1. Transgenic Cotton for Insect Control
Peter C. Ellsworth, Ph.D.
IPM Specialist, University of Arizona
Maricopa Agricultural Center
Maricopa, AZ, USA

2. Disclosure
Those engaged in the dialog on biotechnology should fully disclose their relationships and opinions “up front” so that audiences can consider the context.
Partial support for my research comes from companies with interests in biotechnology.
The balance of support comes from state and federal sources of competitively available public funds.

3. Disclosure (continued)
Biotechnology and its products are neither inherently good nor bad.
The specific process and each of its products should be scientifically and independently evaluated.

4. Transgenic Cotton for Insect Control
What is available now & in the future?
Origin, identity & development
Insect target(s) in the U.S.
Efficacy & utility in the Arizona system (benefits)
Safety (risks)
Resistance
Impact of gene on plant
Biodiversity
non-target effects

5. Products Available for Cotton Insect Control
Only 1 ‘trans’-gene has been commercialized
Based on the crystalline protein produced by Bacillus thuringiensis (Bt)
Developed by Monsanto as Bollgard® and incorporated into commercial varieties by several cotton seed companies (e.g., Delta Pineland Co. & Stoneville Pedigreed Seed Co.)
Sold in the U.S., Australia, Mexico, South Africa, India, China, Argentina, Indonesia

6. Bacillus thuringiensis (Bt)
Common soil bacterium
Present in nature in a variety of forms (species & strains)
Produces proteins that are toxic to insects
Commonly used in garden sprays & for commercial agriculture, including organic farming
Extremely well-known toxin in terms of human health & environmental safety

7. Bacillus thuringiensis (Bt)
Crystalline proteins are classified according to structure & have a specific nomenclature (e.g., Cry1Ac)
Cotton has been transformed with Cry1Ac (narrow spectrum; Lepidoptera only)
Protein binds with receptors in the insect gut causing pores which perforate the midgut & lead to cell leakage & insect death

8. The Transformation Coker 312
The gene of interest is spliced out of the bacterium using a vector, like Agrobacterium tumefasciens, & transferred to cotton cells grown in tissue culture
The cells are grown into a plant & then, after testing, plants are back-crossed into commercial lines to make new varieties
Recurrent back-crossing

9. Spectrum of Activity for BG
Excellent Control
No Control
Tobacco Budworm, the principal pest in the South
Trichoplusia ni
Spodoptera exigua
Heliothis virescens
Spodoptera frugiperda
Spodoptera ornithogalli
Pectinophora gossypiella
Bucculatrix thurberiella
Pink Bollworm (PBW), our principal pest
Beneficial Insects
Estigmene acrea
Helicoverpa zea
(pre-bloom)
Agrotis & Feltia spp.
Helicoverpa zea
(post-bloom)
Pseudoplusia includens
Marmara spp.

10. AZ’s Primary Lepidopteran Pest
Pink Bollworm
Multiple generations
Adult lays eggs on bolls or susceptible squares (SS)
Larvae hatch & penetrate bolls within 24 hrs

11. Alternatives for PBW Control
Repeated, broad-spectrum sprays are required to prevent moths from invading fields
No effective larvicides or ovicides
Biological controls are limited by the biology of this pest
Little impact of parasitoid or predators
Cultural controls can be very effective
Requires early termination & areawide compliance with plowdown requirements

12. Secondary Lepidopteran Pests
Occasional pests
Induced pests
Helicoverpa zea
Heliothis virescens
Trichoplusia ni
Estigmene acrea (Arctiidae)
Bucculatrix thurberiella
Spodoptera exigua

13. Bt Cotton Questions Efficacy & economic studies Agronomic studies
How effective is the gene?
Are oversprays required for lepidopteran control?
If so, are there new scouting & threshold considerations?
Agronomic studies
Impacts (+/-) on yield & fiber qualities?
Product integrity & stability studies
High-dose through life of plant?
High-dose in all varieties?
Purity?
Ecological studies
Impact on non-target organisms (NTO)
Ca. 100% for PBW
Not for PBW
Search for large larvae
No unintended effects
Yes, actively growing
No, some not marketed
> 98% (?)

14. BG Cotton Efficacy Young larvae present regardless of cotton type
Little difference between Bt & non-Bt (-) varieties

15. BG Cotton Kills Small Larvae
PBW larvae must feed in order to be killed.
Large larvae survive mainly in non-Bt varieties.

16. Impact on Arizona Cotton
In 1990, > 6.8 sprays were made against PBW; still, > 5% yield loss
Since 1996 when Bt cotton was introduced, it has never required oversprays for PBW control, AND
Since 1997, only 0.5 sprays have been made against PBW over all cotton acreage (Bt and non-Bt); i.e., an areawide reduction of PBW has occurred
The net reduction in insecticide use has resulted in huge savings to farmers, and large improvements to the agroecosystem in terms of beneficial insect communities & IPM

17. Safety - Resistance
Given time & exposure, insects have the capacity to overcome most insecticides. Bt cotton may be no different, however, there are safeguards:
Refugia
High-Dose Strategy
Development of additional proteins

18. Refugia
Objective: provide harborage for susceptible moth production to reduce the chance of resistant (R) moths mating with each other
U.S. growers are required to plant a proportion of their acreage to non-Bt cotton
5% Refuge, if no lepidopteran-active insecticides are used on it, or else
20% Refuge RR SS RS

19. High-Dose Strategy, Depends on:
Yes
Yes?
Yes, *refuges
No (?)
The production of a dose high enough to kill:
>99.9% of a susceptible (SS) population, and
>95% of the heterozygous (RS) individuals,
A recessive resistance,
Random mating,
A low initial frequency of the ‘R’ allele.

20. Development of Additional Transgenes (Bt’s)
Bollgard II®
2 Bt gene product, original Bollgard (Cry1Ac) + Cry2Ab
Final stages of US-EPA approval
Limited commercial production in 2003
Full replacement of BG varieties by 2008?
Bollgard III
Little information on this available at this time; research stages only
Cry1F
Under development by Dow Agrosciences in combination with Cry1Ac

21. Impact of Gene on Plant
Isogenic lines were developed for testing the impact of the gene(s) on agronomic and efficacy characteristics of the plant
Lines
Cry1Ac+ Cry2Ab
Cry1Ac only
Cry2Ab only
Null
C312B
DP50
DP50B
(Cry1Ac)
DP50II
(Cry1AC+Cry2Ab)
Particle gun
DP50
Cry1Ac
Cry2Ab

22. Isoline Studies of BG & BGII
Replicated studies
Artificial & natural PBW infestations
Sprayed & Unsprayed conditions

23. Warts are often formed at the site of PBW attack
Dead 1st instar in Bt cotton

24. BGII Results - PBW, 1st Instars
Dead 1st Instars
Live 1st Instars

25. BGII Results - PBW, All Instars

26. BGII Results - B. thurberiella
BGII prevented cotton leafperforator development better than BG
Leaves at top of plant (younger) express highest doses of Bt
Older leaves (bottom) have reduced doses of Bt

27. Marmara sp.
Citrus Peel Miner is an incidental lepidopteran that mines the main stem and boll surfaces
Cry2Ab alone (‘X’) is more effective than Cry1Ac (‘B’)

28. Spectrum of Activity for BG (Cry1Ac)
Excellent Control
No Control
Heliothis virescens
Pectinophora gossypiella
Helicoverpa zea
Bucculatrix thurberiella
Spodoptera exigua
Estigmene acrea
Trichoplusia ni
Spodoptera frugiperda
Spodoptera ornithogalli
Pseudoplusia includens
Agrotis & Feltia spp.
Beneficial Insects
Marmara spp.

29. Spectrum of Activity for BGII (Cry1Ac + Cry2Ab)
Excellent Control
No Control
Heliothis virescens
Pectinophora gossypiella
Helicoverpa zea
Bucculatrix thurberiella
Spodoptera exigua
Estigmene acrea
Trichoplusia ni
Spodoptera frugiperda
Spodoptera ornithogalli
Pseudoplusia includens
Agrotis & Feltia spp.
Beneficial Insects
Marmara spp.

30. High Dose and % Efficacy?
Throughout our early work with BG cotton, we often would find low levels of “survivors” from our field plots

31. Source of Survivors Low expression of Bt in plants?
Low levels of non-Bt contaminants?
In the seedbag
From volunteer seed
Resistance?

32. % Efficacy Against PBW Before plants are tested for presence of Bt
After PBW from non-Bt plants are discarded
% Efficacy Against PBW
Cry1Ac 100%
Cry2Ab 99.67%
Both Genes 100%

33. Biodiversity / NTO Studies
The reports of Bt effects on Monarch butterflies have fueled much emotional debate on the use of biotech crops.
Monarch Butterfly, symbol of nature and “wildness” in North America.

34. Non-Target Organisms (NTO)
Over 370 arthropod species have been tracked in 2 years of field studies using a variety of methodologies.
So far, no major or functional differences have been found in Arizona between BG, BGII, and conventional cotton communities…
Except where harsh PBW sprays are needed in conventional cottons.
Thus, Bt cotton ecosystems are not only safe, but safer than conventional cotton ecosystems where insecticidal inputs are higher.

35. Conclusions
The use of Bt cottons in Arizona has provided the first larvicidal and selective approach to controlling PBW.
The control provided by Bt cottons approaches immunity. No survivors have been found in field studies.
Bt cotton has revolutionized our ability to implement IPM in AZ cotton & reduced our insecticide inputs by over 60%.
Future transgenic products for insect control in cotton should be independently & scientifically tested.
Other than new Bt genes/events, there are few, if any, development plans for insect contol products.

36. Information
ACIS
All University of Arizona crop production & crop protection information is available on our web site,
Arizona Crop Information Site (ACIS), at