AU629796B2 - Asexual induction of heritable male sterility and apomixis in plants - Google Patents
Asexual induction of heritable male sterility and apomixis in plants Download PDFInfo
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- AU629796B2 AU629796B2 AU22552/88A AU2255288A AU629796B2 AU 629796 B2 AU629796 B2 AU 629796B2 AU 22552/88 A AU22552/88 A AU 22552/88A AU 2255288 A AU2255288 A AU 2255288A AU 629796 B2 AU629796 B2 AU 629796B2
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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
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- 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)
Description
AU-Al -22552/88 (A'Al- PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT') (51) Internatiomal Patent Classification 4 A01Hl11,,O, A01iN 65/00 C12N 15/00, C07H 15/12 A0IH 1/04 (11) International Publication Number: WO 89/ 00810 Al (43) International Publication Date: 9 February 1989 (K~02.209) (21) International Application Number- PCT/US88/02573 (22) International Filing Date: (31) Priority Application Numbers: 28 July 1988 (28.07.88) 080,505 224,836 31 July 1987 (31.07.87) Z7 July 1988 (27.07.88) (81) Designated States: AT (European patent), AU, BP, 8E (European patent), 130, Bi' (OAPI patent), BR, CF ((.API patent), CG (OAPI patent), CH (European patent), CM (OAPI putant), DE (E~uropean patent), DK, Fl, FR (European patent), GA (OAPI patent), GB (European patent), HU, IT (European patent), JP, KP, KR, LK, LU (European patent), MC, MG, ML (QAPI patent), MR (QAPI patent), MW, NL (European patent), NO, RO, SD, SE (European patent), SN (OAPI patent), SU, TD (OAPI patent), TG (QAPI patent).
(32) Priority Dates: (33) Priority Country:.
Sixric 34 DflX~I SEE FC1JO rt ne lirait for amending the NME DETRED~I the event of the receipt 6n r06 ArPCo-V )~vYCA I 4 APR1989 Avenue of the Americas, New York, NY 10036 AUSTRALIAN 1 -1A tif1989 9 7 9PATENT
OFFICE
(54) Title: ASEXUAL I NDUCTION OF HERITABLE MALE STERILITY AND APOMIXIS IN PLANTS (57) Abstract The present invention relates to methods for asexual induction, of heritable male sterility ap~d apomixis in plants, The i-o ntion is dire?,ted to factors derivable from certain plants which, when applied to certain recipient plants, induce heritable male sterility in the recipient. Such asexually transmi~jsible male sterility factors, termed AMS/vectors, are present in extrzcts of certain male sterile alfalfa plants, where they attascediha unqe Ix16(p x)dlo oeua wei~ht nucleic acid and a 40-110 nanometer particle. The asexually generated mile-sterile plants derived by AMS/vector treatmlent can be used to produce new and valuable hybrids of alfalfa, corn, soybean, sorghum, sunflower, millet, tomato, and other plants.
WO. 8 8 WO 89/00810 PCT/US88/02573 ASEXUAL INDUCTION OF HERITABLE MALE STERILITY AND APOMIXIS IN PLANTS TABLE OF CONTENTS 1. Field of the 2. Background of the 3. Summary of the 3.1. 4. Brief Description of the Detailed Description of the Invention......<.
5.1. Source of AMS/Vector. 5.2. Preparation and Application of AMS/Vector 5.3. Plants Inducible to Male Sterility by AMS/Vector 5.4. Use cf AMS/Vector-Induced Male Sterile Plants to Produce Hybrids......
Induction of 5.5.1. Asexual Reproduction in Higher 5.5.2. Use of Apomixis in Breeding....
5.5.3. AMS/Vector Induction of Page 7 7 14 17 17 23 23 24 26 28 29 29 31 32 36 41 43 47 6. 6.1. Screening for AMS/Vector 6.2. Characterization of Nucleic Acids Associated with AMS/Vector Donors......
6.3. Electron Microscopy of AMS/Vector 6.4. Induction of Male Sterility in Alfalfa.
6.5. Induction of Male Sterility in Soybean.
WO 89/00810 PCT/US88/02573 so induced to increase the number of male-sterile parents WO 89/00810 PCT/US88/02573 6.6. Additional Data on Induction of Male Sterility in 6.7. Induction of Male Sterility in Corn....
6.8. Induction of Male Sterility in Other 6.9. Induction of Apomixis in Soybean.......
6.9.1. Hypocotyl and Flower Color.....
6.9.2. Characteristics of Male 6.9.3. Frequency of Male Sterility in
F
2
-F
5 6.9.4. Podding 6.9.5. Seed 6.9.6. Results and Discussion 6.10. Field Test of AMS/Vector Treatments on Methods..
Corn Plants...
6.10.1.
6.10.1.2.
6.10.1.3.
6.10.1.4.
6.10.1.5.
Materials and Corn Seed Source.....
Field Preparation......
Experimental Design....
Corn Collection and Shipment of Treatment and Control Materials......
Preparation and Application of AMS/Vector Treatments and Control 6.10.1.6.
6.10.1.6.1.
6.10.1.6.2.
6.10.1.6.3.
Treatment Materials and their Sources..
Extraction Procedure..........
Application of it WO 89/0( I
II
)810 PCT/US88/02573 -16associated with such extracts, and treated sterile plants.
Ir) I WO 89/00810 PCT/US88/02573 -3- 6.10.1.7. Collection of 66 6.10.1.8. Statistical Analysis of the 67 6.10.2. Results and Discussion......... 68 6.10.2.1. Statistical Analysis.., 68 6.10,2.1.1. Analysis of Pollen 69 6.10.2.1.2. Analysis of Plant Height 83 6.10.2.1.3. Analysis of Ear Height 6.10.2.1.4. Analysis of Days to 97 6.10.2.1.5. Summary of Statistical Significance of Treatment Differences 104 6.10.2.2. Description of Features of 105 6.11. Growth Room Test of AMS/Vector Treatments on Soybean 108 6.11.1. Materials and 109 6.11.1.1. Soybean Seed Source... 109 6.11.1.2. Collection and Shipment oF Test Materials. 109 6.11.1.3. Growth System and Conditions for Plant Growth. 110 6.11.1.4. Planting and Germination 110 6.11.1.5. Experimental Design and 112 6.11.1.6. Preparation and Application of AMS/Vector Treatmen's and Control 113 6.11.1.6.1. Treatments and WO 89/00810 PCT/US88/02573 -17- AirS/vector, as a transmissible Diant delivery or t WO 89/00810 WO 8900810PCT/US88/02573 -4their Sources 6.11.1.6.2. Extraction Procedure 6.11.1.6.3. Application of Extracts.,. 6.11.1.7. Collection of the D)ata.
6.11.11.8. Statistical Analysis of 6.11.2. Results and Discussion 6.11.2,1. Statistical Analysis 6.11.2.*1.1.
6.11.2.1.2.
6.11.2.1-3.
Analysis of Pollen Rating..
Analysis of Plant Height Analysis of the Number of Flowering Nodes Analysis of the Number off Pods...
Summary of Statistical Significance of Treatment Differences.... 113 114 114 115 116 117 117 118 124 129 134 139 3!3.
ill 146 149 149 151 6.11.2.1.4.
6.11.2.1.5.
6.11.2.2. Description of Features of 6.11.2.3. Addendum to Statistica.
An alysis 6.12. Demonstration of the inheritance of AMS/Vector- induced Male Sterility in a Subsequent Generation of Corn 6.12.1. Materials and methods 6.12.1.1. Corn Seed Sources 6.12.1.2. Preparation of Seed for Pl3anting 6.12.1.3. Characteistics of Field WO 89/008 10 PCT/US88/02573 -16- WO 89/00810 WO 8900810PCT/US88/02573 Site 151 6.12.1.4. Field Preparation and Management 152 6,12.1.5. Experimental Design 152 6.12.1.6. Field Planting 153 6.12.1.7. Experimental Parameters 153 6.12.1.7.1. Visual Rating of Pollen 153 6.12.1.7.2. MicroscopAc observations on Anther and Pollen Characteristics... 154 6.12.1.7.3. Ruting for Plant Height, Ear Heig~ht, and Days to "05% Silking 155 6.12.1.8.' Procedure for Crossing. 155 6.12.1.9, Data Collection and Statistical Analysi 156 6.12.2. Results and Discussion 157 6.12,2.1. Inheritance of Male Sterility 157 6.12.2.11. 6.12.2.1.2. Set 159 6.12.2.1.3. $et 2 161 6.1242.1.4. Set 163 6. 12.,2.1.5. Untreated Controls 165 6.12.2.2. Morphological Features of Pollen Fertility and *Sterility 167 6.12.2.2.1. Visual Features, 167 Set 167 6.12.2.2.1,2. Set 2 167 6.12.2.2.1.3. Set 168 WO 89/00810 PCT/US88/02573 -6- 6.12.2.2.1.4. Set 6.12.2.2.1.5. Untreated Controls.
6.12.2.2.2. Microscopic Features 6.12.2.2.2.1. Set 6.12.2,.2.2.2. Set 2 6.12.2.2.2.3. Set 3 6.12.2.2.2.4. Set 4 6.12.2.2.2.5. Untreated Controls 6.12.2.3. Statistical Analysis of the 6.12.2.3.1. Set 6.12.2.3.2.* Sets 2 and 3 6.12.2.3.3. Set 7. Deposits of 168 168 168 168 169 169 169 169 170 170 170 170 171 WO 89/008 PCT/US88/02573 P4 14 A I- 1. I I WO 89/00810 PCT/US88/02573 -7- 1. FIELD OF THE INVENT1UN 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.
2. BACKGROUND OF THE INVENTION 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, 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 WO 89/00810 PCT'/US88/02573 -21- I WO 89/00810 PCT/US88/02573 -8organs 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 selffertilization. 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. 'ts 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. In general, the greatest heterosis is observed when the least related genotypes are crossed together, crosses between unrelated cultivars tend to produce better hybrids than crosses between related cultivars because of the greater genotypic diferences.
Technically, an I 1 hybrid is the result of a cross between any two genetically distinct parent plants, regardless of their state of homozygosity. In the generally accepted connotation of the art, however, an F hybrid is the product of a cross between two homozygous (but genetically distinct) parents or lines, and all F plants resemble one another exactly. The recognized advantages of F 1 hybrids are: a) greater vigor expressed woc i i ij 89/00810 PCT/US88/02573 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 r;sulting hybrid product.
Because many of the plants that breeders want to make hybrids from are monoecious, capable of undergoing self-fertilization as well as crossfertilization, the desired hybridization is difficult to achieve on a reliable basis particularly on a commercial level. Thus, 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.
In the past, male sterility of parental lines has been achieved in a variety of ways, all fraught with a variety of drawbacks. For example, 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. Alternatively, 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 selffertilization. However, this approach can be costly and/or lead to deleterious environmental effects.
i i; WO 89/00810 PCT/US8$/02573 -23- WO 89/00810 PCT/US86/02573 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, Principles of Plant Breeding, John Wiley Sons, New York, 1960, p. 245; Watts, Flower vegetable Plant Breeding, Grower Books, London, 1980, p. 42). Briefly, in some plants, the 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) ij 15 which yields 50s% homozygous male-sterile progeny and Hi heterozygous male-fer:.le progeny. Care must be taken to use only the homozygous male-sterile progeny as maternal parent for the subsequent hybridizations. Car, must also be taken not to allow the heterozygotes to intercross with one another as that will result in homozygous malefertiles, upsetting the system. Overall the approach is not dependable.
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, supga, 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 malesterile since their cytoplasm is derived entirely from the female gamete (from the male-sterile parent). In other words, the CMS trait is maternally inherited.
Many maize 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, et al., 1984, Plant Science S WO 89/00810 PCT/US88/02573 -24- WO 89/00810 PCT/US88/02573 -11- Letters 34:127-134; Pring, et al., 1977, Proc. Natl.
Acad. Sci. U.S.A. 74:2904; Kemble, et al., 1980, Genetics 95:451; Koncz, 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 bhve a high rate of reversion to male fertility.
Yet another type of biological male sterility is sometimes referred to as cytoplasmic-genetic male sterility (see Allard, supra, at pp. 246-247). It 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 F1 progeny carry genes with the power to restore the pollenproducing 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, onion breeding (See, Jones and Davis, 1944, U.S.D.A. Technical Bulletin 874:1-28).
Creation of a new male-sterile parent for productioj of hybrids by means of cytoplasmic male sterility or cytoplasmic-genetic male sterility requires laborious and time-consuming sexual transmission through backcrdssing. 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 snt 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.
However, sexually transmitted cytoplasmic male sterility WO 89/00810 PCT/US88/02573 WO ,9/00810 PCT/US88/O0573 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.
In addition to the time, effort and expense of multiple breeding generations, 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. For example., in 1970, more than 85% of the corn grown in the United States carried the T-strain of CMS cytoplasm due to the success achieved i using CMS lines in the production of hybrid corn.
i! However, in that same year, an epiphytotic of southern 15 corn leaf blight destroyed a large percentage of the corn crop; this disease is caused by race T of Helminthosporium j maydis, an ascomycete which is particularly virulent on plants with CMS-T cytoplasm.
,Because of ihe nherent drawbacks of breeding programs that rely on iexual transmission of cytoplasmic male sterility, workers in the art have sought asexual means for transmitting cytoplasmic factors responsible for male sterility. 0 asexual means is grai'ting. Male sterility has been shown to be graft trans'missible (although it is not expressed until the F 1 generation) in |uch plants as petunias (Frankel, 1956, Science 124:684- 685; Edwardson and Corbett, 1967, Proc. Natl. Acad. Sci.
U.S.A. 47:390-396; Frankel, 1962, Genetics 47:641-646) and alfalfa (Thompson and Axtell, 1978, J. Hered. 69:159-164).
The problem with this approach is that transmission of male sterility is achieved only at low frequency.
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 WO 89/(6810 PICT/JUS88/02573 -26- Other methods of application are possible includinc. hut no- limi+r-a ,in n WO 89/00810 PCT/US88/02573 -13technique has been used by Belliard and Pelletier in tobacco (1980, Eur. J. Cell Biol. 22(1):605). The majo2 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.).
Such an intermediate host is known in the art as a dodder bridge. The major drawback of this approach is that dodder itself 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. The dsRNA is apparentl.y located in spherical bodies, 70 nanome.'rs in diameter, located in the cytoplasm of the plant, much like a virus. The dsRNA was transmitted to a fertile linj 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. Sixty percent of previously male-fertile plants so treated had become male-sterile and now contained the dsRNA characteristic of the original male-sterile plants.
Though successful, grafting, protoplast fusion and use of dodder bridges as means of asexual transmission of cytoplasmic male sterility are laborious and not wellsuited for large-scale operations.
Cytoplasmic sterility has also been induced by mutagenesis, by exposure to ethidium bromide for pearl c~ r- WO 89/00810 PCT/US88/02573 -14- I millet (Burton, G.W. and Hanna, 1976, Crop Science i 16:731-732), and by treatment with EMS for rice (Mallick, 1980, Genet. Agr. 34:207-213).
comprising double-stranded RNAs of molecular weights 1.9 X 6 6 and 0.5 X 10 have been shown to be associated with the mitochondria in a male-sterile cytoplasm of maize, j termed LBN cytoplasm (Sisco, et al., 1984, Plant Science Letters 34:127-134; U.S. Patent No. 4,569,152 by Gracen et al., filed April 26, 1984). Plasmid-like DNAs have also been detected in the mitochondria of source IS1112C male-sterile sorghum cytoplasm (Pring, et 'i al., 1932, Mol. Gen. Genet. 186:180-184).
t 5 3. SUMMARY OF THE INVENTION It is an object of the present invention to provide a means for inducing heritable male sterility in i plants that overcomes the drawbacks of prior art methods for achieving male sterility. It is thus an object of the present invention to provide a rapid asexual method for inducing male sterility that avoids the laborious, expensive and time-consuming aspects of physical i emasculation, chemical treatments, backcrossing in sexual transmission, grafting, protoplast fusion and intermediate host bridging.
It is a further object of the invention to provide an asexual means for inducing heritable male sterility in plants that is adaptable to large scale generation of new lines useful in and of themselves and new parental lines for the commercial production of new and useful hybrids exhibiting heterosis. In this latter regard, it is an object of the invention to provide a means for asexually inducing male sterility that is subsequently inherited by progeny of the male zterile line WO 89/00810 PCT/US88/02573 -28- Recipient plants inducible to male sterility by r- WO 89/00810 PCT/US88/02573 so induced to increase the number of male-sterile parents for commercial scale hybrid production.
It is also an object of the invention to increase genetic diversity among male sterile parental lines used in hybridizations by inducing male sterility in plants which heretofore were available only as male fertile parental lines. A further object of the invention is to so provide hybrids of agronomic, horticultural, forestry and pomological importance with high yields, dis.,se resistance, pest resistance and/or resistance to adverse environmental conditions.
It is a further object of the invention to provide a versatile asexual means for transferring heritable male sterility between plants not only of different species but of different genera, as well as between dicots and monocots.
It is a further object of the invention to provide a means for inducing apomixis in plants which permits the perpetuation of agromically desirable hybrid lines in a more convenient and efficient manner than has j previously been possible with a large number of plant species. Establishment of apomixis allows the development of seed, identical in genetic composition with the female parent, without the necessity for gametic fusion. In this regard, it is an object of the invention to provide a means for inducing apomitic reproduction, which characteristic is inherited by subsequent progeny, thereby avoiding the need for repeated crossings of selected parental lines in order to continuously produce hybrid seed.
These and other objects can be achieved by the materials and methods provided herein. The invention is directed to asexually transmissible male sterility and apomixis factors, AMS/vectors, present in extracts from certain male sterile alfalfa plants. Characteristically I 1 WO 89/00810 PCT/US88/02573 -16associated with such extracts, and treated sterile plants, are an unique isolatable nucleic acid with a molecular weight of about 1 x 10 daltons; and 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. More specifically, the extracts from alfalfa plants displaying the AMS trait, when applied, 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. Remarkably, 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 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 and which I 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 (approx.) dalton nucleic acid and/or 40-110 nm particle, uniquely associated with extracts containing the _I r_ WO 89/00810 PCT/US88/02573 -17- AMS/vector, as a transmissible plant delivery or expression vector system.
3.1. DEFINITIONS The following abbreviations are used herein and shall have the meanings indicated: AMS asexual male sterility CMS cytoplasmic male sterility DNase deoxyribonuclease RNase ribonuclease kb kilobase pair OBS observation; an experimental treatment group REP replication TRT treatment 4. BRIEF DESCRIPTION OF THE FIGURES Fig. 1A is a photograph of an ethidium brormidestained agarose gel in which nucleic acids extracted from alfalfa AMS/vector source 1.29 PI No. 223386) (lane from an untreated fertile alfalfa maintainer (variety Arc) (lane 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. Lane 2 in Fig. 1A is a HindII 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 bromidestained 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/vectcr source 1.29 PI No. 223386) (lane 3) were run. A single band at approximately 3.5 kb WO 89/00810 PCT/US88/02573 -18associated with the AMS trait is seen in Figure 1B, lane i 3, but not in lane 1. Lane 2 in Fig. 1B is a HindIII digest of bacteriophage lambda DNA, as described for Fig.
I 1A..
Fig. 1C is a photograph of an ethidium bromide- Jstained agarose gel in which nucleic acids extracted from corn (variety B73) converted to male sterility by i treatment with AMS/vector source 1.26 PI No.
221469) (lane 1) and fertile corn (variety B73) (lane 2) were run. A single band at appro>imately 3.5 kb associated with the AMS trait is seen in Fig. IC, lane 1, i but not in lane 2. Lane 3 is a HindIII digest of bacteriophage lambda DNA, as described for Fig. 1A.
Fig. ID is a photograph of an ethidium bromidestained agarose gel in which nucleic acids extracted from soybean (variety Williams 82) converted to male sterility by treatment with AMS/vector source 1.36 PI No.
243223) (lane 1) and fertile soybean (variety Williams 82) S(lane 2) were run. A single band at approximately 3.5 kb I 20 associated with the AMS trait is seen in Fig. 1D, lane 1, jI but not in lane 2. Lane 3 is a HindIII digest of bacteriophage lambda DNA, as described for Fig. 1A.
Fig. 1E 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, HindIII digest of bacteriophage lambda DNA (as described for Fig. 1A); lane 2, restorer alfalfa line Indiana Synthetic lane 3, AMS/vector source 1.26 PI No. 221469); lane 4, AMS/vector source 1.36 PI No. 243223); lane 5, alfalfa maintainer L I I I WO 89/00810 PCr/US88/02573 -32- In recent years, however, it has become apparent WO 89/00810 PCT/US88/02573 -19- (variety Arc); lane 6, AMS/vector source 1.29 PI No. 223386); lane 7, AMS/vector source 1.7 PI No. 173733); lane 8, HindIII digest of bacteriophage lambda DNA (as described for Fig. 1A); lane 9, restorer alfalfa line Indiana Synthetic lane 10, AMS/vector source 1.26 PI No. 221469); lane 11, AMS/vector source 1.36 PI No. 243223); lane 12, alfalfa maintainer (variety Arc); lane 13, AMS/vector source 1.29 PI No. 223386); lane 14, AMS/vector source 1.7 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 malesterile 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 4 20 alfalfa plant, U.S.D.A. PI No. 221469. Magnification: S10,000 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 Sof 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, Magnification: 20,000
X.
Fig. 3 (3A, 3B, 3C, 3D) contains photographs of representative microscopic fields depicting the pollen present in anthers frcn tassels containi ng dehisced pollen for varieties 1-4 of Zea mays L. corn plant, from the field test described in Section 6,9, infra.
mu a i i a~: WO 89/00810 PCT/US88/02573 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. SB contains a photograph of a representative microscopic field depicting tne 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 field 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.
U.
wo dU 89/0 II )810 PCT/US88/02573 -21- 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 vacuolation of anthers as shown in Fig. 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.
"ig. 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 mavs 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 i i' i Ir~i WO 89/00810 PCT/US8$/02573 nucleic acid can be used either in conjunction with, or 7- WO 89/00M10 PCT/US88/02573 -22experiment 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 dehinced 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 i~ i WO PCT/US88/02573 -36prevent confounding sterility with inbreeding depression.
I 1 WO 89/00810 PCT/US88/02573 -23anthers 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 antherc 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 6.11, infra. 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.
5. DETAILED DESCRIPTION OF THE INVENTION 5.1. SOURCES OF AMS/VECTOR Nondomestic alfalfa plants (genus Medicago) of iiddle Eastern origin can serve as sources (donors) of AMS/vectors. Plants obtained from the Seed Increase r, WO 89/00810 PCT/US88/02573 -24- Collection, Reno, Nevada (1979-1984) were screened for insect resistance and reduced seed set. Out of approximately seventeen thousand plants, five were selected as bearing tne AMS/vector trait, 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 f 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 106 i daltons (see Section The specific plants which can I 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 Atg-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.
Other sources of AMS/vectors may exist. They may be determined empirically by following the methods of Sectiomsv 5.2. and 5.3.
5.2. PREPARATION AND APPLICATION OF AMS/VECTOR EXTRACTS 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 *-38 WO 89/00810 PCT/US88/02573 WO 89/00810---T---/US88/07--3 WO 89/00810 PCT/S88/02573 buffer 0.067 M KH 2
PO
4 at pH 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 diatomaceous earth such as Celite) or absorption enhancers dimethylsulfoxide or DMSO). The plant material is macerated by any suitable means, 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, 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 Celite) is preferred.
Recipient plants are to be sprayed at a time when they have foliage, but prior to flowering and seed set. For example, soybean plant recipients may be sprayed at ]east 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. In the case of alfalfa, recipients are cut back about two centimeters above the crown; within a twoweek period of time, the alfalfa recipients may have extracts applied to them.
II_
WO 89/00810 PCT/US88/02573 -26- Other methods of application are possible including, but not limited to, tissue culture (suspension of plant tissue in ir-d i containing AMS/vectors), electroporation of th, MS/vectors into protoplasts for vegetable crops) and injection (p for trees).
5.3. PLANTS INDUCIBLE TO MALE STERILITY BY AMS/VECTORS All plants are potentially inducible to male sterility by the AMS/voctor if genetically predisposed to 4| inducibility. ThJi includes monoecious plants and even dioecious plants 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 fenale plant. Without desiring to be bound by the following proposed theory, it is hypothesized that in inducible recipients, all chromosomes carry the recessive allele for inducibility of male sterility mediated by the AMS/vector. For example, if the recessive allele for inducibility of male sterility is denoted to be an inducible recipient, a tetraploid (eig., alfalfa) would have to be in the "rrrr" state while a diploid soy or corn) would have to be in the "rr" state within the nucleus of the cells of the plant,, 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.
WO 89/00810 PCT/US88/02573 These DNA samples are again thawed and centrifuged in +he desk-top Eppendorf centrifuge for 3 W'O 8/00810 PCT/US88/02573 -27- TABLE I.
MONOECIOUS PLANTS INDUCIBLE TO MALE STERILITY BY AMS/VECTORS Grain Crops Cereals Corn Wheat Barley Sorghum Rye Oats Rice Grain legumes Field beans Peas Peanuts Lentils Seed Propagated Ornamentals Petunias Marigolds Fruits Tomatoes Peppers Watermelons Apples Oranges Grapefruits Lemons Limes Forage Crops Alfalfa Onions Peppers Sugar Beets Turnips Broccoli Cabbage Potatoes Industrial Species Poplar Trees Maple Trees Cotton Tobacco Fibre Flax Kelp Oilseeds Soybeans Sunflower Flax Mustard Safflower Rape ~t WO 89/00810 PCT/US88/02573 -28- 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 plar, 4 may be subjected to restriction endonuclease digestion. Fragments unique to the inducible plants may be identified and serve as a template from which t-o make DNA probes. These probes may then be used to screen, via hybridization methodologies, for other recipients (see Maniatis, et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 'w York). Alternatively, probes may be used to identify induced plants where unique nucleic acids are associated with plants exhibiting the AMS trait.
5.4, USE OF AMS/VECTOR-INDUCED MALE STERILE PLANTS TO PRODUCE HYBRIDS 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', 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 Fl offspring. If the
F
1 offspring plants are desirable in and of themselves as, forage crops or ornamentals,' it is not necessary that the Fl hybrids be male-fertile and hence capable of producing seed. Thus it is not necessary to choose a male O 89/00810 PCT/US88/02573 -42- I| gradient. Approximately 0.5-ml fractions were removed from the tops of the tubes, and each fraction was diluted WO 89/00810 PCT/US88/02573 -29parental line that will result in the F. hybrids being male fertile.
However, if the Fl offspring plants are desired to be seed producers, 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 Fl progeny, are considered restorers. By way of illustration for inbred corn lines, B73 is a known restorer of AMS/vector-induced sterile Mo17; Mo17 is a known restorer of AMS/vector-induced sterile B73; and H95 is a known restorer of AMS/vector-induced sterile A632, Generally, the more unrelated two inbred lines are, the more likely one will act as a restorer for the other and vice versa.
As an alternative to 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, 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, et al., 1979, in Advances in Genetics, Vol. 20, Caspari, ed., Academic Press, New York, pp. 127-216).
INDUCTION OF APOMIXIS 5.5.1. ASEXUAL REPRODUCTION IN HIGHER PLANTS Although, as a rule, higher plants routinely reproduce sexually, by way of gametic fusion, there t- I WO 89/00810 PCT/US88/02573 are, among certain types of plants, episodes of various types of asexual reproduction. Some varieties may typically be reproduced asexually by artificial vegetative propagation. This technique is frequently used by plant breeders in plants with poor seed set; it may also be used to eliminate an undesired genetic variability which may result from seed propagation. Vegetative propagation may be achieved by roots, tubers, stolons, rhizomes, stem or leaf cuttings, or tissue culture; those plants obtained in this manner are, absent a mutation, genotypically and phenotypically identical to the parent plant. A number of well-known commercial crops are routinely produced in this manner. For example, stem sections are frequently used in the propagation of sugarcane, which only rarely produces flowers in non-tropical regions. Similarly, roots and tubers are employed in the production of root crops such as cassava, sweet potatoes, potatoes, and yams.
A very different type of asexual reproduction, which does involve setting of seed, is known as apomixis.
In this form of reproduction, which occurs spontaneously, 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. In certain plant species, apomixis is the only form of reproduction, and these plants are known as obligate apomicts. Frequently, however' the apomictic plant will exhibit both gametic and apomictic reproduction, and these plants are referred to as facultative apomicts. In the latter group, the sexual anu asexual processes may operate simultaneously in an individual plant.
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 WO 89/00810 PCT/US88/02573 -44- I i i WO 89/00810 PCT/US88/02573 -31two 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. Finally, in parthenogenesis, the embryo develops directly from an unfertilized egg and may or may not be haploid, depending on the regularity of meiosis which produces the egg. For purposes of the present discussion, the term 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, Southern Pasture Forage Crop Impr.
Conf. Rep. 26:41-43, 1969) and it has been suggested that may be controlled by a single gene (Harlan et al., Bot.
Gaz. 125:41-46, 1964).
5.5.2. USE OF APOMIXIS IN BREEDING When first discovered, apomixis was considered to be a complete barrier to plant breeding. Hybridization beween 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 developiag new varieties or hybrids, would appear to be severely limited. In fact, early workers genera7.iy considered apomixis as an evolutionary "bli.d (ley" (Darlington, The Evolution of Genetic Systems, p. 149 University Press, Cambridge, 1939) because of the potential for reproductive isolation.
I -~LII: L a WO 89/00810 PCT/US88/02573 TABLE IB.
I
i L i- i' I 0 aw WO 89/00810 PCT/US88/02573 -32- In recent years, however, it has become apparent to plant breeders that the phenomenon may have valuable applications in breeding. If apomixis could be controlled completely, a means is provided whereby a producer would have available a system which provides the consistency and reliability of breeding through vegetative organs, but with the convenience of seed propagation. Further, the breeder attains the advantage of being able to experiment with various parental pairings to isolate superior hybrid combinations, and to simultaneously "fix" the heterosis by obtaining a true breeding F 1 This technique could prove particularly valuable in those crops in which hybrid seed production in commercial quantities has been hampered by low seed set due to inadequate pollination. Such important crops include, for example, wheat, soybean and cotton.
5.5.3. 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 chosen male-sterile plants, used as female parents, produced a white flower and a green hypocotyl; WO 89/00810 PCT/US;88/02573 -33the male fertile plant, used as male parent, produced a purple flower and purple hypocotyl. Each of these is controlled by a single gene. 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 l 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. Surprisingly, however, there was virtually no segregation for flower and hypocotyl color, and again, a significant number of the plants in the F, were male sterile which set seed. This pattern is not only contrary to what would be expected in normal sexual reproduction, but is consistent with a pattern which characterizes apomictic seed production, namely: an absence of the expected segregation among progeny of an F 1 hybrid cross; and the occurrence of male sterile progeny which still set seed. This pattern observed was repeated in subsequent F and F 4 generations, although some breakdown was observed in the F 5 generation.
Nonetheless, it appears clear that apomixis, or an apomixis-like phenomenon, is inducible by application of AMS/vector to a susceptible plant. A similar pattern has also been observed in preliminary trials with wheat and corn.
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 l.
-U II C- L~ar, -r II-- I WO 89/00810 PCT/US88/02573 -34necessary to determine the optimum pattern of application for a given type of plant is well within the skill of the experienced plant breeder.
Folloving application of AMS/vector, 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 i 10 plants are allowed to self. The seed is collected and planted, and the phenotypes of the resulting F 2 generation I 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 potental utility as intracellular plant delivery systems, for delivery of bioactive molecules such as nutrients, pesticides, etc. The 1 x 10 (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 i i I. Il,. i. L_ I J i WO 89/00810 PCT/US88/02573 -48plant. The eight genotypes treated are listed in Table Tn.
WO 89/00810 PCT/US88/02573 nucleic acid can be used either in conjunction with, or without, the 40-110 nm particle.
6. EXAMPLES 6.1. SCREENING FOR AMS/VECTOR DONORS Sterile alfalfa lines (obtained from the Seed Increase Collection, Reno, Nevada) were screened for the presence of the AMS/vector by a grafting experiment. Seventeen sterile alfalfa lines were first identified by visual ratings and acetocarmine staining for pollen, and then confirmed as steriles by crossing with alfalfa plants that later proved to be maintainers.
Fifteen grafts for each of the sterile lines were performed, using different maintainer plants as scions (the upper part of the graft). The 255 grafts were placed in a mist chamber, were allowed to flower, and were selfed (by tripping). The seeds were harvested. In no cases was sterility observed in the graft generation, Plants of the next generation were germinated, and rated at flowering for the presence or absence of sterility. The flower was tripped, and rated as 1, 3, 5, or 7, according to the following: 1 no anthers, no dehisced pollen 3 anthers present, no dehisced pollen anthers present, dehisced pollen present 7 anthers present, abundance of dehisced pollen present That is, ratings of 1 or 3 meant the plant was sterile; ratings of 5 or 7 meant the plant was fertile.
In order to confirm plant sterility, attempts were made to self the sterile plants. Sterile plants were also crossed to a different maintainer plant in order to WO :S9/CS10 PCT/US88/02573 -36prevent confounding sterility with inbreeding depression.
The sterile plants were also crossed to restorer line Indiana Synthetic Two criteria had to be satisfied in order to consider the plant an AMS/vector donor: (i) the maintenance of sterility after the cross with an unrelated maintainer plant; and (ii) the productior of fertile progeny after the cross to the restorer line. Out of the 17 sterile lines screened, 5 lines were identified as AMS/vector donors.
The five identified AMS/vector sources were used again in a grafting experiment. Medicago scutellata, an annual cleistogomous 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 6.2. CHARACTERIZATION OF NUCLEIC ACIDS ASSOCIATED WITH AMS/VECTOR DONORS Alfalfa plants which screened positive for the AMS/vector, as discussed in Section 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 6 approximately 1.1 x 10 daltons (about 3.3 to kilobases) and is postulated to be DNA. By the following procedure for DNA and ZNA extraction from whole plants, 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 WO 89/00810 PCT/US88/02573 m WO 89/00810 PCT/US88/02573 -37procedure 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.
To 5 g of plant tissue in a 50 ml centrifuge tube, 10 ml of lx STE extraction buffer, 0.1 M sodium chloride (NaC1), 0.05 M Tris, 0.001 M ethylenediamine trichloroacetic acid (EDTA), pH 7.0, containing 1% mercaptoethanol is added. The tissue is ground in a Tekmar blender for one minute at 4'C. An additional 10 ml of boiling Ix STE extraction buffer containing 1% mercaptoethanol is then added, whereupon the tube is transferred to a 55'C water bath and stirred manually until the temperature reaches At this point, 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 10"C. The resultant aqueous phase is removed to a new tube to which a volume, equal to one-tenth that of the aqueous phase, of cetyltrimethylammonium bromide (CTAB) solution is added, followed by 20 ml of the 24:1 chloroform:isoamyl alcohol mixture. Again, the tube is centrifuged for minutes at 13,000 rpm.
After centrifugation, 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 25"C) 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 -20'C until needed.
Next, the pellet is resuspended in 5 ml of a solution of 50 mM Tris, pH 8.0; 5 mM EDTA; 50 mM NaCl and W I WO 89/00810 PCT/US88/02573 -38- 200 micrograms per ml (hereinafter "ug/ml") ethidium bromide. To this is added 4.4 g of cesium chloride (CsCl). The resulting mixture is centrifuged for minutes at 28,000 rpm in a Beckman J21C rotor, and the clear supernatant fraction is retained.
Three ml of this supernatant are transferred to centrifuge tubes for a Beckman ultracentrifuge with an SW50.1 rotor and adjusted to 1.390 refractive index with CsCl. Two and a half ml of mineral oil are added to balance the tube to 6.1 g per tube. The tube is spun to equilibrium in a SW50.1 rotor for 60 hours at 23'C at 33,000 rpm. From this, the DNA fraction is removed.
The ethidium bromide is removed from the DNA fraction with three extractions of equal volumes of isopropanol equilibrated with 20x SSC, 0.15 M NaCI, 0.015 M sodium cit:ate, 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 -20'C until further use.
The RNA pellet which restlts after the abovedescribed 60 hour spin is resuspended in 0.5 ml of the 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 FNA 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 -20'C until further use, The DNA and RNA samples are thawed. Tubes with RNA are centrifuged for 10 minutes at 4'C at 10,000 rpm in a Beckman J21C rotor. The supernatmnt fractions are poured off. The RNA pellets remaining in the tubes are allowed to dry ,.nder a stream of nitrogen. Each RNA pellet is resuspended in 250 microliters (hereinafter of Tris borate buffer, 0.089 M Tris, 0.089 M boric 0 89/00810 PCT/US88/02573 -52- S normal anthers present; stainable
W
WO 89/00810 PCT/US88/02573 -39acid, 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 -20'C until needed.
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 CsC1 prea,'°,itate is observed. The DNA solutions are frozen at 20'C until needed.
The 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 ul glycerol/dye (bromophenol blue) mixture. The samples are mixed well and stored at -20"C until needed.
The DNA samples are again thawed. They are centrifuged for 10 minutes at 4'C at 10,000 rpm tn a Beckman J2 C rotor, and resulting supernata.t 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 -70%C for onhour.
These DNA samples are thawed once more and centrif,,ged for 10 minutes at 4C at 10,000 rpm in a Beckman J21C rotor. Supernatant fractions are poured off and the remaining pellets are dried under a stream of nitrogen. The pellets are resuspended in 500 ul Tris borate buffer and these mixtures are transferred to 1.5 ul microfuge tubes. To each tube are added a few drops of 0.3M sodium acetate and 1 ml of ethanol. The samples are frozen overnight at WO 89/00810 PCT/US88/02573 -53- 6.9.1. HYPOCOTYL AND FLOWER COLOR WO 89/00810 PCT/US88/02573 L These DNA samples are again thawed and centrifuged in the desk-top Eppendorf centrifuge for 3 minutes.. Supernatant fractions are poured off and pellets are dried under a stream of nitrogen. The pellets are resuspended in 80 ul Tris borate buffer. Twenty ul of glycerol/dye mixture are added and mixed well.
DNA and RNA samples so prepared are dialyzed overnight and run on a 1% agarose, Ix 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.
Photographs of such a gel .rom the experiment described sura is dresented in Figs. 1A, 1B, 1C, and ID.
16 ?ig. lA depicts the 3.5 kb band present in alfalfa K AMS/vector source 1.21 PI No. 223386), and the absence of the 3.5 kb band in fertile untreated alfalfa maintainer (variety Arc) and fertile untreated nonmaintainer (variety Arc). Fig. 1B depicts the 3.5 kb band 20 present in alfalfa (variety Arc) converted to male sterility by treatment with AMS/vector source 1.29 PI No. 223386). Fig. IC depicts the 3.5 kb band K present in corn (variety B73) converted to male sterility i by treatim;ent with AMS/vector source 1.26 (U.S D.A, PI No.
i 25 221469). Fig. ID depicts the 3.5 kb band present in soy i (vatiety Williams 82) converted ic male sterility by treatment with AMS/vector source 1.36 PT 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 (Dtrase, Boehringer Mannheim, 3000 U/mg) or ribonuclease (RNase, Boehringer Mannheim, 3000 U/mg). 20 l of DNase or RNase (10 U/ul in 50 mM WO 89/00810 WO 90810 PCT/US88/02573 -54grains was apparent. The non-stainable pollen varied in size and shape, was hichlv vacuo.ated, and did not have a r WO 89/00810 PCT/US88/02573 -41- NaC1, 50% glycerol) was added to 60 ul nucleic acid sample, and the mixture was placed at 37'C for 15 minutes.
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.
6.3. ELECTRON MICROSCOPY OF AMS/VECTOR PARTICLES 'The following procedure was used to obtain electron micrographs depicting the 40-110 nm particles associated with the AMS/vector 1.29 (PI No. 223386).
All steps were carried out at 4'C or on ice.
Buffer I consisted of: c nM Tris-HCI (pH 0.4 M sucrose, 10 mM KC1, 5 mM MgCl 2 10% glycerol, and mM 2-mercaptoethanol, Buffer II consisted of 50 mM sodium phosphate buffer, pH A sample of plant tissue was homogenized in a Virtis homogenizer in 6 volumes of Buffer I for 2 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 20,000 x g for 20 minutes.
The resulting supernatant was centrifuged at 180,000 x g for 60 minutes in two tubes.
For further purif'.ation, the small, dark-green pellet was resuspended in 1.5 ml of Buffer II and layered onto a 12-42% gradient of sucrose in Buffer II.
Gradients were centrifuged at 35,000 rpm in ar S '41 rotor at 4'C for 75 minutes. There were no visible bands in the I I 1 L WO 89/00810 PCT/US88/02573 -42gradient. Approximately 0.5-ml fractions were removed from the tops of the tubes, and each fraction was diluted to 0.8 ml for determination of OD254. Fractions from the shoulder region at the leading edge of the peak (at about one-third the distance from the top of the tube) were pooled and stored at 4"C overnight. Samples were dialyzed against Buffer II to remove the sucrose, and were then centrifuged in an SW50.1 rotor at 40,000 rpm for minutes.
For negative staining, the small white pellet was suspended in about 0.2 ml of Buffer II. Five microliters of the suspension was placed on a Formvarcoated grid and allowed to sit for about 2 minutes.
Excess liquid was washed off and the grid (sample side down) was floated on a drop of 2% -ranyl acetate for 2 minutes. Excess liquid was washed off.
The sample was examined by transmission electron microscopy. Some vesicle-like particles were seen, several with dense cores, but these did not have sharply defined structures. Some micrographs, taken of the AMS/vector source male-sterile line, appeared ,o depict 40-110 nm particles. Figure 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/vactor.
WO 89/00810 PCT/US88/02573 -56-
II
WO 89/00810 i PCT/US88/02573 -43- 6.4. INDUCTION OF MALE STERILITY IN ALFALFA The experiment described herein demonstrates the asexual induction of male sterility in alfalfa, mediated by 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 dU tributed as 1 of 8 rows in each plot. The 16 treatments and 8 genotypes tested are listed in Tables IA,
IB.
W 089/00810 I: PCT/US88/02573 -57- TABLE IH i L i~ C~j_ WO 89/00810 PCT/US88/02573 -44- TABLE IA.
Code T1 T2 T3 T4 T6 EXPERIMENTAL TREATMENTS OF ALFALFA Treatment Injection, source 1.7 AMS/vector Injection, Source 1.4 AMS/vector Injection, 1.26 AMS/vector Injection, source 1.36 AMS/vector Injection, source 1.29 AMS/vector Injection, Indiana Synthetic (alfalfa restorer) Injection, naintaineri isolated from Arc variety of alfalfa Injection, buffer only (KH 2
PO
4 pH 6.9) Celite application, source 1.7 AMS/vector Celite application, source 1.4 AMS/vector Celite application, source 1.26 AMS/vector Celite application, source 1.36 AMS/vector Celite application, source 1,29 AM;3/vector Celite application, Indiana Synthetic (C) (alfalfa restorer) Celite application, maintainers isolated from Arc variety of alfalfa Celite application, buffer only (KH 2
PO
4 p] 6.9) T8 T9 T11 T12 T13 T14 H T16 WO 89/00810 It PCT/US88/02573 -58- 6.9.6. RESULTS AND DISCUSSION WO 89/00810 PCT/US88/02573 WO 89/00810 PCT/US88/02573 TABLE lB.
ALFALFA GENOTYPES Genotype Number 1 2 3 4 6 7 8 Genotype Descriptions Source 1.26, AMS/vector Source 1.36, AMS/vect-,,,- Source 1.29, AMS/vector Fertile maintainer Fertile maintainer Fertile maintainer Fertile maintainer Indiana Synthetic restorer U.S.D.A. Plant Introduction (PI) Nos. for each AMS/vector source are listed in Table IC.
WO 89/00810 PCT/US88/02573 -46- TABLE IC.
U.S.D.A. PLANT INTRODUCTION NUMBERS OF ALFALFA AMS/VECTOR SOURCES ii i if if iII if
H
AMS/Vector Source Designation 1.4 1.7 1.26 1.36 1.29 Plant Introduction Number 172429 173733 221469 243223 223386 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 2 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.
Each genotype was rated for sterility three iweeks after treatment. Flowers were tripped on emory cloth, and scored as follows: 1 =.no anthers; no pollen 30 3 anthers present; no pollen a anthers present; small amounts of pollen present 7 anthers present; sufficient pollen present WO 89/00810 1 PCT/US88/02573 and 4. Treatments with corn extract (no AMS/vector), hiiffer alone. alfalfa extract (no AMS/vector). and WO 89/00810 PCT/US88/02573 -47- A rating of 1 or 3 was considered a sterile; or 7 was considered a fer ile. Acetocarmine staining was used to confirm the visual ratings.
The results revealed that treatments T1 through T5, and T9 through T13 (all AMS/vector sources) altered the fertility of genotypes 4 through 7 (maintainers) from a 7 down to a 3, converted the fertile maintainers to a sterile state. Analysis of variance indicated that the treatment effects were highly significant at P 0.01, and that the fertility within the treated generation of the maintainers had been affected and was not significantly different from the AMS/vector sources (genotypes As expected, genotype 8 (restorer) remained fertile with all treatments, and genotypes 1-3 (AMS/vector sources) retained sterility with all treatments. Any variation in replication results was not significant.
INDUCTION OF MALE STERILITY IN SOYBEAN The experiment described herein demonstrates the asexual induction of male sterility in soybeans, mediated by the AMS/vector.
The experimental design was a randomized complete block design, a split-plot, as described in Section 6.4, supra. Treatments T1 through TS, and T9 through T13, were as described in Section 6.4. Treatments TA and T14 were injection and Celite application, respectively, of extracts of the particular genotype beinq 3treated. 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 Wit) 89/00810 PCT/US88/02573 -61- TABLE IT.
WO 89/00810 PCT/US88/02573 -48plant. The eight genotypes treated are listed in Table
ID.
TABLE ID.
SOYA GENOTYPES Genotype Number 1 2 3 Variety Name Williams 82 Wells II Century Hobbit Cumberland McCall Traff Maple Presto Fertility was rated as described in Section 6.4, supra.
The results showed that treatment with all AMS/vector sources (T1 through T5; T9 through T13) affected the fertility of Genotypes 1, 2, and 3, with sterility observed in Williams 82, and 17-30% sterility observed in both Wells II and Century.
Analysis of variance indicated a significant treatment effect (P at 0.01). Any variation in replication results was not significant. Genotypes 4-8 were not altered in their fertility. There were no differences observed between treatment by injection or Celite application.
WO 89/00810 PC/US88/0257 PCT/US88/02573 6.10.1.3. EXPERIMENTAL DESIGN r- L_, WO 89/00810 PCT/US88/02573 -49- 6.6. ADDITIONAL DATA ON INDUCTION OF MALE STERILITY IN SOYBEAN Seeds of male (purple flowers; Wells II) and female (white flowers; Williams 82) soybeans were planted.
27 and 35 days later, a total of 186 female and 24 male plants, at approximately the three-internode stage, were sprayed with AMS/vector extract from alfalfa AMS1.4 or AMS1.36 lines. Spray applications were carried out with Celite, in KH 2
PO
4 pH 6.9 (see Section 6.10.1.6.3, infra), 9-10 days and 19 days after the last spraying, 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.
In all cases, all flowers examined produced large amounts of pollen (fertile) or no pollen of any kind (sterile). This "black and white" rating was in contrast to the situation in alfalfa lines in which sterile plants exhibit a range of phenotypes no pollen, reduced Samount of pollen, aborted pollen, or pollen not released from anthers).
The results of the induction of sterility in soybean are presented in Table IE.
I _I k WO 89/00810o PCT/US88/02573 TABLE IE.
AMS/VECTOR-INDUCED S2TRILITY IN SOYBEANS Percent Sterility Observed in Treated Soybean Plant AMS/Vector AMS/Vector Soybean Recipient Source AMS1.36 Source AMS1.4 White female 19/49 35/126 Purple male 2/10 6/14 (43%) The results shown in Table IE indicate that both 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.
6.7. INDUCTION OF MALE STERILITY IN CORN The experiment described herein demonstrates the asexual induction of male sterility in corn. The 3 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, WO 89/00- P"/ Wo 89/00810 PCT/U88/02573 -51respectively, 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.
TABL IF.
CORN GENOTYPES Genotype Number 1 Genotype Name B73* A632* Mo17* Mo17 (Indiana Crop Improvement Association) B73 (Indiana Crop Improvement Association) VA26* H84* ural Experiment 6 7 8 Obtained from the Purdue Agricult station.
Fertility was rated according to the following: 1 deformed anthers present; no pollen 3 normal anthers present; no pollen; no stainability with acetocarmine WO 89/00810 PCT/USSS/02573 gloves were disposed of after the extraction of each mta 4 -t i 4--1 A TJkfA tt"t 4 jI "1 /^Ptf e- 'ft-"Ae WO 89/00810 PCT/US88/02573 -52normal anthers present; stainable pollen present 7 normal anthers present; abundant pollen present The results showed that the fertility of genotypes 1 through 7 was altered with all treatments with AMS/vector sources. The range of sterility conversion was 15-26% for genotypes 1-7, with treatment effects being highly significant (P less than 0.01). There was no observed effect of treatments on genotype 8. Any variation in replication results was not significant.
6.8. INDUCTION OF MALE STERILITY IN OTHER PLANTS Observational tests were conducted to evaluate the inducibility of male sterility mediated by the 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.
6.9. INDUCTION OF APOMIXIS IN SOYBEAN Hybrid crosses were initiated using an AMS/vector-treated, male sterile, white-flowered "Williams-82" soybean line as female parent, and normal pollen producing fertile, purple-flowered "Wells-II" as a male parent, The F 1 generation produced, as expected, consisted entirely of purple flowered, purple hypocotyl plants. This PF generation was selfed to produce plants of the F 2 generation, which were in turn used as the basis of an inheritance study through the F 5 generation. These plants were examined for flower and hypocotyl color, anther and pollen characteristics (male sterility) and podding status.
I-
WO 89/00810 PCT/US88/02573 -53- 6.9.1. HYPOCOTYL AND FLOWER COLOR Plants having a purple hypocotyl had purple flowers and those having a green hypocotyl had white flowers. There was a predominance of purple hypocotyls I 5 and purple flowers in all four generations (F 2
-F
5 Thirty-seven of the 38 plants rated in F 2 38 of i the 39 plants in F 3 39 of the 40 plants in F 4 and 22 of the 32 plants in Fg had purple Ihypocotyls and purple flowers. The percentages of plants l 10 that bore green hypocotyls and white flowers in the F 2
F
3
F
4 and F 5 generations were and 31.2%, respectively.
6.9,2. CHARACTERISTICS OF MALE STERILITY Two flowers from each plant of F 2
-F
5 generations were examined. Anther, pollen and stigma were stained with acetocarmine and examined under a microscope. Two categories of flowers were observed., In one category, characteristics of anther, pollen, and stigma were very typical of descriptions in the literature for fertile soybean flowers (Albertsen and Palmer, Am. J. Bot.
66:253-265, 1979). Dehiscence of anthers was complete, anthers encircled the stigma, and the dehisced pollen from the anthers was deposited on the stigmatic surface.
Occasionally, pollen tubes were seen on the stigmatic surface Pollen was uniform in size and shape, and stained deep red with acetocarmine. Flowers or plants bearing such flowers belonging to this category were designated as bearing normal pollen.
with a few exceptions of partial dehiscence, the anthers did not dehisce, in the second category of flowers. Pollen grains remained in the anther, and could be liberated only when the anther was crushed, Pollen grains were not uniform in size and shape, and looked abnormal. A mixture of stainable and non-stainable pollen I II I WO 89/00810 PCT/US88/02573 -54grains was apparent. The non-stainable pollen varied in size and shape, was highly vacuolated, and did not have a well defined pollen wall. The stainable pollen was round in shape and looked abnormally large (compared to normal dehisced pollen grains). In a number of preparations, pollen wall development was found to be irregular and incomplete. Occasionally cytoplasm was seen oozing out of such pollen grains. No pollen grains were detected in anthers of two plants belonging to F 5 generation. Pollen grains were not seen on the stigmatic surface. Flowers or plants bearing such flowers were designated "malesterile".
Typically, both flowers from each plant fell into either one or the other of these categories, In subsequent descriptions plants were either designated "normal-pollen-bearing" or "male-sterile".
Remarkable consistency was observed in the morphological features of anthers, pollen and stigma in each of these two categories, across all the four,.
generations.
6,9.3. FREQUENCY OF MALE e ,RILITY IN F -F GENERATIONS 2 5- Most of the plants in each generation bore "sterile pollen" and were designated "male-sterile".
Twenty.-nine of the 38 plants rated in F2 29 of the :79 plants in F 3 and 31 of the 40 plants in F 4 (77.5%)pand 18 of the 32 plants in Fg were "malesterile". As evident from this data male sterility in the
F
2 F3' and F 4 generations was higher than in the F generation. These results are summarized in Table IG.
6.9.4A PODDING STATUS All plants rated as "normal-pollen-bearingg Sformed a number of seed-bearing pods across all the four WO 89/00810 PCT/US88/C!573 Li [1 generations (Table IH). A majority of the pods in this category had 3 seeds per pod, with 2 seeds and 1 seed per pod being of rare occurrence.
A number of plants rated as "male-sterile" also 5 formed seed-bearing pods. Ten out of 29 plants in F 2 18 out of 29 plants in F 3 18 out of 31 plants in F 4 and 9 out of 1 .lants in F 5 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.
The rest of the "male-sterile" plants produced a large number of flowers but had no pods. Percentages of "male-sterile" plants that had no pods in F 2
F
3
F
4 and
F
5 generations were 1i, 3, 3 and 6, respectively.
6.9.5. SEED VIABILITY A sample of seed from each of the four generations was collected from the mature pods, and germinated oi 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 generation were not mature when the germination test was conducted.
WO 89/001810 WO 8900810PCT/US88/02573 -56- TABLE 1G.
SUMMARY OF DATA FOR HYPOCOTYT, COLOR, FLOWER COLOR AND MALE STERILITY IN PLANTS BELONGING TO AND F 5
GENERATIONS.
Number of Plants Rated 38 39 40 32 Plants with Purple 37 38 38 22 Hypocotyl and Purple (68,8%) Flowers Plant with Green 1 1 2 Hypocotyl anid (31.2%) White Flowers Plants Showing 9 10 9 14 Normal Pollen (23.71%) (43.8%) Plgints Showing 29 29 31 18 St'erile Pollen (56.2%) *Percentage of total plants rated in parentheses.
I Y WO 89/00810 PCT/US88/02573 WO 8/00810 PCT/US88/02573 -57- TABLE IH RELATIONSHIP+ BETWEEN FLOWER RATING AND PODDING STATUS IN F 2 F PLANTS Generation Number of Plants 38 39 40 32 Rated Plants with 9 10 9 14 normal pollen Plants containing 9 10 (100%) 9 (100%) 14 (100%) pods with seed Plants with 29 29 31 18 sterile pollen Plants containing 10 18 18 9 pods with seed Plants with no pods 4 1 1 1 +Relationship between flower rating and podding status expressed as percentages, shown in parentheses for each of the two categories of normal pollen and sterile pollen rated plants, itemized separately in the two horizontal columns in the table.
Percentage of normal pollen rated plants, Percentage of sterile pollen ratedi pants.
WO 89/00810 PCT/US88/02573 -58- 6.9.6. RESULTS AND DISCUSSION The foregoing results were consistent with a pattern of apomictic reproduction of hybrid seed, in that there has been demonstrated a failure of segregation in the F 2 and subsequent hybrid generations, and setting of seed in male sterile plants. This pattern has been shown to be maintained over several generations. Although not wishing to be bound by any theory, it may be that thti breakdown in apomixis which has been observed reflects an original induction of a facultative type of apomixis, such as occurs routinely in nature among certain types of apomictic plants. However, any reversion back to normal reproduction can be cured by a reapplication of the AMS/vector to the original hybrids.
I 6.10. FIELD TEST OF AMS/VECTOR TREATMENTS ON CORN PLANTS i The examples described herein demonstrate the induction of male sterility, mediated by the AMS/vector, in corn grown under field conditions.
Four treatments involving AMS/vector sterility I sources and four control treatments were applied to corn, grown under field conditions, to evaluate the effect of the AMS/vector (extracted from alfalfa) on induction of male sterility in corn. Four varieties (inbreds) of corn were used in this study to examine variety-AMS/vector interactions. Pollen presence or absence was noted.
Pollen stainability, plant height, ear height, and time to silking were also observed to determine if there might be any other treatment effects such as plant growth stimulation. The statistical significance of treatment differences was assessed using analysis of variance, and Duncan's multiple range tests.
There was a strong treatment effect for the pollen sterility (pollen absence or presence) variable.
III i WO 89/00810 PCT/US88/02573 -59- Analysis of variance showed significant treatment differences at P less than 0.0001 for this character. The analysis of variance also showed a strong variety effect.
Comparison among treatment means was conducted for three varieties of corn (varieties 1, 2, and 4 of Table II, infra), in which there was strong evidence (P less than 0.0001) that corn plant sterility was effected by the treatments used in the experiment. There was no statistically significant evidence of a treatment effect in variety 3. Duncan's multiple range test revealed that the means of treatments with corn extract (no AMS/vector), buffer only, alfalfa extract (no AMS/vector), and untreated plants were significantly different from that of sources 1, 2, 3 and 4 AMS/vector treatments. In variety 1, means for source 4 AMS/vector treatment were different from the rest, while in variety 4, means for source 2, AMS/vector treatment were different from the rest.
The analysis of variance for plant heighindicated significant (P less than 0.0106) treatment differences and also differences among the four varieties of corn. Duncan's multiple range test indicated that all comparisons of plant height between ary two varieties were significantly different at P less than 0.05.
The analysis of variance results for ear height did not reveal any treatment differences for this character. However Duncan's multiple range test revealed that ear height of corn varieties were significantly different from each other.
No treatment effect was evident in days to silking in any of the corn varieties. However, both analysis of variance and Duncan's multiple 'range test revealed a significant difference among the four corn varieties for this character.
Treatments with sources 1, 2, 3, and 4 AMS/vector showed pollen sterility in corn varieties 1, 2,
I
WO 89/00810 PCT/US88/02573 '1 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.
Microscopic examination of one flower from each of the plants with no dehisced pollen (pollen sterility), revealed that varieties 1 and 2 had non-stainable, abnorrrl, and irregular pollen inside their anthers, which never dehisced. Variety 4 did not produce any pollen, nor did the anthers dehisce. Pollen from all plants in variety 3, and from fertile plants of varieties 1, 2, and 4 showed normal, round, stainable pollen typical of untreated corn plants.
6.10.1. MATERIALS AND METHODS 6.10.1.1. CORN SEED SOURCE Four corn varieties (described in Table II) were selected for this study. The varieties were obtained from the Ohio Foundation Seed Company, Croton, Ohio. The four varieties were received and the identities recorded, by field site personnel. The varieties were coded and provided as knowns to the investigating team. The variety codes are listed in Table II.
WO o 89/00810 PCT/US88/02573 -61- TABLE II.
CORN PROJECT VARIETY CODES Corn Seed Source Variety A632Ht, Lot 950, Grade F 1 B73Ht, Lot 4551 ST, 2 Grade 23-21F Lot 150, Grade MF 3 M017Ht, Lot 055, GLade MF 4 SThe seed was stored in the cold room at 38"F Until the time of planting, 6.10.1.2,. FIELD PREPARATION The experiment was conducted in a field site in S2 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 ar consisted of P 2 0 5 (28 lbs.), K 2 0 (39 ibs.), 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 after emergence. A preemergence herbicide, Lasso (Monsanto, St. Louis), was applied to control the weeds.
The field was rototilled at a shallow depth of four inches Sagain 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.
I I i i~ t C; i;s~~ WO 89/00810 PCT/US88/02573 -62- 6.10.1.3. EXPERIMENTAL DESIGN The design used was a split-plot with treatments as the main plot and varietis as splits. There were four replications, with eight trt7tment plots within each replicate and four varieties ru.domly distributed as one of four rows in each plot. Each variety was planted as a 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.
TABLE III.
CORN TREATMENT AND VARIETY RANDOMIZATIONS T7 Tb T3 T8 T4 T5 T2 T1 R1 4231 1243 1234 4213 4132 1324 3142 2341 T8 T4 T7 T2 T T6 T5 T3 R2 1432 4213 1243 2143 3214 4312 4213 2413 T2 T6 T1 T5 T3 T8 T4 T7 R3 1342 3412 4132 1423 4132 2134 2431 3412 T2 T8 T1 T3 T4 T7 R4 4321 1243 4312 4123 3124 1243 2341 3241 The eight treatments used (TI through T8) in each replication are as listed in Table IV.
I
WO 89/00810 PCT/US88/02573 -63- TAB,' IV.
CORN PROJECT TREATMENT CODES Code 2 Treatment Code 1 (Field Site Code) Corn extract (no AMS/Vector) T1 B2 Source 2, AMS/Vector T2 B6 PI No. 172429) Source 1, AMS/Vector T3 B1 PI No. 221469) Alfalfa extract (no AMS/vector) T4 B8 Untreated T5 B7 Source 3, AMS/Vector T6 B4 PI No. 223386) Source 4, AMS/Vector T7 PI No. 243223) Buffer only T8 B3 6.10.1.4, CORN PLANTING All four corn varieties were hand planted. Each variety was planted in a 20 foot row, with 6 inch spacing between each plant forty plants in each row). The varieties were planted in conformance with the randomization chart (Table III).
6.10.1.5. COLLECTION AND SHIPMENT OF TREATMENT AND CONTROL MATERIALS 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 m
U
WO 89/00810 PCT/US88/02573 -64and 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, 1'2429, 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 Ti, 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.
6.10.1.6. PREPARATION AND APPLICATION OF AMS/VECTOR TREATMENTS AND CONTROL TREATMENTS 6.10.1.6.1. TREATMENT MATERIALS AND THEIR SOURCES The alfalfa material received and stored frozen, was used to prepare four extracts containing AMS/vector and one alfalfa extract free of AMS/vector as a control.
Material from four corn varieties were used to prepare a corn extract control. These varieties were untreated plants, planted in the same field as the full experiment, in eight Srows, two rows per variety, each 90 feet long. Freshly collected plant tissue from each of these varieties was used i 25 to prepare the corn extract control treatment. The otter two Scontrol treatments were one in which only buffer (0.067 M
SH
2
PO
4 pH 6.9) was applied, and another in which neither the i buffer nor plant extract was applied (see Table IV, supra) 6.10.1,6.2. EXTRACTION PROCEDURE 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 11'C. All the extraction procedures were done wearing disposable surgical gloves. The r- I WO 89/00810 PCT/US88/02573 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.
6.10.1.6.3. APPLICATION OF EXTRACTS Personnel conducting the field test were unaware of the identity of the treatments, and the treatment plots were coded by them (see Table IV, supra). Thus, the study was "double-blind." 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 KH2PO 4 buffer (0.067 M, pH 6.9, 11'C), 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.
h~~FC_ ^i'Ttwaws M- S WO 89/00810 PCT/US88/02573 I -66- Corn plants were sprayed when the fifth leaf was fully expanded, four weeks after planting. All plants were sprayed i around the whorl (tip of corn plant) with Celite, using the one gallon tank spraye;:. The six plant extracts and the buffer-only control were sprayed around the whorl using the j one gallon tank sprayers. Treatment T5 plants received no Sbuffer and no plant extracts.
6.10.1.7. COLLECTION OF DATA Data collection included five parameters: pollen presence or absence, pollen stainability, plant height (inches), time (days) to 75% silking, and ear height (inches). The parameters plant height, time to 75% silking, and ear height were observed to determine if there might be any other effects of the treatment applications, such as Splant growth stimulation. An average for plant height, days to 75% silking, and ear height was calculated for each row within each treatment plot. Photographs were taken depicting representative appearance of the pollen rating. Data collection for pollen absence or presence began when anthers first appeared on the tassel. An 8-1/2 inch x 11 inch black i paper was placed below the tassel, At the time of polle, shed (7:30 AM 11:30 AM), the plant was shaken twice.
li Plants were tagged (with colored twine) according to their pollen rating as follows: 1 No dehisced pollen orange tag S2 Presence of dehisced pollen yellow tag 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 tassej, with no dehisced pollen from the field was stained with acetocarmina and observed for normal or abnormal appearance. Pollen staining was done by WO 9/00810 PCT/US88/02573 S WO 89/00810 PCT/US88/02573 -67transferring 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 'epresantative normal and abnormal m croscopic fields were also taken.
6.10.1.8. STATISTICAL ANALYSIS OF THE DATA Four dependent variables were analyzed separately using the tests described below. The four data variables were pollen amount, ear height, plant height, and days to silking. The pollen stainability variable was not statistically analyzed because only one flower was stained in ieach category, namely flowers with dehisced pollen or with no dehisced pollen. The data and trends were tabulated for pollen stainability.
Data was analyzed for this split-plot design, with I appropriate error terms using the Statistical Analysis System (SAS) program. Analysis of variance (F statistic) was used to test for statistically significant differences among the eight treatments. Analysis of variance determines if there is a significant difference between what is observed (treatments) and the expected random values (untreated). To draw inferences from any such differences requires that we have replications to enable us to calculate experimental error, and randomization to ensure a valid measure of experimental error. We satisfied both of these requirements by having four replications. The plants were randomized within the treatments and the treatments were randomized within each of the replications. Significance probabilities less than or equivalent to P 0.01 are considered strong evidences in favor of a treatment effect.
Mean separations were done using Duncan's new multiple range test. 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 WO 89/00810 PT/US88/02573 -81r WO 89/00810 PCT/US88/02573 -68treatments into groups that are significantly different from Seach other. For example, assume that treatments Ti, T2, T3 and T4 are all significant at P less than 0.05. Duncan's 1 test allows us to determine if they are equally different 1 5 (one class) or unequally different, T1 and T2 in class A, and T3 and T4 in class B. Treatment means were compared using critical range values.
6.10.2. RESULTS AND DISCUSSION 6.10.2.1. STATISTICAL ANALYSIS 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 15 randomization of plants within each treatment and of treat- K ments within each replication. (A completely randomized i design would not separate treatments into blocks). In our split-plot design, eight "whole" plots were selected so as to i 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.
Five responses to the treatments were measured: number of sterile plants, number of fertile plants, plant height, ear height, and days to silking. Another response variable was created by dividing the number of sterile plants by the total number of sterile and fertile p FAts, This response variable is the percent of plants t-hat are sterile.
Since data expressed as percentages do adhere to the assumption of constant response variation among plants treated alike, a variance stabilizing transformation was applied which allows for a more appropriate statistical WO 89/00810 PCT/US88/02573 82
I
WO 89/00810 PCT/US88/02573 -69analysis. If in this experiment, p represents the percent sterile, then the appropriate transformation is as follows: z sin-l (p 2 (1) where z represents the new response variable used for analysis.
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. the average of the transformed values was computed, and then these averages were "back-transformed" to their original scale.
6.10.2.1.1. ANALYSIS OF POLLEN RATING (TABLES V THROUGH XIV) Male sterility was observed only in treatments B1 (source 1 AMS/vector), B4 (source 3 AMS/vector), B5 (-source 4 AMS/vector) and B6 (source 2 AMS/vector), in varieties 1, 2 and 4. No male sterility was seen in variety 3 (Table V).
0: 0o~ 71 -a 0 01 0 01 00 0 0 00 0 TABLE V- AVERAGE STERILE", RESPONSES FOR EACH STERILITY TREATMENT (1 8) FOR CORN VARIETIES 1 4.
TREATMENT
BI B 2 B 3 B4 B5 B6 B7 B8 ;Corn Extract: Alfalfa Source 1 (No :Source 3 Source 4 :Source 2 ::Extract, (No: :A145/Vector AHSIVector): Buffer AMS/Vector:AHS/Vector :AMS/Vector Untreated :ANS/Vector):
VARIETY:
2 3 4 17.05: 42.10: 0.00: 21.87: 0.00: 0.00: 0. 00: 0- 00: 0.00 0.00 0.00 0.00 15-97: 9-94: 18..
39.11: 37.60: 29.
0.00: 0.00: 0.' 22.16: 20.65: 14.' 10: 3 3: 00: 72: 0.00: 000: 0. 00: 0.00: 0.00: 0.00: 0.00: WO 89/00810 PCT/US88/02573 -71- The analysis of variance results for the percentage of corn plants that were male sterile show that there was a strong (P less than 0.0001) treatment effect. That is, it is highly improbable, less than 1 in 10,000, that the percentage of corn plants that became sterile in each treatment group is the same for every treatment group (Table VI).
TABLE VI.
ANALYSIS OF VARIANCE: TRANSFORMED PERCENT STERILE (ALL FOUR CORN VARIETIES).
SPLIT PLOT ANALYSIS OF CORN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: ASINQ
SOURCE
MODEL
ERROR
CORRECTED
SOURCE
REP
TRT
REP*TRT
VAR
TRT* VAR,
DF
55 72 TOTAL 127 SUM OF SQUARES 8-41029291 0.35193684 8.76222976 MEAN SQUARE 0.15291442 0-00488801 F VALUE 31 .28 PR F 0.0001 ROOT MSE 0. 06997L4 32
R-SQTJARE
0.959835 c. v- 36.9605 AS'ENQ MEAN 0. 18915975 TYPE I SS 0.00753203 4. 61038623 0.10714971 1.80852032 1.-876704 62 F VALUE 0.51 134.74 1.04 123.33 18.28 PR F DF TYPE III SS 0.-6742 0. 0001 0. 4261 0.0001 0. 0001 0.00753203 4.61038623 0.10714971 1.80852032 1.87670462 F VALUE 0.51 134 .74 1.04 123.33 1a. 28 PR F 0.6742 0.0001 0. 4261 0. 0001 0. 0001 TESTS OF HYPOTHESES USING THE TYPE III VZS FOR RLEP*TRT AS AN ERROR TERM
SOURCE
REP
TRT
DF TYPE 111 SS 3 0.00753203 7 4.61038623 F VALUE 0.49 129-08 PR F 0-6916 0-0001 PCT/US88/02573 WO 89/00810 -73- The analysis of variance results also show a strong (P less than 0.0001) variety effect. However, the magnitude of the difference between any two treatment means is not constant from one variety of corn plant to another. This latter effect, known as an interaction effect, suggests that comparisons among treatment means should be conducted within a single variety.
For varieties one, two, and fo,'r there is strong evidence (P less than 0.0001) that corn p,,ant sterility was affected by the treatmen*s used in the experiment. However, there is no statistically significant evidence of a treatment effect in variety three (T1ble VII, VIII, IX, and X).
TABLE V1I.
ANALYSIS OF VARIANCE: TRANSFORM4ED PERCENT STERILE FOR CORN VARIETY 1.
SPLIT PLOT ANALYSIS OF CORN DATA GENERAL LINEAR MODELS PROCEDURE 31 Ii
C
C
DEPEN'DENT VARIABLE: ASINQ
SOURCE
J4ODEL
ERROR
CORRECTED TOTAL SUM OF SQUARES 1.32087578 0.06522039 1.38609617 MEAN SQUARE 0-13208758 7i310573, F VALUE 42-53 PR F 0.0001 ROOT HSE 0.05572910
R-SQUARE
0-952947 C. V.
27-.9215 ASINQ MEAN 0.19959223
SOURCE
REP
TRT
TYPE I SS 0.01163036 1. 3092454 1 IF VALUE 1-25 60.22 PR, F DF TYPE III SS F VALUE 1.25 60.22 PR F 0.3175 0.0001 0.3175 0. 0001 0.01163036 1.30924541 T1ABLE VIII.
ANALYSIS OF VARIANCE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 2.
SPLIT PLOT ANALYSIS OF CORN DATA VAR=-2 GENERAL LINEAR MODELS PROCEDUES DEPENDENT VARIABLE, ASINQ
SOURCE
MODEL
ERROR
CORRECTED TOTYAL SUM OF SQUARES 3-49566819 0-27777750 3-77344568 MEAN SQUARE 0.34956682 0-01322750 F VALUE 26.43 PR F 0.0001 ROOT HSE 0. 11501087
R-SQUARE
0.926386 C. V.
35.1969 ASINQ MEAN 0. 32676 389 SOURCE TYPE I SS 0 .0933759 3.45633060 F VALUE PR F DF TYPE III SS F VALUE PEZ F 0. 4161 0.0001 0-99 37-33 0. 4161 0.0001 0.03933759 3.45633060 0.99 37.33 -j o I- I TABLE IX- ANALYSIS OF VARIANCE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 3.
SPLIT PLOT ANALYSIS OF CORN DATA VAR =3 GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: ASINQ
SOURCE
MODEL
ERROR
CORRECTED TOTAL SUM OF SQUARES 0 0 MEAN SQUARE F VALUE o 99999.99 0 PR F 0.0 ROOT MSE
R-SQLIARE
0.000000
C.V.
99999.9999 ASINQ MEAN 0
SOURCE
REP
TRT
TYPE I SS F VALUE PR 1- DF TYPE III SS F VALUE PR F TABLE X.
ANALYSIS OF VARIANCE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 4.
SPLIT PLOT ANALYSIS IOF CORN DATA *VAR=4 GENERAL LINEAR MODELS PROCEDURE DEPE4DENT VARIABLE: ASfl
SOURCE
MODEL
ERROR
CORRECTED TOTrAL SUM OF SQUARES 1-74120328 0.05296430 1.79416758 MEAN SQUARE 0-17412033 0.00252211 F VALUE 69-04 PR F 0. 0001 ROOT MSE 0-05022061
R-SQUARE
0. 970480
CV.
21. 8082 ASINQ MEAN 0-23028288
SOURCE
Ralu
TRT
TYPE I SS 0.,01968844 1.72151484 F VALUE 2.60 97.51 Pr" F DF TYPE III 55 F VALUE 2.60 97.51 PR F 0-0790 0-0001 0-0790 0-0001 0-01968844 1.72151484 WO 89/00810 PCT/US88/02573 -78i 1 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, 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 (B1, B6, B4, and 10 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.
hWO 89/00810
I~
PCT/US88/02s73 -92- Oi 00 0 00 TABLE XI.
MULTIPLE RANGE TEST FOR VARIABLE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 1. 0 DUNCAN'S DF=21 MSE=-0031057 NUMBER OF MEANS CRITICAL RANGE 2 3 0-0818478 0-0859649 DUNCAN GROUPING* 4 5 0-0888102 0-0904927
MEAN
0,43940 0-42559 0-41104 0-32070 0-00000 6 7 8 0.0918322 0-09,28782 0-0936988 N TREATMENT 4 B6 4 El 4 B4 0-00000 0-00000 0.00000 4 B8 *MEANS WITH THE SAME LETTER ARE NOT SIGNIFICANTLY
DIFFERENT.
00 00
F-
r TABLE XII.
VARIABLE: TRANSFORMEDr DUNCAN' S MULTIPLE RANGES TEST FOR PERCENT STERILE FOR CORN VAR IET'Y 2.
NUMBER OF MEAS CRITICAL RANGE ,2 3 P.l168913 0,17741 .UNCAN GROUPING*
A
A4
A
A
A
A
A
B
B
B
B
B
B
*lUEANS WITH '11E SAME LT.ERi 4 5 0-183282 0-186754
MEAN
0-70608 0.67560 0.6f010 0-57233 0-00000 0-00000 0.00000 0.00000 ARE NOT SIGNIFICANTLY 6 7 0.189519 0.191677 1 TREMENT a 0.1-93371 4 B7 4 B8
DIFFERENT-
ol 0 in NUMBER OFH CRITICAL R TABLE XIII.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 3.
SPLIT PLOT ANALYSIS OF CORN DATA VAR=3 GENERAL LINEAR MODELS PROCEDURE DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE. ASINQ NOTE: THIS TEST CONTROLS THE TYPE I COMPARISONWISE ERROR RATE, NOT THE EXPERIMENTWISE ERROR RATE DF=21 HSE=O EANS 2 3 4 5 6 7 8 ?JGE 0 0 0 0 0 0 0 00 DUNCAN GROUPING* TREATMENT MEAN
A
A
A
A
A
A
A
A
*MEAN WITH THE SAME LEITERE 4 0 4 B6 0 4 B7 o4 B8 ARE NOT SIGNIFICANTLY DIFFERENT.
0 r
I
C. 3 0 c1 0 0 0 00 TABLE XIV.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: TRANSFORMED PERCENT STERILE FOR CORN VARIETY 4.
DF=21 MSA=.0025221 NUMBiER OF MEANS ZaITICAL RANGE 0.0737576 0.0774678 DUNCAN GROUPING* 4 5 0.01300318 0.081548
MEAN
0.49016 6 7 8 0.0827552 0.0836977 0.0844372 N TREATi4ENT 4 B4 0.48661 0. 47171 0.39379 0.00000 0.00000 0.00000 0.00000 *MHEANS WITH THE SAME LETTER ARE NOT SIGNIFICANTLY DIFFERENT.
WO 89/00810 PCT/US88/02573 -83- 6.10.2.1.2. ANALYSIS OF PLANT HEIGHT (TABLES XV THROUGH XVIII) The analysis of variance for plant height indicated that there were significant differences among treatments (P less than 0.0106). This means that the probability that all of the sterility (including control) treatments had no effect on plant height is about 1 percent or 11 in 1,000. In other words, there is strong evidence that the sterility treatments affected the plant height (Tables XV, XVI).
-d 4- '4 TABLE XV- AVERAGE PLANT HEIGHT RESPONSES FOR EACH STERILITY TREA'THENT (1 8) FOR CORN VARIETIES 1 4.
TREATMENT
BI B2 B3 B4 B5 86 B7 8 :Corn Extract: Alfalfa Source 1 (No Source 3 Source 4 :Source 2 ::Extract, (No: :AMS/Vector AKS/Vector): Buffer :AMS/Vector: AMS/Vector: AMISVector Untreated :AMS/Vector): VARIETY: -PLANT HEIGHT 1 61.53:- 65.18: 67-93: 66.68: 69.90: 65-08: 65-23: 70.20: 2 67.13: 70.38: 71.20: 70.83: 74-18: 68,.13: 70.63: 72.43: 3 53-15: 52.67: 57-45:- 57.65: 57.25: 53-53: 53.87: 57.00: 4 72.33: 76.00: 78.08: 78.28: 80.78: 74.75: 76.10: 80.03: Ij A0 TABLE XVI- ANALYSIS OF VARIANCE: PLANT HEIGHT.
REPEATED MEASURES ANALYSIS OP CORN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: PLNTHT SOURCE Di ERROR 7 CORRECTED TOTAL 121 1558, 1750
F
1979 .385 .J43 010 743 SUM OF SQUARES 10279.30242188 614.14812500 10893-.45054638 TYPE I SS 1017.80335938 645-79867188 539.01601562 7999.83648438 76.84789063 MEAN SQUARF 186. £q64 0767 8.-52983507 F VALUE 21.91
DF
3 7 21 3 21 PR F 0-0001 ROOT MSE 2-92058814 TYPE III SS 1017.80335938 645. 79867188 539. 01601562 7999.-836484 38 76.84789063
R-SQUARE
0.,94 3622 F VALUE 39.77 10.82 3.01 312.62 0.43 C. V.
4,~3359 PLNTHT MEAN 67-35859375 PR F 0.0001 0.0001 0. 0003 0.0 0.9840
SOURCE
REP
TRT
REP*ITfr
VAR
TRT* VAR F VALUE 39-77 10-82 3.01 312-62 0-43 FOR REP*TRT F VALUE 13-22 3.59 PR F 0.00(o11 Olj~oo1 0.0003 0.0 0.9840 TESTS OF HYPOTHESES USING THE TYPE III MS SOURCE .DF TYPE III SS REP 3 1017.80335938 TRT 7 645.79867188 AS AN ERROR TERM PR F 0.0001 0. 0106 r i r WO 89/00810 PCT/U588/02573 -86- In order to determine which treatments were significantly different from one another, the multiple comparison procedure known as Duncan's multiple range test was performed. This statistical analysis allowed us to compare pairs of means without increasing the probability of making the mistake of declaring some comparisons to be significantly different, when in fact they were not.
As indicated in Table XVII, all means within the specific grouping are not significantly different from one another, while any two means from different groups can be declared as significantly different with only a 1 in chance of being wrong. Thus, means of treatments with buffer alone, sources 3 and 4 AMS/vector, and alfalfa extract (no AMS/vector) (B3, B4, B5, and B8, respectively) were significantly different from those of treatments with sources 1 and 2 AMS/vector, corn extract (no AMS/vector), and untreated plants (Bl, B6, B2, and B7, respectively).
-0r '0 0 0 TABLE XVII.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: PLANT HEIGHT.
DF=72 MSE=8.52984 NUMBER OF MEANS CRITICAL RANGE 2 3 2.06032 2.16646 DUNCAN GROUPING* 4 5 2.23575 2.28815
MEAN
70.-525 69.913 68.663 68.356 66.456 66.-056 65.369 63 -531 ARE NOT SIGNIFICANTLY 6 7 2.32836 2.36272 N TREATMENT 16 16 B8 16 B3 16 B4 16 B7 16 B2 16 F6 16 B1
DIFFERENT-
8 2.39075
C
C
C
D C
D
D
*MEANS WITH THE SAME LEFTER m r- WO 89/00810 PCT/US88/02573 -88- The overall analysis of variance also showed significant differences (P less than 0.0001) among varieties of corn plants (Table XVI, supra). The probability of erroneously concluding that the plant height response is different for each corn variety is much less than 0.01 percent or 1 in 10,000. In addition, the differences among treatments is the same for each variety of corn.
Table XVIII contains the results from a Duncan's multiple range test among the means of corn varieties averaged across treatments).
WO 89/008 i i 10 PC/US88/02573 -102- TABLE XVIII.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: PLANT HEIGHT.
DF=72 MSE=B..52984 NUMBER OF MEANS 2 3 CRITICAL RANGE 1-45687 1-53192 DUNCAN GROUJPING* MA A 70-041 B 76-509 C 66.463 D 55-.322 WITH THE SAME LETTE ARE NOT SIGNIFICANTLY 4 1.58091 N VARIETY 32 4 32 2 32 1 32 3
DIFFERENT.
WO 89/008 ii 10 PCT/US88/02573 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 6.10.2.1.3. ANALYSIS OF EAR HEIGHT (TABLES XIX THROUGH XXII) The analysis of variance results for ear height show that there were no statistically significant differences among the sterility treatment means. Ear height appeared to be unaffected by any of the eight treatments used in the experiment (Tables XIX, XX).
i I j i i i ii i
I
WO 8010081.O PCT/US88/02573 -104le Is A 13 1 C Ctter iA)XnV MM V 4' TABLE XIX.
AVERAGE EAR HEIGHT RISPONSES FOR EXCH STERILITY TREATMENT fl 81 FOR CORN VARIETIES 1 4.
TR.ATMENT
BB2B3 B4 B5 56B7 B8 :Corn Extract: Alfalfa Source 1 (No .Source'3 Source 4 :Source 2 :Extract, (No: :PAHS/Vector :AHSIVector): Buffer AMS/Vector:MS/Vector :ANS/Vector Untreated A)4S/Vector: VARIETY: EAR HEIGHT 1 25-18: 27-00: 27.55: 26-95: 27-08: 25-60: 25-30: 28.58: 2 29-37: 30.25: 29-15: 32-60: 34.80: 29.90: 29.23: 31.00: 3 18.50: 19.38: 21-7 5: 22.48: 204.50:. 17.75: 18.87: 22.70:- 4 25-68: 28-73: -29-18: 28-37: 34.37: 28-43.: 20-75: 29-37: 0m TABLE XX- ANALYSIS OF VARIANCE: EAR HEIGHT.
REPEATED MEhSURES ANALYSIS OF CORN DATA GEN~ERAL LINEAR MODELS PROCEDURE DEPENDEN4T VARIABLE: EARHT
SOURCE
HGDEL
ERROR
OF SUM OF SQUARES 55 3131-75742187 72 417-17187500 MEAN SQUARE F VALUE PR F R-SQUARE C.V.
56.94104403 5-79405382 9-83 0.0001 ROOT HSE 2.40708409 0.882451 9-0161 EARHT MEAN 26.69765625 CORRECTED TOTAL 127 3548-92929687
SOURCE
REP
TRT
REP*TRT
VAR
TRT* VAR TYPE I SS 225.27460937 251-36117187 435-.85601562 2055. 63023438 163.63539063 F VALUE 12.96 6.20 3-58 118.26 1.34 PR F DP TYPE III SS 0.0001 0.0001 0. 0001 0. 0001 0- 1773 225.27460937 251-36117188 435-.85601563 2055.63023437 ,163.63539063 F VALUE 12.96 6.20 3.58 118. 26 1.34 PR F 0. 0001 0. 0001 0. 0001 0.0001 0. 1773 TESTS OF HYPOTHESES USING THE TYPE III MS FOR REP*TRT AS AN ERROR TERM SOURCE, DF TYPE III SS F VALUE PR F
REP
TRT
225-27460937 251-3J6117188 3.62, 1-73 0. 0300 0-1560 WO 89/00810 I r- i PCT/US88/02573 -93- Even without a strong treatment effect, a Duncan's multiple range test can be useful in uncovering the patterns and relative magnitude of the differences among treatment means. Table XXI presents the results of the Duncan's multiple range test among treatments.
Fw--- TABLE XXI- DUNCAN' S MULTIPLE RANGE TEST FOR VARIABLE: EAR HEIGHT TREATMENT MEANS.
DF=72 tiSE=5.79405 NUMBER OF MEANS rPITICAL RANGE 2 3 1.69807 1.78555 DUNCAN GROUPING*
A
A
B A 3 A B A
B
B C B C B C D C D C D C D C D
D
*MEANS WITH THE SAME LETTER 4 5 1-84265 1.88419 fKEAN 29-1875 27-9125 27.5000 26. 9062 26.3375 25-5375 25.41,87 24-6813 ARE NOT SIGNIFICANTLY 6 1-.91898 N TRaTMHENT 16 16 8 16 84 16 B3 16 82 16 B7 16 B6 16 B!
DIFFERENT.
7 1. 9473 8 1 ,9704 I c~ihc lar~r i i WO 89/00810 PCT/US88/02573 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.
WO 89/00810 PCT/US88/02573 -109multiple range test was used, means of treatments with source I YUU- -It %q I4h 41 Y al f a I n Mc /urAMQ/ ier if r ot I -4ql TABLE XXII.
DUNCAH'Sf MULTIPLE RANGE TEST FOR VARIABLE: EAR HEIGHT VARIETY MEANS.
DF=72 MSE=5.79405 NUMBER OF MEANS 2 3 CRITICAL RANGE 1.20072 1.26258 DUNCAN GROUP ING* MA A 30-7875 B 29.1094 C 26.6531 i) 20.2406 *)MMIS WITlH THE SAME LE.TTER ARE NOT SIGNIFICANTLY 4 1.30295 N VAR 32 2 32 4 32 1 32 3
DIFFERENT-
I I I WO 89/0( i 10 0810 PCT/US88/02573 -97- 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 I in 6.10.2.1.4. ANALYSIS OF DAYS TO SILKING (TABLES XXIII THROUGH XXVI) The analysis of variance results indicate that none of the treatments have any effect on days to silking, but there is a variety effect. The days to silking differed significantly (P less than 0.0001) from one variety of corn plant to another (Tables XXIII, XXIV)..
it. i TABLE XXIII.
AVERAGE OF DAYS TO SILK RESPONSES FOR EACH STERILITY TREATMENT (1 8) FOR CORN PLANT VARIETIES 1-4.
TREATMENT
BI B2 B3 B4 B5 B: 6 B7 B8 :Corn Extract: Alfalfa :Source (No Source 3 :Source 4 Source 2 :Extract, (No: :AMS/Vector AMS/Vector): Buffer :AMS/Vector: AMS/Vector: AMS/Vector :Untreated AMS/Vector): VARIETY: DAYS TO SILK 1 70.75: 70.00: 70.00: 70.00: 70.00: 70.00: 70.00: 70.00: 2 75.00: 75.00: 75.00: 75.00: 75.00: 75.00: 75.00: 75.00: 3 80.00: 80.00: 80.00: 80.00: 80.00: 80.00: 80.00: 80.00: 4 73.00: 73.00: 73.00: 73.00: 73.00: 73.00: 73.00: 73.00:
T
d TABLE XXIV.
ANALYSIS OF VARIANCE: DAYS TO SILK.
REPEATED MEASURES ANALYSIS OF CORN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: SILKD
SOURCE
MODEL
ERROR
CORRECTED TOTAL
DF
55 72 127 SUM OF SQUARES 1672.86718750 5.06250000 1677.92968750 MEAN SQUARE 30.41576705 0.07031250 F VALUE 432-58 PR F 0.0 ROOT MSE 0.26516504
R-SQUARE
0.996983
C.V.
0.3558 SILKD MEAN 74.52343750
SOURCE
REP
TRT
REP*TRT
VAR
TRT*VAR
TYPE I SS 0.21093750 0.49218750 1-47656250 1669.21093750 1.47656250 F VALUE 1.00 1.00 1.00 7913.30 1.00 PK F 0.3979 0.43t5 0.4743 0.0 0.4743 TYPE III SS 0.21093750 0.49218750 1.47656250 1669.21093750 1.47656250 F VALUE 1.00 1.00 1.00 7913.30 1.00 PR F 0. 3979 0.4385 0.4743 0.0 0.4743 TESTS OF HYPOTHESES USING THE TYPE III MS SOURCE DF TYPE III SS REP 3 0.21093750 TRT 7 0.49218750 FOR REP*TRT AS AN ERROR TERM F VALUE PR F 1.00 0.4123 1.00 0.4586 27 WO 89/00810 PCT/US88/02573 -100- Duncan's multiple range test showed no significanit differences between means of treatments (Table XXV).
~I WO 89/00810 PCT/US88/02573 -114- ("untreated") beyond the Celite application common to all
A
TABLE XXV.
DUNCAK'S MULTIPLE RANGE TEST: TREATMENT MEANS FOR VARIABLE, DAYS TO SILK.
RELATED MEASURES ANALYSIS OF CORN DATA GENERAL LINEAR MODELS PROCEDURE DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: SILKD NOTE: THIS TEST CONTROLS THE TYPE I COMPARISONWISE ERROR RATE, NOT THE EXPERIMENTWISE ERROR RATE DF=72 MSE=.0703125 NUMBER OF MEANS CRITICAL RANGE 2 3 0.18706 0.196697 DUNCAN GROUPIN 4 5 0.202987 0.207563 1G* MEAN A 74.68750
A
A 74.50000
A
A 74.50000
A
A 74.50000
A
A 74.50000
A
A 74.50000
A
A 74.50000
A
A 74.50000 TER ARE NOT SIGNIFICANTLY 6 7 0.211395 0.214516 N TREATMENT 16 B1 16 B2 16 B3 16 B4 16 16 B6 16 87 16 88
DIFFERENT.
8 0.21706 *MEANS WITH THE SAME LEI i", I~ ii\- WO 89/00810 PCT/US8S/02573 -102- 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 i WO 89/00810 PCT/US88/02573 -116flowering nodes,. and number of pods per plant. These TABLE XXVI- DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: DAYS TO SILK.
ALPHA=-05 DF=72 MSE=.0703125 N.UMBER OF MEANS 2 3 CRITICAL RANGE 0.132271 0-139086 DUNCAN GROUPING* MA A 80-00000 B 75-00000 C 73.00000 D 70.09375 WITH THE SAME LETTLER ARE NOT SIGNIFICANTLY 4 0-143534 N VAR 32 3 32 2 32 4 32 1
DIFFERENT.
00 00 w0 0h
I-
*rrrmP*rC~~~*-' WO 89/0810 PCT/US88/02573 -104- 6.10.2.1.5. SUMMARY OF STATISTICAL SIGNIFICANCE OF TREATMENT DIFFERENCES Analysis of variance showed significant treatment differences at P less than 0.0001, for the pollen sterility character. Treatments with sources 1, 2, 3, and 4 AMS/vectors (Bl, B6, 84, and 85, respectively) shower male sterility, while treatments with corn extract (no AMS/vector), buffer, alfalfa extract (no AMS/vector), and untreated plants (B2, 83, B8, and 87, respectively) did not.
A strong variety effect was also apparent. In varieties 1, 2, and 4, there was strong evidence (P less than 0.0001) that corn plant sterility was effected by the treatments used.
There was no statistically significant evidence of a treatment effect in variety 3. Since the analysis of variance showed a strong variety effect, comparison of treatment means were conducted for the three varieties i.e., 1, 2, and 4, which showed sterility. Duncan's multiple range test further revealed that treatments with corn extract (no AMS/vector), buffer, alfalfa extract (no AMS/vector), and untreated plants (B2, B3, B8, and B7, respectively) were significantly different from those with sources 1, 2, 3, and 4 AMS/vectors (81, B6, B4, and 85, respectively) for all the varieties. Ia variety 1, means for treatment with source 4 AMS/vector (B5) were different from the rest of the treatment means, while in variety 4, means of treatment with source 2 AMS/vector (B6) were different from the rest of the treatment means. Differences among treatments were apparent for the "plant height" character, both from the analysis of variance data and the Duncan's multiple range test. Significant differences (P less than 0.05) were also revealed for plant height character among varieties using Duncan's multiple range test.
Ear height was not affected by any of the eight Streatments, but appeared to vary significantly with the WO 89/00810 PCT/US88/02573 -105variety of corn plant. Means of ear height of all four varieties were significantly different from each other.
There was no significant differences between treatizents 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.
6.10.2.2. DESCRIPTION OF FEATURES OF STERILITY Treatments with sources 1, 2, 3, and 4 AMS/vector (B1, B6, B4 and B5, respectively) showed plants without any dehisced pollen across all replicates. In tassels which showed no dehisced pollen, the anthers were covered by the glumes. In tassels which showed pollen when shaken on black paper, the anthers were out of the glumes and were easily seen, Plants showing no dehisced pollen were often seen clustered together, a position effect that was observed among all the treatments showing this fe-ture and across the four replicates.
Microscopic examinations of anthers from tassels that have dehisced pollen were made for all four varieties across treatments and replicates. In all four varieties, the pollen in such tassels was characteristically round and uniform with a dense cytoplasm and was stainable with acetocarmine (Fig. However, anthers from tassels that showed'no dehisced pollen, in varieties 1 and 2, showed no dehiscence, and the abundance of abnormal, irregularly shaped, empty-looking pollen was clearly visible through the anther wall under a microscope (Fig. The pollen from these anthers could be released only when considerable pressure was put on the anthers by pressing the coverslip (Figs. SA, 5B). In variety 4, anthers from tassels that had no dehisced pollen did not form pollen grains, with the exception of a few tassels (Table XXVII). Even after WO 89/00810 PCT/US88/02573 -106crushing the anthers, no sporogenous tissue was apparent (Fig. 6).
TABLE XXVII.
MICROSCOPIC RATINGS OF ANMhERS FROM TASSELS THAT WERE RATED FOR "NO DEHISCED POLLEN" IN THE VISUAL RATINGS.
Variety .1 Varlety 2 Variety 4 Total No pollen, Abnormal Total No pollen,. Abnormal Total No pollen, Abnormal Treat- tassels no pollen. no tassels no pollen, no tassels no pollen, no merit examined dehiscence- dehiscence examined dehiscence dehiscence examined dehiscence dehiscence 17 (100%) 14 (100%) 21, (100%) 4 0 i 43 (96%) 44 (92%) 43 (100%) 32 (97%) 22 (100%) 22 (100%) 16 (80%) 13 (81%j 0 4 3 (19%) B6 21 *Tassels showing this feature **Percentage of total tassels.
WO 89/00810 PCT/US88/02573 -108- 6.11. GROWTH ROOM TEST OF AMS/VECTOR TREATMENT ON SOYBEAN PLANTS The examples described herein demonstrate the induction of male sterility, mediated by the AMS/vector, in soybeans.
Four treatments containing AMS/vector and four control treatments were applied to soybeans, four weeks after emergence (before flowering), to test whether the AMS/vector induces male sterility in soybeans. Microscopic examination of anthers and pollen were made to assess the effects of AMS/vector treatment. Plant height, number of flowering nodes, and number of pods were determined for each plant after complet.ng the pollen examination, in order to see if 't;-ere were any other effects of the treatment applications such as plant growth stimulation. The statistical significance of treatment effects was determined using analysis of variance and Duncan's multiple range test.
Male sterile plants were observed in treatments with sources 1, 2, 3, and 4 AMS/vector. Treatment with soybean extract (no AMS/vector) resulted in one male sterile plant out of 41 plants examined. Treatments with buffer alone, alfalfa extract (no AMS/vector), or no treatment, produced ,o male sterile plants. Statistical analysis was performed across all treatments and replicates using a microscopic rating of 1-7 for polie 'sterility. Analysis of variance indicated that the treatment differences were highly significant IP less than 0.0001) for the microscopic rating of pollen sterility, Duncan's multiple range test revealed that the treatment means with sources 1, 2, 3, and 4 AMS/vector were significantly different from those of treatment with buffer alone, alfalfa extract (no AMS/vector), or no treatment. Analysis of variance did not show any treatment differences for plant height, flowering nodes/plant, and n':mber of pods/plant. When Duncan's WO 89/00810 PCT/US88/02573 -122- The probability of making an error in judgment by concluding A- L.-A i, 1^ .01 -1 ILt_.- WO 89/00810 PCT/US88/02573 -109multiple range test was used, means of treatments with source 1 AMS/vector and with alfalfa (no AMS/vector) differed significantly from the means of treatments with source 2 AMS/vector and with no treatment, for plant height variable, and the mean of treatment with source 3 AMS/vector differed from treatment with alfalfa (no AMS/vector) for number of pods/plant. Duncan's multiple range test did not reveal any treatment differences in flowering nodes/plant.
Three distinct patterns were observed during the microscopic examinationr, of flowers: one in which the flowers had normal looking anthers, abundant pollen that was regular in shape and size and was stainable with acetocarmine; a second in which the anthers were normal looking, but had inside pollen that was a mix of abnormal and normal pollen; and a third in which there were no pollen grains in an apparently normal looking anther.
I 6.11.1. M AIALS AND METHODS 6.11.1.1. SOYBEAN SEED SOURCE 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 38'F until planting. The seed was received in eight batches in seed envelopes designated T1-T8.
6.11.1.2. COLLECTION AND SHIPMENT OF TEST MATERIALS Alfalfa material was cut fresh from the field and immrsed 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 coiltr 1 (an isogeniq non-sterile line). This material arrived in sealed plastic bags, precoded as TI, T2, T3, T4, and T5. A 'record WO 89/00810 PCT/US88/02573 -110was kept of the sources for treatments and controls and their corresponding T numbers. Personnel who did the field test were aware only of the designations, not the nature of the treatments in each case. Those who performed the field test were therefore "blind" to the treatments.
j 6.11.1.3. GROWTH SYSTEM AND CONDITIONS FOR PLANT G,.WTH A sterile, plant growth assembly was used f growing soybeans. The plants were fed a nutrient solution containing macro and micronutrients as follows: 1.08 g CaHPO, 0.2 g K 2
HPO
4 0.2 g MgSO 4 0.2 g NaCl, 0.16 g FeCl 3 l 1000 ml water. One ml of trace elements Bo 0.05%; Mn 0.05%; Zn 0.005%; Mo 0.005%; and Cu 0.002% were added.
This nutrient solution was used at one-tenth strength, and supplemented with KNO 3 as the nitrogen source.
The controlled environment growth room for this experiment was set 14 hour day/10 hour night cycle, with a constant tem)perature regime of 25'C, and a light intensity at -2 -1 the plant canopy of 340 umol m s.
J 6.11.1.4. PLANTING AND GERMINATION The seeds from each packet (T1-TB) 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% HgC1 2 0.5% concentrated HC1 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 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), it it
F'O
j 89/00
I
)810 PCT/US88/02573 -111and 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; 47; T6 -39; T7 33; T8 32. Germination was lower than is usual (usual being greater than so all seedlings in the pots were retained and no thinning was done.
Additional seeds of cultivar Williams-82 (from DeWine Seed Company, Yellow Springs, Ohio) were sterilized and planted into pots with less than three seedlings, using the same procedure as above. Seeds were planted to achieve a minimum of three seedlings per pot. Seedlings corresponding to the original seed batches were labeled to distinguish them from the additional group of Williams-82 seed. Only seeds of Williams-83 of the additional group were thinned whenever the seedlings exceeded three per pot.
Subsequently, 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 'terilization 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 T6 100; T7 90; T8 After ger'.nation of the second planting, pots from the first planting in which none of the original seeds had germinated were discarded. The number of pots discarded were :i c I L~ WO 89/00810 PCT/US88/02573 1 't L I awl 100 co i Me m f l WO 89/00810 PCT/US88/02573 -112as follows: T1 0; T2 6; T3 7; T4 1; T5 2; T6 4; T7 5; T8 3.
6.11.1.5. 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).
There were six replications of eight treatments.
The eight treatments (described in Table XXVIII) included four materials which contained AMS/vector from different alfalfa genotypes as sources, and vari-s controls.
WO 89/00810 I I PCT/US88/02573 126- LA r ON COd U CO I 44 i 000 000 0 ID 0 II i-il(LI WO 89/00810 Tre Sou (1 2: Unt: 10 Soy
A
PCT/US88/02573 -113- TABLE XXVIII.
SOYBEAN PROJECT TREATMENT AND CODES atment rce 1, AMS/vector U.S.D.A. PI No.
21469) reated bean extract, no MS/vector Code 1 Field Site Code B6 Code 2 T1 Source 2, AMS/vector PI No.
172429) Source 3, AMS/vector PI No.
223386) Alfalfa extract, no AMS/vector Buffer only, no plant extract Source 4, AMS/vector PI No.
243223) 6.11.1.6. PREPARATION AND APPLICATION OF AMS/VECTOR TREATMENTS AND CONTROL TREATMENT 6.11.1.6.1. TREATMENTS AND THEIR SOURCES The alfalfa material received and stored frozen, was the source material for the four AMS/vector treatments and the alfalfa extract control. The source material for soybean extract was var. Williams grown at the field site.
The other two control treatments involved application of buffer (0.067 M KH 2
PO
4 pH 6.9) only, or no material applied WO 89/00810 PCT/US88/02573 -127- The probability of erroneously concluding that there was a U WO 89/00810 PCT/US88/02573 -114- ("untreated") beyond the Celite application common to all treatments.
6.11.1.6.2. 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 38'F 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.
6.11.1.6.3. APPLICATION OF EXTRACTS Ec" 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 KH2PO 4 buffer (0.067 M, pH 6.9, 11'C) it, 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- WO 89/00810 PCT/US88/02573 -115buffer 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 aprayed 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 i treatment (no buffer, no extract) had only Celite applied.
At the time of spraying, non-field site personnel noted the number on the pots and their corresponding treatment code. Personnel at the field site then relabeled the T1-T8 pots with randomly assigned designat ons B1-B8.
The field site personnel retained the code relating 'B' numbers to numbers (see Table XXVIII). From this point onwards, the study became a "double-blind" test in that no one could become aware of the nature of each treatment without breaking the codes held by separate parties. After the pots were labeled with the field site codes, they were transferred to the growth room and randomized within four blocks (replications).
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 (Bl-B8), using the same code established for the first planting. These pots were arranged in the growth room and regarded as replicates j and 6, with eight treatments in each replicate. All plants were staked with garden stakes to keep them upright.
6.11.1.7. COLLECTON OF THE DATA Data collection includiad four parameters: pollen stainability, plant height (inche:s) at 1,0 days, number of WO 89/00810 PCT/US88/02573 -129- 6.11.2.1.3. ANALYSIS OF THE NUMBER OF FLOWERING NODES (TABLES XXXVI THROUGH XXXVIII) WO 89/00810 PCT/US88/02573 -116flowering nodes, and number of pods per plant. These parameters were assessed for each plant in all of the treatments. Representative microscopic fields depicting pollen assessment were photographed.
Data collection for pollen stainability began when flowers appeared at the second node. Three flowers were chosen at random from each ,lant for the pollen stainability rating. Stamens from the flowers were transferred to a glass slide using tweezers. One drop of acetocarmne stain was placed on the stamens and covered with a cover slip. The cover slip was tapped gently and the stamens were observed under the microscope and rated for proven stainability as follows: Rating 1 No pollen present.
Rating 3 Less than 5% of the pollen present became stained.
Rating 5 5-95% of the pollen present became stained.
Rating 7 96-100% of the pollen present became stained.
Representative photographs of anthers and pollen qualifying for these ratings were taken as the rating proceeded. The microscopic rating for pollen stainability was completed 120 days after planting, and then plant height, number of Zlowering nodes, and number of pods were measured.
6.1'1.1.8. STATISTICAL ANALYSIS OF THE DATA Data was analyzed as a randomized block design.
The statistical program Statistical Analysis System (SAS) was used for the analysis. Analysis of variance (F statistic) was used to test for statistically significant differences among the eight treatments. Significance probabilities less than or equivalent to a P value of 0.01 are considered strong r,, 00 B .r
LI
I
WO 89/00810 PCT/US88/02573 -117evidences in favor of a treatment effect. Mean separations were done using Duncan's new multiple range test. Treatment means were compared using critical range values.
6.11.2. RESULTS AND DISCUSSION 6.11.2.1. STATISTICAL ANALYSIS One of eight (including four control) treatments was randomly assigned to one of eight groups of potted plants. Each group contained six pots with two plants in each pot. The treatments werei sprayed onto each plant individually. This design was replicated six times.
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, flower rating, plant height, number of .flower nodes, and number of pods.
WO W89/008 10 PC/ US88/02573 -131- O: N C cr 0 r,
I
ii i
I
iI i i i j:l WO 89/00810 PCT/US88/02573 -118- TABLE XXIX.
TREATMENT MEANS FOR RESPONSE VARIABLES Number of Flower Plant Flower Number of Treatment Rating Height Nodes Seed Pods B1 5.80503 52.8340 21.4528 11.1837 B2 5.01389 49.7479 22.6667 8.6977 B3 5.56209 59.9784 21.8824 12.9565 B4 4.91026 51.2077 21.5000 9.1628 5.60819 48.4860 21.6491 12.2642 B6 4.57692 54.6000 23.6731 11.2000 B7 5.43902 49.9732 21.6585 9.8378 B8 4.54167 47.4958 21.9167 9.5745 The means of the response variables of flower rating, plant height, number of flower nodes, and number of pods for each combination of treatment and replicate are given in Tables SXXX, XXXIII, XXXVI, and XXXIX, infra.
6.11.2.1.1. ANALYSIS OF POLLEN RATING (TABLES XXX through XXXII) Three separate flowers were chosen from each plant and rated for pollen sterility. An average of these three ratings was calculated for each plant (Table XXX) and was used as the response variable for analysis.
8 I vo i9/00810 PCT/US88/02573 -132- The probability of making an error in judgment by concluding
A
TABLE XXX_.
,FLOWER RATING MEANS FOR EACH COMBINATION OF TREATMNT AND REPLICATE.
TREATKENT
B1 B2 B3 B4 Bs B6 B 7 B8 Alfalfa :Soybean Source 3 Extract, no: Source 4 Source I Extract, no: Source 2 ;Buffer only: AMS/Vector :AHS/Vector AMS/Vector: Untreated MS/Vector :AIIS/Vector AMS/Vector:
REPLI-
CATION :FLOWER
RATING
1 5-89: 5-93: 5_74: 4-94: 5.,73: 4..39: 5.33: 5.08: 2 6_00:_ 5-30: 5-76: 5.48: 5_38: 4.75: 5-44: 4.50: 3 5.81:- 4.67: 5-67:- 4-33: 5,60: 5.44: 4-44: 4.53: 4 5.37: 5_07: 5-67: 5.37: 5-30: 4.67: 5-92: 4.06: 6_00: 5.00: 5_53: 4-11: 6.00: 4.21: 5-95: 5.83: -6 5.80: 4.17:- 5.24: 4-48: 5.44 :3-67: 5.33: 4.00: I I i b"~ WO 89/00810 PCT/US88/02573 -120- The analysis of variance results (Table XXXI) indicated that there was a highly significant treatment effect (P less than 0.0001).
TABLE XXXI- ANALYSIS OF VARIANCE: FLOWER RATING.
RAIJDOHIZED BLOCK ANALYSIS OF SOYBEAN DATA GEN ERA L LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: FRATING SOURCE DF MODEL 47 ERROR 354 CORRECTED TOTAL 401 SUK OF SQUARES 149.71388344 564-80684624 714.52072968 MEAN SQUARE F VALUE 3.18540178 2-00 1.59549957 PR F 0-0002 ROOT HSE 1-26313086 TYPE III SS 15.04480759 78.33462931 52-41762879
R-SQUARE
0.-209530
C.V.
24 .3422 FRATING MEAN 5-18905473
SOUR~CE
REP
TELT
REP*TRT
TYPE I SS 8-27082938 89-02542528 52-41762879 F VALUE 1.04 7-97 0-94 PR F 0-3957 0-0001 0-5719 F VALUE 1.89 7.01 0.94 PR F 0.0961 0. 0001 0-5719 TE=T OF HYPOTHESES USING THE TYPE III MS' FOR REP*TRT AS AN ERROR TERM SOURCE DF TYPE III SS F VALUE PR F REP 5 15.04480759 2-01 0.1015 TRT 7 78,33462931 7.47 0.0001 WO 89/00810 PCT/US88/02573 -122- The probability of making an error in judgment by concluding that there is a treatment effect when, in fact, there is none, is 0.01 percent or about 1 in 10,000. This is strong evidence that the average flower rating is affected by the range of sterility treatments.
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.
TABLE XXXII.
'DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: FLOWER RATING.
DF=354 MSE=1.5955 CELL SIZES ARE NOT EQUAL.
HARMONIC MEAN OF CELL SIZES=49.8324 NUMBER OF MEANS CRITICAL RANGE 2 3 0-502695 0-528615 DUNCJAN GROUJPING* 0-54529-4 0-557823
MEAN
5.6082 5-5621 5.4390 5.0139 6 7 0.568368 0.576988 1 TREATMENT
BI
B3 B7 B2 8 0.584038
C
C
C
C
C
*MEANS WITH THE SAME LETTER ARE NOT 4.9103 4-5769 4-5417
SIGNIFICANTLY
52 B34 52 B6 48 B8
DIFFERENT'.
a~ill bRI r~ P1- r~^~l WO 89/008110 PCT/US88/02573 -124- As indicated in Table XXXII, all means belonging to the same group are not significantly different at the 0.05 level, but comparisons of any two treatment means between groups can be considered significant. That is, the probability of making an error in judgment by concluding that, say, flower ratings for treatment 2 are significantly different than flower ratings for treatment 3 is less than or equal to 5 percent or about 1 in 20. Treatment means of sources 1, 2, 3, and 4 AMS/vector (B6, B8, B2, and B4, respectively) were significantly different from treatment means of buffer only, alfalfa extract (no AMS/vector), and untreated plants (Bl, B3, and B5, respectively) (Table XXXII).
6.11.2.1.2. ANALYSIS OF PLANT HEIGHT (TABLES XXXIII THROUGH XXXV) The analysis of variance results indicated that there was no significant treatment effect on the h-eight of the soybean plants in the experiment (Tables XXXIII, XXXIV).
TABLE XXXIII.
PLANT-.HEIGHT iEANS FOR EACH COMBINATION TREATMENT AND REPLICATE.
TREATMENT
Bi B2 B 3 B4 5 B6 B7 B8 Alfalfa :-Soybean Source 3 Extract, no: Source 4 Source 1 Extract, no: Source 2 :Buffer only: AMS/Vector): AMS/Vector :AS/Vector: Untreated :AMS/Vector :AMS/Vector AMS/Vector:
REPLI-:
CATION PLANT HEIGHT 1 56-06: 57.96:. 57-90: 47.72: 64.58: 56.01: 47.10: 59.38: 2 5FJ-85: 58-07-. 63-19: 56.64: 54.50: 54.73: 57.40: 44.74: 3 58.74: 47.12: 63.05: 64-70: 45.90: 56.83: 59.28: 45.51: 4 58-31:. 49.53: 90-94: 43,.03: 41.80: 60.10: 47.56: 42.28: 41.15: 44.59: 42-50: 41-27: 41.91: 47.84 -45.31: 49.08 6 44.22: 39-53: 46-51: 48.,72: 43-23: 52.70: 45.72: 48.03:
C,,
00 00 kJ
U'
0 TABLE XXXIV.
ANALYSIS OF VARIANCE: PLANT HEIGHT.
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: PLNTHT
SOURCE
MODEL
ERROR
CORRECTED TOTAL SLFl OF SQUARES 32695 .35078091 63799.92105988 96495.27184080 MEAN SQUARE F VALUE 695.64576130 3.86 I80-22576571 PR F 0.0001 ROOT MSE 13.42481902
R-SQUARE
0. 338829 C. V.
25. 8945 PLNTHT MEAN 51.84427861
SOURCE
REP
TRT
REP*TRT
TESTS OF
SOURCE
REP.
TRT
TYPE I SS 8140.72211101 6553.78476375 18000.84390615 F VALUE 9.03 5.19 2.85 PR F 0-0001 0.0001 0. 0001 DF TYPE III SS 5 7676-88729557 7 5546.33526705 35 18000.84390615 F VALUE 8.52 4.40 2.85 PR F 0.0001 0.0001 0. 0001
HYPOTHESES
5 7 USING THE TiPE III MS TYPE III SS 7676.88729557 5546-.33526705 FOP REP*TRT F 'VALUE 2.99 1.54 AS AN ERROR T7ERM PR F 0.0239 0.-1861 ii a; WO 89/00810 PCT/US88/02573 -127- The probability of erroneously concluding that there was a significant treatment effect is about 18 percent (Table
XXXIV).
Again, a Duncan's multiple range test was performed to assess the pattern and magnitude of the pairwise differences between treatment means. Table XXXV contains the results of this test, and indicated that 9 of the possible 28 different pairs of treatment means could be declared significantly different. The probability that this statement is incorrect is less than or equal to 5 percent or about 1 in Thus the overall main effect for treatments on plant height did not appear to be significant.
7C7-S NUMBER OF MEANS CRITICAL RANGE 5.3427 Dl TABLE XXXV- DUNCAN' S MULTIPLE RANGE TEST FOR VARIABLE: PLANT HEIGHT.
DF=354 MSE=180-226 ,ELL SIZES ARE NOT EQUAL.
HARMONIC MEAN OF CELL SIZES=49.8324 2 3 4 5 6 5 5.618235 5.79549 5.92866 6.04073 6.13 LJNCAN GROUP ING* MEAfN N TREATMEN4T A 59.978 51 B3 B 54.600 52 B6
B
C B 52.834 53 Bi C B C B 51.208 52 B4 C B C B 49.973 41 B7 C B C B 49-748 48 B2 7 :233 8 6. 20728
C
C
C
*MEANS WITH THE SAME LEITER ARE NOT 48. 486 47.496
SYGNIFICANTLY
57 48 8
DIFFERENT-
wo 8900810PCT/US88/02573 -129- 6.11.2.1.3. ANALYSIS OF THE NUMBER OF FLOWERING NODES (TABLES XXXVI THROUGH XXXVIII) The analysis of variance results indicated that there was no evitlence for a treatment effect (Tables XXXVI,
XXXVIIT-
H: V
A.
7L TABLE XXXVI.
MEAN NUMBER OF FLOWER NODES FOR EACH COMBINATION OF TREATMENT AND REPLICATE.
TREATMENT
Bi B2 B3 B4 B5 B6 B7 B8 Alfalfa Soybean Source 3 Extract, no: Source 4 Source 1 Extract, no: Source 2 :Buffer only: AMS/Vector): AMS/Vector AMS/Vector: Untreated AMS/Vector AMS/Vector AMS/Vector REPTi- CATION FLOWER NODES 1 2 3 4 6 22.44: 19.25: 24.67: 23.11: 20.0: 19.10: 28.80: 22.44: 18.17: 22.80: 26.50: 21.00: 20.00: 18-57: 23.12: 20.25: 26.40: 23.36: 19.58: 26.20: 19.00: 19.00: 24.75: 20.60: 18.00: 18.44: 22.67: 22.83: 27.33: 21.44: 21.08: 22.25: 24.44: 23.62: 26.09: 26.00: 16.50: 17.00: 31.5G.
19.25: 28.00: 19.17: 22.75: 16.25: 9.00: 17.08: 24.50: 24.50:
CD
0^ 0 00 0 -TABLE XXXVII.
ANALYSIS OF VARIANCE: FLOWER NODES.
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: &.ODES
SOURCE
MODEL
ERKRR
CORRECTED TOTAL
DF
47 354 401 SUM OF SQUARES 4863.83397945 19111.17099567 23975.00497512 MEAN SQUARE 103.48582935 53-98635874 F VALUE 1.92 PR F 0.0005 ROOT MSE 7.3475410D
R-SQUARE
0.202871
C.V.
33.3226 FNODES MEAN 22.04975124
SOURCE
REP
TRT
REP*TRT
TYPE I SS 1403.70252625 191.28914577 3268.84230744 F VALUE 5.20 0.51 1.73 FR F 0.o0001 0.6298 0.0077 TYPF III SS 1475.41938225 259.9rQ06578 3268.84230744 F VALUE 5.47 0.69 1.73 PR F 0.0001 0.6823 0.0077 TESTS OF HYPOTHESES SOURCE DF REP 5 TRT 7 USING THE TYPE III 4HS TYPE III SS 1475.41938225 259.98006578 FOR REP*TRT AS AN ERROR TERM F VALUE PR F 3-16 0.40 0.0186 0.8972 WO 89/00810 PCT/US88/02573 -132- The probability of making an error in judgment by concluding that the number of flower nodes was significantly affected by the treatments used in this study was nearly 90 percent i (Table XXXVII).
A Duncan's multiple range test also showed no significant pairwise differences among treatment means (Table
XXXVIII).
j j i i o ci 01 o 1 0 TABLE XXXVIII.
00 DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: FLOWER NODES.
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GENERAL LINEAR MODELS PROCEDURE DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE. FNODES NOTE: THIS TEST CONTROLS 'THE TYPE 1 COMPARISONWISE ERROR RATE, NOT THE EXPERIMENTWISE ERROR RATE DF=354 MSE=53.9864 CELL SIZES ARE NOT EQUAL. HARMONIC MEAN OF CELL SIZES=49.8324 NUMBER OF MEANS CRITICAL RANGE 8 3.39731 2.92414 3.07492 DUNCAN GROUPINC* 3.17193 3.24482 MEAr 23.673 22.667 21-917 21.882 21.659 21.549 21.500 21,453 3.30615 3.35629
TREATMENT
86 B2 88 83 87 B4 Bl *MEANS WITH THE SAME LETTER ARE NOT SIGNIFICANTLY DIFFERENT I I I II Il WO 89/00810 PCTUS8802573 -134- 6.11.2.1.4. ANALYSIS OF THE NUMBER OF PODS (TABLES XXXIX THROUGH XLI) Analysis of variance results showed no evidence that the number of pods per plant was significantly affected by any of the sterility treatments (Tables XXXIX, XL).
WO 89/00810 PCT/US88/02573 -148- TABLE XXXIX.
HM! NUMBER OF SEED PODS FOR EACH COFIBINATION OF TREATMENIT AND REPLICATE.
TREA114ENT BI B B3 B4 B5 B6 B7 B8 Alfalfa :::Soybean *Source 3 :Extract, no: Zource 4 ::Source 1 Extract, no: Source 2 :Buffer only: AMS/Vecior): AMS/Vector AMS/Vector: Untreated :AMdS/Vector AMS/Vector AMdS/Vector
REPLI-:
CATION NUMBER OF PODS 1 10-33: 14.80: 14-25: 10.l-: 15-70: 6.67: 9.29: 12.25: 2 3 4 13.56: 7-89: 6-8e: 8.25: 7_00; 13.83: 16-25: 12-12: 11-50: 10.50: 8-22: 13.71: 7_57: 5_ 33: -9-0: 14.86: 12.40: 11.71: 9.50: 9.86: 9.50: 24.87: 10.8B6: 8.91: 7.75: 7.00: 10.00: 10.00: 14.43: 7.00: 3.58: 14.25: 8.1 7 6 8 t 7_
Z
TABLE XL- ANALYSIS OF VARIANCE: PODS.
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GEN~ERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: PODS
SOURCE
MODEL
ERROR
CORRECTED TOTAL SUM OF SQUARES 5023-77617479 20942.13686869 25965.91304348 MEAN SQUARE 106-..888854 78 65.44417771 F VALUE 1.63 PR F 0.0080 ROOT MSE 8. 08975758
R-SQUARE
0.193476 C. V.
75.6359 PODS MEAN 10. 69565217
SOURCE
REP
1TRT
REP*TRT
TYPE I SS 1379.83584045 788. 59635545 2855- 34 397889 F VALUE 4-22 -72 1.25 PR F 0. 0010 0-1032 0. 1668 DF TYPE III SS 1348.45376309 724 .46927394 2855.34397889 F VALUE 4.12 1.58 1.25 PR F 0. 0012 0.-1400 0. '668 TESTS OF~ HYPOTHESES USING THE TYPE III MS FOR REP*TRT AS AN ERROR TERM
SOURCE
REP.
TRT
DF TYPE III SS 5 1348-45376309 7 724.46927394 F VALUE 3-31 1-27 PR F 0.0150 0-294 0 i i WO 89/00810 PCT/US88/02573 -137- 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).
A Duncan's multiple range test indicated that only the differences between the means of treatment B2 (source 3 AMS/vector) and treatment B3 (alfalfa extract, no AMS/vector) can be declared significant, with only a percent probability of being wrong (Table XLI).
L
TABLE -LI.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: PODS.
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GENERAL LINEAR MODELS PROCEDURE DUNCAN'S M4ULTIPLE RANGE TEST FOR VARIABLE: PODS NOTE- THIS TEST CONTROLS THE TYPE I COKPARTSONWISE ERROR RATE, NOT THE EXPERIME4TWISE ERROR RATE DF=320 MSE=65-.4442 CELL SIZES ARE NOT EQUAL. HARMONIC MEAN OF CELL SIZES=45.4984 NUM4BER OF MEANS CRITICAL RANGE 2 3.36938
DUNCAN
3 3.54311
GROUPING*
B A B A B A B A B A B A B A B A B A B A B A
B
B
*jJAAS WITH THE SAME LETTER 3.6549 3-7388
MEAN
12.957 ,12.264 11.200 11.184 9-838 9-574 9.163 8-698 ARE NOT SIGNIFICANTLY 6 7 3-63956 3.86132
TREATMENT
53 B6 B7 B8 8 3.91459 43 B4 43 82
DIFFERENT.
mm wa).
r Li~-- WO 89/00810 PCr/US88/02573 -139- 6.11.2.1.5. SUMMARY OF STATISTICAL SIGNIFICANCE OF TREATMENT DIFFERENCES Treatment differences were highly significant (P less than 0.0001) for the microscopic rating of pollen sterility based on the analysis of variance data. Duncan's multiple range test, performed to review the magnitude of differences between pairs of treatments, revealed that treatment means of sources 1, 2, 3, and 4 AMS/vector (B6, B8, B2, and B4, respectively) were significantly different from treatment means of buffer alone, alfalfa extract (no AMS/vector), and untreated plants (Bi, B3, and respectively).
Analysis of variance results indicated no significant treatment effects on the height of the soybean plant. However, Duncan's multiple range test revealed that means of treatments B6 (source 1 AMS/vector) and B3 (alfalfa extract, no AMS/vector) significantly differed from the means of treatments B8 (source 3 AMS/vector) and B5 (untreated).
There were no significant treatment effects on the number of flowering nodes/plant, either by analysis of variance or Duncan's multiple range test. There was also no evidence that the number of pods was significantly affected by any treatment from the analysis of variance test.
However, Duncan's multiple range test revealed differences between means of treatments B2 (source 3 AMS/vector) and B3 (alfalfa extract, no AMS/vector).
6.11.2.2. DESCRIPTION OF FEATURES OF STERILITY Male sterile plants were observed in treatments with sources 1, 2, 3, and 4 AMS/vector (B6, B8, B2, and B4, respectively). In treatment with soybean extract (no AMS/vector) (B7) only one out of 41 plants was male sterile.
Treatments with buffer alone, alfalfa extract (no AMS/vector), and untreated plants (81, B3, and Srespectively) had no male sterile plants (Table XLII).
WO 89/00810 PCT/US88/02573 -140- TABLE XLII.
SOYBEAN-OVERALL STERILITY PROFILE ACROSS SIX REPLICATES I Plants Examined Sterile* Plants Treatment B1 B2 B3 B4 B6 B7 B8 6 (12%) 0 6 (12%) 0 12 (23%) 1 10 (21%) Fertile Plants 53 (100%) 42 (88%) 51 (100%) 46 (88%) 57 (100%) 40 (77%) 40 (98%) 38 (79%) *Plants had flowers with predominantly 1 rating (no pollen) and a few where less than 5% of the pollen became stained (3 rating). Sterile plants did not have pods.
**Actual number of plants, with percentages in parenthesis.
Representative patterns of sterility observed during microscopic ratings of flowers from treated plants (Figs. 7- 13) revealed three distinct patterns.
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.
The stigmatic surface of such flowers had a mass of pollen grains attached to it (Fig. Soybean is a selfpollinated 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 L WO 89/00810 PCT/U588/02573 -154- WO 89/00810 PCT/US88/02573 -141irregular 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).
6.11.2.3. ADDENDUM TO STATISTICAL ANALYSIS 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. Alternatively, 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 the block-by-try stment interaction is not significant). This was not the case, however, with the results presented in earlier sections. The analysis of variance results for each of the four dependent variables showed statistically significant (P less than 0.05) blockto-block variation, although this effect was much weaker (P 0.085) for flower rating than for the other response variables.
By "pooling" the effects due to blocks and blockby-treatment interaction with the residual sum of squares and using the latter as the "appropriate" term to test for treatment effects, the power of detecting a treatment effect will be reduced. This is true if there is significant block-to-block variation and/or if there is a significant block-by-treatment interaction.
-1 I I I WO 89/00810 PCT/US88/02573 -142- One may consider that blocks (replicates) 5 and 6 were different from blocks 1 and 4, because blocks 5 and 6 were planted later. Plants in these blocks, therefore, were in an earlier stage of development at the end of the experiment than plants in blocks 1-4, and were sprayed when they were one week older than plants in blocks 1-4. A reanalysis of the flower rating data, excluding blocks 5 and 6, was therefore performed (Table XLIII).
WO 89/00810 PCT/US88/02573 -156- TABLE XLIII.
ANALYSIS OF VARIANCE: F LOWER RATING (1-4 REPLICATES).
RANDOMIZED BLOCK ANALYSIS OF SOYBEAN DATA GENERAL LINEAR MODELS PROCEDURE DEPENDENT VARIABLE: FRATING
SOURCE
MODEL
ERROR
CORRECTED TOTAL
DF
31 250 281 SUM OF SQUARES 85-~00052705 383.16810967 468.16863672 TYPE I SS 3.34081032, 49-73290209 31.92681464 MEAN SQUARE 2-.74195249 1-53267244 F VALUE .1-79 PR F 0. 0084 ROOT MSE 1-23601149
R-SQUARE
0. 181560 C. V.
23. 8036 FRATING MEAN 5. 20094 563
SOURCE
REP
TRT
REP*TRT
F VALUE 0.73 4 .64 0.99 PR F DF TYPE III SS OK-5370 0.0001 0. 4738 4.28010506 45-29704776 31.92681464 F VALUE 0.93 4.22 0.99 PR F 0. 4264 0.0002 0. 4738 TESTS OF HYPOTHESES USING THE TYPE III MS FOR REP*TRT AS AN ERROR TERM
SOURCE
REP
TRT
DF TYPE III SS 3 4.28010506 7 45-29704776 F VALUE 0.94 4-26 PR F 0. 4397 0-0045 r7 WO 89/00810 PCT/US88/02573 -144- The analysis of variance results again indicate a strong treatment effect (P 0.003). That is, the probability of erroneously concluding that there is a treatment effect is approximately 0.3 percent or about 3 in 1000. Furthermcre, there does not appear to be any significant block-to-block variation (P 0.47) or block-by-treatment interaction (P 0.48).
It appears that blocks 5 and 6 were responsible for the significant block effect in the earlier analysis.
Table XLIV contains the Duncan's multiple range test results for the new analysis of flower rating which excludes blocks 5 and 6.
WO 89/00810 PCT/US88/02573 1 5 8 0 0 TABLE XLIV.
DUNCAN'S MULTIPLE RANGE TEST FOR VARIABLE: AVERAGE F LOWER RATING (1-4 REPLICATES1).
DF=250 MSE-=1-53267 CELL SIZES ARE NOT EQUAL-. HARMONIC MEAN OF CELL SIZES=34.8684 NUMBER OF MEANS CRITICAL RANGE 8 0.684317 0.589008 0-619378 DUNCAN GROUPING*
A
A
B A B A B A B A B A B A B D A
C
C
C
0..63B92 0-6536n1
MEAN
5-7619 5-7083 5-.5185 5-.3333 5. 1111 5-0667 4-7338 4-4912 RE NOT SIGNIFICANTLY 0-665956 0-676054 N TREAi11MENT 35 BI 32 B3 36 28 B7 36 B2 40 B4 37 B6 38 BB
DIFFERENT-
B,
B
D C D C D C D C *MEANS WITH THE SAME LEITRf A i WO 89/00810 PCT/US88/02573 -146- However, the original analysis (Table XXXII) should be considered as more appropriate than Table XLV for drawing conclusions. Even though there is block-to-block variation when blocks 5 and 6 are included, this effect is accounted for in the earlier analysis and allows the researcher to use all of the data to .estimate the treatin* effect. In fact, the treatment effect is stronger when blocks 5 and 6 are included in the analysis.
6.12. DEMONSTRATION OF THE INHERITANCE OF AMS/VECTOR-INDUCED MALE STERILITY IN A SUBSEQUENT GENERATION OF CORN The study described herein demonstrates the inheritance of AMS/vector-indced male sterility into a subsequent generation of corn. The experiment was conducted on a field site at a research station in Waimanalo, Hawaii. Four sets of corn were planted (Table
XLV).
WO 89/00810 PCT/US88/02573 -147-
I
TABLE XLV.
CORNSEED SOURCES* set Description Seed from crosses of sterile plants from AMS/vector-treated Inbred line x untreated isogenic line.
Seed from self crosses of fertile plants from AMS/vector-treated Inbred line.
Seed from crosses between AMS/vector-treated Inbred line x non-isogenic untreated Inbred line.
Seed of Inbreds 1, 2, 4 from S 2
S
3
S
4 and S generations.** *See section 6.11.1, infra for a more detailed description.
n refers to the nth seed generation.
The codes for inbred lines of corn which were used are described in T'able XLVI.
WO 89/00810 PCT/US88/02573 -148- TABLE XLVI.
INBRED CODES FOR LINES OF CORN Code Seed Company Variety Code i Inbred 1 A632Ht, Lot 950, Grade F Inbred 2 B73Ht, Lot 4551ST, Grade 23-21F Inbred 3 H95Ht, Lot 150, Grade MF Inbred 4 Mol7Ht, Lot 055, Grade MF i 15 Sets 1 and 4 were tested to specifically ii evaluate the heritability of AMS/vector-induced male Ssterility into a subsequent generation of corn.
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.
In Set 3 testing, the goal was to determine if the AMS/vector-Inbred line x non-isogenic, untreated Inbred line derived F 1 seed expressed any sterility.
Results from the data in Set 1 indicated that 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.
In Set 2, sterility was expressed in a few plants of Inbred 1 sterility) and Inbred 3 (3.4% sterility). y In Set 3, no male sterility was expressed.
In Set 4, Inbreds 1, 2 and 4 showed more than male sterility.
L__
WO 89/00810 PCT/US88/02573 -149- The tassels that were rated fertile, in general, had all the anthers fully dehisced. Two exceptions were fertile plants of Inbreds 2 and 4 in Set 1, where the tassels had only 1-10 anthers emerging per spikelet, which were dehiscing and shedding pollen. The rest of the anthers were enclosed in the spikelet rnd did not dehisce.
The tassels of sterile plants showed no dehiscence of anthers, which for the most part were enclosed in the spikelet.
Microscopic observations revealed that anthers from tassels rated fertile had round, stainable pollen grains, while anthers from tassels rated sterile had irregularly-shaped, non-stainable, abnormal pollen. In tassels which were rated fertile and which had only a few anthers dehisced, abnormal pollen was abundant in the non-dehisced anthers, while normal pollen was abundant in dehisced anthers.
6.12.1. MATERIALS AND METHODS 6.12.1.1. CORN SEED SOURCES Prior to silking, the ears of corn plants of all the four genotypes in the experiment described in Section 6.9, supra, were covered with shoot tip bags. Three types of crosses were performed after the ears had silked. the seed derived from the three different crossing patterns constituted the seed for Sets 1-3 of the current experiment. The nature of these three sets and an additional set were as follows: Set 1: After any male steriles were identified for each inbred strain, shoot tip bags were removed and the silks of the ears on the sterile plants were dusted with ,.llen derived from untreated isogenic Inbred S WO 89/00810 PCT/US88/02573 -163- 6.12.2.1.4. SET 3 1 11:- 1-1~ c1 WO 8?/00810 PCT/US88/02573 -150genotypes planted separately in the field.
Ears were harvested at maturity. Seed was separated from cobs and dried to moisture. This seed (referred to as synthetic 1 or S 1 was designated as Set 1.
Set 2: AMS/vrctor-treated plants that produced pollen (fertiles) were selfed. The seed derived from such self-crosses was 1i designated as Set Set 3: Crosses were made between AMS/vectortreated male-sterile Inbred lines and nonisogenic, untreated 'nbred lines. The crosses were as follows: treated Inbred 1 X untreated Inbred 3; treated Inbred 2 X untreated Inbred 4; and treated Inbred 4 X untreated Inbred 2. The seed derived from such crosses was designated Set 3.
Set 4: Seed was generated, comprising four generations of each of three AMS/vectortreated, male-sterile Inbreds 2 and 4) that were crossed to untreated isoaenic lines. This seed, comprising S2 Sg generations, was designated S t 4.
For sets 1-3, seed from each ear was shelled and packed in a seed packet. Each seed packet was given a treatment designation corresponding to the origin of the seed. For example, for Sets 1 and 2, a treatment designation of 11 R 1
B
1 meant Inbred 1, treated with AMS/vector treatment Bl 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 13 corresponding to 'i 11 0 1C WO 89/00810 PCT/US88/02573 i 6 4 WO 89/00810 PCT/US88/02573 -151a cross between AMS/vector-treated (B 1 treatment) Inbred 1 and untreated Inbred 3. Examples of Set 4 treatment designations include 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.
6.12.1.2. PREPARATION OF SEED FOR PLANTING For Sets 1-3, the seed derived from each ear was counted in two lots of 32 seeds each, and each lot was planted in a replicate. In instaices where a single ear did not produce more than 32 seeds, only one replicate was planted.
The seed was coated with 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 nurTJer. The seed was carefully packed and hand carried to Hawaii.
6.12.1.3. CHARACTERISTICS OF FIELD SITE 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 WO 89/00810 PCT/US88/02573 -152f',om the Pacific Ocean at 21 N latitude. The soil was Vertic Haplustoll derived from coral and lal'a 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. RaiLs are more frequent in winter months. Day lengths range from 10.8 to 13.2 tors.
Winds are for the most part continuing and gentle cit 8-15 km/hr but sometimes reach 25-30 km/hr. Incident lLght values average over 540 cal/cm2/day, but can be as low as 220 cal/cm 2/day in cloudy winter months.
II
6.12.1.4. FIELD PREPARATION AND MANAGEMENT A field site 1Q5 feet x 120 feet was plowed, disked and rototilled. A basal fertilizer application consisting of NPK (nitrogen-phosphors-potassium) in the ratio of 150:90:60 kg/h, was made using a fertilizer applicator. Another dose of 80 kgN/ha was applied between rows four weeks after emergence. The field was irrigated on an as needed basis on any Monday, Wednesday, or Friday.
6.12.1.5. 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 waa alternated as 2 feet, 4 feet, 2 feet, 4 feet, and so on. Where two tiers were r WO 89/00810 PCT/US88/02573 -153- V separated by 4 spacing, Pioneer hybrid 304C was planted as ja cross row. No planting was done when the space between I two tiers was 2.
6.12.1.6. FIELD PLANTING 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 Ii one planter, participated in planting at a given time.
Each individual planted one row at a given time. After Seach row, the planters were cleaned by hand to ensure 1i removal of dirt, etc. before moving on to plant another Si 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 i of Set 1 were planted before replicate 2 of Set 1. Set 2 I j 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. After finishing planting of Sets 1-4, 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.
6.12.1.7. EXPERIMENTAL PARAMETERS 6.12.1.7.1. 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,
L~-
WO 89/00810 PCT/US88/02573 -154and the dead plants were discarded prior to rating. The remaining plants were counted and rated for pollen fertility or sterility.
After the tassels had emerged out of the flag leaf (and after the ears had begun to silk), 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 reconf.rm 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 yellowcolored tags are illustrated in Figures 14 and 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., 6.12.1.7.2. MICROSCOPIC OBSERVATIONS ON ANTHER AND POLLEN CHARACTERISTICS Representative examples of fertile and sterile tassels for each Inbred were collected from the field.
Anthers were dissected from the tassel and stained with acetocarmine. Morphological features of anthers and pollen were noted for each representative example.
17- I I I
I
WO 89/0810 CT/US8/0,57 WO 89100810 PCT/US8/02573 -155- 6.12.1.7.3. RATING FOR PLANT HEIGHT, EAR HEIGHT, AND DAYS TO SILKING Plant height, ear height, and days to 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 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.
6.12.1.8. PROCEDURE FOR CROSSING All the ears of plants within Sets 1-4 were covered with white transparent shoot tip bags as soon as the ears were visible and before the ears showed silks.
Silks on each ear were cut with a scissor a day before crossing. At the time of crossing, the top of the shoot tip bag was torn off and the pollen dusted on the silks.
The ears (with the remaining shoot tip bags still attached to the ear) were covered with the pollination bags. The bag was labeled with the Inbred number, the pollen source, the date the cross was made, and the name of the person who performed the cross. The nature of the crosses made in Sets 1-4 are summarized in Table XLVII.
"tr- i L i WO 89/00810 PCT/US88/02573 -169- In Inbreds 1 and 2, tassels which were rated
U
-rn C WO 89/00810 PCT/US88/02573 -156- TABLE XLVII.
i CROSSES MADE IN CORN SETS 1-4 1 Set Crosses 1 i) Sterile plants of Inbred line x non AMS/vector isogenic line.
ii) Self crosses of a few fertile plants.
2 i) Sterile plants of Inbred line x non S1 AMS/vector isogenic line.
ii) Self crosses of a few fertile plants.
3 i) A few self crosses.
4 i) Sterile plants of Inbred line x non AMS/vector isogenic line.
ii) Self crosses of a few fertile plants.
6.12.1.9. DATA COLLECTION AND STATISTICAL ANALYSIS For each treatment row, data on total plant count, total plants rated, number of fertile plants, number of sterile plants, percentage of sterile plants, plant height, ear height, and days to 75% silking were recorded.
Statistical analyses were performed according to a completely randomized block design for Set 1. For Sets 2-4, means and standard deviations were provided for each treatment.
I_
WO 89/00810 PCT/US88/02573 -157- 6.12.2. RESULTS AND DISCUSSION 6.12.2.1. INHERITANCE OF MALE STERILITY Evaluation of the inheritance of AMS/vectorinduced male sterility into a subsequent generation of corn was examined in Sets 1 and 4.
6.12.2.1.1. 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.
OF-
WO 89/00810 PCT/US88/02573 -171- 7. nP- 1nATnT Mr qrM 7T
A.
C3 TABLE3 ICVIII SIGNIFICANCE LEVELS FOR BLOCKING FACTOR (REPLICATE), MAIN EFFECTS (INBRED AND TREATMENT) AND INTERACTION FROM ANALYSIS OF VARIANCE USING RANDOMIZED BLOCK DESIGN. BY DEPENDENT VARIABLE a Percent Plant Ear Days To Sterility Height Height 75% Silking Replicate (Block) (.1245) 0002)** (-0703) (.00l1)** 2) Inbred (.0001)** 2, 4) Treatment (,1817) (-2919) (,1175) (.1786) (BI, B4, B5, BG) Inbred x (,1038) (-2090) (-5284) (.0158)* Treatment.
aHighly significant results (P less than or equal to -01) indicated by fl**fl; P less than or indicated by (Set 1) equal to 0 -i WO 89/00810 PCT/US88/02573 -159- TABLE XLIX.
RESULTS OF DUNCAN'S MULTIPLE RANGE TEST ON CLASSIFICATION VARIABLE INBRED FOR EACH OF THE DEPENDENT VARIABLES Dependent Variable Mean Values For Class Variable Inbred Percent Sterile Plant Height Ear Height Days to 75% Silking Inbred 2 Inbred 4 Inbred 1 (95.4) (85.3) (16.8) Inbred 1 Inbred 2 Inbred 4 (69.4) (63.4) (61.9) Inbred 2 Inbred 1 Inbred 4 (23.8) (21.5) (21.1) Inbred 2 Inbred 4 Inbred 1 (74.2) (73.9) (69.4) aValues underscored by the same bold line were not significantly different at P less than or equal to 0.05. (Set 1) 6,12.2.1.2. SET 4 Inheritance of male sterility was demonrtrated for four generations (S 2
S
3 S4, and Sg) in Inbreds 1, 2, and 4 of Set 4, with more than 90% of the plants from each generation being male sterile (Table L), 1r iY- I S. k -172- The claims defining the invention are as follow t.
TABLE I>.
PERCENT STERILITY, PLANT HEIGHT, EAR HEIGHT., AND DAYS TO SILKING FOR INBREDS 1, 2, AND 4 ACROSS FOUR GENERATIONS (SET 4) NUM4BER STAND PLANT OF COUNT COUNT FERTILES
NUMBER
OF
STERILES
PERCENT
OF
STERILES
PLANT EAR TO HEIGHT HEIGHT STLK INBRED GENERATION REPLICATE 100.0 86-4 88.2 94-.1 93-8 100-0 100.0 91.7 100.0 80.0 100.0 50.0 65.0 66.2 64.2 73.0U 70.0 66.5 64.0 59.3 69-5 66-0 54 .7 12.8 21.0 20.0 19.0 25.0 22-5 23.0 21.0 19.
20-0 17.9 19.5
J
W
ofni PCT/US88/02573 6.12.2.1.3. SET 2 in Set 2. terility was expressed in a few plants of Inbred 1. L.7% sterility) and Inbred 3 (3.4% sterilitty) (Table~ Li). The seed in this Set was derived from self crosses that origir77.ly failed to convert to steriles upon NMS/vector treatmnent.
210 TABLE LI- MEEANS,. STANDARD DEVIATIONS. AND SAMPLE SIZE FOR DATA IN SET 2 FOR RESPONSE VARIABLES BY INBRED GROUP Dependent Variable Percent Plant Ear Days To Inbred Sterility Height Height 75% Silking 1 1.7 6-5 66-9 4.6 20.3 2.7 71.4 1.9 n 31 n =31 n =31 n =31 3 3-4 6-8 58.9 3.7 15.6 2.5 71.9 n =17 n =17 n 7 n =17 4 0.0 0.0 68-2 6-3 19.3 0.8 72.0 0.0 n =2 n =2 n =2 n =2 I, WO 89/00810 PCT/US88/02573 -163- 6.12.2.1.4. SET 3 No male sterility was expressed in the hybrids derived from crosses between AMS/vectcr-treated Inbred genotypes and non-isogenic, untreated Inbred lines (Table
I)
TABLE LII.
MEANS, STANDARD DEVIATIONS, AND SAMPLE SIZE (NW) FOR DATA IN SET 3 FOR RESPONSE VARIABLES BY IN~BRED CROSSES Dep~endent Variable Percent Plant Ear Days To Inbred Sterility Height Height 75% Silking 1 X3 0,0 00 94.1+ 5.8 28.8 +3.2 68.4 n 12 n =12 n =12 n =12 2 X 4 0.0 0.0 91-2 6.1 32.5 3.7 69.3 1.1 n =26 n =26 n =26 n =26 4 X 2 0.0 0,0 92.9 2.9 31.0 3.8 69.3 1.4 n =6 n =6 n =6 n= 6 WO 89/00810 PCT/US88/02573 -165- 6.12.2.1.5. UNTREATED CONTROLS No male sterility was observed in the non- AMS/vector (untreated control) plants off Inbreds (Table
LIII).
A
'a1 TABLE LIII.
PERCENT STERILITY, PLANT HEIGHT. EAR HEIGHT.
AND DAYS TO 75% SILKING IN THE NON-AMS/VEC IOR (UNTREATED CONTROLS) M4ATERIAL OF INBREDS 1, 2. 3, AND 4 TOTAL PLANT EAR DAYS TO PLANTS FERTILES STERILES %STERILE HEIGHT HEIGHT SILKING 1241 241 0 0 69 20 71 2 225 225 0 0 61 20 74 3 310 310 0 0 55 17 74 4 204 204 0 0 64 24 74 r_ I WO 89/00810 PCT/US88/02573 -167- 6.12.2.2. MORPHOLOGICAL FEATURES OF POLLEN FERTILITY AND STERILITY 6.12.2.2.1. VISUAL FEATURES Visual observations of tassel morphology were made in the representative fertile and sterile plants of each inbred (Figs. 16, 17).
6.12.2.2.1.1. SET 1 In Set 1, 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.
In 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. SET 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.
I
WO 89/00810 PCT/US88/02573 -168- 6.12.2.2.1.3. SET 3 All the plants in Set 3 had tassels with dehisced anthers that shed pollen profusely.
6.12.2.2.1.4. SET 4 Tassels which were rated sterile had no anthers emerging out of the spikelet or shedding pollen. Those rated fertile had fully dehisced anthers and profuse pollen shed.
6.12.2.2.1.5. UNTREATED CONTROLS All the tassels had anthers fully dehisced, with profuse pollen shedding.
6.12.2.2.2. MICROSCOPIC FEATURES 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).
6.12.2.2.2.i. SET 1 Tassels where the anthers were fully dehisced and pollen washed profusely, showed typically large, round, normal-looking pollen with cytoplasm densely stained with acetocarmine.
Tassels where only 1-10 anthers emerged out of the spikelet and dehisced, showed normal looking, round pollen grains with dense cytoplasm, only in the anthers that emerged and dehisced. In the same tassel, 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.
i WO 89/00810 PCT/US88/02573 -169- In Inbreds 1 and 2, tassels which were rated sterile had undehisced anthers containing abnormal pollen.
Crushing the anthers with a glass rod facilitated the release of pollen grains from the anthers. However, in Inbred 4, no pollen grains were seen inside the anthers.
Therefore, in all the three Inbreds, the block associated with male sterility appeared to be at the level of differentiation of sporogenous tissue. There was also a good correlation between lack of dehiscence of anthers and presence of abnormal pollen or absence of pollen.
6.12.22.2.2.. SET 2 Microscopic observations revealed that all tassels rated fertile had anthers with large, round pollen grains with densely stained cytoplasm. The anthers from tassels rated sterile did not dehisce and had abnormal, irregularly shaped, non-stainable pollen.
6.12.2.2.2.3. SET 3 All tassels from Set 3 were rated fertile. The anthers dehisced, and pollen grains were round and had densely stained cytoplasm.
6.12.2.2.2.4. SET 4 Tassels rated fertile had anthers showing normal, round pollen with densely stained cytoplasm.
Those tassels that were rated sterile had undehisced anthers and contained abnormal pollen.
6.12.2.2.2.5. UNTREATED CONTROLS The pollen grains from the untreated control plants of all the four Inbreds were round with dense cytoplasm that stained deep red with acetocarmine.
r- I I WO 89/00810 PCT/US88/02573 -170- 6.12.2.3. STATISTICAL ANALYSIS OF THE DATA 6.12.2.3.1. SET 1 Highly significant differences were noticed between Inbreds for the percent sterility variable.
Differences were also noted between Inbreds for plant height, ear height, and days to 75% silking characteristics. A strong to moderate effect between replicate plots (blocks) across all four dependent variables (P 0.0001 to 0.1245) was observed, the least effect being in the percent sterility variable (Table XLVIII). No significant effect of the AMS/vector treatments (B1, B4, B5, and B6 of Section 6.9, supra) applied on plants in the previous generation, was apparent in this generation, for any of the four dependent variables. Duncan's multiple range test also conveyed similar trends (Table XLIV). Means of the percent sterile variable were significantly different for all the three inbreds. Plant height of Inbred 1 was significantly different from Inbreds 2 and 4. For the dependent variable days to silking, Inbreds 2 and Inbreds 4 were significantly different from Inbred 1.
6.12.2.3.2. SETS 2 AND 3 Means and standard deviations were calculated for all the dependent variables for Sets 2 and 3. In Set 2, Inbred I and 3 showed 1.7% and 3.4% male sterile plants, respectively. No male sterile plants were identified in Set 3. No noticeable trends for other dependent variables were apparent (Tables LI, LII).
6.12.2.3.3. SET 4 In the absence of any replication, the actual values were tabulated for this Set (Table L).
WO 89/00810 PCT/US88/02573 -171- 7. DEPOSITS OF SEEDS The following seeds have been deposited with the American Type Culture Collection, Rockville, MD., and have been assigned the listed accession numbers: Seed AMS 1.29 B73-AMS Mo17-AMS A632-AMS Des'cription cross between alfalfa AMS/vector source 1.29 (derived from U.S.D.A.
PI No. 223386) and a maintainer plant male-sterile B73Hit variety of Zea ma s L. corn; asexually induce--to male sterility by treatment with AMS/vector male-sterile Mol7Ht variety of Zea mays L. corn; asexually induced--o mae sterility by treatment with AMS/vector male-sterile A632Ht variety of Zea may L. corn; asexually inducedT male sterility by treatment with AMS/vector Accession Number 40352 40350 40351 .40349 The present invention is not to be limited in scope by the specific seeds deposited since the deposited embodiment is inteinded as a single illustration of one aspect of the invention and any seed which is functionally equivalent is within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Claims (98)
1. An AMS/vector comprising a cytoplasmic factor derived from a donor plant, which factor is capable of asexually inducing heritable male sterility in a recipient plant; is subsequently derivable from the recipient plant; and is present in an extract of the donor plant or recipient plant, which extract characterized by at least one element selected from the group consisting of a nucleic acid of about 1 X dalton molecular weight and a particle of about 40-110 nanometers.
2. The AMS/vector of claim 1 in which the donor plant is an alfalafa plant.
3. The AMS/vector of claim 2 in which the alfalfa plant has U.S.D.A. Plant Introduction No. 172429.
4. The AMS/vector of claim 2 in which the alfalfa plant has U.S.D.A. Plant Introduction No. 173733. The AMS/vector of claim 2 in which the alfalfa plant has U.S.D.A. Plant Introduction No. 221469. 9 i: The AMS/vector of claim 2 in which the alfalfa plant has U.SD.A. Plant Introduction No. 223386.
9. S7, The AMS/vector of claim 2 in which the alfalfa plant has U.S.D.A. Plant Introduction No. 243223. 8. The AMS/vector of claim 2 in which the alfalfa plant comprises plant AMS 1.29, as deposited with the ATCC and assigned accession number 40352. 39 NOB -186- I -173- 9. The AMS/vector of claim 1 plant is a corn plant. The AMS/vector of claim 1 plant is a soybean plant.
11. The AMS/vector of claim 1 plant is a sorghum plant.
12. The AMS/vector of claim 1 plant is a sunflower plant.
13. The AMS/vector of claim 1 plant is a millet plant.
14. The AMS/vector of claim 1 plant is a tomato plant. in which in which in which in which in which in which the recipient the recipient the recipient the recipient the recipient the recipient A plant extract capable of inducing male sterility in a plant, comprising a non-lethal buffer and a cytoplasmic factor derived from a donor plant, which factor is capable of asexually inducing heritable male sterility in a recipient plant; is subsequently derivable from the recipient plant; S: and is present in an extract of the donor plant or recipient plant, which extract characterized by at least one element selected from the group consisting of a nucleic acid of about 1 X 10 dalton molecular weight and a particle of about 40-110 nanometers.
16. The extract of claim 15 in which the donor plant is S. an alfalfa plant, e
17. The extract of claim 15 in which the recipient plant is a corn plant. S 39 1 NOB -174- S18. The extract of claim 15 in which is a soybean plant.
19. The extract of claim 15 in which is a sorghum plant. The extract of claim 15 in which is a sunflower plant.
21. The extract of claim 15 in which is a millet plant.
22. The extract of claim 15 in which is a tomato plant.
23. A male-sterile plant comprising a asexually induced to heritable male AMS/vector of claim 1. 24, A male-sterile plant comprising a asexually induced to heritable male AMS/vector of claim 2.
25. A male-sterile plant comprising a asexually induced to heritable male AMS/vector of claim 3, 4, 5, 6, 7 or 8. the recipient plant the recipient plant the recipient plant the recipient plant the recipient plant plant that has sterility by been the plant that has sterility by plant that has sterility by been the been the 00 0 .0 0 0 A A. A A
26.
27. The plant of claim 23 which is an alfalfa plant. Ti. plant of claim 23 which is a corn plant,
39- NOB NOB -175- 28. The corn plant of claim 27 comprising B73- AMS, as deposited with the ATCC and assigned accession number 40350. 29. The corn plant of claim 27 comprising Mol7-AMS, as deposited with the ATCC and assigned accession number 40351. A632-AMS, accession plant. The corn plant of claim 27 comprising as deposited with the ATCC and assigned number 40349. 31. The plant of claim 23 which is a soybean 32. The plant of claim 23 which is a sorghum plant. 33. The .ant of claim 23 which is a sunflower plant. 34. The plant of claim 23 which is a millet 9 4*~ 9 V I S. I+ i: .5 S SC *9 V 4 plant. The plant of claim 23 which is a tomato plant. 36. The plant of claim 23 which is a wheat plant. 37. The plu.-t of claim 23 which is a cotton plant. C. V *i 38. The plant of claim 23 which 43 a rice 35 plant. -189- nonlethal buffer, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of it" -176- 39. A progeny plant obtained by asexual propagation of the plant of claim 23., 24, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37 or 38.
40. The progeny plant of claim 39, in which the propagation is by cell culture methods.
41. The progeny plant of claim 39, which the propagation ts vegetative.
42. A seed resulting from a cross of the plant of claim 23 or 24 with a maintainer plant.
43. A seed resulting from a cross of the plant of claim 25 with a naintainer plant.
44. A seed resulting from a cross of the plant of cilim 26, 27, 28, 29, 30, 31, 32,, 33, 34, 35, 36, 37 or 38 with a maintainer plant.
45. A progeny plant produced by the seed of claim 42.
46. A progeny plant produced by the seed of 25 claim 43.
47. A progeny plant produced by the seed of claim 44.
48. A method for asexually inducing male sterility in a recipient plant comprising applying the AMS/vector of claim 1 to such recipient plant. 4 4 4 4. 4 4 'I- i pwi 'I J 44 -190- extract is characterized by having at least one element selected from the group consisting of a nucleic acid of about 1 X 3 i -1
49. A method for a sterility in a recipient plan AMS/vector of claim 2 to such
50. A method for a sterility in a recipient plan AMS/vector of claim 8 to such
77- sexually inducing male t comprising applying the recipient plant. sexually inducing male t comprising applying the recipient plant. 51. A method for asexually inducing male sterility in a recipient plant comprising applying the extract of claim 15 to such recipient plant. which the which the 52. The method of claim 48, 49, 50 or 51 application is by injection. 53. The method of claim 48, 49, 50 or 51 application is by spraying. 54. The method of claim 48, 49, 50 or 51 application is by use of tissue culture. The method of claim 48, 49. 50 or 51 application is by electroporation. which the which the which the L q* which the which the 56. The method recipient plant 57. The method recipient plant 58. The method recipient plant 59. The method recipient plant of claim 48, 49, 50 or 51 is an alfalfa plant. of claim 48, 49, 50 or 51 is a corn plant. of claim 48, 49, 50 or 51 is a soybean plant. of claim 48, 49, 50 or 51 is a sorghum plant. which the f" I U -178- The method of claim 48, 49, 50 or 51 in which the recipient plant is a sunflower plant. 61. The method of claim 48, 49, 50 or 51 in which the recipient plant is a millet plant. 62. The method of claim 48, 49, 50 or 51 in which the recipient plant is a tomato plant. 0 63. The method of claim 48, 49, 50 or 51 in which the recipient plant is a wheat plant. 64. The method of claim 48, 49, 50 or 51 in which the recipient plant is a crtton plant. The method of claim 48, 49, 50 and 51 in which the recipient plant is a rice plant. 5 S S S S 66. A method for making an F 1 hybrid crossing a paternal parent plant with the plant 23 or 24 as maternal parent. 67. A method for making an F 1 hybrid crossing a paternal parent plant with the plant 25 as maternal parent. 68. A method for making an F 1 hybrid crossing a paternal parent plant with the plant 45 as maternal parent. 69. A method for making an F 1 hybrid crossing a paternal parent plant with the plant 46 as maternal pareert. comprising of claim comprising of claim comprising of claim comprising of claim 7\f -192- cytoplasmic factor which is capable of asexually inducing male sterility and is expressed in a recipient plant of the eIMaM ft- r- -179- A method for making an F l hybrid comprising crossing a paternal parent plant with the plant of claim 47 as maternal parent. 71. An F 1 hybrid made according to the method of claim 66. 72. An F 1 hybrid made accord!. to the method of claim 67. 73. An F 1 hybrid made according to the method of claim 68. 74. An F 1 hybrid made according to the method of claim 69. An F 1 hybrid made according to the method of claim 76. The F 1 hybrid of claim 71 which is male sterile. 4 77. The F 1 hybrid of claim 72 which is male sterile.
78. The F hybrid of claim 73 which is male sterile.
79. The F 1 hybrid of claim 74 which is male 30 sterile.
80. The F 1 hybrid of claim 75 which is male sterile. i -180-
81. The F 1 hybrid of claim 71 which is male fertile. fertile. fertile.
82. The F 1 hybrid of claim 72 which is male
83. The F 1 hybrid of claim 73 which is male
84. The F 1 hybrid of claim'74 which is male The F 1 hybrid of claim 75 which is male fertile. fertile. a a a. 4
86.
87.
88.
89.
91.
92.
93. seed seed seed seed seed seed seed seed the the the the the the the the hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid claim claim claim claim claim claim claim claim 71. 72. 73. 74. 76. 77. 78. 0 1 "C6 *im-rL. WVO89/00810 'I Ioslal PCT/US88/02573 1/53 -181-
94. A seed of the F 1 hybrid of claim 79. A seed of the F 1 hybrid of claim
96. A seed of the Fl hybrid of claim 81.
97. A seed of the F 1 hybrid of claim 82.
98. A seed of the F 1 hybrid of claim 83.
99. A seed of the F 1 hybrid of claim 84.
100. A seed of the F hybrid of claim
101. A method for inducing apomixis in a recipient plant comprising applying an effective amount of the AMS/vector of claim 1 to such recipient plant.
102. A method for inducing apomixis in.a recipient plant comprising applying the AMS/vector of S claim 2 to such recipient plant.
103. A method for inducing apomixis in a recipient plant comprising applying the AMS/vector of 25 claim 8 to such recipient plant.
104. A method for inducing apomixis in a recipient plant comprising applying the AMS/vector of claim 15 to such recipient plant.
105. The method of claim 101, 102, 103 or 104, in which the application is by spraying.
106. The method of claim 101, 102, 103 or 104 in which the application is by injection. U i -182-
107. The method of claim 101, 102, 103 or 104 in which the application is by use of tissue culture.
108. The method of claim 101, 102, 103 which the application is by electroporation. or 104 in
109. The me%,iod of claim 101, 102, 103 or 104 in which the recipient plant is an alfalfa plant. S 9 .5*
110. The method of which the recipient plant is
111. The method of which the recipient plant is
112. The method of which the recipient plant is
113. The method of 20 which the recipient plant is
114. The method of which the recipient plant is
115. The method of which the recipient plant is
116. The method of which'the recipient plant is
117. The method of which the recipient plant is claim 102, a corn plant. claim 101, 102, a sorghum plant claim 101, 102, a sunflower plar claim 101, 102, a millet plant. claim 101, 102, a tomato plant. claim 101, 102, a wheat plant. claim 101, 102, a cotton plant. claim 101, 102, a rice plant. 103 or 104 in 103 or 104 in 103 or 104 in It. 103 or 104 in 103 or 104 in 103 or 104 in 103 or 104 in 103 or 104 in V.. '4 11, S. *S**S S S
118. A method of making an apomitic hybrid which comprises treating a first parental plant line of said 4, -183- hybrid with an effective amount of the AMS/vector of claim 1, and crossing said first parental line with a second parental plant line to obtain hybrid seed.
119. The method of claim 118 which includes the further step of growing the hybrid seed to produce mature plants, and identifying those plants having apomitic properties,
120. The method of claim 118 which comprises the further step of obtainlIng hybrid seed from the identified plants. S I *r S II S
121. The method of claim 118, wherein the plant is an alfalfa plant.
122. The method of claim 118, wherein the plant is a corn plant.
123. The method of claim 118, wherein the plant is a soybean plant.
124. The method of claim 118, wherein the plant is a sorghum plant.
125. The method of claim 118, wherein the plant is a sunflower plant.
126. The method of claim \18, wherein the plant is a millet plant.
127. The method of claim 118, wherein the plant is a tomato plant. 119 or 120, 119 or 120, 119 or 120, 119 or 120, 119 or 120, 119 or 120, 119 or 120, kf VO, v- NVw089/00810 PCT/USS8/02573 4/53 -184-
128. The method of claim 118, 119 or 120, wherein the plant is a wheat plant.
129. The method of claim 118, 119 or 120, wherein the plant is A cotton plant.
130. The method of claim 118, 119 or 120, wherein the plant is a rice plant.
131. Hybrid seed produced by the method of claim 118.
132. Hybrid seed produced by the method of claim 120.
133. A hybrid plant produced by the method of claim 119, and direct descendants thereof, 134, The seed of claim 131 wherein the plant is 20 selected from the group consisting of corn, alfalfa, soybean, sorghum, sunflower, millet, tomato, wheat, cotton and rice.
135. The seed of claim 132 wherein the plant is selected from the group consisting of corn, alfalfa, soybean sorghum, sunflower, millet, tomato, wheat, cotton and rice.
136. The plant of claim 133 wherein the plant is selected from the group consisting of corn, alfalfa, soybean, sorghum, sunflower, millet, tomato, wheat, cotton and rice.
137. A method of delivering a bioactive molecule intracellularly to a plant comprising applying the abou4 B. B B B.. .9 B B. B It B.. St B B B P r, (Bln u -I i -185- 40-110 nanometer particle associated with the AMS/vector of claim 1, which particle contains a bioactive molecule.
138. A plant delivery system comprising an about 40-110 nanometer particle derivable from an alfalfa plant selected from the group consisting of plants having U.S.D.A. Plant Introduction Nos. 172429, 173733, 221469, 223386, and 243223.
139. A method of expressing a heterologous gene sequence in a plant comprising applying the about 1 X dalton nucleic acid of claim 1, which nucleic acid comprises a heterologous gene sequence capable of being expressed in the plant.
140. A plant expression vector comprising an about 1 X 10 dalton molecular weight nucleic acid derivable from an alfalfa plant selected from the group consisting of plants having U.S.D.A. Plant Introduction Nos. 172429, 173733, 221469, 223386, and 243223. I
141. A mutant, derivative, or fragment of the S" expression vector of claim 140. w -186-
142. The AMS/vector of Claim 1 wherein the nucleic acid has a molecular weight of about 1.1 X 106 daltons.
143. The AMS/vector of Claim 1 wherein the particle is characterized by a dense core surrounded by a bilayer membrane.
144. The AMS/vector of Claim 1 wherein the vector is capable of asexually transmitting male sterility from one species to another species.
145. The AMS/vector of Claim 1 wherein the recipient plant is wheat.
146. An AMS/vector comprising a cytoplasmic factor S. derived from a donor plant which factor is capable of asexually inducing heritable male sterility and is expressed in a recipient plant of the same or different species as the donor plant; and is present in an extract of the donor plant or i. recipient plant, which extract is characterized by having at least S one element selected from the group consisting of a nucleic acid of about 1 X 10 dalton molecular weight and a particle size of about 40-110 nanometers, wherein the donor plant has the -187- identifying characteristics of a plant selected from the group consisting of USDA Plent Introduction Nos. 172429, 173733, 221469, 223386 and 243223.
147. An AMS/vector comprising a cytoplasmic factor derived from a donor plant which factor is capable of asexually inducing heritable male sterility and is expressed in a recipient plant of the same or different species as the donor plant; and is present in an extract of the donor plant or recipient plant, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, 221469, 223386 and
243223. 148. An AMS/vector composition prepared by macerating a donor plant, the donor plant containing a cytoplasmic factor which is capable of asexually inducing heritable male sterility and S: is expressed in a recipient plant of the same or different species t: as the donor plant; and is present in an extract of the donor plant or recipient plant, which extract is characterized by having at least one element selected from the group consisting of a *6 nucleic acid of about 1 X 10 dalton molecular weight and a 0:0: particle size of about 40-110 nanometers, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, S223386 and 243223. 221469, 223386 and 243223. by I I I I II--~ -188- 149. A plant extract for asexually transmitting heritable male sterility in plants obtainable by macerating a donor plant in the presence of a nonlethal buffer, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos, 172429, 173733, 221469, 223386, and 243223. 150. The extract of claim 15 or 149 wherein the extract is combined with an abrador. 151. The method of claims 48-70 and 101-130 wherein the nucleic acil of the AMS/vector has a molecular weight of about 1.1 X 106 daltons. 152, The method of claims 48-70 and 101-130 wherein the particle is characterized by a dense core surrounded by a bilayer membrane. 153. The method of claims 48-70 and 101-130 wherein the vector is transmitted from one species to another species. 154. The method of claims 48-70 and 101-130 wherein the recipient plant is wheat. i *6 155. A method for asexually inducing male sterility in a recipient plant which comprises applying a plant extract a obtainable by macerating a donor plant in the presence of a 39 a 39 39 NOB i _ILL -189- nonlethal buffer, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, 221469, 223386, and 243223, to such recipient plant. 156. A method for asexualty inducing male sterility in a recipient plant which comprises applying an AMS/vector comprising a cytoplasmic factor derived from a donor plant which factor is capable of asexually inducing male sterility and is expressed in a recipient plant of the same or different species as the donor plant; and is present in an extract of the donor plant or recipient plant, which extract is characterized by having at least one element selected from the group consisting of a nucleic acid of about 1 X 106 dalton molecular weight and a particle size of abot 40-110 nanometers, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, Se" 221469, 223386 and 243223, to such recipient plant. S* 157. A method for asexually inducing male sterility in a recipient plant which comprises applying an AMS/vector composition prepared by macerating a donor plant, the donor plant containing a cytoplasmic factor which is capable of asexually inducing male sterility and is expressed 4n a recipient plant of S the same or different species as the doior ,)lant; and is present in an extract of the donor plant r1cipient plant, which 1. -190- extract is characterized by having at least one element selected from the group consisting of a nucleic acid of about 1 X dalton molecular weight and a particle size of about 40-110 nanometers, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, 221469, 223386 and 243223, to such recipient plant. 158. The method of Claim 53 wherein application of the extract is subsequent to or simultaneous with application of an abrador. 159. The method of Claims 48-70, 101-130 and 155-157 wherein the recipient plant is cotton. 160. The method of Claims 48-70, 101-130 and 155-157 wherein the recipient plant is rice. 161. The method of Claims 48-70, 101-130 and 155-157 wherein the donor plant is an alfalfa plant having USDA Plant Introduction No. 172429. 162. The method of Claims 48-70, 101-130 and 155-157 :162. The method of Claims 48-70, 101-130 and 155-157 wherein the donor plant is an alfalfa plant having USDA Plant Introduction No. 173733. US.. i a -191- 163. The method wherein the donor plant is Introduction No. 221469. 164. The method wherein the donor plant is Introduction No. 223386. 165. The method wherein the donor plant is Introduction No. 243223. of Claims 48-70, 101-130, and 155-157 an alfalfa plant having USDA Plant of Claims 48-70, 101-130, and 154-156 an alfalfa plant having USDA Plant of Claims 48-70, 101-130, and 154-156 an alfalfa plant having USDA Plant 4 o *4 4 g4 II 166. A method for asexually inducing male sterility in a recipient plant comprising applying an AMS/vector comprising a cytoplasmic factor derived from a donor plant which factor is capable of asexually inducing male scerility and is expressed in a recipient plant of the same or different species as the donor plant; and is present in an extract of the donor plant or recipient plant, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting of USDA Plant Introduction Nos. 172429, 173733, 221469, 223386 and 243223. 167. A method for asexually inducing male sterility in a recipient plant comprising applying an AMS/vector composition prepared by macerating a donor plant, the donor plant containing a I t ii -192- cytoplasmic factor which is capable of asexually inducing male sterility and is expressed in a recipient plant of the same or different species as the donor plant; and is present in an extract of the donor plant or recipient plant, wherein the donor plant has the identifying characteristics of a plant selected from the group consisting uf USDA Plant Introduction Nos. 172429, 173733, 221469, 223386 and 243223. 168. A AMS/vector according to claim 1 substantially as hereinbefore described with reference to the examples. 169. A method according to claim 48 substantially as hereinbefore described with reference to examples 6.4 to 6.8. 170. A method according to claim 101 substantially as hereinbefore described with reference to example 6.9. DATED: 17 August 1992 3J" 35 333 PHILLIPS ORMONDE FITZPATRICK Attorneys for: AGRIPO BIOSCIENCES INC. AA p; 7434j 39 k .Y NOB
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US224836 | 1981-01-13 | ||
| US8050587A | 1987-07-31 | 1987-07-31 | |
| US080505 | 1987-07-31 | ||
| US22483688A | 1988-07-27 | 1988-07-27 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2255288A AU2255288A (en) | 1989-03-01 |
| AU629796B2 true AU629796B2 (en) | 1992-10-15 |
Family
ID=26763609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU22552/88A Ceased AU629796B2 (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 (en) |
| AU (1) | AU629796B2 (en) |
| BR (1) | BR8807155A (en) |
| DK (1) | DK155189A (en) |
| WO (1) | WO1989000810A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3931969A1 (en) * | 1989-09-25 | 1991-04-04 | Max Planck Gesellschaft | DNA SEQUENCE, WUNL GENE WITH CUT PROMOTOR AND USE THEREOF |
| 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 (en) * | 2018-08-16 | 2018-12-28 | 安徽省农业科学院土壤肥料研究所 | The selection of leaf mustard cytoplasmic male sterile line, breeding system, control method |
-
1988
- 1988-07-28 AU AU22552/88A patent/AU629796B2/en not_active Ceased
- 1988-07-28 BR BR888807155A patent/BR8807155A/en unknown
- 1988-07-28 WO PCT/US1988/002573 patent/WO1989000810A1/en not_active Application Discontinuation
- 1988-07-28 EP EP19880906709 patent/EP0329736A4/en not_active Withdrawn
-
1989
- 1989-03-30 DK DK155189A patent/DK155189A/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0329736A4 (en) | 1991-10-09 |
| BR8807155A (en) | 1989-10-17 |
| DK155189A (en) | 1989-05-31 |
| WO1989000810A1 (en) | 1989-02-09 |
| DK155189D0 (en) | 1989-03-30 |
| EP0329736A1 (en) | 1989-08-30 |
| AU2255288A (en) | 1989-03-01 |
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