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              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.