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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Mushroom breeding!
Until about 1980, button mushrooms had been improved by selection: the discovery and preservation of mutants and of spore cultures that showed improvements relative to their parents. Between 1970 and 1980 several laboratories learned techniques for cross-breeding two strains of A. bisporus to produce a hybrid strain. The first successful commercial hybrid strains were the Horst 'U1' and 'U3' strains, developed by Dr. Gerda Fritsche at the Proefstation voor de Champignoncultuur in The Netherlands. The U1 hybrid now forms the bloodstock of almost all commercial white button mushroom strains grown outside of Asia. Dependence upon a single crop genotype is called 'monoculture'. It raises the risk that a large fraction of the global crop could be susceptible to a new or mutated pathogen. Serious pathogen outbreaks have affected the button mushroom crop previously: LaFrance virus disease in the 1960s, and aggressive Trichoderma harzianum strain types in the 1990s. A basic goal of crop breeding is genetic diversification, to reduce and manage such risks. Further goals of mushroom breeding often include some or all of the following improvements: Resistance to disease Bacterial blotch, mummy (Pseudomonas spp.) Verticillium spp. Trichoderma spp. Mycogone sp. LaFrance virus Better utilization of substrate (compost) nutrients Flavor Appearance Crop reliability Profitability Nutritional or health value Breeding and diversification depend upon the availability of large numbers of genetically different individuals (= strains). Until recently, major culture collections held as few as 12 (or fewer) genetically distinct strains of A. bisporus. The Agaricus Resource Program is the first program designed to increase the amount of Agaricus germ plasm available to mushroom breeders worldwide. In the early 1970's, California scientists first succeeded at splicing viral and bacterial DNAs in the test tube, heralding the birth of the recombinant DNA (rDNA) era, popularly known as genetic engineering, gene transfer technology, gene splicing, molecular biotechnology, and transgenics. This new biotechnology found immediate application in the production of pharmaceuticals, where synthesis by rDNA microbes provided a quantum leap in efficiency over the laborious extraction of miniscule amounts from other sources. Early on it was stated that "the uses of biotechnology are only limited by the human imagination." Today we are witnessing how this broad-based science is impacting virtually every sector of our society. It was during the 1980's when the power and potential of the burgeoning discipline of genetic engineering was first brought to bear on the improvement of agricultural productivity. The discovery of techniques to transfer genes to the major agronomic crops, including corn, soybean, and wheat, from unrelated species provided breeders with new vistas for increasing the efficiency of food crop production. Remarkable progress, far exceeding early predictions, has been made during the last two decades in breeding plants with new traits such as insect, viral, and fungal resistance, herbicide, stress, and cold tolerance, delayed senescence, improved nutritional features, and others. The global demand for transgenic crops is projected to be a $25 billion market by the year 2010. The growth of this industry will be propelled, in part, by "Golden" rice, which was engineered using a daffodil gene to be rich in beta carotene and thereby the promising answer to the vitamin A deficiency problem pervading the developing world. Despite concern for the unforeseeable health and environmental risks posed by genetically-modified (GM) crops, gene transfer technology has irreversibly revolutionized plant breeding. Today, more than 100 plant species have been modified by gene splicing for improved sources of food, fiber, or ornamentation. More than 50 new crop varieties have cleared all federal regulatory requirements and stand approved for commercial retail. Because field testing is an essential step in the commercialization process, the number of permits issued by the U. S. Department of Agriculture, Animal and Plant Health and Inspection Service (APHIS) for GM crops provides a measure of the interest in transgenic breeding. During a 16-year period, more than 8,000 permits and notifications (fast-track permits) were issued, rising from a low of 9 in 1987 to a high of 1,120 in 2001 (Fig. 1). For the first three months of 2002, 536 permits/notifications were recorded by APHIS with 49% involving insect resistance, 33% herbicide tolerance, 7% each for product quality and agronomic properties, and with the balance comprising fungal and viral resistance and other traits. Thus, the "genie out of the bottle" scenario describes the status of agricultural genetic engineering. Despite the anti-GM sentiment expressed by a vocal minority, the potency of the new biotechnology for problem solving has been realized to an extent that is far too compelling for it to be disregarded. Genetically Engineering the Button Mushroom For almost as long as scientists have been introducing genes into crop plants using molecular biotechnology, others have attempted with limited success at developing a gene transfer method for Agaricus bisporus. A major breakthrough came in 1995 with the surprising discovery that the bacterial workhorse, Agrobacterium tumefaciens, used to shuttle genes into plants, also operated with yeast fungi. Shortly thereafter, this method was extended to filamentous fungi, including A. bisporus. Agrobacterium is a common soil bacterium with a worldwide distribution. It causes a disease known as crown gall on hundreds of woody and herbaceous plant species, but most commonly pome and stone fruits, brambles, and grapes. In its normal life cycle, the bacterium transfers a tiny bit of its DNA into the plant DNA resulting in the formation of galls. These galls serve as food factories for the mass production of the bacterium. Over the years, scientists learned how to develop disarmed strains of the bacterium that were incapable of inducing galls, but retained the ability to transfer DNA. In essence, a natural biological process was harnessed to create a bacterial delivery system for moving genes into plants, and now fungi. Though Agrobacterium was shown to be highly promiscuous in shuttling genes into a spectrum of plant and fungal species, the method was still too inefficient to be applied to the breeding of A. bisporus. More recently, we devised a convenient and effective Agrobacterium-mediated 'fruiting body' gene transfer method holding the promise of a powerful tool for the genetic improvement of the mushroom. In our experiments, a small ring of DNA carrying a gene for resistance to the antibiotic, hygromycin, was transferred to a disarmed strain of the Agrobacterium. The antibiotic resistance gene is referred to as a selectable marker, because mushroom cells receiving this gene from the bacterium become marked by the resistance trait and can be selected based on the ability to grow on a hygromycin-amended medium. The end result is a mushroom strain having the newly acquired characteristic of hygromycin resistance. Such a strain has little commercial value, but rather the resistance trait was a research tool that allowed us to easily determine if the bacterium had transferred the gene to the mushroom, and exactly how efficiently it did so under different experimental conditions. Today, and more so in the future, this gene is being replaced or complemented by genes that will confer commercially relevant traits. Figure 2 highlights the steps in the 'fruiting body' gene transfer method. In this procedure, gill tissue is taken from mushrooms approaching maturity, but with the veil intact, so as to ensure some degree of sterility. Next, the tissue is cut into small pieces and vacuum-infiltrated with a suspension of Agrobacterium carrying the antibiotic resistance gene. In a process referred to as co-cultivation, the gill tissue and bacterium are grown together in the laboratory for several days, during which time the bacterium transfers the resistance gene to the mushroom DNA. Because not all mushroom cells receive a copy of the gene, those that have can be distinguished from those that have not by the ability to grow on the antibiotic medium. After 7 days on the medium, mycelium of A. bisporus appears growing at the edges of some of the gill tissue pieces. After 28 days, upwards of 95% of the tissue pieces will have regenerated into visible cultures. At this point, the GM cultures can be transferred to a standard growth medium, and used to prepare grain spawn in the ordinary manner. Figure 3 depicts the first of two cropping trials carried out at the Penn State Mushroom Research Center involving GM mushroom lines. In these trials, all six antibiotic-resistant GM lines mirrored the parental commercial hybrid strain in colonizing the compost and casing layer. Further, the GM lines produced mushrooms having a normal appearance and, in some cases, yielded on a par with the commercial strain (Table 1). Expression of the resistance trait in the mushrooms could be easily demonstrated by placing pieces of the cap or stem tissue on the antibiotic medium and observing for growth (Figure 4). These experiments were crucial, because the results established for the first time that a foreign gene could be introduced into A. bisporus without having a detrimental effect on its vegetative and reproductive characteristics. Table 1. Productivity of genetically-modified (GM) mushroom lines expressing the antibiotic resistance gene that were derived from a commercial off-white hybrid strain. Yield (lbs./sq. ft.) Line Trial I Trial II Commercial hybrid 3.00 a 3.68 a GM-1 2.08 d 0.86 d GM-2 1.73 d 1.45 d GM-3 2.52 bc 2.70 c GM-4 2.12 cd 2.99 bc GM-5 2.90 a 3.63 a GM-6 2.86 ab 3.59 a Means within a column having the same letter are not significantly different according to the Waller-Duncan K-ratio t test at P<0.0001 Impact of Transgenic Breeding on Mushroom Cultivation The overwhelming popularity of the hybrid mushroom strains introduced in the 1980's has created a near global monoculture that is precarious from the standpoint of disease and pest susceptibility, and has limited the choice of production characteristics and the range of tolerance to environmental and cultural stresses. During the last two decades, no notable advances have been made in breeding strains with strikingly improved features. This is due largely to the cumbersome genetics of A. bisporus and a shortage of commercially desirable traits. There is movement afoot in using traditional breeding to explore wild isolates of A. bisporus as a source of new traits. Though this represents an important step towards expanding the genetic base of cultivated A. bisporus, it is not yet clear which traits exist in the wild germplasm collection, and if they can be successfully bred into commercial strains. The advent of a facile gene transfer technique for A. bisporus enables the exploration of genetic solutions to problems confronting the mushroom industry in a realm never before imagined. The awesome power of transgenics lies in what is known as the universality of the genetic code. The biochemical alphabet consisting of the letters G, A, T, and C that spells the DNA sequences of genes controlling traits is identical for all organisms. A scientist blindly handed a gene would have difficulty determining if its source was a mushroom, mouse, or man. It is this unifying feature of genes from all walks of life that makes transgenics so potentially powerful, while it is the tools of molecular biology that unleashes this power so this potential can be realized. Simply stated, the new biotechnology permits the exchange of genetic information between organisms outside the confines of the natural breeding barrier. No longer is the genetic improvement of the mushroom decided by the question of sexual compatibility or traits found within the species. At another level, gene transfer technology will vastly accelerate our understanding of the molecular mechanisms underlying commercially relevant characteristics. It also will serve to strengthen the muscle of our industry's scientific arm, growing from a handful of mushroom researchers to the global workforce of molecular biologists. As one hypothetical illustration, the quest to breed robust resistance to dry bubble disease would not be restricted to a few scientists searching within A. bisporus, where it may or not exist. Instead, it would extend to scores of scientists working on unrelated organisms who have discovered resistance genes to other Verticillium species. Importing these genes to the mushroom for an evaluation against dry bubble is now possible. As farfetched as this may seem, it is precisely this trans-species approach that has met with commercial success. Genetic manipulations of this sort have been carried out on crop plants and include, importing cry genes from the Bacillus thuringiensis bacterium for insect resistance, a synthetase gene from Agrobacterium for glyphosate herbicide resistance, the nitrilase gene from the Klebsiella pneumoniae bacterium for bromoxymil herbicide resistance, a hydrolase gene from the Escherichia coli bacterium for modified fruit ripening, the barnase gene from Bacillus spp. for male sterility, and viral genes for virus disease resistance. It cannot be overstated that gene transfer technology is not a panacea whose arrival marks the departure of traditional breeding. Quite the contrary, it is a new tool at the disposal of the breeder that will complement existing techniques, while offering a far broader range of options for successfully affecting genetic solutions to problems. Gene splicing will expedite the breeding process, transferring much of the time in development from the field to the laboratory. It will enable the introduction of genes with a surgical precision and from exotic sources, which otherwise would be unattainable by more conventional methods. It is important to recognize, however, that in the end, the forces of nature overcoming a trait (e.g., the breakdown of insect resistance) would act with the same intensity on the controlling gene whether introduced by traditional or transgenic breeding. The melding of gene transfer methods with traditional techniques in a mushroom breeding program may take several forms initially, only to be continually refined, streamlined, and improved for higher efficiency and greater effectiveness. Many transgenic manipulations with A. bisporus will require the transfer of the gene to both parental lines so that their offspring mimic the natural inheritance process by carrying a duplicate copy of the gene. For other applications, introducing a single copy of the gene may achieve the desired effect. In either case, the resulting GM lines may require further selection before emerging as worthy commercial strains The Perils of Genetic Engineering If the decision to exploit genetic engineering for agricultural improvement was left to scientists, the cultivation of GM crops would probably be far more widespread and diverse than it is today. But science does not occur in a vacuum. Political forces reflecting the pendulum of public opinion have a strong bearing on the direction and timetable of scientific progress. The early comment that, "the uses of biotechnology are only limited by the human imagination" was used within the context of its seemingly boundless benefit to humanity. In actuality, human imagination has limited biotechnology. That food crops created by genetic engineering are unnatural to the extreme of threatening human health and the delicate balance of the environment is a perception held by a segment of our society. Whether or not these fears are rationale is irrelevant, because their mere existence has hampered the growth of genetic engineering in agriculture. As with many new technologies, the question of acceptance by society will be answered through a distillation of the benefits to be derived for the risks that must be taken. Both transgenic and conventional plant breeding strive to increase yield, improve quality, and reduce production cost. However, the two breeding strategies differ enormously in the manner in which the end is achieved. For many, it is the process of genetic engineering and not the final product that is most disconcerting. Removing the element of compatible sexual crosses from breeding and reducing it to the splicing of genes in the laboratory seems highly unnatural, constituting extreme human intervention. True, only transgenic breeding allows genes from exotic sources to be brought together in unique combinations. This has been criticized for the possibility of creating new and unpredictable food-borne allergies and toxicities. But the conclusions drawn by the American Medical Association, Board on Agriculture and Natural Resources and National Research Council, and Institute of Food Technologists, among other organizations, agree that GM food poses no greater threat to human health than conventional food. Consider the tomato breeder seeking to transfer disease resistance from a wild species to the cultivated species. The traditional approach would be to cross the wild species with the domesticated species producing an offspring having inherited half of its genes from one parent and half from the other. In an effort to filter out the undesirable traits contributed by the wild species, the breeder would repeatedly cross the offspring with the cultivated species. However, this process is imperfect, so the new commercial tomato variety would possess the resistance gene and other contaminating genes from the wild species. Now, in the transgenic approach, the resistance gene alone would be snipped from the DNA of the wild species and transferred to the cultivated variety. Here, the risk of altering the food constituents is greater with the conventionally bred variety than the GM variety. For this reason, a likely backlash of the genetic engineering controversy will be stricter regulation of food crops bred by conventional means. As a matter of fact, GM food is federally regulated and adequate safeguards for quality assurance are in place. There is no logical reason to believe that a GM food product would be any more threatening to human health or any more difficult to evaluate for safety than say, for example, a new drug. Another safety concern with GM food crops revolves around the unintended consequences associated with introduced genes escaping GM crop plants to other species. Science cannot predict with absolute certainty the non-targeted effects of GM crops on the environment, but it can determine which native species could acquire an escaped gene by cross-pollination. More importantly, science is now beginning to appreciate that genetic exchange among unrelated organisms occurs in nature. Therefore, it can be argued that moving genes between unrelated species by transgenic breeding only accelerates this natural evolutionary process. In order for GM food to reach mainstream society, a greater emphasis must be placed on trait improvements that will benefit the consumer. Most of the genetic engineering accomplishments with crop plants have involved input traits, such as herbicide tolerance and disease and insect resistance. Farmers have embraced the new biotechnology with open arms, because GM crops have reduced their workload or increased profit. But what incentives exist for the consumer to choose GM over non-GM produce in the marketplace? The Agbiotech giants now realize this and are redirecting research towards output traits offering greater consumer appeal, as for example, improved shelf life, appearance, color, flavor, nutrition, hypoallergenicity, etc. The Shape of the Future The pace at which genetic engineering is implemented in the mushroom industry will be determined solely by economic factors related to necessity and the resources committed to R & D. If transgenic breeding offered a solution to a problem threatening the livelihood of the mushroom industry today, then the growing of GM strains would become widespread tomorrow. This 'do or die' scenario played out in Hawaii, where a ringspot virus was literally decimating the papaya industry. Fortunately, the fruits of a transgenic breeding effort underway for many years provided a solution. Virtually all papayas now produced in Hawaii are GM for virus resistance. Another economic force driving the rate at which transgenic breeding reaches the mushroom industry is the level of emphasis placed on R & D. Mushrooms lag far behind other crops in molecular biotechnology, so it is likely that the path through public opinion to acceptance of GM food will be forged by these other commodity groups. As the climate for GM food improves, so will the research funding for the transgenic breeding of mushrooms. For the time being, scientific meetings will be punctuated by modest advances in mushroom transgenics contributed primarily by laboratories in Europe and Far East. Early transgenic breeding achievements with mushrooms will likely shadow those on cultivated crop plants. Because of funding constraints and technical ease, traits controlled by single genes will be targeted initially, including viral and fly resistance and possibly resistance to bacterial and fungal pathogens, pesticides, and bruising. With the mapping of the mushroom genome and an increased understanding of genetic mechanisms, complex traits controlled by more than a single gene will be undertaken. Improvements might be expected in the areas of yield, size, color, shelf life, heat and water stress, food constituents, fruiting cycle regulation, sexual compatibility, strain stability, and substrate utilization. Mushrooms will be explored as bioreactors for the synthesis of valuable pharmaceuticals and other bioproducts. The idea of growing the mushroom as a factory rather than a food offers several possible advantages over existing plant-based schemes (i. e., tobacco and corn). A high biomass of mushrooms can be produced on low-cost waste material in a secure containment facility with a controlled, HEPA-filtered environment, and with the option for mechanical harvesting. Further, it may be learned that proteins manufactured by mushrooms have higher specific biological activities in humans than those produced in plant counterparts. By virtue of the foreign gene introduced and its location within the mushroom DNA or, alternatively, through the deliberate introduction of small snippets of DNA as molecular signatures, it will be possible for spawn manufacturers to definitively identify their strains. This ability to fingerprint strains with ease will afford greater patent protection, which, in turn, will provide the resources to expand breeding programs. The economic incentives related to patented strains also may attract new, perhaps venture capital funded parties to strain development and spawn manufacturing. The mushroom industry as a whole would benefit from the increased competition through a greater selection and diversity of mushroom strains. Our industry is on the brink of a new and exciting age of strain improvement of a like never experienced before. Many of the accomplishments being realized for cultivated crop plants through transgenic breeding might now be achieved for mushrooms. The availability of mushroom strains with genuinely novel and obviously improved traits will provide the industry with new options for solving problems, simplifying tasks, increasing the efficiency of production, and usage. Though the timetable for its application to mushroom cultivation remains an uncertainty, to paraphrase, "genetic engineering will be persistent, it will be pervasive, and it will be everlasting." Relevant Resources Altman, A. 1999. Plant biotechnology in the 21st century: the challenges ahead. EJB Electronic Journal of Biotechnology 2:51-55. Available at http://www.ejb.org/content/vol2/ American Medical Association. 2001. Genetically-modified crops and foods. Report 10 of the Council of Scientific Affairs (I-00). Available at http:/www.ama.assn.org/ama/pub/p Animal and Plant Health and Inspection Service. APHIS field test permits. Available at http://www.aphis.usda.gov/ppq/bi Board on Agriculture and Natural Resources and National Research Council. 2002. Environmental effects of transgenic plants: the scope and adequacy of regulation. Committee on Environmental Impacts Associated with Commercialization of Transgenic Plants. National Academy Press. 342 pp. Bundock, P., A. Den Dulk-Ras, A. Beijersbergen, and P. J. J. Hooykaas. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14:3206-3214. Chen, X., M. Stone, C. Schlagnhaufer, and C. P. Romaine. 2000. A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl. Environ. Microbiol. 66:4510-4513. Conway, G., and G. Toenniessen. 1999. Feeding the world in the twenty-first century. Nature 402 (Suppl):C55-C58. Cornell University. 1998. First genetically engineered papaya released to growers in Hawaii. New York Agricultural Experiment Station. Available at http://www.nysaes.cornell.edu/pu De Groot, M. J. A., P. Bundock, P. J. J. Hooykaas, and A. G. M. Beijersbergen. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16:839-842. Food and Agriculture Organization of the United Nations. Statement of biotechnology. Available at http://www.fao.org/biotech/state Food and Drug Administration. Biotechnology main page. Center for Food Safety and Applied Nutrition. Institute of Food Technologists. 2001. Expert report on biotechnology and foods. Institute of Food Technologies, Chicago, IL. 56 pp. Pennsylvania State University. Biotechnology: food and agriculture. Available at http://biotech.cas.psu.edu/. Persidis, A. 1999. Agricultural biotechnology. Nature Biotechnology 17:612-614. Robinson. J. 1999. Ethics and transgenic crops. EJB Electronic Journal of Biotechnology 2:71-80. Available at http://www.ejb.org/content/vol2/ Snow, A. A., and P. M. Palma. 1997. Commercialization of transgenic plants: potential ecological risks. BioScience 47:86-96. U. S. Department of State. 2001. Biotechnology creates a green gene revolution. International Information Programs. Available at http://usinfo.state.gov/topical/ World Health Organization. Genetically modified food main web page. Available at http://www.who.int/fsf/gmfood/in Ye, X., S. Al-Babili, A. Kl?ti, J. Zhang, P. Lucca, P. Beyer, and I. Potrykus. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303-305. 147 Introduction Basidiomycetes, one of the four branches of the monophyletic group of Eumycota (true fungi), account for about 35% of the fungal species currently described, and are of both ecological and industrial importance. Their ecological impact varies according to their different life styles: saprotrophs (feeding on the remains of dead organisms or wastes), which play a central role in the recycling of organic material because of their ability to degrade some molecules especially reluctant to biodegradation (i.e. lignin breakdown by white rot fungi); symbionts, forming ectomycorrhizae with vascular plants, which facilitate nutrient absorption; or fungal, plant or animal pathogens, responsible for crop losses (Ustilago maydis) or serious human diseases (Criptococcus neoformans). Besides, some basidiomycetes have been traditionally used for human consumption because of their organoleptic characteristics (Boletus edulis, Lactarius spp.), their hallucinogenic properties (Amanita muscaria) or, even, as poisons (A. phalloides). There is a growing industry of edible mushroom production based on a process of solid fermentation of pasteurized or sterilized substrates inoculated with the appropriate spawn that proceeds under controlled conditions of temperature and humidity. This control, however, is far from strict and, in practical terms, the overall process shares more characteristics with open-air composting processes, where different populations of microorganisms compete and establish successions, than with industrially-controlled axenic fermentations. The many factors (both biotic and abiotic) involved in this process very often cause instability in yield or in the quality of the product, as it has been the case for the production of the oyster mushroom, Pleurotus ostreatus, over the last few years. Hence, there is a market pressure to improve the yield and quality of the mushrooms currently produced, and to increase the number of cultivable fungi that has fuelled research aimed to develop breeding programs for edible fungi and to formulate appropriate substrates and culture conditions for new species. Furthermore, this pressure is based on three main reasons: (i) the economic value of some highlydemanded fungal species; (ii) their use to produce enzymes or chemicals useful in industry or pharmacy; and (iii) their application in processes aimed to recycle industrial or agricultural wastes. The development of breeding programs for edible basidiomycetes, however, has been hampered by the difficulties in performing directed crosses between fungal strains, due to incompatibility barriers, by the contradictory data about size and organization of the genetic material, and by the lack of linkage maps to localize genes of interest. In our laboratory, Luc?a Ram?rez Luis M. Larraya Antonio G. Pisabarro Department of Agricultural Production, Public University of Navarra, Pamplona, Spain Received 5 May 2000 Accepted 3 July 2000 Correspondence to: Antonio G. Pisabarro. Departamento de Producci?n Agraria. Universidad P?blica de Navarra. 31006 Pamplona. Spain Tel.: +34-948169107 Fax: +34-948169732 E-mail: gpisabarro@unavarra.es REVIEW ARTICLE INTERNATL MICROBIOL (2000) 3:147?152 ? Springer-Verlag Ib?rica 2000 Molecular tools for breeding basidiomycetes Summary The industrial production of edible basidiomycetes is increasing every year as a response to the increasing public demand of them because of their nutritional properties. About a dozen of fungal species can be currently produced for food with sound industrial and economic bases. Notwithstanding, this production is threatened by biotic and abiotic factors that make it necessary to improve the fungal strains currently used in industry. Breeding of edible basidiomycetes, however, has been mainly empirical and slow since the genetic tools useful in the selection of the new genetic material to be introduced in the commercial strains have not been developed for these fungi as it was for other organisms. In this review we will discuss the main genetic factors that should be considered to develop breeding approaches and tools for higher basidiomycetes. These factors are (i) the genetic system controlling fungal mating; (ii) the genomic structure and organisation of these fungi; and (iii) the identification of genes involved in the control of quantitative traits. We will discuss the weight of these factors using the oyster mushroom Pleurotus ostreatus as a model organism for most of the edible fungi cultivated industrially. Key words Pleurotus ostreatus ? Basidiomycetes ? Breeding fungi ? Mating factors ? Fungal genome structure we have used the oyster mushroom Pleurotus ostreatus as a model system to study these three aspects. P. ostreatus is an edible basidiomycete that grows wildly on decaying wood thanks to its lignin-degrading capacity, and is industrially cultivated on a variety of substrates based on agricultural wastes (such as straw, cotton wastes, sawdust, etc.). P. ostreatus is currently the second major mushroom in the world market led by the button mushroom Agaricus bisporus [32]. Besides its importance for food production, it is of interest for industrial applications such as paper pulp bleaching (by the action of its ligninolytic enzymes) and for cosmetics and pharmaceutical industries [2, 3, 12, 13, 20, 21]. The life cycle of P. ostreatus, as well as those of many other higher basidiomycetes, alternates monokaryotic (haploid) and dikaryotic (di-haploid) phases [9]. Two monokaryotic compatible hyphae are able to fuse and give rise to a dikaryotic mycelium in which the two parental nuclei remain independent (dikaryon, heterokaryon) throughout the vegetative growth, and which will fruit under the appropriate environmental conditions. True diploidy occurs at the basidia where karyogamy takes place immediately before the onset of the meiosis giving rise to four uninucleate basidiospores. At this diploid stage, genetic recombination can occur, although some reports have also suggested the occurrence of parasexual somatic recombination in higher basidiomycetes [37]. The basidiospores can germinate when they find the appropriate environmental conditions producing monokaryotic mycelia that reinitiate the fungus life cycle. The monokaryotic or dikaryotic condition of a mycelium can be distinguished by the presence of clamp connections (specialized structures which allow nuclei distribution into daughter cells) in dikaryons and their absence in monokaryons. Genetic structure of mating genes in higher basidiomycetes Monokaryon compatibility and mating is controlled by two multiallelic genetically independent loci that ensure the transmission of the two nuclei of the dikaryotic cell during cell division [4]: the genes in locus A are responsible for controlling the pairing of nuclei in the dikaryon, for the formation and septation of clamp cell, and to coordinate cell division, whereas genes at the B locus control the migration of the nuclei towards the hyphal tip, the dissolution septa, and the fusion of clamp cells to ensure a correct dikaryotic stage after cell division. This system of mating control is referred to as bifactorial (two loci) or tetrapolar (as it generates four different incompatibility types in the monokaryotic offspring of a dikaryon) and is common to most of the edible basidiomycetes industrially cultivated with the exception of the unifactorial button mushroom Agaricus bisporus [1, 8, 31]. Molecular analyses of the A and B genes in Coprinus cinereus and Schyzophillum commune have revealed that A genes code for homeodomain proteins that, to be functional, should form heterodimers (with one subunit coded for by each one of the two nuclei forming the dikaryon), whereas B genes code for pheromones and their receptors [4]. The genetic structure of both factors is complex. The factor A gene complex consists of a central motif of two genes (coding for the two protein types present in the heterodimer) transcribed in divergent directions that appears duplicated one to three times in the different A mating types and species [4, 6, 14, 19, 24, 25]. The gene complex for factor B has a central unit formed by a single gene coding for a membrane pheromone receptor and a variable number of genes (from two to seven) coding for pheromones [4, 35]. Again, a variable number of copies of this central motif can be found in different B factors and species. In P. ostreatus locus A behaves as a single one [16], whereas locus B is a complex of two genes (matB aand matB ?) linked at genetic distances ranging from 17.5 cM to less than 5.0 cM in the different strains, and new B specificities can appear by recombination between the two loci as it occurs in other higher basidiomycetes [9, 26]. The bifactorial mating control system makes it difficult and cumbersome breeding-oriented crossing of monokaryons. In fact, it is first necessary to determine the incompatibility factors present in monokaryons derived from a given strain using testers for the four basic incompatibility types (Ax Bx, Ax By, Ay Bx and Ay By) appearing in the offspring of a dikaryon AxAy BxBy. By using this method, we have studied the mating factors present in P. ostreatus accessions from a variety of origins and have found nine different A and 15 different B mating types, some of which are the result of intra-factorial recombination (Table 1). Moreover, each different strain analysed carried a different pair of A factors, with only one exception, and a different pair of B factors. The determination of the mating type of a given monokaryon is highly facilitated by the use of molecular markers linked to the mating factors. These markers can be identified, in a first step, using a bulked segregant analysis approach [23] to generate Randomly Amplified Polymorphic DNA (RAPD) markers genetically linked to the genes controlling A and B mating factors in P. ostreatus [16] (Table 2). Due to the number of monokaryons analysed in our study, the minimum linkage distance measurable between markers and the corresponding genes is 1.25 cM (centimorgan). RAPD markers behave as dominant and, in the strain under study, they segregate against a null allele. Consequently, only alleles matA1, matB a2, and matB ?1 are directly detectable using these RAPD markers. This limitation, as well as those derived from the RAPD methodology, can be avoided by converting RAPD markers in Restriction Fragment Length Polymorphism (RFLP) markers, which allow a quick and certain identification of monokaryons because they distinguish the two alleles present in a dikaryotic individual. RAPD and RFLP markers, in addition, allow the study of the genomic areas flanking the mating factors that have been reported to be highly conserved [4]. The sequences of the RAPD markers linked to the mating factors in P. ostreatus show no homology with any other entry in the gene databank, and no 148 INTERNATL MICROBIOL Vol. 3, 2000 Ram?rez et al. obvious open reading frames were found to suggest that they may correspond to coding sequences rather than to intergenic regions. Notwithstanding, when those probes were used in Southern experiments on genomic DNA purified from other P. ostreatus strains, a strong hybridization signal was obtained, whereas, in the same conditions, only a weak signal on DNA from other species of the same genus, and no signal on genomic DNA purified from other agaricales appeared; this suggests a high degree of species-specificity [16]. Table 2 RAPD and RFLP alleles genetically linked to the mating alleles identified in Pleurotus ostreatus var. florida. Alleles placed in the same row are in coupling phase Mating allele RAPD marker RFLP marker matA1 S11900 rS11900 a S181300 rS181300 a matA2 ? rS11900 ? ? rS181300 ? matB a1 ? rL313001 matB a2 L31300 rL313002 matB ?1 L61800 rL618003 matB ?2 ? rL618002 149 Breeding of basidiomycetes INTERNATL MICROBIOL Vol. 3, 2000 Table 1 Mating factors found in different Pleurotus ostreatus strains Strain Variety Origin B factor Occurrence Sample Recombination (mating genotype) of B factor size(a) frequency (%) N001 florida USA B1 63 120 15.8 (A1A2 B1B2) B2 38 B3 11 B4 8 N017 florida UPNA(b) B3 45 102 15.7 (A1A2 B3B4) B4 41 B1 8 B2 8 N002 ostreatus Germany B5 39 98 8.2 (A5A6 B5B6) B6 51 B15 6 B16 2 N018 ostretaus UPNA(b) B15 41 105 4.8 (A5A6 B15B16) B16 59 B5 3 B6 2 N003 ostreatus Spain B7 86 170 0.6 (A7A8 B7B8) B8 83 B17 1 N005 colombinus Italy B11 ? ? ? (A8A11 B11B12) B12 ? N006 sajor-caju India B13 ? ? ? (A13A14 B13B14) B14 ? (a)Number of individuals studied. (b)UPNA: Public University of Navarra, Spain. 150 INTERNATL MICROBIOL Vol. 3, 2000 Ram?rez et al. Fig. 1 Molecular karyotype of Pleurotus ostreatus. A) Clamped Homogeneous Electric Field (CHEF) separation of the chromosomes present in the dikaryon (N001) and in each of the two nuclei (PC9 and PC15). B) Idiotype of the two nuclei (PC9 and PC15) indicating the chromosome length polymorphisms. (Figure from Larraya et al. [17]. Reproduced with permission.) Table 3 Characteristics of the molecular karyotype and linkage map of Pleurotus ostreatus Chromosome Sizea (Mbp) Sizeb (cM) Markers number kbp/cM Average Marker Interval (cM) Cross-over events I 4.70 103.0 23 45.6 4.5 0.98 II 4.35 173.6 23 25.1 7.5 1.71 III 4.55 178.7 25 25.5 7.1 1.75 IV 3.55 59.2 14 60.0 4.2 0.59 V 3.45 82.0 13 42.1 6.3 0.81 VI 3.10 76.7 20 40.4 3.8 0.76 VII 3.15 74.4 18 42.3 4.1 0.74 VIII 2.95 85.3 14 34.6 6.1 0.84 IX 2.10 74.5 16 28.2 4.7 0.74 X 1.75 33.8 13 51.8 2.6 0.34 XI 1.45 59.5 10 24.4 5.9 0.59 Average 3.19 91 16.7 35.1 5.3 0.89 Total 35.1 1000.7 189 a Average of the two homologous chromosomes [17]. b Sum of the linkage distances between the markers placed on the corresponding chromosome. Sizes in centimorgans (cM) correspond to the sum of all the distances between adjacent chromosome markers. 151 Breeding of basidiomycetes INTERNATL MICROBIOL Vol. 3, 2000 152 INTERNATL MICROBIOL Vol. 3, 2000 Ram?rez et al. I thought some of us would find this beneficial!
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Da Bud Guru Registered: 07/25/02 Posts: 5,876 Loc: Near Hilo |
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Interesting. I think (and this is only me I'm sure) something far more important then breeding for potency is breeding for contam resistance.
-------------------- All creatures tremble when faced with violence. All creatures fear death, all love life. If we can only see ourselves in others, then how could we possibly hurt another creature? Join us at the Growery! ![]()
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Keep your stickon the ice. Registered: 04/24/04 Posts: 2,101 Loc: lookin at crazy Last seen: 10 years, 6 months |
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Both would be good though!
-------------------- Remember..if the women don't find ya handsome,they should atleast find ya handy! I do what i can with what i don't have!
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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exactly-the best of both worlds in one small package!
Imagine a contam resistant starin of Psilocybe Cubensis,or any active for that matter.Amazing! Oh yeah before you ask for credits,this is where I pasted this from. http://home.alltel.net/kerrigan/ http://www.im.microbios.org/11se
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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The janitor is currently grafting with wild strains with KC under lab conditions, including limits to only cloned standards followed by GC with non o2 carrier to measure increases in C12H17N2O4P, which would be 284.1 - 284.4 on the GC (to keep it simple for time considerations, ignoring all C12H16N2O increases/decreases)
The Darwin thinking is as expected through hypothesis, the strongest survive. Thus the environmental conditions of an outdoor pasture would not be ideal for strains that have been cultivated indoors over and over in sterile conditions. My thinking is that there needs to be grafting occasionally to keep the potency that nature intended. Levels can be increases through limited control groups, but in the end the genetic make up sets the limits (in laymen terms, a baby from Superman and Wonder woman, or a Wonder woman and Doc34 <grin>. Besides the visual fascination of the latter, the first would be the most productive in the most environment conditions. I am sure there are some Myco-Gods on here that will blow me away on the knowledge since they may have been down this road, but maybe this forum isn't the spot for such debates:) Kudos Doc34 for the knowledge and research search materials you provided. -------------------- Will Screw For Shrooms
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Raoul Duke ![]() Registered: 04/14/04 Posts: 1,342 Loc: Amsterdam |
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That post looks like a 20 page essay.
-------------------- Substrate + jars = $20 Magic Mushroom spores = $12 Growing your own Magic Mushrooms = Priceless
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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.."The antibiotic resistance gene is referred to as a selectable marker, because mushroom cells receiving this gene from the bacterium become marked by the resistance trait and can be selected based on the ability to grow on a hygromycin-amended medium."..
I have an idea now with reverse chemistry to medium that will speed the process of strain seperations..need to bump this to advanced since there is too much to explain for basic 101 chemistry. btw, I wonder if anyone has guessed who this is yet -------------------- Will Screw For Shrooms
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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.."The antibiotic resistance gene is referred to as a selectable marker, because mushroom cells receiving this gene from the bacterium become marked by the resistance trait and can be selected based on the ability to grow on a hygromycin-amended medium."..
I have an idea now with reverse chemistry to medium that will speed the process of strain seperations..need to bump this to advanced since there is too much to explain for basic 101 chemistry. btw, I wonder if anyone has guessed who this is yet -------------------- Will Screw For Shrooms
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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" btw, I wonder if anyone has guessed who this is yet"
HMMMMMMM???????LOL
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Hey I tried to move this to the Advance Cultivation forum,but my Moderator button is stuck,maybe I need batteries!
Can I get some assistance?
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Ferret Farmer Registered: 02/10/04 Posts: 18,296 Loc: Zone ate |
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Of course, great thread so far
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Thanks Loki!
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Connoisseur Registered: 01/26/03 Posts: 1,640 Loc: #108768 in line. |
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"let's make them more potent"
Through which means are we discussing here? Selective breeding, strain cross breeding, or genetic manipulation? The first two is possible, but you must know which strains (or sub-strains) produce the most alkaloids in order to breed them together. The latter is pretty much out of bounds for the hobbyist cultivator... -------------------- To give is to live...
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НơĻ ![]() Registered: 06/13/00 Posts: 29,281 Loc: Shroomery B-list Last seen: 13 years, 8 months |
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I'd be most interested in the contamination resistance....
having one super-resistive strain would be a good investment. -------------------- "..all those molecules thrashing their kinky little tails, hot for destiny and the street." Gibson Nuke baby seals for Jesus! (This has been a +1 production.)
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Anonymous |
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There is nothing stagnant in nature. You make a resistant strain and the competitors learn how to beat the resistance, by changing!!!
It is a temporary fix to your problems. Contamination can be excluded, and this is the BEST METHOD AVAILABLE for long term stability.
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Anonymous |
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As far as increasing potency beyond the range of the species, strain you are working with. FIND a WAY to INCREASE THE AVAILABLITY OF PYRUVATE and NADPH to the fungus when it enters the storage phase, prior to fruiting.
The most important FACT I got out of the gartz experiment with adding tryptamine to the substrate, was that of the 3 % total activity ONLY 22% was derived from the Tryptamine. THAT MEANS THE REST OF THE ACTIVITY 2.6% was derived from TRYPTOPHAN manufactured by the mushroom itself. So substrate ammendment increased activity, but the majority of the increase was not directly created from the introduced tryptamine.
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Shagadelic Registered: 05/01/03 Posts: 879 Last seen: 15 years, 10 months |
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Nice read very interesting Doc
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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Does anyone have a link to find out about the chiral center of the phospate group (L or D) of Psilocybin, 4-phosphoryloxy-N,N-dimethyltryptamine. I doubt it would be a 50/50 ratio since it is from fungus, but will continue to search unless someone knows. I don't have the equipment for some wet chemistry, but hopefully more in depth experiments have been done that has the information I am looking for.
-------------------- Will Screw For Shrooms
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Indolealkylamines
All of the hallucinogenic indolealkylamines can be classified as belonging to the family of compounds known as tryptamines and are substituted 3-(2-ethylamino)-indoles. The tryptamines are a most interesting and biologically useful class of compounds. In the human body, serotonin (5-hydroxytryptamine) functions as a vasoconstrictor, inhibits gastric secretion, stimulates smooth muscle, and is naturally present in the central nervous system where it is involved in neurotransmission44. The 5-methoxy homolog of serotonin is considered to be hallucinogenic in humans as is the 5-methoxy homolog of gramine (3-(N,N-dimethylaminomethyl)-indole)41. Melatonin (N-acetyl-5-methoxytryptamine), formed by the mammalian pineal gland, appears to depress gonadal function and is known to cause contractions of melanophores. Bufotenine, the N,N-dimethyl homolog of serotonin, is classified as a very weakly active hallucinogen and is noted to have extremely unpleasant cardiovascular depressive side effects63. The O-methyl homolog of bufotenine, N,N-dimethyl- 5-methoxytryptamine (5-methoxy-DMT), is reported to be an extremely potent hallucinogen, but it, like all other C-5 substituted indolealkylamines, is not active if taken by mouth22. Both DMT and DET are well known for their hallucinogenic activity, just as both of these compounds are also inactive if taken by mouth. The N,N-dipropyl and diallyl derivatives are also hallucinogenic only if used either parenterally or by inhalation at approximately the same level as DET, whereas higher homologs abruptly become inactive148. The compound 6-hydroxy-DET has been determined to be a major metabolite of DET in man149, and it does not possess hallucinogenic activity150. Conversely, the 4-hydroxy-N,N-dimethyltryptamines (psilocin and psilocybin), are very active hallucinogens when taken orally. The activity of psilocybin (O-phosphoryl-4-hydroxy-DMT) when taken by mouth is not related to the phosphoric acid radical since the pharmacological effects of psilocin (4-hydroxy-DMT) are identical67. Pharmacological information for baeocystin (4-hydroxy-N-methyltryptamine) was not found; however, one would expect hallucinogenic activity to parallel that of the N-alkyl-tryptamines and thereby would expect the drug to be weakly hallucinogenic. It is thought that in the past most clandestine syntheses of indolealkylamines used indole as the starting material144. A modest literature search will convince a clandestine chemist that the use of the Fischer indole synthesis affords access to a greater variety of indole derivatives69,119 as it will also reduce the chance that law enforcement will be alerted by his purchases of essential chemicals. Hence, in the production of indolealkylamine derivatives, the covert chemist need not be limited by the commercial availability of appropriate indole precursors. Relative to those which lack an aryl ring substitution, there is no doubt that the activity of psilocybin/psilocin upon ingestion is due to an enhancement of gastrointestinal absorption which, in turn, must be structurally related to the presence of the C-4 hydroxyl substitution. Therefore, if the CsA amendment were not a consideration, derivatives derived from psilocin would be the obvious first choice. These derivatives are the 4-hydroxy-N,N-alkyl homologs starting with N,N-dimethyl, N,N-methyl-ethyl, and on to N,N-diallyl to give a total of 10 possible derivatives. As is also the case for hallucinogenic phenylalkylamines, alkyl substitution, not to exceed a C-3 moiety, at the position alpha to the side chain nitrogen generally will maintain hallucinogenic activity. This brings the total possible number of hallucinogenic CsA's of psilocin to 40. A somewhat removed second choice would be the same series of derivatives in conjunction with either acetylation or methylation of the indole nitrogen. This would then bring the total number of the possible 4-hydroxy-substituted tryptamine CsA's (less one for psilocin) to 119. The 5-methoxy derivatives of gramine and serotonin are first choices for future CsA's when considering the new U. S. amendment. Substitution at the alpha carbon on the side chain will probably maintain psychotropic activity only for serotonin derivatives. Hence, allowing only a methoxy substituent at the aryl C-5 position, and a substitution at the carbon alpha to the nitrogen (the nitrogen being any combination of hydrogen, methyl, ethyl, n-propyl, and allyl) 75 CsA's can be obtained. Then substitution of the indole nitrogen with either methyl or acetyl brings the total number of possible CsA's that can be argumentatively related to serotonin to 225. An additional series of compounds that could serve as future CsA's under U. S. law are those which are substituted with alkyl groups at the carbon alpha to the side chain nitrogen. Recently, a commercially available tryptamine which has an ethyl moiety substituted at the alpha carbon has become the newest U.S. tryptamine CsA. Known as ET in the illicit CsA drug market is 3-(2-amino-butyl)indole (Etryptamine, Monase? by Upjohn; compound 3, Figure 3). Because ET does not appear in either Schedule I or II of the CFR and is a legally marketed product, ET and derivatives thereof are exempted from control under the CsA amendment. Pharmacokenitic data on ET indicates that it is a monoamine oxidase inhibitor45,90 and psycho-energizer31,118. Hence, ET could produce some degree of hallucinogenic activity in man. In 1986 ET was reported as the she causative agent in a fatal overdose in Duesseldorf, Germany30. This may be one of the few times that a CsA has originated outside of the U. S. The sample of ET which was submitted to our laboratory appears to have been obtained from the Aldrich Chemical Company ($48.05/100g). Unfortunately, it is not yet clear if ET is actually the substance which is producing the biological response being sought by the illicit user. It is the case that the sample of ET we examined and the batch of ET which the Aldrich Chemical Company is presently selling contains a major quantity (about 30%) of the agent shown in Figure 4 which could also be a hallucinogen107,158. Nomenclature for this possible hallucinogen can either be 1-methyl-3-ethyl-1,2,3,4-tetrahydroharmane, or 2,2-dimethyl-4-ethyl-2,3,4,5-tetrahydro-β-carboline. The creation of this substance most probably occurred after synthesis and during the purification of ET. Under anhydrous conditions, the reaction of acetone and ET would give the corresponding enamine which could then undergo a Mannich condensation to yield the hallucinogen132,165. The compound 2-methyl-8-methoxy-4,5-dihydro-β-carboline (harmaline) is considered to be a hallucinogen59 as well as a monoamine oxidase inhibitor23. On the other hand, the compound 2-methyl-8-methoxy-2,3,4,5-tetrahydro-β-carboline is classified as a tranquilizer160. We were not able to attain any literature whatsoever on the hallucinogen shown in (Figure 3, Compound 4), much less any pharmacokenetic data. Hence, due to the apparently unpredictable pharmacological behavior of structurally similar β-carboline derivatives, I will not speculate as to the pharmacological properties of said substance. HMMMMMMM?????
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mneumatic device Registered: 11/27/01 Posts: 565 |
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Do you think that niacinamide and pyruvate, each in forms of dietary supplements could be used in this way?
Thanks -------------------- "The real voyage of discovery consists not in seeking new landscapes but in having new eyes." - Marcel Proust I wish you all ceaselessly flowing moments of happiness.
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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the above post is all I found after searching for your request, I hope that helped,
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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thanks!
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Outbreaks of mold and bacterial pathogens during mushroom cultivation are responsible for large losses in potential yields. Typical control methods based on organohalogen pesticides (e.g. prochloraz) must be reevaluated due to new environmental norms strictly regulating pesticide residues and due to growing consumer demands for food products free of pesticide residues. The goal of this project is to develop a novel environmentally sound pathogen control strategy based on the higher tolerance of cultivated basidiomycete mushroom species towards oxidative stress compared to pathogen species. The project will evaluate the tolerance of three commercially important mushroom species (Agaricus bisporus, Pleurotus ostreatus and Lentinula edoles) to reactive oxygen (superoxide, hydrogen peroxide and hydroxy radicals) and compare their tolerance that of typical pathogen species (Trichoderma spp. Verticillium fungicola var. fungicola and Pseudomonas tolaaasii). The ligninolytic enzymes naturally produces by mushroom mycelium will be used to increase the oxidative stress by adding substrates which generate reactive oxygen. The production of the lingiolytic enzymes (laccase, magnese peroxidase and aryl alcohol oxidase) during each stage of mushroom cultivation will be inventoried and mushroom strains with high ligninolytic enzyme activities will be selected. The oxidative stress enhancing additives for mushroom strains producing laccase and manganese peroxidase include organic redow mediators or trace metals (manganese); respectively, in combination with simple organic acids. The oxidative stress enhancing additives for mushroom strains producing acryl alcohol oxidase are various benzylic compounds. The effect of these substrates on the type and concentration of the reactive oxygen species produced will be measured in vivo and in vitro. Redox mediators will be designed that are useful at catalytica concentrations, or biodegradable and are non-toxic after service. A secondary benefit expected from the enhances oxidative stress is improved substrate utilization by mushrooms due to increased lignin degradation. The feasibility of the optimized technology in generation reactive oxygen, lowering incidences of pathogen outbreaks and increasing mushrooms yields will be verified during the cultivation of mushrooms under practical conditions. This research will result in the development of a low cost environmentally sound pathogen control method to be used during mushroom cultivation without any accumulation of harmful chemicals. The users of the developed technology will include mushroom cultivators, spawn and substrate suppliers, biological crop protection compagnies, mushroom cultivation education institutes and white rot fungal bioremediation compagnies.
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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Has anyone tried casing with reed canary grass,Phalaris arundinacae? It is so easy to grow and grows wild in so many places and since it does have DMT, maybe have alkaloids precursors.
-------------------- Will Screw For Shrooms
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Where can this be found?
"reed canary grass,Phalaris arundinacae"
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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http://plants.usda.gov/cgi_bin/plant_profile.cgi?symbol=PHAR3
There should also be a link to state distribution, although Texas is not listed, I know it is grown in wetland reserves around Texas based on a rice university study. Do a search on your area and the key words. -------------------- Will Screw For Shrooms
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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These?
http://www.santarosagardens.com/ Or these? http://www.santarosagardens.com/
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Anonymous |
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- Post History Deleted Upon User's Request -
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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first link
-------------------- Will Screw For Shrooms
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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When I named this post,I did so for a reason.If a mushroom can be "altered" to be resistant to contams-it can also be,higher yeilding,bigger,better in appearance,taste better,and yes of course it can be made more potent,although thats not why I posted this(but it did get your attention sir).
I could change it if you like? And dont thank me,I dont deserve it-I am just a mear peon amongst many! Thank you!
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Fungitarian Registered: 02/14/04 Posts: 2,667 Loc: Myceliaville !!! Last seen: 7 months, 28 days |
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Cant find a distributer for the seeds,only young plants.
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НơĻ ![]() Registered: 06/13/00 Posts: 29,281 Loc: Shroomery B-list Last seen: 13 years, 8 months |
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On a large, ecological scale, i'd have to agree with you.
But for the micro-cultivator (such as myself) having a very contamination resistant strain would be a godsend. Given the current environment, and my massive slaughter of all airborne pathogens between crops, i doubt that anything would survive long enough to make a more potent form. It would give the fungus the headstart against the current stage of invaders, and thru proper control of atmospheric and other contaminants, would stay that way for some time. I'm just interested in the usage for prevention of accidental contaimination (roommate, cat, air vents, etc.) -------------------- "..all those molecules thrashing their kinky little tails, hot for destiny and the street." Gibson Nuke baby seals for Jesus! (This has been a +1 production.)
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mneumatic device Registered: 11/27/01 Posts: 565 |
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One of the shroomery sponsors carries phalaris seeds. www.iamshaman.com
I've heard things about the DMT degrading when it dries though, does anyone have any info on this? -------------------- "The real voyage of discovery consists not in seeking new landscapes but in having new eyes." - Marcel Proust I wish you all ceaselessly flowing moments of happiness.
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Anonymous |
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- Post History Deleted Upon User's Request -
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Day careobserver Registered: 04/09/02 Posts: 1,780 Loc: Oregon |
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I have an outdoor bed of Azures growing on Phalaris and alder right now.We shall see what if any effect the alkaloids(pretty dilute) in phalaris have any effect on potency. As a straw it colonizes quite rapidly.
WR -------------------- To old for this place
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Stranger Registered: 05/05/04 Posts: 4 Last seen: 18 years, 6 months |
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Used to visit very frequently years back. Lost login. . I must say this is a very interesting post. Wish my mind could absorb 100% of that info, aa so littel time to learn all we want. Interesting nonetheless.
Just tell me what to do to help make them more potent. hopefully a position with the word Guiney in it
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Anonymous |
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The problem with adding anything to the substrate for increasing potency is that the mushroom tends to break it up and use it for primary growth. It uses it as food first then what ever it has left over after growth slows and storage increases, it uses for secondary metabolism.
I would think the timing of the application would be critical also.
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always improving Registered: 09/16/03 Posts: 6,056 Loc: El Paso, TX Last seen: 5 years, 10 months |
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why not just grow high potency shrooms like pesa, penis envy, and p. cyan.
-------------------- My YouTube channel Items i use and recommend https://kit.com/MyersMushrooms Pre built mushroom drum sterilizershttp://www.bubbasbarrels.com/cat
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enthusiast Registered: 05/06/04 Posts: 278 Last seen: 19 years, 3 months |
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Might the 'random mutation' / 'accelarated evolution' method be applicable to craeting new variants of shroom ? It's a bit of a hit and miss 'alchemist' type approach, but it would be easy and cheap to try... put simply, you'd grow up a heterogenous bunch of mycelia, then zap it with varying degrees of gamma radiation... then plate out different samples from the zapped concoction... many will be mutated genetic variants. Refine each 'substrain' to a 'monoculture' (pure strain). Then assess these substrains for the desired traights, for example, speed of growth, or levels or certain chemicals. Pick the best ones, or the substrain that has the most desirable mix of traits, and, you guessed it, grow up a big batch and zap it... repeat above process until you have created the super shroom from wonderland... chuckle... compared with, say, plants, shrooms are much more amenable to this approach, cause you can subculture quickly...
Just a thought, would be interested on anyone's opinion... -------------------- Peace, love and organic brown rice...
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enthusiast Registered: 05/06/04 Posts: 278 Last seen: 19 years, 3 months |
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'General' pathogen resistance would actually be very hard to achieve in my humble opinion, as shrooms are so susceptible to a myriad of infections, moulds, other fungi, bacteria, etc... usually the aim is to make a crop resistant or more resistant to a single pathogen that devastates yields of that particular crop, but inducing 'broad spectrum resistance to everything' seems fanciful. One conceivable way would be to insert multiple antibiotic genes using the agarobacterium as is (beautifully!) described above, so that the shroom itself releases antibiotics into the media/ substrate... but even then as also mentioned above, there will always be variant pathogens around waiting for their 'niche' to crop up... Also, might not be good to eat a bunch of antibiotics with your shroom, I guess there's a possibility of allergic reactions and knocking of all your 'natural flora' ('good' bacteria) from your intestine, actually leaving you more susceptible to colonisation by pathogens... Maybe the most realistic way to enhance pathogen resisttance is indirectly, by generating a superfast growing variety... then it will at least have a better chance of outcompeting pathogens...
Again just my humble little ponderings, would be interested in anyone's thoughts... -------------------- Peace, love and organic brown rice...
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mycoexplorer Registered: 05/02/03 Posts: 270 Loc: indonesia Last seen: 11 years, 9 months |
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I have plenty of seeds of P aquatica var Australia
one of the higher potency strains Where i live now is unsuitable for this grass (subtropics) so i want the seeds out there where they will grow e-mail me at seedring@shaman-australis.com we generally accept stamp donations with Oz or US/Euro/pounds 1 outside. Any extra appreciated as i run this as a voluntary non profit thing and there really is nothing left over, sometimes not enough. We usually chuck in extra species if you add enough to justify the extra postage and packing Ideally we want you to grow the plant and then offer up seed for others and keep the planst in circulation AFSR PO box 1417 Byron Bay NSW 2481 Australia -------------------- www.balikebun.blogspot.com www.tropicalfoodforest.blogspot. www.bulelicious.blogspot.com
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enthusiast Registered: 05/06/04 Posts: 278 Last seen: 19 years, 3 months |
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PS, has anyone used the 'series dilution' method before to derive pure strains from single mycelium ? Essentially it just involves making a series of dilutions from a master stock of unknown concentration, then plating a set volume from each dilution out on a different plate... at a certain dilution factor, you should get just a few cultures popping up (plaque forming units they are called when this method is used to derive pure strains of bacteria or viruses on, for example HB agar) on one of the plates, and these can be reasonably be assumed to be monocultures (pure strains derived from a sing mycelium)... should this work? Can a mycelia culture realistically be expected to develop from one single mycelium, or do they really only happy when there's a few of them around, like for example cultured human lymphocytes... Would it be too slow waiting for a culture to develop from a single mycelium ? Any thoughts appreciated...
Lastly, though this may well be answered in the contam' forum where I'll visit next, can anyone advise on a broad spectrum antibiotic that I can add to my agar after autoclaving/ cooling, that will ensure no bacterial contamination, but have zero effect on the mycelial (p. subaeruginosa) growth ? -------------------- Peace, love and organic brown rice...
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mycoexplorer Registered: 05/02/03 Posts: 270 Loc: indonesia Last seen: 11 years, 9 months |
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re that last query
no No antibiotic will inhibit all bacteria However i have found Chloramphenicol useful at a rate of 50mg/L It is heat sensitive but is added before sterilisation. The agar is sterilised for just the time needed, then cooled and pured As soon as possible to reduce the breakdown This is particularly well suited to slants as they cool rapidly why so much bacterial probs anyway? I find aside from those cryptic baterial infestations (cleaned up by reeated subs) that bacteria are not really an issue if the conditions in your workspace are favourable. Mould is more of a bugbear and yes they will reform from single cells in the manner you spoke of. just takes longer. This has been done both in successfully regenerating mushroom protoplast hybrids (eg shiitake x golden oyster) It was also the means by which high yielding Claviceps purpurea cultures were isolated for producing both the lysergic derivatives and ergopeptides and other ergotoxins. Single cells were plated up to form colonies. each colony was grown out and the level of biosysnthesis measured by culturing from half the colony. The other half of High yielding phenotypes were kept and subjected to the same treatment. Generation after generation it was repeated till they had achieved in some cases an increase in biosynthesis from less than 10 mg/l to greater than 2200mg/L. In some cases this happenned in as little as 5 generations with hard selection In the absence of selective pressure these clones revert back to lower yielding phenotypes The process is simple, so simple that when it was presented by the industrial mycologist to their academic colleagues it was thought to be incredible. The key was the large scale and many repetitions needed to carry out all the thousands of experiments If it works with Claviceps it might also work with Cordyceps, and if it worked for them too then maybe the phenomenon can be exploited in many more fungi, possibly even P cubensis, particularly in liquid bioreactor style. from looking at the acquired characteristics of Plant cell lines in Tissue culture it is also apparent that sometimes these traits selected for such as temp tolerance or increased biosynthesis do lead to higher yileding clones when regenerated, but many times they do not Sometimes the mutation is unstable or environment specific, other times there may be other limiting factors like the plants infrastructure acting as a limiting step to biosynthesis (nutrient and water supply for example) In light of this it would probably be best to keep things as whole as possible to avoid adverse selections, either ise dikaryotic mycelium or work with monokaryons Perhaps monokaryotic mycelium is capable of biosynthesis in culture? If so high yielding strains could be mated to inbreed such characteristics. This also has the benfit of heterosis once selection is done and may help the longe term stability and adaptability of the spore race -------------------- www.balikebun.blogspot.com www.tropicalfoodforest.blogspot. www.bulelicious.blogspot.com Edited by Mycena (05/14/04 09:23 AM)
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Stranger Registered: 02/13/04 Posts: 142 Loc: SouthEast Last seen: 16 years, 1 month |
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I think reed canary grass (phalaris) contains 5-OH-DMT, which from what I've heard is not desireable, as opposed to N,N,DMT & 5-Me-DMT.
"Bufotenine (5-OH-DMT) is another DMT relative. This compound is vaguely referred to as "noxious" by Jonathan Ott. Apparently 10mg of pure 5-OH-DMT injected is enough to cause "dramatic circulatory crises." There appears to be debate over the psychedelic qualities of bufotenine. However, McLeod & Sitaram, Shulgin, and Fabins & Hawkings all report the presence of psychotomimetic effects. Bufotenine causes anxiety, circulatory distress, skin flushing, and percieved color distortions. Injected doses of 16 mg (over 3 min. IV drip) have been reported. At this dose, the symptoms of mild skin flushing to extreme purple cast appear. Subjects also report a great deal of psychological distress and fear at this dose. Doses of 8 mg produce mild skin flushing and increased anxiety. Doses of 2-4 mg of bufotenine do not produce hallucinogenic effects. The above discussed negative side effects at 16 mg last for approximately 10 minutes. Other side effects reported are sweating, nausea, yellowed vision, and perception of colored spots. So it appears that bufotenine is a nasty drug to be avoided, Not only does it tend to induce panic, it also appears to have the potential for a fatal overdose, although no case studies to this effect have been found for humans."--Erowid FAQ -------------------- "If King Kong sells drugs, we'll put him in jail"
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Janitor Registered: 05/07/04 Posts: 77 Loc: Texas Last seen: 19 years, 5 months |
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The premises behind using a substrate such as phalaris, would be that the basic elements and elementary compounds such as tryptophan and tryptamine would be present, regardless of quantity.
An analogy to why any amount would be of interest and worth study would be: --would you rather build a house on concrete or on sand. Either one, in theory is feasible, but which one provides the basic foundation for future growth and longevity. In case that analogy is more sound in my thinking I can relate it more to the topic: --We all have seen different degrees of potency in a mushroom and there are many debates on factors involving this, including substrate, casing, temperature, humidity, etc. But hopefully everyone agrees that a mushroom can form with very negligible amounts of resulting psilocin regardless of what caused it, it does happen. So this allows for further thought and exploration into WHY. Prevailing belief of well known mycologists tend to believe that given an ideal growing environment, this does not include substrate, and a resulting low concentration is indicative that either the substrate lacked nutritional value to the mushroom, but not enough to hender growth either by design (using something low in the first place) or expended (possibly by competition or in ability to break down complex molecules present). So even though you could grown a fungus on a sand foundation (if this doesn't make sense or relate, then please read the entire post again) or it can grow on the concrete (more concentrated because there is plenty of foundation for cell growth as well as more reagents to cook up alkaloids inside). The fear of a harmful reaction due to smoking the grass, as indicated on the vault, is arbitrary since fungus doesn't smoke to begin with and the natural filtration and usage of the fungus would be in place (meaning your not going to shit gold by having a gold ring on your finger as much as a mushroom is going to produce a chemical that it does not do in the first place just because it is growing in it, within reason)...does that make sense? There is a lot left to be done in the study of mycology, but some universal laws do exist that allow for some educated guesses towards an hypothesis and future studies. -------------------- Will Screw For Shrooms
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mycoexplorer Registered: 05/02/03 Posts: 270 Loc: indonesia Last seen: 11 years, 9 months |
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Your notes on Bufotenine are in question now due to the experiences of so many smokers of Cebil/Vilca seeds (Anadenathera colubrina)
Bufotenine isnt necessarily the nasty thing ist made out to be The nastiest thinsg in toad poison are other cardiotoxic compounds -------------------- www.balikebun.blogspot.com www.tropicalfoodforest.blogspot. www.bulelicious.blogspot.com
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