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.
In vitro plant material of clones (Q. robur) NL 100 A (adult) and NL 100 R (rejuvenated) received from Germany (A. Meier-Dinkel, 1995) were used in these experiments. WPM medium was used for the multiplication phase. Plantlets were subcultured monthly. Differences in quality and colour of the adult and rejuvenated cultures ind...uced us to follow and compare the changes of mineral- and chlorophyll content and dry weight during the propagation phase. Mineral and chlorophyll content as well as dry weight were measured weekly on three samples during the subculture period.
In the case of propagation rates we stated, they were similar around the year, but both clones had a high peak in April. Examining the cation-content, we detected that, the plantlets had a highest quantity of several elements during the 2nd and 3rd week of subculture. The iron content was the highest in the 1st week and after that it decreased continuously. It is supposed, that the content of iron is not enough in the media. The chlorophyll content of the rejuvenated clone was higher than that of the adult one.
In the rooting experiments it was stated that, after one-week cold treatment the rooting ability was the best.
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.
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.
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 Hungarian cultivar Sorbus redliana 'Burokvölgy' was proliferated on Murashige and Skoog (MS, 1962) medium with half-strength macroelements and 100 mg/1 meso-inositol, 20 g/1 sucrose, 11 g/1 agar-agar. Different combinations of kinetin (KIN), metatopolin (mT), benzyladenine (BA), benzyladenine-ribosid (BAR) and indolebutiric acid (IBA) were... tested, and pH was adjusted to 5.6 every case using KOH. The cultures were incubated at 20-24 °C in 8/16 hours dark/light photoperiod for 50-52 days. The main aim of our research was to find the optimal growth regulator and its optimum concentration. Purthermore, to determine the chlorophyll contents of the in vitro propagated plants' leaves. During the proliferation, the highest number of shoots were observed in the case of using BA + IBA, and on the medium containing 0.75 mg/I BA + 0.05 mg/1 IBA 8.93 shoots were found. The addition of KIN + IBA decreased the number of shoots and increased the sizes of leaves — the widest (11.2 mm) and longest (17.8 mm) leaves were obtained on the medium containing 1.00 mg/I KIN + 0.05 mg/1 IBA. The longest shoots (36.46 mm) were found in the case of applying 0.75 mg/1 BAR + 0.05 mg/I IBA. The BA + KIN + IBA combination resulted the shortest shoots. Sometimes not only shoot regeneration but spontaneous rooting was observed during the multiplication. The highest chlorophyll content (1.569 mg/g total chlorophyll, 1.132 mg/g chlorophyll-a, 0.437 mg/g chlorophyll-b) was obtained in the presence of 1.0 mg/I KIN + 0.05 mg/1 IBA.
Callus cultures derived from young stems of two varieties of Taxus baccata cv. aureovariegata (genotype I) and Taxus baccata L. (genotype II, III) were induced. Gamborg's B5 medium was supplemented with different concentrations of auxin (2,4-D) in combination with cytokinins (kinetin or topolin) and with a phenolic-binding compound (PV...P) to prevent callus darkening and growth inhibition. Stem explants displayed different responses to in vitro culture depending on plant genotype and on the season. Genetic variability was observed in the growth rate of calli initiated from all three genotypes of the same Taxus species. We found the best growth of callus cultures originated from the genotype ill in defined media. After the first subculture the majority of the cream-coloured primary callus turned brown and ceased its growth. However, the long-term culture was initiated.
In vitro shoot multiplication responses of Amelanchier canadensis ‘Rainbow Pillar’ were studied on media solidifi ed with different gelling agents. The media were gelled either with 6.8 g l-1 fi brous agar-agar, or 50.0 g l-1 wheat starch, or 20.0 g l-1 Guar gum, or 15 g l-1 Isubgol or 50.0 g l-1 wheat starch mixed with 0.5 g l-1 Phytagel....Shoot cultures were grown for two months, thereafter the multiplication rates (number of newly developed shoots per explant) were counted and the length of shoots were measured. We found that the highest shoot multiplication of Amelanchier canadensis ‘Rainbow Pillar’ occurred on media gelled with Guar gum, while the longest shoots developed on media with Starch. About four-fold shoot number were obtained on media with Guar gum compared to the weakest results found on media gelled with Isubgol. Finally, considering all factors (shoot growth parameters, costs) the most economical gelling agent for Amelanchier canadensis ‘Rainbow Pillar’ was proved to be wheat starch among the tested alternatives which allows a 75.6% cost reduction.
The production facilities of large-sized microtubers in three potato varieties (cv. Desiree, BorO, Gfilbaba) and the effects of the applied tuberization conditions on the proportion of microtuber tissues, especially on the perimedullary region were investigated in present work. In vitro tuberization was induced on explants wit...h 2 or 5 nodes layered on MS medium supplemented with 8% sucrose. Induced cultures were exposed to short days (8 h) for 2 weeks, then to total darkness for further 11 weeks. For volume calculations of different tissue regions, the formula for ellipsoids (V=4/37c1/8/w2) was used. The number of large-size tubers (> 8 mm, up to 16 mm) reached 53%, 59% and 44% in cvs. Desiree, Giilbaba and Bore, respectively, which indicate that the size of microtubers could be increased by appropriate sucrose support and explant type. Microtubers produced on hormone-free medium have well-developed perimedullary region, and its volume rate seemed to be important in the final size of tubers. The increase in the rate of volume of the perimedulla was connected to the increase of tuber size until tubers reached 12 mm diameter. In microtubers larger than 12 mm in diameter, the volume rate of the pith was increased.
Grapevines are affected by three major bacterial diseases worldwide, such as bacterial blight (Xylophilus ampelinus), Pierce’s disease (Xylella fastidiosa) and crown gall (Agrobacterium vitis). These bacteria grow in the vascular system of their host, thus they invade and colonize the whole plant, independently on symptom development. Latentl...y infected propagating material is a major factor in their spreading. Therefore the use of bacteria-free planting stock has a basic importance in viticulture. Today several innovative diagnostic methods, mostly based on polymerase chain reaction, are available to detect and identify bacterial pathogens of grapevines. For production of bacteria-free plants, the use hot water treatment followed by establishment of in vitro shoot tip cultures is proposed.
In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS ge...ne as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.