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High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)

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Abstract

We developed an efficient culture system for producing cotyledonary embryos from isolated microspores of hot pepper (Capsicum annuum L.) and analyzed the ploidy levels of regenerated plants. Three culture protocols were studied: liquid, double-layer, and two-step culture. In the double-layer culture, cotyledonary embryos were produced more efficiently when the same medium composition was used for the liquid upper-layer and the solid under-layer. The two-step culture system, in which microspores were first incubated on liquid medium and then subcultured on double-layer medium, was most effective for producing cotyledonary embryos. Cotyledonary embryos were produced more efficiently when the isolated microspores were cultured in liquid medium for 1 week in 60 × 15-mm plates at a density of 8–10 × 104/mL and microspore suspensions from two liquid culture plates were combined into a single 100 × 20-mm plate containing solid medium, and the culture was continued for an additional 3 weeks. When cotyledonary embryos obtained from this two-step culture were transplanted into regeneration medium, more than 95 % developed into plants. Only 31 of the 190 analyzed plants (16.3 %) generated by this method were spontaneous doubled haploids. This two-step culture system outperforms all previously reported culture protocols for isolated microspores of hot pepper, and appears to be a promising tool for the production of haploid plants for hot pepper breeding.

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Authors and Affiliations

  1. Department of Life Sciences, Mokwon University, 800 Doan-dong Seo-gu, Daejeon, 302-729, Korea

    Moonza Kim, Eun-Joon Park & Dongju An

  2. Engineering Education Center of Accreditation, Chungnam National University, Daejeon, 305-754, Korea

    Youngwon Lee

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  1. Moonza Kim
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Correspondence to Moonza Kim.

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Supplementary Fig. 1

Effect of different culture systems on embryo production in isolated microspore cultures of hot pepper (C. annuum L.). The yield of total embryos (A) and cotyledonary embryos (B) was determined 4 weeks after culture onset. The bars show the means ± SEs of three experiments, each with 4 replicates. Asterisks above bars indicate significant differences relative to liquid culture (p < 0.05, t-test). (JPG 32 kb)

Supplementary Fig. 2

Effect of plating density used for the L-DL culture step of the two-step culture system on embryo production in isolated microspore cultures of hot pepper (C. annuum L.). The yields of total embryos (A) and cotyledonary embryos (B) were determined 4 weeks after culture onset. For this experiment, 100 × 20-mm plates (black bars) and 100 × 40-mm plates (white bars) were used. The bars show the means ± SEs of three experiments, each with 4 replicates. Asterisks above bars indicate significant differences relative to low-density culture (p < 0.05, t-test). (JPG 33 kb)

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Kim, M., Park, EJ., An, D. et al. High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tiss Organ Cult 112, 191–201 (2013). https://doi.org/10.1007/s11240-012-0222-x

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