On September 21, 1994, a meeting was held at the University of California at Davis to discuss ongoing and future plant breeding strategies to control losses to Beet Yellows Virus (BYV), one of the most important plant pathogens limiting sugarbeet production in California. In particular, the meeting focused on whether it is now prudent for the industry to adopt a molecular biological approach to plant breeding, in order to hasten the development of effective resistance to this and other diseases and pest management problems. The meeting was organized in three parts. The first focused on the effects of BYV on sugarbeet growth and development, and the overall effect of the disease on the industry (Kaffka; Mauk, Godfrey and Limburg). Current efforts to develop resistance to the disease using traditional breeding methods were summarized as well (Lewellen). The second part of the meeting provided an overview of molecular breeding techniques (Lemaux), the benefits, risks and role of molecular techniques in an overall breeding program (Falk), and current efforts in California and elsewhere to use molecular techniques to advance resistance (Creamer). The last part of the meeting was devoted to regulatory, legal, and financial issues associated with molecular approaches. The purpose of the meeting and this review is to encourage informed discussion of the benefits and limitations of molecular methods. Whenever resources are limited, as are research funds within the sugarbeet industry, difficult choices must be made about how to expend them. The better informed we are, the better the choices we can make.
This Sugarbeet Research Review is organized in three parts. Part I reports information presented at the meeting about the sugarbeet industry in California, BYV and its affects on the industry, about current management strategies including the use of classical methods of plant breeding, and an overview in plain language about the techniques used in molecular breeding as well as some considerations associated with its use. Part II is a review of the current literature reporting on the techniques and success to date in the molecular transformation of sugarbeets. It is intended to allow the technically minded reader to assess the current status of work on the crop and lead toinformed consideration of a research effort in California. Part III is a discussion of the possible advantages and disadvantages of creating a public-private effort to improve the sugarbeet cultivars available to farmers in California through biotechnology.
At issue for the California industry is whether it is prudent and timely to provide university and/or USDA/ARS researchers with the resources needed to work at a molecular level on sugarbeet. These resources could be used to engineer sugarbeet for resistance to BYV or provide approaches to solving other difficult problems that have not responded to traditional plant breeding, pest management or agronomic practices. Additionally, molecular techniques can be used to advance understanding of the basic physiology of the crop, leading to new ideas for the improvement of production and plant breeding, or to develop strategies for alternative, valu-added uses for the crop.
Other industries and commodities have made an investment in the public sector (particulalry the University) for developing biotechnological approaches, but not all commodities have as much plant breeding activity within the private sector as the sugarbeet industry, at least on a worldwide basis. There is a major research effort on all aspects of sugarbeet production in Europe, where the crop plays much the same role in farming systems as corn does in the United States. Private industry, particularly the European-based seed companies, have already made progress in molecular breeding of sugarbeet (Büchting, 1995). For example, two companies are field testing glyphosate-resistant beets in Europe and these lines are nearing commercial development. Field tests of other transgenic lines with improved disease resistance to rhizomania also are being conducted. Some have suggested that the prudent course is to allow that work to take place in Europe and then take advantage of any breakthroughs as they occur. The question is whether this serves the long-term interests of the California sugar industry.
There are possible advantages to having a research effort within California. The first is that each crop species, and oftentimes each cultivar, has unique characteristics that require some special technology to be developed for transgenic work. Developing the capacity to work with sugarbeet would allow university researchers and graduate students to apply molecular techniques to current problems and to challenges that will inevitably arise in California. Second, some of the problems experienced in California are more acute here than most other locations in the world. BYV is one example, but rhizomania is another. The future is certain to hold others. Germplasm useful in California must have broad resistance to a number of pathogens that are less important elsewhere, while being tolerant of relatively extreme environmental conditions. Cultivars used elsewhere often are unsuccessful when grown in California. Therefore, new genes would have to be introduced into cultivars adapted to California from European germplasm to be useful. Such redundant crosses may not be important for European companies. One European company has made a strong committment to serving the California market while others, apparently, have not. Lastly, the University of California is one of the leading institutions in the world in the field of molecular biology. The sugarbeet industry in cooperation with the University, should explore ways to benefit from the University’s capacity in molecular technologies and from evolving insights into plant disease suppression and plant physiology that might be derived from these technologies. Engagement of innovative scientists is necessary to keep the commodity competitive with others available to farmers (Staskawicz, et al., 1995).
What is at stake for the industry?
Sugarbeets are grown in California from the Mexican border to the Oregon state line. The state's climate allows for sugarbeet growth and production over a twelve month period. The same benign climate that supports year round crop growth also imposes limits on the industry, especially factory operation. Unlike most other locations where sugarbeets are produced, mature roots cannot be stockpiled near the factories for processing over a sustained period. Rather, they must be harvested within one or two days of processing. Since efficient operation of a sugarbeet factory requires 200 or more days of operation a year, sugarbeets are being harvested somewhere in the state every day except during December through February, when wet soils preclude harvest. Since transportation costs are significant, some beets must be produced and harvested near the factories for extended periods of the year. This emphasizes extending production periods in areas near the factories, where it is most difficult to isolate fields one from another. Because there is never a period when sugarbeets are absent, Beet Yellows Virus (BYV) for example, which is transmitted by aphids from old to young beets, is a more difficult problem to control in California than elsewhere in the world.
Average yields in California are the highest of any major sugarbeet production area in the world. Although yields are high, typically they are not close to the crop's potential and have remained more or less static since 1970 (fig. 1). The sugarbeet crop provides several advantages for California farmers, including its ability to recover residual soil nitrogen and deep soil water (reducing drainage water volume), its tolerance of high levels of salinity and ozone, and its thrifty use of irrigation water when over-wintered. From an environmental perspective, the crop is considered important for raptors such as owls and hawks, which prey on the rodents that occur in sugarbeet fields. Pheasants also thrive and nest in overwintered fields. These economic and environmental advantages notwithstanding, the acreage planted to sugarbeets has declined in recent years (fig. 1) because yields have remained static relative to other crops and costs continue to rise, while sugar prices have remained stable or declined in recent years. When yields per acre are high, California becomes a low-cost producer of sugarbeets in the United States. When they are low, the state becomes a high cost producer (Table 1). Yield advances are essential to the future of the industry in California.
Both the threat and the occurrence of BYV lowers yields and acres in California. The virus reduces rates of photosynthesis and enhances respiration in sugarbeets. When young plants are infected by viriliferous aphids, yield losses can be as high as 50%. Substantial efforts have been made to develop and maintain an isolation system to separate newly planted beets from older crops, to find pesticides effective at reducing transmission of the virus, and to develop resistance using conventional breeding techniques. Despite these efforts, the occurrence and severity of the disease remains unpredictable. A strategy relying on pesticides is uncertain at best because it is difficult to prevent virus transmission by aphids using pesticides and regulatory and environmental concerns also limit reliance on pesticides. Effective genetic resistance remains a distant goal using classical methods.
Besides direct yield losses, BYV affects yields by reducing the ability of growers to identify optimum production dates for their location. Hills et al., (1982) looked at the effects of planting date and virus control on yield. In most trials, beets planted earlier in the spring usually had higher yields than later planted beets. Protecting April-planted beets with aphicides resulted in a yield increase of 10 tons per acre compared to unprotected beets planted in April, and a 16 ton per acre improvement compared to the same cultivar planted in May, even when the May plants were protected with aphicides. Despite these potential yield increases, the industry, wisely, has not relied on pesticides to protect early planted beets. Because reliance on pesticides is only partially successful, the occurrence of BYV would have become widespread and essentially uncontrollable in just a few years, had a pesticide-based strategy been followed. Instead, the beet-free period and classical methods of plant breeding are the bases of the industry’s approach to the problem.
The problem remains that growers are not able to plant and harvest at otherwise optimum times because of aphid-borne viruses, especially BYV, and this results in yield limitations. As long as effective resistance to BYV is unavailable, the industry will be burdened with a significant barrier to improved yields. This yield barrier raises the relative cost of production for California growers compared to elsewhere in the United States, and lowers profitability. Low profit and a continued sense of risk on the part of growers and lenders reduces the acreage available for the crop. Control of BYV is critical to the industry's future. It may now be timely and prudent for the industry to consider the use of biotechnological methods to achieve BYV resistance.
What is beet yellows virus and why is it so difficult to control?
Beet yellows virus (BYV) is a semi-persistent, aphid-borne closterovirus.
It was first identified in California in 1951and since that time has been
a significant limitation to beet production, especially in the central
valley. There are a number of different species of aphids that have been
reported to transmit BYV, but the most important are thought to be Myzus
persicae (the green peach aphid) and Aphis fabae (the black bean aphid).
The primary host of the virus is sugarbeet, although a number of alternate
hosts, mostly in the Chenopodiaceae family (spinach, lambsquarter), are
known. Symptoms of BYV on sugarbeet vary depending on the virulence of
the virus strain. New infections generally induce vein clearing in the
younger leaves and older infected leaves become yellow, thick, leathery,
and brittle. Leaves develop a bronze cast with age.
In 1969, a quarantine program called the “beet-free period” was established
to limit the spread of BYV and inhibit the reintroduction of BYV into new
plantings. It included a 30-day delay in planting from the date of harvest
of nearby sugarbeet fields and minimum critical distances between old,
infected fields and newly planted ones. Growers were not allowed to plant
spring harvested beets until after the flight of the green peach aphid
(after May 1). This program in modified forms, has been followed successfully
in many areas of California since that time. In the late 1970s and periodically
in the 1980s, however, growers in portions of the central valley experienced
outbreaks of the disease. It is unclear if the proximity of the spring
harvested beets to the fall harvested areas is the source of both the aphids
and the virus, if there is an undetermined source (an alternate host) of
BYV, or if the introduction and persistence of the black bean aphid has
caused a periodic breakdown in control.
In the past, the green peach aphid has been considered to be the primary
vector of BYV in California. In the late 1970s, however, there was an apparent
shift in population dynamics of the two aphid species with the black bean
aphid becoming the more prominent vector in the Delta region and surrounding
counties. On the basis of field observations and water-pan trap data, the
black bean aphid may represent 70% to 80% of the aphid population in a
given year (fig. 2). One aspect of the biology of
the black bean aphid that may contribute to the breakdown in the beet-free
period is that the black bean aphid is tolerant of a wider range of temperatures.
This aphid species can begin to fly when temperatures are cool (February)
and will continue to migrate during warm temperatures (June of most years).
With this extended flight, plantings after May 1 are still at some risk
of infection with BYV. Plantings in early May, if infected, can function
as a source of virus for all plantings later that spring, early summer,
or anytime thereafter until the crop is harvested. Aphid behavior
has proven difficult to predict. Previously, plantings after May
1 were not at risk because the flight of the green peach aphid had generally
decreased by that time. Now, the black bean aphid appears to have made
the beet-free period less reliable, and made the maintenance of an effective
beet-free program more difficult.
What progress has been made in developing resistance so far and what are the prospects using traditional plant breeding methods?
Virus yellows has been a problem in sugarbeet production in California for about 50 years. The virus yellows complex is composed of aphid-vectored viruses BYV and BWYV, and sometimes beet mosaic virus (BMV) is included in the complex. These are unique viruses and it appears that resistance (tolerance) to one does not condition resistance to the others. J. S. McFarlane began breeding for beet yellows (BYV/BWYV) resistance at Salinas in 1955. In 1966 the virus yellows breeding program was assigned to R.T. Lewellen. Except for the period 1979-1987 when virus yellows resistance breeding was focused against BWYV only, the program has been continuous for both BYV and/or BYV/BWYV. In this nearly 40-year period, overall losses in field trials to the virus yellows complex have been reduced from greater than 45% in non-resistant cultivars to about 15% in the most resistant breeding lines. This resistance (tolerance) appears to be against both BYV and BWYV. Measured losses attributed to BWYV were reduced from about 25% to about 5% in the most resistant parental lines. Losses due to BYV were reduced from about 45% to about 15%, also in the most resistant parental lines. It has not been possible to obtain significantly higher levels of resistance. The resistance identified appears to be multigenic and additive and has low heritability. These factors make it difficult and very time-consuming to transfer resistance from one source to another, to make improvements within new populations, and to get all parental components of a hybrid to the same level of resistance. Current hybrids are less resistant than the most resistant parental lines. Useful parental lines with other needed characteristics usually are also less resistant than the most resistant base lines or populations. More recently, a search within wild beets (Beta vulgaris subsp. maritima) has shown that some genetic variability for resistance occurs but high levels of resistance do not appear to be present in the wild species. This limits the potential for identifying traits from related species conferring more complete resistance.
Since 1992, cultivar evaluations have been carried out on the Davis campus to expose germplasm to central valley conditions. Commercial hybrids and experimentals have been inoculated with the virus complex and compared to non-inoculated plots. The mean sugar yields of both groups are presented in figure 3. Yield losses have ranged from approximately 35% in a poor growing year, to 45% in a good year.
Virus yellows, as well as other diseases such as curly top, appear to
be the major factors that limit the use in California of high performing,
highly bred germplasm and hybrids that are used in other areas. In the
absence of these diseases, or if these diseases could be controlled easily
with highly heritable host-plant resistance, it would be much easier and
more likely that the high sugar content, high quality germplasm lines and
hybrids used in other regions could be adapted and utilized more quickly
in California. At the present time, the prognosis for finding higher levels
of resistance to BYV in Beta genetic resources by the use of conventional
plant breeding does not appear promising.
What is molecular biology and what makes it different from classical approaches to plant breeding?
Classical breeding or hybridization has been used since the beginnings of agriculture to improve upon natural selection through controlled pollination of selected parent plants to produce offspring with desired characteristics. Virtually every fruit and vegetable available is a product of this type of genetic manipulation. For example, an ancient ancestor of modern-day corn, Tripsacum, does not resemble a modern corn hybrid. It also isn't as productive or nutritious as modern hybrids. If Tripsacum were used for feed stock for animals, hundreds to thousands more plants and acres would be needed to achieve the same yields. Another example of human intervention is the new vegetable - broccoflower. It originated through a cross between broccoli and cauliflower, after years of patient selection by plant breeders. The broccoflower is an example of a wide-species cross that couldn't happen in nature. The fertilized egg from the broccoli/cauliflower cross could not survive. First, it had to be excised from its seed coat and nurtured in the laboratory until it formed a plant that could be grown in the ground.
The genetic information in a cell can be thought of as a recipe that is used to tell the cells what to do and imparts on a plant its characteristics - for example whether a tomato plant bears yellow or red fruit and whether it is resistant or sensitive to a viral disease. That recipe, the genome, is made up of DNA, which is composed of chemical units made up of four different deoxyribonucleic acids (fig. 4). The capacity of these four nucleic acids to form diverse, meaningful patterns (genes) is the basis of genetic variation. If the chemical units forming genes in the cell of a wheat plant, for example, were thought of as alphabetic letters, it would take 1700 books, each of 1000 pages to represent all the genetic information present. If these books were stacked on top of one another they would equal a 20-story building in height.
When genetic information is exchanged in the creation of a new hybrid cultivar or by crossing a broccoli and cauliflower, the number of genes and chromosomes normally do not increase, only one 20-story building can result. To conserve the original size of the genome, a random mixing and deletion of information occurs, some of the cauliflower information is retained and some of the broccoli information is retained, while some is lost. The result is the broccoflower.
Plant breeders have no control over the specific outcome of their crosses. Because they do not, they often encounter genetic drag. Mixing and sorting of tens of thousands of genes from the two parents occurs and undesirable traits can be closely linked to beneficial ones. By watching for desired traits in the offspring, breeders identify individuals they want, but sometimes they also get characteristics they don't want. Consequently, it is necessary to screen large numbers of offspring over many years to find the few plants with desirable traits from each parent. As is the case with BYV, this limited control can be an important constraint on progress when using classical techniques.
Work done at the Univiersity of California, Davis on improving the sweetness of processing tomatoes offers a practical illustration of the contrast between classical and molecular plant breeding approaches to increasing the sugar content of the domesticated tomato (Bennet et al., 1995; Klann et al., 1993). Certain wild tomato relatives, although they look little like our domesticated tomato, are known to have a higher sugar content than domesticated ones. Using the classical approach, plant breeders crossed the wild tomato with the domesticated tomato and over many years of breeding came up with a tomato with higher sugar content. However, the resulting tomato also had fertlity problems. Analogously, they had taken two different stacks of books and mixed them, ending up with a single stack of books that had mostly volumes from the domesticated species, but about 100 pages from the wild species. In that 100 pages was the genetic information for higher sugar and a lot of other information that they had not "read" yet. One of the other pieces of information contained in those 100 pages turned out to cause reduced fertility in the resulting tomato, an instance of genetic drag.
The Davis researchers decided to exchange information between the wild and domesticated species using molecular methods, hoping to try to transfer the wild tomato’s higher sugar characteristic to the domesticated tomato, while retaining all of the domestic tomato’s other desirable characteristics. In the molecular breeding approach, the goal again was the same, to increase the sugar content of the tomato. This time the researchers looked at the "recipe" for the tomato fruit and realized that if a single gene was removed (one responsible for the breakdown of sugar), they could engineer a sweeter tomato. Through genetic engineering technologies they were able to turn off that gene by adding a complement of that gene (that acts like a complementary piece of Velcro) to turn off the machinery that makes the sugar-degrading enzyme (fig.5). Nothing else about the tomato was altered. Instead of changing 100 pages using the classical breeding approach, only 0.5 pages of information in the 1,700,000 pages were changed. The result was a sweeter tomato without fertility problems.
How are molecular techniques being used?
At one time biotechnology was thought to be a science of the future, going on in laboratories but not really affecting the farmer. Now both technologies and products are appearing in the market place that are aiding farmers and providing new products to the consumer. Marker-aided breeding (fig. 6), a technique that surfaced as a result of the development of these new tools, provides the breeder with "road maps" to the genetic information in a crop and allows the generation of disease-resistant varieties in tomato, for example, in 2-3 years, whereas classical breeding approaches require 8-10 years. Diagnostics such as ELISA tests have also had significant impacts that allow the early diagnosis of diseases and earlier application of control agents, thereby significantly reducing the use of chemicals and also allowing the use of other control measures, such as bio-control agents.
Probably the most active area of interest in California for these technologies, including gene introduction via genetic engineering, is in plant protection (Shah, et al., 1995). This interest results from the fact that California farmers are particularly vulnerable to the new environmental laws regarding agrichemicals. Many of the chemicals currently in use for plant protection against fungal, bacterial, viral and insect attack are being removed from the market. It is unlikely that new agrichemicals will be developed or old ones re-registered for all of California’s wide diversity of crops, because although there may be many acres of such crops in California, they have low-acreage nationally and worldwide. Companies are concentrating their efforts on high-acreage crops, in general, in the hopes of recovering the very high expenses associated with registering new chemicals. Sugarbeet is regarded as a low acreage or “minor-use” crop.
Improvement of resistance: fungi and bacteria.
Many plants in the wild have evolved mechanisms to defend themselves against fungal and bacterial attack. The molecular breeding strategies that are currently being explored attempt to exploit these mechanisms by identifying the products that are naturally expressed by plants during attack, so-called natural plant defense compounds. Plants make many natural toxins that defend them against attack or in some way are involved in resistance. In many cases, the ability to make these compounds has been lost over the years during the breeding process. In many genetic engineering strategies, the idea is simply to make these naturally occurring, protective plant compounds in greater amounts, at different times or in different tissues. For example, potatoes have been genetically engineered to be resistant to the fungus Verticillium dahliae that causes a wilt disease. Another example is a tomato which has been engineered with a natural resistance gene against the bacterial pathogen that causes bacterial speck disease. In the process of engineering these resistances, the information in these and other "natural defense genes" has been elucidated and this knowledge will allow the development of yet more effective strategies in the future.
Improvement of resistance: insects
Other plants have been engineered to be prtected against insect damage. There are numerous strategies being investigated, although the one that is the furthest along involves the introduction into plants of a gene for an insecticidal compound that was originally made in a soil bacterium called Bacillus thuringiansis (Bt). The “Bt approach” involves the production of a natural bacterial compound in, for example, a corn plant, allowing it to resist the invasion of European corn borer larvae, which causes approximately $400 million of damage per year in the United States. Bt has also been used to control insect pests in cotton and this product is about to enter the commercial market. Approximately 40% of the pesticides used in the United States are applied to cotton. The introduction of this single gene may reduce pesticide use in the United States by an estimated 10% to 20%. Other strategies are being developed with independent means for controlling insects. Farmers in developing countries can lose 20-40% of their beans to storage pests. Some bean varieties have been engineered with a gene from the common bean that prevents digestion of starches by the predator beetle, starving the larvae. Another approach involves the engineering of the microscopic hairs (trichomes) on the surface of the leaves and stems of plants so that they make sticky substances that entrap unsuspecting insects or make naturally occurring insecticides that kill them.
Improvement of resistance: viruses
Breeders are often limited in the sources of natural resistance available to introduce into a crop using classical methods. This seems to be the case for BYV and sugarbeet, although this problem is not limited solely to sugarbeet. Viral disease and damage is estimated to cause up to 20% of all vegetable crop loss in the United States. Genes from some of the viruses causing melon and squash viral diseases have been inserted into the yellow crookneck squash, making it difficult for the virus to make copies of itself, thereby protecting plants from disease symptoms and crop loss. These methods can provide substantial resistance to four devastating viruses of cucurbits (watermelon mosaic, zucchini yellow, cucumber mosaic, and papaya ringspot). A genetically-engineered, viral-resistant squash is currently entering the marketplace. In other countries, such as China, viral-resistant products are already on the market.
The strategy used for the squash and the Chinese crops is called the
coat protein approach. It is based on the production of a viral component,
the coat protein, in the plant cells. This overproduction of coat protein
does not affect the appearance of the plants. However, expression of the
viral gene by the transgenic plant somehow prevents the extensive replication
of the virus. The efficacy of the coat protein approach
was first demonstrated in 1986 and has since been used to protect plant
species from over 20 different viruses in 10 different taxonomic groups.
The coat protein approach has been used to create transgenic sugarbeets
that are being field tested for resistance to rhizomania (Gassen et al.,
1995; Part II, this review).
Coat proteins are not the only approach being explored to protect crops
against viruses. There are many more that are currently being evaluated
in the laboratory based on the overproduction of other viral components
such as the replicase enzyme, needed for the virus to make copies of itself;
the movement protein, needed for the virus to be able to move from cell
to cell; and the production of other components that interfere with the
ability of the virus to make copies of itself. These methods are
curently being tested in laboratories and greenhouses.
Modification of important plant characterisitcs: food quality
The most widely publicized example in the area of genetically-engineered
plants is in fruit ripening. It is estimated that between 40-60% of fresh
fruits and vegetables are lost to rot in the United States before they
reach the table. Using molecular methods the FlavrSavr© tomato (Calgene,
Inc.) was engineered to extend the shelf life of the tomato. In a
companion approach developed at the University of California, a fresh market
tomato has been modified through anti-sense technologies to turn off a
gene involved in ripening, and can be left on the plant up to 100 days,
long after the time when it is normally ripe. It can then be removed and
ripened by exposure to the chemical compounds (ethylene) released by ripening
bananas and other fruits (Fig. 5). This
tomato (called Endless Summer©) is currently being marketed by DNA
Plant Technologies, Inc.
Cereal grains are being produced with increased nutrition by the boosting
of endogenous proteins with higher lysine and methionine content; rice
has also been genetically engineered to remove allergens, resulting in
a hypoallergenic rice. The amount of soluble solids in tomatoes, a characteristic
important to the processing industry, has also been increased by molecular
breeding. Potatoes have been developed that have approximately 20% increased
starch content. This is accomplished by changing the amount of a single
enzyme that is limiting in the synthesis of starch. The use of a high starch
potato for frying will result in a final fried product that is lower in
oil content.
Modification of important plant characterisitcs: processing quality
of sugarbeet roots
Specific to the sugarbeet industry are factors affecting beet processing quality. These include the formation of betaine and invert sugars, and the tendency of roots to accumulate amino nitrogen. Such characteristics might be selectively reduced, improving sugar recovery and net profits for growers and processors (Nichols, et al., 1992).
How do researchers modify plants using molecular techniques?
The ability to create plants with a single new piece of genetic information is possible because of what is termed "totipotency" of plant cells. Simply stated, this means a single plant cell can be coaxed to undergo repeated divisions to give rise to an entire plant. This is in stark contrast to the situation in mammalian cells. Mammalian cells "know" they are a part of a specific organ or tissue. Epidermal cells, for example, cannot be influenced to become liver or reproductive or any other type of cell. If a plant is wounded, however, one of its responses is to de-differentiate. It returns to an earlier state of differentiation or development called callus. Callus cells can multiply and redifferentiate to form an entire plant. This is comparable to returning to the main menu on a computer. From the main menu you can go down many different pathways to a variety of different outcomes. By giving plant cells the appropriate chemical cues, an entire plant can arise from an original single cell. The importance of this is that each cell in a plant need not be changed for a new plant to have new information in each of its cells. Scientists can simply introduce the information into one cell, coax that cell to divide and form a new plant made up of cells with a copy of the new information (fig. 7).
Once scientists learn how to regenrate plants from callus cells, the next challenge is to insert new genetic information into a single cell. There are many ways to do this but the method used for sugarbeet employs the natural plant pathogen, Agrobacterium tumefaciens (fig. 7). This is a soil bacterium that is able to transfer a portion of its DNA into plant cells. Molecular breeders have learned to exploit this bacteria’s natural capability in order to introduce new genetic information into plant cells. Using this approach, the disease-causing portions of the transferred DNA are removed, leaving its ability to transfer DNA intact. The gene of interest is inserted into the small piece of the bacterial chromosome that is to be transferred into the plant cell. When it is transferred the new genetic material becomes a stable part of the genetic information of the plant.
The remaining challenge is to be able to identify those cells or the plants derived from them that contain the introduced genes. This can be done making use of a selectable gene, allowing only those cells or plants to grow that have received the new information (often using a selectable gene conferring resistance to a particular herbicide or antibiotic).
In sum, the approach to engineering sugarbeet is first to identify the desired new genetic information, such as the coat protein gene from BYV. The gene then is inserted into the transferable portion of the A. tumefaciens DNA and then sugarbeet cells receive the bacterial vector and the new gene. Subsequently, plants are identified that carry the viral resistance gene and then tested for resistance. There are some 80 plant species that can be manipulated by similar molecular breeding strategies. Sugarbeet is a viable target for molecular breeding. The European seed industry has been very aggressive in sugarbeet work and presently has field trials underway with transformed sugarbeet. Molecular techniques are powerful tools that are available to farmers now and increasingly in the future. These strategies are not magic bullets. They are simply new tools that can be used in concert with other elements of classical plant breeding and good farming practice.
Are there special risks associated with the use of molecular techniques?
The use of molecular methods for genetic modification is considered controversial by some of the general public who are fearful of its use and implications. Public opinion surveys, however, indicate that the majority of consumers feel that biotechnology would benefit them personally over the next five years and that it will have a positive effect on food quality and nutrition (Hoban and Kendall, 1992; Hallman and Metcalf 1994). Most scientists familiar with the technology discount the notion of significant risk. Falk and Bruening (1994) discussed one type of hypothetical risk from transgenic virus resistance: the recombination in plant cells of RNA from the invading virus with analogous, related viral RNA carried by transgenic plants. The result might be modified or new virues that would cause new diseases or make existing ones more virulent. The basis for this concern is research in which intermolecular RNA-RNA recombination (between transgenic plant-derived RNA and invading viral RNA) has been reported under laboratory conditions for four groups of plant viruses when selection pressure was artificially strong. Despite the occurrence of recombination under artificial conditions, Falk and Bruening believe that the likelihood of this kind of recombination between viral and viral-analogous RNA from transgenic plants occurring in agricultural fields is vanishingly small. In brief, they maintain that recombination occurs naturally in the field, but that most new viral diseases are the result of slight mutations or variations of existing forms, not novel ones such as those resulting from transgenic recombination. Also, they state that most natural experiments of this kind are failures, i.e., that most new viruses are less fit (less likely to survive and reproduce) than their progenitors . They conclude that while new viral strains can and do evolve, even in response to the development of resistance in cultivars derived from classical plant selection and breeding techniques, the cost to agriculture of such viral strains is much less than the cost of not breeding resistance to viral diseases at all.
There are other concerns associated with the use of transgenic plants. For example, the widespread use of the Bt gene in crops may act as a natural selection pressure on lepidopteran insects now susceptible to Bt toxins, leading to the development of insects insensitive to the toxin. Herbicide resistance has been developed in a number of crops (Sellers and Lemaux, 1996) and is another area of concern (Dyer et al.,1993 ) Also, herbicide-resistant sugarbeets, if allowed to flower and go to seed, may provide pollen to closely related weed species, transferring the herbicide resistance gene to the weedy species in the process (Steen and Pedersen, 1995). Most European scientists and agronomists regard this as a manageable problem (Thomas, 1995). In California, there is one weed species that is a close relative of sugarbeet (Beta vulgaris subsp. macrocarpa) that occurs in isolated areas of the Imperial Valley and there are isolated populations of escaped beets in various locations.
1This report summarizes written or oral remarks made by several
different individuals at a meeting held on September 21, 1994 at the University
of California at Davis. In addition to Kaffka and Lemaux, those making
presentations included Peggy Mauk (UCCE, Sacramento County), Robert Lewellen
(USDA/ARS), Bryce Falk (Plant Pathology, UC Davis), Rebecca Creamer (Plant
Pathology, UC Riverside), Alan Bennett (Divisional Acting Dean, College
of Agriculture and Natural Resources), David Heron (Office of Biotechnology
Permits-USDA/APHIS), Kevin Bastian (a patent attorney from Townsend, Townsend,
Khourie and Crew-San Francisco) and Candace Voelker (Office of Technology
Transfer, University of California).
REFERENCES
Bennet, A.B., Chetelat, R. and E. Klann (1994). Exotic germplasm or engineered genes: comparison of genetic strategies to improve fruit quality. Food Quality Review
Bosemark, N.O. (1993). Genetics and Breeding. p 67-119. In: Cooke, D. A. and R.K. Scott (ed). The Sugar Beet Crop. Chapman and Hall, Cambridge. 675p.
Büchting A. J. (1995). Results of field trials with KWS’s
rhizomania resistant sugarbeets.
(Erfahrungen aus den KWS-Freilandversuchen mit rhizomaniaresistenten
Zuckerrüben). Zucherind. 120 (2):138-142.
Creamer R. (1995). Molecular comparison of California Beet Yellows
Virus isolates. 1995
Sugarbeet Research Review. University of California, Davis. Department
of Agronomy and Range Science.
Dyer, W. E., Hess, F.D., Holt, J.S., and S.O. Duke (1993). Potential benefits and risks of herbicide-resistant crops produced by biotechnology. Horticultural Reviews,15:367-408
Falk B. W. and G. Bruening (1994). Will transgenic crops generate
new viruses and new
diseases? Science, 263:1395-96.
Gassen H-G., Köning B., Sinemus K., and T. Bansgow (1995). Genetic engineering: principles, economic potential and problems with public acceptance. (Gentechnologie: Grundlagen, Wirtschaftspotentiale,und Akzeptanzprobleme). Zuckerind. 120(2):132-137.
Hallman W. K. And J. Metcalfe (1994). Public perceptions of agricultural
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survey of New Jersey residents. Ecosys. Policy Research Center. N.J.
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Hills F. J., Lange W. H., Shepard R. J., and J. S. McFarlane (1982). Sugarbeet pest management: Aphid-borne viruses. Spec. Pub. 3277. DANR, Univ. Calif.
Hoban T. J. IV and P. A. Kendall (1992). Consumer attitudes about the use of biotechnology in agriculture and food production. North Carolina State University, Raleigh.
Nichols J. B., Dalton C. C., Todd G. A., and Broughton N. W. (1992).
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of sugarbeet. Zuckerind. 117(10):797-800.
Sellers, C. and P. G. Lemaux (1996). Development and use of herbicide -resistant crops for California. p212-223 In: Proc. 48th Ann. Calif. Weed Conf., Sacramento, California.
Shah, D.M., Rommens, C.M.T., and R. Beachey (1995). Resistance
to diseases and insects in transgenic plants: progress and applications
to agriculture. Trens in Biotech. 13:362-368
Staskawicz B.J., Ausubel F. M., Baker B. J., Ellis J. G., and J.D. G.
Jones (1995). Molecular
genetics of plant disease resistance. Science 268:661-667.
Steen, P. and H.C. Pedersen (1995). Strategies in creating transgenic herbicide tolerant sugarbeet (Beta vulgaris L.) varieties. p189-196, In: IIRB. Proceedings of the 58th Congress of the International Institute for Beet Research. Brussels, Belgium.
Thomas, T. (1995). Herbicide resistant sugar beet -- a scientist’s view. British Sugar Beet Rev. 63(4):35-36.
FIGURE CAPTIONS
Figure 1. Yearly average sugarbeet root yields and acres planted to sugarbeet on a statewide basis in California (1970 to 1993).
Figure 2. Percent species composition of aphids observed in yellow water pan traps in 1969-1970 and 1992-1993. Percent composition in each year is characteristic for the years when the data was collected. H. Lang and L. Godfrey data. Redrawn with permission.
Figure 3. Sugar yields of experimental lines and commercial hybrids tested for BYV/BWYV resistance (tolerance) at U. C. Davis (1992 to 1995). Comparison is between plants that were inoculated with the viruses and plants that were virus-free for the majority of the growing season. The yield of the susceptible controls is not included in the mean of the inoculated plots. The best yield each year of the inoculated and non-inoculated cultivars are also included for comparison. While increasing resistance to BYV/BWYV is not evident, the lines tested are increasingly more resistant to rhizomania, also a requirement for sugarbeets produced under commercial conditions in California. The need to include a wide range of resistance traits slows the rate of progress in plant breeding programs.
Figure 4. A simplified representation of the DNA double helix. DNA (deoxyribonucleic acid) is made up of a replicated phosphate-sugar backbone and the four nucleic acids (adenine, guanine, thymine, and cytosine). The sequence of nucleic acid bases can vary. By varying, the same four bases can give rise to countless different genes, making countless different proteins. To engineer a transgenic plant, a sequence of bases (a gene) from one source (for example a plant virus) must be inserted into the DNA of the plant in a way which allows the plant to make proteins using the new gene. RNA (ribonucleic acid) differs from DNA in that uracil is substituted for thymine in the sequence of nucleic acids, and the sugar backbone is ribose rather than deoxyribose. Most viruses contain RNA, not DNA.
Figure 5. Representation of the use of an “anti-sense” gene in the Flavr-Savr© tomato. Genes exist as double-stranded complemetary DNA molecules, one strand of which encodes the gene and is used to produce the messenger RNA (mRNA) that is translated to yield the gene product (an enzyme or other complex molecule used to build plant cells and maintain metabolism). In the anti-sense approach, the gene is identified, cloned (copied) and reinserted into the plant cell so that the complementary mRNA is made inside the same plant cell as the coding strand. When this occurs, the two strands join to form a double-stranded structure that prevents the synthesis of the coding mRNA and the gene product cannot be made. In this case, the anti-sense gene keeps the plant from creating the polygalacturosidase enzyme (PG: a protein), which facilitates ripening and decay. A similar approach was used to increase sweetness in the processing tomato.
Figure 6. Representation of the principles of marker-aided breeding. These are techniques to analyze DNA, developed in the last 20 years, which enable plant breeders to locate specific genes or regions of chromosome that correspond to desireable traits. The goal of this technique is to locate readily identifiable markers or regions of the chromosome that are linked with valuable production traits. Sometimes called DNA fingerprinting, this technique is based on the fact that organisms with different characterisitcs have corresponding unique DNA sequences that can be recognized just like a human fingerprint. DNA fingerprinting permits the identification of parts of the DNA pattern associated with particular useful traits. Following genetic crossing, breeders can confirm the presence of the the unique DNA sequences associated with the desired genetic trait, and know that the trait has been successfully transferred to the progeny.
Figure
7. Representation of a method of transferring a viral coat protein
gene to a plant cell and the development of virus resistant plants.
The gall-froming bacteria, Agrobacterium tumefaciens, is used to incorporate
the new gene into sugarbeet plant cells. Gene insertion can occur
one or more times and in any portion of a chromosome. For a
useful plant to occur, gene incorporation must occur in a portion of the
chromosome that does not interfere with normal plant processes and which
allows the expression of the new gene by the plant.
Sugarbeet, Beta vulgaris L., is a naturally cross-pollinating biennial plant and modern genotypes are highly heterozygous. Therefore, the generation of new varieties by conventional breeding practices is slow (Lindsey and Gallois, 1990).
Alternative methods for sugarbeet improvement take advantage of biotechnology to propagate genetically identical plants via tissue culture, speed up classical breeding programs with the use of molecular markers, or transfer desired genes into a transformable sugarbeet line. Genetic transformation allows gene transfer from non-related organisms (including yeast and bacteria) to sugarbeet, unlike conventional breeding which only allows transfer between closely related species. In addition, molecular techniques can be used to manipulate the expression level and tissue specificity of desired genes.
In conventional breeding, it is difficult to breed in a new trait without losing some other existing beneficial traits or without including undesirable traits that are closely linked to the new gene. With current transformation technologies, most sugarbeet lines are difficult to transform, however, when a commercial cultivar can be transformed, a gene which confers disease resistance or herbicide resistance can be added to the cultivar directly without reducing the elite qualities required for sugar production. When a commercial cultivar cannot be directly transformed, the transformed line must then be crossed into an elite line, either a maintainer O-type (cytoplasmic male sterile) or a pollinator.
One of the prerequisites for transformation of a plant species is the demonstration that differentiated plant material (such as leaves, stems, petioles and cotyledons) can be introduced into culture in the laboratory and that the differentiated material can then form undifferentiated tissue, called callus. The callus tissue must contain totipotent cells that are capable of going through all differentiation stages to give rise to a fertile plant. Callus is cued to begin differentiation by treating it with an hormonal regime that allows shoot and root production, ultimately generating a plant. This capability permits the introduction of a new gene into a single cell, that can subsequently form a plant with the introduced gene incorporated into its genome.
It has been demonstrated that shoot regeneration can be triggered from untransformed sugarbeet callus material by several researchers (for a review see Saunders and Shin, 1986). Despite this demonstration, it is not a trivial extension to achieve plant transformation because the methods used to introduce DNA and to identify transformed tissue often have an adverse effect on the ability of the tissue to yield plants. The ability to put plant material into culture, introduce a gene and then get the plant material to reform plants has been the major stumbling block in plant transformation. This difficulty or recalcitrance has been observed in sugarbeet. Compared to many other crop species, sugarbeets have low transformation efficiency.
Transformation
Using a variety of methods, transformation of sugarbeet has been demonstrated
for several genotypes. One successful method used for many dicotyledonous
species involves the co-cultivation of cells with the natural plant transformation
bacterium, Agrobacterium tumefaciens. Agrobacterium in nature
causes crown gall disease by introducing a small portion of its DN into
the plant cell to direct the synthesis of the compounds that help it to
survive. Plant geneticists have taken advantage of this by building
vectors (or carrier DNAs) from the portion of the Agrobacterium DNA that
is transferred. The bacterial vectors are constructed so that the
disease-causing genes are replaced by the genes the researchers wish to
introduce. Using these vectors it is possible for Agrobacterium
to transfer the DNA of choice into the plant cell where it becomes a heritable
part of the plant genome. In addition to the gene of interest, these
Agrobacterium vectors carry a selectable marker, such as antibiotic or
herbicide resistance that allows the identification of transformed cells.
Using such a method (in this case, using dedifferentiated sugarbeet mesophyll
or petiole cells grown in liquid suspension), Kallerhoff et al. (1990)
reported a transformation frequency of approximately 2%. This means
that 2% of the plant cells treated with the Agrobacterium vector integrated
the introduced DNA into their genome and became stably transformed.
A less frequently used method to achieve transformation in sugarbeet
is to introduce DNA directly into plant cells. In order to do this,
plant cell walls must be removed to make protoplasts and then the DNA is
introduced by exposing the cells, bathed in a DNA solution, to an electrical
current. This process, called electroporation, causes holes to form
transiently in the cells thereby allowing the DNA to get in. Using
this method with sugarbeet, the transformation efficiency is quite low,
only two stably transformed cells per 100,000 protoplasts treated (Lindsey
and Jones, 1989). This low frequency likely was due to multiple factors,
including the difficulty in getting protoplasts to regenerate cell walls
and divide normally.
The most common method used to transform sugarbeet utilizes Agrobacterium
tumefaciens or another naturally occurring bacterial strain, Agrobacterium
rhizogenes, which normally causes hairy roots instead of crown galls, to
transfer genes into excised pieces of intact tissue, such as cotyledons,
shoot bases, petioles, leaves, or floral stem tissues. After treatment
with the bacterial strain, these tissues are cultured in the presence of
the selective agent (antibiotic or herbicide) until transformed tissue
is identified which can be regenerated into plants. Several groups
have succeeded in transforming sugarbeet using such a method. Lindsey
and Gallois (1990) have obtained transformed shoots from shoot base pieces
(explants) treated with A. tumefaciens. Four different genotypes
were tested using this method. The best transformation frequencies obtained
(as judged by the numbers of independent events expressing the selectable
markers) were in the genotype Kwerta, where 14% of the tissue explants
gave rise to transformants. Subsequently, Konwar (1994) reported
that, using a similar method, the transformation frequency varied from
2.5% to 10 % depending on the genotype. In this case, the growth of the
transformed tissue was slower than that for tissue from normal plants and
it was demonstrated that 40 to 50 copies of the introduced genes
were integrated into the genome at approximately six sites per genome.
Using cotyledon, hypocotyl, petiole, or leaf explants as starting material
for transformation, D’Halluin et al. (1992) obtained only transformed,
non-regenerable callus or untransformed shoots. However, this group
was able to obtain transformed shoots using callus capable of forming embryos
as the starting material.
Lastly, protoplast fusion may be used to try to improve sugarbeet. It was demonstrated that protoplasts from different genotypes can be fused together with the goal of improving mitochondrial and chloroplast characteristics (for a review see Lindsey, 1992). Jones et al. (1990) have demonstrated that resistance to potato leaf roll virus and potato viruses X and Y can be introduced simultaneously into Solanum tuberosum from the wild relative S. brevidens, which itself does not produce tubers through protoplast fusion. The utility of this method in general, however, is yet to be determined.
Regeneration
Shoot regeneration from undifferentiated callus tissue is crucial for the production of transformed plants from protoplasts or callus derived from excised pieces of tissue. Shoot regeneration is usually achieved by culturing cells or excised tissue on solidified nutrient plates containing plant hormone. The efficiency of regeneration may depend on plant genotype and age, combination of plant hormones, or the culturing environment including the temperature and lighting. A major problem with regeneration from protoplasts is low plating efficiency, because few dividing calli arise even though large numbers of protoplasts are plated. Where plants have been regenerated, the plating efficiency is less than 1% (Hall et al., 1993). Most researchers report efficiencies in the 0.1% to 0.2 % range, but with some genotypes 2% to 4% of the protoplasts form dividing calli.
There are a number of factors that influence the plating efficiency
from protoplasts. Certain chemicals can be added during the culture
phase that aid in the survival of protoplasts (e.g. n-propylgallate, a
lipoxygenase inhibitor crucial for the viability of mesophyll protoplasts).
The plating efficiency can also be improved by approximately 10-fold by
using other extenders, such as feeder cells (cell-wall containing suspension
cells embedded in the agar used to provide certain growth factors for the
cultured protoplasts) or embedding the protoplasts in certain types of
solidifying medium, such as calcium alginate (Hall et al., 1993).
These plating efficiency improvements, however, do not necessarily improve
the ability to produce regenerable calli since the majority of calli recovered
using these methods were not regenerable. In addition, plant regeneration
in general is less likely to occur when using protoplasts derived from
tissue that has been cultured for long periods, since callus tissue and
suspension cells tend to lose their regenerability with time. The
genotype of the source tissue also affects the plating efficiency, although
recently, Schlangstedt et al.(1994) reported that protoplasts from a certain
tissue, i.e. the petiole, gave higher plating efficiencies (4.6%) than
mesophyll protoplasts (1.0%) regardless of the genotypes used.
The second type of regeneration system usually involves either
direct or indirect formation of shoots or embryos from explants directly
or from callus derived from explants. Sugarbeet somatic embryos (derived
from asexual reproduction) can be induced from zygotic embryos (immature
plantlets derived from sexual reproduction) directly, and these somatic
embryos can be induced to form mature plants (Tenning et al., 1992).
When Lindsey and Gallois (1990) compared shoot regeneration from leaf disks,
petioles, and shoot base slices, they found no regeneration of shoots from
leaf tissue and a very low frequency from petioles. Shoot formation
was much more prolific and rapid from shoot base slices in all four genotypes
tested. However, Zhong et al. (1993) have reported that sugarbeet shoots
can be produced directly from the petiole, or indirectly from callus formed
from the base of the petiole. Considerable heritable genetic variation
was observed in petiole-derived shoots. The differences observed
in the regenerability of different tissue sources may depend on the genotype
used. More recently, cotyledons have become the most commonly used
source of tissue for in vitro manipulation. Autonomous cell lines and hormone-treated
cell lines which become habituated to the hormone regime so that they no
longer require the presence of hormones for growth and regeneration have
been identified in several genotypes (Kevers et al., 1981; Saunders and
Doley,1986; Doley and Saunders, 1989). A large study was done in
order to assess the shoot regeneration capability in Beta vulgaris L.
Seventeen germplasm sources including sugarbeet, table beet, fodder beet
and leaf beet were screened for callus-forming and shoot regeneration ability
(Saunders and Shin 1986). Some individual genotypes from each of
the 17 germplasm sources produced callus; shoot regeneration from callus
was seen in 14 of the 17 germplasm sources, indicating that this ability
is widespread in the species. Besides the genotype effect, certain
environmental conditions, such as light intensity and temperature, affected
callus induction frequency. The ability of a particular germplasm to generate
shoots from explants or callus affects the speed with which improvements
via genetic engineering can be brought to commercial usage.
A quick method for producing completely homozygous plants that will uncover all recessive traits simultaneously is to generate haploid plants from unpollinated ovules. In this system chromosome number can be doubled either spontaneously or by applying colchicine to the shoot tip (Doctrinal et al., 1989; Ragot and Steen, 1992). Both genetic and environmental factors were found to affect chromosome doubling ability of sugarbeet haploids (Ragot and Steen, 1992). Using conventional breeding, many years would be required to construct a nearly homozygous genotype. The use of haploids reduces the time required to produce commercially useful cultivars by a number of years.
Rhizomania resistance
Rhizomania is a disease characterized by massive lateral proliferation of rootlets on the main root resulting in severe stunting and consequent reduction in the sugar content of the sugarbeet. The causal agent of the disease is beet necrotic yellow vein virus (BNYVV), a member of the furovirus group, which is transmitted to the plant roots by the fungus, Polymyxa betae. The genome of BNYVV consists of four different single stranded (positive-sense) RNAs. Some Japanese isolates also contain a fifth RNA ( for review, see Gilmer et al. 1992). A rhizomania-resistant sugarbeet variety, called C28, was developed through classical breeding by the USDA-ARS and released in 1989. Based on segregation of resistance in backcrossed lines, the resistance appears to be due to a single dominant gene (Lewellen, 1991). This rhizomania-resistant germplasm does not have other elite qualities for high sucrose output but it has been used in breeding programs to introduce resistance to elite cultivars.
Other approaches to generating rhizomania-resistant germplasm consistently
include using molecular techniques to engineer protection in sugarbeet.
Several molecular strategies for making rhizomania-resistant plants have
been carried out to varying extents and some genetically engineered rhizomania-resistant
lines are already in field trial. One strategy utilizes coat protein
mediated protection (CPMP), in which the gene for the coat protein of the
virus is introduced into the plant, thereby affording protection. The first
successful example of CPMP was in tobacco plants expressing the tobacco
mosaic virus coat protein gene (Abel et al., 1986). This approach
has since been extended to a large variety of other RNA viruses in a wide
variety of plants (Wilson, 1993). The mechanism by which CPMP works
is still not clear. In some cases, making the coat protein itself
is necessary for the protective effect. In other cases the presence
of the coat protein in resistant transgenic plants is not necessary.
Rather, the presence of the coat protein RNA alone is sufficient to confer
resistance, indicating that the mechanism of resistance is associated
with interference with viral RNA replication in transformed cells.
This hindrance may be unrelated to the presence of the actual viral coat
proteins produced by the transformed plant cells.
In sugarbeet, Kallerhoff et al. (1990) successfully transformed suspension
cells (plants growing in a liquid medium) with the BNYVV coat protein gene
using co-cultivation with Agrobacterium tumefaciens. Protoplasts,
isolated from untransformed cells and transformed cells expressing the
viral coat protein, were infected with BNYVV. The coat protein-transformed
protoplasts showed a 73% to 97% reduction in viral titer in six independent
infections. Using a similar strategy, Ehlers et al. (1991) introduced the
BNYVV coat protein gene into sugarbeet roots using A. rhizogenes.
Stable integration into the genome and expression of the coat protein gene
was demonstrated. Even though roots of sugarbeet seedlings can be
infected with the virus, this group was unable to infect transformed roots,
probably due to changed root physiology. Several European seed companies
including Danisco Seed, KWS, SES and Hilleshog all have advanced to the
field testing stage for their own CPMP rhizomania-resistant lines.
KWS reports that transformed lines produce viral coat protein in the absence
of infection when tested using the ELISA technique. If
the resistant plants continue to show promise, within the next 5 to 6 years
there should be varieties available to beet growers that will be practically
immune to rhizomania (Büchting, 1995).
Another molecular strategy for mediating viral resistance involves
the engineering of plant cells to make defective interfering RNAs (DI-RNAs).
DI-RNAs occur naturally in certain plant viral infections and in some way
their presence prevents the efficient replication of the virus, thereby
reducing symptoms. The use of DI-RNAs, made by deleting parts of
the viral RNA so it can no longer replicate by itself, usually lessens
the symptoms by interfering with the replication of the normal virus.
Hehn et al. (1994) reported making DI-RNA transcripts from BNYVV RNA that
were tested for their ability to inhibit replication of viral RNAs in Chenopodium
quinoa (an Andean plant distantly related to the Beta genus).
One of the constructs caused a 90% inhibition of synthesis of the normal
viral RNAs, however, a large excess of DI-RNA is needed relative to the
normal viral RNA in order to cause the inhibition. The feasibility
of employing such DI-RNAs for plant protection will depend on whether the
defective RNA can be made early in infection in amounts sufficient to compete
effectively with the infecting virus.
Currently, molecular markers are being used to assist breeding programs (fig. 6) to transfer naturally occuring BNYVV resistant genes from resistant accessions into breeding material (Scholten et al.,1995; Pelsy et al., 1995; Long, 1995). Molecular markers are similar to co-dominant morphological traits and furthermore they can be made in almost unlimited numbers. Markers linked to the resistant genes can be distinguished by polymorphism (presence or absence of a DNA fragment) between disease-resistant and -susceptible individuals. To date, two chromosome linkage maps of sugarbeet based on RFLP (restriction fragment length polymorphism) markers have been published (Barzen et al., 1992; Pillen et al., 1992). They are certainly non-exhaustive, and more markers need to be incorporated.
Beet Western Yellows Virus
Beet Western Yellows Virus (BWYV) causes a yellowing disease in sugarbeet and other hosts such as lettuce. BWYV is a member of the luteovirus family. Luteoviruses are obligately transmitted by aphids and the virus is mostly confined to the phloem tissue of the host. Most luteoviruses have relatively narrow host ranges, but BWYV is an exception to this rule, infecting a wide range of dicotyledonous and some monocotyledonous plants. BWYV has just a single genomic RNA of about 5.6 kilobases (for a review see Veidt et al., 1992).
Naturally resistant germplasm has been identified in sugarbeet and lettuce. Sugarbeet lines, C31-43 and C31-89, are sister lines that have good to moderate resistance to yellowing disease caused by beet yellows virus and beet western yellows virus but are moderately susceptible to curly top virus (CTV), (Lewellen and Temple, 1992). These lines gave high sugar yield, moderate sucrose concentration, and low soil tare. They yielded better than commercial hybrids under heavy BWYV infection conditions. BWYV-resistant lettuce lines have been identified recently and, in one line, the resistance is due to a single dominant gene (Maisonneuve et al., 1991), while in the other line it is due to a single recessive gene (Pink et al., 1991).
To date genetically engineered BWYV-resistant sugarbeet varieties have
not been reported. Most of the recent research on BWYV has focused
on the identification of the viral genes and their function. Six
potential genes have been identified (for a review see Reutenauer et al.,
1993), with one of them encoding the coat protein (Brault, 1995).
Plant protection using CPMP or alternative strategies, such as antisense,
may provide useful resistance to BWYV since such strategies have been used
successfully with another luteovirus, potato leafroll virus, in transgenic
potatoes (Scholthof et al., 1993).
Beet Yellows Virus
Beet yellows virus (BYV) is a member of the closterovirus group and is transmitted by aphids primarily to sugarbeets and closely related species like spinach. In California, old sugarbeet fields are the primary source of the disease. The large genome of this RNA virus has been sequenced and analyzed (Agranovsky et al., 1994) and the coat protein gene identified. Non-transformed sugarbeet lines C31-43 and C31-89 show limited resistance to BYV. Commercial efforts to genetically engineer resistance to BYV using CPMP have been recently initiated by several European companies. Transformed plants have been created in several locations but there are no reports about the effectiveness of CPMP constructs against BYV infection.
Herbicide Tolerance
An alternative to current weed management in sugarbeet is to grow herbicide resistant/ tolerant sugarbeet plants in the field. The tolerance being engineered is to rapidly degrable herbicides. This allows the use of herbicides that do not persist in the environment. It also provides additional flexibility in the timing of herbicide applications. Different herbicide-tolerant transgenic sugarbeet cultivars have been produced to date. The main herbicides targeted for tolerance breeding are glyphosate, sulfonylurea compounds, and phosphinothricin.
Glyphosate is the active ingredient in Monsanto’s Round Up® herbicide and several other herbicdes. It targets an enzyme (EPSP synthase) which plays a key role in the synthesis of certain amino acids, such as tryptophan, tyrosine and phenylalanine, which in turn are essential for protein synthesis. EPSP synthase, encoded in the nucleus, is imported into the chloroplast where it functions in amino acid synthesis (for a review, see Lindsey, 1992).
Essentially, two strategies have been adopted to engineer tolerance to glyphosate in transgenic plants. In the first strategy, genes encoding mutant EPSP synthase proteins resistant to glyphosate have been introduced into plants. Researchers at Calgene have reported isolation of a mutant bacterial gene that has a lower affinity for glyphosate (Thompson et al., 1987). Transgenic tobacco plants transformed with this gene have enhanced tolerance to glyphosate. To further improve the efficacy of this approach it is presently being modified to allow targeting of the gene to the chloroplast.
In the second approach, a wild-type plant gene is over-expressed thereby confering resistance. Researchers at Monsanto isolated a gene for EPSP synthase from a Petunia hybrida cell line (Della-Cioppa et al., 1986). In vitro assays using isolated chloroplasts demonstrated that the enzyme was transported to the chloroplast, thereby increasing the effective levels of the enzyme in the affected organelle. This same EPSP synthase gene was modified for improved resistance through mutation and transformed into flax plants (Jordan and McHughen, 1988). These plants were found to be resistant to the herbicide. Hilleshog produced glyphosate-tolerant sugarbeet by introducing two genes into their sugarbeet line, one coding for a mutated EPSP synthase enzyme, the other for a different enzyme involved in chloroplast metabolism (Tenning et al., 1995). Monsanto and Danisco Seed have also produced glyphosate-tolerant sugarbeet. Field tests in Europe and the United States indicate that the tolerance is effective (Brants, 1995; Steen and Pedersen, 1995). Monsanto reported at a recent meeting held at UC Berkeley that their glyphosate-tolerant sugarbeets will be available commercially in parts of the United States in the year 2000.
Tolerance to two other herbicides (Basta® and Herbiace®) is also being engineered. Phosphinothricine (PPT), also called glufosinate-ammonium, is the the active ingredient in both and is a glutaimine analogue. PPT and bialaphos (which contains PPT and two alanine residues) are highly toxic to plant cells because PPT acts as a competitive inhibitor of the enzyme glutamine synthetase which uses ammonia to create glutamine. Bacterial cells which naturally synthesize bialaphos are not killed by their own product because they contain the bar gene. This gene codes for an enzyme, phosphinothricin acetyltransferase (PAT) that modifies and inactivates PPT. Resistance to PPT has been transferred from the bacteria to sugarbeet and several other crops by transformation with the bar gene fused to a promoter with strong expression in plants (D’Halluin et al., 1992; De Block et al.,1987). KWS has successfully used glufosinate resistance as a marker gene for transformation. Those plants carrying the transformed gene are tolerant of the herbicide. In 1994 Danisco Seed identified several transgenic sugarbeet lines very tolerant to glufosinate (Steen and Pedersen, 1995).
Sulfonylurea compounds are included in Du Pont’s herbicides Classic®
(chlorimuron ethyl), Glean® (chlorsulfuron), or Harmony® (trifensulfuron
methyl). They block the biosynthesis of the branched chain amino
acids valine, leucine and isoleucine (for reviews see Lindsey,1992).
The target enzyme is acetolactate synthase (ALS) which is similar to EPSP
in that it is encoded in the nucleus but located in the chloroplast.
Mutant tobacco and sugarbeet ALS genes tolerant of sulfonylurea compounds
have been transformed into sugarbeet by D’Halluin et al.(1992) from Plant
Genetic Systems in Belgium. Transformants were tolerant of field
levels of sulfonylurea compounds applied under greenhouse conditions.
Nematode and fungal resistance
In addition to rhizomania and virus yellows, cyst nematodes and pathogenic fungi also cause considerable damage to sugarbeet crops in California. Cyst nematode resistance is found in wild beet species. Both KWS and SES are trying to transfer the resistant gene into breeding cultivars with the aid of molecular markers (Büchting, 1995; Long, 1995). In the same two reports, both companies are also trying to improve the resistance against fungal diseases; KWS is focusing on Cercospora, mildew, and seedling diseases while SES is targeting Cercospora and Aphanomyces (black-leg). SES has extracted a gene from radish that codes for a protein-based fungicide. Other genes active against more than one fungus have also been identified (Long, 1995).
Other opportunities for improvement
There are many more areas of interest for sugarbeet improvement that can benefit from genetic engineering. For example, biotechnology may be used to reduce impurities such as betaine and invert sugar from the storage root, possibly increase sucrose content by overexpressing a sucrose “carrier” protein that has been identified in sugarbeet, or allow novel materials such as biodegradable plastic or fructose polymers to be produced from sugarbeet (Nichols et al., 1992), Carbon metabolism in sugarbeet is being investigated in order to understand basic mechanisms leading to the storage and utilisation of sucrose in the tap root (Monger et al., 1995). Making bolting-resistant sugarbeet would enable early sowing in region where frost or cold weather is a problem in the spring, thus increasing sucrose yield by more efficient use of solar radiation. Van Roggen at IARC-Broom’s Barn, in the United Kingdom is isolating and modifying the expression of sugarbeet genes that affect the transition from the vegetative to the reproductive stage. Flower development genes, cloned from another species- Arabidopsis thaliana, can be used as probes to identify genes with similar function in sugarbeet.
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Why should we care?
Currently, sugarbeet acreage is declining in the Central Valley of California. Low sugar prices, increasing production costs and stagnant yields are the causes. Where yields have improved in recent years (Imperial Valley), there is a greater demand for sugarbeet contracts than the factory can handle. There, the crop fits well into the winter cropping cycle and is tolerant to many of the region’s most important pest problems and its saline soils and irrigation water. Acreage has also expanded in the Upper Klamath Basin, and yields are steadily increasing in that location.
The public benefits from the state’s sugarbeet industry. First, producing sugar in California helps keep the price of this basic commodity stable and low for consumers. While excessive consumption of sugar is poor for health, moderate consumption adds considerably to people’s enjoyment of life. Second, sugar production is a primary industry with a basis in renewable natural resources, including sunlight, soils and water. The reliable, renewable creation of jobs and income is vital to the state’s economy and the general well-being of its people. Third, there are significant potential environmental advantages associated with the inclusion of sugarbeets in farming systems in many parts of the state. This is particularly true where ozone and salinity are problems because sugarbeet is more tolerant of these conditions than most crops. The crop is deep-rooted and can recover nutrients and water lost by other, shallower-rooted crops. Compared to other field crops, fall-planted sugarbeets offer significant savings with respect to irrigation and pest management costs, though not all of the crop can be fall-planted. Fourth, a more diverse crop rotation is easier to manage in an environmentally sound way than a narrower rotation. There is no reason to think that this fact will ever change in agriculture. More broadly, sugarbeet is important in agricultural systems worldwide and is a significant resource in humankind’s battle against hunger. There is ample justification for public involvement in research and extension to support the crop.
Should the industry now undertake, in partnership with the University and/or the USDA, an investment in a new area of research and technology--biotechnology? There are three questions that must be answered for such an investment to make sense:
1. Are there clear benefits from pursuing a biotechnological approach to sugarbeet research and breeding that can be achieved in no other way?
2. Are there reasonable chances that other benefits (some unforeseen) can be derived from a molecular breeding program?
3. Does this work have to be done in California?
With these questions in mind, the arguments against such an undertaking
are contrasted with those supporting the use of biotechnology in sugarbeet
breeding and research in California.
Arguments Against
1. There is a high level of activity in molecular approaches to sugarbeet breeding in Europe. Several companies report work in this area including KWS (the parent company of Beta Seed in Germany), Hilleshog-Mono Hy (Sweden and France), SES (Belgium), and Danisco (Denmark). All of these companies have transformed sugarbeets for resistance to low-impact herbicides, such as glyphosate or glufosinate. Several (KWS, Danisco, Hilleshog Mono Hy) have reported producing beets that have been transformed with the viral coat protein gene intended to provide rhizomania resistance. At least two companies have introduced BYV coat protein genes into sugarbeets and generated plants that can be tested for resistance. Other transformation goals are also referred to in the informal literature, including resistance to Cercospora and Aphanomyces, organisms causing fungal diseases.
The European sugar industry and the seed companies that serve it are well-funded because the industry is considered essential to the material well-being of European farmers and vital for the preservation of the European landscape. These resources and the effort derived from it have served the European industry well by providing it with a low but real rate of yield increase, and a stable, if not decreasing, cost of production. In contrast, the American industry is suffering from marginal prices, relatively flat yields and a low regard for the value of the industry. The resources that can be derived from the production of the crop and directed towards research in the United States, and especially in California, are more limited than in Europe. Given these circumstances, the American and California industries have been willing to wait for results from molecular approaches from abroad. At least one of those companies (Beta-KWS) is agressively pursuing the California market.
2. The possible uses for molecular approaches to breeding are
only limited by basic knowledge of the system and the creative imaginations
of the scientists involved. The creation of fertile, resistant (transformed)
plants, however, is currently limited by another factor--the difficulty
of transforming sugarbeet using current molecular techniques. The
first transformed plant species was tobacco. Many other crop species
have proven amenable to transformation, such as the Flavr-Savr® tomatoes
and Bt-cotton that are currently on the market (fig. 5). Plants in
the cabbage family, particularly oilseed rape (canola), have been modified
with success and these products are approaching commercialization, as is
a transgenic variety of crookneck squash that is protected from viral damage.
Rates of successful transformation with sugarbeet are variable but low
compared to many other species. A transformation rate of 1.0% means
that out of 100,000 attempts, 1000 potentially useful, independently transformed
plants will arise. In practice, from that 1,000 only ten plants (0.01%)
may possess resistance characteristics that are useful in a breeding program.
Given these low levels of success, one scientist engaged in the work characterized
the identification of a useful transgenic plant at present as essentially
a random event.
3. One reason why success from molecular breeding has been slow
to achieve is that little is known about gene expression and how
this affects, for example, the development of disease resistance.
The capacity to transform plants has progressed faster than the understanding
of how basic mechanisms in the cell work at this fundamental level.
Also, some plant traits are influenced by large blocks of genes or by genes
at several loci. Manipulating such traits using molecular techniques
is still problematic. Efficient methods exist only for single genes
or at most a few genes at a time.
Nevertheless, the promise of the technology is so great that efforts to manipulate single traits are going forward in the absence of a full understanding of how the process works. This is not unlike, however, the situation in classical breeding where the reasons for the introgression of certain loci are not completely understood, but their efficacy documented and the movement into commercial germplasm realized nonetheless. Enthusiastic engagement with molecular techniques is not so different in kind from other new, exciting but unproven technologies in an industrial economy. But the chance for success with a difficult species in the short term, when understanding is so incomplete, will be frustratingly low. Without an investment of sufficient resources, success most likely will not be realized in the reasonable near-term. Such funds may not be available within the industry.
In summary, three arguments can be advanced for deferring an investment
in a molecular breeding strategy in California at the present time:
1. The financial resources available may be too limited to allow
for the level of investment needed, particularly that necessary to enlarge
upon the effort made by the European seed companies.
2. The technology is unlikely to lead to short-term successes because sugarbeets are a difficult species to manipulate.
3. Little is understood about the basic biology of sugarbeet and about the transformation process. In the short-term, useful results seem more promising than real.
Arguments in Favor
1. The long-term promise of molecular technologies is very great. Molecular breeding provides precise control because individual genes controlling specific traits can be incorporated without genetic drag. Traits from other plants and organisms, not otherwise found in sugarbeet or closely related species, can be used in breeding programs, greatly broadening the range of resistance genes available. In the last decades, growers have relied on pesticides for plant protection. With time, fewer pesticides will be available and there will be greater restrictions on their use. Resistance will become the cornerstone of plant protection. Molecular biology offers the opportunity to broaden and quicken the development of reliable resistance characteristics. Additionally, the ability to manipulate specific traits also will lead to new insight about the physiology of the crop, resulting in further beneficial developments in both classical and molecular breeding and crop management.
2. Presently, sugarbeet is relatively recalcitrant to the new genetic technologies. Technical progress is rapid, however, and it is likely that within a few years, this difficulty will be overcome. Although the first successful plant transformation (tobacco) occurred in 1987, the first success with a cereal did not occur until 1991 (rice) due to the recalcitrance of grasses. Progress in cereals has been steady since that time and now nearly all cereals can be transformed efficiently. Genetically engineered cereal crops are currently entering the commercialization phase. Research in molecular breeding and tissue culture of the sugarbeet crop in Europe is active, and technical progress is likely.
3. Most of the effort to transform sugarbeets and produce elite germplasm is being carried out in Europe by private firms such as KWS (Beta), Hilleshog-Mono Hy and others. Rhizomania and Beet Yellows Virus are the focus of programs at some of the companies, both problems of importance in California. Most of the transformed sugarbeet cultivars, however, are based on those marketed and adapted in areas other than California. Companies are likely to concentrate on lines with the greatest acreage potential, and less on California, which has a lower overall acreage than other European or U. S. locations, and different conditions for production. This is especially true if acreage declines further in California. Without an effort to manipulate and transform California germplasm, the adoption of advances achieved abroad from molecular breeding programs may be slow to reach California, further disadvantaging the state’s industry. Additionally, pest and disease problems that are serious only in California or which are unique to the state are likely to receive little attention.
4. Knowledge in plant molecular genetics is cumulative. Because research in molecular biology takes place at the fundamental level of genetics, biochemistry and physiology, knowledge can be transferred or adapted between species. Knowledge gained from research on other species can be used to improve sugarbeet provided the basic scientific and technical capability to manipulate the crop is present. For example, much is being learned about pest resistance to insects, viruses, fungi and bacteria in other crops that will facilitate the improvement of sugarbeet through molecular methods. The viral coat protein approach has proven effective on over fifteen plant species and is now being extended to sugarbeets for rhizomania and BYV resistance. Other molecular technologies are being perfected in a diversity of crops and these might be extended to sugarbeet in the future. The University of California, collectively, was the pioneer in the development of the new molecular technologies and probably has the greatest potetial for continued use and improvement of the system of any research institution in the world. This capacity represents a potential advantage for the state’s sugar industry. The state’s sugarbeet industry, however, has not yet benefited directly from that capacity.
In summary:
1. The promise of molecular technology is great. Increasingly, resistance to pests and diseases will become more important as pesticide use declines. Valuable secondary benefits, at this point largely unforeseen, will be derived from a research program.
2. Progress in overcoming the technical difficulties frustrating efforts to transform sugarbeets using single traits is occurring. Other recalcitrant species can now be transformed with reasonable efficiency.
3. For European work to be useful, it must be incorporated into germplasm useful under California conditions and will depend on the availability of the European germplasm.
4. There is a growing capability and understanding in molecular areas and the University of California is a leading institution in the development of biotechnological approaches. The state’s sugarbeet industry should position itself to benefit from this capability.
What We Think
The partnership between farmers, farming-related industries and the University and USDA’s Agricultural Research Service has been a productive and valuable one for more than 100 years in California agriculture. The public’s welfare has been significantly enhanced as a result of this partnership. There is no reason to think that the essential characteristics of that relationship should change in the future. We believe that the use of molecular technology will be essential to the continued, long-term well-being of the state’s sugar industry. Based on the history of agricultural technologies in general, and a careful reading of current literature, we believe that current limitations to success will be overcome soon and that progress will become rapid in molecular breeding efforts in sugarbeet.
A less specific, but equally important consideration is the need to re-create momentum in scientific discovery related to the sugarbeet crop. Classical plant breeding has succeeded in creating sugarbeet lines with high-yield potential. Despite these genetic improvements, chronic diseases and low returns have limited farmers’ abilities to raise sugarbeet yields and improve returns from the crop. To be successful in the future, sugarbeet yields must rise, input costs and the use of pesticides must decline, and crop quality must improve. New pest and disease challenges will inevitably arise. It is hard to imagine achieving all of these goals without the contribution of the powerful tools of biotechnology. Currently, other crops that in essence compete with sugarbeets for a place in California’s farming systems (cotton and tomatoes are examples) are receiving the benefit of that contribution.
One of the most interesting possibilities for molecular breeding is
the prospect that some long-term, chronic problems of sugarbeet in California
can be solved, as opposed to managed. BYV is a prime example.
The short-term outlook for control of BYV is good given careful management
of the beet-free program, the advent of imidicloprid as a seed treatment,
and the relatively few acres of sugarbeets being planted . On the
other hand if sugarbeet plants were effectively immune to BYV, some difficult
planting and harvesting limitations could be removed from the industry.
Solving, rather than managing problems, is one of the most compelling aspects
of the application of molecular biology to plant systems.
We believe that an effort to form a public-private consortium focusing
on the development and application of molecular techniques to sugarbeet
should be undertaken. One outcome might be the creation of a research
team within the University with the capability of advancing the basic understanding
of the biology of sugarbeet and its transformation and, at the applied
level, of introducing resistance genes into sugarbeets.
This would create a support-base in California in which experience with
molecular manipulation of beets would be gained, enabling the state’s industry
to take rapid advantage of future breakthroughs in the field.
The use of molecular technologies will not replace the need for good agronomic research and improved farming practices. Classical plant breeding will remain the most important means of creating new varieties for the foreseeable future and will be essential for the application of new molecular tools. Other types of research, such as cultivar evaluation and the evaluation of new pesticides, will continue to be necessary. However, molecular breeding and other new technologies will be part of the future of the industry, wherever it will have a future. The uses of the sugarbeet crop in farming systems in California are much greater than currently reflected in crop acreage. An industry investment in molecular technology is needed to maintain the competitiveness of the crop in California’s farming systems.
An industry commitment requires a reciprical investment from the University. California has created an unprecedented system of public higher education and research based on the land-grant model. The amount of accumulated capability in the area of molecular biology within the University system is unequaled anywhere in the world and represents a potential advantage to the state’s industry. The industry should try to find ways to utilize this collective capability to generate further economic advantage for the people of California. For its part, the University should find ways to better apply its rapidly accumulating expertise in molecular biology to problems of economic and ecological importance in California.
The rapidly growing human population of our state and the world at large will require an ever increasing effort to sustain adequate food production without requiring the consumption of most natural resources in the process. Maintaining a diverse, increasingly efficient agriculture is of primary importance to all the people of California. For this reason there is a more urgent need for public resources to be applied to crops that are important for the sound agronomic and environmental management of farming systems, but which may generate fewer of the financial resources needed to support research than do crops with larger acreages. Nor is the current comparative advantage of some crops relative to others a permanent phenomenon.
The sugarbeet crop has many characteristics that make it valuable in farming systems throughout California. The application of molecular technologies to solve the most persistent problems of producing the sugarbeet crop, while further improving its useful characteristics, is a worthy use of public and private resources. A public-private consortium should be developed towards this end.
