No abstract

Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca 2 + ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants.

The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A1 plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.

Fusion, protoplasts, high pH and Calcium, complementationHigh frequencies of fusion were induced between isolated tobacco leaf protoplasts when they were incubated at 37 °C in the presence of 0.05 M CaCl2 in 0.4 m mannitol at pH 10.5. Subsequent to the fusion treatment the protoplasts were washed and cultured in a suitable medium and within two weeks, actively dividing cell colonies were observed. The fusion treatment had no irreversible, deleterious effects on the protoplasts.The induced fusion of isolated plant protoplasts with the aim of producing interspecific somatic hybrid plants has drawn the attention of many plant scientists1-5. Induced protoplast fusion refers to the achievement of fusion between protoplasts that are free in suspension and it differs from spon taneous fusion which may occur as a result of plasmodesmatal expansion during isolation and which cannot be utilized for somatic hybridiza tion 6' 7, Carlson et al. 8 have recently reported the form ation of a vegetative, somatic or, as they call it, parasexual hybrid ("burdo" , W inkler9,10) re sulting from the induced fusion of protoplasts from two N ic o tia n a species.Several methods have been reported to induce protoplast fusion. These include m anipulation with a perfusion m icropipetten , treatment with N aN 0 3 12,14, 8, artificial seawater 15,16 lysozyme 17 and deplasmolyzing osmotic shock7. W ith existing methods it has been difficult to induce high fre quencies of fusion and retain the viability of vacuolate protoplasts such as leaf proto plasts 18' 13>17> 8. In the present paper we wish to report evidence for the stimulation of high fre quencies of protoplast fusion by calcium ions at high pH conditions without irreparable damage to the protoplasts. M aterial an d M ethods S o u rce o f p ro to p la stsThe upper expanding leaves of three N ico tia n a tabacu m varieties were utilized for protoplast iso * Present address: Ottawa Research Station, Department of Agriculture, Ottawa, Ontario, Canada. Requests for reprints should be sent to Max-Planck-Institut für Bioolgie, Abteilung Melchers, D-7400 Tübingen, Corrensstr. 41.lation. The varieties were N . ta b a cu m var. " Sam sun" with 48 chromosomes growing in greenhouse conditions and two "haploid" chlorophyll deficient, light sensitive varieties of N . ta b a cu m designated as slj sl2 (from now on called "s" , sublethal) and vi-Aj (called "v" , virescent). The "haploid" forms with 24 chromosomes of the light sensitive varieties were produced by anther culture techniques19,20 and were grown in climate chambers at 700 -800 lx (continuous) at 28 °C and 75% relative humidity. Under these conditions the plants show light green leaves. They are multiplied by cuttings. The Fj hybrids produced by crossing v x s with 48 chromo somes complement for a norm al dark green chloro phyll character and they grow vigorously in high light intensities present in the greenhouse or pro duced artificially. P ro to p la st iso la tio nThe leaves were surface sterilized by a two-min immersion in a 0.3% comme...