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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector

Definitions

• the invention herein described relates to a method for the transfer of exogenous genes in Angiosperms from a selected donor plant to a host plant.
• the method involves incubation of pollen from the parent plant with foreign DNA from the donor.
• the host plant is then pollinated with treated pollen and normal fertilization and development of seed occur.
• a self-pollination system is preferred.
• Transformed offspring generated from seed express genetic traits characteristic of the foreign DNA donor.
• Agrobacterium tumefaciens and the Ti plasmid holds promise, but this system is limited to dicotyledonous plants.
• A. tumefaciens does not infect monocotyledonous plants. This plant group includes grasses and cereals, and indeed most of the world's important food crops [Sci. Amer., 248(6) ;59, 1983].
• Tissue culture techniques are also being investigated. Many dicotyledonous plants can be quite easily regen- erated into intact plants from undifferentiated tis ⁇ sue-culture cells.
• the male gametophyte is a complex structure.
• the male gametophyte (pollen grain) of maize consists of a tube nucleus and a generative cell. Soon after germination the pollen tube protrudes from the pore of the pollen grain and the generative cell divides to produce two sperm.
• the pollen tube then enters the stigma, grows down the style, and enters the female gametophyte where it disposes of its contents into the cytoplasm of the embryosac [Pfahler, P.L. 1978. Biology of the male gametophyte. Iri D.B. Walden (ed.). Maize breeding and genetics. John Wiley and Sons, New York, pp. 517-530; Earle, E. 1982. Gametogenesis, fertilization and embryo development. I I H. Smith and D. Grierson (eds.). Molecular biology of plant development. Bot. Monogs. 1 : 285-305. Univ. Calif. Press, Berkeley; and Linskens, H.F. 1983.
• Mutations and transformations achieved through sexual transfer of exogenous DNA are phenotypically similar to expressions of known mutant loci. If actual gene transfer does take place, it is assumed that incorporation into the genome of the zygote will be at specific sites on one or more chromosomes (Rubin, G.M. , and A,C, Spradling. 1982. Genetic transformation with transposable vectors. Sci. 218: 348-353; Spradling, A.C., and G.M. Rubin. 1982. Transposition of cloned P elements into
• Transformed plants either segregate in a
• OMPI phenotypes and whether loci coding for these genet ⁇ ically mutated phenotypes are located on the expected chromosomes and expected positions on chromosomes arms.
• cultivated maize Zea mays
• arose through natural crossing perhaps first with gamagrass (Tripsacum dactyloides) .
• Hybrids with 36 Tripsacum (Tr) + 10 Zea (Zm) chromosomes are characterized mostly by 18 Tr bivalents and 10 Zm univalents during meiotic prophase [de Wet, J.M.J. and J.R. Harlan. 1974. Tripsacum - maize interaction: A novel cytogenetic system. Genetics 7_8. 493-502; de Wet, J.M.J. et al. 1982. Systematics of . Tripsacum dactyloides (Gramineae) . Amer. J. Bot. 69.* 1251-1257] .
• Tripsacoid maize genotypes so produced carry several traits new to the genome of maize, and are highly desirable in maize improvement (Bergquist, R.L. 1981.
• Diploid Tripsacum taxa produce functional female gametes that are haploid (18 chromosomes) or diploid (36 chromosomes) .
• the ⁇ ytologically non-reduced female gamete may function sexually or develop parthenogenetically to produce a functional embryo.
• Offspring from such crosses were therefore expected to have 18 Tr + 10 Zm, 36 Tr + 10 Zm, or 36 Tr + 0 Zm chromosomes, the last cytotype being maternal. These cytotypes were indeed produced, but some offspring with 36 Tr + 0 Zm chromosomes resembled true hybrids with 36 Tr + 10 Zm in phenotype.
• OMPI _ litter mates that were not genetically altered. Plant embryos were similarly transformed by Sayfer (1980, supra) and by Zhou et a].. (1983, supra) . Pollen may serve as a transfer vector of exogenous DNA (Hess, D. 1980. Investigations on the intra- and interspecific transfer of anthocyanin genes using pollen as vectors. Zeitschr. Dephysiol. Bd. 9J3: 321-337) .
• an object of this invention is to -provide a new and useful method for the transfer of foreign genes among flowering plants using the devel ⁇ oping male gametophyte as a transfer vector.
• a further object is to provide a male gametophyte system for the transfer of genes between maize cultivars.
• Yet another object is to provide a method for the inter-species transfer of genes between gamagr ss and maize using pollen as a vector.
• the male gametophyte of Angiosperms can effectively act as a transfer vector of exogenous genes.
• One of species selected as experimental material for gene transfer is maize (Zea mays) .
• Another experimental species is gamagrass (Tripsacum dactyloides) .
• the genetics of maize is fairly well understood; stocks of marker genes are available; and two genes have been cloned and are available for experimentation.
• pollen can be used as a transfer vector of foreign genes.
• the technique of the invention can be used with flowering plants (Angiosperms) for dicot-dicot or monocot-monocot genetic transfer. This genetic engineering techniques is so simple that it can be used in plant breeding with little refinement.
• the male gametophyte has two major advan ⁇ tages over the use of plasmids as transfer vectors.
• the most important advantage is efficiency. Germinating and incubating of pollen are readily achieved in the field, and self-pollination followed by selection are standard breeding tools for plant im ⁇ provement.
• the usefulness of this technique is further enhanced by the ability to transform zygotes, bypassing problems associated with generating func ⁇ tional plants from protoplasts. Data indicate that germinating pollen grains incubated with alien DNA affect fertilization, and induce directional mutations in the genome of the zygote which are expressed in the resulting offspring and their descendents.
• the technique of the invention can be used to consistently transfer selected marker or other desirable genes from a DNA donor plant to a recipient mother cultivar. However, the mechanisms involved in DNA uptake by the
• OMPI pollen tube transportation of alien DNA to the embryosac by the male gametophyte, and exogenous nucleotide incorporation into the genome of the zygote, as well as the genetics of transferred or mutated genes in the offspring of the recipient mother, are not yet well known.
• the method of the invention comprises the isolation of exogenous DNA from a selected donor plant, removal of mature pollen from the chosen donor plant, germination of this pollen in pollen- germinating liquid medium, incubation of germinating pollen with the foreign DNA, pollination of the mother plant with treated pollen, fertilization of the eggs within mature embryosacs of the mother plant, matura- tion of the ovary, obtain ent of seeds from mother plant and germination of same, and selection of transformed plants from the population obtained from said seeds.
• pollen from a compatible cultivar related to the mother plant- can be treated with exogenous genes and used to pollinate the respec ⁇ tive mother plant.
• OMPI OMPI :4321-4325
• PGM aqueous pollen-germi- nating medium
• PGM comprising carbohydrate, calcium, and boron
• Mature pollen is sprinkled onto a thin layer of PGM. Most of the pollen will begin to germinate within approximately 15 minutes.
• the previously-prepared donor DNA is added to the germinating pollen grains after approximately 10% of the pollen grains have begun germination.
• PGM is poured over germinating pollen and SSC buffer with DNA is added to give a final DNA concentration of approxi ⁇ mately 4-5 g/ml. Pollination is then initiated immediately. The PGM/DNA mixture is then transferred to the stigmatic surface of a receptive female inflorescence. Pollinated flowers are protected from foreign pollen by shoot bags until the PGM evaporates and then are covered with brown paper bags. Fer ⁇ tilization eventually occurs, but embryo and endosperm development is reduced. This effect is due to a reduction of functional pollen and sperm. It is known that several pollen grains are essential for the development of a seed (Klyucharena, M.V. 1962.
• IPO polar cells may come from the same or different male gametophyte as the sperm that fertilizes the egg.
• Pollen grains typically contain a tube nucleus and a generative cell.
• the haploid generative cell divides to form two sperm, the sperm travel down the pollen tube of a germinating pollen grain, traverses the stigmatic surface and the style of a mature female inflorescence, and eventually enters the ovary where one sperm combines in the fertilization process with the haploid egg cell.
• Gametic delivery results in deposition near the egg of two sperm, the vegetative nucleus and cytoplasm by each of several male gametophytes. This is accom ⁇ panied by loss of sperm and egg cell wall components.
• Gametic fusion results in the transmission of nearly the total sperm cytoplasm and organelle complement to the egg. The one sperm plays a role in the develop- ment of endosperm.
• OMPI normal stigma penetration and fertilization occurs, but embryo and endosperm development is greatly reduced. It is also suggested that a critical number of male gametophytes need to deposit their contents into the cytoplasm of the female gametophyte for successful seed development. Increase in quantity of treated pollen used in pollination increases the number of seeds produced.
• Results obtained using the method of the in- vention and maize demonstrate that exogenous genes are incorporated into the genome of the zygote. When and exactly how this occurs is unknown. If the DNA is carried to the female gametophyte by the sperm, incorporation may either be directly from the sperm -genome or indirectly from the sperm cytoplasm. It is also possible that DNA is transported as free frag ⁇ ments in the cytoplasm of the male gametophyte or sperm. Incorporation may then take place during division of the zygote to produce an embryo.
• the Zea mays cultivar B73 was selected for various experiments using the method of the invention.
• the female inflorescence of the standard maize inbred B73 consists of some 500 individual ovules arranged in 8 rows of paired spikelets around a central rachis. Each ovule has its own style with a feathery stigma, and contains a single female gametophyte. Sytles grow to over 15 cm long. Pollen grains are large, and it is possible to pick up individual grains with a fine, moist human hair for transportation to the stigma. Pollen germination and pollen tube growth down the stigma can be followed using fluorescence microscopy. Pollen germination is not severely affected by PGM or DNA incubation but pollen tube growth is retarded and few sperm reach the female gametophyte.
• Maize B73 is self-pollinated with pollen incubat ⁇ ed with DNA obtained from Tripsacum dactyloides or DNA from other maize genotypes carrying specific marker genes using the method of the invention.
• tripsacoid traits, as well as specific genes of the maize DNA donor transferable to maize through sexual transfer of exogenous DNA are similar to those incorporated into the maize genome through introgression (de Wet, J.M.J. et al. 1978.
• Nuclear DNA is extracted from seedling or mature leaves of the donor genotype using a combination of published techniques [J. Mol. Biol. 3:208-219 (1961); Plant Physiol. 6_6_: 1140-1143 (1980) ; Nucl. Acids Res. 8_: 4321-4325 (1980)], using a Trisbase buffer [0.2 M Trisbase (24.22g) , 0.2 M Disodium di-H 2 0 EDTA (74.45g), 4% SDS (40g) in one liter H_0] . Extracted DNA is purified as described in procedures cited above.
• Pollen germination and pollination after incu ⁇ bation with exogenous DNA are the most difficult aspects of the method of the invention using the male gametophyte as a carrier of foreign DNA.
• Pollen germinating medium PGM. comprises a ' solution of approximately 15% sucrose, 0.03% calcium nitrate and 0.01% boric acid in water. Maize, as well as the pollen of other plants, germinates well in the PGM. The base of a large petri dish is covered with a thin layer of pollen germinating medium and sprinkled with mature pollen of the recipient mother. In experiments with maize approximately 27mm of pollen is used for each set of pollinations.
• pollen from a single anther is sufficient to insure seed set.
• pollen starts to germinate within 3 to 10 minutes.
• Approximately 60 to 90% of the pollen is germinated after 15 minutes.
• DNA is obtained from donor plants according to the method of Example 1. Incubation of pollen with exogenous DNA begins after approximately 10% of pollen grains show visible signs of germination. Pollen tubes longer than the diameter of the grains break . during pollination.
• Nine ml of PGM is poured over the germinated pollen and 1 ml of buffer with DNA is added, to obtain a DNA concentration of 4-5 g/ml. Pollination is initiated immediately.
• the 11 ml of solution thus prepared is suffi ⁇ cient, for example, to pollinate three female inflorescences of corn each with approximately 300 to 500 ovules. Pollinating an ear of corn requires approximately one minute. Stigmas are cut to the tip of the cob twelve hours before pollination. The PGM with DNA and pollen is transferred to stigmas with a pasteur pipet. Pollinated ears are protected from foreign pollen by shoot bags until PGM evaporates, and they are then covered by standard brown paper bags. , PGM takes approximately 15 minutes to evaporate. Incubation continues until the developing pollen tube enters the stigma, or until the DNA is destroyed on the stigmas. Pollen tube growth continues during incubation with DNA, and penetration of stigmas proceeds normally. Fertilization takes place, but embryo and endosperm development is greatly reduced. This is believed due to a reduction of functional pollen and sperm. Resultant seeds are then screened for transposed genetic characteristics. Similar procedures are adapted for other experimental plants.
• Maize inbreds B73, DP194 and Zm 1974 produce an average of 425, 368 and 572 caryopses respectively per female inflorescence when they are self-pollinated.
• Various treatments of mature pollen of B73 are per ⁇ formed using the methods described in Example 1 and 2. Results of these experiments are presented in Table I below:
• Zm Zea mays (domesticated maize) ; Z ⁇ mays subsp. parviglumis
• Percentage seedset is negatively correlated with successful germination. Ears were classified into those with 1 to 10, 11 to 20, 21 to 30 and 31 to 40 caryopses. Seedset classes were planted separately and percentage germination recorded. Results of this experiment are presented in Table III below.
• Seedset of 31-40 caryopses per ear resulted in 3.5% germination, of 21-30 caryopses in 31-34% germination, of 11-20 caryopses in 32-43% germination and 1-10 caryopses in 39-63% germination.
• Poor germination from ears with relatively high seedset is due to reduced amounts of endosperm in the small caryopses in relation to caryopses from ears with low seedset.
• Germination is essentially perfect when caryopses are planted in sterile vermiculite and kept in a growth chamber at 75 F.
• Inbred Zea mays DP194 is highly susceptible to common leaf-rust caused by Puccinia sorghi.
• the DNA donor, Zea mays B14-A is resistant to rust. Resistance is dominant over susceptibility, and the genotype of B14-A used as DNA donor was homozygous resistant (Rpl /Rpl) .
• Rpl /Rpl homozygous resistant
• Seedlings are transplanted when the second leaf appears, inoculated with rust starting at the 4-leaf stage. Field germination of the same treatment was 73%. DP194 control planted in vermiculite produced 90% germination within six days, and 100% germination by the eleventh day. These data show that three out of 103 seedlings (No. 74, 102, 103) showed complete resis ⁇ tance after repeated inoculations with rust spores at the four-leaf and later stages. All other seedlings showed disease symptoms within five days after inocu ⁇ lation. Five out of 103 seedlings (No.
• each inflorescence branch is composed of solitary female spikelets, alternately arranged in cavities of an indurated rachis, with the paired male spikelets arranged on the same rachis above the female section.
• Sixteen plants were characterized by soli ⁇ tary female spikelets on tassel branches below the male spikelets.
• Female spikelets in the tassel do " occur in maize as a rare mutation, but they are paired as is typical in the female inflorescence of maize. Five of these robust plants tillered to produce 3 to 6 fertile culms.
• Peduncles of female inflorescences in Zml974 vary from 13 to 57 cm in length.
• trans- formed offspring were three plants with peduncle lengths of 87 cm, 102 cm and 110 cm. Two of these plants tillered while the other was characterized by a single culm.

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• 1984
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