Doubled haploids in eggplant

[EN] This review compiles the most relevant advances made in the production of doubled haploid plants in eggplant, the application of doubled haploid lines in breeding programs, and the future perspectives for the development of alternative technologies for doubled haploid generation in this species. Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies.This research was funded by the Valencian Government, grant number CDEIGENT 2018/023 to RMM and by the Spanish MICINN, grant number PID2020-115763RB-I00 to JMSS. ACS is the recipient of a predoctoral contract from the FPU program of the Spanish Government.Mir Moreno, R.; Calabuig-Serna, A.; Seguí-Simarro, JM. (2021). Doubled haploids in eggplant. Biology. 10(7):1-16. https://doi.org/10.3390/biology10070685S11610

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

Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)

[EN] Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 C, a 35 C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.Thanks are due to all the whole staff of the Cell Biology Group for helping and training AA during his stay in the group. This work was supported by Grant AGL2017- 88135-R to JMSS from Spanish Ministerio de Economı´a y Competitividad (MINECO) jointly funded by FEDER.Asadi, A.; Zebarjadi, A.; Abdollahi, MR.; Seguí-Simarro, JM. (2018). Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.). Euphytica. 214(216):1-17. https://doi.org/10.1007/s10681-018-2297-xS117214216Abdollahi MR, Najafi S, Sarikhani H, Moosavi SS (2016) Induction and development of anther-derived gametic embryos in cucumber (Cucumis sativus L.) by optimizing the macronutrient and agar concentrations in culture medium. Turk J Biol 40(3):571–579Ashok Kumar HG, Murthy HN (2004) Effect of sugars and amino acids on androgenesis of Cucumis sativus. Plant Cell, Tissue Organ Cult 78(3):201–208. https://doi.org/10.1023/b:ticu.0000025637.56693.68Bai B, Su YH, Yuan J, Zhang XS (2013) Induction of somatic embryos in arabidopsis requires local YUCCA expression mediated by the down-regulation of ethylene biosynthesis. Mol Plant 6(4):1247–1260. https://doi.org/10.1093/mp/sss154Claveria E, Garcia-Mas J, Dolcet-Sanjuan R (2005) Optimization of cucumber doubled haploid line production using in vitro rescue of in vivo induced parthenogenic embryos. J Am Soc Hortic Sci 130(4):555–560Corral-Martínez P, Nuez 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):215–228. https://doi.org/10.1007/s10681-010-0303-zDanin-Poleg Y, Reis N, Tzuri G, Katzir N (2001) Development and characterization of microsatellite markers in Cucumis. Theor Appl Genet 102(1):61–72. https://doi.org/10.1007/s001220051618Dong Y-Q, Zhao W-X, Li X-H, Liu X-C, Gao N-N, Huang J-H, Wang W-Y, Xu X-L, Tang Z-H (2016) Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species. Plant Cell Rep. https://doi.org/10.1007/s00299-016-2018-7FAOSTAT (2018) http://faostat.fao.org. Accessed July 2018Ficcadenti N, Sestili S, Annibali S, Di Marco M, Schiavi M (1999) In vitro gynogenesis to induce haploid plants in melon Cucumis melo L. Genet Breed 53:255–257Gałązka J, Niemirowicz-Szczytt K (2013) Review of research on haploid production in cucumber and other cucurbits. Folia Hortic. https://doi.org/10.2478/fhort-2013-0008Hamidvand Y, Abdollahi MR, Chaichi M, Moosavi SS (2013) The effect of plant growth regulators on callogenesis and gametic embryogenesis from anther culture of cucumber (Cucumis sativus L.). Int J Agric Crop Sci 5(10):1089Kurtar ES, Balkaya A, Kandemir D (2016) Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture. Plant Cell, Tissue Organ Cult 127(2):497–511. https://doi.org/10.1007/s11240-016-1074-6Lotfi M, Alan AR, Henning MJ, Jahn MM, Earle ED (2003) Production of haploid and doubled haploid plants of melon (Cucumis melo L.) for use in breeding for multiple virus resistance. Plant Cell Rep 21(11):1121–1128Metwally EI, Moustafa SA, El-Sawy BI, Shalaby TA (1998) Haploid plantlets derived by anther culture of Cucurbita pepo. Plant Cell, Tissue Organ Cult 52(3):171–176. https://doi.org/10.1023/a:1005908326663Mohamed M, Refaei E (2004) Enhanced haploids regeneration in anther culture of summer squash (Curcurbita pepo L.). Cucurbit Genet Coop Rep 27:57–60Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479Parra-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(3):353–360. https://doi.org/10.1007/s11240-012-0242-6Rakha M, Metwally E, Moustafa S, Etman A, Dewir Y (2012) Evaluation of regenerated strains from six Cucurbita interspecific hybrids obtained through anther and ovule in vitro cultures. Aust J Crop Sci 6(1):23–30Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81(24):8014–8018. https://doi.org/10.1073/pnas.81.24.8014Sauton A, Dumas de Vaulx R (1987) Obtention de plantes haploides chez melon (Cucumis melo L.) par gynogenese indute par du pollen irraidié. Agronomie 7:141–148Seguí-Simarro JM (2016) Androgenesis in solanaceae. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis. Methods in molecular biology, vol 1359. Springer, New York, pp 209–244. https://doi.org/10.1007/978-1-4939-3061-6_9Seguí-Simarro JM, Nuez F (2006) Androgenesis induction from tomato anther cultures: callus characterization. Acta Hort 725:855–861Seguí-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(5):1119–1132Seguí-Simarro JM, Nuez F (2008) Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet Genome Res 120(3–4):358–369. https://doi.org/10.1159/000121085Shalaby TA (2006) Embryogenesis and plantlets regeneration from anther culture of squash plants (Cucurbita pepo L.) as affected by different genotypes. J Agric Res Tanta Univ 32(1):173–183Song H, Lou QF, Luo XD, Wolukau JN, Diao WP, Qian CT, Chen JF (2007) Regeneration of doubled haploid plants by androgenesis of cucumber (Cucumis sativus L.). Plant Cell, Tissue Organ Cult 90(3):245–254. https://doi.org/10.1007/s11240-007-9263-ySteward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Am J Bot 45(10):705–708Su YH, Zhao XY, Liu YB, Zhang CL, O’Neill SD, Zhang XS (2009) Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in arabidopsis. Plant J 59(3):448–460. https://doi.org/10.1111/j.1365-313X.2009.03880.xSuprunova T, Shmykova N (2008) In vitro induction of haploid plants in unpollinated ovules, anther and microspore culture of Cucumis sativus. In: Cucurbitaceae 2008: proceedings of the IXth Eucarpia meeting on genetics and breeding of cucurbitaceae, pp 371–374Xie M, Qin L-Y, Pan J-S, He H-L, Wu A-Z, Cai R (2005) Flower morphogenesis and microspore development versus anther culture of cucumber. Acta Bot Boreal-Occid Sin 25(6):1096Zhan Y, Chen J-F, Malik AA (2009) Embryoid induction and plant regeneration of cucumber (Cucumis sativus L.) through microspore culture. Acta Hort Sin 36(2):221–22

Phenological phases of flowering in hop (Humulus lupulus L.) and their correspondence with microsporogenesis and microgametogenesis

[EN] Hop (Humulus Lupulus L.) suffered, as many other crops, a shrinkage of its intraspecific agrobiodiversity. Biotechnological methods of breeding would offer new opportunities to produce improved varieties with interesting phytochemical profiles and adaptable to the challenging conditions of climate change. Doubled haploid (DH) technology could be a useful tool to increase hop agrobiodiversity but, unfortunately, there is a complete lack of information about hop flower biology. For this reason, the main aim of this work is the study of the different phenological phases of flowering in hop and the corresponding developmental stages of microspores/pollen grains contained therein. The results obtained allowed the identification of morphological markers (anther and flower bud length), easy and fast to measure, that would speed up the selection of flower buds containing the highest percentage of vacuolated microspores and young pollen, the stages considered in most species as the most responsive to androgenesis. A further result, derived from the flower bud and anther microscopical observation, evidenced the increase of lupulin glands on bud and anther surface as the bud proceeds in development from microsporogenesis to microgametogenesis.This work was supported by Spanish MINECO [grant number AGL2017-88135-R to JMSS] jointly funded by FEDER.Liberatore, C.; Calabuig-Serna, A.; Rodolfi, M.; Chiancone, B.; Seguí-Simarro, JM. (2019). Phenological phases of flowering in hop (Humulus lupulus L.) and their correspondence with microsporogenesis and microgametogenesis. Scientia Horticulturae. 256:1-6. https://doi.org/10.1016/j.scienta.2019.108639S16256Easterling, K. A., Pitra, N. J., Jones, R. J., Lopes, L. G., Aquino, J. R., Zhang, D., … Bass, H. W. (2018). 3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation. Frontiers in Plant Science, 9. doi:10.3389/fpls.2018.01501Nagel, J., Culley, L. K., Lu, Y., Liu, E., Matthews, P. D., Stevens, J. F., & Page, J. E. (2008). EST Analysis of Hop Glandular Trichomes Identifies an O-Methyltransferase That Catalyzes the Biosynthesis of Xanthohumol. The Plant Cell, 20(1), 186-200. doi:10.1105/tpc.107.055178Parra-Vega, V., González-García, B., & Seguí-Simarro, J. M. (2012). Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.). Acta Physiologiae Plantarum, 35(2), 627-633. doi:10.1007/s11738-012-1104-xPatzak, J., Nesvadba, V., Henychová, A., & Krofta, K. (2010). Assessment of the genetic diversity of wild hops (Humulus lupulus L.) in Europe using chemical and molecular analyses. Biochemical Systematics and Ecology, 38(2), 136-145. doi:10.1016/j.bse.2009.12.023Patzak, J., Nesvadba, V., Krofta, K., Henychova, A., Marzoev, A. I., & Richards, K. (2010). Evaluation of genetic variability of wild hops (Humulus lupulus L.) in Canada and the Caucasus region by chemical and molecular methods. Genome, 53(7), 545-557. doi:10.1139/g10-024Salas, P., Rivas-Sendra, A., Prohens, J., & Seguí-Simarro, J. M. (2011). Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica, 184(2), 235-250. doi:10.1007/s10681-011-0569-9Seguí-Simarro, J. M. (2010). Androgenesis Revisited. The Botanical Review, 76(3), 377-404. doi:10.1007/s12229-010-9056-6Seguí-Simarro, J. M., & Nuez, F. (2005). Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (Solanum lycopersicum) anther cultures. Acta Physiologiae Plantarum, 27(4), 675-685. doi:10.1007/s11738-005-0071-xSHEPHARD, H. L., PARKER, J. S., DARBY, P., & AINSWORTH, C. C. (2000). Sexual development and sex chromosomes in hop. New Phytologist, 148(3), 397-411. doi:10.1046/j.1469-8137.2000.00771.xXie, W., Xiong, W., Pan, J., Ali, T., Cui, Q., Guan, D., … Davis, S. J. (2018). Decreases in global beer supply due to extreme drought and heat. Nature Plants, 4(11), 964-973. doi:10.1038/s41477-018-0263-

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. 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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. 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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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Optimization of the conditions for production of synthetic seeds by encapsulation of axillary buds derived from minituber sprouts in potato (Solanum tuberosum)

[EN] Synthetic seed technology is a convenient alternative to conventional multiplication in potato. In this work, we studied and optimized the process of alginate encapsulation of axillary buds derived from potato minituber sprouts (PMSs). We assayed different concentrations of sodium alginate, CaCl2 and matrix culture media, different explant sizes, different concentrations of 24-epibrassinolide (EBr) applied at different stages of the encapsulation process, different planting substrates and different cold storage periods in order to determine the best conditions for encapsulation in cvs. Sante and Agria. The conditions that resulted in the greatest regrowth rates and speed in both cultivars involved the choice of 2 3 mmlong buds, a 2 days pre-culture of buds in culture medium supplemented with 10-6 M EBr, encapsulation in 3 % sodium alginate with 1 % CaCl2 and full-strength MS culture medium, regrowth in solid MS culture medium and then transfer to coco peat (coir fiber pith) for conversion into plantlets. We also found that buds encapsulated under these conditions maintained the initial viability rates for up to 120 days in Sante and 90 days in Agria , although regrowth speed decreased after 60 days in both cultivars. We demonstrate the possibility of producing synthetic seeds efficiently using axillary buds derived from PMSs.Ghambarali, S.; Abdollahi, MR.; Zolnorian, H.; Moosavi, SS.; Seguí-Simarro, JM. (2016). Optimization of the conditions for production of synthetic seeds by encapsulation of axillary buds derived from minituber sprouts in potato (Solanum tuberosum). Plant Cell Tissue and Organ Culture (PCTOC). 126(3):449-458. doi:10.1007/s11240-016-1013-6S4494581263Adriani M, Piccioni E, Standardi A (2000) Effect of different treatments on the conversion of ‘Hayward’ kiwifruit synthetic seeds to whole plants following encapsulation of in vitro-derived buds. N Z J Crop Hortic Sci 28:59–67Ahmad N, Anis M (2010) Direct plant regeneration from encapsulated nodal segments of Vitex negundo. Biol Plant 54:748–752Azpeitia A, Chan JL, Saenz L, Oropeza C (2003) Effect of 22(S),23(S)-homobrassinolide on somatic embryogenesis in plumule explants of Cocos nucifera (L.) cultured in vitro. J Hortic Sci Biotech 78:591–596Brosa C (1999) Biological effects of brassinosteroids. Crit Rev Biochem Mol Biol 34:339–358Bustam S, Sinniah UR, Kadir MA, Zaman FQ, Subramaniam S (2012) Selection of optimal stage for protocorm-like bodies and production of artificial seeds for direct regeneration on different media and short term storage of Dendrobium Shavin White. Plant Growth Regul 69:215–224Corral-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 195:369–382Divi UK, Rahman T, Krishna P (2010) Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways. BMC Plant Biol 10:151Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42Estrada R, Tovar P, Dodds JH (1986) Induction of in vitro tubers in a broad range of potato genotypes. Plant Cell Tissue Organ Cult 7:3–10Faisal M, Anis M (2007) Regeneration of plants from alginate-encapsulated shoots of Tylophora indica (Burm. f.) Merrill, an endangered medicinal plant. J Hortic Sci Biotechnol 82:351–354Fujioka S, Yokota T (2003) Biosynthesis and metabolism of brassinosteroids. Ann Rev Plant Biol 54:137–164Gantait S, Kundu S, Ali N, Sahu NC (2015) Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix. Acta Physiol Plant 37:1–12Grove MD, Spencer GF, Rohwedder WK, Mandava N, Worley JF, Warthen JD, Steffens GL, Flippenanderson JL, Cook JC (1979) Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen. Nature 281:216–217Hung CD, Trueman SJ (2012a) Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora. Acta Physiol Plant 34:117–128Hung CD, Trueman SJ (2012b) Preservation of encapsulated shoot tips and nodes of the tropical hardwoods Corymbia torelliana × C. citriodora and Khaya senegalensis. Plant Cell, Tissue Organ Cult 109:341–352Jones ED (1988) A current assessment of in vitro culture and other rapid multiplication methods in North America and Europe. Am Potato J 65:209–220Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225:353–364Kitto SL, Janick J (1982) Polyox as an artificial seed coat for asexual embryos. HortScience 17:488Larkin PJ, Davies PA, Tanner GJ (1988) Nurse culture of low numbers of Medicago and Nicotiana protoplasts using calcium alginate beads. Plant Sci 58:203–210Leclerc Y, Donnelly DJ, Coleman WK, King RR (1995) Microtuber dormancy in three potato cultivars. Am Potato J 72:215–223Machii H, Yamanouchi H (1993) Growth of mulberry synthetic seeds on vermiculite, sand and soil media. J Seric Sci Jpn 62:85–87Maguire JD (1962) Speed of germination—aid in selection ane evaluation for seedling emergence and vigor. Crop Sci 2:176–177Micheli M, Pellegrino S, Piccioni E, Standardi A (2002) Effects of double encapsulation and coating on synthetic seed conversion in M.26 apple rootstock. J Microencapsul 19:347–356Murashige T (1977) Plant cell and organ cultures as horticultural practices. Acta Hortic 78:17–30Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479Naik PS, Karihaloo JL (2007) Micropropagation for production of quality potato seed in Asia-pacific. Asia-Pacific Consortium on Agricultural Biotechnology, New DelhiNaik PS, Sarkar D (1997) Influence of light-induced greening on storage of potato microtubers. Biol Plant 39:31–34Nakashita H, Yasuda M, Nitta T, Asami T, Fujioka S, Arai Y, Sekimata K, Takatsuto S, Yamaguchi I, Yoshida S (2003) Brassinosteroid functions in a broad range of disease resistance in tobacco and rice. Plant J 33:887–898Nassar KAM, Kubow S, Donnelly JD (2015) Somatic Embryogenesis for potato (Solanum tuberosum L.) improvement. In: Li X-Q, Donnelly JD, Jensen GT (eds) Somatic genome manipulation: advances, methods, and applications. Springer, New York, pp 169–197Nuñez M, Siqueira WJ, Hernandez M, Zullo MAT, Robaina C, Coll F (2004) Effect of spirostane analogues of brassinosteroids on callus formation and plant regeneration in lettuce (Lactuca sativa). Plant Cell Tissue Organ Cult 78:97–99Nyende AB, Schittenhelm S, Mix-Wagner G, Greef J-M (2003) Production, storability, and regeneration of shoot tips of potato (Solanum tuberosum L.) encapsulated in calcium alginate hollow beads. In Vitro Cell Dev Biol Plant 39:540–544Oh MH, Clouse SD (1998) Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida. Plant Cell Rep 17:921–924Parveen S, Shahzad A (2014) Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange. Acta Physiol Plant 36(3):635–640Pullman GS, Zhang Y, Phan BH (2003) Brassinolide improves embryogenic tissue initiation in conifers and rice. Plant Cell Rep 22:96–104Rai MK, Asthana P, Singh SK, Jaiswal VS, Jaiswal U (2009) The encapsulation technology in fruit plants—a review. Biotechnol Adv 27:671–679Redenbaugh K, Paasch BD, Nichol JW, Kossler ME, Viss PR, Walker KA (1986) Somatic seeds: encapsulation of asexual plant embryos. Nat Biotechnol 4:797–801Rihan HZ, Al-Issawi M, Burchett S, Fuller MP (2011) Encapsulation of cauliflower (Brassica oleracea var botrytis) microshoots as artificial seeds and their conversion and growth in commercial substrates. Plant Cell Tissue Organ Cult 107:243–250Saha S, Sengupta C, Ghosh P (2015) Encapsulation, short-term storage, conservation and molecular analysis to assess genetic stability in alginate-encapsulated microshoots of Ocimum kilimandscharicum Guerke. Plant Cell Tissue Organ Cult 120:519–530Sarkar D, Naik PS (1997) Nutrient-encapsulation of potato nodal segments for germplasm exchange and distribution. Biol Plant 40:285–290Sarkar D, Naik PS (1998) Synseeds in potato: an investigation using nutrient-encapsulated in vitro nodal segments. Sci Hortic 73:179–184Sasaki H (2002) Brassinolide promotes adventitious shoot regeneration from cauliflower hypocotyl segments. Plant Cell Tissue Organ Cult 71:111–116Schafer-Menuhr A, Mix-Wagner G, Vorlop K (2003) Regeneration of plants from cell suspension cultures and encapsulated cell suspension cultures of Solanum tuberosum L. cv. Clarissa. Landbauforsch Völkenrode 53:53–59Sharma S, Shahzad A (2012) Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn. Plant Cell Tissue Organ Cult 111:191–198Sharma SK, Bryan GJ, Winfield MO, Millam S (2007) Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment. 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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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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. 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Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress

[EN] Anther culture can be used as a powerful tool to produce doubled haploid (DH) lines in medicinal plants, thus accelerating breeding programs. In the particular case of borage (Borago officinalis L.), a method to produce DH plants has not been yet published. In this work we evaluated the effect of different culture media and of different chemical (colchicine and n-butanol) and physical stresses (centrifugation and electroporation) on androgenesis induction and plant regeneration in borage anther cultures. We found that the highest response can be obtained with culture medium containing B5 salts and NLN vitamins, the addition of 200 mg/l colchicine during 4 days, a pretreatment of anthers with 0.2% n-butanol for 5 hours, or the application to anthers of single physical stresses (either centrifugation at 300 g or a 100 v electrical shock, but not combined). This is the first report on the production of DH plants in borage. 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