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.
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 recom...mend 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.
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.
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 resistance 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.
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 g...ene 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.
The present paper evaluates the result of irrigation experiments carried out on snap beans sown in spring and summer and grown with and without irrigation. The experiments were run over the course of 12 years. In the average of 12 years, the yield was 2.8t ha-I for spring sown and 1.9 t ha-I in summer-sown plants without i...rrigation. The lowest level of profitable production, the 5.5t ha-I was reached twice in the case of spring sowing and only once in the case of summer sowing. Profitable yield production can be ensured only with regular irrigation and thus the yield may be increased by 4-5 times. In four of the twelve years we determined the canopy surface temperature of snap bean stands with and without irrigation. A Raynger II infrared remote thermometer determined the canopy surface temperature every day at 13.00 hours. The canopy temperature can well characterize the water supply of plant stands. This parameter may be used for describing the degree of drought and the water turnover of plant stands with different water supply. The positive values of foliage-air temperature differences (SDD) numerically express the degree of drought and the water supply of the crops. The results indicated that a 1 °C higher SDD value may cause 90-130 kg/ha yield loss.
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.
Common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli (Xcp). is a major disease problem of common bean (Phaseolus vulgaris L.). The inheritance of resistance in Xrl and Xr2 lines to two isolates of Xcp was studied in the F2 and F3 populations from the crosses between these lines and the Masay...variety (susceptible to Xcp). Segregation patterns indicated that different single recessive genes presumably in coupling phase linkage determined the resistance to the HUN and EK-1 1 strains of Xcp in both lines. The presence of some minor, modifying genes beside the monogenic genetic background of resistance was also observed. Xrl and Xr2 lines represent valuable new monogenic genetic sources in resistance breeding to CBB.
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.
Callus 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.
Callus 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 medi...um 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.
It is an early observation that plants in poor soil are developing roots quicker and more abundantly than on rich one. There is a similar correlation between the nutrient status of medium and adventitious root formation.
In order to throw more light on the background of this strange phenomenon we started a systematic experimental progra...m in which the biological effects of distilled water as model factor was investigated.
The experiments proved that the root formation of Pinto bean (Phaseolus vulgaris L.) cuttings with 3 cm long hypocotyls was promoted by distilled water.
The phenomenon above accompanied with slower decline and faster recovery of total and also water-soluble protein content, more intensive efflux of amino acids, greater amount of tryptophane and increased uptake of water compared to those in control hypocotyls. From other data obtained we may suspect that some additional active substance unknown for us also contributes to the stimulation of root initiation in distilled water.
Our observations regarding the symptoms not fitting into, significantly differing from the hypersensitive defense system, which we noticed during the judgment of several plant species, symptoms provoked on several million plants have constituted a unified entity. They have provided evidence for the existence of a different plant defense system.... We called this so far unknown basic response of plants to biotic effects as general defense system. This system defends them from the attack of numerous microbe species in the environment.
The evolutionary intermediate phase between the general and the specific, the two defense systems is the susceptible host—pathogen relation. The vertical resistance system of plants escaping from the susceptible host—pathogen relation, based on specific hypersensitive reaction also suggested the existence of a more original, general defense system and the susceptible host—pathogen relation developed as a result of the collapse of that system.
The evolutionary relation of the two defense systems is proved by the only recessive inheritance of the older general defense system and in the majority of cases dominant hereditary course of the specific defense system. In our experiences, the modifying genes of the recessive general defense system, in most cases, are behind the specific defense systems, which are known to have monogenic dominant hereditary course and react with hypersensitive tissue destruction. This seemingly striking genetic fact is explained by the following: the general defense system less dependent on environmental effects regulates much faster pathophysiological reaction than the specific resistance genes strongly dependant on environmental effects coding dominant hypersensitive reaction.
The general and specific defense reactions, the processes excluding the microbes attacking plants with compacting of cell growth and tissue destruction, which mean two opposite strategies, building on and regulating each other constitute the entity of resistance to plant disease.
Among diseases that affect walnut, bacterial blight is considered the most important one in all walnut growing areas both from Hungary and Romania. For the determination of susceptibility/resistance of walnut cultivars in our posterior work planned, 61 bacterial isolates were collected from walnuts showing symptoms of blight, with the purpose o...f isolating Xanthomonas arboricola pv. juglandis (Xaj). The characteristic Xaj colonies, frequently present as saprophytes in the infected plant tissues, were separated from other bacteria according to their morphology, yellow colour, hydrolysis of starch, and oxidation of glucose. All isolates were tested for pathogenicity by hypersensitive reaction on tobacco leaves (Nicotiana tabacum L.), bean pods (Phaseolus vulgaris L) and unripe nuts (Juglans regia L.). Determination of taxonomy of the selected isolates denotes a possible subdivision (races, biotypes) of Xaj occurring in different geographical areas, API 20NE and API 50CH kits were used. Hungarian and Romanian strains showed a high degree of similarity of carbohydrate utilization but slightly differed from the type strain. All were Cu-sensitive.
In addition to successes achieved in certain varieties in resistance breeding based on a defense reaction of host plants involving hypersensitive tissue destruction, resistant varieties putting a very strong selection pressure on pathogens have selected more and more aggressive types of pathogens. The never-ending race between plant and pathoge...n resulting from this can only be controlled by a defense system characterised by a different strategy. In each of the plant species that we bred a defence system was found, which contrary to hypersensitive reaction strives to keep the tissues at all costs and is not pathogen specific. This is implied in the term general defense system.