Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).     

Expression of a wild-type GBSS gene introduced into an amylose-free potato mutant by Agrobacterium tumefaciens and the inheritance of the inserts at the microsporic level

Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology.     

Pollen markers for gene-centromere mapping in diploid potato

The utility of two pollen genetic markers for estimating the extent of meiotic recombination between the centromere and a marker gene was tested in 2n pollen of diploid potato clones. One of these markers was the distal locus amylose-free (amf) on chromosome 8 and the other was the isozyme locus alcohol dehydrogenase (Adh-1) on chromosome 4. In the case of the amf locus, the gene-centromere distance was estimated in a normal synaptic and a desynaptic genotype. In both cases the genetic analysis was confined to: (1) a direct estimation of the phenotypic (blue vs red) segregation ratios in FDR (first-division restitution) 2n pollen and (2) a classification of the 4 x progeny from 4x (nulliplex amf) x 2x (Amf/amf) crosses into duplex, simplex and nulliplex classes. The recombination frequency between the centromere and the amf locus in the normal synaptic genotype B92-7015-4 corresponded to a gene-centromere distance of 48.8 cM, whereas this distance amounted to 13.3 cM in the desynaptic genotype RS93-8025-1. Hence desynapsis reduced crossing-over by 73%. The observed genetic distance of 48.8 cM in the normal synaptic clone, B92-7015-4, is the highest gene-centromere distance reported so far in potato and this could be explained on the assumption of absolute chiasma interference. For the Adh-1 locus, it was found that heterozygous 2n pollen grains could be detected in pollen samples of the diploid clones, because of the occurrence of a heterodimeric band of the isozyme. Unlike the amf locus, the genecentromere distance for the Adh-1 locus was estimated only on the basis of the duplex, simplex and nulliplex classes in the progenies from 4x (nulliplex Adh-1 ² )x B92-7015-4 (Adh-1 ¹ /Adh-1 ² )crosses and was found to be 19.4 cM. Because the accurate positions of centromeres in relation to other loci are not available in the existing genetic maps of potato, which are saturated with molecular markers, halftetrad analysis is a promising additional approach to the basic genetics of this crop.     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Cytological analysis of tetraploid hybrids between sweet potato and diploid Ipomoea trifida (H. B. K.) Don.

Summary Tetraploid F₁ hybrids between Ipomoea batatas, sweet potato (2n = 6x = ca. 90), and diploid (2n = 2x = 30) I. trifida (H. B. K.) Don. showed various degrees of fertility reduction. The present study aimed to clarify its causes by cytological analysis of meiotic chromosome behavior in the diploid and sweet potato parents and their tetraploid hybrids. The diploid parents showed exclusively 15 bivalents, and the sweet potato parents exhibited almost perfect chromosome pairing along with predominant multivalent formation. Their hybrids (2n = 4x= 57–63) formed 2.6–5.0 quadrivalents per cell, supporting the autotetraploid nature. The meiotic aberratios of the hybrids were characterized by the formation of univalents, micronuclei, and abnormal sporads (monad, dyad, triad, and polyad). The causes underlying these aberrations were attributed in part to the multivalent formation, and in part to a disturbance in the spindle function. Three hybrids showing serious meiotic aberrations were very low in fertility. The utilization of the sweet potato-diploid I. trifida hybrids for sweet potato improvement is described and, further, the role of interploidy hybridization in the study of the sweet potato evolution is discussed.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Complementation of the amylose-free starch mutant of potato (Solanum tuberosum.) by the gene encoding granule-bound starch synthase

Summary Agrobacterium rhizogenes-mediated introduction of the wild-type allele of the gene encoding granulebound starch synthase (GBSS) into the amylose-free starch mutantamf of potato leads to restoration of GBSS activity and amylose synthesis, which demonstrates thatAmf is the structural gene for GBSS. Amylose was found in columella cells of root tips, in stomatal guard cells, tubers, and pollen, while in the control experiments using only vector DNA, these tissues remained amylose free. This confirms the fact that, in potato, GBSS is the only enzyme responsible for the presence of amylose, accumulating in all starch-containing tissues. Amylose-containing transformants showed no positive correlation between GBSS activity and amylose content, which confirms that the former is not the sole regulating factor in amylose metabolism.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species

Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.     

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.     

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F₁ populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F₁ populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F₁ populations. The mode of inheritance of the R10 allele could not be deduced as only very few F₁ hybrids bearing R10 were obtained. Linkage analysis in two F₁ populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

Segregation patterns of isozyme loci and microsatellite markers show the diploidy of African yam Dioscorea rotundata (2n=40)

The cultivated yam species Dioscorea rotundata (2n=40) has been considered by most authors as a tetraploid species with a basic chromosome number of ten. In this paper, we analysed the segregation of two isozyme loci and six microsatellite markers in the progeny of a self-fertilised monoecious plant. For the eight markers, segregation patterns could be explained by only two genetic models: diploidy or tetraploidy with two null alleles. Given the nature of studied markers, the most parsimonious hypothesis was that the parental plant was diploid. These results, data from a diversity survey and results of other authors led to the conclusion that D. rotundata is a diploid species.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

GBSS T-DNA inserts giving partial complementation of the amylose-free potato mutant can also cause co-suppression of the endogenous GBSS gene in a wild-type background

The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylosefree (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.