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Registered: 02/14/04
Posts: 2,662
Loc: Myceliaville !!!
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Lets make them more potent!
    #2659086 - 05/10/04 11:07 AM (13 years, 11 months ago)

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
Crop reliability
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/issue2/full/1/.

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/print/article/2036-3604.html.

Animal and Plant Health and Inspection Service. APHIS field test permits. Available at http://www.aphis.usda.gov/ppq/biotech/.

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/pubs/press/1998/papayarelease.html.

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.htm.

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/issue2/full/3/.

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/global/biotech/99072900.html.

World Health Organization. Genetically modified food main web page. Available at http://www.who.int/fsf/gmfood/index.htm.

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.

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
INTERNATL MICROBIOL (2000) 3:147?152
? Springer-Verlag Ib?rica 2000
Molecular tools for breeding
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
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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Re: Lets make them more potent! [Re: doc34]
    #2659173 - 05/10/04 12:14 PM (13 years, 11 months ago)

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?

:growingweed: Join us at the Growery! :growingweed:

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Re: Lets make them more potent! [Re: doc34]
    #2659182 - 05/10/04 12:17 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: Magash]
    #2659193 - 05/10/04 12:23 PM (13 years, 11 months ago)

exactly-the best of both worlds in one small package! :cool:
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.





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Re: Lets make them more potent! [Re: doc34]
    #2659237 - 05/10/04 12:50 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: doc34]
    #2659263 - 05/10/04 01:03 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: Transplant]
    #2659274 - 05/10/04 01:05 PM (13 years, 11 months ago)

.."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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Re: Lets make them more potent! [Re: Transplant]
    #2659275 - 05/10/04 01:05 PM (13 years, 11 months ago)

.."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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Re: Lets make them more potent! [Re: Transplant]
    #2659292 - 05/10/04 01:13 PM (13 years, 11 months ago)

" btw, I wonder if anyone has guessed who this is yet"



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Re: Lets make them more potent! [Re: doc34]
    #2659699 - 05/10/04 03:34 PM (13 years, 11 months ago)

Hey I tried to move this to the Advance Cultivation forum,but my Moderator button is stuck,maybe I need batteries! :smile:

Can I get some assistance? :cool:


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Re: Lets make them more potent! [Re: doc34]
    #2659848 - 05/10/04 04:44 PM (13 years, 11 months ago)

Of course, great thread so far :cool:

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Re: Lets make them more potent! [Re: Loki]
    #2659881 - 05/10/04 04:55 PM (13 years, 11 months ago)

Thanks Loki!


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Re: Lets make them more potent! [Re: doc34]
    #2660302 - 05/10/04 06:35 PM (13 years, 11 months ago)

"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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Re: Lets make them more potent! [Re: ATWAR]
    #2660328 - 05/10/04 06:45 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: Randolph_Carter]
    #2660382 - 05/10/04 06:59 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: ]
    #2660447 - 05/10/04 07:09 PM (13 years, 11 months ago)

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. :tongue:

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Re: Lets make them more potent! [Re: doc34]
    #2660747 - 05/10/04 08:23 PM (13 years, 11 months ago)

Nice read very interesting Doc


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Re: Lets make them more potent! [Re: FallenShroom]
    #2660958 - 05/10/04 09:00 PM (13 years, 11 months ago)

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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Re: Lets make them more potent! [Re: doc34]
    #2661266 - 05/10/04 10:04 PM (13 years, 11 months ago)

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-&#946;-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-&#946;-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-&#946;-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 &#946;-carboline derivatives, I will not speculate as to the pharmacological properties of said substance.



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Re: Lets make them more potent! [Re: ]
    #2661929 - 05/11/04 05:38 AM (13 years, 11 months ago)

Do you think that niacinamide and pyruvate, each in forms of dietary supplements could be used in this way?


"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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