Pest Control with Enhanced Environmental Safety - American

Chapter 19

Use of Bacillus thuringiensis Genes in Transgenic Cotton To Control Lepidopterous Insects

Downloaded by PURDUE UNIVERSITY on September 30, 2013 | http://pubs.acs.org Publication Date: March 12, 1993 | doi: 10.1021/bk-1993-0524.ch019

Johnie N. Jenkins Crop Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Mississippi State, MS 39762

Transgenic cotton, Gossypium hirsutum L., plants containing modified, truncated versions of the cryIA genes from Bacillus thuringiensis var kurstaki are resistant to many Lepidopterous insects. Plants containing specific, truncated versions of the cryIA gene were resistant to tobacco budworm, Heliothis virescens Fab., in field and laboratory studies in my laboratory. Other researchers have shown that these transgenic plants are also resistant to bollworm, Helicoverpa zea (Boddie); pink bollworm, Pectinophera gossypiella (Saunders); cotton leafperforator, Bucculatrix thurberiella Busck; and saltmarsh caterpillar, Estigmene acrea (Drury). Others have shown that the proteins coded for by the B. thuringiensis genes bind selectively to sites in the brush borders of epithelial cells in the midgut of the insect and induce the formation of small pores (0.5 to 1.0 nm) in the cell membranes, resulting in a net Influx of ions and outflow of water. The cells swell and lyse. The B. thuringiensis genes in transgenic cotton plants appear to offer an environmentally desirable control method for Lepidopterous cotton pests when used in integrated pest management programs. Introduction to Host Plant Resistance Pest resistant c u l t i v a r s are the most environmentally benign method available for control of many crop pests. For many pests they are the only method a v a i l a b l e . The c u l t i v a r forms the foundation upon which successful integrated pest management programs are b u i l t . Resistance genes are available i n crops for insects, mites, nematodes, and plant diseases caused by fungi, b a c t e r i a , and viruses; however, c u l t i v a r s are not usually resistant to a l l pests that affect the crop. There are several advantages of resistant c u l t i v a r s : 1) pest control i s bought when the seed are purchased, as the control i s bred into the seed; 2) this method of pest control i s compatible with most other methods of control; 3) resistant c u l t i v a r s form a foundation upon which integrated pest management programs can be b u i l t ; 4) few or no adverse effects on the environment are caused

This chapter not subject to U.S. copyright Published 1993 American Chemical Society In Pest Control with Enhanced Environmental Safety; Duke, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


268

PEST CONTROL WITH ENHANCED ENVIRONMENTAL SAFETY

by the genes for resistance; 5) cost of t h i s type of pest control i s usually minimal; and 6) compared to conventional chemical pest c o n t r o l , less effort i s required after the seeds of resistant c u l t i v a r s are planted; however, for cotton, Gossvplum hlrsutum L . , integrated pest management involving control of several pests w i l l be required. Host plant resistance, i . e . , the development of r e s i s t a n t c u l t i v a r s , i s a dynamic process. Most insect pests and pathogens of important crop plants are very diverse and have a wide host and/or geographic range. These pests are dynamic and t h e i r population makeup i s subject to selection pressure from host c u l t i v a r s and other factors i n the environment. A r e s i s t a n t c u l t i v a r can exert selection pressure upon a v a r i a b l e population of insects and, thus may select for a biotype that can successfully colonize and damage the c u l t i v a r . The degree of selection pressure strongly influences how long a gene or set of genes w i l l be useful for control of a p a r t i c u l a r pest. Some genes for resistance to pests l a s t for many years; whereas, others l a s t only a few years. The most dramatic examples of successful control of insects with resistant c u l t i v a r s are the control of hessian f l y , Mavetiola destructor (Say), i n wheat, Triticum spp.; and brown planthopper, Nilaparvata lugens. i n r i c e , Orvza sativa (1). Twenty genes for resistance to hessian f l y have been discovered (2). During the period from 1950 to 1983, 60 c u l t i v a r s of wheat with various combinations of these genes for resistance to hessian f l y were developed and successfully used to control t h i s pest (2). There i s great v a r i a b i l i t y i n the length of time a p a r t i c u l a r gene i s useful for pest control before the selection and development of a pest biotype that is resistant to the gene occurs. For example, the f i r s t gene for resistance to brown planthopper was Bph-1 and was i n the c u l t i v a r IR26 released i n 1973 (3). Widely planted i n the P h i l i p p i n e s , Indonesia, and Vietnam t h i s c u l t i v a r became susceptible to brown planthopper i n just three years after being released (1). This gene was effective but only for a r e l a t i v e l y short time, yet i t was very successful during t h i s time. Plant breeders continued to work and developed r i c e c u l t i v a r s with a different gene for resistance to the new biotype of the brown planthopper. IR36 and IR38 with the bph-2 gene were developed and released (4) and became the dominant r i c e c u l t i v a r i n many areas. This source of resistance has held up for 14 years and i s s t i l l useful i n many areas (1). Continuous research on c u l t i v a r development, discovery, and i d e n t i f i c a t i o n of new genes for resistance to brown planthopper has resulted i n the breeding and release of c u l t i v a r s with additional genes for resistance. This e f f o r t has culminated i n the release of IR56 and IR60 with the Bph-3 gene for resistance which was useful when a resistant biotype was discovered i n small pockets of the P h i l l i p i n e s and Indonesia. IR66 with the bph-4 gene for resistance was released i n 1987 and IR68, IR70, IR72, and IR74, a l l with the bph-4 gene for resistance, were released i n 1988. These c u l t i v a r s are widely grown i n t r o p i c a l and subtropical r i c e growing countries of the world (1). These developments i l l u s t r a t e the continuous e f f o r t necessary to successfully u t i l i z e host plant resistance for pest c o n t r o l . U t i l i z i n g genes made available through recombinant DNA technology probably w i l l not change t h i s . Many factors influence how long a

In Pest Control with Enhanced Environmental Safety; Duke, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

19. JENKINS

Bacillus thuringiensis Genes in Transgenic Cotton

269


p a r t i c u l a r gene for resistance w i l l be u s e f u l . These examples, however, i l l u s t r a t e that i t i s possible to develop resistant c u l t i v a r s for pest control that are f r i e n d l y to the environment; safe, e f f i c i e n t , and economical for the grower; and useful to the consumer. This chapter reviews current research involving transgenic plants for insect c o n t r o l . It deals primarily with cotton and genes from B a c i l l u s thuringiensis because the majority of research with transgenic plants for insect control involves genes from t h i s bacterium and cotton w i l l probably be the f i r s t crop to be grown commercially with these genes.

Conventional Plant Breeding Conventional plant breeding involves crossing plants, usually within a species, and transferring genes for useful t r a i t s . Genes that confer pest resistance are often found i n unadapted, low y i e l d i n g land races of crop plants or i n weedy r e l a t i v e s of crop plants. An arduous and long process i s necessary before an adapted c u l t i v a r can be developed with these gene(s) for resistance. This process has worked very well because plant breeders and associated s c i e n t i s t s have r e a l i z e d that resistant c u l t i v a r s are useful and that pest population gene pools are dynamic and influenced greatly by resistant c u l t i v a r s . Their continuing work has resulted i n discovery of new genes for resistance and the deployment of these through new c u l t i v a r s resistant to pests. Species b a r r i e r s have been major obstacles to this type of research. Plant breeders have been more successful with i n t r a s p e c i f i c crosses. Only a few examples exist where genes from other closely r e l a t e d species have been successfully u t i l i z e d i n breeding programs. F e r t i l e crosses are usually l i m i t e d to plants within the same species and some c l o s e l y related r e l a t i v e s . These have been the constraints within which plant s c i e n t i s t s have worked. Modern c u l t i v a r s of cotton show that plant breeders have been successful i n developing c u l t i v a r s with higher y i e l d s and higher effective l e v e l s of pest resistance ( 5 ) .

Biotechnology and Recombinant DNA Technology The developments i n recombinant DNA technology and other aspects of biotechnology, including genetic engineering, have improved the opportunities for plant s c i e n t i s t s . Genetic engineering complements conventional plant breeding by increasing the d i v e r s i t y of genes available for incorporation into crops. It i s now t h e o r e t i c a l l y possible to move a gene from any organism to a plant and have that gene produce i t s product i n the plant. Presently there are limitations upon which types of genes can be moved and upon how many genes can be moved at one time; however, as t h i s technology advances, many of the present obstacles w i l l be erased or reduced. Genetically changed plants, i n which genetic material from other species i s inserted by biotechnology, are c a l l e d transgenic plants. Transgenic plants have been developed for 47 species of crop plants ( 6 ) . These involve a l l the major food and f i b e r crops of the world.


270


Fraley (6) says that, "Between 1992 and 2030 farmers w i l l have to produce more food--more c a l o r i e s - - t h a n they have done from the beginning of agriculture u n t i l now." He asks and answers the question: "In short, how can we create sustainable agriculture? Invest i n , and develop new a g r i c u l t u r a l technologies." Plant biotechnology i s one of these new technologies that w i l l be developed to help meet these future human needs.


Systems for Producing Transgenic Plants The most advanced systems for producing transgenic cotton plants have involved Agrobacterium tumefaciens as the genetic engineering agent. This bacteria infects many dicotyledonous plants and causes the growth of tumors or g a l l s i n the infected plants. Virulent strains of the bacteria contain large T i (tumor-inducing) plasmids, which are responsible for the DNA transfer and subsequent disease symptoms. These T i plasmids contain two sets of sequences necessary for gene transfer to the plants. One of these sequences i s the T-DNA (transferred DNA) region which i s transferred to the plant. The other sequence, the v i r (virulence genes), i s not transferred during i n f e c t i o n . The T-DNA regions are flanked by border sequences that determine the d e f i n i t i o n of the region transferred to the infected plant. A special type of the bacteria which has the tumor-inducing genes disarmed i s used i n transformation experiments. Disarming the tumor inducing genes allows the bacteria to infect the plant, but does not allow the formation of tumors. The desired genes that have been inserted between the flanked border sequences of the T i plasmid are then inserted into the plant by the bacteria i n a manner s i m i l a r to normal i n f e c t i o n of the plant i n nature. Thus, the i n f e c t i o n process of the A. tumefaciens bacteria i s used to insert the gene of interest rather than the tumor inducing gene and becomes the vehicle by which foreign genes can be inserted into plants. A second major means of producing transgenic plants i s the p a r t i c l e bombardment process. A recent review of gene transfer by p a r t i c l e bombardment describes how this method i s being used with plants and animals, and reports important applications of the process to produce transgenic maize, Zea maize, and soybean, Glycine max. as well as the introduction of DNA into p l a s t i d s and mitochondria (7). One application i s the d i r e c t i n s e r t i o n of genes into organs of l i v i n g animals. This technique i s also being used to transform cotton ( 8 , 9 ) . A recent review of biotechnology of cotton summarizes many of these concepts (10). As plant development i s better understood, we are beginning to achieve a better understanding of how gene regulation occurs. Tissue s p e c i f i c promoters and regulators are now available that when linked with pest resistant genes should allow for tissue s p e c i f i c expression of the pest resistance genes (11). Resistance to the herbicide glyphosate has been enhanced by targeting the product of a mutated form of the EPSPS b a c t e r i a l gene to reside i n the chloroplasts of plants where EPSPS normally occurs (12). There are situations where the tissue s p e c i f i c approach would be desirable from a pest control or safety approach. Gasser and Fraley (10) suggest that i n the near future we w i l l have on hand a large number of regulatory sequences that w i l l allow for accurate


19. JENKINS


271

targeting of gene expression i n s p e c i f i c tissue within transgenic plants. Reducing the expression of a s p e c i f i c gene i n a plant can also be u s e f u l . Through anti-sense RNA technology t h i s i s now possible and has found application i n tomatoes, Lvcopersicon esculentum. where the messengers for polygalacturonase have been altered with anti-sense RNA. As a r e s u l t the altered tomatoes do not produce much of this enzyme and the tomatoes ripen without becoming soft (11, 13).


B. thuringiensis Endotoxin for Insect Control The most widely used genes for insect control i n transgenic plants are those from several strains of B a c i l l u s thuringiensis. This bacteria produces proteins which are l e t h a l to selected insect pests. Many strains of B. thuringiensis are toxic to insects i n the order Lepidoptera, some are toxic to insects i n the order D i p t e r i a ( f l y ) , and some to Coleoptera (beetle). Recently a s t r a i n of B. thuringiensis has been discovered that i s toxic to nematodes (14, 15). Tobacco, Nicotiana tabaccum, plants were f i r s t transformed with the B. thuringiensis delta endotoxin gene i n 1987 (16). Insertion of the 1176 amino acid toxin i n plant c e l l s was phytotoxic; however, Barton et a l . (16) reported that eliminating the protoxin carboxyterminus solved the phytotoxicity problems and produced tobacco plants that expressed the delta endotoxin. Commercial cotton c u l t i v a r s were f i r s t transformed using A. tumefaciens mediated transformation technology (17). Effective selection for kanamycin resistance i n tissue c u l t u r e , regeneration of plants, and expression of the marker enzymes, neomycin phosphotransferase II (npt II) and chloramphenicol acetyltransferase (cat), at the whole plant l e v e l showed that transformation had occurred. Direct evidence of foreign DNA integration was provided by southern blot h y b r i d i z a t i o n . The f i r s t f i e l d test of transgenic cotton plants containing the B. thuringiensis gene encoding for the delta endotoxin was conducted i n 1989; however, these plants did not provide control of tobacco budworm insects i n the f i e l d or i n the laboratory (18). Subsequent f i e l d tests of cotton strains i n 1990, using plants with a different form of the B. thuringiensis gene, did however, provide effective f i e l d control of tobacco budworm (19).

Other Genes for Insect Control in Transgenic Plants A gene encoding a cowpea, Vigna unguiculata. t r y p s i n i n h i b i t o r (CpTI) has been shown to confer a l e v e l of resistance to tobacco budworm when transferred into tobacco (20). Cowpea t r y p s i n i n h i b i t o r s are small polypeptides of around 80 amino acids belonging to the Bowman-Birk type of double-headed serine protease i n h i b i t o r s and are the products of a small gene family (21).

Insecticidal Crystal Proteins Present in B. thuringiensis Nucleotide sequences for 42 c r y s t a l protein genes from B, thuringiensis have been described (22). Several sequences are nearly i d e n t i c a l . There are 14 d i s t i n c t c r y s t a l protein genes of



272


which 13 specify a family of closely related i n s e c t i c i d a l proteins (Cry p r o t e i n s ) . These are divided into a minimum of four major classes and several subclasses specified by s t r u c t u r a l s i m i l a r i t y and i n s e c t i c i d a l spectra of the encoded proteins. The major classes are Lepidoptera s p e c i f i c (I); Lepidoptera and Diptera s p e c i f i c (II); Coleoptera s p e c i f i c ( I I I ) ; and Diptera s p e c i f i c (IV). The Lepidoptera s p e c i f i c c r y s t a l proteins are the best characterized. The 20 different cry I sequences i d e n t i f i e d , can be divided into six different genes. These are crvIA(a). crvIA(b). crvIA(c). crvIB. crvIC. crvID (22). These have 1176, 1155, 1178, 1207, 1189, and 1165 amino acids, respectively. A l l 20 genes encode 130 to 140 kDA proteins which are accumulated i n bipyramidal c r y s t a l l i n e Inclusions during sporulation of B. thuringiensis. These proteins are protoxins and are s o l u b i l i z e d i n the a l k a l i n e environment of the insect midgut and are p r o t e o l y t i c a l l y converted by c r y s t a l associated or l a r v a l midgut proteases into toxic core fragments of 60 to 70 kDa. Strains of B. thuringiensis produce several different c r y s t a l proteins simultaneously and the same or very s i m i l a r c r y s t a l proteins occur i n strains of different subspecies (22). Most of the genes are located on large conjugative plasmids, thus, mobility of c r y s t a l protein genes among subspecies i s expected. This mobility property has been exploited to develop strains of B. thuringiensis with desired genes from two or more s t r a i n s . Genes encoding for i n s e c t i c i d a l proteins for Lepidoptera have been genetically engineered into transgenic cotton plants. The wild type genes are expressed poorly i n plants (21). Therefore, various modifications of the genes are needed to increase expression and increase a c t i v i t y toward Lepidoptera insects. For example, plants with a p a r t i a l l y modified crvIA(b) gene had a 10-fold higher l e v e l of insect control protein than wild plants, and plants with a f u l l y modified crylA(b) had a 100-fold higher l e v e l of the protein (23). Similar results were obtained i n plants with the f u l l y modified crvIA(c) gene. P a r t i a l l y modifying the gene involved s e l e c t i v e l y removing DNA sequences predicted to i n h i b i t e f f i c i e n t plant gene expression without changing the amino acid sequence. F u l l y modifying the gene involved wholesale changes in DNA which required the use of a f u l l y modified synthetic gene (23). This increased gene expression was generic across several plant species--tomato, cotton, and tobacco. These r e s u l t s show that i t i s quite possible to modify wild type genes i n a manner that i s favorable for insect c o n t r o l .

Mode of Action of B. thuringiensis Insect Control Proteins The general c h a r a c t e r i s t i c s exhibited by insects upon ingestion of the B. thuringiensis toxin are: 1) cessation of feeding within one hour, 2) reduced a c t i v i t y within 2 hours, and 3) progressive sluggishness and paralysis within 6 hours. The e p i t h e l i a l c e l l s swell with disrupted m i c r o v i l l i , c e l l l y s i s , and c e l l sloughing occur, and the insects die as a combined r e s u l t of starvation and septisemia. Our experience with transgenic cotton plants with a modified B. thuringiensis gene indicates that neonate tobacco



19. JENKINS


273

budworm larvae feed on transgenic tissue for only a few bites and then stop. They then become lethargic and die within 3 to 6 days. The delta endotoxins are synthesized as large protein molecules and c r y s t a l i z e d as parasporal inclusions. When susceptible insects ingest the c r y s t a l s , they are dissolved i n the midgut of the insect and protoxins are released that are about 140 kDa i n size for the c r v l proteins. They may be further processed by the insect to smaller toxic molecules. The toxic protein molecule then binds with s p e c i f i c a f f i n i t y to receptors i n the midgut e p i t h e l i a l c e l l s , where pores or ion channels develop i n c e l l membranes. This disturbs c e l l u l a r osmotic balance and causes c e l l s to swell and lyse. These effects cause paralysis of the insect midgut and mandibles, and thus, death occurs through a combination of starvation and septicemia. The u l t r a s t r u e t u r a l changes and time course of poisoning varies between insect species and the various c r v l toxins. Limitations to Use of B. thuringiensis Genes It i s well known that pest resistance to conventional insecticides i s widespread. We should expect that genetically engineered plants or biopesticides w i l l also be susceptible to the same problems. Conventional forms of B. thuringiensis pesticides have been used for over 20 years without widespread development of resistance; however, i n the past few years several instances of resistance have been reported. A tobacco budworm s t r a i n has been selected i n the laboratory to be 13X to 20X more resistant to diet incorporated Pseudomonas fluorescens which had been genetically engineered to express the 130kDa protein from the HD-1 s t r a i n of B. thuringiensis (24). These same insects were 4X less susceptible to p u r i f i e d HD-1 endotoxin and to Dipel , which is a commercial formulation of a crystal-spore mixture of B. thuringiensis var k u r s t a k i . Plodia i n t e r p u n c t e l l a . the Indian meal moth, a pest of stored g r a i n , developed resistance to a commercial formulation of B. thuringiensis when stored grain was treated with B. thuringiensis for a few insect generations (25). Resistance increased nearly 30-fold i n two generations in a s t r a i n of this insect reared on diet treated with B. thuringiensis and after 15 generations resistance reached a plateau 100 times higher than the control l e v e l (25). From these reports, we should expect that insect pests of cotton have the genetic c a p a b i l i t y to develop r e s i s t a n t biotypes v i a selection by transgenic c u l t i v a r s . We should at least recognize that the p o s s i b i l i t y for resistance development exists and that the potential for resistance varies with the insect and the c u l t u r a l practices for the crop. This should motivate researchers to develop means of dealing with the p o t e n t i a l for resistance. Mechanisms of Resistance to B. thuringiensis Studies on a s t r a i n of Indian meal moth selected i n the laboratory for resistance to a B. thuringiensis i n s e c t i c i d a l c r y s t a l protein i n D i p e l , provide evidence that resistance i n t h i s insect i s due to an a l t e r a t i o n i n binding of the toxin to c e l l membranes (26). This research provides insight into how the B. thuringiensis protein i s



274


associated with t o x i c i t y i n the insect. Two d i s t i n c t changes have apparently occurred i n the resistant s t r a i n of Indian meal moth. The f i r s t change i s that t h i s s t r a i n i s resistant to insect control protein of the CrvIA(b) type, but there i s no resistance to CrvIC protoxin or CrvIC toxin. This (CrvIC) insect control protein i s not present i n c r y s t a l s of Dipel; whereas, the insect control proteins of CrvIA(b) type are present i n Dipel which was used to select the r e s i s t a n t insects. The second change that has occurred i s a marked increased s e n s i t i v i t y to CrvIC protoxin and CrvIC toxin i n the resistant s t r a i n (26). Van Rie et a l . (26) suggest that when two insect control proteins are available for the same insect, resistance to one insect control protein does not necessarily confer resistance to the second insect control protein; therefore, insect control proteins with different binding properties could be used to delay development of resistance. Research on the molecular mechanisms involved i n resistance i s expanding r a p i d l y and better ways to manage resistance should emerge from these studies. The Indian meal moth population studied by Van Rie et a l . (26) was selected for resistance i n the laboratory. Insect strains selected for resistance i n the laboratory may or may not have the i d e n t i c a l genes for resistance as those selected under f i e l d conditions. In Hawaii, the Diamondback moth, P l u t e l l a x v l o s t e l l a ( L . ) , i s resistant to the i n s e c t i c i d a l spore-crystal protein complex of B. thuringiensis (27). This resistance was developed i n response to commercial f o l i a r applications of B. thuringiensis. In the P h i l l i p i n e s a s t r a i n of diamondback moth r e s i s t a n t to commercial formulations of Dipel was established from pupae (28). Crystal proteins, from recombinant genes CrvIA(b). CrvIB. and CrylC. were obtained as proteins expressed i n Escherichia c o l i and the proteins were evaluated for binding to the brush borders of membrane v e s i c l e s (BBMVs) of the resistant and a susceptible laboratory s t r a i n . A l l three proteins bound to the BBMVs from the susceptible laboratory s t r a i n ; however, CrvIA(b) protein did not bind to the BBMVs from the resistant s t r a i n . These r e s u l t s indicate that the susceptible laboratory s t r a i n has s p e c i f i c receptors for CrvIA(b). CrvIB. and CrylC, whereas i n the r e s i s t a n t s t r a i n , receptors were only detected for CrvIB and CrvIC (28)• Resistance i n the diamondback moth and the Indian meal moth each involved membrane receptor mechanisms, even though one was selected for resistance under f i e l d conditions and one under laboratory conditions. Thus, membrane receptor mechanisms are l i k e l y to be one of the mechanisms involved i n resistance of other insects to B. thuringiensis. Management Strategies f o r B. thuringiensis Transgenic Cotton Various strategies have been proposed for management of B. thuringiensis genes used i n c u l t i v a r s of crops to delay or prevent the development or selection of strains of insects that are no longer susceptible. The strategies must consider that the toxins from B. thuringiensis w i l l be used i n the form of transgenic plants as well as i n the form of commercial applications of spray £ . thuringiensis products. The f i r s t commercial transgenic crop to use modified B. thuringiensis genes i n the plant for control of insects w i l l probably be cotton. Commercialization w i l l probably



19. JENKINS


275

occur i n the mid 1990's. I n d u s t r i a l companies with transgenic cotton germplasm are developing corporate arrangements with established cotton seed breeding companies that w i l l provide growers with transgenic c u l t i v a r s of cotton with selected proprietary £ . thuringiensis genes. Promising results have been obtained from f i e l d tests conducted for two years with transgenic cotton strains for control of Lepidopterous insects. Resistance management i n t h i s crop w i l l employ several techniques not yet f u l l y developed. Several conferences involving Industry, Government, and Academic s c i e n t i s t s have considered strategies for management of B. thuringiensis genes; however, no strategies have been agreed upon by a l l concerned p a r t i e s . As one possible means to manage the development of resistance, a system has been devised for temporally c o n t r o l l i n g the expression of the endotoxin. This has been accomplished i n tobacco. Plants containing a toxin gene driven by a chemically-responsive promoter were developed. When treated with a chemical regulator, chemical induction resulted i n accumulation of toxin mRNA which caused the plants to become insect tolerant (29). This a b i l i t y to control when the toxin gene i s expressed, should be of value i n resistance management. A general scenario for resistance management i n cotton could be as follows: A. Short Term (at commercialization): 1. High dose expression of the gene i n cotton plants to control insects heterozygous for resistance a l l e l e s . 2. Refugia as hosts for sensitive insects. 3. Crop management practices that minimize insect exposure to the gene. 4. The use of integrated pest management t a c t i c s . 5. Monitoring of insect populations for s u s c e p t i b i l i t y to the gene or genes. B. Medium Term (implemented 2 to 5 years after commercialization): 1. Continue a l l short term strategies plus: 2. Combine two B. thuringiensis genes into the same p l a n t , each of which produce toxins that are active on target insects but with different s i t e s of a c t i o n . C. Long Term (more than 5 years after commercialization): 1. Continue a l l short and medium term strategies plus: 2. Incorporate natural cotton host plant resistance genes into the transgenic cotton c u l t i v a r s as the natural cotton genes are proven e f f e c t i v e . 3. Incorporate non B. thuringiensis proteins to provide effective control of Lepidopterous pests. There i s much planning and research effort involved i n developing strategies to successfully u t i l i z e transgenic cotton plants. Similar efforts are under way with other crops being considered for transgenic approaches to pest c o n t r o l . Recent research i s providing new prospects for use of various B. thuringiensis products and a wide range of possible uses for t h i s b i o l o g i c a l pesticide i n forms ranging from transgenic plants to newer types of B. thuringiensis spray products with widened


276


ranges of potencies (15). A l i s t of 18 major companies involved with research on various B. thuringiensis products i s shown. Five of the companies are involved with B. thuringiensis i n plants. Of the 53 U.S. patents on B. thuringiensis granted i n the l a s t 21 years, 39 have been issued i n the l a s t 4 years (15). These newer products may also be useful i n managing resistance to B. thuringiensis endotoxins, including transgenic plants.


Results of Experiments with B. thuringiensis Genes i n Cotton Insect resistant transgenic cotton plants containing B. thuringiensis genes genetically engineered into cotton have been reported (30). F i e l d and laboratory experiments have been conducted to evaluate the effectiveness of several of these transgenic lines of cotton for control of Lepidopterous insects (19, 31-37). Cotton i s a crop i n the U.S. that does not cross p o l l i n a t e with any wild or c u l t i v a t e d plants other than cotton and no wild cottons occurred where these experiments were conducted. Thus, there was no chance for escape of the g e n e t i c a l l y engineered gene to other species v i a p o l l e n . A b i o l o g i c a l sink (buffer) of 24 rows of non-transgenic cotton was grown on a l l sides of the p l o t s . This provided a place for the insects to deposit p o l l e n when they v i s i t e d flowers of the transgenic plants. Seed cotton from the 24 rows of cotton were destroyed. In an experiment i n M i s s i s s i p p i to determine the efficacy of the 24 row border as a p o l l e n sink, we found a s i g n i f i c a n t reduction i n pollen dissemination as distance from the test plot increased. Outcrossing went from 5% to < 1% by 7 m away from the test p l o t . A low l e v e l of pollen d i s p e r s a l of < 1% continued to occur sporadically i n the remaining border rows out to a distance of 25 m. The border rows f u l f i l l e d t h e i r purpose of serving as a pollen sink to s i g n i f i c a n t l y reduce the amount of pollen dissemination from the test plot (38)• Cotton i s normally s e l f p o l l i n a t e d and cross p o l l i n a t e d only by insects. Wind i s not a factor i n cross p o l l i n a t i o n as the cotton pollen i s heavy and not wind-borne. These features provided for a r e l a t i v e l y safe experiment with transgenic cotton i n f i e l d p l o t s . For the two years of f i e l d tests that followed t h i s experiment ( 3 8 ) » regulatory agencies have accepted a 24 row border as s u f f i c i e n t p o l l e n containment for the B. thuringiensis gene for f i e l d experiments with transgenic cotton. A major economic factor i n cotton production i s the cost of insect c o n t r o l . Insects i n the order Lepidoptera, which i n the l a r v a l stage are worms or c a t e r p i l l a r s , are major pests of cotton. In the mid-south about $123.00 US per ha, per year are spent on insect c o n t r o l . Most of this expense i s for control of larva of Lepidoptera. The gene from B. thuringiensis was genetically engineered into the Coker 312 c u l t i v a r of cotton v i a A. tumefaceins technology. The gene expressed i n a l l cotton tissue and was measured i n terminal leaves at a l e v e l of 0.05% to 0.10% of the t o t a l soluble protein (30). Six cotton l i n e s , each with a different i n s e r t i o n of an insect control protein gene from B. thuringiensis. were evaluated i n my laboratory (19). Each time a gene i s engineered into cotton the placement of the gene among the cotton chromosomes i s uncontrolled, but we do not know that i t i s random. Further,



19. JENKINS


111

soma clonal v a r i a t i o n and mutation can occur i n tissue c u l t u r e . Thus, each l i n e i s the r e s u l t of a separate gene i n s e r t i o n and tissue culture processes. Because of t h i s , each transformation event may be expressed d i f f e r e n t l y . This may r e s u l t i n differences i n the l e v e l of insect control protein and insect c o n t r o l . However, each transgenic l i n e and the non-transformed Coker 312 should be considered as a pair of "near isogenic" l i n e s for comparison purposes. The f i e l d experiment conducted by my laboratory (19) involved 6 r e p l i c a t i o n s of 4 row plots each 9 m long separated by 1 m. The four rows were separated by 2 m blank rows. Plots were infested with 10-12 neonate tobacco budworm, H e l i o t h i s virescens. larvae once each week for 5 weeks beginning the second week of squaring. No i n s e c t i c i d e was used for tobacco budworm c o n t r o l ; however, malathion for b o l l weevil, Anthonomus grandis Boheman, and plant bug, Lvgus spp., control was used. An i d e n t i c a l set of plots were planted and not infested with tobacco budworm larvae. These plots were sprayed each week with a pyrethroid i n s e c t i c i d e plus malathion to control a l l major insects. The difference i n y i e l d between the two sets of plots should r e f l e c t the amount of y i e l d l o s t to the tobacco budworm insects and i s an i n d i r e c t measure of resistance to tobacco budworm provided by the genetically engineered insect control protein gene. A second measure of control provided by the B. thuringiensis gene was damaged squares or b o l l s caused by the weekly infestations of tobacco budworm. Results of these f i e l d experiments show that the g e n e t i c a l l y engineered cotton lines were successful i n c o n t r o l l i n g tobacco budworm insects and preventing excessive damage to the cotton plants (19). Transgenic lines never had over 8% damaged squares, whereas the non-transgenic s t r a i n had up to 22% damaged squares. Transgenic strains never had over 5 larvae per 100 squares compared to up to 19 larvae per 100 squares i n Coker 312. The non-transgenic Coker 312 had up to 20% of the b o l l s damaged by tobacco budworm whereas b o l l damage ranged from 2 to 12% i n the transgenic s t r a i n s . Larvae i n b o l l s ranged from 0 to 7.8 per 100 b o l l s i n transgenic s t r a i n s , but up to 22 per 100 b o l l s i n the non-transgenic strains (19). In the plots without pest insects, each of the transgenic strains yielded equal to or s i g n i f i c a n t l y more l i n t than Coker 312. This shows that the foreign gene i n these cotton l i n e s was not detrimental to y i e l d . When grown under tobacco budworm i n f e s t a t i o n , each transgenic s t r a i n yielded s i g n i f i c a n t l y more than Coker 312. This shows that the B. thuringiensis gene i n the transgenic plants was providing a s i g n i f i c a n t and useful l e v e l of protection from damage by tobacco budworm (19). Tobacco budworm larvae were grown i n the laboratory on several plant parts each week beginning at seedling emergence and continuing u n t i l the end of the effective squaring period. The delta endotoxin was expressed i n each plant part (.39) . When neonate tobacco budworm larvae were placed on anther and stigma tissue for 6 days the following results were obtained: Larval s u r v i v a l (mean of transgenics 1.7% + 0.7:Coker 312 62.5% + 7.5); Larval biomass (mean of transgenics 0.2 mg ± 0.2:Coker 312 117 mg ± 14.6) (39). Thus, the small amount of l a r v a l s u r v i v a l on the transgenics was not b i o l o g i c a l l y s i g n i f i c a n t because the larvae



278


were very small and would not survive to pupation. Detailed data from the 1991 experiments cannot be c i t e d due to c o n f i d e n t i a l i t y agreements between my laboratory and the company that provided the transgenic l i n e s . However, the 1991 data f u l l y support the above c i t e d data, and the transgenic cotton l i n e s provided effective f i e l d control of tobacco budworm. Thus, we have shown that the gene from B. thuringiensis which encodes for a delta endotoxin can be genetically engineered into cotton and that the gene product (delta endotoxin) i s expressed i n a form that i s effective i n protecting transgenic cotton plants from damage by tobacco budworm. This represents an environmentally desirable way to control tobacco budworm i n cotton. Although the transgenic l i n e s yielded as much or more than Goker 312 when the insects were c o n t r o l l e d , we measured differences i n some other t r a i t s . Bolls on 4 of the 6 transgenic l i n e s were s i g n i f i c a n t l y smaller than Coker 312. Weight of 100 seed was less on two transgenic lines than on Coker 312 (19). In other research with f i v e of these transgenic l i n e s , each transgenic l i n e had lower l i n t percentages and three had smaller b o l l s than Coker 312 (36)* Effects on f i b e r properties have also been reported (33). At t h i s time we cannot determine i f these differences are due to p l e i t r o p h i c effects of B. thuringiensis genes, p o s i t i o n e f f e c t s , or some (somaclonal) changes that occurred during tissue culture. Other research has shown that these cotton l i n e s also control cotton bollworm, Helicoverpa zea (32-35): pink bollworm, Pectinophora gossvplella: cotton leaf perforator, Bucculatrix t h u r b e r i e l l a Busck; and saltmarsh c a t e r p i l l a r , Extigmene acrea (Drury) (36, 37); and tobacco budworm and cabbage looper, T r i c o p l u s i a n i (33); i n f i e l d p l o t s . These are the major Lepidopterous pests of cotton i n the United States and are among the major worm pests of cotton worldwide. Transgenic cotton with the gene from B. thuringiensis should be available to growers i n the mid 1990 s. These cotton c u l t i v a r s w i l l be marketed through the conventional channels of the existing cotton seed breeding companies. An Experimental Use Permit from the Environmental Protection Agency has been received for 1992 by at least one U. S. company involved i n t h i s research. Approval w i l l be required from APHIS and/or EPA before the cotton c u l t i v a r s can be placed into commercial production. Companies are presently c o l l e c t i n g the research data to request granting of t h i s status by the mid 1990's. Present indications are that cotton w i l l be the f i r s t f i e l d crop to be grown commercially with a gene from B. thuringiensis that encodes for the delta endotoxin expressed at a l e v e l to provide effective control of several major Lepidopterous pests. r

Acknowledgments Transgenic cotton lines used i n the experiments i n M i s s i s s i p p i described under the heading "Results of Experiments with B a c i l l u s thuringiensis Genes i n Cotton" were developed by The A g r i c u l t u r a l Group of Monsanto Company. This research was a cooperative e f f o r t between USDA, ARS, and Monsanto Company under Cooperative Research and Development Agreement No. 58-32U4-0-97.


19. JENKINS Bacillus thuringiensis Genes in Transgenic Cotton

279

Literature Cited 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Khush, G. S.; Brar, D. S. Adv. Agron. 1991, 45, 223-274. Hatchett, J. H.; Starks, K. J.; Webster, J. A. In Wheat and Wheat Improvement; Heyne, E. G., Ed.; ASA, CSSA, SSSA: Madison, WI, 1987; pp. 625-675. Khush, G. S. Ann. NY Acad. Sci. 1977a, 228, 296-308. Khush, G. S. Adv. Agron. 1977b, 29, 265-341. Wharton T.; Jenkins, J. N.; McCarty, J . C., Jr.; Parrott, W. L. Crop Sci. 1991, 31, 29-32. Fraley, R. T. Bio/Technologv 1992, 10, 40-43. Klein, T. M.; Arentzen, R.; Lewis, P. A.; FitzpatrickMcElligott, S. Bio/Technologv 1992, 10, 286-291. Finer, J. J. Plant Cell Reports 1988, 7, 399-402. Finer, J. J.; McMullen, M. D. Plant Cell Reports 1988, 8, 586-589. Stewart, J. McD. CAB International, International Cotton Advisory Committee 1991, 49 pages. Gasser, C. S.; Fraley, R. T. Science 1989, 244, 1293-1299. della Chioppa, G.; Bauer, S. C.; Taylor, M. C.; Rochester, D. E.; Klein, B. K.; Shay, D. M.; Fraley, R. T.; Kishore, G. M. Bio/Technology. 1987, 5, 579-584. Smith, C. J . S.; Watson, C. F.; Ray, J.; Bird, C. R.; Morris, P. C.; Schuch, W.; Grierson, D. Nature 1988, 334, 724-726. Edwards, D. L . ; Payne, J.; Soares, G. G. U. S. Patent 4,948,734. 1990. Feitelson, J. S.; Payne, J.; Kim, L. Bio/Technology 1992, 10, 271-275. Barton, K. A.; Whiteley, H. R.; Yang, Ning-Sun. Plant Physiol. 1987, 85, 1103-1109. Umbeck, P.; Johnson, G.; Barton, K.; Swain, W. Bio/Technology 1987, 3, 263-266. Jenkins, J . N.; Parrott, W. L . ; McCarty, J. C.; Barton, K. A.; Umbeck, P. F. Miss. Agric. Forestry Expt. Stn. Bull. 1991, 174, 6 pp. Jenkins, J . N.; Parrott, W. L . ; McCarty, J. C. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 576. Hilder, V. A.; Gatehouse, A. M. R.; Sheerman, S. E . ; Barker, R. F.; Boulter, D. Nature 1987, 330, 160-163. Gatehouse, A. M. R.; Gatehouse, J. A.; Boulter, D. Phytochemistry 1980, 751-756. Hofte, H.; Whiteley, H. R. Microbiol. Rev 1989, 88, 242-255. Perlak, F. J., Fuchs, R. L . ; Dean, D. A.; McPherson, S. L.; Fischoff, D. A. Proc. Natl. Acad. Sci.(USA)1991, 88, 3324-3328. Stone, T. B.; Sims, S. R.; Marrone, P. G. J. Invertebr. Pathol. 1989, 53, 228-234. McGaughey, W. H. Science 1985, 229, 193-195. Van Rie, J.; McGaughey, W. H.; Johnson, D. E . ; Barnett, B. D.; Van Mellaert, H. Science 1990, 247, 72-74. Tabashnik, B. E.; Cushing, N. L . ; Finson, N.; Johnson, M. W. J. Econ. Entomol. 1990, 83, 1671-1676.



280


28. Ferre, J.; Real, M. D.; Van Rie, J.; Jansens, S.; Peferoen, M. Proc. Natl. Acad. Sci. (USA) 1991, 88, 5119-5123. 29. Williams, S.; Friedrich, L . ; Dincher, S.; Carozzi, N.; Kessmann, H.; Ward, E . ; Ryals, J. Bio/Technology 1992, 10, 541-543. 30. Perlak, F. J.; Deaton, R. W.; Armstrong, T. A.; Fuchs, R. L . ; Sims, S. R.; Greenplate, J. T.; Fischoff, D. A. Bio/Technology 1990, 8, 939-943. 31. Deaton, R. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 576. 32. Williamson, D. R.; Deaton, W. R. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 577. 33. Benedict, J . H.; Altman, D. W.; Sachs, E. S.; Deaton, W. R.; Ring, D. R. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 577. 34. Micinski, S.; Caldwell, W. D. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 578. 35. Gannaway, J. R.; Rummell, D. R.; Owen, D. F. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 578. 36. Wilson, D. F.; Flint, H. M.. In Proceedings Beltwide Cotton Conferences; Herber, D. J., Ed.; National Cotton Council: Memphis, TN, 1991; p. 579. 37. Wilson, F. D.; Flint, H. M.; Deaton, W. R.; Fischoff, D.; Armstrong, T. A.; Fuchs, R. L . ; Berberich, S. A.; Parks, N. J.; Stapp, B. R. J. Econ. Entomol. 1992, In press. 38. Umbeck, P. F.; Barton, K. A.; Nordheim, E. V.; McCarty, J . C., Jr.; Parrott, W. L . ; Jenkins, J. N. J. Econ. Entomol. 1991, 84, 1943-1950. 39. Jenkins, J. N.; Parrott, W. L . ; McCarty, J . C.; Callahan, F. E.; Berberich, S. A.; Deaton, W. R. J. Econ. Entomol. 1992, In press. RECEIVED July 31, 1992


Pest Control with Enhanced Environmental Safety - American

Recommend Documents