ABSTRACT
A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.
ABSTRACT
An early identification of fusion products was based on the presumed vigorous growth of hybrid calluses after fusion between Solanum brevidens and S. tuberosum leaf protoplasts. The S. brevidens protoplasts were unable to form multicellular colonies under the applied culture conditions. Three size groups of calluses were separated and analyzed at two different early phases of culture period. "Squash blot" hybridization with a S. brevidens specific repetitive DNA probe showed that the group of the largest calluses consisted of putative somatic hybrids with a frequency of 80-100% in three independent experiments. Furthermore, approximately 80-95% of the middle sized calluses and 33-90% of the smallest ones were shown to be hybrid. The unexpectedly high percentage of fusion products, even in the case of the smallest calluses, may result from the suppression of the development of parental potato colonies in cultures with mixed cell population. Till this time 120 independent colonies selected as putative hybrids have been regenerated into plants. All of them exhibited hybrid phenotype, and their hybrid origin was proved by cytological and restriction fragment length polymorphism analyses.
ABSTRACT
Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28-40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.
ABSTRACT
Tuber discs of potato (Solanum tuberosum L.) cultivars Desirée and Gracia were infected by Agrobacterium tumefaciens carrying a binary vector with the coat protein gene of potato virus X controlled by the carrot extensin gene long-transcript promoter. Several transgenic potato plants have been obtained by direct regeneration of shoots on culture medium with kanamycin used for selection. The presence of the coat protein gene was proved by Southern hybridization in several transformants. Its low but detectable expression level was shown by Northern and Western analysis. Ethephon treatment resulted in a five-fold increase in the amounts of the coat protein mRNA. The majority of transformants exhibited reduced accumulation of virus RNA in inoculated leaves. Potentials in the use of an ethylene-inducible promoter in the production of virus-resistant transgenic plants will be discussed.
ABSTRACT
Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies.
