Birth of Recombinant DNA Technology
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.)
| 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