Changes in enzyme activity during elongation and tuberization of stolons of Solanum tuberosum L. cultured in vitro

Isolated stolons of Solanum tuberosam L. were cultured in vitroin the presence of kinetin, which induced tuber initiation orgibberellic acid which inhibited initiation. Progressive changes in enzyme activity, at the locus of tuberinitiation, were monitored at specified intervals. In the presenceof kinetin soluble invertase activity was decreased with timewhereas gibberellic acid (GA) evoked substantial increases inactivity. Acid phosphatase activity was enhanced by GA but changedonly slightly under tuber inducing conditions. Enzymic hydrolysisof glucose-6-phosphate and fructose-6-phosphate decreased duringthe course of tuber induction but increased in the presenceof GA. In contrast hydrolysis of 3'AMP was stimulated undertuber inducing conditions. GA evoked substantial increases in peroxidase activity duringthe initial stages of incubation while under tuber inducingconditions increased activity was only observed after 5 days.Substantially higher levels of IAA oxidase activity were associatedwith tuber initiation. RNase activity decreased with time undertuber inducing conditions but showed an initial stimulationin the presence of GA. These results are discussed with referenceto the role of these enzymes in carbohydrate metabolism andthe regulation of hormone levels. (Received February 29, 1972; )     

Cytokinin-cytokinin-Induced tuber formation Tuber Formation on Stolons of Solanum tuberosum

Kinetin-induced tuberization of isolated stolons was investigated with regard to accumulation of labelled kinetin, starch, protein and nucleic acid synthesis. Using kinetin-8-¹⁴C, it was found that more labelled meterial appeared at the locus of tyber formation that in other parts of the stolon. Substantial accumulation was evident before visible signs of tuber formation. The basal portion of the stolon also accumulated substantial amounts of labelled material. Kinetin-treated stolons showed extensive starch accumulation which was not evident in gibberellic acid-treated or untreated stolons. Starch accumulation occurred before any visible sign of tuber formation. There was no marked differences in the ability of the apical 0.5 cm of kinetin-treated and untrated stolons to incorporate ¹⁴ C uridine into RNA and ¹⁴C leucine into TCA precipitable protein. From these results it was concluded that kinetin-induced tuber formation may not involve the synthesis of new proteins. It is suggested that kinetin may be regulating tuber formation by suppressing starch hydrolase activity and stimulating starch synthetase activity whereas gibberellic acid inhibits tuber formation by promoting starch hydrolase activity or by suppressing starch synthesizing enzymes.     

Effect of abscisic acid on elongation and kinetin-induced tuberization of isolated stolons of Solarium tuberosum L.

The response of isolated stolons cultured in vitro, to abscisicacid (ABA) has been studied in the presence and absence of kinetin(6-furfurylaminopurine). ABA alone in concentrations from 7.5x 10^–4 mM to 7.5 x 10^–2 mM, inhibited stolon elongationbut failed to promote tuber initiation. In the presence of kinetin,ABA at concentrations of 3.0 x 10^–2 and 7.5 x 10^–2mM markedly inhibited kinetin-induced tuber initiation and stolonelongation, but at 7.5 x 10^–4 and 7.5 x 10^–3 mMABA did not prevent tuber initiation. When stolons were incubated on a medium containing kinetin andlater transferred to one containing ABA with or without kinetin,the inhibitory effect of ABA decreased appreciably as the timeof incubation on kinetin is increased. The results are discussed in relation to the role of ABA inthe inhibition of nucleic acid and protein synthesis and theinteraction with cytokinins and the possible effect of ABA onkinetin uptake, transport and accumulation at the locus of action. (Received February 26, 1969; )     

Cytokinins antagonize the jasmonates action on the regulation of potato (Solanum tuberosum) tuber formation in vitro

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.     

Effect of carbon dioxide and ethylene on tuberization of isolated potato stolons cultured in vitro

Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C₂H₄ which is by itself also inhibitory of tuberization. Furthermore, C₂H₄ inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C₂H₄. The antagonistic actions of CO₂ and C₂H₄ on tuberization are discussed.     

Effect of physiological age of mother tuber and number of subcultures on in vitro tuberisation of potato (Solanum tuberosum L.)

The physiological age of mother tubers (Solanum tuberosum L. cv. Kennebec) used as a source of material influenced kinetin-induced in vitro tuberisation. Tuberisation significantly increased with physiological age. Kinetin- or ancymidol-induced tuberisation, plantlet and microtuber dry weight decreased with increasing number of subcultures. Single-node segments obtained from tubers stored for more than 9.5 months at 4 °C showed increased kinetin-induced tuberisation rates and earlier tuberisation than those obtained from younger tubers. For any physiological age, material may be safely multiplied using node propagation until the third subculture and bioassayed for tuberisation without variation in the response. Received: 8 July 1996 / Revision received: 28 March 1998 / Accepted: 28 March 1998     

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.     

Hydrolysis of sucrose within isolated vacuoles from Solanum tuberosum L. tubers

The soluble acid invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 ± 0.05) × 10⁻² μl, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2), the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors, confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory role of the reaction products previously proposed for in-vitro assays. Received: 21 July 1997 / Accepted: 31 August 1997     

Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.     

Induction of alcohol dehydrogenase in explants of potato tuber (Solanum tuberosum L.)

Plant Cell Reports - During callus formation a huge increase in alcoholdehydrogenase activity was observed in potato tuber tissue discs. Callus formation was no prerequisite for this increase;...     

Increase in ribonuclease activity following mechanical damage to leaf and tuber tissues of Solanum tuberosum L.

Summary Major increases occurred in the capacity of damaged potato leaf and tuber tissues to hydrolyse ribonucleic acid whilst relatively minor increases were found in the activity of acid phosphomonoesterase and acid phosphodiesterase. Partial purification of homogenates by gel filtration on Sephadex G-100 revealed that much of the increased capacity to degrade ribonucleic acid following damage was due to increased ribonuclease activity. Although appreciable differences in the elution patterns of tuber homogenates subjected to gel filtration were observed before and after the breaking of dormancy the increased ribonuclease activity following damage was a constant feature. Actinomycin D had a relatively small effect on preventing these increases in phosphate-ester hydrolase activities whilst the effect of cycloheximide was very pronounced. Isopycnic equilibrium centrifugation experiments, using deuterium oxide as a density label, provided no evidence that the increased enzyme activity following damage was due to synthesis of new enzyme.     

Callus tissue of Solanum tuberosum L. cultured in the presence of the pyrethroid deltamethrin

The effect of Decis (deltamethrin as active ingredient) on callus tissue of Solanum tuberosum L. cv. Désirée was studied. Decis is an agrochemical currently used on field grown potato plants to control the Colorado beetle, a potato pest. Deltamethrin added to the culture medium interferes with the behaviour of callus tissue. After 5 h of culture, the level of total proteins was higher in treated tissue than in the control, and SDS-PAGE showed that deltamethrin promoted the increase of some soluble proteins. After 24 h and 14 days of culture, the level of total proteins became similar in both treated and control material. This similarity between control and treated tissues, after 14 days of culture, occurred as a result of the treatment with deltamethrin which caused a decrease of soluble proteins, but an increase in insoluble proteins whereas the opposite was observed for control callus. SDS-PAGE of both soluble and insoluble proteins showed that only quantitative dissimilarities occurred. This longer treatment also increased the chlorophyll content. Ultrastructural study of the cells revealed that tissue callus cultured for 14 days in the presence of deltamethrin had plastids containing a more developed membranous system, with a higher number of grana and with more compartments than in control cells. Deltamethrin also promoted the abundance of vesicles associated with dictyosomes. This response of the callus tissue to Decis added to the callus medium parallels the behaviour of potato plants treated with this agrochemical under field conditions (Fidalgo, Santos & Salema, 1993).     

An integrated view of the hormonal regulation of tuber formation in potato (Solanum tuberosum)

The hormonal regulation of the consecutive steps in the formation of tubers on the potato plant ( Solanum tuberosum L.) is described and discussed. An integrated view of the complex regulation of the initiation and growth of stolons and tubers is presented, with special emphasis on the commonly observed lack of synchronization of the various steps in tuber formation within a plant.     

Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The capacity of four bacterial strains isolated from productive soil potato fields to solubilize tricalcium phosphate on Pikovskaya agar or in a liquid medium was evaluated. A bacterial strain was selected to evaluate in vitro capacity of plant-growth promotion on Solanum tuberosum L. culture. Bacterial strain A3 showed the highest value of phosphate solubilization, reaching a 20 mm-diameter halo and a concentration of 350 mg/l on agar and in a liquid medium, respectively. Bacterial strain A3 was identified by 16S rDNA analysis as Bacillus pumilus with 98% identity; therefore, it is the first report for Bacillus pumilus as phosphate solubilizer. Plant-growth promotion assayed by in vitro culture of potato microplants showed that the addition of bacterial strain A3 increased root and stems length after 28 days. It significantly increased stem length by 79.3%, and duplicated the fresh weight of control microplants. In this paper, results reported regarding phosphorus solubilization and growth promotion under in vitro conditions represent a step forward in the use of innocuous bacterial strain biofertilizer on potato field cultures.Key words: Bacillus sp., phosphorus soluble, Pikovskaya agar, potato rhizosphere, plant growth promoting rhizobacteria     

In vitro microtuberization in potato (Solanum tuberosum L.) cultivars

Mechanism of microtuberization in three elite cultivars kufri badhsha (KB), kufri chandramukhi (KCM) and kufri jawahar (KJ) of potato was studied. Sprouts of all the three cultivars were used to obtain in vitro shoot cultures. MS medium supplemented with chlorocholine chloride was found to be most suitable for all the cultivars. Maximum tuberization was obtained under incubation conditions of continuous darkness at 20 degrees +/- 1 degrees C. The highest number of micro-tubers per plant basis was produced under continuous darkness and KCM recorded the highest yield of micro-tubers and was found significantly superior to KJ and KB.     

Effect of Calcium on Tuberization in Potato (Solanum tuberosum L.)

Tuberization response of single-node leaf cuttings from induced potato plants (Solanum tuberosum L.) was reversed when pretreated with 5 millimolar ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) + 50 micromolar calcium ionophore (A23187) and resumed when transferred to a CaCl(2)-containing medium. Tuberization was inhibited by LaCl(3), chlorpromazine, and trifluoperazine at 5 to 10 micromolar. These results suggest a role for calcium in the tuberization process.     

Inheritance of electrophoretic variants of tuber proteins in Solanum tuberosum haploids

Soluble tuber proteins were separated by discontinuous polyacrylamide gel electrophoresis on vertical slabs. Banding patterns of proteins stained with Coomassie Blue in 7.5% acrylamide gels (pH 4.3) were few and distinctive for haploids (2n = 2x = 24) derived from several cultivars (2n = 4x = 48). Katahdin and Chippewa haploids have only three different banding patterns for the eight fastest moving bands. The haploids have either the parental pattern (all eight bands) or one of two complementary banding patterns (four bands). The frequency of these patterns among the haploids indicates that the eight bands are controlled by one locus which is duplex (A¹A¹A²A²) in the parents. Haploids with the genotype A¹A² have eight bands. A¹A¹ haploids have four bands, and A²A² have the other four bands. Tawa haploids have in equal numbers either the eight (A¹A²) or four (A²A²) band pattern. Thus the genotype of Tawa is A¹A²A²A². The control of four bands by one allele could be explained by assuming that these alleles are involved in posttranslational modification or assembly of one or two protein species. Another explanation is that pseudoalleles or redundant genes produce the groups of protein bands. The eight proteins studied apparently are of similar molecular weight but differ in charge.