simarro JM (2015) Androgenesis in solanaceae. In Germanà MA, Lambardi M (eds), In vitro embryogenesis. Springer Science + Business Media, The NetherlandsSeguí-Simarro JM, Nuez F (2006) Androgenesis induction from tomato anther cultures: callus characterization. Acta Hort 725:855–861Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Sgamma T, Thomas B, Muleo R (2015) Ethylene inhibitor silver nitrate enhances regeneration and genetic transformation of Prunus avium (L.) cv Stella. 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Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes

[EN] Notwithstanding the importance of eggplant in global horticulture, doubled haploid production in this species is still far from being efficient. Although acknowledged to have a role in the efficiency of androgenesis induction, factors such as the growth conditions of donor plant or the in vitro culture environment have not been deeply explored or not explored at all in eggplant, which leaves room for further improvement. In this work, we investigated the effects of different in vivo and in vitro parameters on the androgenic performance of different eggplant genotypes, including two hybrids and a DH line. The in vivo parameters included the exposure of donor plants to different temperature and light conditions and to increased levels of boron. The in vitro parameters included the use of different concentrations of NLN medium components, sucrose and growth regulators, and the suspension of microspores at different densities. Our results showed that whereas greenhouse temperature variations or boron application did not to have a positive influence, greenhouse lighting influenced their viability, thereby conditioning the embryogenic response. Changes in different sucrose, salts and hormone levels had different effects in the genotypes studied, which correlated with their genetic constitution. Finally, we determined the best microspore density, different from that previously proposed. Our work shed light on the role of different factors involved in eggplant microspore cultures, some of them not yet studied, contributing to make microspore culture a more efficient tool in eggplant breeding.This work was supported by Grant AGL2017-88135-R to JMSS from Spanish MICINN, respectively, jointly funded by FEDER. ARS and CCF were supported by predoctoral fellowships from the FPI Programs of Universitat Politecnica de Valencia and Generalitat Valenciana, respectively.Rivas-Sendra, A.; Corral Martínez, P.; Camacho-Fernández, C.; Porcel, R.; Seguí-Simarro, JM. (2020). Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes. Euphytica. 216(11):1-15. https://doi.org/10.1007/s10681-020-02709-4S11521611Abdollahi MR, Corral-Martinez P, Mousavi A, Salmanian AH, Moieni A, Seguí-Simarro JM (2009) An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiol Plant 31:1313–1317Aulinger IE (2002) Combination of in vitro androgenesis and biolistic transformation: an approach for breeding transgenic maize (Zea mays L.) lines. Swiss Federal Institute of Technology, Zurich, p 115Borderies G, le Bechec M, Rossignol M, Lafitte C, Le Deunff E, Beckert M, Dumas C, Matthys-Rochon E (2004) Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development. Eur J Cell Biol 83:205–212Bueno MA, Gómez A, Sepúlveda F, Seguí-Simarro JM, Testillano PS, Manzanera JA, Risueño MC (2003) Microspore-derived embryos from Quercus suber anthers mimic zygotic embryos and maintain haploidy in long-term anther culture. J Plant Physiol 160:953–960Camacho-Fernández C, Hervás D, Rivas-Sendra A, Marín MP, Seguí-Simarro JM (2018) Comparison of six different methods to calculate cell densities. Plant Methods 14:30Chambonnet D (1988) Production of haploid eggplant plants. Bulletin interne de la Station d’Amélioration des Plantes Maraichères d’Avignon-Montfavet, France, pp 1–10Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Custers J (2003) Microspore culture in rapeseed (Brassica napus L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 185–193Dunwell JM (1976) A comparative study of environmental and developmental factors which influence embryo induction and growth in cultured anthers of Nicotiana tabacum. 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Plant Cell Rep 22:365–370Jouannic S, Champion A, Seguí-Simarro JM, Salimova E, Picaud A, Tregear J, Testillano P, Risueno MC, Simanis V, Kreis M, Henry Y (2001) The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division. Plant J 26:637–649Kim M, Jang I-C, Kim J-A, Park E-J, Yoon M, Lee Y (2008) Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep 27:425–434Kim M, Park E-J, An D, Lee Y (2013) High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:191–201Lantos C, Juhasz AG, Vagi P, Mihaly R, Kristof Z, Pauk J (2012) Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.). Plant Biotechnol Rep 6:123–132Liu L, Huang L, Li Y (2013) Influence of boric acid and sucrose on the germination and growth of areca pollen. Am J Plant Sci 4:1669–1674Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L). Plant Cell Rep 15:391–395Paire A, Devaux P, Lafitte C, Dumas C, Matthys-Rochon E (2003) Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation. Plant Cell Tissue Organ Cult 73:167–176Parra-Vega V, Seguí-Simarro JM (2013) Improvement of an isolated microspore culture protocol for Spanish sweet pepper (Capsicum annuum L.). In: Lanteri S, Rotino GL (eds) Breakthroughs in the genetics and breeding of Capsicum and Eggplant. Universita degli Studi di Torino, Torino, Italy, pp 161–168Peñaloza P, Toloza P (2018) Boron increases pollen quality, pollination, and fertility of different genetic lines of pepper. J Plant Nutr 41:969–979Rivas-Sendra A, Corral-Martínez P, Camacho-Fernández C, Seguí-Simarro JM (2015) Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell Tissue Organ Cult 122:759–765Rivas-Sendra A, Calabuig-Serna A, Seguí-Simarro JM (2017a) Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena. Front Plant Sci 8:1177Rivas-Sendra A, Campos-Vega M, Calabuig-Serna A, Seguí-Simarro JM (2017b) Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica 213:89Rivas-Sendra A, Corral-Martínez P, Porcel R, Camacho-Fernández C, Calabuig-Serna A, Seguí-Simarro JM (2019) Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer. 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Genetic, quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured ms1035 tomato anthers

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 35 gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 35 mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.We want to acknowledge Drs. Alicia Sifres and Begona Renau for their excellent technical work, as well as the staff of the COMAV greenhouses for their valuable help. Thanks are also due to the editor and the anonymous reviewers for their valuable comments to improve the final version of the paper. This work was supported by grants AGL2006-06678 and AGL2010-17895 from Spanish MICINN to JMSS.Corral Martínez, P.; Nuez Viñals, F.; Seguí-Simarro, JM. (2011). Genetic, quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured ms1035 tomato anthers. Euphytica. 178(2):2151-228. doi:10.1007/s10681-010-0303-zS21512281782Areshchenkova T, Ganal MW (1999) Long tomato microsatellites are predominantly associated with centromeric regions. Genome 42:536–544Bal U, Abak K (2005) Induction of symmetrical nucleus division and multicellular structures from the isolated microspores of Lycopersicon esculentum Mill. Biotechnol Biotec Eq 19:35–42Bal U, Abak K (2007) Haploidy in tomato (Lycopersicon esculentum Mill.): a critical review. Euphytica 158:1–9Dao NT, Shamina ZB (1978) Cultivation of isolated tomato anthers. 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Hilgardia 18:599–633Seguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404Seguí-Simarro JM, Nuez F (2005) Meiotic metaphase I to telophase II is the most responsive stage of microspore development for induction of androgenesis in tomato (Solanum Lycopersicum). Acta Physiol Plant 27:675–685Seguí-Simarro JM, Nuez F (2007) Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers. J Exp Bot 58:1119–1132Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134:1–12Seguí-Simarro JM, Otegui MS, Austin JR, Staehelin LA (2008) Plant cytokinesis—insights gained from electron tomography studies. In: Verma DPS, Hong Z (eds) Cell division control in plants. 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Plant Cell Rep 18:312–317Smulders MJM, Bredemeijer G, RusKortekaas W, Arens P, Vosman B (1997) Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species. Theor Appl Genet 94:264–272Summers WL, Jaramillo J, Bailey T (1992) Microspore developmental stage and anther length influence the induction of tomato anther callus. Hortscience 27:838–840Touraev A, Pfosser M, Heberle-Bors E (2001) The microspore: a haploid multipurpose cell. Adv Bot Res 35:53–109Varghese TM, Gulshan Y (1986) Production of embryoids and calli from isolated microspores of tomato (Lycopersicon esculentum Mill.) in liquid media. Biol Plant 28:126–129Zagorska NA, Shtereva A, Dimitrov BD, Kruleva MM (1998) Induced androgenesis in tomato (Lycopersicon esculentum Mill.)—I. Influence of genotype on androgenetic ability. 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Chaperone-like properties of tobacco plastid thioredoxins f and m

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein

Embryogenic competence of microspores is associated to their ability to form a callosic, osmoprotective subintinal layer

[EN] Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multi-disciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.Thanks are due to the Electron Microscopy Service of Universitat Politecnica de Valencia, Marisol Gascon (IBMCP Microscopy Service), Dr Kim Boutilier (WUR, Wageningen) for hosting ARS at her lab, and Dr Samantha Vernhettes (INRA Versailles) for kindly providing us with S4B. This work supported by grants AGL2014-55177-R and AGL2017-88135-R to JMSS from MINECO jointly funded by FEDER.Rivas-Sendra, A.; Corral Martínez, P.; Porcel, R.; Camacho-Fernández, C.; Calabuig-Serna, A.; Seguí-Simarro, JM. (2019). Embryogenic competence of microspores is associated to their ability to form a callosic, osmoprotective subintinal layer. Journal of Experimental Botany. 70(4):1267-1281. https://doi.org/10.1093/jxb/ery458S12671281704Abramova, L. I. (2003). Russian Journal of Plant Physiology, 50(3), 324-329. doi:10.1023/a:1023866019102Adkar-Purushothama, C. R., Brosseau, C., Giguère, T., Sano, T., Moffett, P., & Perreault, J.-P. (2015). Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants. The Plant Cell, 27(8), 2178-2194. doi:10.1105/tpc.15.00523Cordewener, J., Bergervoet, J., & Liu, C.-M. (2000). 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Induction of embryogenesis in Brassica napus microspores produces a callosic subintinal layer and abnormal cell walls with altered levels of callose and cellulose

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.01018The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.We want to express our thanks to the staff of the Electron Microscopy Service of Universitat Politecnica de Valencia. Thanks are also due to Dr. Kim Boutilier (PRI Wageningen, The Netherlands) for her help during the stays of VPV and ARS at her lab, to Dr. Samantha Vernhettes (INRA Versailles, France) for her kind gift of S4B staining, and especially to Prof. L. A. Staehelin for his help and friendship during the stay of JMSS at UC Boulder. This work was supported by grants BEST/20081154 from Generalitat Valenciana and AGL2014-55177-R from Spanish MINECO to JMSS.Parra Vega, V.; Corral Martínez, P.; Rivas-Sendra, A.; Seguí-Simarro, JM. (2015). Induction of embryogenesis in Brassica napus microspores produces a callosic subintinal layer and abnormal cell walls with altered levels of callose and cellulose. Frontiers in Plant Science. 6(1018):1-17. https://doi.org/10.3389/fpls.2015.01018S1176101

Filosofía, darwinismo y evolución

Los ensayos recogidos en este texto son una muestra del aporte de investigadores de tres países latinoamericanos a la reflexión teórica y filosófica sobre la naturaleza y los alcances de la selección natural. / Contenido. Preliminares; Capítulo 1 - Selección natural; Capítulo 2 - Cognición y evolución; Capítulo 3 - El Origen del hombre; Anexo 01 - índice de autores; Anexo 02 - índice de conceptos