Mojgan Parvandi; Mohammad Farsi; Mohsen Ashrafi
Abstract
Introduction: The white button mushroom does not produce remarkable yield in the third flash. Nutritional deficiency and the inability of this mushroom to efficient use of compost are mentioned as its reasons. Basically, compost includes two major food components, lignocellulose and microbial biomass. ...
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Introduction: The white button mushroom does not produce remarkable yield in the third flash. Nutritional deficiency and the inability of this mushroom to efficient use of compost are mentioned as its reasons. Basically, compost includes two major food components, lignocellulose and microbial biomass. But this microbial biomass provides just 10% of button mushroom food needs. According to research studies, differentenzymes in both white button mushroom and oyster mushroom are responsible for decomposition of lignin compounds in compost media, from begin of mycelium grows to the end of fruiting. Lacasse, manganese peroxidase, lignin peroxidase, glyoxal oxidase enzymes contribute to degradation of lignin compounds in degradation mushroom has proven by researchers however itis dependent on mushroom types. Manganese peroxidase enzyme (EC. 1.11.1.13) is an extracellular parser lignin enzyme that has a central peroxidase core. Manganese peroxidase enzyme oxidizesMn2+ to Mn3+ and then Mn3+ oxidizes phenolic structure to fonoxile radical. Produced Mn3+ is very active and makes complex by chelating organic acids that is produced by mushrooms such as oxalate or malate. Mn3+ ions become stable by helping of these chelates and it can penetrate through materials such as wood. On the other hand, in recent years, plant biotechnology provides new solutions for old problems such as use of microorganisms, particularly using bacteria for gene transfer and improvement of superlatives. For a sample of this method, Agrobacterium-mediated transformation system can be noted. In addition, the use of suitable promoters for heterologous genes expression in suitable hosts is an important strategy in functional biotechnology that has been raised in edible mushroom genetic engineering. The lack of efficient and sufficient use of compost, low power of white button mushroom in competition with other rivals, lack of yield per area unit due to production costs, pests and diseases, low flexibility and adaptability with environmental conditions changes are some of the problems that the mushroom reformers are faced. Unlike the great efforts made by researchers, conventional breeding techniques to produce the A. bisporus mushroom only have been led to produce a few new races. Therefore, todays some problems associated with traditional methods of breeding of edible mushrooms, including the need to provide races that have desired characteristics, the traditional method performance tests and low chances of success in the transfer of important agronomic characteristics such as functionality and disease resistance. So, they almost have been replaced with new biotechnology methods. Anexample of this method is to manipulateproperties transformation for the particular purpose. Modification of both expression or type of lignin degrading enzyme are possible solutions to deal with this problem, but these are not applicable or are difficult to be done with traditional breeding programs. In recent years, gene transformation mediated with Agrobacterium routinely is used for gene transformation to mushrooms and is proposed as a method for removing limitations of white button mushroom breeding.
Materials and Methods: In this research, the oyster mushroom strain Florida was used as the source of manganese peroxidase (mnp) gene and white button mushroom strain 737 gill and cap tissue were used as transformation host. Agrobacterium strain LBA4404 harbors p133H88-FM plasmid thatcontainsmnp gene of oyster mushroom and also hph gene under control of gpdII promoter of the button white mushroom strain IM008 was used as a transformer. Selection medium containing 30 mg/ml Hygromycin B and was used for selecting transformed explants. To confirm transformation, PCR with specific primers of mnp and hph genes was performed on genomic DNA of selected colonies.
Results and Discussion: Results showed the gill tissue explants, with transformation rate 5%, have a better response to applied transformation method than cap tissue explants, with transformation rate zero percent. As expected, polymerase chain reaction with specific primers ofhph and mnp genes amplified 1049 and 1086 bp fragments and verified the transformation of mycelium's grown on selection medium. It seems that Bacterial strain and also used plasmid were one of the responses for observed low rate transformation which is in accordance with leach and co-workers study. Finally, we could propose that cap tissue is more suitable for further gene transformation of this mushroombecause of high transformation rate of cap tissue.
Mahdi Ghabouli; Ahmadreza Bahrami; Farajollah Shahriari; Jafar Zolala; Ali Mohammadi
Abstract
Plant tissue culture techniques are used as basic requirement of common plant transformation systems. In most cases of plant transformation, a reproducible regeneration protocol is the limiting step due to long time lasting, specialized facilities and well experienced persons. Furthermore, tissue culture ...
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Plant tissue culture techniques are used as basic requirement of common plant transformation systems. In most cases of plant transformation, a reproducible regeneration protocol is the limiting step due to long time lasting, specialized facilities and well experienced persons. Furthermore, tissue culture procedures induce somaclonal variation among regenerated transgenic plants. Therefore, recently current studies in plant molecular biology prefer plant transformation procedures avoiding tissue culture phase. Various in plant a transformation procedures have been explored, among which the floral dip method is the most reliable in vivo transformation method. In this research, with the aim of evaluating the ability of floral dip method for genetic transformation of some Apiaceae plants, we studied Dill (Anethum graveolens), Fennel (Foeniculum vulgare), Coriander (Coriandrum sativum), Carrot (Daucus carrota), Parsley (Petrocelium sativum) and Celery (Apium graveolens). Arabidopsis thaliana was used as a model plant of experimental procedure. Flowers, in different stages of inflorescence development, were immersed in different suspension of Agrobacterium tumefaciens carrying the plant binary vector pBI121. This vector carries plant reporter gene uidA (gus) and the plant selectable marker gene npt II. Although, producing transgenic Arabidopsis plants with a high transformation rate of 4% verified the accuracy of experimental procedure, floral dip method was not successful for transformation of Apiaceae plants. Only one transformed celery plantlet, carrying nptII gene with no expression of GUS, was obtained bby screening more than 10000 seeds produced by treated plants from all the species. Transgenic Arabidopsis plants expressing gus reporter gene were confirmed through PCR and histochemical assays.
