, Volume 256, Issue 1, pp 13–24 | Cite as

Enhancement of microspore embryogenesis induction and plantlet regeneration of sweet pepper (Capsicum annuum L.) using putrescine and ascorbic acid

  • Ali Akbar Heidari-Zefreh
  • Mehran E. ShariatpanahiEmail author
  • Amir Mousavi
  • Sepideh Kalatejari
Original Article


Production of doubled haploid (DH) plants is an efficient tool in genetic and plant breeding programs; however, sweet pepper (Capsicum annuum L.) is recalcitrant to microspore embryogenesis and DH production. Trying to break the barrier of DH production, three independent experiments were conducted on microspore embryogenesis of sweet pepper. In the first experiment, the effect of cold (4 °C) and heat (32 °C) pretreatments were investigated on microspore embryogenesis of three genotypes of sweet pepper including “Inspiration F1,” “Maratus F1,” and “Magno F1” cultivars in a factorial design with three replications. Heat shock (32 °C for 7 days), applied to mannitol-starved anthers of “Inspiration F1,” showed higher multinuclear microspore percent, number of multicellular structures, total embryos, cotyledonary embryos, and regenerants. In the second experiment, the effect of different concentrations of putrescine (0, 0.5, 1, 2, and 5 mg l−1) was evaluated on microspore embryogenesis of the three aforementioned cultivars of sweet pepper. The highest mean number of multicellular structures, cotyledonary embryos, and regenerants were achieved by applying 0.5–1 mg l−1 putrescine during the mannitol starvation and heat shock (32 °C) treatments of isolated microspore culture of “Inspiration F1” cultivar. Significant decrease in microspore embryogenesis efficiency was observed when high levels of putrescine (2 and 5 mg l−1) were used. Microspore embryogenesis was prevented completely at 5.0 mg l−1 putrescine. In the third experiment, the effect of different concentrations of ascorbic acid (0, 20, 50, 100, and 200 mg l−1) was investigated and the results showed that the application of ascorbic acid (20 and 50 mg l−1) during mannitol starvation and heat shock treatment (32 °C) caused remarkable improvement in the number of produced cotyledonary embryos and their regeneration ability compared to control treatment. However, the application of higher levels of ascorbic acid (100 and 200 mg l−1) inhibited microspore cell divisions and embryogenesis. In conclusion, the results indicated that both putrescine and ascorbic acid have significant effect on microspore embryogenesis efficiency of sweet pepper when they are used in appropriate concentrations.


Ascorbic acid Capsicum annuum L. Microspore embryogenesis Putrescine Temperature stress 



Ascorbic acid


1-aminocyclopropane-1-carboxylic acid


Cotyledonary embryo


Doubled haploid


Multicellular structure


Multinuclear microspores


Reactive oxygen species


Total embryo


Completely randomized design



The authors would like to express their deepest appreciation to Dr. Mohsen Niazian and Dr. Maryam Jafarkhani-Kermani for their excellent manuscript editorial helps and critical reading. This research was supported by grants from Agricultural Biotechnology Research Institute of Iran (ABRII) Project No. 14-05-05-9154-91002.

Authors’ contributions statement

Ali Akbar Heidari Zefreh contributed in designing the experiment, practical process of microspore culture, practical process of flow cytometery, cytogenetic analysis, plant adaptation, greenhouse management, data analysis, and writing of the manuscript. Mehran E. Shariatpanahi (corresponding author) contributed in supervising the whole practical process of microspore culture, data analysis, and writing of the manuscript. Amir Mousavi contributed in the data analysis and editing of the manuscript. Sepideh Kalatejari contributed in providing the plant material seeds and materials needed for cytogenetic analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Ali Akbar Heidari-Zefreh
    • 1
    • 2
  • Mehran E. Shariatpanahi
    • 2
    Email author
  • Amir Mousavi
    • 3
  • Sepideh Kalatejari
    • 1
  1. 1.Department of Horticultural Science, College of Agricultural Science and Natural Resources, Tehran Science and Research BranchIslamic Azad UniversityTehranIran
  2. 2.Department of Tissue and Cell Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO)KarajIran
  3. 3.Department of Molecular Plant BiotechnologyNational Institute of Genetic Engineering and Biotechnology (NIGEB)TehranIran

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