Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 106 ml−1) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 106 ml−1). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Cytoplasmic effects on plant performance are well-documented and result from the intimate interaction between organellar and nuclear gene products. In plants, deletions, mutations, or chimerism of mitochondrial genes are often associated with deleterious phenotypes, as well as economically important traits such as cytoplasmic male sterility used to produce hybrid seed. Presently, genetic analyses of mitochondrial function and nuclear interactions are limited because there is no method to efficiently produce mitochondrial mutants. Cucumber (Cucumis sativus L.) possesses unique attributes useful for organellar genetics, including differential transmission of the three plant genomes (maternal for plastid, paternal for mitochondrial, and bi-parental for nuclear), a relatively large mitochondrial DNA in which recombination among repetitive motifs produces rearrangements, and the existence of strongly mosaic (MSC) paternally transmitted phenotypes that appear after passage of wild-type plants through cell cultures and possess unique rearrangements in the mitochondrial DNA. We sequenced the mitochondrial DNA from three independently produced MSC lines and revealed under-represented regions and reduced transcription of mitochondrial genes carried in these regions relative to the wild-type parental line. Mass spectrometry and Western blots did not corroborate transcriptional differences in the mitochondrial proteome of the MSC mutant lines, indicating that post-transcriptional events, such as protein longevity, may compensate for reduced transcription in MSC mitochondria. Our results support cucumber as a model system to produce transcriptional “knock-downs” of mitochondrial genes useful to study mitochondrial responses and nuclear interactions important for plant performance.

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10-400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200-400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50-100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm −1 ). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot. Key messageSalt stress applied to carrot protoplasts generates variability manifested in differences in cellularresponse and variation in ploidy. The adaptation of carrot regenerants to soil salinity was associatedwith accumulation of anthocyanins and increased hairiness.

Protoplasts of six cabbage accessions were isolated from leaf mesophyll and cultured in the presence of 0.01, 0.1 and 1.0 µM phytosulfokine-α (PSK-α) and in a PSK-free control medium. PSK-α was applied for 10 days and later, protoplast-derived cells were cultured in the PSK-free medium. Supplementation of the culture medium with PSK-α showed a dose-dependent effect on the mitotic activity of cultured cells. On the 15th day of culture, the highest mitotic activity of protoplast-derived cells was observed in cultures treated with 0.1 µM of PSK-α, and ranged from 14 to 60% dependent on the accession. The number of multi-cell structures was also higher (90-93%) on this medium compared to the control (77-80%). Analysis of cellulose regeneration in cultured protoplasts after Calcofluor White staining showed that this process was not synchronous, but depended instead on the presence of PSK-α in the culture medium, and was more pronounced in the low-responding accession. Sustained cell divisions led to formation of microcallus colonies, subjected to regeneration on solid media. Supplementation of the regeneration media with 0.1 µM of PSK significantly increased shoot regeneration compared to the control media. Moreover, enhanced regeneration was observed from calluses developed from cells treated with PSK-α at the early stages of development and later transferred for regeneration onto the media supplemented with 0.1 µM of this peptide.