In vitro rooting and anatomical study of leaves and roots of in vitro and ex vitro plants of Prunus x davidopersica 'Piroska'

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.

The in vitro and in vivo anatomical structure of leaves of Prunus x Davidopersica ‘Piroska' and Sorbus rotundifolia L. ‘Bükk szépe'

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'.

Further information to the acclimatization of "in vitro" 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.


Nutrition of the micropropagated fruit trees in vitro and ex vitro

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 acclimatization.

Development of in vitro propagation system for Atriplex halimus L.

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 was 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.

New in vitro micrografting method for apple by sticking

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. Scion 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.

In vitro sensitivity of Monilinia laxa to fungicides approved in integrated and organic production systems

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.

Model experiments for establishment of in vitro culture by micrografting in apple

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.

Cytokinins affect the stomatal conductance and CO2 exchange of in vitro apple leaves

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. Cytokinin 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.

The effect of different biostimulators on morphological and biochemical parameters of micropropagated Hosta ’Gold Drop’

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 peroxidase 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.

Isolation of living sperm cells and in vitro fusion of Torenia fournieri gametes

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 Mol, 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.

Testing the virulence of some Hungarian Erwinia amylovora strains on in vitro cultured apple rootstocks

A useful method was improved to test and to evaluate the susceptibility of plants to fire blight and the virulence of E. amylovora strains. Six Hungarian strains from different host plants were tested on in vitro cultured apple rootstocks. Disease rating was used for the characterization of the process of disease development. The different strains had different capacity to cause disease, mainly in the first period of incubation. There were significant differences between the virulence of the strains.

Ultrastructural and biochemical aspects of normal and hyperhydric eucalypt

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.

Rhizogenesis in in vitro shoot cultures of passion fruit (Passiflora edulis f. flavicarpa Deg.) is affected by ethylene precursor and by inhibitors

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 ethylene 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.


Pollen tube growth in sweet cherry (Prunus avium L.) styles following fully compatible, half compatible and incompatible pollinations

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.

Dynamics of the uptake of nutrient elements from the medium of in vitro cultured apple rootstocks

Cation uptake of J-TE-F apple rootstocks propagated in vitro was studied by the analysis of culture medium. Test-plants were grown on liquid medium under different light regimes. Samples for tests were taken twice a week.

Media without plants served as controls. The analysis of those showed, that only the uptakeable iron-content changed depending upon light treatment. The concentration of all other cations was considered unaltered.

As a result of analysis, it could be established, that elements present in the media were taken up in different rates by plantlets: Cu, P and Zn were utilized totally, but only 50% of K and 20 to 40% of Ca and Mg were taken up under the light treatments applied.

The dynamics of the uptake process was also observed. It was registered that they differed in the case of some cations. So Ba was utilized at the beginning of subculture, others for example B in the later phases. Some elements disappeared unevenly so K, P but the whole quantity is taken up during subculture.

In vitro multiplication and hardening of grapevine plants in aeriated media

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. Microcuttings 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.

In vitro effect of different cytokinin types (BAP, TDZ) on two different Ocimum basilicum cultivars explants

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.

Results with the establishment of in vitro culture of Leucojum aestivum

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.

Genetic engineering of apple (Malus domestica Borkh.) for resistance to fungal diseases using g2ps1 gene from Gerbera hybrida (Asteraceae)

In the present study, g2ps1 gene from Gerbera hybrida coding for 2-pyrone synthase which contribute for fungal and insect resistance was used. The aim was to work out an efficient approach of genetic transformation for apple cvs. ‘Golden Delicious’, ‘Royal Gala’ and ‘MM111’, ‘M26’ rootstocks for improving their fungal resistance using genetic engineering techniques. Adventitious shoot formation from leaf pieces of apples studied was achieved using middle leaf segments taken from the youngest leaves from in vitro-grown plants.
Optimum conditions for ‚direct’ shoot organogenesis resulted in high regeneration efficiency of  0%, 95%, 92%, 94% in the studied apples respectively. Putative transgenic shoots could be obtained on MS media with B5 Vitamins, 5.0 mg l-1 BAP, or 2.0 mg l-1 TDZ with 0.2 mg l-1 NAA in the presence of the selection agent “PPT” at 3.0-5.0 mgl-1. Shoot multiplication of transgenic shoots was achieved on: MS + B5 vitamins + 1.0 mg l-1 BAP + 0.3 mg l-1 IBA, 0.2 mg l-1 GA3+1.0 g/l MES+ 30 g/l sucrose + 7.0 g/l Agar, with the selection agent PPT at 5.0 mg l-1 and were subcultured every 4 weeks in order to get sufficient material to confirm transformation of the putative shoots obtained. Six, seven, one and six transgenic clones of the apples studied respectively have been obtained and confirmed by selection on the media containing the selection agent “PPT” and by PCR analysis using the suitable primers in all clones obtained for the presence of the selection” bar gene (447 bp) and the gene-of- interest “g2PS1” (1244 bp), with transformation efficiency of 0.4%, 0.6%, 0.1% and 0.3% respectively. These transgenic clones were multiplied further in vitro in the presence of the selection agent ‘PPT’ and rooted in vitro. Rooted transgenic plantlets were successfully acclimatized and are being kept under-containment conditions according to the biosafety by-law in Syria to evaluate their performance for fungal resistance .

Jerusalem artichoke (Helianthus tuberosus L.): A review of in vivo and in vitro propagation

Jerusalem artichoke (Helianthus tuberosus L.) is an old tuber crop with a recently renewed interest in multipurpose improvement. It is a perennial tuberous plant rich in inulin and is a potential energy crop. During food shortages in times of war Jerusalem artichoke received more attention by scientists and farmers because of its multiple uses as a vegetable, medicinal plant, forage plant and source for biofuel. The energy crisis of the 1970s motivated research on Jerusalem artichoke for biofuel as the aboveground plant biomass and the tubers can be used for this purpose. There are different methods to propagate Jerusalem artichoke using tubers, rhizomes, slips (transplants derived from sprouted tubers), stem cuttings, seeds and tissue culture. So, this review was presented to highlight on propagation of Jerusalem artichoke via in vivo and in vitro techniques.

Investigation of the in vitro regeneration of mericlones in the caribe variety of carnation

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 gibberelic 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.


Impact of sodium-selenate on the growth of radish (Raphanus sativus L.) seedlings in vitro

Selenium (Se) is an essential trace element for animals, microorganisms and some other Eukaryotes. It has become increasingly evident that Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Although it is well known that some species among higher plants are able to accumulate selenium in their tissues, but others are not able to do so, and there is evidence that selenium is needed for the growth of algae, meanwhile the question of essentiality of Se in vascular plants is unresolved. We aimed to study the in vitro growing and to characterise some physiological properties in radish (Raphanus sativus L.) seedlings treated with 0 to 200 mg/1 sodium-selenate. The results showed that lower (2 mg/1) concentration sodium-selenate increased the biomass as well as the total antioxidant capacity of seedlings. The seedling's selenium content showed linear correlation with the sodium-selenate content of the medium.

Luminescence variations in cucumber (Cucumis sativus L.) leaves derived from different regeneration systems

Plants obtained from in vitro culture can show increased susceptibility to environmental stress conditions. In the process of their adaptation to natural conditions it requires monitoring of their physiological state. The methods used to check this phenomenon should estimate quickly and exactly the tolerance to suboptimal environmental factors. Such requirements are satisfied by the methods of measuring chlorophyll luminescence in vivo, e.g. fluorescence induction and delayed luminescence. The objects of our studies were cucumber plants regenerated from cultures of callus and embryogenic cell suspension, as well as the plants obtained from seeds. The plants derived from in vitro cultures displayed a poor physiological condition at the early phase of adaptation characterised by higher susceptibility both to stress caused by increased density of the light flux and low temperature (4 °C) in comparison with the plants obtained from seeds.


Some aspects of reduced disease management against Monilinia spp. in sour cherry production

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.

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