Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat

  • 1137 Accesses

  • 33 Citations


The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4



Foreign species ovary co-culture


Fluorescein diacetate




  1. Bárány I, González-Melendi P, Fadón B, Mitykó J, Risueno MC (2005) Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development. Biol Cell 97:709–722. doi:10.1042/BC20040142

  2. Bruins MBM, Rakoczy-Trojanowska M, Snijders CHA (1996) Isolated microspore culture in wheat (Triticum aestivum L.): the effect of co-culture of wheat or barley ovaries on embryogenesis. Cereal Res Commun 24:401–408

  3. Buyukalaca S, Comlekcioglu N, Abak K, Ekbic E, Kilic N (2004) Effects of silver nitrate and donor plant growing conditions on production of pepper (Capsicum annuum L.) haploid embryos via anther culture. Eur J Hortic Sci 69:206–209

  4. Chaim AB, Borovsky Y, De Jong W, Paran I (2003) Linkage of the A locus for the presence of anthocyanin and fs10.1, a major fruit-shape QTL in pepper. Theor Appl Genet 106:889–894

  5. Cistue L, Soriano M, Castillo AM, Valles MP, Sanz JM, Echavarri B (2006) Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant Cell Rep 25:257–264

  6. Coronado MJ, Hensel G, Broeders S, Otto I, Kumlehn J (2005) Immature pollen-derived doubled haploid formation in barley cv. Golden Promise a tool for transgene recombination. Acta Physiol Plant 27(4B):591–599. doi:10.1007/s11738-005-0063-x

  7. Coventry J, Kott L, Beversdorf WD (1988) Manual for microspore culture technique for Brassica napus. University of Guelph, Guelph, pp 1–35

  8. Custers JBM, Cordewener JHG, Nollen Y, Dons HJM, Campagne MMV (1994) Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Rep 13:267–271. doi:10.1007/BF00233317

  9. Davies PA, Morton S (1998) A comparison of barley isolated microspore and anther culture and the influence of cell culture density. Plant Cell Rep 17(3):206–210. doi:10.1007/s002990050379

  10. Derera FN, Nagy N, Hoxha A (2005) Condiment paprika research in Australia. J Bus Chem 2:4–18

  11. Dolcet-Sanjuan R, Claveria E, Huerta A (1997) Androgenesis in Capsicum annuum L. Effect of carbohydrate and carbon dioxide enrichment. J Am Soc Hortic Sci 122:468–475

  12. Doležel J, Binorova P, Lucretti S (1989) Analysis of nucler-DNA content in plant-cells by flow-cytometry. Biol Plant 31:113–120. doi:10.1007/BF02907241

  13. Dumas de Vaulx R, Chambonet D, Pochard E (1981) Culture in vitro d’anthères du piment (Capsicum annuum L.): amélior des taux d’obtention de plantes chezdifférents génotypes par des traitments ŕ + 35 C. Agronomie 1:859–864. doi:10.1051/agro:19811006

  14. Ercan N, Sensoy FA, Sensoy S (2006) Influence of growing season and donor plant age on anther culture response of some pepper cultuvars (Capsicum annuum L.). Scientia Hortic 110:16–20. doi:10.1016/j.scienta.2006.06.007

  15. Eudes F, Amundsen E (2005) Isolated microspore culture of Canadian 6× triticale cultivars. Plant Cell Tissue Org 82:233–241. doi:10.1007/s11240-005-0867-9

  16. Gémes Juhász A, Sági Zs, Salamon P, Somogyi N, Zatykó L, Vencel G (1988) Experiences and results of in vitro haploid methods application in pepper breeding programme. Xth EUCARPIA Meeting on Genetics and Breeding of Capsicum and Eggplant Avignon, France, September 7–11. Proceedings:201–203

  17. Gémes Juhász A, Vencel G, Sági Zs, Gajdos L, Kristóf Z, Vági P, Zatykó L (2006) Production of doubled haploid breeding lines in case of paprika, eggplant, cucumber, zucchini and onion. Acta Hortic 725:845–854

  18. George L, Narayanaswamy S (1973) Haploid capsicum through experimental androgenesis. Protoplasma 78:467–470. doi:10.1007/BF01275781

  19. Gyulai G, Gémesné JA, Sági Z, Vencel G, Pintér P, Kristóf Z, Törjék O, Heszky L, Bottka S, Kiss J, Zatykó L (2000) Doubled haploid development and PCR analysis of F-1 hybrid derived DH-2-R paprika (Capsicum annuum L.) lines. J Plant Physiol 156:168–174

  20. Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol 45:115–120

  21. Kasha KJ, Simion E, Oro R, Yao QA, Hu TC, Carlson AR (2001) An improved in vitro technique for isolated microspore culture of barley. Euphytica 120:379–385. doi:10.1023/A:1017564100823

  22. Kim M, Kim J, Yoon M, Choi DI, Lee KM (2004) Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum L.). Plant Cell Tissue Org 77:63–72. doi:10.1023/B:TICU.0000016506.02796.6a

  23. Kim M, Jang IC, Kim JA, Park EJ, Yoon M, Lee Y (2008) Embryogenenesis and plant regeneratio of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep 27:425–434. doi:10.1007/s00299-007-0442-4

  24. Kristiansen K, Andersen SB (1993) Effect of donor plant-temperature, photoperiod, and age on anther culture response of Capsicum annuum L. Euphytica 67:105–109. doi:10.1007/BF00022732

  25. Kuo JS, Wang ZZ, Chien NF, Ku SJ, Kung ML, Hsu HC (1973) Investigation on the anther culture in vitro of Nicotiana tabacum L. and Capsicum annuum L. Acta Bot Sin 15:43–47

  26. Lantos C, Jancsó M, Pauk J (2005) Microspore culture of small grain cereals. Acta Physiol Plant 27(4B):631–639. doi:10.1007/s11738-005-0067-6

  27. Letarte J, Simion E, Miner M, Kasha K (2006) Arabinogalactans and arabinogalacta-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture. Plant Cell Rep 24:691–698. doi:10.1007/s00299-005-0013-5

  28. Li H, Devaux P (2001) Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep 20:475–481. doi:10.1007/s002990100368

  29. Mejza SJ, Morgant V, DiBona DE, Wong JR (1993) Plant regeneration from isolated microspores of Triticum aestivum. Plant Cell Rep 12:149–153. doi:10.1007/BF00239096

  30. Mittykó J, Andrásfalvy A, Csilléri G, Fáry M (1995) Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.). Plant Breed 114:78–80. doi:10.1111/j.1439-0523.1995.tb00764.x

  31. Mitykó J, Gémes Juhász A (2006) Improvement in the haploid technique routinely used for breeding sweet and spice pepper in Hungary. Acta Agron Hung 54:203–219. doi:10.1556/AAgr.54.2006.2.8

  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cutures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

  33. Nageli M, Schmid JE, Stamp P, Büter B (1999) Improved formation of regenerable callus in isolated microspore culture of maize: impact of carbohydrates, plating density and time of transfer. Plant Cell Rep 19:177–184. doi:10.1007/s002990050730

  34. Ouyang JW, Jia SE, Zhang C, Chen X, Fen G (1989) A new synthetic medium (W14) for wheat anther culture. Annual report. Institute of Genetics, Academia Sinica, Beijing, pp 91–92

  35. Pauk J, Mihály R, Monostori T, Puolimatka M (2003) Protocol of triticale (x Triticosecale Wittmack) microspore culture. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants, a mannual. Kluwer Academic Publishers, Dordrecht, pp 129–134

  36. Sibi M, Dumas de Vaulx R, Chambonnet D (1979) Obtention de plantes haplod’des par androgeňcse in vutro chez le piment (Capsicum annuum L.). Ann Amclior Plantes 29:583–606

  37. Sugita T, Yamaguchi K, Kinoshita T, Yuji K, Sugimura Y, Nagata R, Kawasaki S, Todoroki A (2006) QTL analysis for resistance to phytophthora blight (Phytophtora capsici Leon.) using an intraspecific doubled-haploid population of Capsicum annuum. Breed Sci 56:137–145. doi:10.1270/jsbbs.56.137

  38. Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006a) 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. doi:10.1007/s00299-005-0028-y

  39. Supena EDJ, Muswita W, Suharsono S, Custer JBM (2006b) Evaluation of crucial factors for implementing shed-microspore culture of Indonesian hot pepper (Capsicum annuum L.) cultivars. Scientia Hortic 107:226–232. doi:10.1016/j.scienta.2005.08.006

  40. Szarka B, Dévényi M, Mórocz S (2001) Fertile maize lines obtained from isolated microspores. Euphytica 122:53–60. doi:10.1023/A:1012699332546

  41. Thomas WTB, Forster BP, Gertsson B (2003) Doubled haploids in breeding. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants, a manual. Kluwer Academic Publishers, Dordrecht, pp 95–102

  42. Touraev A, Ilham A, Vicente O, HeberleBors E (1996) Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep 15:561–565. doi:10.1007/BF00232453

  43. Wang YY, Sun CS, Wang CC, Chien NF (1973) The induction of the pollen plantlets of triticale and Capsicum annuum from anther culture. Scientia Sin Vol. XVI 1:147–151

  44. Zheng MY, Weng Y, Sahibzada R, Konzak CF (2003) Isolated microspore culture in maize. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants a manual. Kluwer Academic Publishers, Dordrecht, pp 95–102

Download references


This work was supported by the Agency for Research Fund Management and Research Exploitation (KPI) and the National Office for Research and Technology (NKTH), project number DA_TECH_05-FPFTT005. The authors thank Erzsébet Juhász Fehér, Éva Kótai, Petra Majer, Mária Olasz, Zsuzsanna Kun and Zsuzsanna Ábrahám Táborosi for their conscientious work and Maria Isabel Garcia Pomar, Council of Extremadura Region Agricultural Research Service, “La Orden” Experimental Station, Badajoz, Spain, for the Spanish pepper genotypes.

Author information

Correspondence to János Pauk.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lantos, C., Juhász, A.G., Somogyi, G. et al. Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tiss Organ Cult 97, 285–293 (2009).

Download citation


  • Capsicum annuum L.
  • Embryogenesis
  • Microspore culture
  • Ovary co-culture
  • Pepper