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Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion, regeneration and isozyme analysis

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Abstract

Mesophyll protoplasts ofSolanum commersonii, a frost tolerant wild species not crossable with the cultivated potato, were fused with either dihaploid or tetraploid S.tuberosum. Protoplasts were aggregated by means of alternating current (AC) or polyethylene glycol (PEG), and electrofused with three direct current (DC) pulses. The treatments with PEG/DC generally resulted in very low heterofusion frequency and protoplast viabiity. On the other hand, AC/DC fusion conditions were optimized by increasing the fusion density of protoplasts and adding CaCl2 to fusion medium. When a density of 4.8 × 105 protoplasts ml−1 was used in the fusion medium containing 0.2 mM Ca++, AC/DC treated protoplasts showed heterofusion frequencies and plating efficiencies of about 10 and 3%, respectively. Fast growing calli from AC/DC fusion experiments were further cultured for regeneration. Fifty-seven plants were regenerated and clonedin vitro as shoot cultures. Compared to parents they showed heterotic vigor and could be identified as hybrids, based on isozyme analysis.

Compendio

Protoplastos mesofílicos deSolatium commersonii, una especie silvestre de papa resistente a las heladas que no admite cruzamiento con las papas cultivadas, fueron fusionados ya sea conS. tuberosum diploide o con tetraploides de esta misma especie. Los protoplastos fueron agregados por medio de corriente alterna (AC) o con glicopolietileno (PEG), y electrofusionados con tres pulsaciones de corriente directa (DC). Los tratamientos con PEG/DC dieron generalmente como resultado muy baja frecuencia de heterofusión y viabilidad de protoplastos. De otro lado, las condiciones de fusión AC/DC fueron optimizadas incrementando la densidad de fusión de los protoplastos y adicionando CaCl2, al medio de fusión. Cuando se utilizó una densidad de 4.8 × 105 protoplastos/cm3 en el medio de fusión conteniendo 0.2 mM Ca++, los protoplastos tratados con AC/DC mostraron frecuencias de heterofusión y eficiencia en platos de alrededor de 10 y 3%, respectivamente. Los callos de rápido crecimiento de los experimentos de fusión AC/DC fueron posteriormente cultivados para su regeneración. Cincuenta y siete plantas fueron regeneradas y se obtuvieron clonesin vitro mediante cultivo de brotes. Comparados con los progenitores ellos mostraron vigor híbrido (heterosis) y pudieron ser identificados como híbridos basándose en el análisis de isoenzimas.

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Abbreviations

AC:

alternating current

DC:

direct current

FDA:

fluorescein diacetate

MDH:

malate dehydrogenase

PEG:

polyethylene glycol

POX:

peroxidase

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Authors and Affiliations

  1. Research Centre for Vegetable Breeding, CNR, via Università, 133, 80055, Portici, Italy

    T. Cardi

  2. Center for Plant Breeding and Reproduction Research, CPRO, Postbox 16, 6700 AA, Wageningen, The Netherlands

    K. J. Puite & K. S. Ramulu

  3. Department of Agronomic Sciences and Plant Genetics, University of Naples, via Università, 100, 80055, Portici, Italy

    F. D’Ambrosio & L. Frusciante

Authors
  1. T. Cardi
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  2. K. J. Puite
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  3. K. S. Ramulu
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  4. F. D’Ambrosio
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  5. L. Frusciante
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Contribution no. 106 from Research Centre for Vegetable Breeding.

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Cardi, T., Puite, K.J., Ramulu, K.S. et al. Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion, regeneration and isozyme analysis. American Potato Journal 70, 753–764 (1993). https://doi.org/10.1007/BF02849103

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