The process of in vitro rooting and the anatomical characters of in vitro and ex vitro leaves and roots of Prunus x davidopersica 'Piroska' were studied. Best rooting percentage (50%) and highest root number (5.0) was achieved in spring on a medium containing 0.1 mg/I NAA + 30 g/1 glucose. At the end... of rooting the parenchyma of the in vitro leaves was more loose and spongy, than during the proliferation period. In the first newly developed leaf of an acclimatised plant, the parenchyma was much more developed, contained less row of cells and less air space too, compared to the leaves developed in the field. The in vitro developed root had a broad cortex and narrow vascular cylinder with less developed xylem elements, but at the end of the acclimatisation the vascular system became dominant in the root.
Immature in vitro leaves showed similar structure of the mesophyll tissue to the immature field-grown (in vivo) leaves of Prunus x davidopersica `Piroska'. Mature leaf anatomical characteristics of in vitro plantlets differ from the field-grown plants. The mesophyll tissue of in vitro plantlets were thinner than the in vivo plants and consisted... of only one layer palisade parenchyma, the shape of the cells and the structure of spongy parenchyma basically differed from the field-grown plants. In the case of Sorbus rotundifolia similar anatomical differences were found both in vitro and in vivo as in the case of Prunus x davidopersica `Piroska'.
Effects of different cytokinin supplies including two types of aromatic cytokinins, such as benzyl-adenine, and 3-hydroxybenzyladenine applied at two different concentrations (2.0 and 6.0 μM) were studied on water and gas exchange parameters in in vitro apple leaves of ‘Royal Gala’ and ‘Freedom’ scions after 3 weeks of culture. Cytokin...in supply affected the stomatal conductance of water vapour, transpiration rate and the sub-stomatal CO2 concentration of leaves. Effect of cytokinin depended on its applied type and concentration, moreover on the apple scion. According to the results, the rate of CO2 exchange itself is not usable for characterization of function of photosynthetic apparatus of in vitro leaves. However, measurements of stomatal conductance of water vapour and transpiration rate seemed to be good indicators for stomatal behaviour of in vitro apple leaves.
Some experience or details are introduced in connection with the nutrient uptake of micropropagated fruit trees in the different phase of the in vitro or ex vitro development. It can be stated, that the plants during the micropropagation procedure are overfed. Special careful nutrient supply is necessary during the acclimatiza...tion.
The requirements for in vitro micrografting in apple are described. In vitro multiplicated shoots of cv. Royal Gala were the sources of rootstocks and scions after different pre-treatment, respectively. Oxidative browning of cut surfaces could be inhibited by the use of antioxidant mixture during grafting process. Sci...on base cut in v-shape was stuck by 1% agar-agar solution into the vertical slit of rootstock. There was no any displacement and the rate of fused and further developed grafts was 95 percent. Agar-agar between the rootstock and scion made the transport of different materials possible and hold the graft units together until the fusion took place. Fusion was proved also by histological studies. Some of in vitro micrografts were planted and acclimatisated and the survival was 100 percent.
During in vitro multiplication of Hosta ‘Gold Drop’, 20 g l-1 sucrose, 5.5 g l-1 agar and 4 concentrations (0.1-0.8 ml l-1) of Ferbanat L, Kelpak, Pentakeep-V were added to half-strength Murashige and Skoog (MS) basal medium. As compared to the control and other biostimulators, plants with lower per...oxidase activity, larger fresh weight, more, longer shoots and roots, larger leaves were developed on medium containing Kelpak. The best concentration was 0.4 ml l-1 for in vitro rooting, shoot formation, plant weight and ex vitro chlorophyll, carotenoid level, peroxidase activity. Pentakeep was the less efficient biostimulator, increasing of its concentration mostly decreased root and shoot values (furthermore, abnormal callus formation was observed, as non-wanted effect), chlorophyll content and sizes (length, width) of leaves, not only during in vitro propagation but also (as after-effect) acclimatization because of the high mortality and weakly developed survivor plants.
The experiment was carried out with in vitro propagated 'MM 106' apple-rootstock plantlets. The transpiration of the plantlets was examined, and the changes followed by SEM analysis.
Data about the transpiration intensity of the acclimatized plants, of its value under different conditions of relative humidity and influenced by ...the existence of roots, as well as by the degree of acclimatization are presented.
Leaves were also examined and it was found, that stomata of in vitro developed leaves closed slowly, and the number of stomata of newly developed leaves decreased.
It is also shown, that in vitro propagated roots, generally, lose their hairs during acclimatization, but these roots are all the same important, as new roots of full value develop out of them.
Micrografting was used in our experiments for establishment of in vitro culture from one rootstock (`JTE-F') and three scion cultivars (`Remo', 'Rewena' and `Reanda') of apple. Shoot tips of these cultivars were harvested from field and grafted onto in vitro rootstock cultivars. Their survival and development were studied. 42-...93% of shoot tips survived and developed further depending on cultivar. Impermanent browning of sticking agar-agar could be observed in 21-25% of the micrografts depending on cultivars but discolouration of agar-agar ceased within one week and did not cause any death of shoot tips. We used micrografting successfully for establishment of in vitro culture from cultivars, from which earlier with conventional methods the culture establishment was not possible because of hard tissue browning. However, further studies are necessary to ensure the survival and development of shoots after removing them from micrografts.
The effects of the ethylene precursor ACC and two inhibitors, AgNO3 and AVG, on root formation were tested in in vitro shoots of passion fruit (Passiflora Midis f.flavicalpa Deg.). The organogenic response was assessed on the basis of percentage of shoot-forming. roots, root number and length. The time course of eth...ylene production was also monitored. ACC inhibited root formation by delaying root emergence and increasine, callus formation at the basis of the shoots. In addition, ACC caused a marked increase in ethylene production, coupled to leaf chlorosis and senescence with lower rooting frequencies, number and length of roots. IAA supplementation increased ethylene production. Both ethylene inhibitors, AgNO3 and AVG, at appropriate concentrations reduced callus formation at the basis of shoots. AVG increased the number of roots per shoot, but drastically reduced length of differentiated roots. Regarding to leaf pigments, ACC promoted a marked reduction on carotenoids and total chlorophyll, whereas AVG and AgNO3 delayed explant senescence and pigments degradation, not differing from IAA supplemented and non-supplemented control treatments. The results confirm previous reports on the beneficial effects of ethylene inhibitors on in vitro rooting and suggest its reliability to be used as an alternative approach to evaluate sensitivity of Passiflora species to ethylene.
Explants excised from adult shrubs were surface sterilized and cultured on Murashige and Skoog (MS) basal medium in the presence of plant growth regulators (PGRs) at different concentrations. A high multiplication rate of 7.2-fold was achieved every four weeks on MS medium supplemented with 4.44 μM BA, 0.49 μM IBA and 0.58 μM GA3. Rooting wa...s achieved with 73% efficiency within 2-4 weeks on agar-gelled MS basal medium free of PGRs. Rooted plantlets were gradually acclimatized to field conditions over 5-6 weeks with 65% efficiency. For in vitro selection for salt tolerance, MS medium was supplemented with increasing concentrations of NaCl ranging between 25 and 1000 mM. This study has demonstrated that in vitro shoots could tolerate up to 600 mM NaCl with optimal growth at 200 mM, while higher concentrations of NaCl affected growth negatively. Growth and shoot number decreased with increasing NaCl concentration with all plantlets died at 1000 mM NaCl.
The aim of this study was first to test the in vitro effeicacy of some fungicides against brown rot of sour cherry, and secondly to evaulate the effectiveness of reduced spray programmes against brown rot in integrated and organic sour cherry orchards. In vitro efficacy of 7 fungicides (Champion 50 WP, Kocide 2000, Nordox 75 WG, Olajos rézkén..., Kumulus S, Rézkén, Rézoxiklorid) and another 6 fungicides (Score 25 EC, Efuzin 500 SC, Systane, Folicur Solo, Zato Plusz, Rovral) approved in organic and integrated production systems, respectively, were tested against brown rot of sour cherry. Altogether four spray programmes were performed i) standard integrated: sprays followed by forecasting systems during the season, ii) reduced integrated: sprays followed by forecasting systems but only 75% of the spray numbers used during the season-long spray programme, iii) standard oragnic: sprays applied every 7–14 days during the season and iv) reduced organic: 60% of the spray numbers used during the season-long spray programme. In vitro results showed that fungicides (with active ingredients of copper and sulphur) applied in organic production showed relatively high percent growth capacity of Monilinia fungus. Rézkén showed the highest and Kumilus S the lowest efficacy against brown rot. Fungicides applied in integrated production showed relatively low percent growth capacity of Monilinia fungus. Score 25 EC showed the highest and Rovral the lowest efficacy against brown rot. Field study showed that reduced spray programmes did not increase significantly brown rot incidence in the integrated field. However, brown rot incidence increased significanly (above 30%) in the reduced spray programme for the organic orchard.
In contrast to most angiosperms, Torenia contains a naked embryo sac and therefore has been considered since many years as an exciting model plant to study the double fertilization process of flowering seed plants. It is thus not surprising that the isolation of protoplasts from the female gametophyte has been reported already 20 years ago by M...ol, the isolation of megaspores and megagametophytes has been published by the authors of this manuscript in 1996 and in 1999. The isolation of the male gametophyte and of sperm cells was published by the authors in 2004. The isolation of viable Torenia sperm cells is a crucial part of the elaboration of an in vitro fertilization system. Torenia sperm cells were isolated from in vivo — in vitro cultured pollen tubes. In this system pollen tubes first grow inside a cut style then follow their elongation in a solid isolation medium. The medium contained agarose in order to detain pollen tube contents. Released sperm cells and enzymatically isolated egg cells were collected and handled using glass micropipettes and transmitted to an electrofusion apparatus or polyethylene glycol containing media for fusion probes.
Still a large amount of pesticides and spary applications are used in environmentally friendly fruit production systems; therefore, the aim of our study was first to test the in vitro effeicacy of some fungicides against a key apple disease (apple scab), and secondly to evaulate the effectiveness of reduced spray programmes against apple s...cab, powdery mildew and coding moth in integrated and organic apple orchards. In vitro efficacy of 7 fungicides (Champion 50 WP, Kocide 2000, Nordox 75 WG, Olajos rézkén, Kumulus S, Rézkén, Rézoxiklorid) and another 6 fungicides (Score 25 EC, Efuzin 500 SC, Systane, Folicur Solo, Zato Plusz, Rovral) approved in organic and integrated production systems, respectively, were tested against apple scab. Altogether four spray programmes were performed i) standard integrated: sprays followed by forecasting systems during the season, ii) reduced integrated: sprays followed by forecasting systems but only 75% of the spray numbers used during the season-long spray programme, iii) standard oragnic: sprays applied every 7–14 days during the season and iv) reduced organic: 60% of the spray numbers used during the season-long spray programme. In vitro results showed that fungicides (with active ingredients of copper and sulphur) applied in organic production showed relatively high percent growth capacity of the apple scab fungus. Rézkén showed the highest and Kumilus S the lowest efficacy against apple scab. Fungicides applied in integrated production showed relatively low percent growth capacity of the apple scab fungus. Score 25 EC showed the highest and Rovral the lowest efficacy against apple scab. Field study showed that reduced spray programmes did not increase significantly scab incidence in the integrated field. However, scab incidence increased significanly (above 30%) in the reduced spray programme for the organic orchard.Mildew incidence was low (below 5%) in both integrated and organic spray programmes. Mildew incidence on both shoots and fruits increased significanly in the reduced spray programme for the organic orchard. Incidence of codling moth damage was affected the most by standard vs. reduced spray programmes. Though incidence remained below 10% in the integrated plots, the incidence was significantly higher in the reduced spray programme compared to the standard programme. Similar results were obtained for organic spary programmes, but the incidence was 10 times higher and the differences among the two programmes were larger.,
Hyperhydricity was observed throughout in vitro multiplication phase of a Eucalyptus grandis clone. Ultrastructural approach of tissue and cell differentiation, izoenzyme patterns, binding protein (BiP) expression, and pigment content were performed. Hyperhydric tissues showed a reduction in cell wall deposition, reduction of ...membranous organelles, higher cell vacuolation, and more intercellular spaces than its normal counterpart. Additionally, several vesicles were present in hyperhydric cells suggesting the occurrence of organelle autophagy by autophagic vacuole. Lower pigment content, intercellular spaces on the epidermis and the induction of a molecular chaperone (BiP) were observed in hyperhydric phenotype. Evidences of schizolysigenous process of intercellular space formation are compatible with a stress condition. Although plastoglobulli were observed in normal and hyperhydric chloroplasts, they were more evident in the normal ones. Abnormal stomata also reflected a disruptive situation and morphogenesis disturbances which would difficult plant acclimatization. Further observation of the epidermis ultrastructure allows us to conclude that the presence of intercellular spaces on its surface may be constraining the recovery and development of hyperhydric plants. Similarly to BiP, other proteins such as esterase (EST), acid phosphatase (ACP), malate dehydrogenase (MDH) and peroxidase (PDX) are possible to be used as stress markers in in vitro conditions. Our results confirm earlier findings about negative effects of hyperhydricity on in vitro plant morphogenesis and ultrastructure, which in eucalypt is associated with a stressful condition contributing to lower propagation ratios.
In vivo as well as in vitro pollen tube growth studies along the style were performed, each with two pairs of sweet cherry cultivar combinations by means of fluorescence microscopy. In vivo studies showed that the percentage of pollen tubes penetrating the middle and basal section of the style was higher in the fully ...compatible 'Margit' x 'Alex' combination than in the half compatible `Germersdorfi 3' x `Alex' cross. The year effect was significant at P=0.] probability level. All pollen tubes in vitro stopped at the upper third of the style in the incompatible 'Vera' x 'Van' cross, whereas in the half compatible 'Alex' x 'Van' 50% of the pollen tubes penetrated to the lower third of the style. By in vitro fluorescence microscopy, it was possible to distinguish half compatible combinations from incompatible ones. Results obtained by in vivo technique only were much ambiguous.
The aim of this study was to test the in vitro sensitivity of two isolates of Monilinia laxa to fungicides approved in integrated and organic production systems. In vitro efficacy of 7 fungicides (Champion 50 WP, Kocide 2000, Nordox 75 WG, Olajos rézkén, Kumulus S, Rézkén, Rézoxiklorid) and another 6 fungicides (Score 25 EC, Efuzin 500 SC,... Systane, Folicur Solo, Zato Plusz, Rovral) approved in organic and integrated production systems, respectively, were tested against brown rot of sour cherry. The three isolates showed differences in sensitivity to fungicides. Fungicides (with active ingredients of copper and sulphur) applied in organic production showed relatively high percent growth capacity of M. laxa. Rézkén and Kocide 2000 showed the highest and Kumilus S the lowest efficacy against brown rot. Fungicides applied in integrated production showed relatively low percent growth capacity of M. laxa. Score 25 EC showed the lowest and Rovral and Folicur solso the highest efficacy against M. laxa.
In vitro culture conditions were experimented for the relatively sensitive, but very esthaetic "Caribe" variety of carnation with uniformly dark violet flowers. Regeneration of new plants from shoot apex meristems can be significantly improved by the combined addition of very low amounts of indolebutiric acid, benzyladenine and gibbere...lic acid, dissolved in the Murashige-Skoog nutrient medium. Callus formation as a prerequisite for the induction of somaclonal variability can be achieved successfully with certain molar ratios between 2,4-dichlorophenoxyacetic acid and benzyladenine. Acclimation of the obtained mericlones to the ex vitro conditions was also evaluated.
In vitro cultures have widely been used in horticulture for rapid multiplication of new varieties and clones as well as to produce pathogen-free stock material. To improve efficient hardening and transfer in vitro grown grapevine plants were multiplied by cutting them into single-node internodes with the whole leaf. Microcutti...ngs including the shoot tips were rooted in granulated perlite moisted with tapwater under sterile conditions. After 2-3 weeks the rooted microcuttings were supplied by nutrients and hardened by gradual opening and finally by complete removal of the lids of jars or plastic boxes used for growth. Using this method microcuttings of Vitis vinifera cvs. „Chardonnay", „Cabernet franc", „Riesling" and „Sauvignon blanc" and the rootstock varieties Vitis riparia x Vitis cinerea cv. „Barrier" and Vitis berlandieri x Vitis rupestris cv. „Richter 110" formed new roots and shoots and 100% of the tested plants survived the acclimatization procedure. Similar results were obtained when perlite was replaced with rockwool-, or pit-pot blocks. This method may highly increase the efficiency of producing pathogen-free propagating material and new transgenic lines.
Rooting ability of in vitro apple shoots of 'Red Fuji' grown on proliferation media with different hormone content were tested at three IBA levels in root induction media. Rooting percentage could be slightly increased with an increase in IBA concentration in proliferation media. The highest IBA concentration (3.0 mg 1-1) in root ind...uction media showed strong inhibitory effect on rooting capacity of in vitro shoots. The highest rooting percentage (95%) could be achieved by shoots grown on proliferation media containing TOP or BA+KIN as cytokinins before rooting.
Ocimum basilicum L. (sweet basil) is an economically and ethnobotanically important aromatic, medicinal, ornamental and culinary herb, with a very wide gene pool, that is sensitive to cold and prone to several plant pathogens that can demolish harvest and lessen yield. In this research, the effects of BAP (6-Benzylaminopurine) and TDZ ...(Thidiazuron) on different genotypes for in vitro cloning were determined, in order to provide a detailed protocol guide concerning Ocimum basilicum L. propagation. The results from the O. basilicum seed propagations revealed that the best condition for the secondary shoot growth is with 5.0 mg/l TDZ or 1.5 mg/l BAP on all types of explants except the root, the secondary root growth can be obtained on all types explant with any BAP concentration and all cytokinins can induce callus on all types of explants. On the whole, it shows that multiple secondary shoot induction and regeneration in Ocimum basilicum L. is regulated by appropriate cytokinin concentration.
Two strains of Pseudomonas syringae subsp. savastanoi were isolated from Forsythia sp. and Nerium oleander in Hungary in 1997. The effects of growth regulators produced by the bacteria were studied in different experiments. The strains were co-cultured with Sorbus redliana in vitro shoots without being in contact with the plant on solid media. ...Further culture filtrates in different concentrations were added to the culture medium. The growth regulators presented in the agar caused knot formation on the shoots and on the leaves in both kinds of culture. There were significant differences in the cultural and physiological characters, auxin and cytokinin activity of the strains of different origin.
Leucojum aestivum is a native, protected ornamental and medicinal plant in Hungary and in Ukraine too. The aim of our work was to establish in vitro cultures of this bulbous plant. Prior to surface sterilisation the old leaves and roots were dissected from the bulbs and they were stored in a refrigerator (2-3°C) for different periods ...(1 week for the first starting experiment and 5 weeks for the second one). After sterilisation, bulbs, bulb scales and leaves of the bulbs were placed on Murashige and Skoog's (1962) medium with 1 mg/1 benzyl-adenine (BA) and 0,1 mg/1 naphthalene acetic acid (NAA). At the first starting experiment 81,3%, and at the second one 92,3% of the explants turned to be sterile. Bulblets and roots were developed on the explants in the case of using bulb plates together with bulb scales and leaves as inoculua. The best result was achieved after 5 weeks chilling and it was possible to gain little bulbs from the bulb leaves too.
The effects of different aromatic cytokinins applied in different concentrations and combinations were investigated on the histology of in vitro apple leaves and their post-effects on subsequent shoot regeneration from these leaves were studied. Great differences in the anatomical structure of leaves could be detected originating from ...media containing different types and concentrations of aromatic cytokinins. The number of regenerated shoots per explant and the organogenetic index were used for the evaluation of the post-effect of aromatic cytokinins on shoot regeneration. The histological structure of leaves used for regeneration and their regeneration response showed a good correlation. When the pre-treatment caused a juvenile-like or less-differentiated structure, the number of regenerated shoots per explant increased and often vitrification also decreased and consequently the organogenetic index also increased. A strong interaction between cytokinin-content (type and concentration) of the pre-treatment medium and that of the regeneration medium could also be detected.
Rooting of in vitro 'Royal Gala' shoots was studied under different conditions of root induction and root elongation phase. The rooting capacity was affected by both rooting phases. Very high rooting percentage could be reached with both liquid and solid root induction media. Raising the temperature from 22 °C to 26 °C during root in...duction phase increased the rooting percentage. Presence of activated charcoal in root elongation media can affect the number of roots per rooted shoots and can increase the rooting percentage, the length of roots and the rate of survival depending also on other conditions during rooting. Presence of NAA in root elongation media reduced the number and the length of roots considerably. Favourable effect of activated charcoal on rooting was mainly due to adsorption of NAA.
Phaseolus vulgaris L. is the most important economic species within the genus Phaseolus. It is grown in all parts of the world. Genetic improvement by conventional breeding has met considerable success, although production of hybrids between species within the genus has been limited due to sexual incompatibility. Recent advanc...es in tissue culture have offered the opportunity to produce cultivars, which could not be obtained by conventional breeding methods. The use of tissue culture and genetic engineering is viewed as a logical approach to improve bean production. Gene transfer techniques will have a great impact on legumes. Although the concept of cell totipotency is widely proved, in vitro morphogenesis has not yet been achieved for a large number of cultivated beans. Regeneration protocols are strongly influenced by the genotype. In tissue and cell culture of beans, the factors controlling shoot morphogenesis and somatic embryogenesis are still unknown. The reported data suggest a possible way for future research.