BASIC ORGANISM MODULE/GENERAL/PLANTS RICE EXAMPLES SOURCE #1 test organisms Oryza sativa L. (RICE) /end donor organisms transposable element Activator from maize (Zea mays) /end Vectors Vector Agent: Agrobacterium tumefaciens Vector: Disarmed Ti plasmid /end other genetic sequences Two other genes, besides the transposable element Activator, are incorporated into chromosomal DNA after transformation. The first. encoding the enzyme, hygromycin B phosphotransferase (hphB), detoxifies the aminocyclitol antibiotic hygromycin B by phosphorylating the antibiotic (Gritz and Davies, 1983: Kaster et al., 1984). The second marker gene, neomycin phosphotransferase (NPT II), confers resistance to the common aminoglycoside antibiotic, kanamycin, by phosphorylating the molecule and thereby inactivating it (Fraley et al., 1986). Both genes were isolated from Escherichia coli. Neither the marker genes nor the resultant enzymes have any plant pest characteristics. There is no evidence that these genes can be transferred to other plants during the field test. /end location The field test will be conducted on a research plot of agricultural land owned by (institution name). It is located on a secondary road in (country}, (state), (county). This farm is 0.75 miles from the nearest highway (name), 2.25 miles from (city), the nearest population center, and 2.0 miles from the nearest commercially grown rice. Summary The recipient organism is rice, O. sativa L. which has been modified to contain the transposable element Activator from maize (Zea mays). The gene was inserted into the plant genome by a chemical method. The introduction of the transposable element Activator into rice is intended to studying developmental and mutational processes in rice. /end Purpose The purpose of this test is to evaluate the performance under field conditions of the selfed progeny of transgenic rice plants. /end reproductive cycle Rice is an annual (sometimes perennial in the tropics) erect grass, 50-150 cm tall. Culms cylindrical, smooth, 6-10 mm diameter, with solid nodes and hollow internodes, buds in axils of lower leaves produce tillers. Leaves alternate, two-ranked, made of sheath and lamina, and bearing a ligule and auricles. Inflorescence a terminal panicle, 14-42 cm long, each with (50)-100-(500) spikelets, erect or drooping, base of panicle enclosed in modified leaf (flag). Spikelets usually borne singly, laterally compressed, on a short pedicel, and with two glumes and a palea and an awned lemma; stamens six, anthers versatile; gynoecium monocarpellate, with single ovule, styles two, with plumose stigmas. Fruit a caryopsis, retained in palea and lemma; grain white to translucent, sometimes red, brown or black (Purseglove, 1988). The spikelets begin to open on the day of panicle emergence, or the day after. Blooming continues in sequential fashion and is completed in six to ten days. Weather, photoperiod, and cultural conditions may influence anthesis. Anthesis is generally in the morning. Pollen is shed about the time of spikelet opening. It remains viable from five minutes to about 50 hours. Pollen tubes emerge about three minutes after deposition on a receptive stigma. Fertilization occurs about 12 hours thereafter (Adair and Jodon, 1973). Because of the physical proximity in the same spikelet of fertile stamens and receptive stigmas, most rice is self- pollinated, but small and varying amounts of cross- pollination by wind do occur. This percentage is varies from 0-4.5 percent, rarely as much as 30 percent, with an average of 0.45 percent; most cross-pollination occurs within two meters (Grist, 1975; Purseglove, 1988). Because of this constant inbreeding, rice maintains true-breeding homozygous lines. Certified Seed Regulations, 7 CFR 201.76, require an isolation distance of ten feet. Additional distance is required for aerial seeding or ground broadcast seeding. /end DNA sequence The donor organism and the vector agent were developed by the (institution name and address). /end DNA insertion The primary plasmids used in rice transformation were pTRA131/132 (see Figure X, page XX). It is composed of sequences derived from plasmid pUC12, which allows its replication in E. coli, and a plant-expressible chimeric gene composed of the 35S CaMV promoter (for plasmid pTRA132) or nopaline synthase promoter (for plasmid pTRA131) and hygromycin phosphotransferase. When the chimeric gene is introduced into nucleus and expressed, resistance to hygromycin is expressed constitutively in the plants. Expression of this resistance gene allows the selection of transformed cells from their nontransformed counterparts. The second plasmid, pTRA137 or 137R (see Figure X), has the transposable element Activator, inserted in plasmid pTRA132 between the promoter sequences and the hygromycin phosphotransferase gene. This insertion results in inactivation of the resistance gene. However, if the transposon Activator excises from the recombinant gene and inserts itself at another site in the genome, the functional resistance marker genes is restored. Plasmid pTRA137R differs from pTRA137 in that the transposon is inserted in the reverse orientation. The orientation of insertion of the transposable element Activator has apparently minimal effect on the frequency of excision. The third plasmid (see Figure X), pTRA139R, has the NPT II gene (with bacterial regulatory sequences) inserted upstream from the transcription initiation site of the transposase gene but downstream from the inverted terminal repeat of plasmid pTRA137R. Presumably, insertion of the NPT II gene interferes with excision of the transposon. Plants containing this construct were made for use as experimental controls. Each of the recombinant plasmids was introduced into rice plants by polyethylene glycol treatment of protoplasts. After treatment, protoplasts were allowed to divide and placed under hygromycin selection. After callus formation, mature plants were regenerated. /end amount and nature Southern hybridization analysis of genomic DNA from the transgenic rice plants indicated that one to ten copies of the hygromycin resistance genes were present. The hygromycin resistance trait was transferred from transgenic rice to the progeny in a Mendelian pattern. (Inheritance was analyzed by germinating and growing seeds in the presence of hygromycin for 10 days). Of 27 plants examined, 7 plants showed at segregation ratio of 3:1 suggesting that the resistance gene(s) is locate at one closely-linked chromosomal loci. Six of the plants revealed segregation ratios of this trait between 3:1 and 15:1, while 14 plants revealed segregation ratios of less than 3:1. The exact interpretation of the segregation ratio which do not support a single loci, await further analysis of the progeny of these plants. The intact Ac element in pTRA137 appeared to excise from the this construct with high frequency in transgenic rice protoplasts (frequency rate was up to 20%). Southern hybridization data on select plants showed that the excised Ac element reintegrated into the rice genome. /end containment procedures All research and procedures used in the production of the donor organism, recipient organism, vector and/or vector agent and the transgenic plants were done utilizing level BL2 containment according to approved guidelines. Research facilities were inspected and approved by Institutional, State and Federal authorities. /end viability of the pollen Pollen is shed about the time of spikelet opening. It remains viable from five minutes to about 50 hours. Pollen tubes emerge about three minutes after deposition on a receptive stigma. Fertilization occurs about 12 hours thereafter (Adair and Jodon, 1973). Oryza is a genus of about 18 species of the grass family (Gramineae or Poaceae). Two closely related, and perhaps even conspecific, species of the genus, O. glaberrima Steud. and O. sativa, are cultivated. On a worldwide basis, the cultivation of Oryza glaberrima, also known as African rice, is insignificant (Cobley and Steele, 1976). Related to Oryza are those members of the grass tribe Oryzeae, including the genera Leersia, Zizania, Zizianiopsis, Luziola, and Hydrochloa (Gould, 1968). There are no species of Oryza native to the United States. Oryza sativa is the only species cultivated in the United States. Other members of the Oryzeae occur in the United States, but they do not interbreed with Oryza. There are innumerable cultivated varieties within Oryza sativa. These cultivars can be roughly divided into three groups; japonica, indica, and bulu; and are distinguished by strong sterility barriers between them (Adair and Jodon, 1973). /end inserted gene The foreign gene(s) remains structurally stable through meiosis and is transmitted in the seed. The gene(s) is expressed as a dominant marker and is inherited in a Mendelian manner (De Block et al., 1984; Horsch et al., 1984). Of course, any DNA sequence in plant chromosomes bears some degree of instability. This is evidenced in nature and in plant breeding by gene amplification, by such phenomena as unequal crossovers or chromosomal disjunction, and transposon mediated instability. As fully integrated pieces of plant chromosomes, recombinant marker genes are subject to the same rules governing chromosomal rearrangements and gene stability as are other plant genes. Once integrated into plant chromosomes DNA, becomes no different than naturally occurring plant genes in terms of stability or any potential ability to persist in the environment outside of direct progeny of transformed plants. Therefore, the term "stable insertion" implies a degree of stability that is similar to naturally occurring plant genes. Any slight instability that could be demonstrated would not be a cause for real concern, except for the loss of the utility of the insertion giving expression to the desired trait. There is no indication that such an instability could in some way be deleterious to anything except the transformed plants themselves. Transposons, by their nature, are more unstable than other genes. However, this does not to imply that their movement in the chromosomal DNA is not regulated. McClintock reported a classic property of maize transposons was their ability to cycle between active (i.e., moving) and inactive states, changing both their timing and frequency of movement. Recent evidence suggests that Ac activity is regulated by the degree of methylation of its DNA sequence. Thus, the movement of Ac in maize genome is strictly regulated (Schwartz and Dennis, 1986). In nature, chromosomal genetic material can only be transferred to other sexually compatible plants by cross-pollination. This is also true for transposons. Recent molecular probing of tomato and tobacco genomes support this, maize transposon Ac has not been detected by molecular probes in tobacco or tomato, two plant species that Ac has been introduced by transformation techniques (Baker et al., 1986; Yoder et al., 1988). The recombinant marker genes and the transposons are transmitted through mitosis and meiosis as an inherent part of the plant genome. The integrated foreign DNA is now a new and novel locus. Stable incorporation of the genes into the plant genome can be further confirmed by the demonstration of standard Mendelian genetics for the inheritance of these traits. Rice does not possess any special weedy characteristics. Some kinds of Oryza, called red rice, are a problem in rice fields because they are carried with cultivated rice and lower its value and agronomically desirable characteristics, but this is a phenomenon peculiar to the cultivation of the crop and does not reflect on any general trend of weedy aggressiveness of red rice into other crops. Cultivated rice is occasionally adventive in the United States along the coast from Virginia to Florida and Texas (Hitchcock and Chase, 1951). /end good agronomic practices Pollen and/or plants and/or grain will be transported according to 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 (name, affiliation, address, phone number) to (city), (state). /end Description Example Seed shipping 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 18. (Shipping) Seed or propagation material will be shipped according to USDA/APHIS regulations. The seeds will be packaged as required in Title 7 CFR part 340.6(b) (52 FR 22892-22915, June 16 1987). /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 Pollinating insects are not of concern in the cultivation of rice. /end design of the experiment Field Test Design The total size of the field plot will be 50 feet wide by 120 feet long. Plants will be spaced one foot apart in 100 foot long rows. Each row will be separated by 4 foot. The total number of transgenic plants to be introduced will be not exceed 835. The specific constructs used in the transformations and the exact numbers of each type to be introduced are as follows: pTRA131 (100 plants), pTRA132 (100 plants), pTRA137 (250 plants), pTRA137R (375 plants), and pTRA139R (10 plants). Equal numbers (200 of each) of nonengineered control plants consisting of seed-derived and protoplast-derived Nipponbare rice plants will be planted as control plants. Transgenic plants will be separated from nonengineered control by at least 2 meters. Dissemination of pollen will be prevented by placing two plastic bags over the growing panicles, starting at one week before flowering until two weeks after flowering. To prevent dissemination of seed by insects or birds, insect nets will be placed over and around the transgenic plants. Recovery of mature seeds from the plants will be facilitated by placing seed bags over each panicle and enclosing the bottoms of the bags with string from the second until the eighth week. Tentative schedule: - Field transplanting: Approximately June 1, 19XX - Experiment termination: Approximately October 15, 19XX The proposed field test will be conducted for a period of xxx days observation. Final Disposition of Test Plants After seed harvesting, the remaining plants will be sprayed with glyphosate. /end consequences Impact on Nontarget Organisms Exposure of Threatened and Endangered Organisms The plot will be surrounded by agricultural land which should reduce visitation by native animals. There are no threatened or endangered organisms in this parish (50 CFR 17.11 and 17.12). No factor unique to this field test has been identified that would have an effect on any plant or animal species. Alteration in Susceptibility to Plant Pathogens or Palatability to Insects 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 significantly different in the transformed and untransformed plants. The only physiological changes in the transformed plants are presumed to be the synthesis of up to three additional proteins, these are not expected to have any effect on plant disease organisms or insects. The random insertion of the transposon into a gene encoding plant pest resistance could affect the rice plants susceptibility to fungal, bacterial, or viral pathogens. If there were any changes in disease susceptibility, these effects should be confined to a few plants in the test plot. Impact on the Immediate Physical Environment Due to the nature of the transformed and control rice plants and the safeguards built into this field test, upon termination of this experiment no rice plant will survive to cause an effect on the physical environment. Impact on Human Health No rice will be available for human consumption. No potential impact on people living in the area of the field test, or any other human population, can be identified. The test has been designed with safety factors to minimize the possibility of adverse ecological effects. At the conclusion of the experiment, all of the plants will be killed, the field will be tilled, and then monitored during the subsequent season for any volunteer plants. Should unanticipated effects arise, the isolation of the test site and manner of conducting the test indicate that the effects can be readily contained and would have no permanent effect on the environment. /end monitored University personnel will be on site during working hours. The agronomic traits to be monitored are: plant height, tiller numbers, average panicle length, average spikelet numbers per panicle, and seed fertility. /end border rows The test area will be marked to monitor reemergence of volunteer rice plants the following season. The plot will not be planted the following season but will be plowed several times to destroy and any plant material. If any volunteer rice plants emerge in the marked test area, they will be removed by rouging or glyphosate application. We feel that such steps are sufficient to guarantee the termination of this experiment and prevent any unplanned releases. /end sprayed with disinfectant This would be an extraordinary precaution to prevent pollen or seed from escaping the area on tools or equipment. /end REFERENCES Adair, C. R., Jodon, N. E. 1973. Distribution and Origin of Species, Botany, and Genetics. pp. 6-21. In USDA. Rice in the United States: Varieties and Production. Agriculture Handbook No. 289. Agricultural Research Service, U. S. Department of Agriculture. Washington, D.C. 154 pp. Baker, B., Schell, J., Lorz, H., Federoff, N. 1986. Transposition of the maize controlling element "Activator" in tobacco. Proceedings of National Academy of Sciences (USA) 83:4844-4848. Cobley, L. S., Steele, W. M. 1976. An Introduction to the Botany of Tropical Crops. Second Edition. Longman, London and New York. 371 pp. Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffman, N. L., Woo, S. C. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences (USA) 80:4803-4807. Fraley, R. T., Roger, S. G., Horsch, R. B. 1986. Genetic transformation in higher plants. CRC Critical Reviews in Plant Science 4:1-46. Gould, F. W. 1968. Grass Systematics. McGraw Hill, New York et alibi. 382 pp. Grist, D. H. 1975. Rice. Fifth Edition. Longman, London and New York. 601 pp. Gritz. L., Davies, J. 1983. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisae. Gene 25:179-188. Hitchcock, A. S., Chase, A. 1951. Manual of the Grasses of the United States. U. S. Government Printing Office, Washington, D.C. 1051 pp. Kaster, K. R., Burgett, S. G., Inogolia, T. D. 1984. Hygromycin B resistance as dominant selectable marker in yeast. Current Genetics 8:353-358. Purseglove, J. W. 1988. Tropical Crops: Monocotyledons. Longman Scientific & Technical, Essex, England. 607 pp. Schwartz, D., Dennis, E. 1986. Transposase activity of the Ac controlling element in maize is regulated by its degree of methylation. Molecular and General Genetics 205:476-482. Yoder, J, I., Palys, J., Alpert, K., Lassner, M. 1988. Ac transposition in transgenic tomato plants. Molecular and General Genetics 213:291-296.