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 weeks,... 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.
The Pseudomonas savastanoi pv. phaseolicola (PS) is one of the most significant stressors of bean (Phaseolus vulgaris L.). Chemical and agrotechnical treatments have minor importance, so breeding has great part in the protection against this pathogen. Most of the cultivars are susceptible to PS. The genetic background of resistance in the plant is... a complex system. Leaf resistance is a monogenic system, but there are some modifier genes. The pathogen species can be divided into different races.
To understand the functioning of this resistance gene, experiments were carried out using bean varieties with different genotypes and near isogenic lines of bean. Eight lines were tested. Our main objective was to test bean lines with PS with high virulence.
The experiment was made in greenhouse and on field. The virulent bacterium strain has been isolated in Hungary.
The changes of carbohydrates were tested after infection. In homeostasis the level of carbohydrates (especially glucose and fructose) were higher in susceptible lines. In case of artificial and natural infection the decrease of glucose were more significant in susceptible lines than in resistant lines. In the leaf samples from systemic chlorosis the level of this carbohydrate increased.
These changes are connected with the level of resistance, but more experiments are needed to verify this assumption.
About 600-year-old plant seeds were discovered in a well of a mediaeval cellar in the course of an excavation in Budapest. After the archaeobotanic purification seed of 16 species were found in large quantities. Seeds preserved in the best state were selected from each group. The existence of endosperm was analysed in these subfossils, which turne...d to be successful mainly in the case of grapes (Vitis vinifera) and cornels (Cornus mas). Seeds of these two species contained the most endosperm and remains of the embryo. DNA was extracted with the help of DNEasy Plant Mini Kit and analysed by RAPD-PCR method. The amplification of DNA extracted from cornel seeds resulted in detecting a 1500 by fragment, which makes the comparison of these samples with present-day cornels possible.
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 gene as a screenable ma...rker. 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.
RAPD markers were used to reveal genetic diversity between nine varieties of Cucumis melo L. and to identify the studied varieties. Of the 60 primers tested 12 primers produced polymorph patterns. A set of 4 primers was sufficient for distinction the nine investigated melon varieties.
Some parameters involved in Agrobacterium-mediated transformation in muskmelon Hales best (HBS) were studied. Cotyledon explants excised from 3.5-day-old seedlings were co-cultivated with Agrobacterium tumefaciens harbouring binary vectors which contained GUS and BAR genes. After co-cultivation on a low pH medium, explants were transferred to sele...ctive medium, with higher pH, containing Claforan and Finale. The medium was changed every two weeks till shoots were induced. All shoots rooted on MS medium supplemented with 0.3 mg/L IBA. These parameters combined as a whole led to successful transformation. The expression of the introduced gene construct was confirmed by GUS staining of shoot segments.
We have found that 50 mg/I kanamycin and 0.8 Mo1/1 mannitol concentration was sufficient to kill the control callus of bean (Phaseolus vulgaris L.) and differentiate transgenic from the non-transgenic cells. The GeneBooster particle delivery system was used for the bombardment of bean callus. The kanamycin resistance gene was used as a selectable ...marker. The test was made by transferring the healthy white callus, subcultured for three months on selective and non-selective medium. After selection on kanamycin containing media, several kanamycin resistant calli had been obtained, survived and grew. After selection on mannitol containing media no drought resistant calli had been obtained. Resistance of the selected calli were verified by their ability to grow repeatedly on selective medium containing 150 mg/I kanamycin. Selective pressure was maintained over a period of 8 months.
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 wit...h 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.
Tissue cultured Cymbidium PLBs (protocormlike body) were used as starting material to induce embryogenic callus which could serve as objects of genetic transformation. We obtained callus using two methods. The first method was culturing the PLB segments for one month in liquid MS medium in the presence of 0.5 mg/1 benzyladenine and 0.05 mg/1 napht...ylacetic acid followed by cultivation on the same composition solid medium with 0.5 g/l activated charcoal for an additional month. Callus formation was observed on 30% of the explants. The second way was to propagate the PLB segments on solid MS medium supplemented with 1 mg/1 thidiazuron. In these cultures we also observed callus formation on 20% of the explants.
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 acid (IAA), and benzyladen...ine (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.
We 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 recommen...d 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.
The Pseudomonas savastanoi pv. phaseolicola is one of the most expressive biogen stressors of the bean (Phaseolus vulgaris L.) in Hungary. The chemical and agrotechnological defence is inefficient, so breeding is the only workable way. The conventional cultivars are susceptible to PS while most of the new industrial varieties have genetic resistan...ce to the pathogen. The genetic background of resistance is, however, a complex system in the bean. Leaf resistance is a monogenic system, but this gene is not expressed in juvenile stage of the host. The pathogen species can be divided into different races. After inoculation with virulent strains, typical symptoms appeared on the leaves. To understand the details of host-pathogen relationships, there were carried out experiments using bacterial strains with altered virulence. Six transposon mutants of the PS were tested. Our main objective was to test these modified bacterial strains on bean cultivars of known genetic background. First we analysed the symptoms, and then the correlation between the symptoms and the multiplication of mutant bacteria. Three cultivars (Cherokee, Inka and Főnix) were tested.
The infection by the virulent PS isolate produced typical symptoms on the three cultivars tested. Mutant bacteria (except strain 756) did not cause any significant symptoms on the hosts. The mutant 756 induced visible symptoms on the cultivars Cherokee and Inka. On Cherokee there were small watersoaked lesions, and HR (hypersensitivity reaction) was detected on Inka, but this was restricted to some cells only (mikro HR). The rate of multiplication of the wild type strain was much higher than the multiplication of the mutants. Bacteria were detected in the cotyledons and primordial leaf, but there is not any substantial number of bacteria in leaves, except for strains 757, 1212 and 1213. The rate of multiplication of strain 756 was intermediate. These, and other experiments can help to understand the genetic background of resistance and the host-pathogen relationship in the Pseudomonas-bean pathosystem.