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Agrobacterium transformation of Rhodiola sp.: current status and limitations
37-42.Views:404The 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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The role and current state of gene reservation of medicinal and aromatic plants in Hungary an overview
19-21.Views:425As it well known the decrease of biodiversity is a large problem all over the world. In case of medicinal plants, where the huge majority of drugs are collected from natural ecosystems, the sustainable, utilization of the populations and reservation of their gene-pools has an increased interest.
In Hungary, the major background of 'in situ' reservation of medicinal plant species, their natural plant associations and ecosystems is the official protection by law. Successful examples are known for the controlled utilization of medicinal and aromatic plant species grown in protected areas. Assuring the naturally occuring high degree of biological and chemical diversity of species is a special task in this field: only maintenance of valuable intraspecific races can form the real genetic basis of natural biologically active compounds.
Maintenance of chemotaxonomical gardens and gene bank collections (seed banks, tissue banks) as 'ex situ' methods of reservation is carried out on an extended range in Hungary. As the required information on storage and maintenance of many medicinal and aromatic plant species is yet missing, exhaustive research is carried out at both genebanks in Hungary, which are specialized for medicinal plant reservation (RIMAP- Budakalász, SZIU- Budapest).
Beside the static conservation methods, 'quasi-production systems' are intended to assure an up-to-date and economically possible way of dynamic reservation with sustainable utilization.
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Identification of ripening-related genes in strawberry fruit by cDNA-AFLP
33-41.Views:331An RNA fingerprinting study of strawberry receptacle and achene tissue was performed to identify candidate genes involved in fruit ripening. Quantitative cDNA-AFLP was used to detect differential gene expression in green, white, pink and red stages of fruit ripening. Based on hierarchical average linkage clustering the differentially expressed genes formed three major groups, genes expressed only in green receptacle, genes expressed mainly in white, pink and red receptacle, and in achene. 130 transcript-derived fragments (TDFs) were isolated and sequenced. Most TDFs did not show any homology to sequences with known functions, others were homologous to genes involved in oxidative stress response, signal transduction, regulation of development and cell-wall metabolism. Novel genes, so far not associated with strawberry ripening and ripening in general, were identified, such as genes encoding a bHLH protein, putative nitrilase-related protein, putative HD-zip protein. The differential pattern of gene expression draws the attention to the significance of ripening induced-or repressed promoters in strawberry fruit, whose isolation and characterization can be useful tool for functional genomics. For this purpose nine cDNA-AFLP fragments related either to ontogeny or senescing were completed with 5'UTR aiming at more precise annotation and future promoter isolation. Although tens of potentially important transcriptome changes were identified, the function of many ripening induced genes remain unknown.
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Susceptibility of sour cherry cultivars to isolates of Monilia laxa (Ehrenbergh) Saccardo et Voglino
83-87.Views:534In this study, the susceptibility of 7 commercially important sour cherry cultivars to Monilinia laxa was studied. Artificial inoculation was made with M. laxa isolates, which were isolated from different woody plants. Artificial inoculation was prepared in the laboratory and in the field. In laboratory, flowers of sour cherries while in the field, the two-year old twigs were inoculated in 2006 and 2007. According to results of stigmata inoculation, there were infection ability differences among the isolates originated from five different stone fruit host. Cultivars could be sorted into two susceptibility groups. In the field, twig inoculation in 2007 was made at blossom period and in 2007 at harvest. Seven sour cherry cultivars were inoculated with 8-day-old mycelial culture of M. laxa originated from sour cherry and almond. The agressivity and pathogenicity of the two isolates were measured by the degree of floem death: Results showed that year and phenological stage considerably influenced the degree of symptoms caused by the fungus. After artificial inoculation, tissue death progression was studied by fluorescent microscope. According to results, sour cherry cultivars were sorted into disease susceptibility groups. Susceptibility orders were identical to results on stigmata inoculation.
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Anatomical relations of root formation in strawberry
71-75.Views:597Anatomical relations of root formation are traced throughout the life cycle of the strawberry plant from the germinating seed up to the runners of the adult plant. Histological picture of the root changes a lot during the development of the plant. First the radicle of the germ grows to a main root, which makes branches into side roots and later adventitious roots are formed on the growing rootstock or rhizome. The anatomy of the different types of roots is also conspicuously different. First tiny branches appear relatively early after germination on the seedling's radicle, but soon the hypocotyl of the seedling thickens and develops side roots, which are already somewhat stronger. During this interval, the first true leaves are formed. The 4th or 5th of them being already tripartite, and the initiation of new roots extends into the epicotylar region of the shoot. The second years growth starts with the development of reproductive structures, inflorescences and runners starting from the axils of the new leaves. Near the tips of the runners below the small bunch of leaves, new root primordia are initiated. The tiny radicle of the germ develops a cortical region of 5-6 cell layers. Cells of the central cylinder are even smaller than the cortical parenchyma and include 3-4 xylem and 3-4 phloem elements as representatives of the conductive tissue. Roots originating from the shoot region are much more developed; their cortical zone contains 17-20 cell layers, whereas the central cylinder is about half as large. In the next year, new roots are formed at the base of the older leaves. These roots differ hardly from those of the last season in size and volume, however, they are recognised by colour and their position on the rhizome. The roots of the last year are dark, greyish-black, and grow on the lower third length of the rhizome, on the contrary, the new ones, on the upper region, are light brown. Roots starting from the shoot or rhizome are, independently from their age or sequence, mainly rather similar in size and diameter, thus being members of a homogenous root (homorhizous) system, i.e. without a main root. Plants developed and attained the reproductive phase develop in the axils of the leaves runners being plagiotropic, i.e. growing horizontally on the surface of the soil. The runners elongate intensely, become 150-200 mm, where some long internodes bear a bunch of small leaves and root primordia on short internodes and a growing tip. Runners do not stop growing, generally, further sections of 15-25 cm length are developed according to the same pattern, with small leaves on the tip. The growing tip of the runners is obliquely oriented, and small, conical root primordia are ready to start growing as soon as they touch the soil. The roots penetrate the soil, quickly, and pull, by contraction, the axis of the runner downwards, vertically, developing a new rhizome. The short internodes elongate a little and start developing adventitious roots. At the end of the growing season, the plantlets arisen on the rooted nods of runners are already similar to the original plants with homogenous root system. On the side of the adventitious roots, new branches (side-roots) are formed. The root-branches are thinner but their capillary zone is more developed being more active in uptake of water and nutrients. The usual thickening ensues later.
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Anatomical study of the leaves and petioles of scab resistant and susceptible apple cultivars
53-57.Views:393Anatomic studies have been performed on the leaf blade, petiole and annual shoot on six apple cultivars by means of scanning electron as well as light microscope. Four of the cultivars examined are resistant to scab (Florina, Freedom, MR-10, MR-11), whereas two of them are susceptible (Jonathan and Idared). Preliminary results suggest that differences in the width of cross sections of leaf blades, in hairyness, in the shape and size of epidermal cells, moreover, in the cross sections of petioles and shoots are considerable. Some of the anatomical properties seem to be correlated with scab resistance or susceptility of the respective cultivars. Therefore, further studies extending to other cultivars may corroborate our claims to find causal relations between anatomical traits of the leaves and disease, especially scab resistance of apple cultivars.
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Callus induction on standard type Cymbidium cultivars
108-110.Views:371Tissue 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 naphtylacetic 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.
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Frost induced changes in enzyme activities and carbohydrate content in the spurs of some pear cultivars during the dormancy
41-44.Views:607Frost tolerance of pear cultivars was checked after artificial cold treatment in 2003-2005. Limbs collected during the endodormancy were exposed in a climatic chamber for 24 hours to —25; —28 °C, while those collected in the ecodormancy were kept at —15 and —18 °C. Frost damages of buds were registered according to a visually defined scale, then peroxidase (POD), polyphenol oxidase (PPO) enzyme activities and carbohydrate contents were checked in buds and spur-part below the buds. POD activity of untreated control in tissue below buds was higher than in the buds, which were increasing continuously during the endodormancy and decreased at the end of the ecodormancy. During endodormancy, cold treatment of —25 and —28 °C effected different changes of enzyme activity in buds of the cultivars. In the ecodormancy, enzyme activities increased after a cold treatment of —15 °C, whereas the activities decreased significantly after —18 °C. `Kaiser' — susceptible to frost — with its higher values of both enzyme activities marked out from other cultivars, which is correlated with its stress response. Changes in carbohydrate components — especially in glucose — of buds monitored well the different stress responses of tolerant and resistant pear cultivars induced by frost stress.
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Study of different factors of grapevine regeneration systems and genetic transformation
33-36.Views:375The 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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Shoot induction and plant regeneration from cotyledon segments of the muskmelon variety "hógolyó"
61-64.Views:350Cotyledonary 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.
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Micropropagation of Rudbeckia hirta L. from seedling explants
105-108Views:453We 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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Burrowing nematode (Radopholus similis) abundance, distribution and agronomic impact on bananas in Kirinyaga County, Kenya
7-14.Views:226Burrowing nematodes (Radopholus spp.) are the most significant parasites of bananas (Musa spp.) world over. They feed on the root tissue of bananas resulting to toppling disease at the fruit bearing stage. This study assessed the impact of farmers selected agronomic practices that influenced distribution of burrowing nematodes. We also assessed the abundance of Radopholus spp. across four AEZs namely; UM 2, UM 3, LM 3 and LM 4 of Kirinyaga County, Kenya. Purposive sampling was done on April, 2024 targeting farmers that grew ‘dwarf Cavendish’ variety of bananas, those that grew an average of 25 banana stems and those whose farms that were 1km apart. A structured questionnaire was issued to ninety farmers to collect demographic information and to determine their awareness of burrowing nematodes and toppling disease. Soil and banana root samples were also collected to determine abundance and distribution of burrowing nematodes. Data from the questionnaire and samples was subjected to R -statistical software and analysed at p≤ 0.02. The study revealed that majority of banana farmers practised mixed cropping and were not aware of toppling disease associated with Radopholus spp. It also revealed that majority of banana farmers were male and of ˃ 50 years of age. The findings also showed Radopholus spp was rarely distributed in lower AEZs of LM 3 and LM 4 compared to Pratylenchus spp. which was pre- dominantly distributed in upper AEZs of UM 2 and UM 3 in both soil and root samples.
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High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells
99-102.Views:334We 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 evaluation of grape vine decline pathogens in the experimental field of the Georgikon Faculty of Agriculture in Cserszegtomaj
19-22.Views:368Vine decline causes important economic loss in viticulture, especially in longer view. Several causal pathogen were described lately, however little is known about the etiology or epidemiology of these pathogens on grapevine rootstock. It is well known that these diseases affect grafted and rooted grapevines and are not specific to any scion-rootstock combinations. Our aim was to determine what pathogens are presents in the experimental field, especially causal agents of the rootstock decline. Different grapevine rootstocks and scion varieties were tested in our trial. Isolations were made from the wood tissue and pathogenity tests were done with isolated Cylindrocarpon destructans. The possibility of infection during the propagation process was studied as well. Most commonly Cylindrocarpon sp. and Phomopsis sp. species were identified from the examined varieties. Cylindrocarpon destructans was able to spread to apical (shoot) and basal (root) direction from the point of infection with uneven speed. Callus development is not inhibited by the fungi causing the leaf symptom of the vine decline. Shoot development is reduced if unhealthy parts are grafted to each other.
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Improved clonal approaches to growing black locust (Robinia pseudoacacia L.) in Hungary: a case study
53-56.Views:537In Hungary black locust (Robinia pseudoacacia L.) is considered as an important exotic stand-forming tree species and due to climate change effects its importance is increasing in many other countries. It has some desirable characteristics from both the practical and research standpoints. As a result of a partly new black locust selection programme new black locust clones were improved and a technology was developed for mass clonal micropropagation of juvenile trees. Clone trials with micropropagated plants were established in the country for evaluating the juvenile growth and the stem form of promising black locust clones under marginal site conditions. Significant differences (P<5%) were found for stem form value which partly verified the genetic gain of the selected clones against the common black locust. It was also proved that tissue culture could offer partly new prospects for the rapid mass cloning of selected genotypes.
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Attempting Regeneration from Cultured Cotyledons and Plant Regeneration from Cotyledonary Nodes in Common Bean (Phaseolus vulgaris L.)
57-60.Views:371Dry 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 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.
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Factors affecting rhizogenesis in vitro and acclimatisation of three cherry rootstocks
40-46.Views:274Factors affecting rhizogenesis in vitro and acclimatisation of three rootstocks of cherry, i.e. Mahaleb, Maxma-14 and Weiroot -10 were investigated.
Rooting was easily achieved within 2-4 weeks on MS-based liquid or agar-gelled media containing auxins IBA at conc. 0.49 or 2.45 pM or NAA at conc. of 0.49 pM. On liquid media with 2.45 pM IBA, a maximum rooting efficiency of 95-100% was obtained. However, high concentrations of auxin delayed the time of root initiation for 3-5 days.
Rooted plantlets were transplanted into pots with a mixture of 3:1 (v/v) peat:perlite and acclimatised gradually to field conditions with efficiency of 60%.
