Development of Bt-cry5 Insect- resistant Potato Lines ‘Spunta-G2’ and ‘Spunta-G3’

The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L.) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic ‘Spunta’ clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to ‘Spunta’. These Bt-cry5 transgenic potato plants with high potato tuber moth resistance have value in integrated pest management programs. Received for publication 5 July 2001. Accepted for publication 7 Jan.2002. This publication was made possible through support provided by the office of USAID/CAIRO/AGR/A, under grant no. 263-0152A-00-3036-00, and by the Michigan Agriculture Experiment Station. The authors would like to thank Zeneca for providing the codon-modified Bt-cry5 gene. To whom reprint requests should be addressed. Phone: 517-353-3145; fax 517-353-5174; e-mail address: [email protected] Visiting Scholar. Insect damage to tubers is often a serious problem in cultivated potato; one such destructive insect in the subtropic and tropical regions is the potato tuber moth. The larvae mine the foliage, stems, and tubers in the field and tubers in storage. Tuber infection by various other insects and secondary diseases that subsequently attack damaged tubers can cause dramatic losses. Annual losses in storage alone range from 30% to 70% in India and similar losses occur in the Middle East, North Africa, and South America (Raman and Palacios, under the revised nomenclature as stated by Crickmore et al., 1998) from B. thuringiensis exhibits activity against both Lepidoptera and Coleoptera (Tailor et al., 1992), and was codon-modified to increase its expression level in plants. Douches et al. (1998) transformed this gene into potato and, subsequently, Mohammed et al. (2000) identified high Btcry5 expressing potato lines using tuber bioassays. The development of this germplasm was supported under the Agricultural Biotechnology Support Project (ABSP; http:// www.iia.msu.edu/absp), funded by the U.S. Agency for International Development (USAID). ABSP supports the use of biotechnology to develop crops with improved agronomic traits for developing countries and, in this case, collaborative research between MSU and the Agricultural Genetic Engineering Research Institute (AGERI) in Egypt. This paper describes the development of the Bt-cry5 transgenic lines ‘Spunta-G2’ and ‘Spunta-G3’, which were subjected to potato tuber moth bioassays (foliar and tuber), Colorado potato beetle detached-leaf bioassays, agronomic field studies, and field and storage evaluations of tuber damage from potato tuber moth. Materials and Methods ‘Spunta’ is a long, white, tablestock cultivar bred in the Netherlands and grown widely in subtropical regions, such as North Africa and South America. ‘Spunta’ was transformed via Agrobacterium tumefaciens LBA4404 with a Bt-cry5 construct (Fig. 1) according to Douches et al. (1998). The vector was the binary pBI121 (Clontech, Palo Alto, Calif.) with the codon-modified Bt-cry5 gene (Tailor et al., 1992) supplied by Zeneca (Berkshire, U.K.). The pBI121 vector was digested with SmaI and EcoICRI, and self-ligated. The resulting plasmid, pBIML4, was then cleaved with BamHI and ligated with the Bt-cry5 fragment that was cut from a Bluescript SK based plasmid (Stratagene, La Jolla, Calif.), resulting in the plasmid pBIML5. Two Bt-cry51982). Currently, available means to control the potato tuber moth and avoid major crop losses have left farmers with no alternatives to chemical control and, in some cases, the pesticides are applied directly to the potatoes in storage (Raman, 1988). The general utility of insecticides, however, is limited by high cost, persistence of residue in tubers and the environment, and development of insecticide-resistant pests. The Colorado potato beetle is the most important pest of potato production in northern latitudes. This insect has shown the ability to adapt to many insecticides over the past half-century (Casagrande, 1987). Currently, it is reported to be resistant to 37 insecticides worldwide, including organophosphates, carbamates, organochlorines, pyrethroids, and hydrogen cyanide (Georgiou and LagunesTejeda, 1991). Thus, if the Colorado potato beetle is to be properly managed, host plant resistance needs to be combined with integrated pest management practices. The Bt-cry5 toxin gene (designated cry1Ia1 Fig. 1. pBIML5, plant transformation vector for Btcry5. Important functional region of the vector originates from plasmid pBI121, containing a chimeric neomycin phosphotransferase II gene and CaMV 35S promoter. 6953, p. 1103-1107 11/21/02, 1:06 PM 1103 HORTSCIENCE, VOL. 37(7), DECEMBER 2002 1104 BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES Spunta lines, G2 and G3, with high insecticidal activity were selected from more than 20 lines for advanced testing because of their performance in preliminary detached-leaf bioassays. Insect bioassays. Young, fully expanded leaves from pesticide-free, greenhouse-grown plants were collected for potato tuber moth and Colorado potato beetle detached-leaf bioassays. The petiole with leaf was removed from the plant using a new single-edged razor blade. A petiole leaf was inserted through a pre-moistened sponge (1 cm) into each 3.5mL glass vial full of water. The single detached leaf was then placed horizontally in a petri dish (15 cm × 2 cm) containing a 15-cmdiameter 3MM Whatman paper. One leaf was used per replication and the bioassay was replicated four times per potato line. Ten neonate larvae were placed near the mid-rib of each leaf. The petri dish was covered with the lid and placed at 25 ± 2 °C and 25 μE·m·s fluorescent light. Mortality of the larvae was determined after 72 h. For Colorado potato beetle leaf bioassays, leaf defoliation as a percentage of feeding of the susceptible cultivar Russet Burbank was also estimated. RBN15 and RBN20 (Bt-cry3A-transgenic lines from John Kemp, New Mexico State Univ.) were used as positive controls. ‘Russet Burbank’ and the ‘Spunta’ lines were negative controls. NYL235-4, a line with glandular trichomebased insect resistance (Plaisted et al., 1992), was also tested. For tuber bioassays, a greenhouse-grown tuber (5–40 g) from ‘Spunta-G2’ or ‘SpuntaG3’ was placed in a Phytatray II box (Sigma, St. Louis). Holes were punched through the top for aeration. Five potato tuber moth neonates were placed on each tuber and kept at 23 ± 2 °C in the dark. The number of pupae, adults, and larvae in each box was counted after 4 weeks and percent mortality calculated. The tuber bioassay included four replications for ‘Spunta-G2’ or ‘Spunta-G3’. Field experiments were conducted at the International Potato Center (CIP) field station, Kafr-el-Zyat, Egypt, and AGERI, Giza, Egypt, to evaluate resistance to tuber moth for ‘Spunta’, ‘Spunta-G2’, ‘Spunta-G3’, and the glandular trichome-based insect resistant line, NYL235-4 (Plaisted et al., 1992) (tested in 1999 only). Seed tubers used in the field trials were obtained from seed plots at the Lake City Experiment Station, Lake City, Mich. In each trial, 10-plant (85.4-cm spacing) one-row plots Table 1. Results from tuber and leaf bioassays of ‘Spunta-G2’ and ‘Spunta-G3’ transgenic lines and Spunta by feeding with potato tuber moth