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Plant Cell Reports

, Volume 25, Issue 1, pp 1–10 | Cite as

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

  • E. D. J. Supena
  • S. Suharsono
  • E. Jacobsen
  • J. B. M. Custers
Cell Biology and Morphogenesis

Abstract

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4°C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9°C and thereafter at 28°C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 μM zeatin and 5 μM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

Keywords

Capsicum annuum Hot pepper Shed-microspore culture Defective shoots  

Notes

Acknowledgements

The authors thank Dr K.S. Ramulu, Dr A.H.M. van der Geest and Dr K.A. Boutilier for critically reading the manuscript, Mr J.H.W. Bergervoet for flow cytometry, Dr P.K. Agarwal for useful suggestions during the initial period of investigations, Mr A.H.J. Hermsen and Mr A. Kooijman for plant care. This work was supported by the research program on “Biotechnology Research Indonesia-Netherlands (BIORIN)”, with financial aid from the Royal Netherlands Academy of Arts and Sciences (KNAW) and the fellowship program on “Quality for Undergraduate Education Project (QUE)”, Bogor Agricultural University (IPB), Bogor, Indonesia (IBRD LOAN No.4193-IND)

References

  1. Abak K, Pochard E, Dumas de Vaulx R (1982) Transmission of resistance to Phytophthora capsici on roots and stems of pepper plants: study of doubled haploid lines issued from the cross ‘PM217’ × ‘Yolo Wonder’ through anther culture. Capsicum Nwsl 1:62–63Google Scholar
  2. Anonymous (2000a) Survey Pertanian, Produksi Sayuran dan Buah-buahan di Indonesia, 1999 (Agriculture Survey, Production of Season Vegetables and Fruit, 1999). Badan Pusat Statistik (BPS/Statistics Indonesia), JakartaGoogle Scholar
  3. Anonymous (2000b) Food and Agriculture Organization of the United Nations (FAO) Yearbook: Production 1999, vol 53. FAO, RomeGoogle Scholar
  4. Berke T, Shieh SC (2000) Chilli peppers in Asia. Capsicum Eggplant Nwsl 19:38–41Google Scholar
  5. Büyükalaca S, Mavituma F (1996) Somatic embryogenesis and plant regeneration of pepper in liquid media. Plant Cell Tiss Org Cult 46:227–235CrossRefGoogle Scholar
  6. Caranta C, Palloix A, Gebre-Selassie K, Lefebvre V, Moury B, Daubèze AM (1996) A complementation of two genes originating from susceptible Capsicum annuum lines confers a new and complete resistance to pepper veinal mottle virus. Phytopathology 86:739–743CrossRefGoogle Scholar
  7. Christensen HM, Bamford R (1943) Haploids in twin seedlings of pepper, Capsicum annuum L. J Heredity 34:99–104Google Scholar
  8. Cooper GM, Hausman RE (2003) The cell: a molecular approach, 3rd edn. Sinauer Associates, Sunderland USA, pp 739Google Scholar
  9. Custers JBM, Snepvangers SCHJ, Jansen HJ, Zhang L, Van Lookeren Campagne MM (1999) The 35S-CaMV promoter is silent during early embryogenesis but activated during nonembryogenic sporophytic development in microspore culture. Protoplasma 208:257–264CrossRefGoogle Scholar
  10. Daubèze AM, Palloix A, Pochard E (1990) Resistance of androgenetic autodiploid lines of pepper to Phytophthora capsici and tobacco mosaic virus under high temperature. Capsicum Nwsl 8–9:47–48Google Scholar
  11. Dolcet-Sanjuan R, Claveria E, Huerta A (1997) Androgenesis in Capsicum annuum L.-Effects of carbohydrate and carbon dioxide enrichment. J Am Soc Hort Sci 122:468–475Google Scholar
  12. Dumas de Vaulx R, Chambonnet D, Pochard E (1981) Culture in vitro d’anthères du piment (Capsicum annuum L.): amélioration des taux d’obtention de plantes chez différents génotypes par des traitements à +35°C. Agronomie 1:859–864CrossRefGoogle Scholar
  13. Dumas de Vaulx R, Pochard E (1986) Parthénogenèse et androgenèse chez le piment. Role actuel dans les programmes de sélection. Le Sélectionneur Francais 36:3–16Google Scholar
  14. George L, Narayanaswamy S (1973) Haploid Capsicum through experimental androgenesis. Protoplasma 78:467–470CrossRefGoogle Scholar
  15. Gémesné JA, Sági ZS, Salamon P, Somogyi N, Zatykó L, Venzcel G (1998) Experiences and results of in vitro haploid methods application in pepper breeding programme. In: Proceedings of the Xth Meeting on Genetics and Breeding of Capsicum and Eggplant, Avignon, France, Sept. 7–11, 1988, pp 201–205Google Scholar
  16. Gyulai G, Gémesné JA, Sági ZS, Venczel G, Pintér P, Kristóf Z, Törjék O, Heszky L, Bottka S, Kriss J, Zatykó L (2000) Doubled haploid development and PCR-analysis of F1 hybrid derived DH-R2 paprika (Capsicum annuum L.) lines. J Plant Physiol 156:168–174Google Scholar
  17. Harini I, Lakshmi-Sita G (1993) Direct somatic embryogenesis and plant regeneration from immature embryos of chilli (Capsicum annuum L.). Plant Sci 89:107–112CrossRefGoogle Scholar
  18. Hendy H, Pochard E, Dalmasso A (1985) Transmission de la résistance aux nématodes Meloidogyne chitwood (Tylenchida) portée par 2 lignées de Capsicum annuum L.: Étude de descendances homozygotes issues d’androgenèse. Agronomie 5:93–100CrossRefGoogle Scholar
  19. Jo JY, Choi EY, Choi D, Lee KW (1996) Somatic embryogenesis and plant regeneration from immature zygotic embryo culture in pepper (Capsicum annuum L.). J Plant Biol 39:127–135Google Scholar
  20. Johansson L, Andersson B, Eriksson T (1982) Improvement of anther culture technique: activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration. Physiol Plant 54:24–30CrossRefGoogle Scholar
  21. Kristiansen K, Andersen SB (1993) Effect of donor plant temperature, photoperiod, and age on anther culture response of Capsicum annuum L. Euphytica 67:105–109CrossRefGoogle Scholar
  22. Kyo M, Harada H (1986) Control of the developmental pathway of tobacco pollen in vitro . Planta 168:427–432CrossRefGoogle Scholar
  23. Lanteri S, Portis E, Bergervoet HW, Groot SPC (2000) Molecular markers for the priming of pepper seeds (Capsicum annuum L.). J Hort Sci Biotechnol 75:607–611Google Scholar
  24. Ltifi A, Wenzel G (1994) Anther culture of hot and sweet pepper (Capsicum annuum L.): Influence of genotype and plant growth temperature. Capsicum Eggplant Nwsl 13:74–77Google Scholar
  25. Maheswary V, Mak C (1993) The influence of genotypes and environments on induction of pollen plants for anther culture of Capsicum annuum L. AsPac J Mol Biol Biotechnol 1:43–50Google Scholar
  26. Mitykó J, Andrásfalvy A, Csilléry G, Fári M (1995) Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.). Plant Breed 114:78–80CrossRefGoogle Scholar
  27. Morrison RA, Koning RE, Evans DA (1986) Anther culture of an interspecific hybrid of Capsicum. J Plant Physiol 126:1–9Google Scholar
  28. Munyon IP, Hubstenberger JF, Phillips GC (1989) Origin of plantlets and callus obtained from chile anther cultures. In Vitro Cell Dev Biol 25:293–296CrossRefGoogle Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  30. Mythili JB, Thomas P (1995) Some factors influencing the in vitro establishment and callusing of anthers in Capsicum (Capsicum annuum L. var. Grossum Sendt). Indian J Plant Physiol 38:126–130Google Scholar
  31. Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–85PubMedCrossRefGoogle Scholar
  32. Ochoa-Alejo N, Ramírez-Malagón R (2001) In vitro chili pepper biotechnology. In Vitro Cell Dev Biol Plant 37:701–729CrossRefGoogle Scholar
  33. Pechan PM, Keller WA (1988) Identification of potentially embryogenic microspores in Brassica napus. Physiol Plant 74:377–384CrossRefGoogle Scholar
  34. Pochard E, Dumas de Vaulx R (1979) Haploid parthenogenesis in Capsicum annuum L. In: Hawkes JG, Lester RN, Skelding AD (eds) The biology and taxonomy of the Solanaceae. Academic Press, London, pp 455–472Google Scholar
  35. Pochard E, Selassié KG, Marchoux G (1983) Oligogenic resistance to potato virus Y pathotype 1-2 in the line ‘Perennial’. Capsicum Nwsl 2:137–138Google Scholar
  36. Polsoni L, Kott L, Beversdorf WD (1987) Large scale microspore culture technique for mutation-selection studies in Brassica napus. Can J Bot 66:1681–1685Google Scholar
  37. Qin X, Rotino GL (1993) Anther culture of several sweet and hot pepper genotypes. Capsicum Eggplant Nwsl 12:59–62Google Scholar
  38. Regner F (1994) Microspore culture of Capsicum annuum. Capsicum Eggplant Nwsl 13:72–73Google Scholar
  39. Regner F (1996) Anther and microspore culture in Capsicum. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol. 3. Kluwer Academic Publishers, Dordrecht, pp 77–89Google Scholar
  40. Sibi M, Dumas de Vaulx R, Chambonnet D (1979) Obtention de plantes haploïdes par androgenèse in vitro chez le piment (Capsicum annuum L.). Ann Amélior Plantes 29:583–606Google Scholar
  41. Steinitz B, Küsek M, Tabib Y, Paran I, Zelcer A (2003) Pepper (Capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot. In Vitro Cell Dev Biol Plant 39:296–303CrossRefGoogle Scholar
  42. Touraev A, Pfosser M, Vicente O, Heberle-Bors E (1996) Stress as the major signal controlling the developmental fate tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis. Planta 200:144–152CrossRefGoogle Scholar
  43. Touraev A, Vicente O, Heberle-Bors E (1997) Initiation of microspore embryogenesis by stress. Trend Plant Sci 2:297–302CrossRefGoogle Scholar
  44. Touraev A, Heberle-Bors E (1999) Microspore embryogenesis and in vitro pollen maturation in tobacco. In: Hall RD (ed.) Methods in molecular biology, vol.111: Plant cell culture protocols. Humana Press Inc. Totowa, NJ, pp 281–291CrossRefGoogle Scholar
  45. Vagera J, Havránek P (1985) In vitro induction of androgenesis in Capsicum annuum L. and its genetic aspects. Biol Planta 27:10–21Google Scholar
  46. Wang YY, Sun CS, Wang CC, Chien NF (1973) The induction of the pollen plantlets of triticale and Capsicum annuum from anther culture. Scientia Sinica 16:147–151Google Scholar
  47. Wolf D, Matzevitch T, Steinitz B, Zelcer A (2001) Why is it difficult to obtain transgenic pepper plants? Acta Hortic 560:229–233Google Scholar
  48. Ziauddin A, Simion E, Kasha KJ (1990) Improved plant regeneration from shed microspore culture in barley (Hordeum vulgare L.) cv. Igri. Plant Cell Rep 9:69–72CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • E. D. J. Supena
    • 1
    • 2
  • S. Suharsono
    • 1
  • E. Jacobsen
    • 2
  • J. B. M. Custers
    • 2
  1. 1.Research Center for BiotechnologyBogor Agricultural University (IPB)BogorIndonesia
  2. 2.Plant Research InternationalWageningen University and Research CentreWageningenThe Netherlands

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