The 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 ran...ge 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.
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.
The most limitating factor for successful transformation is the absence of high-yielding regeneration protocols. However, the anther-derived embryogenic culture is an optimal technique for genetic transformation and it has been widely applied in many important cultivars, but the necessity of further development of regeneration systems has been ...proved. We attempted to produce somatic embryos on a wide range of genotypes from various tissues; leaves, petioles, stem segments. We started the examination of grapevine regeneration via organogenesis, succeeded in inducing shoot from the meristematic tissue of the base of bud by testing induction medium contained different concentrations of two types of hormones. To optimize the conditions of the Agrobacterium-mediated transformation, we studied the effectiveness of different Agrobacterium-treatments, the use of antioxidants and the sufficient quantity of kanamycin for selection of transformed cells.
Dry seeds from two cultivars of common bean (Phaseolus vulgaris L.) were germinated on sterile cotton and sterile deionized distilled water. Cotyledonary node tissue of seedlings were cultured on Murashige and Skoog(MS)-based media supplemented with different combination of N6-benzyl-aminopurine (BAP) and indole-3-acetic aci...d (IAA), and benzyladenine (BA) and a-naphthaleneacetic acid (NAA). The results revealed that the regeneration percent and the average number of buds and shoots per explant were influenced by the type of explants and exogeneously added hormones. Multiple shoot induction on dry bean cotyledonary node that contain 4-5 mm from cotyledons and hypocotyl on a medium containing full concentration of MS inorganic salts supplemented with 0.5mg/1 BA and 0.1mg/1 NAA was feasible and the method can be applied in transformation experiments.
Cotyledonary segments of the casaba type muskmelon variety "Hógolyó" were used to induce organogenesis. Fifty different hormone combinations were applied to enhance the induction of shoot formation on the edge of the segments. The phases of organogenesis were followed with light- and scanning electron microscope. Shoot induction was achieved ...with high frequency. The shoots were transferred to hormone free media for root induction. The rooted plantlets were planted out to soil.
NAA was feasible and the method can be applied in transformation experiments.
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 .
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.
Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four wee...ks, then the shoots were transferred to hormone free medium for root induction.
This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.
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.
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.
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.
Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the et...hylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.
The effects of kanamycin and hygromycin-based selection and co-culture temperature ranging from 22 to 28 °C upon eggplant transformation efficiency were evaluated. Both morphogenic pathways, somatic embryogenesis and organogenesis, were adopted using cotiledonary and hypocotyl explants, respectively. Somatic embryos were recovered in the prese...nce of both antibiotics, although lesser escapes were observed in hygromycin-supplemented medium. Indeed, selection provided by this antibiotic was more efficient compared to kanamycin, nevertheless, shoot regeneration was not observed with hygromycin. Significant difference on the frequency of cotiledonary explants displaying callus (FEC) was observed as embryogenesis was concerned, although a higher number of embryos was observed in hygromycin selective media. The frequency of explants presenting callus (FEC), embryos (FEE) and shoots or buds (FERG) did not differ statistically for the tested co-culture temperatures, although higher regenerant number was observed at 24 °C.
The 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 mis...sing 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.
Callus formation, as a prerequisite for the induction of somaclonal variability, was achieved successfully with certain molar ratios between 2,4-dichlorophenoxyacetic acid and benzyladenine. Regeneration of new plants from shoot apex meristems could be significantly improved by the combined addition of very low amounts of indolebutiric acid, be...nzyladenine and gibberelic acid, dissolved in the Murashige-Skoog nutrient medium. These in vitro treatments may contribute to a more efficient micropropagation of the Rimini variety of carnation.
Eustoma 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.