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; )     

Effect of kinetin on tuber formation on isolated stolons of Solanum tuberosum L. cultured in vitro

Kinetin-induced tuber formation was investigated with regardto the role of temperature, sucrose concentration, time of kinetinapplication, and inhibitors of protein and nucleic acid biosynthesis.Low temperatures (15°C and 20°C) failed to promote tuberformation in the absence of kinetin, and at 15°C K-inducedtuber formation was partially prevented. Similarly, sucrose concentration per se did not promote tuberformation, however, K-induced tuber formation required a 6%or greater concentration of sucrose in the medium. Stolons preincubated in K prior to incubation on a basal mediumwithout K failed to form tubers but tubers were formed if theywere incubated on a basal medium with K. In order to inducetuber formation, K is only required in the basal medium for3–4 days. Thereafter tuber formation can progress unimpairedon a basal medium only. The inhibitors of protein and nucleic acid synthesis (ACTD,PFA, 2-TU, CHL, 5-FUDR) delayed tuber formation but failed toinhibit the process. The results are discussed in relation to the possible existenceof a tuber forming hormone related to cytokinins and the possibleeffect of temperature on its action. The possibility that tuberformation may be independent of protein and nucleic acid synthesisis also discussed. ¹Present address: Plant Hormone and Regulator Pioneering ResearchLab., U.S. Dept. Agric., Crops Res. Div., Beltsville, Md., U.S.A. (Received October 13, 1969; )     

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.     

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     

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.     

Numerical taxonomic studies on Solarium L. section Solarium (Maurella)

Population differentiation was studied in 32 accessions belonging to Solarium L. section Solarium (Maurella); these accessions represented 20 species of four differing ploidy levels. A study of discordance demonstrated that the species exhibit considerable inherent discordant variation; it also provided an estimate of the relative variabilities of characters within these populations, and the consistency with which they differentiate populations.
Hierarchic clusters, obtained using the single-link cluster method, confirmed the relative importance of the different classes of characters used for differentiation within this section. Similarly, the hierarchic clusters generally supported earlier taxonomic decisions.
The use of a non-hierarchic cluster method provided the most interesting data on differentiation within the section Solarium, particularly for genome relationships of the polyploid taxa. This clustering pattern fitted well with known genome relationships, while also suggesting putative ancestors for other allopolyploids; suggestions deriving retrospective support from the hierarchic dendrograms. The progenitors suggested by tliese two clustering methods supported data derived from orthodox taxonomy, comparative morphology, hybridisation studies, the artificial synthesis of polyploids and seed protein band patterns.     

An ultrastructural study of bruising in stored potato (Solarium tuberosum L.) tubers

Potato tubers of the cultivar Pentland Dell were investigated for ultrastructural changes following impact. Studies were carried out on tubers after 12 and 24 wk of storage using a falling bolt delivering 0.7 J of energy. Ultra-thin sections were examined by light and transmission electron microscopy. Tubers stored for 12 wk exhibited no visual bruise formation, whereas visible bruising resulted following impact of tubers stored for 24 wk. An ultrastructural sequence of events during bruising was established as (1) a collapse of intracellular compartmentation, (2) increased ribosomal and mitochondrial abundance within the cytoplasm, (3) increased density of cytoplasm adjacent to the cell wall and surrounding amyloplasts, and (4) the development of melanin in bruised cells. These observations are discussed in relation to tuber turgor and physiology during storage.     

Reduction of abscisic acid content and induction of sprouting in potato,Solanum tuberosum L., by thidiazuron

The effect of [(N-phenyl-N-1,2,3-thidiazol-5-ylurea)] (thidazuron) on sprouting of potato (Solanum tuberosum L.) tubers and the role of ABA in bud break and subsequent bud growth were studied. Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) from the peel of potato tubers. The ELISA results were also validated by gas chromatography-electron capture detector and confirmed by gas chromatography-mass spectrometry and by a lettuce hypocotyl bioassay. The degree of rest in the tubers was associated with ABA content in the peel. Basal portion (where tuber was attached to mother plant) contained the highest amount of ABA. Thidiazuron reduced ABA content and induced potato tuber sprouting. Exogenously applied ABA stimulated growth of buds that had emerged from dormancy.On leave from the Department of Horticulture, South China Agricultural University, Guangzhou, People's Republic of China.     

The effect of abscisic acid on flowering inChenopodium rubrum L.

Flowering in the short-day speciesChenopodium rubrum L. was stimulated by treatment with abscisic acid (ABA) in concentrations from 1×10^?3 M to 1×10^?7 M only in plants partly induced by two dark periods. We assume that ABA weakens the inhibitory effect of continuous light (similarly as do some other substancese.g nucleic acid inhibitors) and thus enables the expression of the evoked floral state. ABA was ineffective in promoting flowering in photoperiodically non-induced plants.     

The Effect of Vanadate on Phosphate Transport in Aged Potato Tuber Tissue 2Solarium tuberosum)

The effect of orthovanadate on the uptake of phosphate by agedpotato tuber tissue was investigated to study the relationshipwith plasma membrane ATPase activity. Vanadate inhibited therate of phosphate uptake by aged discs with a maximum effectat 500 µM (58% inhibition). When vanadate was added tothe ageing medium for 24 h, the subsequent rate of phosphateuptake was also markedly decreased (68% inhibition). The resultsshow that the inhibition by vanadate was not due to enhancedleakage of phosphate nor to a non-specific toxic effect. Furthermore,complementary experiments with erythrosin B and molybdate wereconsistent with the hypothesis that vanadate acts specificallyon the plasma membrane ATPase and that this enzyme is involvedin maintaining the driving force for active uptake of phosphate(via co-transport with protons) by storage cells of potato tubers. Key words: Proton-phosphate co-transport, vanadate, plasma membrane ATPase, unloading     

Interaction between ethylene,abscisic acid and gibberellic acid in elongation of rice mesocotyl

Summary Combined application of ethylene, abscisic acid and gibberellic acid produced marked partly synergistic stimulation of mesocotyl growth of japonica rice in darkness.     

Effect of jasmonic acid on developmental morphology during in vitro tuberization of Dioscorea alata (L.)

A developmental morphology study was performed during different stages of in vitro yam microtuber formation on both hormone-free medium (HF) and medium supplemented with 10 µM of jasmonic acid (JA). The axillary protuberance, considered as the first morphological evidence for microtuber formation, became visible after one-week of culture in JA-medium and after three weeks of culture in HF-medium. In addition, the formation of the axillary protuberance in JA-cultures preceded the stem elongation, whereas in HF-medium stem elongation was visible before the formation of the axillary protuberance. Tuberization improvement in medium supplemented with JA is likely to be the result of morphogenetic modifications occurring during the early stages of microtuber formation. At the molecular level, 2D-PAGE analysis of HF- and JA-cultures allowed the identification of four differentially expressed polypeptides, including two putative pathogenesis-related proteins and one putative glutathione S-transferase. For JA-cultures, three additional differentially expressed polypeptides were identified.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Stimulation of potato (Solanum tuberosum L.) tuberization by semicontinuous liquid medium surface level control

A procedure for the mass propagation of potato (Solanum tuberosum L.) tubers using a laboratory scale jar fermentor is described. Tuberization was clearly suppressed when the shoots were completely submerged in the liquid medium. Tubers were mainly formed at the medium surface. When shoots were cultured by the semi-continuous medium surface level control method, in which the medium surface level was raised or lowered throughout the culture period, tubers were induced and developed in every area in the jar fermentor. Tubers propagated by this method contained about 18 % (w/w) dry matter, slightly less than in field grown tubers, but most of the tubers weighing more than 0.2 g(FW) sprouted under room condition, without any acclimatization, during 3 month after the culture.     

Enhancement of Tuberization of Axillary Shoot Buds of Potato (Solarium tuberosum L.) Cultivars Cultured In Vitro

Factors controlling growth and tuberization of axillary budsin shoots of plantlets of potato (Solarium tuberosum L.) culturedin vitro were investigated. Correlative inhibition restrainedgrowth and tuberization of the axillary buds. Exposure of intactplantlets for various periods (4 to 48 h) to low (2 or 12C)or high (30 C) temperatures as comparedto 18C, did not alleviatecorrelative inhibition. Removal of the apical part of the shoot,the roots or both was generally ineffective Elevating sucroseconcentration from 30 to 80 g dm^–3 promoted tuberizationon axillary buds, and the cytokinin 6-(-dimethylallylamino)purine (2iP), alleviated correlative inhibition and enhancedtuberization in intact plantlets. In the whole plantlet mostof the tubers were formed on the basal nodes, however, oncecorrelative inhibition was eliminated by the dissection of theshoot to single node sections, tubers were formed on every axillarybud. The single most effective factor inducing tuberizationin single node sections was the growth retardant ancymidol,an inhibitor of giberellin biosynthesis. Key words: Potato, Solanum tuberosum L., in vitro tuberization, correlative inhibition     

Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers     

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     

Ultrastructural study of the effect of abscisic acid on cell elongation in plant cells

Summary An ultrastructural study is presented of the effect of abscisic acid (ABA) on the elongation of cells in the cortical area ofAllium cepa roots between 3 and 4 mm from the tip (the area of differentiation). If we take the size ultimately attained by these cells as our point of reference, the elongation is inhibited to the extent of about 2.6 times. The dictyosomes appear to be as numerous and as active in treated as in untreated cells. There is a demonstrably marked increase in the thickness of the longitudinal cell wall due to the equal supply of Golgi vesicles to cover a smaller surface, and the postulate is advanced that ABA mainly affects the absorption of water and diminishes intracellular swelling, to which cell elongation is linked.     

Effect of water stress on metabolism of abscisic acid in Populus robusta× schnied and Euphorbia lathyrus L.

Abstract. Dihydrophaseic acid (DPA) has been identified in leaves from Euphorbia lathyrus L and Populus robusta x schnied. The formation of DPA from abscisic acid (ABA) was demonstrated using ¹⁴C-ABA. Measurements of ABA, DPA and phaseic acid (PA) concentrations were made in Euphorbia subjected to drought and waterlogging and in Populus subjected to rhythmic water stress. The results are consistent with the proposition that ABA concentrations are controlled by de novo biosynthesis and by metabolism via the PA /DPA pathway. The findings are discussed in relation to effects of the type of stress and its duration and to behaviour during stress relief.