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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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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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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.

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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 the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.

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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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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.