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Study of different factors of grapevine regeneration systems and genetic transformation
33-36.Views:350The 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.
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In vitro regeneration from cotyledons of watermelon
96-98.Views:287Cotyledonary 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.
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Examinations of 600-year-old seeds by means of archaeobotanical and genetical methods
79-80.Views:312About 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 turned 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.
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Characterization of quince (Cydonia oblonga Mill.) cultivars using SSR markers developed for apple
7-10.Views:1212Quince (Cydonia oblongaMill.) is a minor fruit crop, which is primarily used for marmalade, jam and sauce.Very few quince cultivars are known all over the world and in many cases similar names are used for presumably different cultivars. The aim of the present study was to evaluate and characterize the genetic diversity of 36 quince cultivars and selections with SSR markers. Seven out of 8 SSR markers designed from apple sequences could successfully yield amplification also in quince cultivars. Number of alleles per locus ranged from 2 to 3 alleles. These allele numbers are quite low when compared to apple. It is supposed to be the consequence of a genetic bottleneck. In spite of the low allele number per locus, the 36 quince cultivars formed 30 different genotypes. The ratio of homozygosity was low, which might be coupled with the self-(in)compatibility phenotype of quinces. SSR markers proved unable to differentiate putatively closely related cultivars (e.g. ‘Bereczki’ and ‘Bereczki bôtermő’). In general, the level of polymorphism among the tested quince genotypes was much restricted due to the low allele number detected. However, it must be considered that the number of analysed SSR loci is not enough high to estimate the overall heterozygosity of the quince genome. Further experiments are needed and the SSR markers proved to be a reliable and useful tool for such analyses.
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Production of transgenic carnation with a heterologous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme cDNA
75-79.Views:256Transgenic carnations were produced with a modified mammalian bifunctional enzyme cDNA coding 6-phosphofructo-2- kinaseffructose 2,6-bisphosphatase. Relative activity of this enzyme determines the fructose 2,6-bisphosphate (fru 2,6-P2) cytosolic concentration. This metabolite — as a signal molecule — is one of the carbohydrate metabolism regulators. The regenerated Dianthus chinensis and Dianthus caryophyllus shoots were selected on MS basal medium containing 150 mg/1 kanamycin. Transgene integration was proven by PCR analysis with cDNA specific primers followed by Southern hybridization of DNA isolated from selected green shoots, which survived on kanamycin containing medium, so 3 D. chinensis and 20 D. caryophyllus transgenic plants were produced. Transgene expression were examined by RT-PCR. Transformed and control plants were potted in glasshouse to evaluate the effect of modified fru 2,6-P2 on development, growth and carbohydrate metabolism.
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Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer
76-78.Views:284We 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.
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Self-incompatibility alleles in Esatern European and Asian almond (Prunus dulcis) genotypes: a preliminary study
23-26.Views:432Almond [Prunus dulcis (Mill.) D. A. Webb.] as one of the oldest domesticated plants is thought to have originated in central Asia. Gametophytic self-incompatibility of almond is controlled by the highly polymorphic S-locus. The S-locus encodes for an S-ribonuclease (S-RNase) protein in the pistils, which degrades RNA in self-pollen tubes and hence stops their growing. This study was carried out to detect S-RNase allelic variants in Hungarian and Eastern European almond cultivars and Turkish wild growing seedlings, and characterize their S-allele pool. Five new alleles were identifi ed, S31H, S36-S39 in Eastern European local cultivars. The village Bademli and Akdamar island are two distinct places of almond natural occurrence in Turkey. Trees growing wild around Bademli city showed greater genetic diversity than those originated on Akdamar island. Many of the previously described 45 S-RNase alleles have been also detected in these regions. Homology searches revealed that Turkish almonds carried some P. webbii alleles indicating hybridization between the two cultivars and massive introgression events. Our results supply long-awaited information on almond S-allele diversity from regions between the main cultivation centres and the centre of origin of this species; and are discussed from the aspect of methodological developments and evolution of the cultivated almond.
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Somatic embriogenesis and regeneration of Vitis sp.
41-45.Views:289Somatic embriogenesis and regeneration of Vitis sp.
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Agrobacterium transformation of Rhodiola sp.: current status and limitations
37-42.Views:332The study of secondary metabolites has led to the discovery of new drugs for treating human diseases. However, consistent plant supply can be challenging, leading to the use of plant tissue culture techniques such as hairy root culture. Hairy roots have stable genetics, lateral branching, and can produce secondary metabolites, including alkaloids, flavonoids, and terpenoids. Research on hairy roots as a subject began in the late 19th century, and for the last four decades, hairy roots have been utilized for producing secondary metabolites and recombinant proteins. This article focuses on Rhodiola species - genus of perennial plants that belongs to the family Crassulaceae - and its potential as a source of secondary metabolites using hairy root culture techniques. Rhodiola sp. is widely distributed throughout the Arctic regions of the Northern Hemisphere, with several species having significant medicinal properties. The article discusses the possible use of hairy root cultures for the production of Rhodiola secondary metabolites, including salidroside and rosavins, which have demonstrated significant pharmacological activity in various studies. The use of elicitation and genetic engineering techniques to boost secondary metabolite production in Rhodiola hairy roots is also explored. Overall, the article highlights the potential of Rhodiola hairy root cultures as a valuable source of secondary metabolites with medicinal properties. However, despite some studies Rhodiola hairy root induction and culturing still remains highly unexplored.
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Nutritional and seasonal requirements for callus growth in Taxus baccata
111-114.Views:205Callus 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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Bean tissue culture and genetic transformation with Agrobacterium
32-35.Views:332In 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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In vitro multiplication and hardening of grapevine plants in aeriated media
15-18.Views:380In vitro cultures have widely been used in horticulture for rapid multiplication of new varieties and clones as well as to produce pathogen-free stock material. To improve efficient hardening and transfer in vitro grown grapevine plants were multiplied by cutting them into single-node internodes with the whole leaf. Microcuttings including the shoot tips were rooted in granulated perlite moisted with tapwater under sterile conditions. After 2-3 weeks the rooted microcuttings were supplied by nutrients and hardened by gradual opening and finally by complete removal of the lids of jars or plastic boxes used for growth. Using this method microcuttings of Vitis vinifera cvs. „Chardonnay", „Cabernet franc", „Riesling" and „Sauvignon blanc" and the rootstock varieties Vitis riparia x Vitis cinerea cv. „Barrier" and Vitis berlandieri x Vitis rupestris cv. „Richter 110" formed new roots and shoots and 100% of the tested plants survived the acclimatization procedure. Similar results were obtained when perlite was replaced with rockwool-, or pit-pot blocks. This method may highly increase the efficiency of producing pathogen-free propagating material and new transgenic lines.
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Resistance Gene Analogs (RGA) as a tool in fruit tree's breeding
7-15.Views:367Breeding for pest and disease resistance comes as a major objective behind the fruit traits. To increase the effectiveness of fruit resistance breeding application of the Marker Assisted Selection ( MAS) is advantageous. For generating molecular markers which enable the following of interesting traits basically two methods are available: targeted marker design based on conservative region of already known Resistance ( R) gene sequences or randomly generated markers. The creation and the application of these homology based markers are the object of this review in the main temperate zone fruit species.
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The effects of ACS (1-aminocyclopropane-l-carboxylate synthase) gene down regulation on ethylene production and fruit softening in transgenic apple
65-70.Views:244A detailed examination of the production of ethylene and other ripening parameters during storage period has been undertaken in transgenic apple fruits, where the ethylene biosynthesis was inhibited by antisense ACS (l-aminocyclopropane-l-carboxylate synthase) gene. Data indicate down regulation of ethylene production, softening and spoilage in some transgenic lines. In some cases ethylene production was inhibited for over 90 percent, considerable reduction of softening and spoilage was observed probably due to the reduced activity of cell wall degradable enzymes. ACS activity was also monitored during ripening. The fruits of the best transgenic lines could be stored for minimum 4-5 months longer under 5 °C cold room storage conditions and one month longer at normal room temperature. This molecular approach can provide an alternative way to replace the commonly used and costly atmospheric storage of fruits.
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Analyses of Hungarian sour cherry germplasm with simple sequence repeat markers
27-31.Views:1203Twenty-four sour cherry cultivars (genotypes), belonging to four cultivar groups were fingerprinted using microsatellite markers. All genotypes have been arisen from the Carpathian basin, which could be secondary gene centre of sour cherry, since its progenitor species, ground cherry and sweet cherry overlap here. Five SSR primer pairs, earlier used for fingerprinting Turkish sour cherry germplasm were tested. None of the five primer pairs showed any polymorphism within the cultivar groups. The primer pairs were able to distinguish between the cultivar groups. The Oblacsinszka and the Cigánymeggy cultivar groups were the most difficult to separate, while the Pándy cultivar group was the most distinguishable.
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Luminescence variations in cucumber (Cucumis sativus L.) leaves derived from different regeneration systems
50-52.Views:272Plants 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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Determination of the cold tolerance off sour cherry cultivars with frost treatments in climatic chamber
49-54.Views:847Nowadays, sour cherry buds can be seriously damaged by spring and winter frosts. Unlike other fruit species threatened by high frost damage, sour cherry cultivars have not been assessed for frost tolerance. The aim of .our survey was to establish the relative cold tolerance of the Hungarian cultivars after treatment in a climatic chamber, and to optimize the methodology formerly elaborated for the frost treatment of apricot. Fourteen cultivars of Hungarian sour cherry (Prunus cerasus) were used in the experiments, which spanned the winters of 2005/2006 and 2006/2007. Our data were used to rank cultivars in two groups according to their levels of cold resistance. We also recommend critical temperatures and treatment times for the testing of sour cherry cultivar resistance to cold in climatic chambers.
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General defense system in the plant kingdom III.
45-54.Views:304Our 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.
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Review of the molecular background of self-incompatibility in rosaceous fruit trees
7-18.Views:439This review gives a presentation of the gametophytic self-incompatibility system in the roscaeous fruit trees. Studies to discover the pistil (S-ribonucleases) and pollen-part components (F-box molecules) are summarized and models for the self-incompatibility reactions as well as their molecular background are discussed. We describe how mutations within the S-RNase or F-box genes can contribute to the transition from self-incompatibility to the self-compatible phenotype in many fruit tree crops. The current state of the arts is compared to the information obtained in other plant species possessing similar incompatibility system.
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Evaluation of different sources of resistance for breeding powdery mildew resistant grapevine varieties
35-40.Views:330The appearance of resistance to powdery mildew was investigated on seedling progenies of interspecific crossings. Hybrids of American and East-Asian wild species and one Vitis vinifera variety (Janjal kara) were used as source of resistance in the crossings. The resistance of foliage and of berries were tested in the field at the time of vintage. The degree of resistance was ranked into six classes.
In American and Asian hybrid derivatives there were few highly resistant progenies, but in the hybrids of Janjal kara variety more than 90 % of the progenies proved to be highly resistant to powdery mildew. According to the distribution of classes of resistance in the populations the resistance of American and Asian sources are mostly influenced by additive gene affects. The resistance of Janjal kara seemed to be mono or digenic.
No close correlation was found between the powdery mildew resistance of the foliage and that of the berries.
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Transmethylation and the general defense reaction of plants
35-40.Views:366Plant breeding for resistance, namely building specific resistance genes into cultivated plants to ensure resistance against certain pathogen species, is a several-decade-long practice. While looking for purposes of failures appearing during the cultivation of varieties created in this way, a plant feature that ensures non-specific reactions against effects which evoke biotic stress attracted our attention. We named this plant defense form the general defense reaction. The general defense reaction is a fundamental attribute of the plant kingdom, fulfils the role of plant immune system and manifests itself in cell enlargement and cell division. Plants with a high level general defense reaction endure abiotic stresses as well.
In studying the biochemical background of the interaction of the general defense reaction and transmethylation, we found that transmethylation has important role in warding off both biotic and abiotic stresses. According to our observations, plants possessing high level general defense system are suitable for thorough examination of the process and plant physiological role of transmethylation. Biochemical studies also strengthened our observation, which has been taken on the basis of phenotype, that the general defense system can not be ignored during future plant breeding.
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Effects of methyl jasmonate, salicylic acid and phenylalanine on aloe emodin and aloin in diploid and tetraploid Aloe barbadensis
Views:440Aloe vera is one of the most famous medicinal plants. Aloin and aloe emodin are the most important active compounds in this plant. The purpose of this research was the comparison of aloin and aloe emodin production after the elicitation by methyl jasmonate, salicylic acid, and phenylalanine in diploid and tetraploid Aloe vera plants in greenhouse conditions. The plants were treated with the concentrations of 25, 50, and 100 µM. The amounts of aloin and aloe emodin were determined 24 and 48 hours after application of the treatment. HPLC analysis showed that the leaves of the control diploid plants (without applying elicitors) had more aloin (1.20 fold) and aloe emodin (1.14 fold) than the control tetraploid plants. The maximum concentration of aloin (1.15 ± 0.07 µg mg-1 dry weight) was obtained after the elicitation by 25 µM methyl jasmonate, 24 hours after treatment, in diploid plants) 6.36 fold compared to the control (0.18 µg mg-1 dry weight (. In addition, the maximum concentration of aloe emodin (0.28 µg mg-1 dry weight) was obtained after the elicitation by 25 µM salicylic acid, 24 hours after treatment, in diploid plants) 6.18 fold compared to the control (0.04 µg mg-1 dry weight)). The long-term effect of three studied elicitors (after 240 days) on plant health and survival was also studied. This investigation showed that only methyl jasmonate at a concentration of 100 µM was resulted in the death of Aloe vera plants.
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Preliminary evaluation of selected Prunus spinosa and P. insititia genotypes for their nutraceutical properties
19-22.Views:1142Fruits of nine Prunus spinosa and P. insititia selections were compared in their ferric reducing antioxidant power and total phenolic content. The antioxidant capacity and total phenolic content ranged between 6.36 and 29.26 mmol AA/L, and 5.04 and 29.71 mmol GA/L, respectively. These ranges cover an almost 5-times variation among the tested genotypes. The Pearson’s coefficient was very high (0.92) indicating a major contribution of polyphenolics to the antioxidant capacity of the tested Prunus fruits. Conserving resulted in an approximate 20 % loss of antioxidant power and slightly increased phenolic contents. Our results led us to the conclusion that fruit of Prunus spinosa and P. insititia might be considered as rich sources of antioxidants. In addition, procession with heat treatment caused only a slight decrease in the antioxidant capacity without loss in the total polyphenolic content.
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Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer
49-53.Views:231Callus 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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Evaluation of some Achillea L. accessions based on morphological, cytological and chemical characteristics
69-74.Views:329Taxonomical evaluation of six taxa of the Achillea aggregate was carried out to determine their potential for breeding and cultivation. We used complex morphological, cytological and chemical aspects of characterisation. Three taxa of tetraploid genom and high chamazulene contents (33-40% ess.oil) proved to be A. collina Becker. A strain of wild origin had also high -bisabolol content and high oil level, comparable with the selected cultivars. A population was identified as new chemotype of A. pannonica Scheele with typical morphological and cytological traits, a wide sesquiterpene spectrum but lacking of 1.8 cineole and -pinene. We found a mixed population which consisted of both A. collina and A. pannonica plants. A tetraploid, azuleneless taxon could be concluded as A. pratensis Saukel and Langer not described before in Hungary. The diverse performance of the populations calls the attention to the significance of controlled plant raw in phytopharmaceutical products.
