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Model experiments for establishment of in vitro culture by micrografting in apple
47-49.Views:185Micrografting 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.
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Dr. Ottó Orsós, the forgotten Hungarian pioneer in plant tissue culture
9-13.Views:161The knowledge of tissue culture deserves attention in respect of understanding the development of universal biology. This study intends to contribute to the past of the plant tissue culture by such data of the history of science which have been unprocessed so far. It seems that the life-work of the Hungarian biologist, Dr. Ottó Orsós is a missing and essential link between those early plant hormone researchers and the representatives of the pioneers of tissue culture schools who have contributed substantially to the development of the modern in vitro plant morphogenesis and plant cell biology. Orsós cultured kohlrabi tuber cubes on White culture medium in a sterile manner. This way, he could efficiently direct the in vitro morphogenesis of the kohlrabi, the regeneration of its shoot and root, and the formation and steps to subculture of pure callus tissues in 1938. He supported the correctness of its statements by means of detailed anatomical examinations. Orsós successfully rooted and aclimatized complete regenerated plants. We may as well call the above system — in remembrance of the creators of the original concept — "Haberlandt-Orsós model". Between the publishing of his main paper in 1938 and 2003, a period of 65 years has lapsed. On the occasion of this anniversary, we bow before this forgotten pioneer.
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Micropropagation of Rudbeckia hirta L. from seedling explants
105-108Views:163We conducted experiments for developing an in vitro micropropagation protocol starting from meristems of Rudbeckia hirta L seedlings. We pre-soaked the seeds in sterile ion-exchanged water for 17 hours, and then achieved surface disinfection in two separate steps. First, we used concentrated household sodium-hypochloride solution for 20 minutes and, also for 20 minutes, we applied hydrogen peroxide of 10%, which was followed by washing with sterile ion-exchanged water three times. For the propagation of seedling meristems, the combination of half-strenght solid Murashige and Skoog (1962) culture medium containing 10 mg/1 of kinetin or 2 mg/I of kinetin + 0.1 mg/1 of 2iP proved to be the most suitable. The average number of shoot-buds developed from the seedling axillary meristem in the best culture media varied between 5 and 17. Without separating them, we inoculated the shoot-bud clusters on MS culture medium containing 2 mg/1 of IAA. After four weeks of incubation we obtained elongated shoots which we separated and inoculated into a new culture medium and we obtained elongated roots. The rooted plants were gradually acclimatised in the cultivation room, potted and carried to a greenhouse, and then planted in open field for subsequent observation. By adopting this method, our laboratory started the micropropagation of the superior and/or elite genotypes of the Rudbeckia hirta L. being of special value in respect of breeding.
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Knot formation by Pseudomonas syringae subsp. savastanoi on the in vitro shoots of Sorbus redliana
59-62.Views:158Two 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.
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In vitro plant regeneration from immature embryo axis and cotyledons of common bean (Phaseolus vulgaris L.)
93-97.Views:127Phaseolus 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 advances 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.
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Dynamics of the uptake of nutrient elements from the medium of in vitro cultured apple rootstocks
50-53.Views:143Cation 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.
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Results with the establishment of in vitro culture of Leucojum aestivum
67-71.Views:219Leucojum 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.
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Cytokinins affect the stomatal conductance and CO2 exchange of in vitro apple leaves
25-28.Views:249Effects 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.
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Impact of sodium-selenate on the growth of radish (Raphanus sativus L.) seedlings in vitro
113-115.Views:158Selenium (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.
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Luminescence variations in cucumber (Cucumis sativus L.) leaves derived from different regeneration systems
50-52.Views:154Plants 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.
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Rhizogenesis in in vitro shoot cultures of passion fruit (Passiflora edulis f. flavicarpa Deg.) is affected by ethylene precursor and by inhibitors
47-54.Views:167The 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.
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Genetic engineering of apple (Malus domestica Borkh.) for resistance to fungal diseases using g2ps1 gene from Gerbera hybrida (Asteraceae)
15-12.Views:267In 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
131-136.Views:490Jerusalem 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.
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Investigation of the in vitro regeneration of mericlones in the caribe variety of carnation
87-89.Views:130In 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.
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Nutritional and seasonal requirements for callus growth in Taxus baccata
111-114.Views:114Callus 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 (PVP) 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.
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Effect of aromatic cytokinins and explant position on shoot multiplication of Asparagus offi cinalis L.
67-70.Views:239Asparagus offi cinalis has been widely studied, but little information is available about its in vitro response to exogenous cytokinin during shoot multiplication. To study the effects of different cytokinins on shoot multiplication of A. offi cinalis ‘Grolim’, in vitro culture was initiated from shoot segments cultured on media with Murashige and Skoog medium. Effects of different aromatic cytokinins (6-benzylaminopurine, 6-benzylaminopurine riboside and meta-topolin) applied in four concentrations (0.5, 1.0, 1.5, 2.0 mg/l) on shoot multiplication of ‘Grolim’ were tested. Effect of explant position (vertically or horizontally) on the shoot multiplication outcome was also studied. Both the length and the number of newly developed shoots were signifi cantly affected by explant position and cytokinin content of the medium. The highest numbers of shoots (4.9) were produced in the presence of 0.5 mg l-1 6-benzylaminopurine riboside when explants were paced horizontally onto the medium. Although the longest shoots (41.5 mm) developed on explants placed vertically onto medium supplemented with 2.0 mg l-1 meta-topolin, the lengths of shoots developed on medium with 0.5 mg l-1 6-benzylaminopurine riboside were also adequate in both explant position (29.5 and 33.6 mm placed horizontally and vertically, respectively).
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In vitro comparative study of two Arundo donax L. ecotypes’ selenium tolerance
119-122.Views:286Selenium tolerance of two somatic embryo-derived Arundo donax L. ecotypes (Blossom, 20SZ) were compared in in vitro culture. Sodium-selenate (1 – 100 mg L-1) as known the most phytoavailable selenium form and the less studied red elemental nanoselenium (100 mg L-1) were applied as selenium treatments. Basis on the results Blossom ecotype seemed to be more sensitive to the sodium-selenate than 20SZ. Inhibiting effect of selenate was effectuated above 10 mg L-1 in case of Blossom, which was manifested in decreased survival rate and growing parameters. Contrast to this 20SZ could tolerate the selenate ≤ 20 mg L-1 without any toxic symptoms. Lower selenate tolerance of Blossom could be explained with higher selenium accumulation. Both of two ecotypes could also uptake and accumulate the red elemental nanoselenium however in much less extent compared to selenate.
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In vitro propagation of 'Echo' cultivars of Eustoma grandiflorum (Raf.) Shinn.
87-91.Views:133Eustoma grandiflorum (Raf.) Shinn. 'Echo' Fl cultivars ('Echo White', 'Echo Rose', 'Echo Blue', 'Echo Blue Picotee') were used and multiplication of shoots was evaluated on Murashige and Skoog (1962) basal medium with 11 g/1 agar-agar and 20 g/1 sucrose. To test the effect of BA different concentrations were added: 0.10, 0.25 mg/1 and a culture medium without BA. Differentiation of roots was examined on Jámbor-Benczúr and Marta (1990) basal medium with the same concentration of agar-agar and sucrose. To examine the effect on rooting, various concentrations of NAA were used: 0.5, 1.0, 2.0, 3.0 mg/l. The pH was adjusted to 5.6 in every case using KOH. We studied the after-effect of different concentrations of BA during the acclimatisation. During the multiplication, the cultivar 'Echo White' formed the most shoots and the smallest leaves on the medium with 0.10 mg/1 BA. Fortunately, in the case of this cultivar, the number of shoots was reduced and the length of leaves was increased succesfully on the medium without BA. The other three cultivars developed the longest leaves on the medium containing 0.10 mg/1 BA. Sometimes not only shoot regeneration but spontaneous rooting was observed during the multiplication. Examining the rooting, the highest percent of roots was found on the medium with 1.0 mg/1 NAA, and the cultivar 'Echo Rose' formed the most roots on this medium. Higher concentration (2.0 and 3.0 mg/1) of NAA already reduced the number of roots in all of the cultivars. During the acclimatisation, the percentage of survival was 76.3% and the tallest plants with the longest leaves were found on the multiplication medium with 0.25 mg/1 BA. 'Echo Blue Picotee' gave the best results with the tallest pieces and longest leaves on this medium.
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Results in the vitro propagation acclimatization of Pontederia lanceolata
47-49.Views:113This paper gives an outline of micropropagation of Pontederia lanceolata. Pontederia l. is a widely used aquatic plant, therefore there is an increasing demand for them, which can be satisfied only by in vitro culture. Research was carried out to find the best nutrient media conditions for micropropagation and acclimatisation of Pontederia lanceolata.
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Factors affecting rhizogenesis in vitro and acclimatisation of three cherry rootstocks
40-46.Views:127Factors affecting rhizogenesis in vitro and acclimatisation of three rootstocks of cherry, i.e. Mahaleb, Maxma-14 and Weiroot -10 were investigated.
Rooting was easily achieved within 2-4 weeks on MS-based liquid or agar-gelled media containing auxins IBA at conc. 0.49 or 2.45 pM or NAA at conc. of 0.49 pM. On liquid media with 2.45 pM IBA, a maximum rooting efficiency of 95-100% was obtained. However, high concentrations of auxin delayed the time of root initiation for 3-5 days.
Rooted plantlets were transplanted into pots with a mixture of 3:1 (v/v) peat:perlite and acclimatised gradually to field conditions with efficiency of 60%.
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Bean tissue culture and genetic transformation with Agrobacterium
32-35.Views:117In 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 gene 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.
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Micropropagation of an orchid Dendrobium strongylanthum Rchb.f.
61-64.Views:317A simple and reliable procedure for in vitro propagation of an orchid Dendrobium strongylanthum Rchb.f. was studied. Protochorm was induced from seed explants on 1/2 MS medium supplemented with 0.2 mg/L NAA. A mass of protochorm could be multiplied on proliferated medium of 1/2 Ms containing 0.5 mg/L V-6-BA. And bud differentiation of green global body was cultured in the same media, 2-2.5 cm shoots were formed after 30 day of culture. Addition of mashed banana and 0.5 mg/L NAA to 1/2 MS medium promoted root formation and vigorous growth. The plantlets were acclimatized and transplanted to compound materials of humus:sawdust (1:1) in greenhouse, the survival rate was more than 98%.
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Comparative investigations on protoplast culture of some Brazilian and Hungarian sweet pepper cultivars and hybrids
39-45.Views:177Cotyledon protoplasts were isolated from 16-18-day-old in vitro grown seedlings of 9 Brazilian and 3 Hungarian pepper varieties and hybrids. Large numbers (average 9.59 X 106 protoplasts g 14 fresh weight) of highly viable (average 87.0%) protoplasts were released using a pectocellulolytic enzyme mixture. Protoplasts were cultured in K8p mediuni using an alginate disc embedding method. The osmotic pressure of the medium surrounding the alginate-embedded protoplasts was reduced by replenishing the liquid medium at K8p:K8 ratios of 1:0. 2:1, 1:1 in the first. second, and third week, respectively. Initial plating efficiency (IPE) average was 38.5% and after 21 days protoplasts reached microcolonies (15-20 cells) stages. Microcolonies were transferred after 3-4 weeks to a MS-based medium supplemented with 1.0 mg I-1 zeatin, 3.0% (w/v) sucrose, 0.24% (w/v) phytagel and pH 5.8, whereupon they formed callus. Final plating efficiency (FPE) average was 0.29% at a plating density of 1.0 x 105 protoplasts Protoplast-derived calli were cultured on a range of MS-based media supplemented with either BAP, IAA, TDZ; and zeatin. No morphogenic response was observed in any genotype investigated.
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A complex system for the production of pathogen-free grapevine propagating material
59-62.Views:207The use of pathogen-free planting stock for new vineyard establishment is a key component in the maintenance and expansion of vine and quality table grape production. The success of the necessary changes in the structure of the grape industry is forced by the globalization process, the climate change, the rediscovery of autochton varieties as well as breeding of new tolerant and resistant varieties. The renewal of vineyards largely depend on the availability of planting stocks. Serbia and Hungary found a common interest in establishing pathogen-free stock materials from newly breed resistant varieties and clonal selections of varieties which are traditional in the Serbian-Hungarian border area. During a cross-border cooperation program a complex system for the production of pathogen-free grapevine propagating material was established. Using heat therapy, in vitro shoot tip culture and traditional and molecular diagnostic techniques new pathogen-free stock materials were established from 26 varieties. They have been or will be tested for the presence of most important grapevine viruses, phytoplasmas, as well as bacterial and fungal pathogens. The complex system applying green grafting for indexing on grapevine indicators can shorten the duration of the procedure from 4 years to two-three years.