In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

In vitro conservation of Mandevilla moricandiana (Apocynaceae): short-term storage and encapsulation–dehydration of nodal segments

In vitro conservation of Mandevilla moricandiana was performed by slow-growth storage and encapsulation–dehydration. For slow-growth storage, half- and full-strength Murashige and Skoog (MS) medium and Woody Plant Medium, with or without sorbitol, mannitol, or glucose, were used to test the development of nodal segments and maintenance of plant viability after 6 mo. Recovery was performed using MS medium. The basal medium and carbon source did not interact, and only the carbon source had a significant effect on slow-growth storage and recovery. Sorbitol and glucose, individually or in combination, promoted development of plants with a low multiplication rate during the slow-growth period and a high multiplication rate during the recovery period. For encapsulation–dehydration, nonencapsulated and sodium alginate-encapsulated nodal segments were evaluated to determine their viability after storage at different temperatures. Nonencapsulated nodal segments gave 16.6% recovery after 60 d at 25°C. The effects of preculturing encapsulated nodal segments in MS medium with 0.4 or 0.75 M sucrose followed by dehydration were also tested. Capsules precultured for 48 h in the presence of 0.40 M sucrose and dehydrated to 40% moisture content showed 93.3% recovery. These conditions were then used to store capsules under different temperatures and for different lengths of time. The precultured capsules showed ca. 30% recovery after storage for 30 d at 4°C. Well-developed plantlets regenerated from encapsulated, stored nodal segments were rooted and acclimatized successfully, with 100% survival.     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

Formation of onion bulblets in vitro and viability during medium-term storage

 The influences of light conditions, sucrose and ethylene on in vitro formation and storability of onion (Allium cepa L.) bulblets were studied in various accessions. Light, sucrose and ethylene influenced bulb formation. Storability was primarily enhanced by a high sucrose concentration (100 g/l) in the culture medium. The bulbing process was characterised by changes in bulbing ratio, leaf length, number of leaves and leaf development time. The viability of bulbs after 1 year of in vitro storage at low temperatures was determined by their growth reaction in subsequent subcultures, growth after transfer into the greenhouse and tetrazolium staining. Sufficient sprouting of bulblets previously stored at –1  °C demonstrated the possibility of storing them in a low-temperature, slow-growth culture. Received: 8 June 2000 / Revision received: 5 October 2000 / Accepted: 5 October 2000     

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

 Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml^–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l^–1 mannitol and 40 g l^–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml^–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium. Received: 2 September 1998 / Revision received: 20 November 1998 / Accepted: 12 December 1998     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

Isolation and culture of suspension-derived protoplasts ofBeta vulgaris L.

Sugar beet protoplasts (Beta vulgaris L.) were isolated from hypocotyl-derived suspension cells and cultured on modified Murashige and Skoog medium supplemented with 5 μM naphthaleneacetic acid (NAA) and 2 μM 6-benzyl-aminopurine (BAP). Protoplasts were plated at a density 1.0–1.5×10⁵ cm⁻³ and incubated in either liquid medium or in medium solidified by 1.2% agarose, at 25°C in the dark. Comparison of two methods of culture unequivocally showed the second to be superior. Immobilizing the protoplast in agarose proved to be essential for obtaining sustained protoplast division and reproducible colony formation. The plating efficiency after two weeks of culture, expressed as the percentage of protoplasts which developed to form colonies, reached 40%. Subsequent subcultures of protoplast-derived callus to regeneration media with different concentrations of BAP (5 μM, 10 μM, 20 μM, 30 μM) resulted in very good callus proliferation at the three lowest concentrations, although organogenesis was not achieved.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Effects of “media osmotic agents” on the growth and morphogenesis ofActinidia deliciosa cv “hayward” callus

Summary The influence of various osmotic agents (carbohydrates) on the morphogenesis and growth of callus ofActinidia deliciosa cv Hayward was studied. Sucrose supported the highest level of growth and the lowest was supported by the sugar alcohols used in the experiments (glycerol, mannitol, sorbitol). The growth and survival of callus were evaluated with different osmotic sources in media containing glycerol, mannitol, or sorbitol at a concentration of 0.2M each for an extended period of eight subcultures (360 days). Two crucial points were identified: until the third subculture (135 days) the vitality seemed to be elevated; whereas the fifth (225 days) seemed to be a “point of no return” for tissues grown in glycerol and mannitol. Pretreatment with osmotic carbohydrates was shown to increase the magnitude of the morphogenetic events of callus subsequently transferred to sucrose-containing medium. Callus grown in the presence of mannitol and sorbitol showed a similar frequency of morphogenetic response. With respect to the media containing glycerol and sucrose, these induced more intense regeneration of shoots. When glycerol was present in the medium, however, we observed a synchronization of the morphogenetic response. Our results suggest that it is possible both to stimulate and to synchronize morphogenesis utilizing osmotic conditioning subcultures.     

Control of Embryoid Development in Tissue Cultures of Celery

Scanning electron microscope photographs of the embryoids showedglobular embryoids attached to the surface of aggregates inliquid medium and also some free floating. The surface structureof the unattached embryoids was very irregular, but, with thechange to polarized growth in the heart and torpedo forms, thesurface of the embryoid became smoother. The stage of developmentof the embryoids could be controlled by modifying the compositionof the medium to the extent that the majority of the embryoidsin the culture were either globular or torpedo forms. One ofthe most effective compounds in controlling development was2,4–dichlorophenoxy acetic acid (2,4–D). At high2,4–D concentrations, embryogenesis in the callus wasrestricted to the globular stage and after two subcultures itwas totally repressed, while after ten subcultures the potentialfor embryogenesis was lost and could not be regained even aftersubculture on a normal medium. On the normal agar medium thecallus always continued to show embryogenesis, but when it wastransferred to liquid medium of the same composition, embryoidswere produced in the first subculture but the potential haddeclined by the third subculture, when only roots were produced,and after ten subcultures cell growth and all differentiationwas totally it hibited. However, in the first subculture inliquid medium, embryogenesis was sequential with the whole cultureprogressing from globular to torpedo forms. This was particularlyeffective when the callus inoculum had been maintained on ahigh 2,4–D concentration for the two subcultures priorto inoculation of the liquid medium. By making use of this sequentialchange in embryoid development, a large number of embryoidscould be obtained at any particular stage. Apium graveolens, celery, tissue culture, embryoids, 2,4–D     

Enhancement of embryogenic culture initiation from tissues of mature sweetgum trees

 Male inflorescences, female inflorescences, and leaves collected from dormant buds of three sweetgum (Liquidambar styraciflua) trees were tested for induction of somatic embryogenesis following treatment with thidiazuron, naphthaleneacetic acid (NAA) or different combinations of the two. Explants were placed into culture either within a few days after collection or following 2 months of storage at –15  °C. Although embryogenic cultures were obtained from all three trees, embryogenesis induction was strongly affected by genotype (source tree), with 100% of the staminate inflorescence explants from one tree producing embryogenic cultures in one experiment. Embryogenesis induction was also influenced by explant type, with staminate inflorescences up to five times more likely to produce an embryogenic culture than female inflorescences. No embryogenic cultures were obtained from leaf explants. While treatment with plant growth regulators was not required for embryogenesis induction from inflorescence explants, culture on medium with NAA alone resulted in the highest production of repetitively embryogenic cultures and cultures producing proembryogenic masses. Dormant buds stored for 2 months at –15  °C were still able to produce embryogenic cultures, although frozen storage decreased this ability by over one-half for staminate inflorescences. Received: 20 January 1999 / Revision received: 18 April 1999 / Accepted: 29 April 1999     

Establishment of adventitious root culture of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7 μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6 months with regular subcultures after 4 weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7 μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25 ml), culture period (4 weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2 g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

The role of calcium nutrition in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) microplants in relation to minimal growth over prolonged storage <Emphasis Type="Italic">in vitro</Emphasis>

The present study investigated the effect of different levels of Ca_[ext] (0.3, 3.0, 5.0, 7.0, 9.0 and 11.0 mM) on potato over minimal growth in vitro in relation to varying water stress levels and moisture vapour transmission regimes using ⁴⁵Ca as an isotopic tracer. Ca nutrition was substantially limited when the microplants were grown at enhanced water stress level (MS + 40 g l^-1 sucrose + 20 g l^-1 mannitol) under minimal growth. Ca_[ext] in excess of standard level (3.0 mM), however, resulted in a significant increase in Ca content in microplants. The differential Ca uptake in microplants in relation to water stress and moisture vapour transmission has been discussed in terms of transpiration stream and root pressure water flow under minimal growth. The study showed that poor microplant quality at standard Ca_[ext] over prolonged storage under minimal growth was due to limiting Ca nutrition, and this could be improved by using Ca_[ext]-enriched (5.0-7.0 mM) minimal growth medium for conservation of potato microplants. The role of high Ca_[ext] in reducing the phenotypic abnormalities such as vitrification, flaccidity, hyperhydricity, etc. in potato microplants over extended storage under minimal growth has also been discussed.     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers

Use of high levels of growth regulators during micropropagation results in undesirable clonal variability in important commercial crops such as banana. The present study investigated the effects of high levels of cytokinins on micropropagation in banana (genotype AAB), and the genetic stability of plantlets was assessed using RAPD and ISSR markers. Cytokinins, such as BA and kinetin were added to the routine shoot multiplication medium at concentrations up to 10 mg l⁻¹. After 12 weeks of culture involving three subcultures, the maximum number of shoot buds were produced in cultures receiving either 5 mg l⁻¹ BA (80 shoot buds) or 4 mg l⁻¹ kinetin (62 shoot buds). Certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro. To check the genetic stability, RAPD and ISSR profiles of micropropagated plantlets obtained from different cytokinin-treatments were compared with control microplants maintained on MS medium as well as the field-grown mother plant. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands. Thus a total of 17,400 bands were generated showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation in all the plantlets analysed. Based on these results a protocol for high rate shoot multiplication was worked out leading to uniform shoot production.     

Microtuber formation in micropropagated Jerusalem artichoke (Helianthus tuberosus)

Clonal micropropagation of Jerusalem artichoke (Helianthus tuberosus L.) was initiated from axillary meristems of lateral shoots of field-grown plants on medium with MS salts, 2% sucrose, 1 mg l-1 thiamine-HCl, 1 mg l-1 IAA and 0.6% agar. Plantlets were cut into nodal sections and used for subsequent subcultures and for microtuber induction. Microtubers were induced from axillary meristems on medium with half-strength MS salts, 8% sucrose and 0.5 mg l-1 BA in darkness at 18 °C. They had near to 30% of dry matter. Microtubers resumed growth in light room at 23 °C after 4–6 months of cold storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Cunila galioides,a popular medicinal plant of south Brazil

Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.     

Induction of thermotolerance in potato microplants by acetylsalicylic acid and H2O2

Potato microplants propagated as nodal explants were subjected to heat treatments in vitro similar to those employed in the thermotherapy step of virus eradication procedures. Low concentrations (10^-6-10^-5 M) of acetylsalicylic acid (ASA) in the culture medium improved (by 3.7-fold) tolerance of a 5-week high-temperature (35C) treatment. Furthermore, tissues subcultured on to ASA-free medium following several weeks of growth on ASA were more thermotolerant (by 3.8-fold) of a 7 week 35C treatment, and (by 38-fold) of a 15 h 42°C heat-shock. Stems of microplants grown on ASA contained significantly less catalase activity and higher levels of H2O2 than controls. Explanting and heat treatment, however, reduced catalase activity to similar levels in ASA-treated and control microplant tissues. To investigate whether H2O2 could be involved in signal transduction during the induction of thermotolerance, nodal explants were incubated for 1 h in H2O2 (0.1-50 mM), and then cultured under standard conditions. The microplants that grew from the H2O2-treated explants showed concentration-dependent decreases in stem height, but were significantly more thermotolerant than controls, more than 1 month after the H2O2 treatment. Thus, thermotolerance induced in these conditions was extremely stable. It is concluded that both salicylate and H2O2 treatments can induced thermotolerance in this system.Keywords: Acetylsalicylic acid, heat-shock, hydrogen peroxide, potato, microplant, thermotolerance.