Calcium and cell wall dynamics during microspore embryogenesis and double haploid production in rapeseed and eggplant

Tesis por compendioAndrogenesis induction is an experimental procedure by which microspores are diverted from their original gametophytic pathway towards embryogenesis by applying specific stresses in vitro. It allows for the production of doubled haploid (DH) pure lines through anther culture or isolated microspore culture followed by chromosome doubling. DH technology is interesting for both basic research and plant breeding. In this Thesis, we studied microspore embryogenesis with two parallel approaches: (I) an applied study directed to the development of the first eggplant (Solanum melongena) highly embryogenic line and the improvement of the efficiency of eggplant microspore cultures; and (II) a fundamental research study focused on the relationship between microspore embryogenesis ability, intracellular Ca2+ levels and the dynamics of callose and cellulose deposition for cell wall formation in microspore-derived structures, using rapeseed (Brassica napus) as a model species. As an applied research, we developed and evaluated an eggplant DH population from a commercial hybrid, and identified and characterized the first eggplant highly androgenic DH line (DH36), which may be used to facilitate the study of eggplant androgenesis and for both basic and applied research. In addition, we evaluated different factors involved in microspore embryogenesis induction efficiency in eggplant and optimized the regeneration protocol for DH production via microspore culture. Together, the applied research on eggplant microspore embryogenesis made in this Thesis resulted in the most efficient protocol existing to date for DH production in eggplant. As a fundamental research, we studied the dynamics of Ca2+ during in vivo microsporogenesis and microgametogenesis, as well as during the first stages of in vitro-induced microspore embryogenesis, establishing a link between microspore embryogenesis and changes in Ca2+ levels and subcellular distribution. In addition, we studied the deposition of callose and cellulose during the first stages of microspore embryogenesis and demonstrated that the abnormally increased callose deposition and the inhibition of cellulose deposition observed in embryogenic microspores is most likely caused by a transient increase in the intracellular Ca2+ levels that occurs right after microspore induction. We also found that this particular dynamics of callose and cellulose deposition is related to microspore embryogenesis ability, and is essential for proper progression and success of microspore embryogenesis. In summary, the research made in this Thesis helps to further understand the basis underlying microspore embryogenesis and cell totipotency, and to apply the powerful DH technology to an economically important crop such as eggplant.La inducción de androgénesis es un procedimiento experimental en el cual las microsporas se desvían de su vía gametofítica original hacia embriogénesis, mediante la aplicación de estreses específicos in vitro. Este fenómeno permite la producción de líneas puras dobles haploides (DH) mediante cultivo de anteras o cultivo de microsporas aisladas seguidos de duplicación cromosómica. La tecnología DH es interesante tanto para la investigación básica como para su aplicación a la mejora genética vegetal. En esta Tesis se estudia la embriogénesis de microsporas y la obtención de DHs con dos enfoques paralelos: (I) un estudio aplicado dirigido al desarrollo de la primera línea de berenjena (Solanum melongena) con alta respuesta androgénica y a la mejora de la eficiencia de los cultivos de microsporas de berenjena; y (II) un estudio de investigación básica centrado en la relación entre la habilidad para la embriogénesis de microsporas, los niveles intracelulares de Ca2+ y la dinámica de la deposición de calosa y celulosa para la formación de paredes celulares en estructuras derivadas de microsporas, utilizando como especie modelo la colza (Brassica napus). Como investigación aplicada, se desarrolló y evaluó una población DH de berenjena a partir de un híbrido comercial, y se identificó y caracterizó la primera línea DH altamente androgénica de berenjena (DH36), que puede usarse para facilitar el estudio de la androgénesis en berenjena y para otros estudios aplicados o básicos. Además, se evaluaron diferentes factores implicados en la eficiencia de la inducción de embriogénesis de microsporas en berenjena, y se optimizó el protocolo de regeneración para la producción de DH mediante cultivo de microsporas. En conjunto, la investigación aplicada sobre la embriogénesis de microsporas realizada en esta Tesis proporciona el protocolo más eficiente existente hasta la fecha para la producción de DH en berenjena. Como investigación fundamental, se estudió la dinámica del Ca2+ durante la microsporogénesis y la microgametogénesis in vivo, así como durante las primeras etapas de la embriogénesis de microsporas inducida in vitro, y se estableció un vínculo entre la embriogénesis de microsporas y los cambios en el nivel y distribución intracelular de Ca2+. Además, se estudió la deposición de calosa y celulosa durante las primeras etapas de la embriogénesis de microsporas y se demostró que la excesiva deposición de calosa y la inhibición de la deposición de celulosa, exclusivas de las microsporas embriogénicas, están causadas por el aumento transitorio de Ca2+ intracelular que se produce justo tras la inducción. Hemos demostrado que esta particular dinámica de la deposición de calosa y celulosa está relacionada con la capacidad androgénica, y que es fundamental para la correcta progresión y éxito de la embriogénesis de microsporas. En resumen, la investigación realizada en esta Tesis ayuda a comprender mejor la base de la embriogénesis de microsporas y de la totipotencia celular, y a aplicar la potente tecnología DH a un cultivo económicamente importante como es la berenjena.La inducció d'androgènesi és un procediment experimental en el qual les microspores es desvien de la seua via gametofítica original cap a un nou destí embriogènic, mitjançant l'aplicació d'estressos específics in vitro. Aquest fenomen permet la producció de línies pures dobles haploides (DH) mitjançant cultiu d'anteres o cultiu de microsporas aïllades seguits de duplicació cromosòmica. La tecnologia DH és interessant tant per a la recerca bàsica com per a la seua aplicació a la millora genètica vegetal. En aquesta Tesi s'estudia l'embriogènesi de microspores i l'obtenció de DHs amb dos enfocaments paral·lels: (I) un estudi aplicat dirigit al desenvolupament de la primera línia d'albergina (Solanum melongena) amb alta resposta androgènica i a la millora de l'eficiència dels cultius de microspores d'albergina; i (II) un estudi de recerca bàsica centrat en la relació entre la capacitat per a l'embriogènesi de microspores, els nivells intracel·lulars de Ca2+ i la dinàmica de la deposició de cal·losa i cel·lulosa per a la formació de parets cel·lulars en estructures derivades de microsporas, utilitzant com a espècie model la colza (Brassica napus). Com a recerca aplicada, es va desenvolupar i avaluar una població DH d'albergina a partir d'un híbrid comercial, i es va identificar i caracteritzar la primera línia DH altament androgènica d'albergina (DH36), que pot usar-se per a facilitar l'estudi de l'androgènesi en albergina i per a altres estudis aplicats o bàsics. A més, es van avaluar diferents factors implicats en l'eficiència de la inducció d'embriogènesi de microspores en albergina, i es va optimitzar el protocol de regeneració per a la producció de DH mitjançant cultiu de microspores. En conjunt, la recerca aplicada sobre l'embriogènesi de microspores realitzada en aquesta Tesi proporciona el protocol més eficient existent fins avui per a la producció de DH en albergina. Com a recerca fonamental, es va estudiar la dinàmica del Ca2+ durant la microsporogènesi i la microgametogènesi in vivo, així com durant les primeres etapes de l'embriogènesi de microspores induïda in vitro, i es va establir un vincle entre l'embriogènesi de microspores i els canvis en el nivell i distribució intracel·lular de Ca2+. A més, es va estudiar la deposició de cal·losa i cel·lulosa durant les primeres etapes de l'embriogènesi de microspores i es va demostrar que l'excessiva deposició de cal·losa i la inhibició de la deposició de cel·lulosa, exclusives de les microspores embriogèniques, estan causades per l'increment transitori del Ca2+ intracel·lular que es produeix just després de la inducció. Hem demostrat que aquesta particular dinàmica de la deposició de cal·losa i cel·lulosa està relacionada amb la capacitat androgènica, i que és fonamental per a la correcta progressió i èxit de l'embriogènesi de microspores. En resum, la recerca realitzada en aquesta Tesi ajuda a comprendre millor la base de l'embriogènesi de microspores i de la totipotència cel·lular, i a aplicar la potent tecnologia DH a un cultiu econòmicament important com és l'albergina.Rivas Sendra, A. (2017). Calcium and cell wall dynamics during microspore embryogenesis and double haploid production in rapeseed and eggplant [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/85548TESISCompendi

Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena

[EN] Calcium is widely known to have a role as a signaling molecule in many different processes, including stress response and activation of the embryogenic program. However, there are no direct clues about calcium levels during microspore embryogenesis, an experimental process that combines a developmental switch toward embryogenesis and the simultaneous application of different stressing factors. In this work, we used FluoForte, a calcium-specific fluorescent vital dye, to track by confocal microscopy the changes in levels and subcellular distribution of calcium in living rapeseed (B. napus) and eggplant (S. melongena) microspores and pollen grains during in vivo development, as well as during the first stages of in vitro-induced microspore embryogenesis in rapeseed. During in vivo development, a clear peak of cytosolic Ca2+ was observed in rapeseed vacuolate microspores and young pollen grains, the stages more suitable for embryogenesis induction. However, the Ca2+ levels observed in eggplant were dramatically lower than in rapeseed. Just after in vitro induction, Ca2+ levels increased specifically in rapeseed embryogenic microspores at levels dramatically higher than during in vivo development. The increase was observed in the cytosol, but predominantly in vacuoles. Non-embryogenic forms such as callus-like and pollen-like structures presented remarkably different calcium patterns. After the heat shock-based inductive treatment, Ca2+ levels progressively decreased in all cases. Together, our results reveal unique calcium dynamics in in vivo rapeseed microspores, as well as in those reprogrammed to in vitro embryogenesis, establishing a link between changes in Ca2+ level and subcellular distribution, and microspore embryogenesis.This work was supported by grant AGL2014-55177-R to JS from Spanish Ministerio de Economia y Competitividad (MINECO) jointly funded by FEDER. AR was supported by a predoctoral fellowship from the FPI Program of Universitat Politecnica de Valencia.Rivas-Sendra, A.; Calabuig-Serna, A.; Seguí-Simarro, JM. (2017). Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena. Frontiers in Plant Science. 8. doi:10.3389/fpls.2017.01177

Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response

[EN] We developed an eggplant doubled haploid (DH) population from a commercial hybrid through androgenesis in microspore culture. Morphological variation, reproductive ability and androgenic responsiveness were evaluated. The DH population showed segregation in vegetative traits related to leaf, flower and fruit, and in reproductive traits such as fruit and seed setting or germination rate. The DH population and subsequent generations also presented variation in the androgenic response, with null, low and high response lines. From this population, we were able to identify the first eggplant highly androgenic DH line (DH36), remarkably similar to the donor hybrid in terms of morphology and reproductive ability, but stably producing four times more calli than the hybrid. The segregating DH population is potentially useful for genetic studies and mapping of several traits, whereas the highly androgenic line DH36 may be used as a model line to facilitate the study of eggplant androgenesis and embryogenesis for both basic and applied research.We would like to thank the reviewers of this manuscript for their critical and helpful comments. This work was supported by Grant AGL2014-55177-R to JMSS from Spanish Ministerio de Economia y Competitividad (MINECO) jointly funded by FEDER. ARS is supported by a Predoctoral Fellowship from the FPI Program of Universitat Politecnica de Valencia.Rivas-Sendra, A.; Manuel Campos-Vega; Calabuig-Serna, A.; Seguí-Simarro, JM. (2017). Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica. 213(4):1-14. doi:10.1007/s10681-017-1879-3S1142134Alpsoy HC, Seniz V (2007) Researches on the in vitro androgenesis and obtaining haploid plants in some eggplant genotypes. Acta Hortic 729:137–141Başay S, Şeniz V, Ellialtioğlu Ş (2011) Obtaining dihaploid lines by using anther culture in the different eggplant cultivars. J Food Agric Environ 9:188–190Bohanec B (2002) Doubled-haploid onions. In: Rabinowitch HD, Currah L (eds) Allium crop science: recent advances. CABI Publishing, Wallingford, pp 145–157Corral-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 195:369–382Corral-Martínez P, Parra-Vega V, Seguí-Simarro JM (2013) Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning. J Exp Bot 64:3061–3075Daghma DES, Hensel G, Rutten T, Melzer M, Kumlehn J (2014) Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging. Front Plant Sci 5:675Doğramacı-Altuntepe M, Peterson TS, Jauhar PP (2001) Anther culture-derived regenerants of durum wheat and their cytological characterization. J Hered 92:56–64Dumas 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 2:983–988Dumas de Vaulx R, Chambonnet D, Pochard E (1981) Culture in vitro d’anthères de piment (Capsicum annuum L.): amèlioration des taux d’obtenction de plantes chez différents génotypes par des traitments à +35°C. Agronomie 1:859–864Eudes F, Shim Y-S, Jiang F (2014) Engineering the haploid genome of microspores. Biocatal Agric Biotechnol 3:20–23FAOSTAT (2016). http://faostat.fao.org . Accessed Dec 2016Ferrie A, Caswell K (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104:301–309Ferrie AMR, Keller WA (1995) Microspore culture for haploid plant production. In: Gamborg OL, Phillips GC (eds) Plant cell, tissue and organ culture: fundamental methods. Springer, Berlin, pp 155–164Ferrie A, Möllers C (2011) Haploids and doubled haploids in Brassica spp. for genetic and genomic research. Plant Cell Tissue Organ Cult 104:375–386Ferrie AMR, Palmer CE, Keller WA (1995) Haploid embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer Academic Publishers, Dordrecht, pp 309–344Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375Frary A, Doganlar S, Daunay MC (2007) Eggplant. In: Kole C (ed) Vegetables. Springer, Berlin, pp 287–313Germanà MA (2011a) Anther culture for haploid and doubled haploid production. Plant Cell Tissue Organ Cult 104:283–300Germanà MA (2011b) Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell Rep 30:839–857Hoekstra S, Vanzijderveld MH, Louwerse JD, Heidekamp F, Vandermark F (1992) Anther and microspore culture of Hordeum vulgare L. cv. Igri. Plant Sci 86:89–96Immonen S, Robinson J (2000) Stress treatments and Ficoll for improving green plant regeneration in triticale anther culture. Plant Sci 150:77–84Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188Kasha KJ, Simion E, Miner M, Letarte J, Hu TC (2003) Haploid wheat isolated microspore culture protocol. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 77–82Li H, Soriano M, Cordewener J, Muiño JM, Riksen T, Fukuoka H, Angenent GC, Boutilier K (2014) The histone deacetylase inhibitor Trichostatin A promotes totipotency in the male gametophyte. Plant Cell 26:195–209Malik MR, Wang F, Dirpaul J, Zhou N, Hammerlindl J, Keller W, Abrams SR, Ferrie AMR, Krochko JE (2008) Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis. J Exp Bot 59:2857–2873Miah MAA, Earle ED, Khush GS (1985) Inheritance of callus formation ability in anther cultures of rice, Oryza sativa L. Theor Appl Genet 70:113–116Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.). Plant Cell Rep 15:391–395Nitsch JP (1972) Haploid plants from pollen. Z Pflanzenzücht 67:3–18Nitsch C, Nitsch JP (1967) Induction of flowering in vitro in stem segments of Plumbago indica L. I. Production of vegetative buds. Planta 72:355Nuez F, Llácer G (2001) La horticultura española. Ediciones de Horticultura, S.L., ReusOleszczuk S, Rabiza-Swider J, Zimny J, Lukaszewski AJ (2011) Aneuploidy among androgenic progeny of hexaploid triticale (XTriticosecale Wittmack). Plant Cell Rep 30:575–586Petolino JF, Jones AM, Thompson SA (1988) Selection for increased anther culture response in maize. Theor Appl Genet 76:157–159Quimio CA, Zapata FJ (1990) Diallel analysis of callus induction and green-plant regeneration in rice anther culture. Crop Sci 30:188–192Rivas-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 122:759–765Rizza F, Mennella G, Collonnier C, Shiachakr D, Kashyap V, Rajam MV, Prestera M, Rotino GL (2002) Androgenic dihaploids from somatic hybrids between Solanum melongena and S. aethiopicum group Gilo as a source of resistance to Fusarium oxysporum f. sp. melongenae. Plant Cell Rep 20:1022–1032Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Kluwer Academic Publishers, Dordrecht, pp 115–141Rotino GL, Restaino F, Gjomarkaj M, Massimo M, Falavigna A, Schiavi M, Vicini E (1991) Evaluation of genetic variability in embryogenetic and androgenetic lines of eggplant. Acta Hortic 300:357–362Rotino GL, Sihachakr D, Rizza F, Vale G, Tacconi MG, Alberti P, Mennella G, Sabatini E, Toppino L, D’Alessandro A, Acciarri N (2005) Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives. Acta Physiol Plant 27:723–733Salas P, Prohens J, Seguí-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184:235–250Sanguineti MC, Tuberosa R, Conti S (1990) Field evaluation of androgenetic lines of eggplant. Acta Hortic 280:177–182Seguí-Simarro JM (2016) Androgenesis in Solanaceae. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis. Springer, New York, pp 209–244Seguí-Simarro JM, Nuez F (2008) Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet Genome Res 120:358–369Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Snape JW, Sitch LA, Simpson E, Parker BB (1988) Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the miah system. Theor Appl Genet 75:509–513Soriano M, Li H, Jacquard C, Angenent GC, Krochko J, Offringa R, Boutilier K (2014) Plasticity in cell division patterns and auxin transport dependency during in vitro embryogenesis in Brassica napus. Plant Cell 26:2568–2581Toppino L, Mennella G, Rizza F, D’Alessandro A, Sihachakr D, Rotino GL (2008) ISSR and isozyme characterization of androgenetic dihaploids reveals tetrasomic inheritance in tetraploid somatic hybrids between Solanum melongena and Solanum aethiopicum group Gilo. J Hered 99:304–315Uzun S (2007) Effect of light and temperature on the phenology and maturation of the fruit of eggplant (Solanum melongena) grown in greenhouses. N Z J Crop Hortic Sci 35:51–59van der Weerden GM, Barendse GWM (2007) A web-based searchable database developed for the EGGNET project and applied to the Radboud University Solanaceae database. Acta Hortic 745:503–506Veilleux RE (1998) Gametoclonal variation in crop plants. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Springer, Dordrecht, pp 123–133Yamagishi M, Yano M, Fukuta Y, Fukui K, Otani M, Shimada T (1996) Distorted segregation of RFLP markers in regenerated plants derived from anther culture of an F1 hybrid of rice. Genes Genet Syst 71:37–41Zhang FL, Takahata Y (2001) Inheritance of microspore embryogenic ability in Brassica crops. Theor Appl Genet 103:254–25

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. Plant Cell Tissue Organ Cult 120:79–88Shivaraj G, Rao S (2011) Rapid and efficient plant regeneration of eggplant (Solanum melongena L.) from cotyledonary leaf explants. Indian J Biotechnol 10:125–129Tuberosa R, Sanghineti MC, Conti S (1987) Anther culture of eggplant Solanum melongena L. lines and hybrids. Genética Agrária 41:267–274Veen H, van de Geijn S (1978) Mobility and ionic form of silver as related to longevity of cut carnations. Planta 140:93–96Xing Y, Yu Y, Luo X, Zhang JN, Zhao B, Guo YD (2010) High efficiency organogenesis and analysis of genetic stability of the regenerants in Solanum melongena. Biol Plant 54:231–236Zhang P, Phansiri S, Puonti-Kaerlas J (2001) Improvement of cassava shoot organogenesis by the use of silver nitrate in vitro. Plant Cell Tissue Organ Cult 67:47–5

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. Environ Exp Bot 16:109–118Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8:377–424Dutta SS, Pale G, Pattanayak A, Aochen C, Pandey A, Rai M (2017) Effect of low light intensity on key traits and genotypes of hilly rice (Oryza sativa) germplasm. J Exp Biol Agric Sci 5:463–471Esteves P, Clermont I, Marchand S, Belzile F (2014) Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism. Plant Cell Rep 33:871–879 (in press)Gaillard A, Vergne P, Beckerte M (1991) Optimization of maize microspore isolation and culture conditions for reliable plant regeneration. Plant Cell Rep 10:55–58Höfer M (2004) In vitro androgenesis in apple—improvement of the induction phase. 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. J Exp Bot 70:1267–1281Robert HS, Grunewald W, Sauer M, Cannoot B, Soriano M, Swarup R, Weijers D, Bennett M, Boutilier K, Friml J (2015) Plant embryogenesis requires AUX/LAX-mediated auxin influx. Development 142:702–711Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Kluwer Academic Publishers, 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 182:261–274Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184:235–250Satpute G, Long H, Seguí-Simarro JM, Risueño MC, Testillano PS (2005) Cell architecture during gametophytic and embryogenic microspore development in Brassica napus. Acta Physiol Plant 27:665–674Saxena N, Johansen C (1987) Adaptation of chickpea and pigeonpea to abiotic stresses. Proceedings of the consultants’ workshop held at ICRISAT Center, India, 19–21 December 1984, ICRISAT, Patancheru, IndiaSeguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404Seguí-Simarro JM (2016) Androgenesis in solanaceae. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis. Springer, New York, pp 209–244Seguí-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, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Sinha R, Eudes F (2015) Dimethyl tyrosine conjugated peptide prevents oxidative damage and death of triticale and wheat microspores. Plant Cell Tissue Organ Cult 122(1):227–237Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25:1–10Touraev A, Heberle-Bors E (2003) Anther and microspore culture in tobacco. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 223–228Touraev A, Ilham A, Vicente O, Heberle-Bors E (1996a) Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep 15:561–565Touraev A, Indrianto A, Wratschko I, Vicente O, Heberle-Bors E (1996b) Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperatures. Sex Plant Reprod 9:209–215Tsay H-S (1981) Effects of nitrogen supply to donor plants on pollen embryogenesis in cultured tobacco anthers. J Agric Res China 30:5–13Tsay H-S (1982) Microspore development and haploid embryogenesis of anther culture with five nitrogen doses to the donor tobacco plants. J Agric Res China 31:1–13Tuberosa R, Sanguineti MC, Toni B, Cioni F (1987) Ottenimento di aploidi in melanzana (Solanum melongena L.) mediante coltura di antere. Sementi Elette 3:9–14Żur I, Dubas E, Krzewska M, Janowiak F (2015) Current insights into hormonal regulation of microspore embryogenesis. Front Plant Sci 6:42

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). Changes in Protein Synthesis and Phosphorylation during Microspore Embryogenesis in Brassica napus. Journal of Plant Physiology, 156(2), 156-163. doi:10.1016/s0176-1617(00)80300-4Corral-Martínez, P., García-Fortea, E., Bernard, S., Driouich, A., & Seguí-Simarro, J. M. (2016). Ultrastructural Immunolocalization of Arabinogalactan Protein, Pectin and Hemicellulose Epitopes Through Anther Development inBrassica napus. Plant and Cell Physiology, 57(10), 2161-2174. doi:10.1093/pcp/pcw133Fortes, A. M., Testillano, P. S., Del Carmen Risueño, M., & Pais, M. S. (2002). Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget. Physiologia Plantarum, 116(1), 113-120. doi:10.1034/j.1399-3054.2002.1160114.xFurch, A. C. U., Hafke, J. B., Schulz, A., & van Bel, A. J. E. (2007). Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba. Journal of Experimental Botany, 58(11), 2827-2838. doi:10.1093/jxb/erm143Grewal, R. K., Lulsdorf, M., Croser, J., Ochatt, S., Vandenberg, A., & Warkentin, T. D. (2009). Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant Cell Reports, 28(8), 1289-1299. doi:10.1007/s00299-009-0731-1Hoekstra, S., van Bergen, S., van Brouwershaven, I. ., Schilperoort, R. ., & Wang, M. (1997). Androgenesis in Hordeum vulgare L.: Effects of mannitol, calcium and abscisic acid on anther pretreatment. Plant Science, 126(2), 211-218. doi:10.1016/s0168-9452(97)00096-4Hong, Z., Delauney, A. J., & Verma, D. P. S. (2001). A Cell Plate–Specific Callose Synthase and Its Interaction with Phragmoplastin. The Plant Cell, 13(4), 755-768. doi:10.1105/tpc.13.4.755Jacobs, A. K., Lipka, V., Burton, R. A., Panstruga, R., Strizhov, N., Schulze-Lefert, P., & Fincher, G. B. (2003). An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation. The Plant Cell, 15(11), 2503-2513. doi:10.1105/tpc.016097Jacquard, C., Mazeyrat-Gourbeyre, F., Devaux, P., Boutilier, K., Baillieul, F., & Clément, C. (2008). Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression. Planta, 229(2), 393-402. doi:10.1007/s00425-008-0838-6Jensen, W. A. (1968). Cotton embryogenesis: The zygote. Planta, 79(4), 346-366. doi:10.1007/bf00386917Joosen, R., Cordewener, J., Supena, E. D. J., Vorst, O., Lammers, M., Maliepaard, C., … Boutilier, K. (2007). Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development. Plant Physiology, 144(1), 155-172. doi:10.1104/pp.107.098723KAY, R., CHAN, A., DALY, M., & MCPHERSON, J. (1987). Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes. Science, 236(4806), 1299-1302. doi:10.1126/science.236.4806.1299Ochatt, S., Pech, C., Grewal, R., Conreux, C., Lulsdorf, M., & Jacas, L. (2009). Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae). Journal of Plant Physiology, 166(12), 1314-1328. doi:10.1016/j.jplph.2009.01.011Park, E., Díaz-Moreno, S. M., Davis, D. J., Wilkop, T. E., Bulone, V., & Drakakaki, G. (2014). Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation. Plant Physiology, 165(3), 1019-1034. doi:10.1104/pp.114.241497Parra-Vega, V., Corral-Martínez, P., Rivas-Sendra, A., & Seguí-Simarro, J. M. (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. doi:10.3389/fpls.2015.01018Paul, D. C., & Goff, C. W. (1973). Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis. Experimental Cell Research, 78(2), 399-413. doi:10.1016/0014-4827(73)90085-2Pauls, K. P., Chan, J., Woronuk, G., Schulze, D., & Brazolot, J. (2006). When microspores decide to become embryos — cellular and molecular changesThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology. Canadian Journal of Botany, 84(4), 668-678. doi:10.1139/b06-064Reynolds, T. L. (1990). Interactions between calcium and auxin during pollen androgenesis in anther cultures of Solanum carolinense L. Plant Science, 72(1), 109-114. doi:10.1016/0168-9452(90)90192-qReynolds, T. L. (2000). Effects of calcium on embryogenic induction and the accumulation of abscisic acid, and an early cysteine-labeled metallothionein gene in androgenic microspores of Triticum aestivum. Plant Science, 150(2), 201-207. doi:10.1016/s0168-9452(99)00187-9Rivas-Sendra, A., Calabuig-Serna, A., & Seguí-Simarro, J. M. (2017). Dynamics of Calcium during In vitro Microspore Embryogenesis and In vivo Microspore Development in Brassica napus and Solanum melongena. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.01177Rivas-Sendra, A., Campos-Vega, M., Calabuig-Serna, A., & Seguí-Simarro, J. M. (2017). Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica, 213(4). doi:10.1007/s10681-017-1879-3Rivas-Sendra, A., Corral-Martínez, P., Camacho-Fernández, C., & Seguí-Simarro, J. M. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture (PCTOC), 122(3), 759-765. doi:10.1007/s11240-015-0791-6Saidi, Y., Finka, A., Muriset, M., Bromberg, Z., Weiss, Y. G., Maathuis, F. J. M., & Goloubinoff, P. (2009). The Heat Shock Response in Moss Plants Is Regulated by Specific Calcium-Permeable Channels in the Plasma Membrane. The Plant Cell, 21(9), 2829-2843. doi:10.1105/tpc.108.065318Samuels, A. L., & Staehelin, L. A. (1996). Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma, 195(1-4), 144-155. doi:10.1007/bf01279193Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676-682. doi:10.1038/nmeth.2019Schl�pmann, H., Bacic, A., & Read, S. (1993). A novel callose synthase from pollen tubes of Nicotiana. Planta, 191(4). doi:10.1007/bf00195748Shi, X., Sun, X., Zhang, Z., Feng, D., Zhang, Q., Han, L., … Lu, T. (2014). GLUCAN SYNTHASE-LIKE 5 (GSL5) Plays an Essential Role in Male Fertility by Regulating Callose Metabolism During Microsporogenesis in Rice. Plant and Cell Physiology, 56(3), 497-509. doi:10.1093/pcp/pcu193Slewinski, T. L., Baker, R. F., Stubert, A., & Braun, D. M. (2012). Tie-dyed2 Encodes a Callose Synthase That Functions in Vein Development and Affects Symplastic Trafficking within the Phloem of Maize Leaves. Plant Physiology, 160(3), 1540-1550. doi:10.1104/pp.112.202473Sun, F., Fan, G., Hu, Q., Zhou, Y., Guan, M., Tong, C., … Wang, H. (2017). The high-quality genome ofBrassica napuscultivar ‘ZS11’ reveals the introgression history in semi-winter morphotype. The Plant Journal, 92(3), 452-468. doi:10.1111/tpj.13669Tan, H., Yang, X., Zhang, F., Zheng, X., Qu, C., Mu, J., … Zuo, J. (2011). Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds. Plant Physiology, 156(3), 1577-1588. doi:10.1104/pp.111.175000Töller, A., Brownfield, L., Neu, C., Twell, D., & Schulze-Lefert, P. (2008). Dual function of Arabidopsis glucan synthase-like genes GSL8 and GSL10 in male gametophyte development and plant growth. The Plant Journal, 54(5), 911-923. doi:10.1111/j.1365-313x.2008.03462.xVerma, D. P. S. (2001). CYTOKINESIS ANDBUILDING OF THECELLPLATE INPLANTS. Annual Review of Plant Physiology and Plant Molecular Biology, 52(1), 751-784. doi:10.1146/annurev.arplant.52.1.751Verma, D. P. S., & Hong, Z. (2001). Plant Molecular Biology, 47(6), 693-701. doi:10.1023/a:1013679111111Vithanage, H. I. M. V., Gleeson, P. A., & Clarke, A. E. (1980). The nature of callose produced during self-pollination inSecale cereale. Planta, 148(5), 498-509. doi:10.1007/bf00552666Waldmann, T., Jeblick, W., & Kauss, H. (1988). Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells. Planta, 173(1), 88-95. doi:10.1007/bf00394492WHITE, P. J. (2003). Calcium in Plants. Annals of Botany, 92(4), 487-511. doi:10.1093/aob/mcg164Xie, B., Deng, Y., Kanaoka, M. M., Okada, K., & Hong, Z. (2012). Expression of Arabidopsis callose synthase 5 results in callose accumulation and cell wall permeability alteration. Plant Science, 183, 1-8. doi:10.1016/j.plantsci.2011.10.015Ling You, X., Seon Yi, J., & Eui Choi, Y. (2006). Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis. Protoplasma, 227(2-4), 105-112. doi:10.1007/s00709-006-0149-3Yu, Y., Jiao, L., Fu, S., Yin, L., Zhang, Y., & Lu, J. (2016). Callose Synthase Family Genes Involved in the Grapevine Defense Response to Downy Mildew Disease. Phytopathology®, 106(1), 56-64. doi:10.1094/phyto-07-15-0166-rZhang, C., Guinel, F. C., & Moffatt, B. A. (2002). A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3. Protoplasma, 219(1-2), 59-71. doi:10.1007/s00709020000

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

Unravelling massive crocins transport and accumulation through proteome and microscopy tools during the development of saffron stigma

Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined crocinoplast ) were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-D-xylulose 5-phosphate synthase (DXS), 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., -carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted.This work was supported by the Spanish Ministerio de Economia y Competitividad (BIO2013-44239-R), participates in the IBERCAROT network (112RT0445) and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN). The proteomics analysis LC-MS/MS by LTQ Orbitrap Velos was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network. We wish to thank Javier Argandona and Carmen Cifuentes for technical assistance and Kathy Walsh for language revision.Gómez Gómez, L.; Parra Vega, V.; Rivas Sendra, A.; Seguí Simarro, JM.; Molina Romero, RV.; Pallotti Sagripanti, CG.; Rubio Moraga, Á.... (2017). Unravelling massive crocins transport and accumulation through proteome and microscopy tools during the development of saffron stigma. International Journal of Molecular Sciences. 18(1)(76):1-22. https://doi.org/10.3390/ijms18010076S12218(1)7

Protective intraoperative ventilation with higher versus lower levels of positive end-expiratory pressure in obese patients (PROBESE): study protocol for a randomized controlled trial

Background: Postoperative pulmonary complications (PPCs) increase the morbidity and mortality of surgery in obese patients. High levels of positive end-expiratory pressure (PEEP) with lung recruitment maneuvers may improve intraoperative respiratory function, but they can also compromise hemodynamics, and the effects on PPCs are uncertain. We hypothesized that intraoperative mechanical ventilation using high PEEP with periodic recruitment maneuvers, as compared with low PEEP without recruitment maneuvers, prevents PPCs in obese patients. Methods/design The PRotective Ventilation with Higher versus Lower PEEP during General Anesthesia for Surgery in OBESE Patients (PROBESE) study is a multicenter, two-arm, international randomized controlled trial. In total, 2013 obese patients with body mass index ≥35 kg/m2 scheduled for at least 2 h of surgery under general anesthesia and at intermediate to high risk for PPCs will be included. Patients are ventilated intraoperatively with a low tidal volume of 7 ml/kg (predicted body weight) and randomly assigned to PEEP of 12 cmH2O with lung recruitment maneuvers (high PEEP) or PEEP of 4 cmH2O without recruitment maneuvers (low PEEP). The occurrence of PPCs will be recorded as collapsed composite of single adverse pulmonary events and represents the primary endpoint. Discussion To our knowledge, the PROBESE trial is the first multicenter, international randomized controlled trial to compare the effects of two different levels of intraoperative PEEP during protective low tidal volume ventilation on PPCs in obese patients. The results of the PROBESE trial will support anesthesiologists in their decision to choose a certain PEEP level during general anesthesia for surgery in obese patients in an attempt to prevent PPCs. Trial registration ClinicalTrials.gov identifier: NCT02148692. Registered on 23 May 2014; last updated 7 June 2016. Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-1929-0) contains supplementary material, which is available to authorized users

Obtención de doble haploides en berenjena mediante cultivo de microsporas aisladas: organogénesis y regeneración a partir de callos androgénicos

[ES] Las líneas puras son la base de la producción de semilla híbrida. La producción de doble haploides es una metodología alternativa a las técnicas de mejora genética clásica, que reduce considerablemente el tiempo y los recursos necesarios para la obtención de líneas puras. Mediante la inducción de un fenómeno conocido como androgénesis, es posible obtener líneas puras (doble haploides) en algunas especies de forma mucho más rápida y barata. Este método consiste en desviar la microspora de la ruta gametofítica e inducirla in vitro a formar un embrión o callo haploide. Existen dos métodos para obtener individuos doble haploides de origen androgénico a partir de microsporas: el cultivo de anteras y el cultivo de microsporas aisladas. Los trabajos existentes demuestran que la inducción de la androgénesis en cultivos de microsporas aisladas de berenjena tiene una eficiencia muy superior a la obtenida en cultivos de anteras. Sin embargo, el proceso de regeneración de individuos completos a partir de los callos androgénicos obtenidos de las microsporas en cultivo está muy poco estudiado y es el principal paso limitante en la obtención de haploides o doble haploides. En este trabajo se pretende mejorar la eficiencia de regeneración de plantas completas a partir de microsporas aisladas mediante la optimización del proceso de inducción de organogénesis y regeneración. De esta forma, podrá confirmarse que el cultivo de microsporas aisladas es una forma fácil y rápida de obtener líneas puras. Se ha utilizado el genotipo Bandera para obtener callos androgénicos a partir de cultivo de microsporas aisladas. Con estos callos se ha estudiado la inducción de organogénesis, la elongación y el enraizamiento de los brotes hasta formar plantas completas. Como resultado de este trabajo se ha establecido un primer protocolo para la regeneración de plantas completas a partir de callos androgénicos formados en cultivo de microsporas de berenjena. De esta forma, podrá confirmarse que el cultivo de microsporas aisladas es una forma fácil y rápida de obtener líneas puras en esta especie.[EN] Pure lines are the basis of hybrid seed production. Doubled haploid technology is an alternative methodology to the classical breeding techniques, which greatly reduces the time and resources necessary to obtain pure lines. By inducing a phenomenon known as androgenesis, it is possible to obtain pure lines (doubled haploids) in some species much more quickly and cheaply. This method consists in deviating the microspore from the gametophytic pathway and inducing the in vitro formation of an haploid embryo or callus. There are two methods to obtain androgenic doubled haploid individuals from microspores: anther culture and isolated microspore culture. Previous studies demonstrated that androgenesis induction through isolated microspores culture of eggplant has a much higher efficiency than that obtained by anther culture. However, the process of regeneration of entire individuals from androgenic calli obtained through isolated microspore culture is poorly studied and is the main limiting step for haploid or doubled haploid production. This work aims to improve the efficiency of plant regeneration from isolated microspores by optimizing the process of organogenesis and regeneration. We used the genotype Bandera to obtain androgenic calli through isolated microspore culture. With these calli, we studied the induction of organogenesis, elongation and rooting of shoots to form whole plants. As a result of this study, we have established an initial protocol to regenerate plants from androgenic calli formed in cultures of isolated eggplant microspores. In this way, we could confirm that isolated microspore culture is a quick and easy way to obtain pure lines.Rivas Sendra, A. (2012). Obtención de doble haploides en berenjena mediante cultivo de microsporas aisladas: organogénesis y regeneración a partir de callos androgénicos. http://hdl.handle.net/10251/27311Archivo delegad