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Gene Transfer Technology for Mushrooms: The Power and Potential for Significant Crop Improvement


Birth of Recombinant DNA Technology

In the early 1970’s, California scientists first succeeded at splicing viral and bacterial DNAs within the take a look at tube, heralding the beginning of the recombinant DNA (rDNA) generation, popularly known as genetic engineering, gene switch generation, gene splicing, molecular biotechnology, and transgenics. This new biotechnology discovered instant utility within the manufacturing of pharmaceuticals, where synthesis by way of rDNA microbes equipped a quantum jump in efficiency over the exhausting extraction of miniscule amounts from different assets. 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 just about each sector of our society.

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It was once right through the 1980’s when the facility and possible of the burgeoning discipline of genetic engineering was first brought to endure at the growth of agricultural productivity. The discovery of tactics to transfer genes to the major agronomic plants, together with corn, soybean, and wheat, from unrelated species provided breeders with new vistas for expanding the potency of food crop production. Remarkable development, a long way exceeding early predictions, has been made throughout the closing two decades in breeding crops with new traits comparable to insect, viral, and fungal resistance, herbicide, stress, and chilly tolerance, behind schedule senescence, advanced nutritional options, and others. The international call for for transgenic vegetation is projected to be a $25 billion market by the 12 months 2010. The growth of this business will be propelled, partially, by “Golden” rice, which was once engineered the usage of a daffodil gene to be wealthy in beta carotene and thereby the promising solution to the diet A deficiency drawback pervading the creating world.

Despite concern for the unforeseeable health and environmental dangers posed via genetically-modified (GM) plants, gene transfer generation has irreversibly revolutionized plant breeding. Today, more than 100 plant species had been changed by means of gene splicing for stepped forward assets of food, fiber, or ornamentation. More than 50 new crop types have cleared all federal regulatory requirements and stand authorized for commercial retail. Because field testing is an essential step within the commercialization process, the number of lets in issued by means of the U. S. Department of Agriculture, Animal and Plant Health and Inspection Service (APHIS) for GM vegetation provides a measure of the hobby in transgenic breeding. During a 16-year duration, more than eight,000 allows and notifications (fast-track allows) were issued, rising from a low of 9 in 1987 to a prime of one,120 in 2001 (Fig. 1). For the primary three months of 2002, 536 lets in/notifications were recorded through APHIS with 49% involving insect resistance, 33% herbicide tolerance, 7% every for product high quality and agronomic properties, and with the stability comprising fungal and viral resistance and different characteristics. Thus, the “genie out of the bottle” situation describes the standing of agricultural genetic engineering. Despite the anti-GM sentiment expressed through a vocal minority, the efficiency of the new biotechnology for downside solving has been realized to an extent this is a ways too compelling for it to be dismissed.

Genetically Engineering the Button Mushroom
For virtually as long as scientists were introducing genes into crop plants the use of molecular biotechnology, others have tried with limited success at creating a gene transfer means for Agaricus bisporus. a Big leap forward got here in 1995 with the sudden discovery that the bacterial workhorse, Agrobacterium tumefaciens, used to trip genes into vegetation, also operated with yeast fungi. Shortly thereafter, this system used to be prolonged to filamentous fungi, together with A. bisporus.

Agrobacterium is a not unusual soil bacterium with a world distribution. It causes a illness referred to as crown gall on hundreds of woody and herbaceous plant species, however maximum often pome and stone end result, brambles, and grapes. In its commonplace existence cycle, the bacterium transfers a tiny little bit of its DNA into the plant DNA resulting in the formation of galls. These galls serve as food factories for the mass manufacturing of the bacterium. Over the years, scientists learned increase disarmed strains of the bacterium that were incapable of inducing galls, but retained the facility to switch DNA. In essence, a natural biological process was once harnessed to create a bacterial delivery gadget for transferring genes into plants, and now fungi.

Figure 1. Total number of permits and notification

Though Agrobacterium used to be shown to be highly promiscuous in shuttling genes into a spectrum of plant and fungal species, the method was once still too inefficient to be carried out to the breeding of A. bisporus. More not too long ago, we devised a handy and efficient Agrobacterium-mediated ‘fruiting body’ gene transfer means conserving the promise of a formidable software for the genetic growth of the mushroom. In our experiments, a small ring of DNA wearing a gene for resistance to the antibiotic, hygromycin, was once transferred to a disarmed pressure of the Agrobacterium. The antibiotic resistance gene is known as a selectable marker, as a result of mushroom cells receiving this gene from the bacterium grow to be marked by the resistance trait and will also be decided on based on the facility to grow on a hygromycin-amended medium. The finish result is a mushroom pressure having the newly received function of hygromycin resistance. Such a strain has little business price, but somewhat the resistance trait was once a research tool that allowed us to easily resolve if the bacterium had transferred the gene to the mushroom, and exactly how successfully it did so beneath different experimental conditions. Today, and more so in the future, this gene is being replaced or complemented by means of genes that can confer commercially relevant characteristics.

Figure 2 Steps in the Agrobacterium

Figure 2 highlights the steps in the ‘fruiting body’ gene switch approach. In this procedure, gill tissue is taken from mushrooms approaching adulthood, however with the veil intact, with the intention to be sure that some degree of sterility. Next, the tissue is minimize into small pieces and vacuum-infiltrated with a suspension of Agrobacterium wearing the antibiotic resistance gene. In a process known as co-cultivation, the gill tissue and bacterium are grown in combination in the laboratory for several days, all over which time the bacterium transfers the resistance gene to the mushroom DNA. Because no longer all mushroom cells receive a replica of the gene, those who have can also be prominent from those who have not via the facility to grow at the antibiotic medium. After 7 days at the medium, mycelium of A. bisporus appears rising on the edges of probably the most gill tissue items. After 28 days, upwards of 95% of the tissue pieces may have regenerated into visual cultures. At this point, the GM cultures may also be transferred to a normal growth medium, and used to prepare grain spawn in the unusual method.

Agrobacterium is a not unusual soil bacterium with a global distribution. It reasons a illness referred to as crown gall on hundreds of woody and herbaceous plant species, however maximum regularly pome and stone end result, brambles, and grapes. In its standard existence cycle, the bacterium transfers a tiny little bit of its DNA into the plant DNA resulting within the formation of galls. These galls serve as food factories for the mass manufacturing of the bacterium. Over the years, scientists learned how you can increase disarmed strains of the bacterium that had been incapable of inducing galls, however retained the facility to switch DNA. In essence, a herbal organic process used to be harnessed to create a bacterial supply machine for moving genes into crops, and now fungi.

Though Agrobacterium used to be proven to be highly promiscuous in shuttling genes into a spectrum of plant and fungal species, the method was once nonetheless too inefficient to be carried out to the breeding of A. bisporus. More just lately, we devised a handy and efficient Agrobacterium-mediated ‘fruiting body’ gene switch way protecting the promise of a powerful device for the genetic development of the mushroom. In our experiments, a small ring of DNA sporting a gene for resistance to the antibiotic, hygromycin, used to be 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 also be selected in line with the facility to grow on a hygromycin-amended medium. The finish result is a mushroom pressure having the newly got function of hygromycin resistance. Such a strain has little business worth, but somewhat the resistance trait was once a research tool that allowed us to simply decide if the bacterium had transferred the gene to the mushroom, and precisely how successfully it did so below different experimental conditions. Today, and more so at some point, this gene is being changed or complemented by way of genes that may confer commercially related characteristics.

Figure 3 Production trail with Six GM Line

Figure 3 depicts the first of 2 cropping trials carried out at the Penn State Mushroom Research Center involving GM mushroom lines. In these trials, all six antibiotic-resistant GM strains reflected the parental business hybrid strain in colonizing the compost and casing layer. Further, the GM strains produced mushrooms having an ordinary appearance and, in some instances, yielded on a par with the economic strain (Table 1). Expression of the resistance trait within the mushrooms could be easily demonstrated via placing pieces of the cap or stem tissue at the antibiotic medium and staring at for enlargement (Figure four). These experiments had been an important, since the effects established for the primary time that a international gene could be offered into A. bisporus with no need a adverse effect on its vegetative and reproductive traits.

Table 1. Productivity of genetically-modified (GM) mushroom traces expressing the antibiotic resistance gene that were derived from a business off-white hybrid pressure.
Yield (lbs./sq. feet.)

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 approval for the hybrid mushroom strains introduced within the 1980’s has created a close to global monoculture that is precarious from the viewpoint of disease and pest susceptibility, and has limited the selection of production traits and the variety of tolerance to environmental and cultural stresses. During the remaining 20 years, no notable advances have been made in breeding strains with strikingly advanced features. This is due in large part to the cumbersome genetics of A. bisporus and a shortage of commercially fascinating traits. There is movement afoot in the usage of conventional breeding to explore wild isolates of A. bisporus as a supply of new characteristics. Though this represents an important step against increasing the genetic base of cultivated A. bisporus, it isn’t but transparent which traits exist within the wild germplasm collection, and if they are able to be successfully bred into commercial lines.

The introduction of a facile gene transfer methodology for A. bisporus permits the exploration of genetic solutions to problems confronting the mushroom trade in a realm by no means sooner than imagined. The awesome energy 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 characteristics is the same for all organisms. A scientist blindly handed a gene would have difficulty determining if its source was once a mushroom, mouse, or guy. It is that this unifying feature of genes from all walks of existence that makes transgenics so potentially robust, while it is the tools of molecular biology that unleashes this power so this doable will also be discovered. Simply said, the brand new biotechnology permits the trade of genetic data between organisms outdoor the confines of the herbal breeding barrier. No longer is the genetic improvement of the mushroom made up our minds by the query of sexual compatibility or characteristics found throughout the species.
At every other stage, gene transfer generation will hugely boost up our figuring out of the molecular mechanisms underlying commercially relevant characteristics. It additionally will serve to make stronger the muscle of our industry’s medical arm, rising from a handful of mushroom researchers to the global group of workers of molecular biologists. As one hypothetical representation, the search to reproduce tough resistance to dry bubble illness would now not be restricted to a couple of scientists looking inside A. bisporus, the place it is going to or now not exist. Instead, it would extend to ratings of scientists operating on unrelated organisms who have came upon resistance genes to other Verticillium species. Importing these genes to the mushroom for an analysis in opposition to dry bubble is now possible. As farfetched as this will likely appear, it is precisely this trans-species method that has met with business success. Genetic manipulations of this sort have been performed on crop crops and come with, 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 changed fruit ripening, the barnase gene from Bacillus spp. for male sterility, and viral genes for virus illness resistance.

It cannot be overstated that gene switch technology is not a panacea whose arrival marks the departure of traditional breeding. Quite the contrary, this can be a new instrument at the disposal of the breeder that will supplement current techniques, while providing a far broader vary of options for effectively affecting genetic solutions to issues. Gene splicing will expedite the breeding process, transferring a lot of the time in development from the sphere to the laboratory. It will enable the introduction of genes with a surgical precision and from exotic sources, which another way would be unattainable through extra typical strategies. It is essential to recognize, alternatively, that after all, the forces of nature overcoming a trait (e.g., the breakdown of insect resistance) would act with the same depth at the controlling gene whether introduced by means of traditional or transgenic breeding.

The melding of gene transfer strategies with traditional techniques in a mushroom breeding program might take several paperwork initially, most effective to be continually subtle, streamlined, and stepped forward for higher efficiency and bigger effectiveness.

Many transgenic manipulations with A. bisporus will require the switch of the gene to both parental traces so that their offspring mimic the herbal inheritance process through carrying a duplicate replica of the gene. For different packages, introducing a single reproduction of the gene might succeed in the desired effect. In both case, the ensuing GM strains would possibly require additional variety ahead of emerging as worthy industrial traces



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