Gibberellic acid and the inhibition of aerial tuberisation in Solanum tuberosum L.

The sensitivity of aerial and subterranean tuberisation to photoperiod was studied in potato (Solanum tuberosum cv. Aracy). Although photoperiodic sensitivity varied with the position along the stem, all buds could be induced to develop tubers under SD. Gibberellic acid (GA₃) applied to induced (30 short days) cuttings inhibited the photoperiodic effect. No tubers were formed and orthotropic shoots developed instead. The GA₃ caused a reduction in starch content in induced buds, lowering it to the same level as found in long-day treated plants. However, -amylase activity of buds of induced plants was not affected by GA₃, suggesting that GA₃ does not inhibit tuberisation by promotion of starch hydrolysis.     

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     

Somatic hybridization of amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) by electrofusion

Summary Intraspecific somatic hybridization between amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) has been carried out following electrofusion of protoplasts. In initial analytical electrofusion experiments (1 mm electrode separation) optimal fusion conditions were determined by changing the fusion medium (addition of Ca and/or spermine) and the electrical parameters. Subsequently, in large scale experiments, cell suspension protoplasts of aec-1, a variant resistant to AEC, were fused with the same type of protoplasts of 5mt-26 or 5mt-27, both variants resistant to 5MT and cross-resistant to 3 FT. After an extensive selection procedure only somatic hybrid lines of aec-1 + 5mt-26 were obtained. The resistance traits of aec-1 and 5mt-26 were expressed fully, indicating that the variant characters involved are transmitted dominantly. Quantitative examination of the free amino acid content revealed characteristics of both the parental cell lines in most of the somatic hybrids. However, initially selected double resistant colonies from fusions of aec-1 + 5mt-27 lines appeared not to be somatic hybrids.Abbreviations AEC S-aminoethylcysteine - 3FT 3-fluorotyrosine - 5MT 5-methyltryptophan     

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 and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.     

Effect of photoperiod on in vitro tuberization of potato (Solanum tuberosum L.)

Single-node cuttings of potato cultivars Jemseg, Katahdin, Russet Burbank and Superior were cultured on a multiplication medium containing MS salts and no growth regulators. Cultures were exposed to 8 h (SD) and 16 h (LD) photoperiodic regimes. The subsequent plantlets were excised and single node cuttings from each photoperiodic regime were placed under SD or LD on a second medium containing growth regulators which promoted tuberization. Production of microtubers was strongly influenced by genotype and by photoperiodic treatments. Superior produced stunted plantlets and some microtubers under SD conditions in the multiplication medium. The number of microtubers formed by Jemseg was not influenced by photoperiod. However, Katahdin and Russet Burbank formed fewer microtubers under LD-LD conditions compared to LD-SD, SD-SD and SD-LD regimes. Compared with the other regimes, LD-SD photoperiod generally promoted microtuber formation with larger diameters and significantly (p<0.05) greater fresh weight. The intensity of the tuberization stimulus was affected by daylength, and this was characterized by microtubers with secondary tubers, the growth of more than one axillary microtuber, and microtubers subtended by stolons. The maturity group of the potato cultivars and photoperiodic regime in vitro strongly influenced the production of microtubers. These results can be employed to adapt light regimes for multiplication and tuberization to the specific requirements for cultivars from different maturity groups, and thus increase the efficiency of potato multiplication protocols.     

Potato cold hardiness development and abscisic acid. I. Conjugated abscisic acid is not the source of the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

Free and conjugated abscisic acid (ABA) levels in stem-cultured plantlets of potato ( Solanum commersonii Dun, PI 458317) during cold acclimation were measured. The levels of free and conjugated ABA were measured by an enzyme immunoassay (EIA) with rabbit anti-ABA-serum. The use of immunoglobulin G fraction purified from rabbit antiserum and the methylated form of ABA resulted in an improved measuring range (0.01 to 10 pmol ABA) and precision (slope of logit-log plot, −1.35) of EIA, compared to the use of antiserum and free ABA. Estimates of the EIA were consistent with those resulting from a commercial EIA. Under a 4/2°C (day/night) temperature regime, the potato plantlets increased cold hardiness from −5°C (warm-grown control) to −10°C by the 7th day. During the same period, there were two transitory increases in free ABA, the first one three-fold from 1.5 to 5.3 nmol (g dry weight)⁻¹ on the 2nd day and the second one five-fold from 1.5 to 7.6 nmol (g dry weight)⁻¹ on the 6th day. Each increase in ABA concentration was followed by an increase in cold hardiness. There was no significant change in conjugated ABA content (4.2±0.6 nmol [g dry weight]⁻¹) throughout the cold acclimation period. The lack of an interrelationship between levels of free and conjugated ABA suggested that the transitory increase in free ABA during cold acclimation was not a result of the conversion of conjugated ABA. The increase in free ABA due to biosynthesis of ABA during potato cold acclimation is discussed.     

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.     

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.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

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     

Effects of ethylene on the tuberization of potato (Solanum tuberosum) cuttings

Ethylene, applied as ethephon, inhibited the elongation of etiolated, axillary potato shoots cultured in vitro and it stimulated radial growth along the whole length of these shoots. The same phenomena were observed when ACC, the precursor of ethylene, was added to the medium, whereas silver ions reversed these effects. However, tuber formation in vitro was suppressed by ethephon. This indicates a dual role of ethylene in the induction of tuber formation in potatoes: it had a positive effect by blocking the elongation of stolons and it suppressed tuber initiation.     

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.     

The effect of the recombinant human interleukin-2 Gene in potato (Solanum tuberosum cv. superior)

To examine the effect of the T-cell growth factor (human interleukin-2), we constructed a binary vector, pSSK-1, carrying the recombinant human interleukin-2 (rhlL-2) gene, and transferred it intoAsrobacterium tumefaciens. Using this construct, we then transformed potato explants(Solanum tuberosum cv. Superior), achieving 100% regeneration of shoots on a modified MS medium. Of the putative transformed shoots, 81% rooted and were selected on 200 ms/L kanamycin. Both Southern and northern analyses verified the transformation events. An ELISA test also indicated that the rhlL-2 protein was produced from rhlL-2-transformed potatoes. To determine whether this protein was biologically active in the potato cells, we performed a biological assay using the 11.-2 dependent cell line, CTLL-2. The suspension containing extract from the transformants showed significant proliferation of the 11.-2 dependent CTLL-2 cells, whereas cells did not proliferate in the nontransformed potato. We then grew the verified rhlL-2 transgenic potatoes in soil, and compared their performance with that of nontransgenic potatoes as well as those that had been transformed with GUS. Growth rates, as calculated from plant heights, were up to 50% higher than for either the nontrans-genic or the GUS-transformed potatoes. Similar patterns were found withArabidopsis thaliana plants treated in the same manner. All of these results suggest that rhlLo2 may function as a growth factor in potato.     

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens

Summary Leaves of the in vitro grown potato cultivars Bintje, Berolina, Desiree, and Russet Burbank were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For Russet Burbank, it was necessary to include AgNO₃ in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants.     

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.