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Micropropagation of Rudbeckia hirta L. from seedling explants
105-108Views:195We 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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Studies on the alkaloid production of genetically transformed and non-transformed cultures of Lobelia inflata L.
65-71.Views:152The investigations of the growth and alkaloid production of cell suspension-, callus-, organized- and hairy root cultures from Lobelia inflata L. proved that these cultures are able to synthesize the characteristic piperidine alkaloids of the intact plant. Alkaloid precursor amino acids (Phe, Lys) and plant growth regulators affect not only the growth and differentiation of tissue cultures but also their secondary metabolism. The synthetic regulator Sz/I I combined with Phe increased the total alkaloid content considerably in callus- and organized cultures; regulator Sz/28 especially increased the lobeline content (in organized cultures in response to Lys, in callus tissues as a result of Phe application). With the aim of optimizing growth and alkaloid production of the genetically transformed hairy root cultures of Lobelia inflata L. we studied the effect of some growth regulators (NAA, IAA, kinetin) and precursor amino acids (Lys, Phe). The kinetin had inhibiting effect on the growth and lobeline production of the hairy roots. The IAA and NAA increased the biomass formation and lobeline production. The highest lobeline level was detected in tissues cultivated on hormone-free medium containing Phe.
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High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells
99-102.Views:129We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.
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The effect of different carbohydrates on the multiplication of Hosta cultivars
115-117.Views:159The effect of seven concentrations of two carbohydrate sources were compared to determine the best source and the most suitable source and concentration for micropropagation of some Hosta cultivars: H. 'Gold Haze', H. 'Gold Drop' and H. 'Dew Drop'. 0, 5, 10, 20, 30, 40 and 50 g/1 sucrose or glucose were added to a MS basic medium supplemented with 3 mg/1 kinetin and 0.1 mg/1 IAA. For 'Gold Haze' 40 g/1 sucrose proved to be the best source and concentration, the proliferation ratio was 15 shoots per explant. Thirty g/1 sucrose concentration was the optimum for 'Gold Drop', the proliferation rate was 14.6 shoots per explant. In 'Dew Drop,' the best results were obtained with 30 g/1 sucrose but 40 g/l sucrose gave good results too. Both cultivars rooted well on these media. On glucose containing media, very low propagation rates were found in all concentrations and all examined cultivars.
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Influence of aromatic cytokinins on shoot multiplication and their after-effects on rooting of apple cv. Húsvéti rozmaring
84-87.Views:242Different aromatic cytokinins (BA, BAR, TOP and KIN) were tested alone or in combination for the shoot proliferation response of ‘Húsvéti rozmaring' apple scion. The best multiplication rate was achieved by dual cytokinin application (1 mg 1-1 BA + 1.5 mg 1-1 KIN). The rooting capacity was affected considerably by the position of shoots: transfer of the three-week-old shoots to the same or other proliferation medium in vertical position inhibited the following rooting totally. Post-effects of different cytokinins (BA and TOP) on subsequent rooting could be detected: BA increased the number of roots markedly, while TOP resulted in significantly longer roots.
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Effects of different cytokinins on the shoot regeneration from apple leaves of 'Royal Gala' and 'M.26'
69-75.Views:220The effects of different types of cytokinins on the shoot regeneration from leaf explants of apple scion 'Royal Gala' and apple rootstock 'M.26' were evaluated. Regeneration media contained either thidiazuron, or 6-benzylaminopurine, or meta-topolin, or zeatin, or kinetin, or their N9-ribosides, respectively, in the concentration range 0.5 to 8.0 mg 1-1. Effects of 'these cytokinins were evaluated on the percentage of regeneration (R%) and that of vitrification (V%) and on the number of regenerated shoots per explant (SN). Organogenetic index (0I) calculated from these data was used for the evaluation of efficacy of cytokinins. The course of shoot organogenesis also was followed using stereomicroscope. Types and concentrations of cytokinins applied in the regeneration media influenced each parameter significantly and the regeneration answer was strongly genotype-dependent. The best regeneration (SN: 11.08, 01: 7.5) was achieved in `Royal Gala' by using TDZ in concentration of 0.5 mg 1-1 (2.271,1M). There was a clear relationship between the effect on the regeneration efficacy and the chemical structure of cytokinins considering classical cytokinins, namely N9-ribosides applied in less concentration than nonribosides have the same or best regeneration effects except for 6-benzylaminopurine riboside. However, similar relationship could not be detected in the case of 'M.26'. SN was the highest (3.22) using 6.5 mg 1-1 (18.2011M) 6-benzylaminopurine riboside or 8.0 mg 1-1 (21.44 µM) meta-topolin riboside (3.18). SN was not significantly lower (3.12) by using 2.0 mg 1-1 (9.08 1M) TDZ, however, OI was about half as big (0.63 compared to 1.29 or 1.74 with 6-benzylaminopurine riboside or meta-topolin riboside, respectively). 'Royal Gala' had higher organogenetic ability, than `M.26': 3.5-fold higher shoot number per explant and more than 4-fold higher organogenetic index was reached with this cultivar than with 'M.26'. Moreover, the similar developmental stage of shoots could be observed 3-5 days earlier than in 'M.26' and if explants of 'Royal Gala' were further cultured with 3 weeks, SN increased from 11.08 to 24.42 on TDZ-containing regeneration medium, which might suggest higher organogenetic ability, too.
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Single and dual effects of different cytokinins on shoot multiplication of different apple scions
76-78.Views:205Shoot multiplication responses of three apple scions to different concentrations of BA and BAR as single source of cytokinins and in combination with two concentrations of KIN were studied. The effects of hormones depended on genotype, type and interactions of different cytokinins. Use of BAR significantly enhanced the shoot multiplication of cv. Jonagold (6.5 shoots per explant). The multiplication rate of cv. Jonagold could not be improved by using the combination of BAR and KIN. The best proliferation was achieved by 1.0 mg 1-1 BA combined with 1.0 mg 1-1 KIN of cv. Prima..(8.1) and of cv. Galaxy (10.4).The effect of 0.5 mg 1-1 BA along with 1.5 mg 1-1 KIN was similar on multiplication rate (10.9) of cv. Galaxy.
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Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer
49-53.Views:130Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.
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Morphological, physiological features and differences of Vriesea splendens ’Fire’ plants during in vitro multiplication and rooting
Views:134During in vitro multiplication and rooting of Vriesea splendens ’Fire’, 0.1, 0.2, 0.4 and 0.8 mg l-1 benzyladenine (BAP), benzyladenine-riboside (BAPR), kinetin (KIN), meta-topoline (MT), indole-butyric acid (IBA) and naphthalene-acetic acid (NAA) were added to basal Murashige and Skoog (1962) MS medium. As compared to the hormone-free control, plants developed significantly more shoots on medium supplemented with almost all cytokinins (excepting KIN), especially BAP resulted the highest multiplication up to almost 26 shoots. Enhancement of cytokinin concentrations increased shoot number (and in case of BAP, peroxidase activity) but decreased plant height and rooting parameters. Regarding root production, both auxins were definitely beneficial (0.2 mg l-1 NAA resulted more than 7.5 roots and higher auxin concentrations efficiently stimulate root elongation); however, KIN had similar effects. After a three-month duration time of acclimatization, we observed that plants which were previously cultured on medium containing certain cytokinins (KIN in all doses and 0.1 mg l-1 MT) or both auxins had greater survival, moreover, as negative after-effect, higher cytokinin concentrations reduced the number of survived specimens.
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Results in the vitro propagation acclimatization of Pontederia lanceolata
47-49.Views:120This 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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Production of transgenic bean callus via genetic transformation by DNA-coated tungsten particles
43-47.Views:121Callus cultures were induced from hypocotyl of young bean seedlings. The B5 medium completed with 1 mg/1 KIN and 2mg/1 2,4-D proved the best. Callus developed and maintenaned on B5 medium supplemented with 1mg/1 kinetin and 2mg/I 2,4-D. The B5 medium supplemented with 1mg/1 KIN and 2mg/1 2,4-D induced much more callus than half strength MS medium supplemented with 0.5 or 0.75mg/1 BA and 0.1 mg/1 NAA. The results demonstrate that GeneboosterTM is convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus shot by GeneboosterTM can be transformed to get (kanamycin-resistant and stress mannitoltolerant) calli. The presence of mannitol-dehydrogenase gene (mt/) was verified by PCR, showing the integration of mt/ gene carried by two plasmids. Co-transformed calli were selected after bombardment on kanamycin, mannitol and (kanamycin+mannitop-containing media. Data of molecular analysis (PCR) confirmed the insertion of mtl gene in the genome of mannitol-tolerant callus lines.
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The effect of different cytokinins on chlorophyll content and morphological features of in vitro Nidularium ’Kertész Jubileum’
47-51.Views:258During in vitro multiplication of Nidularium ‘Kertész Jubileum’, 20 g/l sucrose, 5 g/l agar, 100 mg/l inositol, and different concentrations of benzyladenine (BA), benzyladenine-riboside (BAR), kinetin (KIN), meta-topolin (mT) were added to the MKC (Knudson, 1946) basal medium. Furthermore, 0.1 mg/l naphthaleneacetic acid was used to every medium. Number of shoots, length of leaves, number and length of roots, chlorophyll (a+b) content were examined and evaluated with Ropstat statistical software. As compared to the other cytokinin, significantly most shoots were obtained in the case of applying BA. Increasing of BA-concentration (as far as 2 mg/l) enhanced shoot number (from 10.92 to 19.26) but 4 mg/l BA resulted only 6.63 shoot. The less efficient cytokinin was KIN, in most cases no more than about 2 shoot was achieved. Regarding the length of leaves, the higher level of BA effected averagely the shorter leaves (from 24,46 to 7.31 mm). KIN effected significantly the longest leaves (43.4-61.29) in inverse proportion to the concentration. The same cytokinin resulted the most (and the longest) roots with the highest rooting percentages, but more KIN decreased the number and length of roots (from 7.95 to 4.4 and from 38.49 to 22.73 mm). There were no definite correlation between cytokinin concentration and chlorophyll (a+b) content, but the highest doses resulted decreasing (except of meta-topolin which leads to the lowest values). Summarizing, BAR effected the highest contents (mostly more than 1400 μg/g), particularly in the case of 1 mg/l (1807.3 μg/g).
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The effects of growth regulators in proliferation of Sorbus redliana 'Burokvölgy'
77-83.Views:216The 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.
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Nutritional and seasonal requirements for callus growth in Taxus baccata
111-114.Views:125Callus 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.