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Ruth S. Kobayashi, Stephen L. Sinden, and Lind L. Sanford

Leptine glycoalkaloids found in certain genotypes of Solanum chacoense, a wild potato relative, are resistance factors against the Colorado potato beetle (CPB). To efficiently introgress CPB resistance through leptine production into the cultivated potato, an understanding of leptine inheritance is important. Analysis of sibs within PI lines revealed a wide segregation for level of leptines. Leptine levels ranged from not detectable to 120 mg/100 g fresh weight among six sibs in a PI line, suggesting leptine production may be controlled by a few major genes. TLC analysis of F2 and backcross progeny, however, indicated that several genes probably control leptine level. This apparent multigene control of leptine level may make it difficult to incorporate leptine synthesis into the cultivated potato. Therefore, we are presently identifying microsatellite and RAPD markers associated with leptine synthesis to enable marker-assisted selection and facilitate the incorporation of leptine synthesis into the cultivated potato.

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Catherine M. Ronning, Lind L. Sanford, and John R. Stommel

Colorado Potato Beetle (Leptinotarsa decemlineata Say., CPB) is a destructive pest of the cultivated potato, Solanum tuberosum L. Certain glycoalkaloids in potato leaves are effective deterrents to this insect; however, in tubers these compounds can be toxic to humans. Leptines are foliar-specific glycoalkaloids produced by the related species, S. chacoense. These compounds have been shown to confer resistance to CPB. We are studying the inheritance of leptine production in segregating F1 and F2 populations derived from two S. chacoense accessions, 55-1 and 55-3, which are (respectively) high and low leptine producers. The F1 segregates 1:1 for high (>70% of total glycoalkaloids) and low (<20% of TGA) leptine content. Segregation data from the F1 and F2 populations suggest a twogene model for leptine production: a dominant repressor and a recessive inducer. Using two bulked DNA samples composed of highand low-leptine individuals from the F1 population, we are using various types of molecular markers (RAPDs, SSRs, DS-PCR, and AFLPs) to search for markers linked to leptine production. We have identified a RAPD band that appears to be closely associated with low leptine content and supports the two-gene model. The use of such a marker in a breeding program will facilitate the development of CPB resistant potato varieties.

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Anusuya Rangarajan, A. Raymond Miller, and Richard E. Veilleux

Leptine glycoalkaloids in leaves of the weedy diploid potato, Solanum chacoense Bitt., have been shown to reduce feeding by Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). Development of cultivated potatoes with natural resistance to CPB has the potential to reduce costs and environmental impacts of production by reducing pesticide use. Through efforts to move the genes controlling leptine biosynthesis into cultivated potato, a series of hybrids was generated between the high leptine producing S. chacoense and a cultivated type, S. phureja Juz. and Buk. These hybrids were evaluated for solanine (+chaconine), leptinins, leptines, and total steroidal glycoalkaloid content. All hybrids contained leptines, but at different levels (ranging from 117 to 802 mg·g-1 dry weight of leptine aglycon). Some hybrids appeared to convert solanine (+chaconine) to leptinine and leptine efficiently and had no detectable solanine in sampled leaves. To verify the biological significance of these glycoalkaloids, leaf tissue was subjected to feeding assays with second instar CPB. CPB feeding rate ranged from 38 to 87 mm2·d-1 and was most closely correlated with leptine concentration. A minimum leptine level of 300 mg/100 g fresh leaves suppressed feeding by 50%, and levels below this had no effect on CPB feeding.

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D.S. Douches, T.J. Kisha, J.J. Coombs, W. Li, W.L. Pett, and E.J. Grafius

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is the most serious insect pest of potatoes throughout the eastern and north central United States. Host plant resistance to the Colorado potato beetle has been identified in wild Solanum species and Bt-transgenic potato lines. Detached-leaf bioassays (72 h) were conducted on insecticide-resistant, first instar Colorado potato beetles to study the effectiveness of individual and combined host plant resistance traits in potato. Potato lines tested include non-transgenic cultivars (`Russet Burbank', `Lemhi Russet', and `Spunta'), a line with glandular trichomes (NYL235-4), a line with high foliar leptines (USDA8380-1), and transgenic lines expressing either codon-modified Bt-cry3A or Bt-cry5 (Bt-cry1Ia1). Bt-cry3A transgenic lines, foliar leptine line, and foliar leptine lines with Bt-cry5 had reduced feeding compared to non-transgenic cultivars. Glandular trichome lines and glandular trichome lines with Bt-cry5 did not reduce feeding in this no-choice feeding study. Some Bt-cry5 transgenic lines, using either the constitutive promoters CaMV35s or (ocs)3mas (Gelvin super promoter), were moderately effective in reducing larval feeding. Feeding on Bt-cry5 transgenic lines with the tuber-specific patatin promoter was not significantly different than or greater than feeding on the susceptible cultivars. Mortality of first instars was highest when fed on the Bt-cry3A lines (68% to 70%) and intermediate (38%) on the Bt-cry5 `Spunta' line SPG3 where the gus reporter gene was not included in the gene construct. Host plant resistance from foliar leptines is a candidate mechanism to pyramid with either Bt-cry3A or Bt-cry5 expression in potato foliage against Colorado potato beetle. Without multiple sources of host plant resistance, long-term sustainability is questionable for a highly adaptable insect like the Colorado potato beetle.

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Richard E. Veilleux and A. Raymond Miller

F1 hybrids between high leptine-producing clones (8380-1, PI 458310 and 55-1) of Solanum chacoense Bitt. and anther culture competent or anther-derived clones of S. phureja Juz. & Buk. that did not produce leptines were generally weak plants that grew slowly and died before flowering. Exceptional hybrids could be found that were capable of completing a life cycle, especially during the hot summer months in the greenhouse. All F1 hybrids produced leptines in the leaves but not the tubers, albeit at lower levels than in the S. chacoense parent. Anther-derived monoploids from the F1 hybrids exhibited a range of leptine production from none to levels approaching the S. chacoense parent. Backcross populations of an F1 hybrid to the S. chacoense and S. phureja parents were examined for leptine production. Backcross hybrids were generally much more vigorous than the F1 hybrids. All of the S. chacoense backcrosses produced leptines ranging from intermediate to high levels; four of the twelve S. phureja backcrosses exhibited low leptine levels. A general dominance of leptine synthesis was therefore exhibited, although the nonleptine-producing parent affected the expression of leptines in the hybrids.

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Anusuya Rangarajan, A. Raymond Miller, and Richard Veilleux

Leptine (LP) glycoalkaloids have been demonstrated to confer natural resistance to the Colorado potato beetle (CPB) in Solanum chacoense (chc). Development of cultivated potatoes with natural resistance to CPB has the potential to reduce both costs and environmental impacts of production by reducing pesticide use. To introgress the genes conferring leptine production from chc into S. tuberosum (tbr), clones of chc have been crossed with clones of S. phureja. Leaf disks from eight hybrids were subjected to a CPB second instar feeding bioassay to determine if extent of feeding was related to LP levels. Most hybrids contained leptinidine (LD, the aglycone of LP) levels intermediate to chc and tbr, and insect feeding was suppressed 30% to 50% in hybrids containing >10 mg·g–1 DW LD. One hybrid displaying feeding suppression contained a very low level of LD, whereas another hybrid that contained higher levels of LD had higher feeding rates. The presence of LD at “threshold” levels in these hybrids will suppress feeding of CPB, but other factors affecting resistance are also present and need to be explored.

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C.M. Ronning, S.P. Kowalski, L.L. Sanford, and J.R. Stommel

The Colorado potato beetle is a serious pest of the cultivated potato. Natural resistance has been found in a few wild species, including Solanum chacoense Bitter, in which resistance is attributed to the presence of foliar specific leptine glycoalkaloids. Production and accumulation of these compounds within S. chacoense varies widely and appears to be inherited in a quantitative fashion, but high leptine producing clones occur rarely. In the present study, 15 different accessions from various locations and altitudes of origination were analyzed for foliar glycoalkaloid content in order to determine the frequency and distribution of genes for leptine production/accumulation, and to see if we could find a center, or core, of leptine production. Leptines were detected in eight of the 15 accessions, and the amounts within each accession varied widely, but none of the individuals produced high amounts of leptine (defined as greater than 62% of total glycoalkaloids). All of the leptine-containing accessions originated from western Argentina. There was no relationship between elevational level and leptine, but there was a negative trend with total glycoalkaloids and elevation; this was due to levels of solanine and chaconine decreasing with increasing elevation. In addition, nine unidentified glycoalkaloids were detected, in very high proportions in some individuals and accessions. AFLP marker frequency and diversity were used to compare subpopulations of these accessions. AFLP markers revealed substantial diversity among clones. The relationship of marker distribution to glycoalkaloid content is discussed. The results raise interesting questions about glycoalkaloid biosynthesis and inheritance, and point the direction for new avenues of leptine and glycoalkaloid research.

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Abbas M. Lafta and James H. Lorenzen

Growth chamber and greenhouse experiments were conducted to investigate the effect of temperature and irradiance on foliar glycoalkaloids of three potato genotypes (Solanum tuberosum L.) that differ in glycoalkaloid content. Two genotypes (ND4382-17 and ND4382-19) produced the acetylated glycoalkaloids, leptine I and II, that contribute resistance to the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). The glycoalkaloids were separated and quantified by high performance liquid chromatography. Exposure of plants to high temperature (32/27 °C, 14-hour day/10-hour night) for 3 weeks under a 14-hour photoperiod with an irradiance of 475 μmol·m-2·s-1 significantly increased the levels of leptines I and II, solanine, and chaconine compared to that at low temperature (22/17 °C). Increases in foliar leptines and total glycoalkaloids at high temperature were 90% and 169%, respectively. Growing potato plants at low irradiance (75% reduction) for 2 or 4 weeks resulted in a significant reduction in the levels of leptine I and II (46%), solanine (43%), and chaconine (38%) compared to nonshaded plants. Transferring plants from high to low irradiance or from low to high irradiance for 2 weeks caused a decrease and an increase in glycoalkaloid concentration, respectively. Therefore, both temperature and irradiance influenced foliar levels of glycoalkaloids in potato plants without changing the leptines and solanine to chaconine ratios. Thus, irradiance and temperature influenced glycoalkaloid compounds that can effect resistance to CPB, especially leptine I and II.

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Shelley Jansky, Sandra Austin-Phillips, and Corine McCarthy

The Colorado potato beetle (CPB) is a major insect pest that is controlled mainly through the use of pesticides. Development of potato clones with multiple forms of host plant resistance may provide a stable alternative or supplemental form of CPB control. Tetraploid hybrids were developed by somatic fusion of diploid interspecific Solanum clones with different forms of resistance to CPB. Hybrids were created between a clone containing leptine glycoalkaloids and four clones producing glandular trichomes. One fusion produced vigorous hybrids that were analyzed for CPB resistance traits. Somaclonal variation among hybrids was detected for trichome density and resistance to feeding by adult and larval beetles. Somatic hybrids were less resistant than the parents in adult feeding preference trials, but several were more resistant than either parent in larval feeding trials. Future studies are needed to determine whether clones producing both glandular trichomes and leptines express resistance that is more stable than that of clones with only one resistance factor.

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Joseph J. Coombs, David S. Douches, Wenbin Li, Edward J. Grafius, and Walter L. Pett

The Colorado potato beetle [Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)] is a destructive pest of the cultivated potato (Solanum tuberosum L.) in northern latitudes. Combining resistance mechanisms of leptine glycoalkaloids and glandular trichomes with the synthetic Bacillus thuringiensis Berliner (Bt) cry3A gene in potato may be an effective strategy for controlling the Colorado potato beetle. Bt-cry3A transgenic plants were developed for three potato lines with differing levels of resistance to Colorado potato beetle ['Yukon Gold' (susceptible control), USDA8380-1 (leptine glycoalkaloids), and NYL235-4 (glandular trichomes)]. Polymerase chain reaction, and Southern and northern blot analyses confirmed integration and transcription of the cry3A gene in the transgenic lines. Detached-leaf bioassays of the cry3A engineered transgenic lines demonstrated that resistance effectively controlled feeding by first instar Colorado potato beetles. The susceptible `Yukon Gold' control suffered 32.3% defoliation, the nontransformed high foliar leptine line (USDA8380-1) had 3.0% defoliation, and the nontransformed glandular trichome line (NYL235-4) had 32.9% defoliation. Mean percentage defoliation for all transgenic lines ranged between 0.1% and 1.9%. Mean mortality ranged from 0.0% to 98.9% among the Bt-cry3A transgenic lines, compared to 20% for the susceptible `Yukon Gold' control, 32.2% for USDA8380-1, and 16.4% for NYL235-4. Results indicate that genetic engineering and the availability of natural resistance mechanisms of potato provide the ability to readily combine host plant resistance factors with different mechanisms in potato.