Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Occurrence of jasmonic acid in organs of Solanum tuberosum L. and its effect on tuberization

The aims of this study were to demonstrate the endogenous presence of jasmonic acid (JA) in roots, stolons and periderm of new formed tubers, by means of bioassays, ELISA and GC-MS, and to test a microdrop bioassay using the leaflets of potato cuttings cultured in vitro. Our results confirm the existence of JA by bioassays and GC-MS in foliage, stolons, roots and tuber periderm.Abbreviations DW dry weight - GC-MS gas chromatography-mass spectrometry - JA jasmonic acid - MeOH methanol - SD short day     

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

Summary An amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.     

Protein kinase activity in different stages of potato (Solanum tuberosum L.) microtuberization

In vitro culture was used to study morphogenetic aspects of the tuberization process under controlled conditions in potato (Solanum tuberosum L.) plants. This paper accurately defines four stages of tuber development and their correlation to external morphological characteristics and histological structures. Protein kinase activity, assayed in each stage using Historic HAS as substrate, was differentially expressed during the tuberization process. Phosphorylation was maximum in the first stages of tuber formation. The incorporation of [³²PO₄ ^–1] to endogenous peptides containing serine/threonine amino acidic residues followed the same pattern that the protein kinase activity did.Abbreviations EDTA Ethylenediaminetetraacetic acid - EGTA ethylenebis (oxyethylenenitrilo) tetraacetic acid - MOPS 4-morpholine-propanesulfonic acid     

Molecular changes associated with tuberisation in Solanum tuberosum. Differential expression of α-tubulin isotypes

Changes in gene expression that occur in the stolon tips of potato ( Solanum tuberosum L.) cv. Record during tuberisation were investigated. Protein extracts from stolon tips at various stages in the tuberisation process were analysed by two-dimensional gel electrophoresis. A number of quantitative and qualitative changes in polypeptide composition accompanied the very early stages of tuberisation. In vitro translation of RNA extracted from stolon tips also revealed quantitative and qualitative changes associated with tuberisation. Immunoblotting of protein extracts with monoclonal antibodies raised against α- and β-tubulin showed quantitative changes in the relative level of β-tubulin, but not α-tubulin, as the stolon tips tuberised. Changes in the pattern of α-tubulin isotype expression were shown to occur at early stages in the tuberisation process.     

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.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

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.     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solanum tuberosum L

Freshly isolated mesophyll and suspensions-cell protoplasts of S. tuberosum cvs. Desiree and Maris Piper were cultured in different media i.e. modified MS, V-KM and MS-KM. Protoplast plating efficiencies were higher in MS-KM medium. Resulting protoplast-derived calluses were transferred either onto the medium of Bokelmann and Roest (1983) or that of Lam (1977) for shoot regeneration. Calluses derived from mesophyll cell protoplasts differentiated about 2 weeks earlier than calluses derived from suspension-cell protoplasts. Shoot initiation was also about 2 weeks earlier from calluses subcultured onto the former medium as compared to the latter.     

Aphid repellent sesquiterpenes in glandular trichomes of Solanum berthaultii and S. tuberosum

Exudate from glandular trichomes of the wild potato species, Solanum berthaultii Hawkes, and the cultivated potato, S. tuberosum L., contained volatile substances including sesquiterpenes when examined with gas chromatography/mass spectrometry procedures. Sesquiterpenes were the major volatile constituents of Type A trichome exudate but were a minor constituent of Type B exudate. Sesquiterpene mixtures of the two potato species differed qualitatively and quantitatively. -Caryophyllene and E--Farnesene were major components identified in Type A trichome exudate of the cultivated potato. Sesquiterpene mixtures of both potato species were repellent to the green peach aphid, Myzus persicae (Sulzer). The role of trichome sesquiterpenes as a determinant of aphid behavior on the two potato species and in the expression of host resistance is discussed.

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Résumé L'examen en chromatographie gazeuse et en spectométrie de masse a révélé la présence de substances volatiles contenant des sesquiterpènes dans l'exsudat des trichômes glandulaires des pommes de terre sauvage (Solanum berthaultii) et cultivée (S. tuberosum). Les sesquiterpènes étaient le principal composé volatile de l'exsudat de type A, tandis qu'ils n'étaient que faiblement représentés dans l'exsudat de type B. Les mélanges de sesquiterpènes des deux espèces différaient qualitativement et quantitativement. Le -Caryophyllène et le E--Farnesène constituaient les principaux composés identifiés dans l'exsudat de trichôme de type A de S. tuberosum. Les mélanges sesquiterpéniques des deux espèces ont repousée Myzus persicae Sulzer. La discussion a porté sur l'influence des sesquiterpènes des trichômes dans la définition du comportement des pucerons sur ces deux plantes et sur la manifestation de la résistance de l'hôte.

     

Characterization of proteins associated with self-incompatibility in Solanum tuberosum

Summary The gametophytic self-incompatibility system of Solarium tuberosum is controlled by a single locus, designated as the S-locus. Protein extracts from potato styles of defined S-genotypes have been analysed by two-dimensional gel electrophoresis, and found to contain a group of basic glycoproteins. Each genetically determined allele S ₁ to S ₄ was associated with the presence of one of a number of these polypeptides differing slightly in isoelectric points (in the range 8.3–>9.1) and/or apparent molecular weight (ranging from 23,000 to 29,000). Two abundant basic polypeptides, one of which is apparently not glycosylated, were present in all genotypes examined. Amino-terminal protein sequence determinations revealed homologies of the S. tuberosum stylar proteins S₂, S₃ and S₄ with SI-associated polypeptides from Nicotiana alata and Lycopersicon peruvianum. With an oligonucleotide generated to the potato-S₂ N-terminal protein sequence, it was possible to detect a style-specific RNA species of 920 nucleotides. The oligonucleotide also behaved as an allele-specific probe when hybridized to total RNA of different S-genotypes.     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Inheritance of three electrophoretically determined protein bands in potato (Solanum tuberosum L.)

Summary A modified polyacrylamide gel electrophoresis technique is employed to resolve proteins for use as biochemical gene markers in potato. Dominant, duplicate dominant and complementary gene action are three modes of inheritance that adequately explain the segregation of three respective protein bands in two generations of crossing within diploid Phureja X haploid Tuberosum families.Scientific Journal Seires Article 10,171 of the Minnesota Agricultural Experiment Station     

Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer

Summary Five diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.     

Photosynthetic enhancement and conductance to water vapor of field-grown Solanum tuberosum (L.) in response to CO2 enrichment

The photosynthetic responses of potato [Solanum tuberosum (L.)] to CO₂ enrichment were studied in open-topped field chambers. Plants were raised in 2.4 m² plastic enclosures over three growing seasons from 1996 to 1998. Plots were continuously fertilized with 1, 1.5 and 2 times ambient daytime CO₂. These were the low (L), medium (M) and high (H) CO₂ treatments, respectively. Tuber dry matter yields were increased 9 and 40%, respectively, in the M and H treatments compared to the L CO₂ treatment. Net photosynthesis (P _n ) and conductance to water vapor (g _s) of upper canopy leaves were measured at 1 or 2-week intervals at the growth CO₂ partial pressure and then P _n of plants in the L treatment was determined at 70 Pa CO₂ (L₇₀). Leaflet P _n rates averaged over all measurement dates were 28, 49 and 84% greater, respectively, in the M, H and L₇₀ CO₂ treatments, compared to plants in the L treatment. Changes of P _n in response to the L, M and H CO₂ treatments were proportional to increases of internal CO₂ (C_i) and at low leaf-to-air vapor pressure deficits mid-day g _s was inversely related to growth CO₂. The ratio of P _n at H compared to L₇₀ was 0.81 when averaged over all measurement dates. Leaf soluble protein, Rubisco protein and chlorophyll (a + b) levels were unaffected by CO₂ treatment. Total Rubisco activity was decreased by CO₂ enrichment in 1998, but percent activation was similar in the L, M and H plots. Leaf starch was increased but sucrose, glucose and fructose were unaffected by CO₂ treatment. The above findings indicated that a down regulation of P _n in response to elevated CO₂ was consistently observed in field-grown potato. This was attributed to a decrease of total Rubisco activity that was potentially due to the presence of inhibitory compounds bound to the active site of the enzyme. The amount of photosynthetic acclimation observed here did not preclude a persistent enhancement of P _n under the elevated CO₂ growth conditions.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Crossability between the diploid species S. circaeifolium subsp. circaeifolium (crc) and other diploid species, primarily diploid S. tuberosum subsp. tuberosum (tbr-2x), was studied. Forty-seven hybrids were obtained from crosses between crc as female parent and tbr-2x and some other species from series Tuberosa as male parents. Of these hybrids 17% were diploids; the other 83% were triploids, probably carrying two genomes of crc. Female fertility was sufficient to obtain offspring from backcrosses with the cultivated parent. Pollen stainability of the f₁ varied, and micro-pollen as well as unreduced pollen occurred. During meiosis of the diploids and triploids a rather high proportion of univalents was found, and in the triploids on average two or three trivalents per cell were found. All hybrids were resistant to Globodera pallida pathotypes 2 and 3, and 75% of the tested genotypes were highly resistant to Phytophthora infestans. Solanidine, tomatidine, tomatidenol, and demissidine glycosides were found in tubers of the hybrids. Comparisons with somatic hybrids between crc and tbr-2x are made. It is concluded that crc is a valuable Solanum species that can and should be included in potato breeding programs.