1The University of Georgia, Georgia Station, Griffin, GA 30223-1797 USA
2 University of North Carolina, Greensboro, NC 27402-6174 USA
ABSTRACT
Canola cultivars ‘Oscar’ and ‘Westar’ were transformed with a truncated synthetic Bacillis thuringiensis insecticidal crystal protein gene (Bt cryIAc) gene using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. Expression in transformed lines ranged from 0 to 0.4% cry as a percentage of total extractable protein. In greenhouse studies, the diamond back moth (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni H??bner), were completely controlled by low-, medium-, and high-expressing lines. Control of the corn earworm (Helicoverpa zea Boddie) was nearly complete, and the beet armyworm (Spodoptera exigua H??bner) showed a dose response that had a negative association between defoliation and cry expression. In field tests artificially infested with neonates of diamondback moth or corn earworm, transgenic plants proved highly resistant to both diamondback moth and corn earworm larvae and had very low defoliation levels. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Six Oscar and three Westar Bt lines with high cry expression were advanced to the T3 generation by the pedigree method and evaluated for seed yield and other agronomic traits in field trials at two locations. Four of the Oscar Bt T1 families produced similar seed yields to Oscar at both locations while two Oscar Bt T1 families produced statistically lower seed yields than Oscar. All three of the Westar Bt T1 families produced seed yields similar to Westar at both locations. Our results suggest that Bt canola could be used for effective management of many Lepidopterous pests of canola.
KEYWORDS Brassica napus, transformation, insecticidal gene, field study
INTRODUCTION
Canola quality oil produced from oilseed rape, Brassica napus L. is a relatively new crop in the United States. The production of canola quality oilseed rape in the United States has increased dramatically in the past five years with more than one-half million hectares grown in 1998 (Raymer et al., 1998) (Gumpert, 1999). As canola production increases, insect problems associated with the crop are also likely to increase (Lamb, 1989). Insect problems associated with canola may be more serious in the southeastern United States due to the mild climate and presence of crucifer specialists including diamondback moth (DBM), Putella xylostella (L.) and cabbage looper (CBL), Trichoplusia ni (H??bner) . Beet armyworm (BAW), Spodoptera exigua (H??bner) and corn earworm (CEW), Helicoverpa zea (Boddie) may increase in importance where canola is grown in warmer regions (Buntin and Raymer, 1994). Transgenic canola cultivars with insecticidal properties will certainly play a major role in integrated pest management strategies for canola pests (All et al., 1999) (Evans and Scarisbrick, 1994).
Scientists at The University of Georgia successfully transformed Brassica napus L. cultivars 'Oscar' and 'Westar' with a truncated synthetic Bacillis thuringiensis (Bt) insecticidal crystal protein gene (Bt cry1Ac) gene using Agrobacterium tumefaciens mediated transformation. A total of 57 independently transformed lines were produced, containing 1 to 12 copies of the transgenes. Expression in transformed lines ranged from 0 to 0.4% cry as a percentage of total extractable protein ( Stewart et al., 1996).
In greenhouse studies, DBM and CBL were completely controlled by high-, medium-, and low-expressing lines. Control of CEW was nearly complete, and BAW showed a dose response that had a negative association between defoliation and cry expression (Ramachandran et al., 1998a). In field tests artificially infested with neonates of DBM or CEW, transgenic plants had very low defoliation levels and proved highly resistant to both DBM and CEW larvae (Ramachandran et al., 1998b). After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. For a more detailed review of the many insect evaluations conducted on this plant material refer to the paper by G. D. Buntin et al. in the entomology section of these proceedings.
Six of the original Oscar and three of the original Westar transformed Bt lines with varying levels of cry expression were advanced to the T3 generation in the greenhouse using the pedigree method. The objective of this study was to evaluate this plant material for seed yield and other agronomic traits in field trials and to determine if any of this material was suitable for commercialization.
MATERIALS AND METHODS
T3 seed from single T2 generation plants were harvested from greenhouse grown plants. Since seed quantities were limited, a single replicate of each T3 line was compared to multiple replicates of the parental lines (Oscar and Westar) in field trials at two locations. A total of 70 T3 Oscar Bt lines and 12 T3 Westar Bt lines were compared in each trial. These field evaluations were fall-seeded and grown as a winter crop at Griffin and Tifton, Georgia in 1997-98. The Griffin and Tifton locations are typical of the Southern Piedmont and Coastal Plain regions, respectively. Tifton has an elevation of 110 m and is located at 31.28 ?? N latitude. Griffin has an elevation of 270 m and is located at 33.28 ?? N latitude. Individual plots consisted of seven rows, 3 m in length, and spaced 18 cm apart. Data were collected on date of 50 percent bloom and harvest maturity, plant height, lodging, and seed yield at both locations. Total oil content was determined using NMR on seed collected from the Tifton trial only.
RESULTS AND DISCUSSION
Table 1 provides a summary of seed yield and other agronomic characteristics of T3 lines when averaged over the two test locations. Seed yield for the 70 Oscar Bt lines tested ranged from 1248 to 3034 kg ha -1 compared to Oscar, which averaged 2554 kg ha -1 . Seed yield of the 12 Westar Bt lines tested ranged from 1205 to 1858 kg ha -1 compared to Westar, which averaged 1386 kg ha -1 . In spite of the wide range of values, few Oscar Bt lines were statistically different from Oscar and none of the Westar Bt lines were statistically different from Westar. Values presented in Table 1 also indicate substantial variability for the other traits measured, but it is obvious that further testing with more replication will be necessary to detect true differences among these lines.
Table 1. Average Performance of Oscar and Westar Bt T3 Lines Grown at Griffin and Tifton, Georgia in 1997-98.
T3 Family/Parent Yield Oil Bloom Maturity Height Lodging1
kg/ha % month/day month/day cm score
T3 Oscar Bt
Lines (range) 1248-3034 34.9-40.9 2/18-3/6 5/9-5/16 107-133 0.0-2.5
Oscar 2554 38.9 2/28 5/15 116 0.5
T3 Westar Bt
Lines (range) 1205-1858 35.4-41.7 2/19-2/28 5/07-5/13 97-127 0.5-2.0
Westar 1386 41.1 2/21 5/12 99 0.5
1. Lodging rated on a 0-5 scale with 0 = all plants erect and 5 = all plants prostrate.
In order to gain a more reliable comparison, data was summarized by location and data pooled within T1 families. Average performance of T1 families at Griffin and Tifton are presented in Tables 2 and 3, respectively. Four of the six Oscar T1 Bt families produced similar seed yields to Oscar at both locations while the O3Bt and O63Bt families produced statistically lower seed yields than Oscar. All three of the Westar Bt families produced seed yields similar to Westar at both locations. Few differences from Oscar or Westar parental lines were observed for total oil content or other traits at either location.
Our results suggest that many of the Bt lines developed are comparable in agronomic performance to the parental line from which they were derived. We acknowledge that few of these lines are likely to be commercially competitive by the time they could be released due to the rapid progress in cultivar development in most production regions of the world. However, our research clearly demonstrates the feasibility of developing high-yielding, well-adapted canola cultivars with highly effective insecticidal properties for use in controlling Lepidopterous pests of canola.
Table 2. Seed Yield and Agronomic Traits of Oscar and Westar Bt Lines Grown at Griffin, Georgia in 1997-98.
T1 Family/Parent Yield Oil Bloom Maturity Height Lodging1
kg/ha % month/day month/day cm score
Oscar Lines
Oscar 2532 02/26 05/21 111 1.0
096Bt 2368 02/23 05/19 106 1.0
095Bt 2365 02/25 05/19 108 1.0
056aBt 2294 02/24 05/20 116 1.0
052Bt 2267 02/25 05/20 113 1.0
03Bt 1916 02/27 05/20 104 1.0
063Bt 1327 02/25 05/21 102 1.0
Average 21532 02/26 05/20 109 1.0
Westar Lines
W1Bt 1624 02/16 05/18 94 1.0
W58Bt 1404 02/19 05/16 115 1.0
Westar 1285 02/20 05/14 96 1.0
W55aBt 1083 02/22 05/14 86 1.0
Average 13493 02/20 05/16 98 1.0
Bolded values are not statistically different from Oscar or Westar, respectively.
1. Lodging rated on a 0-5 scale with 0 = all plants erect and 5 = all plants prostrate.
2. C.V. for seed yield = 18.9%.
3. C.V. for seed yield = 12.9%.
Table 3. Seed Yield and Agronomic Traits of Oscar and Westar Bt Lines Grown at Tifton, Georgia in 1997-98.
T1 Family/Parent Yield Oil Bloom Maturity Height Lodging1
kg/ha % month/day month/day cm score
Oscar Lines
Oscar 2583 38.9 03/02 05/08 123 1.6
096Bt 2500 38.4 03/02 05/07 113 1.4
052Bt 2399 39.2 03/04 05/07 123 1.7
095Bt 2372 36.2 03/04 05/07 118 1.7
056aBt 2311 38.7 03/05 05/05 121 1.9
063Bt 2292 37.5 02/28 05/11 122 1.0
03Bt 1980 39.3 03/07 05/08 120 1.5
Average 23482 38.3 03/04 05/08 120 1.5
Westar Lines
W58Bt 1855 38.9 02/28 05/08 126 2.3
W1Bt 1605 40.7 02/28 05/03 115 3.0
Westar 1603 41.1 02/27 05/05 112 3.0
W55aBt 1344 40.4 02/28 05/03 112 3.0
Average 16033 40.3 02/28 05/05 116 2.8
Bolded values are not statistically different from Oscar or Westar, respectively.
1. Lodging rated on a 0-5 scale with 0 = all plants erect and 5 = all plants prostrate.
2. C.V. for seed yield = 14.7%.
3. C.V. for seed yield = 18.0%.
REFERENCES
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2. Buntin, G.D., and P.L. Raymer. 1994. Pest status of aphids and other insects in winter canola in Georgia. Journal of Economic Entomology 87:1097-114.
3. Evans, K.A., Scarisbrick, D.H. 1994. Integrated insect pest management in oilseed rape crops in Europe. Crop Protection 13:403-412.
4. Gumpert, Jim. 1999. The U.S. Canola Craze: The New Gold Rush. Seed World. March, 1999.
5. Lamb, R.J. 1996. Entomology of oilseed Brassica crops. Annual Review Entomology 34:211-229.
6. Ramachandran, S., G. D. Buntin, J. N. All, B.E. Tabashnik, P. L. Raymer, M. J. Adang. D.A. Pullinam, and C.N. Stewart, Jr. 1998a. Survival, development, and oviposition of resistant diamondback moth (Lepidoptera: Plutellidae) on a transgenic canola producing a Bacillus thuringiensis toxin. Journal of Economic Entomology 91:1239-1244.
7. Ramachandran, S., G. D. Buntin, J. N. All, P. L. Raymer, and C. Neal Stewart, Jr. 1998b. Greenhouse and field evaluations of a transgenic canola against diamondback moth, Plutella xylostella and corn earworm, Helicoverpa zea. Entomologia Experimentalis et Applicata. 88:17-24.
8. Raymer, P.L., G.D. Buntin, M.J. Weiss, and J. McCaffery. 1998. Canola Production in the United States. GCIRC Bulletin 15: 47-49.
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