EVALUATION AND PERFORMANCE OF TRANSGENIC OILSEED

RAPE CONTAINING cry1Ac GENE FOR RESISTANCE TO DIAMONDBACK MOTH

Suresh Ramachandran¹, G. D. Buntin², J. N. All¹, P. L. Raymer³, and C. N. Stewart, Jr.⁴

¹ Dept. of Entomology, University of Georgia, Athens, GA 30602, USA

² Dept. of Entomology, Georgia Experiment Station, Griffin, GA 30223, USA

³ Dept. of Crop and Soil Sciences, Georgia Experiment Station, Griffin, GA 30223, USA

⁴ Dept. of Biology, University of North Carolina, Greensboro, NC 27402, USA

ABSTRACT

Oilseed rape (canola), Brassica napus L., expressing toxic protein(s) from the bacteria, Bacillus thuringiensis Berliner (Bt) could be utilized for management of the insect pest diamondback moth, Plutella xylostella L.  Canola cultivars 'Oscar' and 'Westar' were genetically engineered with a synthetic cry1Ac gene.  Three out of the 57 recovered transgenic lines recovered were tested under greenhouse (O52‑6 and W58‑3) and field (O52‑6) conditions for resistance to diamondback moth larvae.  Transgenic lines provided complete mortality of diamondback moth neonates at all growth stages and recorded low levels of defoliation under greenhouse conditions.  Transgenic Oscar expressed high levels of resistance against diamondback moth both as a pure stand and in seed mixtures under field conditions.  In seed mixtures, no competitive advantage occurred for either transgenic or nontransgenic plant types when there was no diamondback moth infestation. However, except for neonates, larvae began moving from the transgenic plants within 24 h, but required at least 48 h exposure to transgenic plants to acquire lethal doses of the toxin.  Thus, seed mixtures are not the best strategy for deployment of transgenic canola against diamondback moth.  A diamondback moth colony expressing >3000 folds of resistance to sprayable formulations of Bt survived successfully on transgenic plants without any abnormal effects.  The ability of a Bt-resistant diamondback moth population to overcome resistance of transgenic plants expressing high levels of toxin emphasizes the importance of proper resistance management strategies for the deployment of transgenic canola.

KEYWORDS: canola, Bacillus thuringiensis, seed mixtures, insect resistance, Plutella xylostella

INTRODUCTION

In the United States, oilseed rape (canola), Brassica napus (L.), is grown in the northern Great Plain region, Pacific Northwest  region and southeastern parts of the country.  Canola oil consumption has grown rapidly after its approval for consumption in 1985.  Current consumption of canola oil is about 1.1 billion pounds per year, which account for 5% of the total edible oil consumption.  Corresponding with the oil consumption, canola acreage also has increased rapidly and in 1997 more than 700,000 acres were planted with canola.  The demand for canola oil and consequently area under canola is poised for further growth in the coming years (Stanton 1993).

            Among the many insect pests that attack canola, the lepidopteran crucifer specialist, diamondback moth, Plutella xylostella (L.) (Plutellidae), is expected to be a major problem.  Diamondback moth is also an important pest of summer canola in the Pacific Northwestern U.S.  Non-availability of diamondback moth resistant canola cultivars and resistance of diamondback moth to many of the synthetic insecticides make it as a difficult pest to manage.  Further, attempts to identify diamondback moth resistant B. napus sources also have been unsuccessful (Ramachandran et al.1998d).  Thus, it is essential to devise alternative strategies to manage diamondback moth on canola.

            Crops genetically engineered with genes from the bacteria, Bacillus thuringiensis (Bt) Berliner, are being used to manage key pests on cotton, corn, and potato.  We genetically engineered a synthetic cry1Ac gene into canola cultivars ‘Oscar’ and ‘Westar’ and the transgenic lines were reported to confer high levels of resistance against diamondback moth under laboratory conditions (Stewart et al. 1996).  The objective this study was to evaluate the performance of selected transgenic lines under greenhouse and field conditions.  We also tested the feasibility of seed mixtures, a deployment strategy to minimize resistance development by insects, against diamondback moth.  Further, survival and development of a Bt resistant diamondback moth strain also was tested on transgenic canola.

GREENHOUSE AND FIELD EVALUATIONS OF TRANSGENIC CANOLA

            Fifty-seven transgenic lines were recovered from the transformation process with varying levels of Cry1Ac toxin expression (Stewart et al. 1996).  A line with high levels of toxin expression (O52-6; transgenic Oscar) and another with moderate levels of toxin expression (W58-3; transgenic Westar) were tested under greenhouse and field conditions.  Transgenic lines provided 100% mortality of diamondback moth neonates, in greenhouse antibiosis tests, at seedling, vegetative, bolting, and flowering stages of the plant.  In greenhouse preference assays, conducted at vegetative and flowering plant stages, transgenic plants recorded less than 4% of defoliation as compared to up to 30% of defoliation in non-transgenic plants (Ramachandran et al. 1998b).

            In field tests conducted during 1995-1997, plots were artificially infested with diamondback moth neonates.  Generally, the damage to plants was higher in the 1995-1996 season as compared to 1996-1997 season.  But, transgenic plants recorded significantly lower damage compared to non-transgenic plants in both seasons (Table 1).  Non-transgenic plants harbored at least 10 times more of diamondback moth larvae as compared to transgenic plants.  Further more, transgenic plants had a better plant stand and produced more pods and seeds at the end of the season than non-transgenic plants (Ramachandran et al. 1998b).

Table 1.  Percent defoliation of transgenic and non-transgenic canola plants in field plots with artificial infestations of diamondback moth neonates¹

Percent defoliation

Plant type	1995-1996	1996-1997
Transgenic	6.5 ± 0.3b	0.5 ±  0.2b
Non-transgenic	59.9 ±  13.2a	22.4 ± 4.7a

Means (±  SE) followed by different letters with in a column are significantly different from one another in a Students t-test (P < 0.05). ¹ from Ramachandran et al. (1998b)

FEASIBILITY OF SEED MIXTURES OF CANOLA AGAINST DIAMONDBACK MOTH

            Mixtures of resistant (transgenic) and susceptible (non-transgenic) plants have been suggested as a strategy to avoid or minimize the resistance development by insects against transgenic plants (Tabashnik 1994, Hokkanen and Wearing 1995).  In seed mixtures adults produced from transgenic plants would be in close proximity to the adults produced from the non-transgenic plants, thus providing more chances for random mating and dilution of insect resistance.  However, there is a possibility that transgenic plants (due to their improved or lowered fitness) could out-compete non-transgenic plants in seed mixtures.  Also, the movement of insects feeding on the susceptible plants in their early stages and later moving to the transgenic plants to feed and grow would not only result in crop loss, but also could expedite the evolution of insect  resistance.  So, it is essential to understand these 2 factors to implement a successful seed mixture strategy.

Competitive advantage of transgenic canola

            Competitive interaction between transgenic and non-transgenic plants was tested by planting 100:0, 25:75, 50:50, 75:25 and 0:100 ratio of transgenic and non-transgenic plants in the field at different locations.  Plots were either infested with diamondback moth neonates or left without any insect infestation.  Percent defoliation of plants in different mixtures was recorded at weekly intervals.  At the end of the season, transgenic and non-transgenic plants were harvested separately and total biomass and seed weights were recorded.

            Transgenic plants recorded significantly lower levels of defoliation compared to the non-transgenic plants in all mixtures and in pure stands.  This demonstrates the high  levels of resistance by transgenic canola against diamondback moth in seed mixtures also (Ramachandran et al. 1999).  In plots where there was no diamondback moth infestation, relative competitive coefficient (RCC), a measure of competitive interaction between plants, were not significantly different from 1 for both biomass and seed weight indicating no competitive advantage occurred for either plant type in seed mixtures.  In plots where there was diamondback moth infestation, RCC values were significantly different from 1 indicating transgenic plants out performed non-transgenic plants.  Significant RCC values in insect infested plots implied transgenic plants were able to better withstand diamondback moth infestation.

Diamondback moth larval movement in seed mixtures

            To study the larval movement patterns, transgenic and non-transgenic plants were arranged at 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 ratios in closed cages in greenhouse.  Plants were infested with diamondback moth neonates and numbers of larvae surviving on each plant on day 6 were enumerated.  The experiment indicated that the larvae moved from plant to plant during their growth.  When 2^nd, 3^rd, and 4^th instar larvae were exposed to transgenic plants for 48 h and subsequently transferred to non-transgenic plants no larvae were able to complete the life cycle.  These results suggest that the larvae need to feed on transgenic plants for at least 48 h to acquire a lethal dose of the toxin.  But, in an another experiment concerned with larval movement, larvae were observed to move from the infested plant within 24 h (Table 2).  This shows that larvae would move from the transgenic plants before acquiring lethal doses of the toxin.  Thus, we conclude that seed mixtures of canola may not be the best strategy to minimize rate of resistance development by diamondback moth against transgenic canola.  Instead of seed mixtures, strip planting could be practiced with adequate row spacing between transgenic and non-transgenic plants to avoid larval movement between the rows.

Table 2.  Mean ± SE percentage of 2^nd, 3^rd, 4^th instar stages of diamondback moth recovered from infested transgenic and uninfested non-transgenic canola plants, 24 h after infestation¹

Percent live larvae recovered

Larval instar	On infested transgenic plant	On uninfested non-transgenic plant	t value*
II	71 ± 9	29 ± 9	2.3
III	52 ± 5	48 ± 5	0.5
IV	55 ± 5	45 ± 5	1.1

* No significant differences were recorded in the larval recovery from transgenic and non-transgenic plants. ¹ from Ramachandran et al. (1998c)

BT RESISTANT DIAMONDBACK MOTH ON TRANSGENIC CANOLA

            Diamondback moth has already evolved resistance to sprayable Bt products in various parts of the world.  Thus, some resistance of these populations to the transgenic canola is expected.  So, we evaluated the survival and development of a Bt resistant diamondback moth strain on transgenic canola.  The resistant strain was originally collected from the field and intensively selected with Bt sprays in laboratory and the tested colony expressed > 3000 folds of resistance to Bt sprays.

            Larval and pupal durations, pupal weights, and adult emergence were similar for both resistant and susceptible strains on non-transgenic canola.  Transgenic canola killed all larvae tested from the susceptible strain.  In contrast, for the resistant strain, no significant differences occurred between transgenic and non-transgenic canola in larval survival, percentage pupation, pupal weight, and percentage adult emergence (Ramachandran et al. 1998a).  Further, larvae from the resistant strain were able to damage transgenic and non-transgenic plants equally well.  This shows that transgenic canola did not have any effect on the resistant strain, and also proves that a pest can overcome high levels of resistance exhibited by transgenic plants.

            When oviposition preference by adults and feeding initiation by neonates were tested for resistant and susceptible strains, no significant differences occurred between transgenic and non-transgenic canola (Ramachandran et al. 1998a).  This suggests that oviposition preference by adults and larval susceptibility is unrelated.  Further, transgenic plants did not alter the oviposition by adults and feeding initiation by neonates.

CONCLUSIONS

            In conclusion, tested transgenic canola provided high levels of resistance against diamondback at all growth stages under greenhouse conditions.  Transgenic canola also provided excellent control of diamondback moth under field conditions both as a pure stand and in mixtures.  No competitive advantage occurred for either transgenic or non-transgenic plant type in seed mixtures.  However, in seed mixtures there is a possibility that larvae would move off from the transgenic plants before acquiring lethal doses of the toxin, thus accelerating the possibility of resistance development.  To minimize resistance development, strip planting could be practiced with adequate row spacing between transgenic and non-transgenic plant rows.  A strain of diamondback moth resistant to Bt sprays was able to overcome the toxin expressed by the transgenic plants.  Further, transgenic plants did not have any measurable effect on the resistant strain.

            Our project clearly demonstrates the power of transgenic technology to manage a key pest.  However, it also underlines the fact that transgenic technology is also vulnerable for failures if used improperly.  So, it is essential to use transgenic crops, including canola, judiciously to manage the target pests.

ACKNOWLEDGMENTS

            We thank Drs. M. J. Adang, K. E. Espelie, and B. E. Tabashnik for their helpful suggestions and support for this project.  We also extend our thanks to K. J. Lance and B. Slaughter, Jr., for their technical assistance.

REFERENCES

Hokkanen, H. M. T., and C. H. Wearing. 1995. Assessing the risk of pest resistance evolution to Bacillus thuringiensis engineered into crop plants: a case study of oilseed rape. Field Crops Research 45:171-179.

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. J. Econ. Entomol. 91: 1239-1244.

Ramachandran, S., G. D. Buntin, J. N. All, P. L. Raymer, and C. N. Stewart, Jr. 1998b.  Greenhouse and field evaluations of transgenic canola against diamondback moth, Plutella xylostella, and corn earworm, Helicoverpa zea. Entomol. Exp. Appl. 88:17-24.

Ramachandran, S., G. D. Buntin, J. N. All, P. L. Raymer, and C. N. Stewart, Jr. 1998c.  Movement and survival of diamondback moth (Lepidoptera: Plutellidae) larvae in mixtures of nontransgenic and transgenic canola containing a cry1Ac gene of Bacillus thuringiensis. Environ. Entomol. 27:649-656.

Ramachandran, S., G. D. Buntin, J. N. All, and P. L. Raymer. 1998d. Diamondback moth (Lepidoptera: Plutellidae) resistance of Brassica napus and B. oleracea lines with differing leaf characteristics. J, Econ. Entoml. 91: 987-992.

Ramachandran, S., G. D. Buntin, J. N. All, P. L. Raymer, and C. N. Stewart, Jr. 1999. Intraspecific competition of a insect resistant transgenic canola in seed mixtures. Agron. J. (Submitted).

Stanton, J. 1993. Canola on the United States. Cereal Foods World 38: 340-346.

Stewart, Jr., C. N., M. J, Adang, J. N. All, P. L. Raymer, S. Ramachandran, and W. A. Parrott.1996. Insect control and dosage effects in transgenic canola, Brassica napus L. (Brassicaceae), containing a synthetic Bacillus thuringiensis cryIAc gene. Plant Physiol. 112:115-120.

Tabashnik, B. E. 1994. Delaying insect adaptation to transgenic plants: seed mixtures and refugia reconsidered. Proc. R. Soc. Lond. B 255:7-12.