Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary 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.     

Nuclear-organelle interaction in Solanum: interspecific cybridizations and their correlation with a plastome dendrogram.

Summary Alloplasmic compatibility, namely the functional interaction between the nuclear genome of a given species with plastomes and chondriomes of alien species, is of considerable relevance in plant biology. The genus Solanum encompasses a wide spectrum of species and is therefore suitable for a study of this compatibility. We thus chose the nuclear genome of Solanum tuberosum (potato) and organelles (chloroplast and mitochondria) from 14 other Solanum species to initiate an investigation of intrageneric nucleus/organelle interactions. An assessment of the diversity of the chloroplast DNAs from these 15 species resulted in the construction of a plastome dendrogram (phylogenetic tree). In parallel we extended a previous study and performed ten additional fusion combinations by the donor-recipient protoplast fusion procedure, using potato protoplasts as recipients and protoplasts from any of ten other Solanum species as donors. We found that two fusion combinations did not yield cybrids and that the chloroplasts of S. polyadenium and the mitochondria (or mitochondrial components) from S. tarijense could not be transferred to cybrids bearing potato nuclei. In general, there is a correlation, albeit not perfect, between the cybridization data and the plastome dendrogram. These results furnish valuable information toward future transfer of plasmoneencoded breeding traits from wild Solanum species into potato. This information should also be useful for the planning of asymmetric protoplast fusion between potato and wild accessions for the improvement of pathogen and stress resistance of potato cultivars.     

Somatic hybridization in the genus Solanum: S. tuberosum and S. brevidens

Somatic hybrid plants were regenerated from fused mesophyll protoplasts of an albino potato (Solanum tuberosum spp. tuberosum) variant and Solanum brevidens, a non-tuber bearing species which is sexually incompatible with S. tuberosum. These somatic hybrid plants represent the first example of direct hybridization between potato and members of the taxonomic group Etuberosa, and offer the potential for introgressing valuable germplasm from Solanum species outside the sexually compatible range into a worldwide crop 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.     

Ethylene and in vitro culture of potato: suppression of ethylene generation vastly improves protoplast yield,plating efficiency and transient expression of an alien gene

Ethylene release by potato shoots cultured in closed boxes was suppressed by the addition of silver thiosulfate to the culture medium. Shoots cultured in the presence of silver thiosulfate produced appreciably more tissue and the yield of protoplasts per unit tissue mass was vastly increased, resulting in an 8 fold increase of protoplast yield per shoot. Exposure of pricked leaves to macerating enzymes facilitated ethylene generation. Leaves of shoots which were previously cultured in silver thiosulfate containing medium generated much less ethylene than leaves from control shoots and this generation could be further reduced by the addition of acetylsalicylic acid during maceration. The capability of polyethylene glycol treated potato protoplasts to produce microcalli was vastly increased by the addition of silver thiosulfate during exposure of protoplasts to Ca(NO₃)₂ following the polyethylene glycol treatment. Similarly, when a plasmid (pCAP212) containing an expressible gene for chloramphenicol acetyltransferase was introduced into potato protoplasts through a polyethylene glycol treatment, the transient expression of acetyltransferase was very much increased by the addition of a short incubation of the protoplasts with silver thiosulfate.Abbreviations AOA (aminooxy)acetic acid - ASA acetylsalicylic acid - AVG aminoethoxyvinyl-glycine - CAT chloramphenicol acetyltransferase - MV methyl viologen - PEG polyethylene glycol - STS silver thiosulfate     

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis,S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.Abbreviations BAP 6-benzylaminopurine - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalenacetic acid On leave from: Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brasil     

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species,Sonneratia alba,S. caseolaris and S. ovata: development of a bioassay method for allelopathy,the protoplast co-culture method

A bioassay method for allelopathy, the ‘protoplast co-culture method’ was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl₂, MgSO₄ and CaCl₂) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3 % sucrose and 0.6–0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the ‘sandwich method’ with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.     

Biological performance of Myzus persicae and Macrosiphum euphorbiae (Homoptera: Aphididae) on seven wild Solanum species

Resistance level of seven accessions of wild Solanum species (Solanaceae) to Myzus persicae and Macrosiphum euphorbiae (Homoptera: Aphididae) was evaluated by measuring survival and fecundity during sleeve cage experiments and population growth on whole plants in a controlled environment. The survival was lowest on the Solanum circaeifolium spp. capsicibaccatum, Solanum pinnatisectum and Solanum trifidum accessions for M. persicae and on the S. circaeifolium spp. capsicibaccatum, Solanum okadae and S. trifidum, accessions with M. euphorbiae. Plant species significantly influenced the fecundity of both aphid species. Aphid population growth on whole plants was negatively affected by the age of the plant, but generally followed the levels of net reproductive rate on different plant species observed during the sleeve cage experiment. The population of M. persicae varied among the seven wild potato accessions with the lowest growth rates on S. circaeifolium spp. capsicibaccatum, Solanum polyadenium, Solanum tarijense and S. trifidum. The number of M. euphorbiae also varied among accessions but the smallest population was collected from S. polyadenium. The results can be used to identify sources of resistance to aphids within those accessions already known to be resistant to the Colorado potato beetle. This study highlights the difficulties involved in developing a high-throughput screening test for aphid resistance compatible with a potato breeding programme.     

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK₃-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK₃-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.     

Introgression of resistance to root-knot nematodes from wild Central American Solanum species into S. tuberosum ssp. tuberosum

 Crossing experiments were conducted to introduce resistance to the root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from various polyploid Central American Solanum spp. into the cultivated potato, S. tuberosum ssp. tuberosum. The most effort was put into producing tetraploid hybrids through inter-EBN (Endosperm Balance Number) crosses. From the crosses of tetraploid S. tuberosum (4 EBN) with tetraploid S. stoloniferum and S. fendleri (both 2 EBN), few seeds were derived that led to viable plants. In vitro culture of immature seeds also yielded several hybrid plants. From crosses of diploid S. tuberosum (2 EBN) with hexaploid S. hougasii (4 EBN) four hybrids were obtained through in vitro culture. Backcrosses were made with selected hybrids and a variable number of seeds was produced depending on the hybrid genotype. The successful introgression of resistance into backcross populations is shown. A scheme is presented for the introgression of traits at a tetraploid level from allotetraploid Solanum species into autotetraploid S. tuberosum through sexual crosses. The relevance of EBN for potato breeding is discussed. Received: 25 November 1996 / Accepted: 14 February 1997     

Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding

The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.Abbreviations BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) - 2,4-d dichlorophenoxyacetic acid     

Molecular,cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens

Summary Fifty-eight somatic hybrid plants, produced both by chemical (11) and electrical fusion (47) of protoplasts of dihaploid Solanum tuberosum and S. brevidens, have been analysed by molecular, cytological and morphological methods. The potentially useful euploid plants constituted 34% of the total, of which 20% were tetraploid and 14% hexaploid; the remainder were aneuploid at the tetraploid, hexaploid and octoploid levels. Analysis of chloroplast DNA showed that 55% of hybrids contained chloroplasts from S. brevidens and 45% from S. tuberosum. Hexaploids, the products of three protoplasts fusing together, were analyzed with specific DNA probes, and this revealed that nuclear genome dosages could be either 21 S. tuberosumS. brevidens, or vice-versa. Chloroplast types of hexaploids were not influenced by nuclear genome dosage, and all six possible combinations of genome dosage and chloroplast types were found amongst tetraploids and hexaploids. To examine the morphology of the hybrid population and its possible relation to the chromosome number and chloroplast DNA type, 18 morphological characteristics were measured on greenhouse-grown plants and analyzed by principal component and canonical variate analyses. Both analyses showed that nuclear ploidy has the most prominent influence on the overall morphology of the hybrids. Differential parental genome expression in the morphology of the hybrids is discussed. These results provide useful data on the range of genetic combinations that can be expected to occur amongst somatic hybrid plants.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary 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.     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Role of host‐plant selection in resistance of wild Solanum species to Macrosiphum euphorbiae and Myzus persicae

Damage to potatoes by Macrosiphum euphorbiae (Thomas) and Myzus persicae (Sulzer) (both Hemiptera: Aphididae) can be controlled through plant resistance. We used ethological experiments and electric penetration graph (EPG) analysis to evaluate the role of host selection in the previously assessed resistance levels of Solanum accessions: Solanum circaeifolium Bitter subsp. capsicibaccatum (Cárdenas) (PI210036), S. chomatophilum Bitter (PI243340 and PI310990), S. okadae Hawkes & Hjert. (PI458367), S. oplocense Hawkes (PI473368), S. pinnatisectum Dunal (PI186553), S. polyadenium Greenm. (PI230463), S. tarijense Hawkes (PI414150), and S. trifidum Correll (PI255538), to M. euphorbiae and M. persicae. Through multivariate analysis, we grouped behavioural variables into factors, which we related to host selection behaviours, and then evaluated whether factors varied between each accession and the susceptible S. tuberosum. None of the factors obtained by ethological experiments differed among accessions. Four of six and three of five factors obtained through EPG varied among accessions for M. euphorbiae and M. persicae, respectively, and were used to suggest resistance characteristics. The resistance to M. persicae of both S. chomatophilum accessions was associated with pathway activity disturbance. Solanum tarijense and S. polyadenium resistance to M. persicae resulted from leaf surface characteristics, which may be trichomes. Solanum oplocense and S. trifidum resistance to M. euphorbiae resulted from the wound response system, whereas S. pinnatisectum resistance may stem from nutritionally unbalanced or toxic phloem sap. Solanum polyadenium resistance to M. euphorbiae was phloem‐based. Solanum circaeifolium ssp. capsicibaccatum resistance to M. persicae, and the resistance of PI243340 S. chomatophilum and S. tarijense to M. euphorbiae were not related to host selection and therefore were presumably due to physiologically active compounds.     

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.     

Plant regeneration from in vitro culture of anthers of Solanum chacoense Bitt. and interspecific diploid hybrids S. tuberosum L. x S. chacoense Bitt.

Summary Production of plants from cultured anthers of Solanum chacoense clone IP 33, of its interspecific diploid hybrids with S. tuberosum clones IP 354 and IP 372, and of a complex Solanum hybrid containing in its genome S. ajanhuiri is reported. Genotypic differences were found to influence both the induction phase and the regeneration process. Hybrids derived from clone IP 354 of S. tuberosum were much more responsive in culture than hybrids from clone IP 372. Altogether, 507 plants were regenerated and 309 were cytologically analyzed. Of these, 52% were haploid, 47% diploid and 1% mixoploid or tetraploid. A number of diploid plants probably originated from unreduced microspores and some genetic consequences of this event are discussed.