In Vitro Mutagenesis followed by Polymorphism Detection Using Start Codon Targeted Markers to Engineer Brown Spot Resistance in Kalanchoe

in Journal of the American Society for Horticultural Science

Kalanchoe (Kalanchoe blossfeldiana) is a common potted flower that is popular throughout the world. Brown spot (caused by Stemphylium lycopersici) is one of the common foliage diseases in kalanchoe. This disease tends to infect leaves of kalanchoe plants in hot and humid environments, reducing their aesthetic value. The current investigation aimed to generate mutations resistant to brown spot in ‘Mary’ kalanchoe through chemical mutagenesis followed by molecular marker identification. Putative mutants were developed by treating embryogenic calluses with ethyl methanesulfonate (EMS) at median lethal doses (LD50)–either a 0.8% concentration for 2 hours or a 1.0% concentration for 0.5 hours. Brown spot crude toxin solution was used as the selection agent to identify disease-resistant calluses during tissue culture. The optimal crude concentration (60%) was determined by soaking calluses with different concentrations of crude pathogen: 0%, 20%, 40%, 60%, and 80% (v/v). A total of 32 anti-brown spot lines were regenerated and tested for disease resistance with detached leaves. Three regenerated EMS mutant lines showed no obvious brown spot lesions on their leaves after the disease resistance assay and were subjected to polymorphism identification by start codon targeted (SCoT) molecular markers. Three (SCoT40, SCoT71, and SCoT72) of 45 selected primers were chosen to identify the mutants. This work may lay the foundation for further development of new disease-resistant cultivars of kalanchoe.

Contributor Notes

This research was financially supported by the Chongqing Municipal Education Project, China (grant no. CY140206); and the Chongqing Research Program of Basic Research and Frontier Technology (no. cstc2016jcyjA0153).

These authors contributed equally to this work.

Corresponding authors. E-mail: Liu19830222@163.com or sszcq@swu.edu.cn.

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Article Figures

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    Foliar symptoms on kalanchoe. (A) Brown spot was shown on the whole plant (a), partial leaves (b), and a single leaf (c). Toxic effect of crude toxin derived from Stemphylium lycopersici on kalanchoe calluses were photographed after 3 d of cultivation. (B) Calluses treated without crude toxins (d) and calluses treated with 100% crude toxins for 48 h (e). (C) Calluses treated with crude toxin at various concentrations for 48 h: 0% (f), 20% (g), 40% (h), 60% (i), and 80% (j). Differentiation was observed after 20 d; scale bars = 1 cm.

  • View in gallery

    Outline of three groups for developing mutagenic lines of kalanchoe resistant to brown spot (A) and symptom comparison after 5 d of resistance detection with detached leaves (B), including the untreated (CK), ethyl methanesulfonate (EMS)-mutagenized, and crude toxin-induced plants. Each plant was inoculated twice; scale bars = 1 cm.

  • View in gallery

    (A) Three ethyl methanesulfonate (EMS) mutant lines of kalanchoe with brown spot resistance grown for 2 months: (a) 1, (b) 2, and (c) and 3, respectively. (B) Start codon targeted (SCoT)–polymerase chain reaction amplification of the three EMS mutant lines with three primers: (d) SCoT40, (e) SCoT71, and (f) SCoT72, respectively. Dots refer to band placement within the gel. DNA markers of 500 to 2000 bp (e and f) and 1000 to 15,000 bp (d) were used to compare the DNA band. M = DNA marker; CK = untreated; scale bars = 1 cm.

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