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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Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm(-3) in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2-30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).

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INDEX WORDSPetunia hybrida, petunia, cytoplasmic male sterility, esterase activity, esterase isoenzymes, histochemical analysis. SUMMARYAnthers of male fertile, cytosterile and restored male fertile clones of Petunia hybrida were compared for esterase activity and composition in subsequent stages of microsporogenesis. Three methods were applied (i) ultra-thin layer isoelectric focussing on polyacryl amide gels, (ii) quantitative spectrophotometricat assay, (iii) histochemical determination of total esterase activity associated to the azo-coupling method (PEARSE, 1972).In male fertile and restorer idiotypes the isozyme patterns were quite similar. Both the number and intensity of bands increased gradually till the tetrad stage. In contrast, esterase activity in cytosterile anthers remained at a low level and hardy any new bands showed up in later stages. This unvariable, low activity level in cytosterile anthers was also found in the spectrophotometric assay. Histochemical determinations revealed that in male fertile anthers esterase activity is concentrated in the outer tapetal layer at late prophase and that it accumulates there till the early microspore stage. In male sterile anthers esterase accumulation in the tapetal cells stops at the moment that tapetal breakdown becomes visible. This suggests that differences in esterase activity and composition are an effect rather than a cause of the failing pollen formation. GENERAL INTRODUCTIONAs yet cytoplasmic male sterility (CMS) is the most widely adopted tool for hybrid seed production. Male sterility is necessary to prevent the maternal line (seed line) from selfing or sibbing where hand emasculation is not feasible. Nevertheless, its application is limited by a number of severe drawbacks: (i)the non-availability of the cms characteristic in many crops and their wild relatives; (ii) the necessity of a time-consuming series ofbackcrossings to introduce cms in breeding stocks; (iii) the need for fertility restorer genes in seed or fruit producing hybrids; (iv) the complexity of seed production and maintenance of parental lines. Breeders are therefore looking for systems of artificial prevention of selfing which can be applied on call. In connection with this the 77 G. A. M. VAN MARREW1JK~ R. J. B1NO AND L. C. J. M. SUURS main attention has been directed at developing gamete killing chemicals (gametocides) such as maleic hydrazide, gibberellins, Ethrel (2-chloroethane phosphonic acid) and (sodium 2,3-dichloroisobutyrate). Wide scale use of gametocides has hitherto been impeded by serious shortcomings, of which female sterility and unreliable activity are the most notorious. Only in one single case (wheat) have hybrids, that were produced on the basis of gametocides, been released or have been put on trial (JENSEN, 1985).