The growing public and political pressure to minimize the use of pesticides has led to increasing emphasis on the breeding of cultivars with resistance or tolerance to disease and pest attack. Notable success in resistance breeding was achieved in controlling onion pink root, a soil-borne disease of warm regions. Early studies indicated the presence of a single recessive allele conferring resistance to this disease in Bermuda-type cultivars. This was successfully transferred to other sweet types suitable for the southern USA. Later research has indicated susceptibility to other strains of the fungus in resistant cultivars (Pike, 1986).
This illustrates the important point that resistance, particularly the sort conferred by a single gene, may break down in the face of new strains of the pathogen. Disease and pest organisms usually have short generation times and rapid rates of evolution, factors that favour the early breakdown of varietal resistance, particularly if introduced on a widespread and continuous basis.
Various degrees of resistance, indicating the influence of a number of genes of small effect, have also been observed for pink root disease. Such polygenic resistance may not be as complete as single-gene resistance, but it is less liable to catastrophic breakdown in the face of a new, virulent strain of the pathogen than is single-gene resistance.
Allium fistulosum is highly resistant to pink root as well as to the other important onion diseases such as neck rot, anthracnose, smut and leaf rot due to Botrytis squamosa (see Table 5.5). In view of this, many interspecific hybrids between A. fistulosum and common onion have been produced and shown to be resistant to pink root. One or two of these hybrids are grown as bunching onions, but backcrossing the hybrids to onion to incorporate resistance into an acceptable bulb onion variety has proved difficult because the hybrids are largely sterile. This sterility results from various abnormalities in meiosis in the hybrids, and these have been analysed in considerable detail (Kik, 2002).
In an effort to overcome sterility and therefore to be able to transfer some of the disease-resistance genes found in A. fistulosum to A. cepa, some crosses between the two species have been followed by the deliberate induction of a doubling of the number of chromosomes. The resulting pairs of homologous chromosomes from A. fistulosum and from A. cepa can pair with each other during meiosis and normal crossing over, leading to viable pollen and egg cells, can occur (Jones and Mann, 1963). Such plants are termed amphidiploids, an example of which is the cv. ‘Beltsville Bunching’ that is grown from seed as a minor crop in the USA. An amphidiploid from a cross between a shallot and A.
fistulosum, which was backcrossed to the shallot, gave rise to the vigorous, pink root-resistant, shallot-like ‘Delta Giant’, which is grown for bulbs in Louisiana.
This is a triploid with 24 chromosomes, two sets of eight derived from shallot and one set from A. fistulosum. The oriental Wakegi onion is a diploid hybrid between shallot and A. fistulosum, but it is propagated vegetatively.
Several groups have demonstrated some exchange of genetic material between homologous chromosomes of the two species in backcrosses of the hybrid to A. cepa (Kik, 2002). More recently, it has been shown that both A. cepa and A.
fistulosum will cross with the wild species A. roylei to produce fertile hybrids. Allium roylei is a diploid with an amount of DNA in the chromosomes that is intermediate between the two crop species. In a cross between A. cepa and an A. fistulosum Alium roylei hybrid to form the three-way hybrid, it was possible to show that crossing over, and therefore genetic exchange, occurs between homologous chromosomes from all three species. Moreover, recombination points were randomly distributed over the chromosomes and not confined to a limited region near the centromere, as occurs in A. fistulosum (Kik, 2002). Thus A. roylei can act
as a genetic ‘bridge species’ that can make the introgression of useful genes from A.
fistulosum into common onion a practical possibility.
Interest in A. roylei arose when it was found to be completely resistant to onion downy mildew and partially resistant to onion leaf blight, B. squamosa. The hybrid between A. roylei and A. cepa produced pollen with few chromosomal abnormalities and was therefore fertile, and could be backcrossed to A. cepa.
Downy mildew resistance was shown to be due to a single dominant gene, which has been named ‘Pd’ and which is located at one end of chromosome 2 in A.
roylei. This was shown from a genetic linkage map based on molecular markers and by direct visualization of a DNA marker for the Pd gene on the chromosome using a fluorescent stain linked to probe DNA.
A DNA sequence of 20 base pairs closely linked to Pd and which can be amplified for ease of identification has been developed (termed a ‘sequence-characterized amplified region’ (SCAR) marker). Genetic mapping of the crosses between A. roylei and onion showed that the mildew resistance gene was located at the end of chromosome 3 (Scholten et al., 2007). The discriminating power of the SCAR marker to show only downy mildew-resistant genotypes declined during the programme of repeated back-crossing of resistance-carrying genotypes with onion to produce resistant, good-quality onions. Hence, new AFLP markers for the resistance gene had to be developed (Scholten et al., 2007). Furthermore, it was found that a region of the A. roylei fragment introduced into onion was lethal when homozygous within an onion genetic background.
However, one individual in which the A. roylei genetic fragment was shorter carried the mildew resistance gene but had lost the lethal recessive gene (Scholten et al., 2007). From this, breeding lines homozygous for mildew resistance that could transmit resistance to all their progeny were developed.
Several companies that have developed downy mildew-resistant onion cvs are using genetic markers to follow the incorporation of the Pd resistance gene into their breeding lines. This avoids having to perform time-consuming assess-ments for resistance to the disease at every step, and represents a good example of marker-assisted breeding. Commercial downy mildew resistant cvs are expected to be available in 2008–2009, some 20 years after the original identification of resistance in A .roylei (Scholten et al., 2007).
The potentiality exists to introduce resistance to many diseases using genetic transformation (Eady, 2002). Incorporation of engineered viral protein genes into plant genomes has conferred resistance to viral disease in a number of crops.
The DNA sequences that code for the coat-proteins of allium carlaviruses and potyviruses, groups that include a number of serious diseases (see Table 5.3), have been determined. Using this information it should be possible to engineer and express these sequences in alliums to induce resistance.
Many onion fungal pathogens cause damage by hyphal invasion, and this can be combated by various resistance genes. A number of these have been identified and shown to act by preventing the growth of fungal cell walls,
inhibiting the plant-damaging enzymes produced by invading fungi or by detoxifying fungal toxins. An example of the latter type is the gene encoding the enzyme oxalic oxidase, which produces an enzyme that breaks down the fungal toxin oxalic acid to carbon dioxide and hydrogen peroxide. This stops oxalic acid decreasing tissue pH which, in turn, prevents the fungal pathogenic enzymes from working effectively. This gene has been introduced into other plant species and found to inhibit fungal invasion. It has now been introduced into onions using Agrobacterium-mediated transformation (Eady et al., 2005). Oxalic acid is the toxin produced by Sclerotium cepivorum, the causal agent of onion white rot, when it invades roots. Another intriguing possibility for preventing this intractable disease is to use ‘gene silencing’ techniques to ‘switch off ’ the genes responsible for producing the volatile sulfur compounds in allium roots.
Sclerotium cepivorum sclerotia are stimulated to germinate by the release of these substances in soil. A root-specific alliinase enzyme has been identified and, by
‘silencing’ the gene coding for this enzyme, the release of the sulfur compound germination signal to the disease fungus might be prevented, thereby avoiding infection (Eady, 2002).
An interesting example of durable resistance to a pest is provided by that of onion to Thrips tabaci, the onion thrip, probably the most severe pest of the crop worldwide. Resistance was found in a cultivar with a wide angle of divergence of the innermost leaves, and in which successive leaf sheaths elongate beyond the older ones that enclose them. The pest normally shelters and proliferates in the crevice between the youngest leaves, so this morphological adaptation denies the pest its usual habitat. Another external change, ‘glossy foliage’ – caused by a lack of wax on the outside of the leaf – confers thrips resistance. This is caused by a single recessive gene allele. However, plants with this characteristic have increased susceptibility to downy mildew and purple blotch disease.
This illustrates another general problem in developing resistance. Sometimes the properties conferring resistance may increase susceptibility to other pests or pathogens, as in the last example, and sometimes they may conflict with other requirements for the crop. For example, studies on resistance to attack by the onion fly indicated that onion varieties containing high concentrations of volatile sulfur compounds were most susceptible to attack (Soni and Ellis, 1990). The fly locates onions by these compounds. However, these compounds are those that confer the flavour and pungency to onions, so a conflict between pest resistance and a requirement for a strongly flavoured onion can be foreseen.
Transgenic shallot and garlic plants have been engineered to be resistant to the beet army worm Spodoptera exigua, an important tropical pest. The resistant plants contain a protein toxic to the pest that is coded by a gene derived from Bacillus thuringiensis. This gene has been incorporated into the allium chromosomes using the Agrobacterium-mediated genetic transformation of callus tissue cultures of shallot and garlic. Normal plants containing the novel protein have resulted and been shown to be resistant to the pest (see Fig. 3.7b;
Zheng et al., 2004, 2005).
Several laboratory techniques have been developed to challenge breeding lines with a disease or pest organism, and to measure the degree of susceptibility or resistance. For example, Currah and Maude (1984) tested resistance to leaf rot caused by B. squamosa by giving a standard dose of B. squamosa to small discs of leaf on damp wadding. The discs were then incubated at a constant 15°C at 100% RH. The average time for threads of fungus mycelium to appear on the leaf disc surface was determined. There were significant differences between cultivars in the time for infection to develop. Using such ‘screening tests’ it is possible to test for resistance in a standard way in many lines. Resistant lines exposed by these rapid tests can then be included in larger scale and more time-consuming tests for improved resistance in field conditions. Screening tests have been reported for onion white rot, pink root disease, neck rot, onion fly and others (see relevant chapters in Rabinowitch and Brewster, 1990b).
Much of the resistance to pests and diseases has been derived by mass selection under the pressure of attack by these organisms. This still remains an important breeding strategy, and field resistance is the ultimate test for resistance developed by more sophisticated means. The landraces of cultivated alliums that have been developed in the face of the worldwide diversity of pests and diseases are probably the most important reservoir of resistance genes.
Therefore, as discussed in Chapter 1, it is vital for future resistance breeding that these old varieties are conserved (Astley, 1990).
CONCLUSIONS
The breeding of edible alliums has been relatively unsophisticated compared with many important crops, because of a comparative lack of fundamental genetic and molecular genetic information. This is now changing, and particularly in the area of disease resistance – for example, in the transfer of downy resistance from A. roylei, new techniques are enabling the commercial development of resistant cvs (Kik, 2002).
Genetic transformation has already produced herbicide- and insect pest-resistant alliums, the use of which could reduce the need for pesticides and simplify crop management. However, despite the environmental benefits from the use of less and safer pesticides and the unlikelihood of any risks from transgenic alliums (Eady, 2002), there remains some hostility to transgenic crops, and this has prevented their commercialization in many countries. The development of hybrid cultivars seems to have been the dominant trend in onion breeding for the last 50 years, first in the USA and then in Europe and Japan. Despite this, Dowker and Gordon (1983) pointed out that there were little published data showing that hybrid cultivars are higher yielding, more uniform or of better quality than the best open-pollinated cultivars.
A comparison of hybrid and open-pollinated (OP) short- and intermediate-day cvs in New Mexico, USA found that the OP cvs performed better than the
hybrids for marketable quality, disease (pink root) resistance and yield, the latter mainly because the hybrids produced more bolters (Cramer, 2001).
However, this difference may simply reflect the importance of selection for local adaptation with onions, since many of the OP varieties tested derived from a long-standing programme of breeding OP cvs at New Mexico State University, and the hybrids were from California-based breeders.
Part of the attraction of hybrid cultivars lies in the control that the breeder and seed producer maintains over them: they cannot be reproduced from farmer-saved seed, so the work invested in their development cannot be pirated.
On the other hand, low and erratic seed yields have sometimes made hybrid cultivars unavailable and seeds of hybrids are two or three times the price of OPs (Cramer, 2001). Also, hybrids take a long time to develop and need sophisticated breeding and seed production facilities. The genetic base of hybrid cultivars is narrow and they contain less genetic variability than open-pollinated cultivars, so they may be less adaptable in abnormal, stressed conditions (Pike, 1986).
For all of these reasons they are not necessarily the best route to crop improvement, particularly in some of the poorer, tropical countries where there is great need for onion improvement. In these countries, as well as in wealthier temperate regions, old, open-pollinated varieties are being replaced by newer ones, often hybrids, from transnational seed companies (Currah and Proctor, 1990). The old varieties were often perpetuated by farmer-saved seed.
The same trends have been noted for Japanese bunching onion varieties in Japan and for leeks in Europe (de Clercq and van Bockstaele, 2002).
As pointed out previously, it is important that the genetic diversity represented by the traditional varieties is conserved and not lost. These tendencies in allium crop breeding are typical of the situation in many crops.
There is an interesting and ongoing debate centred on whether the commercial interests directing crop breeding correspond with long term public interests, particularly those of the poorer fraction of the world’s population (Mooney, 1979). Partly as a result of this debate, various publicly and charitably funded agencies are involved in the genetic conservation of allium crops coordinated by the International Board for Plant Genetic Resources (Astley, 1990). It is apparent, then, that many possibilities and conflicts arising from the new biotechnologies, the globalization of breeding and seed production companies and the geopolitical debate on the conservation and use of genetic resources are well illustrated by trends in allium breeding and seed marketing.
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© J.L. Brewster 2008. Onions and other Vegetable Alliums, 85 2nd Edition (J.L. Brewster)