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


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.


Capsicum annuum Hot pepper Shed-microspore culture Defective shoots  



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)


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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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