EP0329736A1 - Induction asexuee de la sterilite male heritable et de l'apomixie chez les plantes - Google Patents

Induction asexuee de la sterilite male heritable et de l'apomixie chez les plantes

Info

Publication number
EP0329736A1
EP0329736A1 EP19880906709 EP88906709A EP0329736A1 EP 0329736 A1 EP0329736 A1 EP 0329736A1 EP 19880906709 EP19880906709 EP 19880906709 EP 88906709 A EP88906709 A EP 88906709A EP 0329736 A1 EP0329736 A1 EP 0329736A1
Authority
EP
European Patent Office
Prior art keywords
plant
ams
vector
hybrid
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19880906709
Other languages
German (de)
English (en)
Other versions
EP0329736A4 (en
Inventor
Ellen Jones Maxon
Norman Patrick Maxon
Original Assignee
MAXELL HYBRIDS Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MAXELL HYBRIDS Inc filed Critical MAXELL HYBRIDS Inc
Publication of EP0329736A1 publication Critical patent/EP0329736A1/fr
Publication of EP0329736A4 publication Critical patent/EP0329736A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A01H1/023—Male sterility
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • This invention relates to asexual induction of heritable male sterility in plants. This phenomenon of induction and inheritance is hereinafter referred to as "asexual male sterility" or "AMS”.
  • the invention also relates to a method for induction of an apomictic-like phenomenon in plants, a phenomenon which may be associated with, but which is distinct from, male sterility. More particularly, this invention relates to factors derivable from certain plants which when applied to certain recipient plants induce heritable male sterility and/or apomixis in the recipient. These factors are hereinafter referred to as "AMS/vectors”.
  • the invention further relates to the use of such AMS/vectors in a rapid, asexual method for generating genetically diverse male sterile plants. Such plants can be used to produce new hybrids of importance in agronomy, horticulture, pomology and forestry.
  • Monoecious plants are those in which male (staminate) and female (pistillate) organs are borne separately on the same individual plant.
  • the male and female organs may be located in separate flowers, as in corn plants, or they may be in close physical juxtaposition, as in soybean plants.
  • Monoecious plants occur widely in nature and are well represented among cultivated species, including important agricultural crops, horticultural varieties, as well as lumber, fruit and nut-bearing trees. Because monoecious plants have both. male and female sex organs, they are capable of self-fertilization, i.e., pollen from the male organ can pollinate the female organ, giving rise to seed. Even in those monoecious plants which normally reproduce by cross-fertilization such as corn, where male and female organs are located apart from each other on a plant, self-fertilization is possible.
  • While monoecy may be advantageous in nature, it can represent a problem in cultivar production. Indeed, it is frequently desirable that a cultivated monoecious plant be male-sterile so that it is incapable of self- fertilization. Situations in which male sterility is advantageous include the production of parthenocarpic fruits; the non-seed-setting of ornamentals thus giving long retention of flowers; and the production of doubleness in flowers where male sterility results in the transformation of anthers into petals. However, the most important instance by far in which male sterility is used advantageously as a breeding tool is in the production of hybrids, particularly F 1 (first filial generation) hybrids.
  • Hybridization is the cross-fertilization of one genetically unique plant by another. Its main virtues are to increase the genetic variation of plants and their progeny, to keep the population stable and to increase plant vigor.
  • the increased plant vigor resulting from hybridization is referred to in the art as heterosis.
  • the greatest heterosis is observed when the least related genotypes are crossed together, e.g., crosses between unrelated cultivars tend to produce better hybrids than crosses between related cultivars because of the greater genotypic diferences.
  • an F 1 hybrid is the result of a cross between any two genetically distinct parent plants, regardless of their state of homozygosity.
  • an F 1 hybrid is the product of a cross between two homozygous (but genetically distinct) parents or lines, and all F 1 plants resemble one another exactly.
  • F 1 hybrids are: a) greater vigor expressed as, inter alia, improved yield, flower or seed production, earlier germination, disease resistance, insect resistance and other manifestations of heterosis; b) greater adaptability to varying environmental conditions because the majority of genes are present in the heterozygous state; c) the expression of advantageous characters when these are controlled by dominant alleles; and d) control by the breeder over the resulting hybrid product.
  • the desired hybridization is difficult to achieve on a reliable basis particularly on a commercial level.
  • the goal in any hybridization program involving monoecious plants is to control or facilitate cross-fertilization by minimizing, or preferably eliminating, self-fertilization.
  • One way to attain this goal is to use a male-sterile plant as one of the parents in the breeding scheme.
  • male sterility of parental lines has been achieved in a variety of ways, all fraught with a variety of drawbacks.
  • monoecious plants may be made male-sterile by physically (either manually or mechanically) removing the male flowers, organs or pollen-bearing anthers from the plant.
  • This approach can be labor-intensive and, given human and machine error, not particularly fail-safe. Physical emasculation in the field is weather-dependent and can result in loss of tissue and yield.
  • monoecious plants may be treated with chemicals such as gametocides, which destroy the ability of the plant to yield viable pollen, or chemical hybridizing agents, which do not affect pollen viability but prevent pollen from causing self- fertilization.
  • gametocides which destroy the ability of the plant to yield viable pollen
  • chemical hybridizing agents which do not affect pollen viability but prevent pollen from causing self- fertilization.
  • the most frequently encountered approach to male sterility in monoecious plants is through biological means which result in an inability of the plant to produce viable pollen.
  • One type of biological male sterility is known in the art as genetic male sterility (Allard,
  • male-sterile or male-fertile state is dependent on a single gene. Plants homozygous for the recessive allele are male-sterile and can be used as parental lines for hybrid production. The homozygous male-sterile line is maintained by crossing it with a known heterozygote (for the sterility/fertility alleles) which yields 50% homozygous male-sterile progeny and 50% heterozygous male-fertile progeny.
  • cytoplasmic male sterility Another type of biological male sterility is known in the art as cytoplasmic male sterility, or CMS, and is dependent on cytoplasmic factors. See Allard, supra, at pp. 245-246. Plants carrying particular types of cytoplasm are male-sterile and can be used as parental lines to make F 1 hybrids. These F 1 hybrids are all male- sterile since their cytoplasm is derived entirely from the female gamete (from the male-sterile parent). In other words, the CMS trait is maternally inherited.
  • CMS cytoplasmic male sterility
  • S cytoplasms which can confer the trait of male sterility belong to the S group, which has been shown to contain three plasmid-like DNAs in the mitochondria (Sisco, P.H., et al., 1984, Plant Science Letters 34:127-134; Pring, D.R., et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:2904; Kemble, R.J., et al., 1980, Genetics 95:451; Koncz, C., et al., 1981, Mol. Gen. Genet. 183:449). S cytoplasms do not show stable male sterility (Laughnan, J.R. and Gabay-Laughnan, 1983, Ann. Rev. Genet. 17:27-48) and in some genetic backgrounds have a high rate of reversion to male fertility.
  • cytoplasmic-genetic male sterility differs from cytoplasmic male sterility only in that the offspring of male-sterile (maternal) plants are not necessarily male-sterile but can be made male-fertile if plants of a certain genetic make-up are used as the paternal parent.
  • These paternal parents that produce male-fertile F 1 progeny carry genes with the power to restore the pollen- producihg ability of plants with male-sterile cytoplasm. These genes are known as restorer genes and the plants that carry them, restorers.
  • Such cytoplasmic-genetic male sterility has been put to use in, e.g., onion breeding (See, Jones and Davis, 1944, U.S.D.A. Technical Bulletin874:1-28).
  • cytoplasmic male sterility for production of hybrids by means of cytoplasmic male sterility or cytoplasmic-genetic male sterility requires laborious and time-consuming sexual transmission through backerossing.
  • the scheme for sexual transmission of cytoplasmic male sterility which may be more accurately described as the transfer of a genotype or nuclear component to a male-sterility-producing cytoplasm, is set forth in Allard, supra, at pp. 246-247.
  • the seed industry has long used sexually transmitted cytoplasmic male sterility for pollination control in the production of hybrid seed products.
  • sexually transmitted cytoplasmic male sterility is carried in very few varieties of any one species, and, as mentioned previously, transmission is a time-consuming and expensive process requiring numerous generations of breeding to arrive at a new male sterile parental line.
  • the use of sexually transferred cytoplasmic male sterility has led to a very narrow cytoplasmic base as the cytoplasms are not genetically altered by conventional pollination. This has had deleterious consequences.
  • Cytoplasmic male sterility factors have also been asexually transmitted by means of somatic fusions. Protoplasts from different plants are fused in culture to form hybrids, sometimes called 'cybrids'. Such a technique has been used by Belliard and Pelletier in tobacco (1980, Eur. J. Cell Biol. 22(1):605). The major drawbacks of somatic fusion as an asexual means of cytoplasmic male sterility transmission are very low regeneration frequencies and the need for appropriate screens or markers for selecting the fused hybrids in vitro. Another asexual technique that has been used for the transfer of cytoplasmic male sterility is transmission through an intermediate host such as dodder (Cuscuta sp.).
  • dodder bridge Such an intermediate host is known in the art as a dodder bridge.
  • the major drawback of this approach is that dodder itself is considered a noxious parasite, both a weed and a disease, and therefore is not a likely candidate for large-scale field use. Grill and Garger (1981, Proc. Natl. Acad. Sci.
  • U.S.A. 78(11):7043-7046) have used the dodder bridge with Vicia faba (fava bean plant). They identified and characterized a high molecular weight double-stranded RNA (dsRNA) associated with cytoplasmic male sterility in Vicia faba.
  • dsRNA double-stranded RNA
  • the dsRNA is apparently located in spherical bodies, 70 nanometers in diameter, located in the cytoplasm of the plant, much like a virus.
  • the dsRNA was transmitted to a fertile line of V. faba by first growing dodder on the CMS V. faba and then contacting this dodder with a male fertile plant. After removing the dodder from the recipient, its flowering was observed.
  • Cytoplasmic sterility has also been induced by mutagenesis, by exposure to ethidium bromide for pearl millet (Burton, G.W. and Hanna, W.W., 1976, Crop Science
  • LBN cytoplasm male-sterile cytoplasm of maize
  • Plasmid-like DNAs have also been detected in the mitochondria of source IS1112C male-sterile sorghum cytoplasm (Pring, D.R., et al., 1982, Mol. Gen. Genet. 186:180-184).
  • a further object of the invention is to so provide hybrids of agronomic, horticultural, forestry and pomological importance with high yields, disease resistance, pest resistance and/or resistance to adverse environmental conditions.
  • Establishment of apomixis allows the development of seed, identical in genetic composition with the female parent, without the necessity for gametic fusion.
  • the invention is directed to asexually transmissible male sterility and apomixis factors, AMS/vectors, present in extracts from certain male sterile alfalfa plants. Characteristically associated with such extracts, and treated sterile plants, are (1) an unique isolatable nucleic acid with a molecular weight of about 1 x 10 6 daltons; and (2) particles, about 40-110 nanometers in diameter, consisting of a dense core surrounded by a bilayer membrane, as observed microscopically. The invention is further directed to such extracts and their use in asexually inducing male sterility and/or apomixis in recipient plants.
  • the extracts from alfalfa plants displaying the AMS trait when applied, e.g., by spraying, to susceptible recipient plants, induce or impart male sterility in the recipient. These extracts have also been demonstrated to induce or impart an apomictic form of reproduction in plants so treated.
  • the AMS/vector extracts are effective in inducing male sterility or apomixis across species and genera as well as between dicots and monocots.
  • the invention further provides improved methods for the production of F 1 hybrid plants wherein the improvement comprises using asexually AMS/vector-induced male sterile plants as the maternal parent in F 1 crosses.
  • the invention also provides a method of producing hybrid seed in which the improvement comprises crossing two parent lines, one of which has been treated with AMS/vector, to produce F 1 hybrid progeny, using the F 1 progeny to produce F 2 progeny, identifying those F 2 plants which are identical in phenotype to the F 1 and which set seed, propagating such plants, and collecting hybrid seed therefrom.
  • the invention further provides hybrid seed capable of producing apomictic plants, as well as the apomictic plants derived therefrom.
  • the invention also contemplates the use of the 1 X 10 6 (approx.) dalton nucleic acid and/or 40-110 nm particle, uniquely associated with extracts containing the AMS/vector, as a transmissible plant delivery or expression vector system.
  • AMS asexual male sterility
  • CMS cytoplasmic male sterility
  • DNase deoxyribonuclease
  • FIG. 1A is a photograph of an ethidium bromidestained agarose gel in which nucleic acids extracted from alfalfa AMS/vector source 1.29 (U.S.D.A. PI No. 223386) (lane 1), from an untreated fertile alfalfa maintainer (variety Arc) (lane 3), and from an untreated fertile alfalfa non-maintainer (lane 4) were run.
  • a single band at approximately 3.5 kb associated with the AMS/vector source is seen in lane 1, but not in lanes 3 or 4.
  • FIG. 1A is a Hindlll digest of bacteriophage lambda DNA, with molecular weights of (from top to botom) 23.6 kb, 9.6 kb, 6.6 kb, 4.3 kb, 2.2 kb, and 1.9 kb.
  • Fig. 1B is a photograph of an ethidium bromide- stained agarose gel in which nucleic acids extracted from fertile alfalfa (variety Arc) (lane 1) and from alfalfa (variety Arc) converted to male sterility by treatment with AMS/vector source 1.29 (U.S.D.A. PI No. 223386) (lane 3) were run.
  • FIG. 1B A single band at approximately 3.5 kb associated with the AMS trait is seen in Figure 1B, lane 3, but not in lane 1.
  • Lane 2 in Fig. 1B is a Hindlll digest of bacteriophage lambda DNA, as described for Fig. 1A.
  • Fig. 1C is a photograph of an ethidium bromide- stained agarose gel in which nucleic acids extracted from corn (variety B73) converted to male sterility by treatment with AMS/vector source 1.26 (U.S.D.A. PI No. 221469) (lane 1) and fertile corn (variety B73) (lane 2) were run.
  • a single band at approximately 3.5 kb associated with the AMS trait is seen in Fig. 1C, lane 1, but not in lane 2.
  • Lane 3 is a Hindlll digest of bacteriophage lambda DNA, as described for Fig. 1A.
  • Fig. 1D is a photograph of an ethidium bromide- stained agarose gel in which nucleic acids extracted from soybean (variety Williams 82) converted to male sterility by treatment with AMS/vector source 1.36 (U.S.D.A. PI No. 243223) (lane 1) and fertile soybean (variety Williams 82) (lane 2) were run.
  • AMS/vector source 1.36 U.S.D.A. PI No. 243223
  • lane 2 fertile soybean
  • Lane 3 is a Hindlll digest of bacteriophage lambda DNA, as described for Fig. 1A.
  • Fig. IE depicts the results of the experiment described infra in Section 6.2, demonstrating the DNA nature of the approximately 3.5 kb nucleic acid associated with extracts containing the AMS/vector.
  • Nucleic acids extracted from alfalfa were subjected to treatment with either DNase (lanes 1-7) or RNase (lanes 8-14) before agarose gel electrophoresis and ethidium bromide staining.
  • Fig. 1E is a photograph of the ethidium bromide staining pattern.
  • Lane 1 Hindlll digest of bacteriophage lambda DNA (as described for Fig. 1A); lane 2, restorer alfalfa line Indiana Synthetic (C); lane 3, AMS/vector source 1.26
  • AMS/vector source 1.36 U.S.D.A. PI No. 243223
  • lane 12 alfalfa maintainer (variety Arc)
  • lane 13 AMS/vector source 1.29 (U.S.D.A. PI No. 223386)
  • lane 14, AMS/vector source 1.7 U.S.D.A. PI No. 173733.
  • the approximately 3.5 kb band (indicated by the arrow) present in AMS/vector sources remains after RNase treatment, but is absent after DNase treatment.
  • Fig. 2A is an electron micrograph of the 40-110 nanometer particles present in a crude extract of a male- sterile alfalfa plant, U.S.D.A. PI No. 223386. Magnification: 20,000 X.
  • Fig. 2B is an electron micrograph of the 40-110 nanometer particles observed in an ovule of a male-sterile alfalfa plant, U.S.D.A. PI No. 221469. Magnification: 10,000 X.
  • Fig. 2C is an electron micrograph of a thin section of a seed from a cross between an alfalfa maintainer plant and a formerly fertile alfalfa plant that was converted to male sterility by treatment with extracts of an alfalfa AMS/vector source, U.S.D.A. PI No. 223386.
  • the white inclusion bodies exhibiting dark spots may contain the approximately 3.5 kb nucleic acid associated with extracts of the AMS/vector.
  • Fig. 3 (3A, 3B, 3C, 3D) contains photographs of representative microscopic fields depicting the pollen present in anthers from tassels containing dehisced pollen for varieties 1-4 of Zea mays L. corn plant, from the field test described in Section 6.9, infra.
  • Fig. 4 contains a photograph of a representative microscopic field depicting the anthers from tassels that showed no dehisced pollen, for variety 2 of Zea mays L. corn plant, from the field test described in Section 6.9, infra.
  • Fig. 5A contains a photograph of a representative microscopic field depicting the release of pollen from anthers of variety 1 as shown in Fig. 4, after the application of pressure.
  • Fig. 5B contains a photograph of a representative microscopic field depicting the release of pollen from anthers of variety 2 as shown in Fig. 4, after the application of pressure.
  • Fig. 6 contains a photograph of a representative microscopic fieid depicting the absence of observable sporogenous tissue in anthers with no dehisced pollen, after the application of pressure, in variety 4 of Zea mays L. corn plant, from the field test described in Section 6.9, infra.
  • Fig. 7 is a photograph of a representative microscopic field depicting anthers with abundant pollen grains of uniform size and shape, in a treated soybean plant (Glycine max var. Williams 82) from the growth room test described in Section 6.10, infra.
  • Fig. 8 is a photograph of a representative microscopic field depicting the red staining with acetocarmine of anthers as shown in Fig. 7.
  • Fig. 9 is a photograph of a representative microscopic field depicting the characteristic mass of pollen grains from anthers, as shown in Fig. 7, attached to stigma.
  • Fig. 10 is a photograph of a representative microscopic field depicting anthers containing a mix of non-stainable, abnormally shaped pollen grains and normal pollen, from a treated soybean plant (Glycine max var. Williams 82) from the growth room test described in Section 6.10, infra.
  • Fig. 11 is a photograph of a representative microscopic field depicting the irregular shape, lack of staining with acetocarmine, and high degree of vacuolatlon of anthers as shown in Fig. 10.
  • Fig. 12 is a photograph of a representative microscopic field depicting anthers which lack any pollen grains, from a treated soybean plant (Glycine max var. Williams 82) from the growth room test described in Section 6.10, infra.
  • Fig. 13 is a photograph of a representative microscopic field depicting the absence of any observable pollen grains in anthers as shown in Fig. 12, after the application of pressure.
  • Fig. 14 is a photograph of representative sterile tassels of "inbred 1" Zea mays L. corn plant, from the experiment described in Section 6.11, infra. There is no visible dehiscence of anthers. Such tassels did not shed pollen, and were rated sterile.
  • Fig. 15 is a photograph of representative sterile tassels of "inbred 2" Zea mays L. corn plant, from the experiment described in Section 6.11, infra. There is no visible dehiscence of anthers. Such tassels did not shed pollen, and were rated sterile.
  • Fig. 16 contains photographs of representative tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows a fertile tassel of inbred 1, exhibiting dehisced anthers. (The mass of pollen on the blue paper is apparent.)
  • Part B shows a tassel of inbred 1, rated sterile.
  • Part C shows a tassel of inbred 2, rated fertile.
  • Part D shows a tassel of inbred 2, rated sterile.
  • Fig. 17 contains photographs of representative tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows a tassel of inbred 4, rated fertile.
  • Part B shows a tassel of inbred 4, rated sterile.
  • Part C shows a tassel of inbred 2 rated fertile, but showing only one dehisced anther.
  • Part D shows a tassel of inbred 4 rated fertile, showing up to ten dehisced anthers.
  • Fig. 18 contains photographs of representative microscopic fields depicting the results of acetocarmine staining of anthers from tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows normal, round, and stainable pollen of inbred 1 from tassels rated fertile.
  • Part B shows pollen from tassels of inbred 2, rated fertile.
  • Part C shows pollen from tassels of inbred 3, rated fertile.
  • Part D shows pollen of tassels of inbred 4, rated fertile.
  • Fig. 19 contains photographs of representative microscopic fields depicting the results of acetocarmine staining of anthers from tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows a fully dehisced anther from tassels of inbred 1, rated fertile. The anther wall and a single pollen grain are apparent.
  • Part B shows a fully dehisced anther from tassels of inbred 2, rated fertile.
  • Part C shows a fully dehisced anther from tassels of inbred 3, rated fertile.
  • Part D shows a fully dehisced anther from tassels of inbred 4, rated fertile.
  • Fig. 20 contains photographs of representative microscopic fields depicting the results of acetocarmine staining of anthers from tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows abnormal pollen in the anthers from a tassel of inbred 1, rated sterile.
  • Part B shows abnormal pollen in the anthers from a tassel of inbred 2, rated sterile.
  • Part C shows abnormal pollen in the anthers from a tassel of inbred 3, rated sterile.
  • Part D shows no detectable pollen in the anther from a tassel of inbred 4, rated sterile.
  • Fig. 21 contains photographs of representative microscopic fields depicting the results of acetocarmine staining of anthers from tassels of inbred Zea mays L. corn plants, from the experiment described in Section 6.11, infra.
  • Part A shows anthers from a tassel of inbred 1, rated sterile, crushed to reveal abnormal, irregularly shaped, non-stainable pollen.
  • Part B shows anthers from a tassel of inbred 2, rated sterile, crushed to reveal abnormal, irregularly shaped, non-stainable pollen.
  • Part C shows anthers from a tassel of inbred 3, rated sterile, crushed to reveal abnormal pollen.
  • Part D shows anthers from a tassel of inbred 4, rated sterile, revealing no pollen after crushing.
  • Fig. 22 contains photographs of representative microscopic fields depicting the results of acetocarmine staining of anthers from tassels of inbred Zea mays L. corn plants, from the experiment described in Section
  • Part A shows anthers from tassels of inbred 1, rated sterile, exhibiting a few stainable, normal looking pollen, which were uncommon.
  • Part B shows predominantly abnormal and a few normal looking pollen in an undehisced anther from a tassel of inbred 2, rated fertile.
  • Parts C and D show anthers as described in Part B, revealing bulged portions which lodged predominantly normal looking pollen.
  • Nondomestic alfalfa plants (genus Medicago) of Middle Eastern origin can serve as sources (donors) of AMS/vectors. Plants obtained from the Seed Increase Collection, U.S.D.A., Reno, Nevada (1979.-1984) were screened for insect resistance and reduced seed set. Out of approximately seventeen thousand plants, five were selected as bearing the AMS/vector trait, i.e., all possessed an extractable factor which when applied to susceptible recipients imparted male sterility. All the AMS/vector-bearing plants were characterized as being male sterile, tetraploid, purple-flowered perennials.
  • Extracts of these plants characteristically contained particles about 40-110 nanometers in diameter and an isolatable nucleic acid with a molecular weight of about 1.1 x 10 6 daltons (see Section 6.2.).
  • the specific plants which can serve as a source of the AMS/vector in a particular embodiment as described herein had the following Plant Introduction numbers (PI Nos.) when obtained from the Seed Increase Collection: PI No. 172429, PI No. 173733, PI No. 221469, PI No. 223386, and PI No. 243223. Seeds from plants resulting from crosses between each of the five sources and Arc-derived maintainer plants (Medicago sativa var. Arc developed at the U.S.D.A. Labs, Beltsville, MD) can be used to generate plants which can also serve as sources of AMS/vectors.
  • Arc-derived maintainer plants Medicago sativa var. Arc developed at the U.S.D.A. Labs, Beltsville
  • AMS/vectors may exist. They may be determined empirically by following the methods of Sections 5.2. and 5.3.
  • Preparations containing the AMS/vector may be prepared by a simple extraction procedure. Donor alfalfa plants are harvested when they have fully developed crowns, usually at one-tenth bloom or a week before.
  • Leaves and stems are used fresh or stored frozen for future use.
  • the plant material is suspended in any suitable non-lethal buffer such as potassium phosphate buffer (e.g., 0.067 M KH 2 PO 4 at pH 6.9). Typically, for every five to seven ml of buffer, about one gram of plant tissue is suspended therein.
  • Other ingredients may be added to the extract such as abradors (e.g., diatomaceous earth such as Celite) or absorption enhancers (e.g., dimethylsulfoxide or DMSO).
  • the plant material is macerated by any suitable means, e.g., blending in a high speed blender to form a homogenate . Residual plant debris is removed by filtration, decantation or other suitable means.
  • the extraction procedure need not be performed under sterile conditions and the resulting filtrate or extract need not be stored in sterile containers.
  • the extract may be kept refrigerated for periods up to approximately three hours before use. Otherwise, it may be stored frozen, e.g., in liquid nitrogen, until use.
  • AMS/vector extracts thus prepared are sprayed on recipient plants using standard field equipment. In general, only one application of the extract is necessary. In a particular embodiment, about 5 to 25 milliliters (ml) can be applied per plant. The extract is sprayed onto the leaves of the recipient. The inclusion of an abrador (e.g., Celite) is preferred.
  • an abrador e.g., Celite
  • Recipient plants are to be sprayed at a time when they have foliage, but prior to flowering and seed set.
  • soybean plant recipients may be sprayed at least about two weeks after germination; earlier application does not result, or results poorly, in induction of male sterility.
  • Corn plant recipients may be sprayed when the fifth leaf is exposed, at the beginning of the grand growth stage, approximately three weeks after germination.
  • alfalfa recipients are cut back about two centimeters above the crown; within a two- week period of time, the alfalfa recipients may have extracts applied to them.
  • tissue culture suspension of plant tissue in media containing AMS/vectors
  • electroporation of the AMS/vectors into protoplasts e.g., for vegetable crops
  • injection e.g., for trees
  • All plants are potentially inducible to male sterility by the AMS/vector if genetically predisposed to inducibility. This includes monoecious plants and even dioecious plants (i.e., plants in which male and female organs occur on different individuals) where, as a result of inducing male sterility, a male plant is transformed into a female plant.
  • dioecious plants i.e., plants in which male and female organs occur on different individuals
  • a tetraploid e.g., alfalfa
  • a diploid e.g., soy or corn
  • Plants which are of greatest interest are those of agronomic and horticultural importance, including, but not "limited to, grain crops, forage crops, seed propagated fruits, seed propagated ornamentals and industrial species.
  • Representative monoecious plants which may be used as recipients of the AMS/vectors to create new male sterile plants are listed in Table I. The table is presented by way of illustration and is by no means exhaustive. Recipient plants inducible to male sterility by AMS/vectors may be identified by applying extracts as described in Section 5.2. and visually rating the recipient plant with regard to pollen production and seed set. Those which do not produce pollen and/or seed are inducible recipients.
  • This invention contemplates the use of DNA probes to identify inducible recipients.
  • the DNA of known inducible and non-inducible plants may be subjected to restriction endonuclease digestion. Fragments unique to the inducible plants may be identified and serve as a template from which to make DNA probes. These probes may then be used to screen, via hybridization methodologies, for other recipients (see Maniatis, T., et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Alternatively, probes may be used to identify induced plants where unique nucleic acids are associated with plants exhibiting the AMS trait.
  • the AMS/vector-induced male sterile plants may be used as the maternal parental line in hybridization schemes known in the art. Such male-sterile maternal lines may be maintained or expanded in number by crossing them with 'maintainers', i.e., the genetically identical, non-AMS/vector-treated plant.
  • the choice of paternal lines for crossing with the AMS/vector-induced male sterile maternal lines varies, depending on the intended use of the F 1 offspring. If the F 1 offspring plants are desirable in and of themselves as, e.g., forage crops or ornamentals, it is not necessary that the F 1 hybrids be male-fertile and hence capable of producing seed. Thus it is not necessary to choose a male parental line that will result in the F 1 hybrids being male fertile.
  • a restorer may be used as the male parental line. Restorers are identified by performing the cross and observing the percent fertility of the F 1 progeny. Male parental plants, which when crossed with the male-sterile female parental plants yield fertile F 1 progeny, are considered restorers.
  • B73 is a known restorer of
  • AMS/vector-induced sterile Mo17 is a known restorer of AMS/vector-induced sterile B73; and H95 is a known restorer of AMS/vector-induced sterile A632.
  • restorers male fertile plants, which when crossed with male-sterile maternal plants yield F 1 progeny that are vegetatively propagated through seed, may be used as the paternal plant for production of seed-bearing F 1 hybrids.
  • Both the F 1 progeny and the male-sterile plants containing the AMS/vector can, in addition to other methods, be propagated vegetatively.
  • the stem of the plant can be cut off at the base, placed in rooting medium and allowed to root, before being transplanted to soil.
  • Tissue culture methods of propagation are also envisioned for use (for review, see Vasil, I., et al., 1979, in Advances in Genetics, Vol. 20, Caspari, E.W., ed., Academic Press, New York, pp. 127-216).
  • stem sections are frequently used in the propagation of sugarcane, which only rarely produces flowers in non-tropical regions.
  • roots and tubers are employed in the production of root crops such as cassava, sweet potatoes, potatoes, and yams.
  • apomixis In this form of reproduction, which occurs spontaneously, i.e., without human intervention, in hundreds of plant species.
  • the sexual organs and related structures take part in reproduction, but the seeds which are formed are produced without union of gametes.
  • apomixis is the only form of reproduction, and these plants are known as obligate apomicts.
  • the apomictic plant will exhibit both gametic and apomictic reproduction, and these plants are referred to as facultative apomicts.
  • the sexual and asexual processes may operate simultaneously in an individual plant.
  • apomixis In both obligate and facultative apomicts, there may be several mechanisms or combinations of mechanisms involved in the asexual process. There are four basic types of apomixis. In apogamy, the embryo develops from two haploid nuclei other than the eggs; .frequently it results from the fusion of two cells of the embryo sac, either synergids or antipodal cells. In apospory, the embryo sac develops directly from a somatic cell without reduction and formation of spores; the embryo develops from the diploid egg without fertilization. In diplospory, the embryo develops from the megaspore mother cell without reduction.
  • apomixis will be used generically to .apply to any or all of these phenomena, or any variation which produces the same end result. It is generally believed that apomixis is controlled genetically (Taliaferro, CM., Southern Pasture Forage Crop Impr. Conf. Rep. 26:41-43, 1969) and it has been suggested that it may be controlled by a single gene (Harlan et al., Bot. Gaz. 125:41-46, 1964).
  • apomixis When first discovered, apomixis was considered to be a complete barrier to plant breeding. Hybridization between obligate apomicts is virtually impossible except in the rarest of circumstances. In most types of apomixis, the embryo has the same genetic constitution as the maternal plant, and is a true clone. Thus, the possibility of introducing variation into an apomictic line for the purpose of developing new varieties or hybrids, would appear to be severely limited. In fact, early workers generally considered apomixis as an evolutionary "blind alley" (Darlington, The Evolution of Genetic Systems, p. 149 University Press, Cambridge, 1939) because of the potential for reproductive isolation. In recent years, however, it has become apparent to plant breeders that the phenomenon may have valuable applications in breeding.
  • AMS/VECTOR INDUCTION OF APOMIXIS In addition to the observed effect on male sterility which can be obtained by treatment of plants, it has also been unexpectedly discovered that AMS/vector has the ability to induce apomixis in treated plants. In the process of study of the pattern of inheritance of male sterility in AMS/vector-treated plants, certain initial observations in the inheritance of other phenotypic characteristics indicated that some treated plants were not exhibiting a pattern which would be expected from normal hybrid production between sexually reproducing parents. For example, crosses were performed between phenotypically distinct soybean parent lines, one parent of which had been treated with AMS/vector, and were shown to be male sterile. Soybean is not known to be naturally apomictic.
  • the F 1 produced by this cross all exhibited, as expected, the purple flower, purple hypocotyl phenotype; among these plants were a substantial percentage of male steriles, of which a small percentage set seed.
  • the F 1 plants were then selfed to produce an F 2 generation. If normal patterns of sexual propagation and inheritance were occurring, it would be expected that the resulting F 2 generation should segregate for flower and hypocotyl color.
  • the treatment of plants to induce apomixis can be achieved in much the same manner as is the Induction of male sterility.
  • a usual method of application is spraying the subject plants at a time prior to flowering, but at a time when the plant is sufficiently mature to have developed foliage. Alternately, it may be desirable to spray shortly after flower initiation, in order to attempt to directly affect the developing seed.
  • the manipulations necessary to determine the optimum pattern of application for a given type of plant is well within the skill of the experienced plant breeder.
  • the seeds resulting from the cross are planted and grown to maturity, this group constituting the F 1 hybrid generation.
  • All members of the F 1 should be identical in phenotype. Among these will usually be a number of male sterile plants resulting from the treatment. The F 1 plants are allowed to self. The seed is collected and planted, and the phenotypes of the resulting F 2 generation observed. If no induction of apomixis has occurred, the plants of the F 2 will show traits in a 3:1 ratio, and various combinations of parental characteristics, due to segregation of traits during meiosis. However, if apomixis has occurred, the resulting F 2 will substantially all be phenotypically identical to the F 1 generation. Additionally, there will be a number of male sterile plants, many of which will set seed. The existence of both these characteristics indicates that apomixis is occurring and that the seed being produced is identical to that produced by the original hybrid cross.
  • the AMS/vector can also be valuable as a plant vector system.
  • the 40-110 nm (approx.) particles associated with extracts containing the AMS/vector have potential utility as intracellular plant delivery systems, e.g., for delivery of bioactive molecules such as nutrients, pesticides, etc.
  • the 1 x 10 6 (approx.) dalton nucleic acid associated with extracts containing the AMS/vector, or a derivative, mutant, or fragment thereof, also has potential value as a transmissible expression vector.
  • the nucleic acid when comprised of a heterologous gene sequence, can be used as a vehicle for the expression of the heterologous gene sequence. Such a nucleic acid can be used either in conjunction with, or without, the 40-110 nm particle.
  • ratings of 1 or 3 meant the plant was sterile; ratings of 5 or 7 meant the plant was fertile.
  • Medicago scutellata an annual cleistogomous (i.e., a plant that self-pollinates before flowering) that is very similar to soybean, was grafted on as scion for each of the five AMS/vector lines. The grafts did not alter fertility in the graft generation, which was all fertile; the next generation, however, contained male sterile plants at a frequency of approximately 10%.
  • Alfalfa plants which screened positive for the AMS/vector, as discussed in Section 6.1., possess a unique nucleic acid. When extracts of such plants are applied to recipients (maintainers), the same nucleic acid is subsequently extractable from the recipient (now asexually induced to male sterility). The nucleic acid is not extractable from untreated isogenic maintainers.
  • This nucleic acid has a molecular weight of approximately 1.1 x 10 6 daltons (about 3.3 to 3.5 kilobases) and is postulated to be DNA.
  • the unique nucleic acid has been isolated from leaves, stems and/or primary callus tissue derived from ovules of the alfalfa plants described in Section 6.1.
  • the same procedure has been performed on alfalfa, soybean, and corn plants induced to male sterility by treatment with AMS/vector extracts and the unique nucleic acid was isolated from these plants as well.
  • an equal volume (20 ml) of a 24:1 chloroform:isoamyl alcohol mixture is added and the tube is centrifuged for 10 minutes at 13,000 rpm with a Beckman J21C rotor, in a Sorvall centrifuge at 10oC.
  • the resultant aqueous phase is removed to a new tube to which a volume, equal to one-tenth that of the aqueous phase, of 10% cetyltrimethylammonium bromide (CTAB) solution is added, followed by 20 ml of the 24:1 chloroform: isoamyl alcohol mixture.
  • CTAB cetyltrimethylammonium bromide
  • the resultant aqueous phase is transferred to a new tube to which an equal volume of STE buffer is added.
  • the tube is allowed to stand at room temperature (about 25oC) for 30 minutes and is then centrifuged for 5 minutes at 4,000 rpm in a Beckman J21C rotor. The supernatant fraction is removed and the remaining pellet is dried under a stream of nitrogen. The pellet can be stored frozen at -20oC until needed.
  • the pellet is resuspended in 5 ml of a solution of 50 mM Tris, pH 8.0; 5 mM EDTA; 50 mM NaCl and 200 micrograms per ml (hereinafter "ug/ml") ethidium bromide.
  • ug/ml a solution of 50 mM Tris, pH 8.0; 5 mM EDTA; 50 mM NaCl and 200 micrograms per ml
  • CsCl cesium chloride
  • the ethidium bromide is removed from the DNA fraction with three extractions of equal volumes of isopropanol equilibrated with 20x SSC, 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8.
  • the DNA fraction is diluted two-fold with a solution of 10 mM Tris, pH 7.6 and 1 mM EDTA, adjusted to 0.3 M sodium acetate.
  • the DNA is precipitated with two volumes of ethanol. The precipitate is frozen at -20oC until further use.
  • RNA pellet which results after the above- described 60 hour spin is resuspended in 0.5 ml of the 10 mM Tris, pH 7.6, 1 mM EDTA buffer and the ethidium bromide is extracted with two extractions of equal volumes of isopropanol equilibrated with 20x SSC.
  • the RNA is diluted two-fold with 10 mM Tris, pH 7.6, 1 mM EDTA, adjusted to 0.3 M sodium acetate. Two volumes of ethanol are added and the mixture is frozen at -20oC until further use.
  • RNA samples are thawed. Tubes with RNA are centrifuged for 10 minutes at 4oC at 10,000 rpm in a Beckman J21C rotor. The supernatant fractions are poured off. The RNA pellets remaining in the tubes are allowed to dry under a stream of nitrogen. Each RNA pellet is resuspended in 250 microliters (hereinafter "ul") of Tris borate buffer, 0.089 M Tris, 0.089 M boric acid, 2.5 mM EDTA, pH 8.3, and a few drops of 0.1 M sodium acetate and then one volume of ethanol is added. These mixtures are frozen at -20oC until needed.
  • ul 250 microliters
  • the thawed DNA samples are transferred to 45 ul centrifuge tubes. Five ul of the Tris 10 mM, pH. 7.6 1 mM EDTA buffer are added and mixed until the precipitate dissolves. Ten ul of ethanol and a few drops of 0.1 M sodium acetate are added. No CsCl precipitate is observed. The DNA solutions are frozen at -20oC until needed.
  • RNA samples are again thawed and centrifuged in a desk-top Eppendorf centrifuge for 3 minutes. The supernatant fractions are poured off and the pellets in the tubes are allowed to dry under a stream of nitrogen. The pellets are resuspended in 8 ul Tris borate buffer and 20 ul glycerol/dye (bromophenol blue) mixture. The samples are mixed well and stored at -20o C until needed.
  • the DNA samples are again thawed. They are centrifuged for 10 minutes at 4oC at 10,000 rpm in a Beckman J21C rotor, and resulting supernatant fractions are poured off. The pellets are dried under a stream of nitrogen and then resuspended in 5 ml of 10 mM Tris, pH 7.6, 1 mM EDTA buffer to which sodium acetate is added to a concentration of 0.3 M. Then 10 ml of ethanol are added. The DNA is allowed to precipitate at -70o C for one hour.
  • DNA and RNA samples so prepared are dialyzed overnight and run on a 1% agarose, lx Tris-Borate-EDTA (TBE) gel. The DNA and RNA are pooled before running the gel. After ethidium bromide staining, the band characteristic of plants carrying the AMS/vector is seen at approximately 3.5 kb.
  • Fig. 1A depicts the 3.5 kb band present in alfalfa
  • AMS/vector source 1.29 (U.S.D.A. PI No. 223386), and the absence of the 3.5 kb band in fertile untreated alfalfa maintainer (variety Arc) and fertile, untreated non- maintainer (variety Arc).
  • Fig. IB depicts the 3.5 kb band present in alfalfa (variety Arc) converted to male sterility by treatment with AMS/vector source 1.29 (U.S.D.A. PI No. 223386).
  • Fig. IC depicts the 3.5 kb band present in corn (variety B73) converted to male sterility by treatment with AMS/vector source 1.26 (U.S.D.A. PI No. 221469).
  • Fig. 1D depicts the 3.5 kb band present in soy (variety Williams 82) converted to male sterility by treatment with AMS/vector source 1.36 (U.S.D.A. PI No. 243223).
  • the approximately 3.5 kb nucleic acid associated with extracts containing the AMS/vector appears to be comprised of DNA (Fig. 1E). This was shown by digesting nucleic acid samples from alfalfa, prepared as described supra, with deoxyribonuclease (DNase, Boehringer Mannheim, 3000 U/mg) or ribonuclease (RNase, Boehringer Mannheim, 3000 U/mg). 20 ul of DNase or RNase (10 U/ul in 50 mM NaCl, 50% glycerol) was added to 60 ul nucleic acid sample, and the mixture was placed at 37oC for 15 minutes.
  • DNase deoxyribonuclease
  • RNase Ribonuclease
  • the reaction was stopped by adding 15 ul of 0.4 M EDTA before subjecting samples to agarose gel electrophoresis.
  • the resulting ethidium bromide-stained bands are shown in Fig. 1E.
  • the approximately 3.5 kb band associated with AMS/vector extracts is discernible in the RNase-treated samples, but is absent from the DNase-treated samples.
  • the 3.5 kb nucleic acid thus appears to be comprised of DNA, as evidenced by its susceptibility to DNase digestion.
  • Buffer I consisted of: 50 mM Tris-HCl (pH 7.5), 0.4 M sucrose, 10 mM KCl, 5 mM MgCl 2 , 10% (v/v) glycerol, and 10 mM 2-mercaptoethanol.
  • Buffer II consisted of 50 mM sodium phosphate buffer, pH 7.0.
  • a sample of plant tissue was homogenized in a Virtis homogenizer in 6 volumes (v/s) of Buffer I for 30 seconds on slow speed and 30 seconds on fast speed.
  • the homogenates were filtered through 4 layers of Miracloth and centrifuged at 2,000 x g for 5 minutes.
  • the supernatant was centrifuged at 0,000 x g for 20 minutes.
  • the resulting supernatant was centrifuged at 180,000 x g for 60 minutes in two tubes.
  • the small, dark-green pellet was resuspended in 1.5 ml of Buffer II and layered onto a 12-42% (w/w) gradient of sucrose in Buffer II.
  • FIG. 2A shows the 40-110 nm particles present in a crude extract (prepared as herein described) of a male-sterile alfalfa plant, AMS 1.29 (PI No. 223386).
  • Figure 2B depicts the 40-110 nm particles present in an ovule of a male-sterile alfalfa plant, AMS 1.26 (PI No. 221469).
  • Figure 2C depicts a thin section of a seed from a cross between an alfalfa maintainer plant and a formerly fertile alfalfa plant that was converted to male sterility by treatment with extracts of AMS/vector source 1.29.
  • the white inclusion bodies exhibiting dark spots may contain the approximately 3.5 kb nucleic acid associated with extracts of the AMS/vector.
  • the experimental design was a randomized complete block design, as a split-plot, with treatments as the main plot, and varieties as splits. There were four replications, with 16 treatment plots within each replicate, and 8 genotypes (varieties) randomly distributed as 1 of 8 rows in each plot. The 16 treatments and 8 genotypes tested are listed in Tables IA, IB.
  • Treatments consisted of injection of soluble extracts, or Celite (diatomaceous earth, grade III, Sigma Chemicals, Cat. No. D5384) application. Injections were done with a 28-gauge needle. The needle was passed through the stem of the plant, a drop of extract was exuded on the other side, and the needle was pulled back through the stem. Approximately 5 stems were injected per plant. Any stem that was not injected was trimmed back. Celite application was carried out basically as described in Section 6.9.1.6.3, infra.
  • the experimental design was a randomized complete block design, a split-plot, as described in Section 6.4, supra.
  • Treatments T1 through T5, and T9 through T13, were as described in Section 6.4.
  • Treatments T6 and T14 were injection and Celite application, respectively, of extracts of the particular genotype being treated.
  • Treatments T7 and 15 were untreated plants.
  • Treatments T8 and T16 were, injection and Celite application, respectively, with buffer (KH 2 PO 4 , pH 6.9) only. Injections were done at the nodes and into the petioles (stem tissue between the stem and leaf) of each plant.
  • the eight genotypes treated are listed in Table ID.
  • treated soybean plants producing flowers were rated for fertility based on the presence or absence of pollen in anthers excised from flowers. At least three flowers per plant were rated.
  • AMS/vector source extracts were capable of inducing male sterility, with the purple-flowered soybean line responding more strongly to the AMS1.4 extract, while the white-flowered line responded more strongly to the AMS1.36 extract. 50 of the female (white-flowered) plants out of a total of 175 rated were sterile, and 8 of the male (purple-flowered) plants out of a total of 24 rated were sterile. Therefore, sterility was induced in 58 out of 199 or 29% of the plants rated.
  • the experiment described herein demonstrates the asexual induction of male sterility in corn.
  • the experimental design and treatments 1-5 and 9-13 were as described in Section 6.4, supra.
  • Treatments 6 and 14 were injection and Celite application, respectively, of extracts of the particular genotype being treated.
  • Treatments 7 and 15 were untreated plants.
  • Treatments 8 and 16 were injection and Celite application, respectively, with buffer alone. Celite applications were carried out as described in Section 6.9.1.6.3, infra. Injection was by use of a 28 gauge needle inserted into the pith of the plant. Genotypes which were subjected to experimental treatments are listed in Table IF.
  • Fertility was rated according to the following:
  • AMS/vector in sorghum, sunflower, pearl millet, and tomato Treatment with AMS/vector sources appeared to result in reduced seed set in sorghum, sunflower, and millet, and reduced fruit set in tomatoes.
  • a number of plants rated as "male-sterile" also formed seed-bearing pods. Ten out of 29 plants in F 2 (34%), 18 out of 29 plants in F 3 (62%), 18 out of 31 plants in F 4 (58%) and 9 out of 18 plants in F 5 (50%) had seed-bearing pods. A majority of the plants in this category had three seeds per pod. In some plants one seed per pod and two seeds per pod were also common.
  • F 5 generations were 14, 3, 3 and 6, respectively.
  • a sample of seed from each of the four generations was collected from the mature pods, and germinated on moist filter paper. Germination was more than 99% overall. Seed produced from both normal pollen bearing plants in F 2 -F 5 generations and "male-sterile" plants in the F 2 -F 4 generations, had excellent seed viability. Seeds from male-sterile plants of the F 5 generation were not mature when the germination test was conducted.
  • Treatments with sources 1, 2, 3, and 4 AMS/vector showed pollen sterility in corn varieties 1, 2, and 4.
  • Treatments with corn extract (no AMS/vector), buffer alone, alfalfa extract (no AMS/vector), and untreated plants showed no pollen sterility in any corn variety.
  • Variety 3 showed no pollen sterility under any treatment. There was a position effect in the expression of sterility, with plants showing sterility occurring in clusters among the fertile plants.
  • the seed was stored in the cold room at 38oF until the time of planting.
  • the experiment was conducted in a field site in West Jefferson, Ohio.
  • the field site 150 feet x 120 feet was plowed, disked and rototilled.
  • a basal fertilizer application was made using a fertilizer applicator and consisted of P 2 O 5 (28 lbs.), K 2 O (39 lbs.), and urea (140 lbs.), as phosphorus, potassium, and nitrogen sources, respectively.
  • Another dose of 140 lbs. of urea was applied between rows, by hand, four weeks afte'r emergence.
  • a preemergence herbicide, Lasso was applied to control the weeds.
  • the field was rototilled at a shallow depth of four inches again after the fertilizer and herbicide application.
  • the experimental plot was surrounded on two sides by ecology experiments and on two sides by fields leased to farmers. Those fields were in oats for the period of the experiment. 6.10.1.3.
  • EXPERIMENTAL DESIGN The design used was a split-plot with treatments as the main plot and varieties as splits. There were four replications, with eight treatment plots within each replicate and four varieties randomly distributed as one of four rows in each plot. Each variety was planted as a 20 foot row, with 6 inches in-row and 3 feet between-row spacing. All plots were separated by a matrix of 6 foot wide alleys. The treatment and variety randomizations were as outlined in Table III.
  • Alfalfa material was cut fresh from the field, immersed in liquid nitrogen, and the liquid nitrogen frozen material shipped overnight to the field site.
  • the alfalfa material was used as a basis for five of the eight treatments, one of which was the control without AMS/vector and four of which contained AMS/vector.
  • the AMS/vector sources were four different male sterile genotypes developed from four alfalfa lines, namely PI Nos. 221469, 172429, 223386, and 243223.
  • the alfalfa control extract (without AMS/vector) was a maintainer isogenic non-sterile line.
  • the frozen alfalfa material arrived in sealed plastic bags, precoded as T1, T2, T3, T4, and T5 (see Table IV, supra). A record was retained elsewhere of the control and AMS/vector treatment materials and the corresponding codes on the plastic bags. The treatment codes were therefore "blind" for those who performed the field test.
  • Phosphate buffer (KH 2 PO 4 , 0.067 M, pH 6.9) was prepared three days before the extraction of the plant material and was stored at 11o C All the extraction procedures were done wearing disposable surgical gloves. The gloves were disposed of after the extraction of each treatment material was completed. A new pair of gloves was used for each treatment.
  • the frozen alfalfa plant material was transferred to the West Jefferson field facility in an ice box under dry ice.
  • the ice box was kept in a cold room at 38 "F until the extractions began.
  • For each treatment extract a total of 310 g of plant material and 2200 ml of buffer was used. Because of the availability of only one centrifuge, the extraction was done in two batches for each treatment, each using 155 g of plant material and 1100 ml buffer.
  • the buffer and the plant material were macerated for 2-3 minutes in a Waring heavy duty blender. The homogenate was filtered through four layers of sterile cheesecloth to remove the plant debris.
  • the filtrate was collected in sterile 250 ml centrifuge bottles and centrifuged at 2,000 rpm for five minutes at 11'C in a GSA rotor.
  • the supernatant was decanted into sterile flasks, labeled, and put in the cold room at 38"F until used for spraying.
  • Freshly collected leaf material from the four corn varieties was similarly extracted.
  • the supernatants derived from the above procedure were used as the treatment materials for spraying corn plants.
  • Celite diatomaceous earth, grade III, Sigma Chemicals Cat. No. D5384
  • KH 2 PO 4 buffer 0.67 M, pH 6.9, 11oC
  • the Celite-buffer mix was vigorously shaken, to ensure a uniform dispersion of Celite in the buffer for spraying.
  • Corn plants were sprayed when the fifth leaf was fully expanded, four weeks after planting. All plants were sprayed around the whorl (tip of corn plant) with Celite, using the one gallon tank, sprayer.
  • the six plant extracts and the buffer-only control were sprayed around the whorl using the one gallon tank sprayers.
  • Pollen stainability was rated in one of each of the two types of results, one each for each variety in each treatment.
  • One fertile flower for each variety treatment was stained.
  • a portion of the tassel with no dehisced pollen from the field was stained with acetocarmine and observed for normal or abnormal appearance.
  • Pollen staining was done by transferring the anther to a glass slide, applying a drop of acetocarmine and covering the stamen with a coverslip. Pollen stainability was observed immediately. Photographs of representative normal and abnormal microscopic fields were also taken.
  • Duncan's multiple range test does not in itself determine if there is a "significant difference" from the null hypothesis, but allows us to break our treatments into groups that are significantly different from each other. For example, assume that treatments T1, T2, T3 and T4 are all significant at P less than 0.05. Duncan's test allows us to determine if they are equally different (one class) or unequally different, e.g. T1 and T2 in class A, and T3 and T4 in class B. Treatment means were compared using critical range values.
  • split-plot design The experimental design established for this study is called a split-plot design.
  • the split-plot helps reduce error by keeping treatment blocks together. There is still randomization of plants within each treatment and of treatments within each replication. (A completely randomized design would not separate treatments into blocks).
  • eight "whole" plots were selected so as to be linearly contiguous in space. One of eight treatments (some of which were controls) were randomly assigned to these whole-plot units. Each whole-plot unit was then subdivided or "split" into split-plots. Each split-plot unit was randomly assigned one of four varieties of corn plants to be planted in that split-plot. Finally, this design was replicated four times.
  • the means of percent sterile, plant height, ear height, and days to silking for each combination of treatment and variety are given in Tables V, XV, IX, XXIII, respectively, infra. Each mean is an average of the four replications.
  • the means of percent sterile and days to silking were really "back-transformed" means. That is, each of these two variables was transformed (eq. 1), the average of the transformed values was computed, and then these averages were "back-transformed" to their original scale.
  • Tables XI, XII, XIII and XIV contain the Duncan's multiple range test results for the comparisons of pairs of the sterility treatment means for corn varieties 1, 2, 3, and 4, respectively. Again, all treatment means specified as belonging to the same group, are not significantly different from one another, i.e., the percentage of plants that become sterile in each group of corn plants treated alike, do not differ between groups. Thus, means of treatments with sources 1, 2, 3, and 4 AMS/vector (BI, B6, B4, and B5, respectively) are significantly different from those of treatment with corn extract (no AMS/vector), buffer only, alfalfa extract (no AMS/vector), and untreated plants (B2, B3, B8, and B7, respectively), in varieties 1, 2 and 4. No male sterility was observed in variety 3.
  • Table XVlIl contains the results from a Duncan's multiple range test among the means of corn varieties (i.e., averaged across treatments).
  • Table XVIII as with all subsequent tables of Duncan's multiple range test results, can be interpreted as explained for Table XVII above.
  • Table XVIII indicates that all comparisons of plant height between any two varieties were significantly (P less than 0.05) different. The probability of erroneously concluding that the average plant height for one variety differs significantly from any other is less than or equal to 5 percent or 1 in 20.
  • Ear height appeared to vary with the variety of corn plant.
  • the analysis of variance results show that the probability of erroneously concluding that ear height varies significantly among varieties of corn plants was less than 0.01 percent or 1 in 10,000.
  • Table XXII contains the Duncan's multiple range test results for the corn plant variety means.
  • Table XXII shows that each corn variety was significantly (P less than 0.05) different from each of the other three varieties. The probability that this conclusion is wrong is less than or equal to 5 percent or 1 in 20.
  • Table XXVI contains the results of the Duncan's multiple range test for comparisons of corn plant variety means for days to silking variable. All possible comparisons of the two varieties are significantly (P less than 0.05) different. The probability that this statement is in error is less than or equal to 5 percent or 1 in 20.
  • AMS/vectors (B1, B6, B4, and B5, respectively) showed male sterility, while treatments with corn extract (no
  • AMS/vector AMS/vector
  • buffer AMS/vector
  • alfalfa extract no AMS/vector
  • untreated plants B2, B3, B8, and B7, respectively
  • Ear height was not affected by any of the eight treatments, but appeared to vary significantly with the variety of corn plant. Means of ear height of all four varieties were significantly different from each other. There was no significant differences between treatments for the "days to silking" variable. Both analysis of variance (P less than 0.0001) and Duncan's multiple range test P less than 0.05) revealed a significant varietal difference.
  • Seeds of soybean used in the experiment described herein were Williams-82 variety. The seeds were shipped overnight to the field test site. Seed was stored in a cold room at 38oF until planting. The seed was received in eight batches in seed envelopes designated T1-T8.
  • Alfalfa material was cut fresh from the field and immersed in liquid nitrogen. The liquid nitrogen frozen material was shipped overnight in dry ice to the field test site.
  • Four of these materials (from four male sterile alfalfa lines, PI Nos. 221469, 172429, 223386, and 243223) contained AMS/vector, and one was an alfalfa control (an isogenic non-sterile line).
  • This material arrived in sealed plastic bags, precoded as T1, T2, T3, T4, and T5.
  • a record was kept of the sources for treatments and controls and their corresponding T numbers. Personnel who did the field test were aware only of the 'T' designations, not the nature of the treatments in each case. Those who performed the field test were therefore "blind" to the treatments.
  • GROWTH SYSTEM AND CONDITIONS FOR PLANT GROWTH A sterile, plant growth assembly was used for growing soybeans. The plants were fed a nutrient solution containing macro and micronutrients as follows: 1.08 g
  • the seeds from each packet were emptied into sterilized jars, and surface sterilized by rinsing momentarily with 50 ml of 95% ethanol and then treating for 1.5 minutes with acidified mercuric chloride solution (0.2% HgCl 2 , 0.5% concentrated HCl in water).
  • the mercuric chloride solution was decanted and the seeds rinsed 10 times with sterile distilled water. After the final rinse, the seeds were left to imbibe in sterile distilled water for one hour before planting.
  • Three seeds were planted per pot at 1.5 to 2 cm depth in the soil.
  • the surface of the soil was covered with one inch of sterile aquarium gravel after planting to prevent bacterial and fungal contamination.
  • the entire assembly was wrapped with brown paper to shield the soil and root system from light. Pots were labeled (T1-T8), and transferred to the growth room. A total of 24 pots were planted with each of the eight batches of seed.
  • Germination data were recorded six days after planting. Germination percentages noted for each batch of seed were as follows: T1 - 44; T2 - 42; T3 - 35; T4 - 54; T5 - 47; T6 -39; T7 - 33; T8 - 32. Germination was lower than is usual (usual being greater than 90%), so all seedlings in the pots were retained and no thinning was done.
  • cultivar Williams-82 from DeWine Seed Company, Yellow Springs, Ohio
  • Seeds were planted to achieve a minimum of three seedlings per pot. Seedlings corresponding to the original seed batches were labeled to distinguish the from the additional group of Williams-82 seed. Only seeds of Williams-82 of the additional group were thinned whenever the seedlings exceeded three per pot.
  • a second set. of pots was planted with soybean seeds, as eight batches of seed from packets labelled T1-T8 containing 20 seeds each. These seeds were kept in a cold room at 38'F until planting. Two seeds were planted in each of 80 pots, 10 pots per packet of seeds.
  • the plant growth system and planting procedures were similar to the first planting, except that the seeds in the second planting were not surface sterilized. The seeds in the second planting were not surface sterilized because there were fine cracks in the seed coat from shipping damage, and the sterilization procedure was penetrating the seed through these fine cracks and reducing viability and germination. Germination percentages of the seed in the second planting were as follows: T1 - 80; T2 - 100; T3 - 70; T4 - 80; T5 - 60; T6 - 100; T7 - 90; T8 - 65.
  • pots from the first planting in which none of the original seeds had germinated were discarded.
  • the number of pots discarded were as follows: T1 - 0; T2 - 6; T3 - 7 ; T4 - 1; T5 - 2; T6 - 4; T7 - 5; T8 - 3.
  • EXPERIMENTAL DESIGN AND TREATMENTS The experimental design was a split-plot with replications as the whole plot and treatments as the split. The split-plot design helps reduce error by keeping treatment blocks together. There is still randomization of plants within each treatment, and of treatments within each replication. (A completely randomized design would not separate treatments into blocks).
  • the eight treatments included four materials which contained AMS/vector from different alfalfa genotypes as sources, and various controls.
  • EXTRACTION PROCEDURE Phosphate buffer (KH 2 PO 4 , 0.67 M, pH 6.9) was prepared three days before the extraction of plant material and was kept stored at 11'C. All of the extraction procedures were performed while wearing disposable surgical gloves. A new pair of gloves was used for each treatment to avoid cross-contamination.
  • the frozen alfalfa plant material was taken out of the freezer and weighed. For each extract, 160 g of material was macerated in 800 ml of KH 2 PO 4 buffer (0.067 M, pH 6.9, 11'C), in a waring heavy duty blender for 2-3 minutes. The homogenate was filtered through four layers of sterile cheese cloth to remove the plant debris. The filtrate was collected in sterilized 250 ml centrifuge bottles, and centrifuged at 2,000 rpm for 5 minutes using a GSA rotor in a refrigerated Sorvall centrifuge. The supernatant was decanted into sterile flasks, labeled, and stored at 38oF until used for spraying. The resultant supernatant constituted the extract for spraying the soybean plants.
  • Soybean extract was prepared in the same manner, from soybean plants grown at the field site.
  • Celite (diatomaceous earth, grade III, Sigma Chemicals Cat. No. D5384) was used as an abrador.
  • One hundred grams of Celite was added to 1000 ml of KH 2 PO 4 buffer (0.067 M, pH 6.9, 11oC) in a one gallon garden tank sprayer.
  • the Celite-buffer mix was vigorously shaken, to ensure a uniform dispersion of Celite in the buffer for spraying.
  • the soybean plants were sprayed at a stage when the fifth internode appeared but no floral primordia were visible to the eye. All plants were sprayed first with the Celite buffer mixture. Then plants were taken out of the growth room and sprayed with one treatment (extract) at a time. The six treatments involving plant extracts and the buffer-only control were sprayed using an aerosol spray unit (Sigma Chemicals Cat. No. S4885) and an aerosol propellant refill (Sigma Chemicals Cat. No. A4532). Each soybean plant was sprayed starting from the first node and proceeding up to the shoot tip. The soybean plants were rotated during application. Approximately 25 ml of plant extract (or buffer only) were applied to each plant. Plants in one control treatment (no buffer, no extract) had only Celite applied.
  • Extract preparation and spraying of soybeans in the second planting of the additional seed was performed in the same manner as the first planting.
  • the pots were then coded with the corresponding field site number (B1-B8), using the same code established for the first planting.
  • These pots were arranged in the growth room and regarded as replicates 5 and 6, with eight treatments in each replicate. All plants were staked with garden stakes to keep them upright.
  • SAS Statistical Analysis System
  • the experimental design described above is the familiar randomized-block design in which the eight treatments comprise the treatment main effect and each replicate constitutes a block.
  • Table XXIX contains the overall means of each of the four response variables measured in this investigation, i.e., flower rating, plant height, number of flower nodes, and number of pods.
  • a Duncan's multiple range test was performed at the 0.05 level, to review the pattern or magnitude of the differences between pairs of treatment means.
  • Table XXXII contains the results of the Duncan's multiple range test.
  • the probability of erroneously concluding that the number of pods was significantly affected by the sterility treatments is about 30 percent or 3 in 10 (Table XL).
  • the first pattern showed flowers with anthers that had abundant pollen grains (Fig. 7) which were uniform in size and shape and were stained red with acetocarmine (Fig. 8).
  • the stigmatic surface of such flowers had a mass of pollen grains attached to it (Fig. 9).
  • Soybean is a self- pollinated species and Figures 7-9 show characteristic features that could be seen in a normal soybean flower.
  • the second pattern was characterized by flowers that had normal looking anthers, but the anther contents were a mix of non-stainable, abnormally shaped pollen grains and normal pollen (Fig. 10).
  • the abnormal pollen was of irregular shape, non-stainable and highly vacuolated (Fig. 11).
  • the normal to abnormal ratio varied from flower to flower in this pattern.
  • the third pattern was characterized by flowers that had normal looking anthers, but the anthers lacked any pollen grains (Fig. 12). The stigma on such a flower lacked pollen and stigmatic hairs on its surface. Such anthers, even after being crushed, did not reveal any pollen grains inside them (Fig. 13).
  • a randomized block design was used as the basis for the analysis of variance for flower rating, plant height, number of flower nodes, and number of seed pods in the sections supra.
  • the data could be viewed as a one-way, completely randomized design.
  • the latter approach could be valid if one assumes that there are no significant differences among blocks (replicates) and that differences between treatments are constant from one block to another (i.e., the block-by-treatment interaction is not significant). This was not the case, however, with the results presented in earlier sections.
  • the study described herein demonstrates the inheritance of AMS/vector-induced male sterility into a subsequent generation of corn.
  • the experiment was conducted on a field site at a research station in
  • the objective in testing Set 2 material was to determine if sterility is expressed in subsequent generations of selfed corn plants that originally failed to convert to steriles upon AMS/vector treatment.
  • AMS/vector-induced male sterility in corn was inherited into a subsequent generation of corn. Inbreds 2 and 4 showed more than 80% male sterility in this set.
  • Set 2 AMS/vector-treated plants that produced pollen (fertiles) were selfed.
  • the seed derived from such self-crosses was designated as Set 2.
  • Set 4 Seed was generated, comprising four generations of each of three AMS/vectortreated, male-sterile Inbreds (1, 2 and 4) that were crossed to untreated isogenic lines. This seed, comprising S 2 - S 5 generations, was designated Set 4.
  • Each seed packet was given a treatment designation corresponding to the origin of the seed.
  • a treatment designation of I 1 R 1 B 1 meant Inbred 1, treated with AMS/vector treatment B 1 from replicate 1 of the experiment described in Section 6.9.
  • An example of treatment designation for Set 3 is T I 1 B 1 X UT I 3 corresponding to a cross between AMS/vector-treated (B. treatment) inbred 1 and untreated Inbred 3.
  • Set 4 treatment designations include I 1 S 2 , or I 2 S 4 corresponding to the S 2 generation of Inbred 1 or the S 4 generation of Inbred 2, respectively.
  • Sets 1 to 3 were prepared for ear to row planting with two replicates.
  • Set 4 was also ear to row, but was planted only as one replicate.
  • the seed derived from each ear was counted in two lots of 32 seeds each, and each lot was planted in a replicate. In instances where a single ear did not produce more than 32 seeds, only one replicate was planted.
  • Captan a wettable fungicide (Dragon Chemical Corporation, Roanoke, Virginia).
  • Captan was mixed with water to make a thin paste.
  • Four full tablespoons of Captan were mixed in 1 liter of water, and the solution was kept agitated with a magnetic stirrer.
  • Thirty-two seeds from each packet were emptied into a tea strainer, which was dipped in the Captan-water mix. Excess Captan was strained off, the seed was placed on a paper towel to remove the excess moisture on the surface and was allowed to dry for 2 hours.
  • the Captan-coated seed was then packed in paper bags, previously labeled with the appropriate treatment number. The seed was carefully packed and hand carried to
  • the field site was at Waimanalo research station, which is within the Waimanalo Village boundary in Hawaii.
  • the research station is located at an elevation of 20 meters above the sea level, on a plane three miles from the Pacific Ocean at 21 N latitude.
  • the soil was Vertic Haplustoll derived from coral and lava intrusions and is considered prime farmland.
  • the pH of the soil averages 6.0.
  • Mean annual temperature at the station is 24'C, with monthly averages ranging from 22 to 27'C.
  • Average annual rainfall is 1320 mm but monthly averages range from 10 to 180 mm. Rains are more frequent in winter months. Day lengths range from 10.8 to 13.2 hours. Winds are for the most part continuing and- gentle at 8-15 km/hr but sometimes reach 25-30 km/hr. Incident light values average over 540 cal/cm 2 /day, but can be as low as 220 cal/cm 2/day in cloudy winter months.
  • EXPERIMENTAL DESIGN The design was a randomized complete block design with ear to row planting done in two replicates for Sets 1, 2 and 3. Set 4 was also ear to row, but planted only in one replicate. Within each block or replicate, treatments (ear to row) were completely randomized. Each treatment was planted as a 10 foot row in each replicate. Thirty-two seeds were planted per row, with approximately 4 inch spacing between each seed. Planting within each block was done in tiers, 20 rows per each tier. Within each tier, the rows were separated by 3 foot spacing. The spacing between each tier was alternated as 2 feet, 4 feet, 2 feet, 4 feet, and so on. Where two tiers were separated by 4 spacing, Pioneer hybrid 304C was planted as a cross row. No planting was done when the space between two tiers was 2.
  • One seed packet was assigned for each row according to the designated random number. Planting was done by hand using a push planter. Six persons, each with one planter, participated in planting at a given time. Each individual planted one row at a given time . After each row , the planters were cleaned by hand to ensure removal of dirt, etc. before moving on to plant another row. Planting was done on tier after tier, for example, rows 1-20 in the tier 1 were planted first before moving on to rows 21-40 in the tier 2. All rows in replicate 1 of Set 1 were planted before replicate 2 of Set 1. Set 2 planting commenced only after completion of Set 1 planting. Similarly, Set 3 was planted after completion of Set 2. Set 4 was planted in one tier of 12 rows, treatments being randomized within these 12 rows.
  • the untreated Inbreds 1-4 were planted in rows (one Inbred in each row), each row 100 feet long, to serve as pollen source for crossing. Another four rows of untreated Inbreds 1-4 were planted a week later as pollen source. On three sides of the experimental plots, a six row border was planted with Pioneer 304C. The fourth side was planted to corn four weeks later.
  • VISUAL RATING OF POLLEN Stand counts were made a week after the emergence of corn plants. Plants that were severely dwarfed and/or heavily infested with virus or diseases, and the dead plants were discarded prior to rating. The remaining plants were counted and rated for pollen fertility or sterility.
  • a black cardboard paper was placed under the tassel and the latter was shaken. If the tassels were shedding pollen on the black paper, the tassel was rated as fertile. Those tassels that did not show visible pollen on the black paper were rated as sterile. The fertile tassels were tagged with a red twine and the sterile tassels with a yellow twine. Those tassels that were deemed doubtful as to their pollen shedding were not rated, but were tagged with both yellow and red twines.
  • the pollen rating was done from 8 AM to 12:30 PM every day, and was continued for three weeks. All the 4 sets were surveyed and rated every day, and all the tassels were checked to reconfirm their previous days' ratings. Those tassels with both yellow and red tags were rated as fertile or sterile, when the rating criteria were clearly met. Field observations on sterile tassels of Inbreds 1 and 2 with the yellow- colored tags are illustrated in Figures 14 and 15, respectively. At the end of the rating period, plants with yellow tags (steriles) and plants with red tags (fertiles) and total number of plants were counted in each row.
  • Plant height, ear height, and days to 75% silking were recorded for each row. Plant height was measured on one average plant within a row. Plant height was measured in inches as the height from the ground level to the top of the tassel. Ear height was measured in inches from the ground level to the ear base. Ear height was measured on one average plant within a row. When 75% of the plants within a row showed silks on the ears, the date and month of that particular day was noted. Number of days from planting to 75% silking within a row constituted the number for days to 75% silking.
  • SET 1 Seed was derived from male sterile plants (induced by AMS/vector) crossed with pollen from an untreated isogenic Inbred genotype. Inheritance of AMS/vector-induced male sterility was evident in Inbreds 2 and 4 with more than 80% of the plants being sterile (Tables XLVIII, XLIX). In Inbred 1, only 17% of the total plants were male sterile.
  • Inbreds 2 and 4 formed three, different types of tassels. In the first type, no anthers emerged out of the spikelet and the tassels showed no pollen shedding when they were shaken. Such tassels were rated as sterile. In the second type, only 1-10 anthers emerged out of a tassel, dehisced, and shed pollen. These were rated as fertile. In the third type, all the anthers in a tassel emerged out of the spikelets, dehisced, and shed profuse pollen. These were also rated as fertile. There were only two tassels of each of Inbred 2 and 4 in Set 1, which fell into the latter category.
  • Inbred 1 plants rated sterile had no visible anthers on the tassel and there was no pollen shed. The plants rated fertile had anthers which had emerged out of the spikelet, and exhibited profuse pollen shedding from the anthers. Pollen shedding was delayed in some plants of Inbred 1, which necessitated revision of rating in some instances.
  • 6.12.2.2.1.2 Plants belonging to all the Inbreds in this set which were rated fertile had tassels with anthers dehisced, and shed pollen profusely. Those that were rated sterile had all the anthers enclosed within the spikelet, and exhibited no pollen shedding. 6 .12.2.2.1.3. SET 3. All the plants in Set 3 had tassels with dehisced anthers that shed pollen profusely.
  • Anthers from representative examples of tassels rated fertile or sterile were stained with acetocarmine, and the preparations were examined for differences in the characteristics of anthers and pollen (Figs. 18-22).
  • anthers which remained in the spikelet and failed to dehisce showed abnormal, irregularly shaped pollen, with very little stainable cytoplasm.
  • a few of the undehisced anthers were bulged in the middle and the bulge was filled with normal-looking pollen grains, while the rest of the anther had abnormal pollen.
  • Inbreds 1 and 2 tassels which were rated sterile had undehisced anthers containing abnormal pollen.
  • AMS 1.29 cross between alfalfa AMS/vector 40352 source 1.29 (derived from U.S.D.A. PI No. 223386) and a maintainer plant
  • B73-AMS male-sterile B73Ht variety of Zea 40350 mays L. corn; asexually induced to male sterility by treatment with AMS/vector
  • Mo17-AMS male-sterile Mol7Ht variety of Zea 40351 mays L. corn; asexually induced to male sterility by treatment with AMS/vector
  • A632-AMS male-sterile A632Ht variety of Zea 40349 mays L. corn; asexually induced to male sterility by treatment with

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Environmental Sciences (AREA)
  • Botany (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention se rapporte à des procédés permettant l'induction asexuée de la stérilité mâle héritable et de l'apomixie chez les plantes. La présente invention utilise des facteurs dérivés de certaines plantes qui, lorsqu'on les applique à certaines plantes réceptrices, induisent une stérilité mâle héritable chez la plante réceptrice. Ces facteurs de stérilité mâle transmissibles par voie asexuée, appelés AMS/vecteurs, sont présents dans les extraits de certaines plantes de luzerne stériles mâles, où ils sont associés à un seul acide nucléique dont le poids moléculaire est de 1 x 106 (approximativement) dalton avec une particule de 40 à 110 nanomètre. Les plantes stériles mâles produites par voie asexuée, qui sont obtenues par traitement à l'AMS/vecteur, peuvent être utilisées pour produire de nouveaux composés valables de luzerne, maïs, de soja, de sorgo, de tournesol, de millet, de tomate, et d'autres plantes.
EP19880906709 1987-07-31 1988-07-28 Asexual induction of heritable male sterility and apomixis in plants Withdrawn EP0329736A4 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US224836 1981-01-13
US8050587A 1987-07-31 1987-07-31
US80505 1987-07-31
US22483688A 1988-07-27 1988-07-27

Publications (2)

Publication Number Publication Date
EP0329736A1 true EP0329736A1 (fr) 1989-08-30
EP0329736A4 EP0329736A4 (en) 1991-10-09

Family

ID=26763609

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19880906709 Withdrawn EP0329736A4 (en) 1987-07-31 1988-07-28 Asexual induction of heritable male sterility and apomixis in plants

Country Status (5)

Country Link
EP (1) EP0329736A4 (fr)
AU (1) AU629796B2 (fr)
BR (1) BR8807155A (fr)
DK (1) DK155189A (fr)
WO (1) WO1989000810A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3931969A1 (de) * 1989-09-25 1991-04-04 Max Planck Gesellschaft Dna-sequenz, wunl-gen mit gestutztem promotor sowie verwendung derselben
US5710367A (en) * 1995-09-22 1998-01-20 The United States Of America As Represented By The Secretary Of Agriculture Apomictic maize
GB9610044D0 (en) * 1996-05-14 1996-07-17 Sandoz Ltd Improvements in or relating to organic compounds
US6046385A (en) * 1997-10-09 2000-04-04 Midwest Oilseeds, Inc. Mutant male sterile gene of soybean
US9210847B2 (en) * 2011-05-06 2015-12-15 Kenavis Corporation, Llc Soybean varieties and methods for producing said varieties
CN109089874A (zh) * 2018-08-16 2018-12-28 安徽省农业科学院土壤肥料研究所 芥菜细胞质雄性不育系的选育方法、选育系统、控制方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Plant Molecular Biology, Vol. 10, 1988, pages 489-497, Kluwer Academic Publishers, Dordrecht, NL; T. TURPEN et al.: "On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba", the whole document. *
Proc. Natl. Acad. Sci., Vol. 78, No. 11, November 1981, pages 7043-7046, US; L.K. GRILL et al.: "Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in vicia faba", Abstract, introduction, discussion. *
See also references of WO8900810A1 *

Also Published As

Publication number Publication date
EP0329736A4 (en) 1991-10-09
BR8807155A (pt) 1989-10-17
DK155189A (da) 1989-05-31
WO1989000810A1 (fr) 1989-02-09
DK155189D0 (da) 1989-03-30
AU629796B2 (en) 1992-10-15
AU2255288A (en) 1989-03-01

Similar Documents

Publication Publication Date Title
Tatlioglu Cucumber: Cucumis sativus L.
Kalloo Eggplant: Solanum melongena L.
e Silva et al. Banana breeding program at Embrapa
US20050188437A1 (en) Brassica napus with early maturity (Early Napus) and resistance to an AHAS-inhibitor herbicide
Norman et al. Reproductive biology of Asimina parviflora (Annonaceae)
Sidhu et al. An outlook in hybrid eggplant breeding
Lu et al. Interspecific hybridization between Vitis rotundifolia and Vitis vinifera and evaluation of the hybrids
EP4048064A1 (fr) Procédés de fabrication de graines de cannabis sativa et compositions associées
AU629796B2 (en) Asexual induction of heritable male sterility and apomixis in plants
Potts et al. Reproductive biology and controlled pollination of Eucalyptus-a review
Madhusoodanan et al. Botany, crop improvement and biotechnology of cardamom
Singh et al. Genetic improvement of cauliflower
Driscoll et al. Nuclear male sterility systems in seed production of hybrid varieties
JPH05503836A (ja) 植物類における遺伝性雄性不稔および単為生殖の無性生殖性誘導
Dedio et al. Sunflower
Zarei et al. Biodiversity, germplasm resources and breeding methods.
KR20010101540A (ko) 종간특이적 니코티아나 하이브리드 및 그의 자손
EP1465475B1 (fr) TOURNESOLS RéSISTANTES à L'OROBANCHE
CA2453984C (fr) Resistance a l'infection d'oidium et absence de necrose dans le concombre, cucumis sativus
EP1973397A2 (fr) Nouvelles cucurbitacées
Irwin Male competition and outcrossing rate in a hermaphrodite plant
Jingyang Taigu genic male-sterile wheat
Yu The dwarf curly leaf tomato mutant
Muoki et al. Potential male gametophyte competition among Camellia sinensis genotypes in isolated biclonal seed orchards
US20210259193A1 (en) Cotton variety bx 2116gltp

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE DE FR GB IT

17P Request for examination filed

Effective date: 19890807

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: AGRIPRO BIOSCIENCES INC.

A4 Supplementary search report drawn up and despatched

Effective date: 19910819

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): BE DE FR GB IT

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MAXON, NORMAN PATRICK

Owner name: MAXON, ELLEN JONES

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MAXON, NORMAN PATRICK

Inventor name: MAXON, ELLEN JONES

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19940201