Somatic hybridization of an atrazine resistant biotype of Solanum nigrum with Solanum tuberosum

Summary Representative regenerated clonal plants from protoplast fusion of Solanum tuberosum L. and an atrazine resistant biotype of S. nigrum L. were studied to ascertain which plastomes each clone contained. DNA was isolated from fractionated chloroplasts, restricted with DNAases XHO-1, BGL-1, PVU-2 and BAM-H1, and the fragments separated by agarose gel electrophoresis for comparison. No difference could be found between resistant and susceptible biotypes of S. nigrum with all four enzymes. XHO-1, BGL-1, BAM-H1 differentiated between S. nigrum and S. tuberosum. All atrazine resistant regenerants, despite plant morphology, had the plastid DNA pattern of S. nigrum while all sensitive ones resembled S. tuberosum, even the subclone 38S having a S. nigrum morphology and chromosome number.     

Isoenzyme Patterns of Solanum Nigrum and the Cybrid Plant containing S. Nigrum Genome and S. Tuberosum Plastome

Transfer of chloroplasts from Solanum tuberosum into S. nigrum cell resulted in an atrazine sensitive cybrid plant. The shoots of this cybrid were bleached under atrazine stress. The cybrid displayed identical isoenzyme patterns that have been found in S. nigrum, and thus nuclei of the cybrid plant cells did not integrate any chromosomes or chromosome fragments from S. tuberosum nuclei. Under atrazine stress, differences in the isoenzyme expression were found in both the cybrid and original plants. In the peroxidase patterns, POX-4 was detected while POX-1 disappeared. Esterase patterns were less influenced, EST-4 was expressed in both plants under the influence of atrazine. Atrazine treatment of both cybrid and original plant shoots had specific effects on carbonic anhydrase, malate dehydrogenase, and glutamate oxaloacetate transaminase. The number and/or the staining intensity of bands corresponding to these isoenzymes decreased under atrazine stress more in cybrid plants (atrazine sensitive) than in S. nigrum (atrazine resistant).     

Protective effects of proline against cadmium toxicity in micropropagated hyperaccumulator, Solanum nigrum L.

Solanum nigrum is a newly discovered Cd-hyperaccumulator. In the present study, the protective effects of proline against cadmium toxicity of callus and regenerated shoots of S. nigrum are investigated based on a high frequency in vitro shoot regeneration system. Proline pretreatment reduces the reactive oxygen species levels and protects the plasma membrane integrity of callus under cadmium stress, and therefore improves the cadmium tolerance in S. nigrum. Inductively coupled plasma mass spectroscopy analysis shows that exogenous proline increases the cadmium accumulation in callus and regenerated shoots of S. nigrum. Further analysis indicates that the improvement of cadmium tolerance caused by proline pretreatment is correlated with an increase of superoxide dismutase and catalase activity and intracellular total glutathione content. The interaction between proline and enzymic or non-enzymic antioxidants is discussed.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

Biological containment of potato (Solanum tuberosum): outcrossing to the related wild species black nightshade (Solanum nigrum) and bittersweet (Solanum dulcamara)

The biological containment of the potato (Solanum tuberosum) was assessed by establishing the crossability of this tuberous crop with the related wild non-tuberous species in The Netherlands, black nightshade (S. nigrum) and bittersweet (S. dulcamara). To circumvent crossability barriers, genotypes with different ploidy number were employed and crosses were performed under different environmental conditions. S. dulcamara was shown to be incongruent with potato at all ploidy levels, while S. nigrum displayed unilateral incompatibility. If S. nigrum was emasculated and used as female, fertilization by potato pollen resulted in berry set and seed development. Emasculation of S. nigrum was essential in this cross, because analysis of the fertilization process demonstrated that this species is highly self-compatible and potato pollen was outcompeted by pollen of S. nigrum. The hybrid seeds derived from this cross did not mature and appeared not to be viable. By application of the technique of embryo rescue of immature embryos, hybrid plants could be obtained. However, these hybrid plants proved to be sterile. These data demonstrate that gene flow by pollen dispersal from potato to its most common wild relatives in Western Europe is highly unlikely. The potato is thus a naturally contained species in this part of the world.     

Differences in Nitrogen Metabolism During Growth of Plants from Aged Potato (Solanum tuberosum L.) Meristems

The effect of advanced meristem age on growth and accumulationof plant nitrogen (N) in potato (Solanum tuberosum L.) was studied.Etiolated plantlets, excised from sprouted, single-eye-containingcores from 7 and 19-month-old seed-tubers, were transplantedinto aerated nutrient culture. Rates of shoot and root dry matterand shoot soluble-N (which included nitrate-N) accumulationwere similar for plants from both meristem ages over a 30 dinterval of log-linear growth. The rate at which nitrate-N accumulatedwas consistently 17 per cent higher in shoots from 19-month-oldcompared to those from 7-month-old meristems. However, accumulationof free amino-N and soluble protein-N were 21 and 15 per centlower, respectively in shoots from 19-month-old meristems. Abuild-up of shoot nitrate, along with lower rates of accumulationof amino-N and soluble protein-N, suggests a lower capacityfor nitrate reduction during early growth of plants from oldermeristems. Furthermore, these effects can be attributed to age-inducedchanges in the meristem or bud tissue as the plants were separatedfrom the tuber tissue initially in the study. Long-term ageingof seed-potatoes apparently affects changes within meristemsthat translate into a lower capacity to accumulate reduced formsof nitrogen during early plant growth. Potatoes (Solanum tuberosum L.), meristem age, nitrogen metabolism, plant growth potential     

Strategy differences of two potato species in response to nitrogen starvation. Do plants have a genetic switch for nitrogen signalling?

Survival responses to nitrogen starvation are well known in micro‐organisms but little studied in plants. To construct a framework for study of the plant responses, we investigated the strategy differences of tubers from two closely related potato species. Solanum tuberosum conserves tuber nitrogen by inhibiting shoot growth, but S. phureja mobilizes tuber nitrogen to grow shoots, flowers and seeds. Genetic analysis of progeny from S. phureja–haploid S. tuberosum crosses uncovered segregation of a single dominant gene for the S. tuberosum inhibition strategy. Within S. tuberosum, haploid progeny closely resembled their tetraploid parents, suggesting strong genetic control of the inhibition. Growth of the inhibited shoots was proportional to sub‐optimal levels of added nitrate, and was triggered by exogenous gibberellic acid (GA₃). These observations support the notion that potato plants can closely tie shoot growth to ambient nitrogen levels – probably by a root–shoot nitrogen signal transduction pathway, and that this can be overridden by emergency mobilization of nitrogen reserves, perhaps by GA signalling from the tuber. Furthermore, genes for such developmental switches can be identified by classical genetic analysis of closely related species, such as S. tuberosum and S. phureja, that exhibit opposite survival strategies.     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Cd Hyperaccumulative Characteristics of Australia Ecotype Solanum Nigrum L. and Its Implication in Screening Hyperaccumulator

A pot culture experiment was used to determine the differences in uptake characteristics of a cadmium hyperaccumulator Solanum nigrum L. discovered in China, an ecotype from Melbourne, Australia and a non-hyperaccumulator Solanum melogena. Australian ecotype was not significantly different to the China ecotype. In particular, Cd concentration in leaves and shoots of S. nigrum collected from Australia were 166.0 and 146.3 mg kg^?1 respectively when 20 mg kg^?1 Cd spiked, and were not significantly different to the ecotype imported from China which had 109.8 and 85.3 mg kg^?1 respectively, in the stems and leaves. In contrast, the tolerance of the eggplant to Cd was significantly less than the two S. nigrum ecotypes. Although some morphological properties of S. nigrum collected from Australia were different from that of the plants collected from China, Cd hyperaccumulator characteristics of two ecotypes were similar. The results suggested that the tolerance and uptake of Cd may be a constitutive trait of this species.     

Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum and S. bulbocastanum

The mesophyll protoplasts were isolated from the Solanum tuberosum (S. tbr) clones of different ploidy level (4x Bzura cv., 2x H-8105, and 2x ZEL-1136) as well as from the wild species: S. bulbocastanum (S. blb, 2x) and two accessions of S. nigrum (S. ngr, 6x). Additionally, the protoplasts were isolated from the cell suspensions of Bzura cv. and H-8105 clone. The conditions of protoplast isolation as well as the media for their culturing and regeneration, were selected and optimized for the studied genotypes. For mesophyll protoplasts, the shooting calli were produced by all the cultured protoclones except that of S. bulbocastanum. The shoots excised from the protoplast-derived calli developed into whole plants in all the studied potato clones but only in one accession of S. nigrum, i.e. S. ngr var. gigantea. As for suspension-cell-derived protoplasts, only H-8105 clone produced the regenerative type of calli, though normal shoots could not be obtained. The regenerative capacity of the protoplasts isolated from leaves and cell suspensions is compared and discussed. We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Compound amino acids added in media improved Solanum nigrum L. phytoremediating CD-PAHS contaminated soil

Cd hyperaccumulator Solanum nigrum L. was a promising plant used to simultaneously remediate Cd-PAHs combined pollution soil through its extra accumulation capacity and rhizosphere degradation. This article compared the strengthening remediation role of cysteine (Cys), glycine (Gly) and glutamic acid (Glu) with EDTA and TW80. The results showed that the addition of 0.03 mmol L^?1 Cys, Gly, and Glu didn't significantly impact (p < 0.05) shoot biomass of S. nigrum, but obviously increased Cd concentration. Therefore, Cd capacity (µg pot^?1) in shoots of S. nigrum was significantly increased (p < 0.05) by 37.7% compared to the control without reagent added. At the meantime, the PAHs degradation ratio in rhizoshpere was increased by 34.5%. Basically, the improving role of Cys, Gly, and Glu was higher than EDTA and TW80. The main reasons of enhanced the accumulation of S. nigrum to Cd might lie in the addition of Cys, Gly, and Glu which reduced pH and increased extractable Cd concentration in rhizosphere and phytochelatines (PCs) concentration in leaves. As for the degradation of PAHs in rhizosphere, increased microorganism number might be play important role.     

In vitro regeneration of Solanum nigrum with enhanced solasodine production

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm⁻³ benzylaminopurine and 1.5 mg dm⁻³ kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm⁻³ indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.     

Characterisation of somatic hybrids between Solanum tuberosum and its frost-tolerant relative Solanum commersonil

 Somatic hybrids between three dihaploid Solanum tuberosum (2n=2x=24) genotypes and the frost-tolerant, diploid, relative Solanum commersonii (2n=2x=24) were analysed for variation in morphological traits, fertility and frost tolerance. The somatic hybrids were more vigorous than their parents and in many ways resembled a tetraploid S. tuberosum. All of the hybrids flowered profusely, although the male fertility was largely dependent on the S. tuberosum genotype used. In one hybrid combination all plants were both male- and female-fertile, while in the other two combinations the majority of plants were male-sterile but female-fertile. The somatic hybrids showed an increase in direct frost tolerance when compared with the dihaploid S. tuberosum parents, and to a varying extent had gained the capacity to cold acclimate. These somatic hybrids will be used in breeding programmes involving repeated cycles of anther culture and somatic hybridisation. Received: 20 May 1997 / Accepted: 12 June 1997     

Selection of hydroxyproline-resistant cell lines from Solanum Tuberosum L. Callus. II. Plant regeneration and frost tolerance of regenerated plants

The calluses of two hydroxyproline-resistant lines (D20-1 and D30-1) of Solanum tuberosum L. were transferred to a solidified MS medium containing 1.0 mg/I IAA, 2.0 mg/l zeatin, 40.0 mg/l adenine sulphate, 1 g/l casein hydrolysate, 20 g/l sucrose and 10 g/l agar for plant regeneration. The shoot regeneration was only achieved from the callus of line D20–1. Regenerated shoots exhibited morphological variability. The degrees of frost tolerance were higher in the leaves of the regenerated plants compared with the leaves of the non-selected control plants, but lower than that of the callus from which they were regenerated.     

Nitrogen Nutrition and Cytokinin Activity in Solanum tuberosum

In water culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of continuous nitrogen nutrition (constant supply of NO₃^?) and discontinuous nitrogen nutrition (interruption of NO₃^? supply, i. e., nitrogen withdrawal for 6 days) on the endogeneous cytokinin level in the roots, shoots and exudate of decapitated plants was studied. Harvests took place at intervals of 3 days. The chlorophyll formation test (cucumber cotyledons) and soya callus test were used to determine the cytokinin activity. With continuous nitrogen, the cytokinin activity decreased slightly with time in both roots and shoots but rose in the exudate. With discontinuous nitrogen, the nitrogen withdrawal led to a temporary, pronounced increase in cytokinin activity in the roots; at the same time, the cytokinin activity in the exudate decreased sharply. It is assumed that this temporary increase in cytokinin activity in the roots is a reflection of increased meristem activity in the roots. In the shoots, the cytokinin activity decreased during the nitrogen withdrawal period. These nitrogen-induced changes in cytokinin activity in the roots and shoots of potato should presumably have an important influence on the physiological age of the shoot, with all its consequences in the further development of the plant. Zeatin riboside was likely the main cytokinin component involved.     

A Preliminary Study on the Effects of Nitrogen Supply on the Growth In Vitro of Nine Potato Genotypes (Solanum spp)

Cultures of nine potato genotypes (seven Solanum tuberosum oneS. sparsipilum and one S. oplocense genotypes) were examinedfor their response to growing on medium containing either 60mol m^–3, 40 mol m^–3 or 20 mol m^–3 nitrogen.Genotypes differed in their response to nitrogen. Reducing thenitrogen regime tended to produce taller plants with longerinternodes, shoots had larger leaves but contained less chlorophyll.No change in fresh weight or number of nodes was observed. Genotypex nitrogen interactions were significant for chlorophyll content,shoot length and internode length. Results suggest that thechanges observed were as a result of changes in the total nitrogenlevel rather than changes in the ammonium : nitrate ratio. Thisstudy suggests that for certain potato genotypes, nitrogen levelsin MS medium are too high for producing desirable microplantsin terms of leaf area and shoot length Key words: Solanum tuberosum, S. sparsipilum, S. oplocense, micropropagation, morphogenesis     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

Cadmium accumulation in relation to organic acids in leaves of Solanum nigrum L. as a newly found cadmium hyperaccumulator

The influence of various cadmium concentrations on organic acid levels in leaves of the Cd hyperaccumulator, Solanum nigrum L. and a closely related species, Solanum melongena L., were investigated. In particular, the relationship of organic acids with Cd accumulation in the two plants was investigated. The results showed that Cd accumulation in the shoots of S. nigrum was significantly higher than that of S. melongena. The accumulation of Cd in the leaves of S. nigrum ranged from 2.0 to 167.8 μg g⁻¹ dry weight (DW), but only from 1.2 to 64.0 μg g⁻¹ DW in S. melongena. Solanum melongena was considerably less tolerant to Cd than S. nigrum. Approximately 20% of the total Cd in S. nigrum leaves was water-soluble, suggesting that some accumulated Cd was associated with water-soluble compounds such as organic acids. Malic acid in the leaves of S. nigrum was the most abundant organic acid [up to 115.6–145.7 μmol g⁻¹ fresh weight (FW)], but this acid was not significantly affected by the Cd concentration in soil. However, the level of malic acid in S. melongena plants was much lower, only 16.3–75.4 μmol g⁻¹ FW. The significant positive correlations between total Cd and water-soluble Cd concentrations and both acetic and citric acid concentrations in the leaves of S. nigrum were observed. In contrast, there was no correlation between concentrations of the two acids and Cd concentrations in the leaves of S. melongena. These results indicated that acetic and citric acids in the leaves of S. nigrum might be related to its Cd hyperaccumulation.     

Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum

Leaf protoplasts of two wild species, Solanum nigrum var. gigantea (S. ngr gig) and S. bulbocastanum Dun. (S. blb), were electrofused with leaf protoplasts of two diploid potato clones, H-8105 and ZEL-1136, respectively, in order to confer the late blight-resistance from the wild species to the cultivated potato. The S. ngr gig mesophyll (+) H-8105 mesophyll combination resulted in regenerants of mostly normal ngr phenotype. Two regenerants from this combination were proved to be true hybrids by RAPD analysis but they rooted poorely in vitro and did not survive the transfer to soil. The S. ngr gig (+) H-8105 fusion combination was also performed with H-8105 cell suspension derived protoplasts enabling an easy identification of interspecific fusants on basis of their intermediate morphology. From the S. ngr gig mesophyll (+) H-8105 cultured cell combination, many abnormal shoots were regenerated. The two lines which survived had normal ngr phenotype but the presence of tuberosum (tbr) genome in those regenerants was not confirmed by RAPD analysis. No plants with tbr phenotype were obtained from both of S. ngr gig (+) H-8105 combinations. On the contrary, when S. blb mesophyll protoplasts were electrofused with ZEL-1136 mesophyll protoplasts, all regenerated plants had tbr phenotype, indicating much lower morphogenetic potential of S. bulbocastanum in comparison with that of S. nigrum var. gigantea. However, the hybridity of those regenerants has not been confirmed by RAPD analysis with two different primers. The efficiency of the applied fusion procedure and analysis of the regenerants is discussed.