Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration

• Published in: Plant cell reports 1987-06, Vol.6 (3), p.172-175
• Main Authors: Tan, M.L.M.C, Rietveld, E.M, Marrewijk, G.A.M. van, Kool, A.J
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Biotechnology; Cell cultures. Hybridization. Fusion; cultivars; Eukaryotic cell cultures; Fundamental and applied biological sciences. Psychology; Genetics and breeding of economic plants; Haploidy, in vitro culture applications, somatic hybrids; in vitro; Laboratorium voor Plantenveredeling; Laboratory of Plant Breeding; leaves; methodology; Methods. Procedures. Technologies; micropropagation; Molecular and cellular biology; Plant Breeding; Plant breeding: fundamental aspects and methodology; Plant cells and fungal cells; Protoplast preparation, cell fusion and regeneration; protoplasts; Solanum lycopersicum var. lycopersicum
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/bf00268470

Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 μl droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).