Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization.

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.

Distribution of novel and known repeated elements of Solanum and application for the identification of somatic hybrids among Solanum species.

Species-specific repetitive DNA probes are a useful tool for the molecular identification of somatic hybrids. Therefore, the distribution of three repetitive DNA elements of Solanum was investigated in Solanum wild species, Solanum breeding lines, and in more distantly related species of the genera Lycopersicon, Nicotiana, and Datura. The clone pSCH15, obtained from S. circaeifolium, represents a new 168-bp repetitive element; it shows 73-79% sequence similarity to repetitive elements of S. brevidens and Lycopersicon species. The 163-bp element in pSBH6, cloned from S. bulbocastanum, turned out to be very similar (95% sequence homology) to the Lycopersicon element pLEG15/TGRI previously regarded to be present only in species of the genus Lycopersicon and in S. lycopersicoides. Lower sequence similarity of approximately 80% was observed to repetitive elements of S. brevidens which are organized differently. The repeats exhibited different degrees of specificity: by Southern hybridization the element represented by the clone pSBH6 could be detected in almost all Solanum species investigated here but only after long exposure to X-ray film. The previously described "Solanum-specific" element represented by the clone pSA287 was also found, although in a very low copy number, in Lycopersicon esculentum. Therefore, detection of the repetitive elements pSA287 and pSBH6 in those species in which the respective repeat is less represented depends on exposure time. In contrast, the element pSCH15 is prominently present only in a small number of Solanum wild species and - to some extent - in the diploid breeding lines as revealed after long exposure. Use of these repeated elements for the identification of specific genomes in protoplast-fusion hybrids between Solanum wild species and Solanum breeding lines, or between two breeding lines, was evaluated.

A specific oligonucleotide of the 5S rDNA spacer and species-specific elements identify symmetric somatic hybrids between Solanum tuberosum and S. pinnatisectum.

The nucleotide sequences of the 5S rRNA genes (5S rDNA) of two Solanum tuberosum breeding lines (R1 and B15) and of the Mexican wild species S. pinnatisectum were determined and compared with each other and to the 5S rDNA of other Solanaceae species (Lycopersicon esculentum, Nicotiana rustica and Petunia hybrida). The 5S rDNA repeats of the Solanum species are 324-329 bp in length, and they exhibit 91-95% sequence identity. Sequence variability is mainly located in a short region of the spacer separating the 5S rRNA coding regions. A synthetic 28-mer oligonucleotide constructed according to this region can be used as a specific hybridization probe to distinguish symmetric somatic hybrids between S. tubersosum breeding line B15 and S. pinnatisectum produced by protoplast fusion. Interestingly, the two Solanum breeding lines R1 and B15 differ also in this spacer region.

Genetic control of embryo formation in anther culture of diploid potatoes.

A diploid potato clone AH 78/8015.37a with androgenetic ability was crossed with a root-knot nematode resistant diploid clone 381320.23 which did not have this ability.Among 19 F1 progenies tested a wide range of continuous variability was found for androgenetic capability. Four F1 clones with different level of embryo formation capability were backcrossed to 381320.23 to produce 4 F1BC1 families to further clarify the genetic control of androgenetic capability.From the wide range of continuous variability for androgenetic ability observed, it can be inferred that this character is controlled by more than one major gene.The occurrence of plants with androgenetic ability derived from parents both lacking this character, indicated that the androgenetic ability is controlled by recessive genes or results from complementation of various factors singly present in the parents. The present results demonstrated that the androgenetic ability could be transferred through sexual crosses, making it possible to successfully apply anther culture to other useful genetic material lacking this ability.

Role of the cell wall in the ability of tobacco protoplasts to form callus.

Addition of cellulose to the culture medium inhibited cell wall regeneration and nuclear and cell division in tobacco (Nicotiana tabacum L.) mesophyll protoplasts. When the activity of celllase was inhibited by cellobiose, the inhibition of nuclear and cell division was overcome. Cell wall formation thus appears to be a prerequisite for nuclear and cell division in tobacco protoplasts.