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%).

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Reports on direct gene transfer have dealt with either the obtention of stable transformants and transgenic plants, or described the use of reporter genes to analyse different aspects of gene expression in plant protoplasts and conditions for their use in transient gene expression assays. In this paper we present comparisons between several transformation techniques, show species‐specific differences in efficiencies of stable transformants and in the levels of transient gene expression, and report on the identification of major parameters responsible for DNA uptake as judged from transient chloramphenicol acetyl transferase (CAT) expression levels and from efficiencies of transformation based on kanamycin‐resistance. The described procedures have been simplified, optimized and standardized and should allow routine use with a great variety of plant species.

The growth (fresh and dry weight increase) of potato tuber (Solanum tuberosum L. cv. Bintje) callus discs was stimulated by incubation in air with 500 ppm 2,5‐norbornadiene (NBD, a competitive inhibitor of ethylene action) and inhibited by incubation in air with 4 000 ppm NBD. Ethylene formation by the callus was stimulated by NBD. The development of the alternative pathway, measured in isolated mitochondria was inhibited by NBD in a concentration‐dependent way. The alternative pathway capacity, measured in vivo, was inhibited by 4 000 ppm NBD, but not by 500 ppm. Uninhibited in vivo respiration, which consists of cytochrome path activity and alternative path activity, was stimulated by the treatment with 500 ppm NBD. The main contribution to this stimulation was made by the cytochrome pathway. In 4 000 ppm NBD‐treated callus, uninhibited respiration seemed to be unaffected as a consequence of an inhibited cytochrome path activity, which was compensated by a stimulated alternative path activity. Both in 500 and 4 OIK) ppm NBD‐treated callus the alternative path activity in vivo was stimulated. The regulatory role for endogenous ethylene in potato tuber callus is discussed in relation to: 1) The induction of respiratory pathways, 2) the supply of reduction equivalents in vivo and 3) growth.