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Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment

  • Genetic Transformation and Hybridization
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Abstract

To the best of our knowledge, this is the first accurate and reliable protocol for hop (Humulus lupulus L.) genetic transformation using particle bombardment. Based on the highly productive regeneration system previously developed by us for hop var. Eroica, two efficient transformation protocols were established using petioles and green organogenic nodular clusters (GONCs) bombarded with gusA reporter and hpt selectable genes. A total of 36 hygromycin B-resistant (hygr) plants obtained upon continuous selection were successfully transferred to the greenhouse, and a first generation group of transplanted plants was followed after spending a complete vegetative cycle. PCR analysis showed the presence of one of both transgenes in 25 plants, corresponding to an integration frequency of 69.4% and an overall transformation efficiency of 7.5%. Although all final transformants were GUS negative, the integration frequency of gusA gene was higher than that of hpt gene. Petiole-derived transgenic plants showed a higher co-integration rate of 76.9%. Real-time PCR analysis confirmed co-integration in 86% of the plants tested and its stability until the first generation, and identified positive plants amongst those previously assessed as hpt + only by conventional PCR. Our results suggest that the integration frequencies presented here, as well as those of others, may have been underestimated, and that PCR results should be taken with precaution not only for false positives, but also for false negatives. The protocols here described could be very useful for future introduction of metabolic or resistance traits in hop cultivars even if slight modifications for other genotypes are needed.

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Acknowledgments

This work was supported by a grant from Programme PRAXIS XXI (GGP/XXI/BD/3112/96), and in part by project PRAXIS XXI (2/2.1/BIO/1142/95), from Foundation for Science and Technology (FCT), Ministério da Ciência, Tecnologia e Ensino Superior, Portugal, whose funding is gratefully acknowledge.

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Authors and Affiliations

  1. Unit of Molecular Biology and Plant Biotechnology, Institute of Applied Science and Technology (ICAT), Science Faculty, Lisbon University, Campo Grande, 1749-016, Lisbon, Portugal

    Dora Batista, Sandra Fonseca, Susana Serrazina, Andreia Figueiredo & Maria Salomé Pais

  2. Estação Florestal Nacional, Quinta do Marquês, Av República, 2780-159, Oeiras, Portugal

    Dora Batista

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  1. Dora Batista
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  2. Sandra Fonseca
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  3. Susana Serrazina
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  4. Andreia Figueiredo
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  5. Maria Salomé Pais
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Correspondence to Dora Batista.

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Communicated by H. Ebinuma.

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Batista, D., Fonseca, S., Serrazina, S. et al. Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment. Plant Cell Rep 27, 1185–1196 (2008). https://doi.org/10.1007/s00299-008-0537-6

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  • DOI: https://doi.org/10.1007/s00299-008-0537-6

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