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Production of “super-males” of asparagus by anther culture and its detection with SSR-ESTs

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Abstract

Anther culture is used to develop asparagus “super-male” (di-haploids) in asparagus, which can be used to develop “all-male” varieties, by crossing them with suitable females; their progenies will be formed only by males which is advantageous for producers. This report describe a new anther culture protocol adapted to “Morado de Huétor”, a Spanish tetraploid landrace, and studied the different factors involved in callus proliferation success from anther explants such as the microspore development stage, or the type of stress used to induce the symmetric division of the microspores, to obtain a high success rate (90 %). For plantlets regenerates from anther culture (PRACs) regeneration we develop a proliferation media supplemented with a combination of pCPA and BA able to induce callus proliferation and plantlet regeneration in the same step in a 50 % of calli, simplifying the procedure. The high percentage of heterozygous male recovery, originated from somatic cells, is an important problem in the anther culture, and to elucidate the origin of PRACs we have combined different tools: ploidy analysis, characterization with the linked sex-marker Asp1-T7 and with EST-SRRs. We can establish that 50 % of PRACs obtained in this work were regenerated from diploid microspores of “Morado de Huétor”, regenerating diploid, di-diploid and tetra-diploid plantlets. The di-diploids males (MMmm) would generate a ratio male:female of 5:1 (83.3 %) and the tetra-diploid males (MMMMmmmm) a ratio male:female of 69:1 (98.6 %), so the tetra-diploid males could be considered “super-males” and be used to develop “all-male” varieties of “Morado de Huétor”.

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References

  • Aionesei T, Touraev A, Heberle-Bors E (2005) Pathways to microspore embryogenesis. In: Palmer CE, Keller WA, Kasha KJ (eds) Haploids in crop improvement II, vol 56. Springer, Berlin, pp 11–34

    Chapter  Google Scholar 

  • Aulinger IE, Peter SO, Schmid JE, Stamp P (2003) Rapid attainment of a doubled haploid line from transgenic maize (Zea mays L) plants by means of anther culture. In Vitro Cell Dev Biol Plant 39:165–170

    Article  Google Scholar 

  • Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173

    Article  CAS  Google Scholar 

  • Benmoussa M, Mukhopadhyay S, Desjardins Y (1996) Optimization of callus culture and shoot multiplication of Asparagus densiflorus. Plant Cell Tissue Organ Cult 47:91–94

    Article  Google Scholar 

  • Benmoussa M, Mukhopadhyay S, Desjardins Y (1997) Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri. Plant Cell Rep 17:123–128

    Article  CAS  Google Scholar 

  • Boonen P (1988) Espárrago: variedades, investigación científica y producción comercial de la semilla en los Países Bajos. II Jornadas técnicas del espárrago. Pamplona, En, pp 267–275

    Google Scholar 

  • Braak JP, Zeilinga AE (1957) Production of a colchicine-induced tetraploid asparagus. Euphytica 6:201–272

    Article  Google Scholar 

  • Brettin TS, Sink C (1992) Allozyme variation and genetics in Asparagus. J Hered 83:383–386

    CAS  Google Scholar 

  • Cao HB, Biswas MK, Lu Y, Amar MH, Tong Z, Xu QA, Xu JA, Guo WW, Deng XX (2011) Doubled haploid callus lines of Valencia sweet orange recovered from anther culture. Plant Cell Tissue Organ Cult 104:415–423

    Article  Google Scholar 

  • Carmona-Martin E, Regalado JJ, Padilla IMG, Westendrop N, Encina CL (2014) A new efficient micropropagation method and its breeding applications in Asparagus genera. Plant Cell Tissue Organ Cult 119:479–488

    Article  CAS  Google Scholar 

  • Carmona-Martin E, Regalado JJ, Raghavan R, Encina CL (2015) In vitro induction of autooctoploid asparagus genotypes. Plant Cell Tissue Organ Cult 121:249–254

    Article  CAS  Google Scholar 

  • Caruso M, Federici CT, Roose ML (2008) EST-SSR markers for asparagus genetic diversity evaluation and cultivar identification. Mol Breed 21:195–204

    Article  CAS  Google Scholar 

  • Castro P, Gil J, Cabrera A, Moreno R (2013) Assesment of genetic diversity and phylogenetic relationship in Asparagus species related to Asparagus officinalis. Genet Resour Crop Evol 60:1275–1288

    Article  Google Scholar 

  • Chani E, Veilleux RE, Boluarte-Medina T (2000) Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants. Plant Cell Tissue Organ Cult 60:101–112

    Article  CAS  Google Scholar 

  • Cho M, Zapata FJ (1988) Callus formation and plant regeneration in isolated pollen culture of rice (Orysa Sativa L., cv. Taipei 309). Plant Sci 58:239–244

    Article  Google Scholar 

  • Corriols L, Doré C, Rameau C (1990) Commercial release in France of Andreas, the first asparagus all male F1 hybrid. Acta Hortic 271:249–252

    Article  Google Scholar 

  • Delaitre C, Ochatt S, Deleury E (2001) Electroporation modulates the embryogénie responses of asparagus (Asparagus officinalis L.) microspores. Protoplasma 216:39–46

    Article  CAS  PubMed  Google Scholar 

  • Doré C (1974) Production de plantes homozygotes mâles et females à partir d’anthères d’asperge, cultivées in vitro. C R Acad Sci 278:2135–2138

    Google Scholar 

  • Doré C (1990) Asparagus anther culture and field trials of dihaploids and F1 hybrids. In: Bajaj YPS (ed) Haploids in crop improvement. I. Biotechnology in agriculture and forestry, vol 12. Springer, Berlin, pp 322–345

    Chapter  Google Scholar 

  • Eimert K, Reutter G, Strolka B (2003) Fast and reliable detection of doubled-haploids in Asparagus officinalis by stringent RAPD-PCR. J Agric Sci 141:73–78

    Article  CAS  Google Scholar 

  • Ellison JH (1986) Asparagus breeding. Breeding vegetable crops. AVI Publishing Co, Westport, pp 521–569

    Google Scholar 

  • Ellison JH, Kinelski JJ (1985) Jersey Giant, an all-male asparagus hybrid. HortScience 20:1141

    Google Scholar 

  • Ellison JH, Garrison SA, Kinelski JJ (1990) Male asparagus hybrids—Jersey Gem, Jersey General, Jersey King, Jersey Knight, and Jersey Titan. HortScience 25:816–817

    Google Scholar 

  • Encina CL, Caro E, Padilla IMG, Westendorp N, Carmona-Martín E, Barceló-Muñoz A, Vidoy Mercado I (2008) Procedimiento para la propagación in vitro del espárrago. Patent No.: P200803544 (ESP). Spain. Owner CSIC

  • Falavigna A, Tacconi MG, Soressi GP (1983) Recent progress in asparagus (Asparagus officinalis L.) breeding by anther in vitro culture. Acta Hort 131:215–222

    Article  Google Scholar 

  • Falavigna A, Casali PE, Battaglia A (1999) Achievement of asparagus breeding in Italy. Acta Hort 479:67–74

    Article  Google Scholar 

  • Feng XR, Wolyn DJ (1991) High frequency production of haploid embryos in asparagus anther culture. Plant Cell Rep 10:574–578

    Article  CAS  PubMed  Google Scholar 

  • Feng XR, Wolyn DJ (1993) Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhaced by ancymidol. Plant Cell Rep 12:281–285

    Article  CAS  PubMed  Google Scholar 

  • Ferrie AMR, Epp DJ, Keller WA (1995) Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line. Plant Cell Rep 14:580–584

    Article  CAS  PubMed  Google Scholar 

  • Flory WS (1932) Genetic and cytological investigations on Asparagus officinalis L. Genet Princenton 17:432–467

    CAS  Google Scholar 

  • Galli L, Viégas J, Augustin E, Eckert MI, Da Silva JB (1998) Meiosis of anther culture regenerants in asparagus (Asparagus officinalis L.). Genet Mol Biol 21:93–97

    Article  Google Scholar 

  • Geoffriau E, Denoue D, Rameau C (1992) Assessment of genetic variation among asparagus (Asparagus officinalis L.) population and cultivars: agromorphological and isozymatic data. Euphytica 61:169–179

    Article  CAS  Google Scholar 

  • Germana MA, Chiancone B (2003) Improvement in Citrus clementina ort.Ex Tan. microspore derived embryoids induction and regeneration. Plant Cell Rep 22:181–187

    Article  CAS  PubMed  Google Scholar 

  • Gesteira AS, Otoni WC, Barros EG, Moreira MA (2002) RAPD based detection of genomic instability in soybean plants derived from somatic embryogenesis. Plant Breed 121:269–271

    Article  CAS  Google Scholar 

  • Hartmann HD (1985) Possibility of predicting asparagus yields in central Europe. In: Lougheed C, Tiessen H (eds) Proceedings of the 6th International Asparagus Symposium. University of Guelph, Canada, pp 218–323

    Google Scholar 

  • Heberle-Bors E (1983) Induction of embryogenic pollen grains in situ and subsequent in vitro pollens embryogenesis in Nicotiana tabacum L. by treatments of the pollen donor plants with feminizing agents. Physiol Plant 59:67–72

    Article  CAS  Google Scholar 

  • Höfer M, Gomez A, Aguiriano E, Manzanera JA, Bueno MA (2002) Analysis of simple sequence repeat markers in homozygous lines of apple. Plant Breed 121:159–162

    Article  Google Scholar 

  • Jamsari A, Nitz I, Reamon-Büttner SM, Jung C (2004) BAC-derived diagnostic markers for sex determination in asparagus. Theor Appl Genet 108:1140–1146

    Article  CAS  PubMed  Google Scholar 

  • Jin S, Mushke R, Zhu H, Tu L, Lin Z, Zhang Y, Zhang X (2008) Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers. Plant Cell Rep 27:1303–1316

    Article  CAS  PubMed  Google Scholar 

  • Khandka DK, Nejidat A, Golan-Goldhirsh A (1996) Polymorphism and DNA markers for asparagus cultivars identified by random amplified polymorphic DNA. Euphytica 87:39–44

    Article  CAS  Google Scholar 

  • Kunitake H, Nakashima T, Mori K, Tanaka M (1998) Somaclonal and chromosomal effects of genotype, ploidy and culture duration in Asparagus officinalis L. Euphytica 102:309–316

    Article  Google Scholar 

  • Labbani Z, Richard N, De Buyser J, Picard E (2005) Chlorophyllian durum wheat plants obtained by isolated microspores culture: importance of the pre-treatment. C R Biol 328:713–723

    Article  PubMed  Google Scholar 

  • Lallemand J, Briand F, Breulis F, Denoue D, Rameau C (1994) Identification of asparagus varieties by isozyme patterns. Euphytica 79:1–4

    Article  CAS  Google Scholar 

  • Lapitan VC, Redona ED, Abe T, Brar DS (2009) Molecular characterization and agronomic performance of DH lines from the F-1 of indica and japonica cultivars of rice (Oryza sativa L.). Field Crop Res 112:222–228

    Article  Google Scholar 

  • Litt M, Luty JA (1989) A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet 44:397–401

    PubMed Central  CAS  PubMed  Google Scholar 

  • López-Anido F, Cointry E (2008) Asparagus. In: Prohens J, Nuez F (eds) Vegetables II: Fabaceae, Liliaceae, Umbelliferae, and Solanaceae. Series: handbook of plant breeding. Springer, New York, pp 87–119

    Google Scholar 

  • Löptien H (1979) Identification of the sex choromosome pair in asparagus (Asparagus officinalis L.). Zeitschrift für Pflanzenzüchtung 82:162–173

    Google Scholar 

  • Malik MR, Wang F, Dirpaul JM, Zhou N, Polowick PL, Ferrie AMR, Krochko JE (2007) Transcript profiling and identification of molecular markers for early microspore embryogenesis in Brassica napus. Plant Physiol 144:134–154

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Moreno R, Espejo JA, Cabrera A, Millan T, Gil J (2006) Ploidic and molecular analysis of ‘Morado de Huetor’ asparagus (Asparagus officinalis L.) population; a Spanish tetraploid landrace. Genet Resour Crop Evol 53:729–736

    Article  CAS  Google Scholar 

  • Moreno R, Espejo JA, Cabrera A, Gil J (2008a) Origin of tetraploid cultivated asparagus landraces inferred from nuclear ribosomal DNA internal transcribed spacers polymorphisms. Ann Appl Biol 153:233–241

    CAS  Google Scholar 

  • Moreno R, Espejo JA, Moreno MT, Gil J (2008b) Collection and conservation of “Morado de Huetor” Spanish tetraploid asparagus landrace. Genet Resour Crop Evol 55:773–777

    Article  Google Scholar 

  • Moreno R, Espejo JA, Gil J (2010) Development of triploid hybrids in asparagus breeding employing a tetraploid landrace. Euphytica 173:369–375

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nehra NS, Kartha KK, Stushnott C, Giles KL (1992) The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry. Plant Cell Tissue Organ Cult 29:257–268

    Article  CAS  Google Scholar 

  • Nomizu T, Niimi Y, Han DS (2004) Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis. Plant Cell Tissue Organ Cult 79:307–331

    Article  Google Scholar 

  • Okada H, Ohashi Y, Sato M, Matsuno H, Yamamoto T, Kim H, Tukuni T, Komori S (2009) Characterization of fertile homozygous genotypes from anther culture in apple. J Am Soc Hortic Sci 134:641–648

    Google Scholar 

  • Perera PIP, Perera L, Hocher V, Verdeil JL, Yakandawala DMD, Weerakoon LK (2008) Use of SSR markers to determine the anther-derived homozygous lines in coconut. Plant Cell Rep 27:1697–1703

    Article  CAS  PubMed  Google Scholar 

  • Pontaroli AC, Camadro EL (2005) Somaclonal variation in Asparagus officinalis plants regenerated by organogenesis from long-term callus cultures. Genet Mol Biol 28:423–430

    Article  Google Scholar 

  • Qiao YM, Falavigna A (1990) An improved in vitro anther culture method for obtaining double-haploid clones of asparagus. Acta Hortic 271:145–150

    Article  Google Scholar 

  • Raimondi JP, Camadro EL, Masuelli RW (2001) Assessment of somaclonal variation in asparagus by RAPD fingerprinting and cytogenetic analyses. Sci Hortic 90:19–29

    Article  Google Scholar 

  • Regalado JJ, Gil J, Castro P, Moreno R, Encina CL (2014) Employment of molecular markers to develop tetraploid “supermale” asparagus from andromonoecious plants of the landrace ‘Morado de Huétor’. Span J Agric Res 12:1131–1136

    Article  Google Scholar 

  • Regalado JJ, Carmona-Martín E, Castro P, Moreno R, Gil J, Encina CL (2015a) Micropropagation of wild species of the genus Asparagus L. and their interspecific hybrids with cultivated A. officinalis L., and verification of genetic stability using EST-SSRs. Plant Cell Tissue Organ Cult 121:501–510

    Article  CAS  Google Scholar 

  • Regalado JJ, Carmona-Martín E, Castro P, Moreno R, Gil J, Encina CL (2015b) Study of the somaclonal variation produced by different methods of polyploidization in Asparagus officinalis L. Plant Cell Tissue Organ Cult 122:31–44

    Article  CAS  Google Scholar 

  • Riccardi P, Casali PE, Mercati F, Falavigna A, Sunseri F (2011) Genetic characterization of asparagus doubled haploids collection and wild relatives. Sci Hortic 130:691–700

    Article  CAS  Google Scholar 

  • Rodrigues LR, Terra TD, Bered F, Bodanese-Zanettini MH (2004) Origin of embryo-like structures in soybean anther culture investigated using SSR marker. Plant Cell Tissue Organ Cult 77:287–289

    Article  CAS  Google Scholar 

  • Salas P, Prohens J, Segui-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274

    Article  CAS  Google Scholar 

  • Seguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404

    Article  Google Scholar 

  • Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the reprogramming of plant microspores towards in vitro embryogenesis. Physiol Plant 127:519–534

    Article  CAS  Google Scholar 

  • Shiga I, Uno Y, Kanechi M, Inagaki N (2009) Identification of polyploidy of in vitro anther-derived shoots of Asparagus officinalis L. by flow cytometric analysis and measurament of stomatal length. J Jpn Soc Hort Sci 78:103–108

    Article  Google Scholar 

  • Simmonds DH, Keller WA (1999) Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus. Planta 208:383–391

    Article  CAS  Google Scholar 

  • Smykal P (2000) Pollen embryogenesis—the stress mediated switch from gametophytic to sporophytic development. Current status and future prospects. Biol Plant 43:481–489

    Article  CAS  Google Scholar 

  • Sneep J (1953) The significance of andromonoecy for the breeding of Asparagus officinalis L. Euphytica 2:89–95

    Article  Google Scholar 

  • Snow R (1963) Alcoholic-hydrochloric acid carmine as a stain of chromosomes in squash preparations. Stain Tech 38:9–13

    CAS  Google Scholar 

  • Supena 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 25:1–10

    Article  CAS  PubMed  Google Scholar 

  • Touraev A, Pfosser M, Heberle-Bors E (2001) The microspore: A haploid multipurpose cell. Adv Bot Res 35:53–109

    Article  Google Scholar 

  • Vanwynsberghe L, De Witte K, Coart E, Keulemans J (2005) Limited application of homozygous genotypes in apple breeding. Plant Breed 124:399–403

    Article  Google Scholar 

  • Veilleux RE, Shen LY, Paz MM (1995) Analysis of the genetic composition of anther-derived potato by randomly amplified polymorphic DNA and simple sequence repeats. Genome 38:1153–1162

    Article  CAS  PubMed  Google Scholar 

  • Wang JJ, Hu DF, Wang HM, Tang YL (1981) Studies on increasing the induction frequency of pollen callus in wheat. Hereditas (Beiging) 3:28–29

    Google Scholar 

  • Wolyn DJ, Feng X (1993) Genotype, temperature, and sampling date affect embryogeneis in Asparagus anther culture. HortScience 28:216–217

    Google Scholar 

  • Zaki MAM, Dickinson HG (1991) Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis I to embryogenic development. Sex Plant Reprod 4:48–55

    Article  Google Scholar 

  • Zhang C, Wang H, Ma Y, Kang Y (1994) Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.). Plant Cell Rep 13:637–640

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was funded by the Project of Excellence AGR3648, managed by the Junta de Andalucia (Spain).

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Authors and Affiliations

  1. Laboratorio de Cultivo de Tejidos y Biotecnología, Instituto de Horticultura Subtropical y Mediterránea La Mayora, CSIC-UMA, 29750, Algarrobo-Costa, Málaga, Spain

    J. J. Regalado, E. Carmona Martín & C. L. Encina

  2. Institute for Sustainable Agriculture, CSIC, Apdo 4084, 14080, Córdoba, Spain

    E. Madrid

  3. Dpto. Genética, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C-5, 14071, Córdoba, Spain

    R. Moreno & J. Gil

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  1. J. J. Regalado
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  2. E. Carmona Martín
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  3. E. Madrid
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  4. R. Moreno
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  5. J. Gil
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  6. C. L. Encina
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Correspondence to J. J. Regalado.

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Regalado, J.J., Martín, E.C., Madrid, E. et al. Production of “super-males” of asparagus by anther culture and its detection with SSR-ESTs. Plant Cell Tiss Organ Cult 124, 119–135 (2016). https://doi.org/10.1007/s11240-015-0880-6

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