POTENTIAL GENE FLOW BETWEEN CULTIVATED POTATO AND ITS WILD TUBER-BEARING RELATIVES: IMPLICATIONS FOR RISK ASSESSMENT OF TRANSGENIC POTATOES
S. A. Jackson1 and R.E. Hanneman, Jr.2
1Research Assistant, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, Wisconsin 53706; email@example.com; and
2Research Geneticist, USDA, Agricultural Research Service, Vegetable Crops Research Unit, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, Wisconsin 53706; firstname.lastname@example.org
Crossability between the cultivated tetraploid potato and its wild tuber-bearing and close non-tuber-bearing relatives was examined. Crosses were made using wild species both as males and females to the cultivars. Crossability was analyzed both on a geographic and taxonomic basis. Generally seed set increased both as taxonomic distance between the cultivated and wild species decreased and as the location of the wild species moved further south through the Andes of South America. Thus, the species from the southwestern United States and Mexico had lower seed sets (6.8 seeds/fruit) as compared to those from South America (15.2 seeds/fruit). Further crossability analysis with the hybrids revealed greater fertility among those derived from species more closely related to the cultivated potato. The implications for risk assessment of transgenics is discussed.
Key words: Solanum, crossability, 2n gametes, EBN, transgenic
Potato is one of the most commonly transformed and tested crops worldwide. Together with oilseed rape and maize, the potato constitutes 60% of worldwide transgenic trials. The number of transgenic field trials in Central and South America climbed from two to 20 between 1990 and 1993. Nearly 2% of all transgenic potato field trials worldwide were in Central and South America, and that number is likely to increase with the release of transgenic cultivars in North America. Approximately 8% of transgenic potato field trials were for herbicide tolerance; 58% were for various insect, virial, bacterial and fungal resistances, and the remainder were for quality improvements and marker genes (Goy and Duesing, 1995).
The commonly cultivated potato (Solanum tuberosum) is a tetraploid (2n = 4x = 48). The wild relatives of the cultivated potato exist in a polyploid series from diploid to hexaploid, with 12 as the basic chromosome number. Geographically the wild species are found from the southwestern USA through Mexico and Central America into South America, generally following the mountain chains. Hawkes (1990) describes 235 wild species of which seven are cultivated. The wild relatives are found in very diverse ecological conditions ranging from arid desert-like conditions, to very wet and humid environs, to mountain forests, to weeds of cultivation. Likewise, the wild species also exhibit very diverse agronomic and horticultural traits. Resistances to most pathogens can be found in the wild species, as can other desirable traits, such as processing, storage, 2n gametes, etc.
An analysis of crossability of the cultivated potato with its wild relatives is complicated by many pre- and post-fertilization events, some of which are environmentally related and some are genetic. Stylar barriers are present in some interspecific crosses, as reported by Fritz and Hanneman (1989). This is evidenced by pollen tube stoppage either in the stigma or in the upper quarter of the style. Ploidy barriers may exist in interploidy crosses preventing hybridization. Endosperm Balance Numbers (EBN) also play an important role in interspecific seed production. For successful seed set, the EBN's of the respective parents should be in a 2:1 maternal to paternal ratio in the endosperm (Johnston et al., 1980). EBN's are assigned based on crossability to standard testers and are independent of ploidy. The presence of 2n gametes may allow a species to overcome stylar, ploidy and EBN barriers. 2n gametes permit two S-alleles to be present in the gamete from a diploid species which may lead to a "competitive interaction" (Lewis and Modlibowska, 1942) in crosses to tetraploids, thus weakening or eliminating the stylar barriers. 2n gametes can overcome ploidy barriers. Since EBN's are additive genetic factors, a 2n gamete will carry the EBN of the parent (Ehlenfeldt and Hanneman, 1984); thus, inter-EBN crosses may be possible.
Therefore, to assess crossability between the cultivated potato and its wild relatives, all of the above have to be measured. In this study, 2n pollen frequencies were determined for most wild species used. In vivo pollen tube growth measurements were taken for many of the crosses made. Parental EBN's were taken into account when determining cross success or failure.
Prerequisite to any discussion of the risk of transgene escape in areas of natural diversity is an assessment of crossability between the transformed line and its wild relatives. It is assumed that the first transgenics to be tried commercially will be transformations of common tetraploid cultivars. The crossability between the cultivated tetraploid potato and its wild relatives has been assessed under artificial conditions. Groups of species, based on putative evolutionary relationships to the cultivated potato, are assessed for relative seed production in these crosses, and classified by geographic distribution, allowing the identification of those groups of species and geographic areas wherein lie the greatest potential for hybridization.
MATERIALS AND METHODS
Five hundred seventeen accessions (PI's) representing 121 species and 17 of the 19 series were obtained from the Potato Introduction Station, NRSP-6, Sturgeon Bay, Wisconsin (Table 1). Plants were obtained as true seed and germinated in a greenhouse. Twelve to 24 plants of each PI were transplanted to peat pots and held in the greenhouse for three weeks before transplanting to the field. Between six and 18 seedlings per PI were tranplanted to the field at 12 inch spacing. The plants were maintained under drip irrigation using standard cultural practices at the UW-Lelah Starks Potato Breeding Farm, Rhinelander, Wisconsin. Seeds were planted, and plants were transplanted and maintained in greenhouses in Madison, Wisconsin for winter and spring crossing. The tetraploid cultivars used in this study (Atlantic, Langlade, Ontario, Katahdin, Kennebec, W1005, W870) were grown in the field and greenhouse using similar cultural practices as those used for the species.
Pollen was collected from the PI's by removing at least 20 open flowers from at least six plants within a PI. Pollen was collected and stored for up to three months in gelatin capsules over anhydrous CaCl2 at -10C. 2n pollen frequencies were determined by staining pollen with acetocarmine-glycerol and viewing at 400x with a standard light microscope. In 1994, four fields of view with approximately 50 grains each were counted for a total of 200 pollen grains. In 1995, two to four fields of view with between 25 and 50 grains were counted for a total of 100 pollen grains. 2n pollen frequencies were determined from these data.
Crossing in Rhinelander, Wisconsin was performed using the cut-stem method (Peloquin and Hougas, 1959). Stems were collected from the field and open flowers were removed and buds were emasculated. Pollen was applied and crosses were tagged. The pollinated cut-stems were placed in a shadehouse during fruit development. Fruit were harvested four to six weeks after pollination and stored in paper bags for at least one month at room temperature before seed was extracted. The number of fruit and seeds were recorded. Crossing procedures were the same in the greenhouse except that pollinations were done on plants rather than using cut-stems.
Forty-eight to 72 hours after pollination, styles were removed and placed in 3:1 (95% ethanol:glacial acetic acid). Styles were stored in a refrigerator at 7C for several months until evaluated. The method of Martin (1958) was used to stain and view pollen tubes. Styles were removed from the 3:1, rinsed in water, treated in 8N NaOH for 8-24 hours and rinsed in water again for 1 hour. Styles were placed on a slide in a drop of aniline blue solution (0.05% aniline blue in 0.1N K2PO4) and gently squashed with a coverslip. Fluorescence of the pollen tubes was observed using a Zeiss microscope with a Zeiss HBO 50W high pressure lamp, a G365 excitation filter with a dichroic reflector FT460, and a LP520 barrier filter.
Crossing data are presented using the wild species both as males and females in crosses with tetraploid cultivars. The number of pollinations, fruit and seed are combined over two years. Tables 2 and 3 indicate the number of pollinations, fruit, seed, seed/fruit (s/f) and seed/pollination (s/p) produced by series and superseries using wild species as both males and females. The superseries are associated with specific geographic areas as presented in Table 1. Series Morelliformia, Olmosiana, Maglia and Ingifolia were not used in crosses because they were either unavailable from the genebank, or did not flower in this study. Hawkes (1990) describes the evolution of the potato as progressing from the 'primitive' to the 'advanced' Stellata and then to the 'primitive' and 'advanced' Rotata.
There was limited seed production in the series Etuberosa when used either as males or females. S. fernandezianum (PI 473463) was particularly fertile in these crosses accounting for all the seed produced. When used as a male it produced 6 fruit and 5 seeds from 23 pollinations, and as a female, it produced 87 seeds and 1 fruit from 24 pollinations. S. fernandezianum, as well as all the Etuberosa, are diploid species with EBN's of 1 (Table 1). This series when used as males had an overall seeds/fruit (s/f) ratio of 0.6 and a seeds/pollinations (s/p) ratio of 0.02 (Table 2), and when used as females the s/f ratio was 87 and the s/p ratio 1.2 (Table 3).
The 'primitive' North American Stellata were entirely unsuccessful in seed production in either direction. However, the 'primitive' South American Stellata were successful when used as males. Two series, Lignicaulia and Circaeifolia, produced a total of 29 seeds from 5 fruit and 211 pollinations (Table 2). Series Commersoniana also produced fruit but they were devoid of seed. The overall s/f ratio for this group of South American 'primitive' Stellata was 3.6, and the overall seeds/pollination ratio was 0.07 (Table 2).
The 'advanced' South American Stellata was successful only when used as males, as with the 'primitive' Stellata. Thirty-four seeds were produced in 31 fruit from 457 pollinations. The s/f was lower than that of the 'primitive' Stellata at 1.1s/f (Table 2); however, the s/p ratio was the same at 0.07.
The 'primitive' Rotata are also South American in distribution, and as with the previous two superseries these were successful only when used as males. Two series were used in crossing but only series Megistacroloba produced seed. Out of 980 pollinations for this group, 41 fruit were produced which contained 19 seeds (Table 2). As is expected from the greater number of fruit and pollinations, and the reduced number of seeds, the s/f and s/p ratios were lower than for the previous superseries at 0.5 and 0.02, respectively.
The South American 'advanced' Rotata had two series, Conicibaccata and Acaulia, out of three used that were successful in crosses both as males and females. The seed set in these particular series was much higher when the species were used as females (Table 3) than when they were used as males (Table 2). When the species were used as females, 496 pollinations produced 36 fruit and 1,222 seeds, for a s/f ratio of 33.9 and a s/p ratio of 2.5. However, when these species were used as males, 723 pollinations produced 17 fruit and 2 seeds for a s/f ratio of 0.1 and a s/p ratio of 0.003.
The 'advanced' North American Rotata were much more successful than their South American counterparts in crosses with the cultivars. Two series represent this superseries, but both series were successful in setting seed. Six hundred forty-two pollinations set 72 fruit and 2,090 seeds when these species were used as females (Table 3), and when used as males (Table 2), 1,785 pollinations set 36 fruit and 293 seeds. The s/f ratio was 29 when used as females versus 8.1 s/f when used as males. The s/p ratio for use as females was 3.3, and 0.2 as males.
The wild series Tuberosa are a very diverse group of species not only morphologically, but geographically as well (Hawkes, 1990). There are three recognized species endemic to Mexico (Solanum verrucosum, S. leptosepalum and S. macropilosum), but only S. verrucosum has an appreciable distribution; however, for South America, 77 species and approximately nine hybrid species are recognized. The crossability with this group of species was appreciable. When used as males (Table 2), 5,000 pollinations resulted in 333 fruit and 3,233 seeds, a s/f ratio of 9.7 and s/p ratio of 0.6, but when used as females (Table 3) 2,306 pollinations resulted in 26 fruit and 505 seeds, a s/f ratio of 19.4 and a s/p ratio of 0.2.
The cultivated series Tuberosa are almost entirely located in South America, with the exception of ssp. andigena, which has some distribution in Mexico and Guatemala (Hawkes, 1990). This series was quite successful in seed production in these crosses. When used as males (Table 2), they produced 96 fruit and 3,623 seeds from 600 pollinations for a s/f ratio of 37.7 and a s/p ratio of 6.0. When used as females (Table 3), they produced 2 fruit and 52 seeds from 155 pollinations for a s/f ratio of 26 and a s/p ratio of 0.3. The combined crossing for the series Tuberosa, both wild and cultivated, resulted in a s/f of 16.0 and s/p of 1.2 as males, and a s/f of 19.9 and a s/p of 0.2 as females.
2n pollen was found in every series and every species. However, the frequency of 2n pollen production was variable both within a series and within a species. Also, stylar barriers were found in almost every series, although, the incidence of stylar barriers seemed to increase as putative evolutionary distance from the cultivated potato increased.
The crossability data suggest that seed set, and ease of seed production increased as putative evolutionary distance decreased. Thus, the series Etuberosa and superseries Stellata had the lowest crossability, while crossability increased through the superseries Rotata and into the wild series Tuberosa and peaked with the cultivated series Tuberosa (Figure 1). This is expected, as the ploidy and EBN's of the wild species increase from series Etuberosa [2x(1EBN)] to the superseries Stellata, to the Rotata and into the cultivated series Tuberosa [4x(4EBN)].
Geographically, the crossability statistics are informative. Crossability by two major geographic regions, Mexico and South America, suggest that seed production is greater in those species located outside of Mexico (Mexico and USA: 6.8 s/f; S. America: 15.2 s/f). This would be predicted by ploidy and EBN. Most Mexican species are either 2x(1EBN) or 4x(2EBN). The first should not hybridize with the tetraploid cultivars, and the latter only as a function of the presence of 2n gametes. The hexaploid species crossed readily with the cultivars. This might also be expected as the Chilean and Peruvian Andes are a major center of diversity for the cultivated potato (Budin and Gavrilenko, 1994) and its species thought to be more closely related to the cultivated Tuberosum. Crossability would be expected to be significantly higher for those species which are the closest relatives of cultivated potato because of reduced incidences of stylar, ploidy, chromosomal and EBN barriers (Hawkes, 1990; Hermsen, 1994; Matsubayashi, 1991).
There were several species that produced seed in spite of suspected EBN and ploidy barriers. The hybrid seed germinability was not always high, but seed could be produced. Of the 1EBN species, accessions of S. capsicabaccatum, S. lignicaule and S. fernandezianum produced seed. These crosses need to be attempted again to generate more seed to be able to determine germinability and confirm introgression through hybridization using molecular markers. Many 2EBN species, especially those more closely related to the cultivars, produced seed. These species are dependent on 2n gametes for successful endosperm development. 2n pollen counts were made for almost all of the wild species used in this study. At least one accession of every wild species produced a measurable amount of 2n pollen. Introgression into natural diploid populations is dependent on the production of 2n gametes. The production of 2n gametes to hybridize first with the transgenic cultivar, and subsequently with the tetraploid progeny, is necessary until enough tetraploid progeny are formed to create a separate crossability group. Thus, the risk implications associated with wild diploid populations may be directly related to 2n gamete production.
An evaluation of risk assessment should not proceed on the above data alone. There are many constraining factors on this experiment that need to be considered before a comprehensive understanding of the risk of transgene introgression is known. In this experiment, thousands of pollinations were made by hand under favorable conditions. Is this paralleled in nature? Are the necessary insect pollinators present, and in sufficient numbers? The cut-stem technique (Peloquin and Hougas, 1959) used in much of this experiment promotes fruit retention. There is nothing in nature to emulate this. Species and cultivars were planted at varying times to provide a continuous source of flowers and pollen. In natural settings, are equal flowering times between species and a transgenic cultivar likely to occur? It is necessary that an understanding of all of these factors be considered and evaluated in natural settings to establish in situ crossability.
Furthermore, if hybridization can, and does occur, what is the outcome, or potential hazards and how can it be evaluated? The first thing that needs to be ascertained is gene flow. An analysis of hybrids to determine transmission of chromosomal segments and their stability in natural populations is imperative. If it can be established that chromosome segments are transferred to natural populations, then will they be expressed, lost in future generations, or turned off? It has been shown that epigenetic silencing can occur as a result of changes in ploidy (Scheid et al., 1996), and that transgenes, in certain genetic backgrounds can be silenced (Matzke et al., 1994). Lastly, what is the impact of a particular transgene on natural populations? Is there an increase in weediness? It is possible that certain transgenes may effect changes in diversity, if they cause changes in fertility, fecundity, resistance or susceptibility to pests. Therefore, a transgene in a wild species may not cause it to become a noxious weed, but may have an impact on the natural diversity in a particular region, or on a species through natural selection.
Risk analysis with transgenic cultivated potato has been addressed previously for other geographic locations. Love (1994) evaluated the risk associated with growing transgenic potatoes in Canada and the U.S. He concluded that "given the number and potency of barriers to hybridization, and more especially to introgression, and stabilization, the only sound conclusion is that transgene introgression into wild Solanum species will not occur under natural conditions." McPartlan and Dale (1994) made crosses with two solanaceous wild species endemic to the United Kingdom, Solanum nigrum and S. dulcamara. No evidence of cross-pollination between the two wild species and transgenic cultivars was found. Eijlander and Stiekma (1994) determined that stylar, ploidy and possibly EBN barriers contributed to the lack of crossability between the cultivars and S. nigrum and S. dulcamara. Only through embryo rescue were they able to get hybrid seed; however, these hybrids were sterile. They concluded that the potato is 'a naturally contained species in this part of the world [Western Europe].' Transgenic pollen flow has been measured between fields of cultivated potato and been found to travel as far as 1000 meters (Skogsmyr, 1994). The distance is effectively limited by the pollinator. Generally bumblebees, a natural pollinator of potato, only travel short distances, but may travel as much as five kilometers (Heinrick, 1979); however, in Skogsmyr's study a small beetle, Meligethes aeneus, was the pollinator explaining the longer pollen flow.
A comprehensive study of the crossability, introgression, stability and ecological impact of transgenic potato with its wild relatives in Mexico, Central and South America has not been done prior to this study. One reason is the large number of species endemic to these regions and the complexity of their sexual relationships. This study provides the first piece--an analysis of crossability. It has yet to be determined whether transgenes will introgress into natural populations, be stably expressed and what the impact on the ecology of the related species and the biodiversity as a whole will be. In conclusion, there exists a degree of crossability within all tested geographic locations and taxonomic series; however, whether or not this level is significant and should deter or allow the release of transgenic potatoes into regions of diversity remains to be answered.
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Table 1. The ploidy, Endosperm Balance Number (EBN), geographical distribution and number of species and accessions of
series and superseries of the tuber-bearing and closely related non-tuber-bearing Solanums. (adapted from Hawkes, 1990)
|Superseries (corolla groups)||Taxonomic series||Ploidy (EBN)||Distribution||Species||PI's|
|Etuberosa||2x (1EBN)||Central Chile, South Argentina, Islands of Juan Fernandez||3||13|
|Stellata (primitive)||Morelliformia||2x (?EBN)||Southwestern USA, Mexico, Central||1||4|
|Stellata (primitive)||Lignicaulia||2x (1EBN)||South America||1||4|
|Stellata (advanced)||Yungasensa||2x (2EBN)||South America||5||19|
|Rotata (primitive)||Cuneolata||2x (2EBN)||Southern to central regions of South America||2||5|
|Rotata (advanced)||Piurana||2x (2EBN), 4x (2EBN)||Central to northern America||8||21|
|Conicibaccata||2x (2EBN), 4x (2EBN), 6x (4EBN)||14||40|
|Acaulia||4x (2EBN), 6x (4EBN)||Southwestern USA, Mexico and Central||2||18|
|Tuberosa (wild)||2x (1EBN), 2x (2EBN), 4x (4EBN)||Mexico, South America||47||205|
|Rotata||Tuberosa (cultivated)||2x (2EBN), 4x (4EBN)||Mexico, Central and South America||5||30|
Table 2. Crossability of the wild species used as males in crosses with tetraploid cultivars, grouped by series and superseries.
|Stellata (primitive) 2||Lignicaulia||53||3||4||1.3||0.08|
|Stellata (advanced) 2||Yungasensa||457||31||34||1.1||0.07|
|Rotata (primitive) 2||Cuneoalata||126||0||0||0||0|
|Rotata (advanced) 2||Piurana||106||0||0||0||0|
|Rotata (advanced) 1||Longipedicellata||1219||22||180||8.2||0.1|
|Rotata (4)||Tuberosa (wild)||5000||333||3233||9.7||0.6|
1Southwestern USA, Mexico and Central America
3Poll. = pollinations, s/f = seed/fruit and s/p = seed/pollination
4Primitive and advanced
Table 3. Crossability of the wild species used as females in crosses with tetraploid cultivars, grouped by series and superseries.
|Stellata (primitive) 2||Lignicaulia||17||0||0||0||0|
|Stellata (advanced) 2||Yungasensa||117||0||0||0||0|
|Rotata (primitive) 2||Cuneoalata||3||0||0||0||0|
|Rotata (advanced) 2||Piurana||101||0||0||0||0|
|Rotata (advanced) 1||Longipedicellata||330||19||44||2.3||0.1|
|Rotata (4)||Tuberosa (wild)||2306||26||505||19.4||0.2|
1Southwestern USA, Mexico and Central America
3Poll. = pollinations, s/f = seed/fruit and s/p = seed/pollination
4Primitive and advanced
Figure 1. Crossability between the cultivated tetraploid (2n=4x=48) S. tuberosum ssp. tuberosum and the wild tuber-bearing and non-tuber-bearing related species, as given by seed/fruit.
*Series Etuberosa's 87 s/f as a female, due to the success of one PI of S. fernadezianum [2x(1EBN)], was dropped from the figure.