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Use of Sorghum bicolor leaf whorl explants to expedite regeneration and increase transformation throughput

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Abstract

Sorghum is considered a challenging species for transformation due to the limited choice in genotypes that can be transformed and the reliance on callus derived from immature embryos that need to be teased out of developing seeds. We have developed a more practical tissue culture procedure to generate embryogenic callus from leaf whorl explants obtained from 4 week-old seedlings. This procedure offers greater ease of obtaining explants, enables a more rapid turnover of growth chamber or greenhouse plants that can be used for explant collection, and eliminates the dependency on obtaining flowering plants with adequate numbers of seeds. Callus derived from these somatic embryos was amenable to biolistic transformation with DNA-coated gold particles and transformants were shown to express the Cas9 transgene. A comparison between genotypes RTx430 and P898012 indicated that the former was more suitable to tissue culture from leaf whorls based on greater callus induction and regeneration. Callus from both genotypes secreted colored compounds into the medium that we determined to be phenolic in nature, likely 3-deoxyanthocyanidins, based on UV–Vis absorbance profiles and thin layer chromatography. In order to reduce negative impacts of these compounds on regeneration, the addition of activated charcoal (AC) or polyvinylpyrrolidone (PVP) to the culture medium was examined. The addition of 0.5 g L−1 of AC after 4 weeks on callus induction medium accelerated callus regeneration, whereas culturing leaf whorl explants on medium containing 1 g L−1 PVP reduced the production of colored compounds and enhanced callus growth.

Key message

The use of leaf whorl explants from developing sorghum plants serves as a practical alternative to immature embryos as a source of embryogenic callus and doesn’t require plants to flower.

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Acknowledgements

TNS gratefully acknowledges a CAPES (Coordination for the Improvement of Higher Education Personnel) graduate fellowship provided by the Brazilian Ministry of Education (BEX 11883-13-8), and funding from the University of Florida Genetics Institute. MEK is grateful for financial support from the UF Plant Molecular & Cellular Biology program.

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Author notes

  1. Tallyta N. Silva

    Present address: Joint BioEnergy Institute, Emeryville, CA, USA

  2. Tallyta N. Silva

    Present address: Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Authors and Affiliations

  1. Graduate Program in Genetics & Genomics, University of Florida, Gainesville, FL, USA

    Tallyta N. Silva

  2. UF Genetics Institute, University of Florida, Gainesville, FL, USA

    Tallyta N. Silva, Megan E. Kelly & Wilfred Vermerris

  3. Graduate Program in Plant Molecular & Cellular Biology, University of Florida, Gainesville, FL, USA

    Megan E. Kelly

  4. Department of Microbiology & Cell Science, University of Florida, Gainesville, FL, USA

    Wilfred Vermerris

  5. Florida Center for Renewable Chemicals and Fuels, University of Florida, Gainesville, FL, USA

    Wilfred Vermerris

  6. University of Florida Genetics Institute, Cancer/Genetics Research Complex 302, 2033 Mowry Road, Gainesville, FL, 32610, USA

    Wilfred Vermerris

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  1. Tallyta N. Silva
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Correspondence to Wilfred Vermerris.

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Silva, T.N., Kelly, M.E. & Vermerris, W. Use of Sorghum bicolor leaf whorl explants to expedite regeneration and increase transformation throughput. Plant Cell Tiss Organ Cult 141, 243–255 (2020). https://doi.org/10.1007/s11240-020-01783-9

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