PLANTS - WALNUT MODULE EXAMPLES Quantity of the regulated article: The plot will include 40 genotypes: Delta Franquette as a control (1 tree); Sunland-2 as a control (1 tree); Delta Franquette containing pCGN200 (4 trees); Sunland-2 containing pCGN7314 (8 trees); Sunland-2. /end test organisms Juglans regia (English Walnut) Juglans hindsii (Northern California Black Walnut) J. hindsii x J. regia hybrid (Paradox Walnut) /end donor organisms Bacillus thuringiensis /end Vectors Vector agent: Agrobacterium tumefaciens Vector: disarmed Ti plasmid /end other genetic sequences Enhanced CaMV 35S promoter Signal sequences for transcription termination and polyadenylation derived from 3''-termini of a variety of Ti plasmid derived genes. Gene encoding the enzyme APH(3'')II from transposon Tn5 (functions as a genetic marker in the initial cell selection process following transformation). GUS gene, from E. coli, encoding a hydrolase that catalyses the cleavage of beta-glucuronides. /end location The field trial will take place on a small plot (approximately 0.50 acre) on agricultural land in (county), (city), (state). There are no walnuts growing near the experimental plot. /end Summary Walnut trees have been modified to express Bacillus thuringiensis insecticidal crystal protein (ICP) (formerly delta-endotoxin). The gene encoding delta-endotoxin has been inserted into the walnut chromosome using a vector system that utilizes the ability of A. tumefaciens to transfer DNA to plant cells. The natural components of A. tumefaciens were modified so that it can no longer cause plant tumors or other disease symptoms on the trees. Adequate biosafety procedures, described in this application, will be followed to prevent escape of modified material thus preventing risk to the environment and/or human health. /end Purpose This limited field release will provide information necessary for the scientific evaluation of the efficacy of the inserted ICP gene against the larvae of certain lepidopteran pests of walnuts. The trees have been tested in the greenhouse to obtain initial data relating to genetic stability and preliminary efficacy data. Controlled field tests in the environment using standard agricultural practices are necessary to validate greenhouse results. /end reproductive cycle The walnut belongs to the amentiferous family Juglandaceae, genus Juglans (10-22 species). Genes may be transfered from walnut trees as vegetative material, pollen or seeds. Crosses of J. regia with other species of Juglans are common in nature and to plant breeders. J. hindsii, in particular, is receptive to pollen of other Juglans species. The only report of intergeneric crosses within the Juglandaceae is a J. regia cross with Petrocarya sp (McGranahan et al., 1986), which required embryo rescue under laboratory conditions. /end DNA sequence A disarmed binary plasmid is used to transform walnut embryos in vitro. The incorporation of the foreign DNA into the plant genome can be confirmed by Southern hybridization analysis. Five binary vector plasmids were used to transform walnuts. First, plasmid pGCN200 is a cointegrate of two previously described plasmids (Houck et al., 1988; Dandekar et al., 1987). This plasmid contains two functional chimeric APH(3'')II genes - one containing CaMV 35S promoter and the other octopine synthase promoter. The next two plasmids pCGN7001 and pCGN7314 were developed by XYZ, Inc. and are described in detail in Appendix xxx. Plasmid pGCN7001 contains two expressed chimeric genes. The first is APH(3'')II, whose production is driven by the CaMV 35S promoter, and the second is GUS whose production is under the direction of a mannopine synthase promoter. Plasmid pGCN73414 has the same two expressible genes as pCGN7001 except that GUS has enhanced CAMV 35S promoter and APH(3'')II under the direction of a mannopine synthase promoter. The final pair of related plasmids, pWB139 and WB149, are described in detail in Appendix XX. Plasmid WB139 contains a wound inducible gene that encodes a fusion protein containing the insecticidal crystal protein from B. thuringiensis strain HD-73 with the kanamycin resistance gene APH(3'')II. To obtain high expression levels of fusion protein, two promoters were used: CaMV 35S and nopaline synthase. Two termination/polyadenylation signal sequences in the fusion protein construct were also used from tomato protease inhibitor I gene and nopaline synthase. Plasmid pWB149 is identical to pWB139 except the former lacks the ICP gene. In addition to ICP, two marker genes (APH(3'')II and GUS) are incorporated into the chromosomal DNA after transformation. We have used the N-terminal region ICP (approximate size 68,000) and fused the truncated polypeptide to the coding region of the kanamycin resistance marker from Tn5. /end DNA insertion A disarmed Ti plasmid is a wild-type Ti plasmid whose tumor- inducing genes have been removed from the central core of the T-region and replaced with exogenous DNA. Removal of the tumor-inducing genes allows efficient DNA transfer and is essential for plant genetic engineering since these genes interfere with the regeneration of normal fertile transgenic plants (Joos et al., ...1980). Only 25 bp repeats on the Ti plasmid are required for insertion, therefore, the additional interstitial DNA sequences inserted between these 25 bp repeats are cotransferred and integrated into the plant nuclear genome (Hernalsteens ... 1980). The T-region appears to integrate randomly into either unique or highly repetitive DNA. /end amount and nature The disarmed binary plasmid system is used to transform walnut embryos in vitro. The incorporation of the foreign DNA into the plant genome can be confirmed by Southern hybridization analysis (Fraley ...1986). The foreign gene(s) remains structurally stable through meiosis and is transmitted in the seed. The gene(s) is inherited in a Mendelian manner (De Block...1984). Regenerated transformed plants are phenotypically normal and fertile. As fully integrated pieces of plant chromosomes, T-DNAs are subject to the same rules governing chromosomal rearrangements and gene stability as are other plant genes. The T-DNA is transmitted through mitosis and meiosis as an inherent part of the plant genome. The integrated foreign DNA is not a new and novel locus. /end containment procedures Seeds for planting or seedlings will be produced in greenhouses that are in compliance with "BL##-P Growth Conditions for Plants" (NIH Guidelines for Research involving recombinant DNA molecules). /end viability of the pollen When the trees are sexually mature with both female and male flowers (catkins), female flowers will be isolated from other sources of pollen in flower isolation bags. These bags are specifically designed to prevent pollen dissemination and developed specially for breeding purposes. They are made of polyester materials and obtained from commercial sources. The catkins will be harvested prior to anther dehiscence. Pollen will be collected in closed containers in the laboratory and transported in sealed syringes to the field site. Each syringe will contain pollen sufficient to pollinate one flower bag. Pollen will be injected into the bags and the hole taped. Each syringe will be used only once. After use each syringe will be placed in a sealed bag and transported to the laboratory for autoclaving. The isolation bags will remain on the flowers until they are no longer fertile and the pollen no longer viable (approximately 4 weeks). The bags will be autoclaved and may be reused. Isolation bags will be replaced with plastic net "onion" bags to allow the developing nuts to mature and to make sure no pollinated nuts escape. /end inserted gene Only the T-region from A. tumefaciens is transferred and integrated into the plant genome. The sequence that is integrated includes the genes which are contained between certain short, well-characterized segments of the Ti plasmid essential for incorporation into the plant genome. Border sequences (the 25 base pairs required for transfer) are lost during the process of insertion of T-DNA into the plant cell genome. Therefore, the inserted DNA is no longer a functional T-DNA capable of being transferred by the same mechanism that originally inserted the T-DNA into the plant genome. The vector does not survive in the plants. The vector agent, the bacterium used to deliver the vector DNA and the marker genes into the plant has shown to be eliminated and no longer associated with the transformed walnut plants. There is minimal risk of the inserted genes from this experiment surviving in these or other organisms in the environment beyond the termination of the experiment or becoming mixed with the gene pool of other populations outside of the experimental site, or with other nontarget plants. There is no evidence that the gene sequences, incorporated into the chromosomal DNA after transformation to serve as marker genes, can be transferred to other plants during the field test. /end good agronomic practices Pollen, plants, and seeds will be transported according to USDA/APHIS regulations in an adequately sealed container to prevent dissemination, i.e., in a lockable, refrigerated container for mail or carrier. /end shipment of the test organism Seed sent back to (institution) will be packaged in 2 heavy duty industrial weight burlap bags and then enclosed inside a woven polypropylene shipping bag. The seed will be hand carried and transported by (institution) personnel to (city), (state). /end Description Example Seed shippping container: Seeds will be sealed in plastic bags of at least 5 mil thickness, inside a sealed metal container, which will be placed inside a second sealed metal container. Shock absorbing cushioning material shall be placed between the inner and outer metal containers. Each set of metal containers shall then be enclosed in a corrugated cardboard box or other shipping container of equivalent strength. /end Shipping Seed will be shipped according to USDA/APHIS regulations inside a plastic bag in a sealable, sturdy container. /end moving a material Seeds obtained from the transgenic plants will be transported from (institution) to the designated field test site via common carrier. The return shipment of seed from (sending source) to (institution) will be hand carried. The (institution) personnel directly responsible for supervising the transportation will be: Name: Title: Institution: Street address: City, State Zip code: Telephone number: /end pollinating insects Pollen transfer will be measured throughout the experiment using marker genes and/or glandless walnut border rows. /end design of the experiment The plot will include 40 genotypes: Delta Franquette as a control (1 tree); Sunland-2 as a control (1 tree); Delta Franquette containing pCGN200 (4 trees); Sunland-2 containing pCGN7314 (8 trees); Sunland-2. Each genotype will be transplanted 10 feet apart. Rows will be 18 feet apart and 36 feet from any other planting. The approximate field plot size will be 0.50 acre. Sexually mature trees will be isolated, hand-pollinated, and kept in isolation until nuts are mature. Mature nuts will be brought to the laboratory for drying and subsequent germination in the greenhouse. Seedlings will be grown, in containment, for further analysis. During the field test, university scientists will monitor the field plots regularly. Trees will also be examined to ensure that they are receiving proper horticultural care. /end consequences The expression of the gene encoding an insecticidal crystal protein (ICP) from Bacillus thuringiensis does not provide the transformed walnut trees with any apparent selective advantage over nontransfromed walnuts in their ability to be disseminated or to become established in the environment. With the exception of ICP there has been no intentional change in these plants to affect their susceptibility to disease-causing organisms or palatability to insects, and there is no reason to believe that these characteristics are different in the transformed and untransformed plants. If there were any changes in disease susceptibility, the effects should be confined to these plants and the test plot. The only physiological changes in the transformed plants are the addition of the two marker enzymes and the ICP. These changes are not expected to have any effect on plant disease organisms or insects other than the target insects. /end monitored Example ##1: The two rDNA genes in the plant strains that will undergo field tests have been analyzed for expression levels by measurement of mRNA in northern hybridizations and for protein content using immunoblotting techniques. Example ##2: Presence of the desired modified trait will be monitored through insect feeding studies. /end border rows At the conclusion of the experiment, trees will be dug and roots pulled; both will be burned in an area near the test site. Soil will be fumigated and tilled according to normal nursery practices. Mature fruit will be removed, in appropriate containers, to the laboratory for analysis and further testing. Any plant material removed from the field will be autoclaved prior to its disposal. The site will be monitored for two growing seasons to ensure that no volunteer saplings appear. /end sprayed with disinfectant This would be an extraordinary precaution to prevent pollen or seed from escaping the area on tools or equipment. /end 26. Effect on environment example The insecticidal crystal protein is a polypeptide which, upon ingestion, kills only select lepidopteran insects. It is not toxic to other insects, wild or domestic birds, fish or mammals. Because of its safety, its topical application on vegetable crops is permitted up to harvest date. No potential impact on people living in the area of the field test, or any other human population, can be identified. None of the walnuts will be available for human consumption. /end