Patent Application
20090217409
This invention relates to the seed-to-seed perpetuation of hybrid vigor and other traits through apomixis (asexual seed formation) in flowering plants (angiosperms). More particularly, it provides predictable methods for producing, from sexual or facultatively-apomictic plants, progeny plants that express an increased percentage of ovules in which normal sexual development is replaced by aposporous or diplosporous (apomictic) embryo sac formation, parthenogenesis (embryo formation from an egg without fertilization), adventitious embryony (embryo formation from cells other than the egg), or endosperm formation of the autonomous (central cell not fertilized) or pseudogamous (central cell fertilized) types. It also provides descriptions of embryological phenotypes necessary for mapping and cloning the genes responsible for apomixis. This invention uses: plant cyto-embryology procedures to identify and select a plant or group of plants that possess appropriate genetic variability for initiation times and durations of megasporogenesis (female meiosis), embryo sac formation (including egg and central cell maturation), fertilization, embryony and endosperm formation; plant breeding procedures to produce numerous and divergent genetically-recombined progeny; and plant cyto-embryology or progeny test procedures to select segregant plants that express an increased frequency of aposporous or diplosporous embryo sac formation, parthenogenic or adventitious embryo formation, and/or autonomous or pseudogamous endosperm formation.
Apomixis is a natural but rare anomaly that occurs in less than 1% of angiospermous genera (Carman 1997). It does not occur in most of the world's important food and fiber crops, including rice, wheat, maize, barley, millet, sorghum, soybeans, potatoes, most vegetable and oil crops, cotton and many others (Asker and Jerling 1992). It is among such crops that apomixis holds it's greatest potential for providing commercial and humanitarian benefits. Conferring apomixis to world crops could benefit crop production in at least three ways.
First, inbred crops, such as wheat, rice and soybeans, could be converted to superior-yielding hybrid crops such that hybrid vigor is permanently inherited from seed to seed. Today, wheat hybrids yield up to 15% more grain than inbred varieties, but the vast majority of wheat grown internationally is varietal—not hybrid. The high cost of producing hybrid seed, compared to the low cost of producing varietal seed, currently limits the use of hybrid wheat seed to the very highest wheat production areas in the world (Guillen-Portal et al 2002).
It is the anatomy, physiology and genetics of most world crops that currently prevent the economic production of mass quantities of hybrid seed. These economics continue to prohibit a world-wide conversion from inferior varieties to superior hybrids. Apomixis could eliminate this economic bottleneck. For rice, the full exploitation of hybrid vigor could raise rice yields by 30% to 50% over yields of inbred varieties currently grown on the vast majority of rice acreage worldwide (Yuan 1993). By conferring apomixis to hybrids of rice and other major world crops, hybrid seed would be as cheap to produce as varietal seed. This is because apomictic hybrids clone themselves asexually from seed to seed, i.e. from one seed generation to the next. In essence, apomictic seed production systems do not require costly cross-pollination procedures for producing hybrid seed.
Second, apomixis could enhance crop production by reducing costs associated with producing hybrid seed of crops currently grown as hybrids. For example, hybrid seed of corn is produced by identifying genetically-divergent inbred parent lines that when crossed or double-crossed with each other produce superior-yielding hybrid progeny. Once appropriate parent lines are identified or bred, mass cross-pollinations are required to produce commercial quantities of hybrid seed. Apomixis could eliminate most of the cross-pollination costs, i.e. once an apomictic hybrid is produced, it clones itself through it's own seed, generation after generation. Seed companies in the U.S. currently spend about $600 M per year to produce hybrid corn seed. Apomixis could eliminate the cross-pollination procedures and save U.S. corn seed producers more than $300 M annually.
Third, apomixis could be used to transfer biotechnological and productivity advances to marginal farmlands in the developed world and to resource poor farmers in developing nations (Toenniessen 2001). Currently, high costs associated with producing hybrid seed or conferring value-added agbiotech traits to crops prohibit the use of hybrids or value-added traits in resource poor areas of the world. Because apomixis perpetuates such value-added traits (hybridity or agbiotech modifications) from seed to seed, apomixis could become a cost-effective vehicle for delivering these traits to resource-poor farmers in poor nations.
Before the major benefits of apomixis can be realized, methods for inducing apomixis and enhancing its expression in major crops must be developed and perfected. The instant specification provides novel methods for such inductions and enhancements.
The methods of the instant specification increase the expression of apomixis in plants. These methods are not related to nor are they extensions of approaches currently pursued by other scientists. The following discussion of current approaches is provided so as to clearly identify the critical factors that differentiate the methods of the instant specification from all other approaches currently being pursued.
Approaches currently being explored to confer apomixis to sexual crops by other scientists consist of:
Though progress has been made with each of these approaches, none has yet succeeded in converting a sexual species to a commercially-viable apomict (Spielman et al 2003, Estrada-Luna et al 2002, Richards 2003). These four approaches are based on a common belief that apomixis is conferred by one to a very few apomixis gene(s) that evolved through mutation or through epigenetic changes to regulatory genes (Koltunow and Grossniklaus 2003). Hence, it is believed that:
The approach taken by the inventor of the instant specification is not based on a belief in one to a very few apomixis genes of mutagenic origin, which can be manipulated or created by conventional or molecular breeding procedures; nor is it based on a belief that apomixis is the result of epigenetic changes in gene regulation. Instead, it is based on a series of novel discoveries, made by the inventor, that place apomixis in the category of genetically regulated traits, which are stabilized by structural heterozygosity at the genome level.
The inventor discovered that extensive genetic variability exists among plants of the same species, genus or family for initiation times and durations of megaspore mother cell (MMC) differentiation, megasporogenesis, embryo sac formation, fertilization, embryony, and endosperm formation (referred to hereinafter as components of the germline development sequence, GDS, or ovule development sequence, ODS) relative to the maturity level of the nongametophytic (sporophytic) tissues of the ovule. This variation has now been characterized in the inventor's lab for several species including Antennaria (
The instant specification describes an important advancement to the inventor's state-of-the-art methods for predictably producing apomictic plants from sexual plants. Before describing this advancement in detail, it is important to note that the inventor's methods (WO 98/33374, WO 01/32001) are the only methods published to date for predictably producing apomictic plants from sexual plants and genetically stabilizing them. In this respect, WO 98/33374 and WO 01/32001 teach that apomixis can be induced by creating F1 hybrids in which developmentally-disruptive competition occurs between asynchronously-expressed developmental programs (
It is further taught in WO 01/32001 that sexually-derived progeny from a “genetically-unstable apomict” usually will have largely reverted to sexuality due to genetic recombination at the multiple loci responsible for apomixis. Genetic segregation during facultative sexual reproduction in a genetically-unstable facultative apomict tends to produce progeny in which the allelic combinations that cause the asynchronous competition responsible for apomixis are disrupted. Accordingly, sexually-derived progeny of a genetically-unstable facultative apomict tend to express less penetrance for apomixis. WO 01/32001. In contrast, Applicant surprisingly discovered that a low percentage of sexually-derived progeny of certain genetically-unstable facultative apomicts will actually express a higher level of apomixis due to specific and infrequent recombination events the frequency of which can be predicted by plant breeders. In this context, the inventor has discovered that genes regulating initiation times and durations of:
The significance of this discovery is that initiation times and durations of the various GDS components, and even processes within each GDS component, are not controlled exclusively by the same genes. Based on these discoveries, the inventor theorized that:
Starting with a genetically-unstable facultative apomict, the percentage of sexually-derived progeny (genetically recombined) that express an enhanced level of apomixis will generally be low, but within the scope of screening procedures common to the plant breeding industry. This novel approach for increasing the frequency of apomixis expression in angiosperms has been tested and reduced to practice (
The instant specification discloses methods that increase the frequency of apomixis expression in angiosperms. These newly disclosed methods were made possible by the inventor's surprising discoveries concerning the genetic control of initiation times and durations of GDS components. The inventor discovered that a low percentage of sexually-derived progeny of certain genetically-unstable facultative apomicts will actually express a higher level of apomixis due to specific and infrequent recombination events. Further study and development based on this surprising discovery lead to the presently disclosed methods of producing and/or enhancing apomicitic plants.
It is an object of the instant specification to provide methods for producing apomictic plants from sexual angiospermous plants or from angiospermous plants that express a lower frequency of apomixis expression than the apomictic plants produced. Another object of the instant specification is to provide new methods for producing apomixis-enhanced plants that express a higher frequency of one or more of the various components (or elements) of apomixis relative to the plant materials from which said apomixis-enhanced plants were produced. Elements of apomixis include unreduced embryo sac formation (aposporous or diplosporous), parthenogenesis or adventitious embryony, and autonomous or pseudogamous endosperm formation. A further object of the instant specification is to provide novel methods for enhancing genetic variability within individual plants for initiation times and durations of various GDS components including megasporogenesis, embryo sac formation (including egg and central cell formation and maturation), fertilization, embryo formation and endosperm formation.
Additional objects and advantages of the present invention are set forth in the detailed description or will be appreciated by the practice of the invention.
To address the foregoing objects, and in accordance with the invention as described herein, the instant specification provides methods for:
It will be appreciated that numerous plants may be produced by the methods of the instant specification, and that the numerous plants produced will range in apomixis expression from less expression to more expression relative to the starting plant materials.
In one embodiment, the present invention is directed to a method of producing an apomictic plant having a frequency apomictic seed set exceeding that of the parent plant from which the apomictic plant was produced. The invention is also directed to a method of producing a plant that expresses an increased frequency of one or more of the various elements of apomixis. The elements of apomixis preferably include unreduced embryo sac formation (aposporous or diplosporous), parthenogenesis or adventitious embryony, and autonomous or pseudogamous endosperm formation. Generally, these methods include the steps of: (a) obtaining a parent plant that expresses one or more elements of apomixis and is not genetically stable for the elements of apomixis; (b) self fertilizing the parent plant or sib-mating the parent plant with another related parent plant that also expresses elements of apomixis, but is not genetically stable for the elements of apomixis; (c) obtaining seed from the parent plants; (d) sowing the seed obtained; (e) raising progeny plants there from; (f) screening the progeny plants for an increased frequency of apomictic seed set as compared to the parent plants; and (g) isolating the progeny plant expressing the increased frequency of apomictic seed set.
Preferably the frequency of apomictic seed set in the isolated progeny plant produced is at least 5% greater than the parent plants, more preferably at least 20% greater than the parent plants, and most preferably 40% greater than the parent plants.
In this embodiment, steps (b) through (e) can optionally be repeated at least one time to obtain second generation or higher generation progeny having an increased frequency of apomictic seed set compared to the previous generation.
Preferably the sib-mating is a full sib-mating or a half sib-mating, but other broad sib-matings are envisioned and intended to be within the scope of the invention.
In yet another embodiment, the parent plant is obtained by:
The invention is also directed to a method for selecting a group of plants to be used as a breeding population for producing plants that express apomixis. This method preferably includes the following steps:
The present invention is further directed to a method for selecting parent plants from a breeding population to produce plants that express an increased frequency of apomictic embryo sac formation and to a method of selecting parent plants from a breeding population for the purpose of producing plants that express an increased frequency of parthenogenesis, adventitious embryony, pseudogamous endosperm formation, or autonomous endosperm formation. These methods preferably include the steps of identifying ecotypes or breeding lines from the breeding population that represent extremes in GDS timing; and selecting from the identified ecotypes or breeding lines pairs of plants to be used as parents such that the mean onset time for embryo sac formation in one parent occurs early relative to the maturity level of sporophytic ovule or ovary tissues while the mean onset time for female meiosis (megasporogenesis) in the other parent occurs late.
The present invention also encompasses a method of producing an apomictic or apomictic-enhanced progeny plant from sexual or facultatively-apomictic parent plants comprising the steps of:
(a) selecting a first and second sexual or facultatively apomictic parent plant from an angiospermous plant species, genus, or family, wherein the mean onset time for embryo sac formation in the first parent plant occurs at about the same time as or before the mean onset time for megasporogensis in the second parent plant relative to the maturity level of sporophytic ovule or ovary tissue;
(b) hybridizing the first and second parent plants;
(c) obtaining seed from the first or second plants;
(d) sowing the seed obtained;
(e) raising progeny plants there from;
(f) identifying progeny plants that expresses elements of apomixis and is not genetically stable for the elements of apomixis;
(g) self-fertilizing or sib-mating one or more progeny plants identified;
(h) obtaining second generation seed from the progeny plants;
(i) sowing the obtained second generation seed obtained;
(j) raising second generation plants there from; and
(k) screening and identifying the second generation plants that are apomictic.
In yet another embodiment of the invention, the invention is a method of producing an apomictic progeny plant from sexual or facultatively-apomictic parent plants comprising the steps of:
(a) selecting genetically-divergent ecotypes or breeding lines of the same angiospermous species, genus or family;
(b) characterizing the ecotypes or breeding lines according to germline development sequence (GDS) relative to the maturity level of sporophytic ovule or ovary structures;
(c) producing a breeding population that includes ecotypes or breeding lines that represent extremes in GDS timing comprising:
(d) identifying ecotypes or breeding lines from the breeding population that represent extremes in GDS timing;
(e) selecting parent plants from the identified ecotypes or breeding lines, wherein the selected parent plants have:
(f) crossing the parent plants, obtaining seed there from, sowing the seed, raising F1 progeny plants, self fertilizing or intercrossing the F1 progeny, obtaining F2 or double cross seed from the F1 plants, sowing the F2 or double cross seed, raising the F2 or double cross progeny there from; and
(g) screening F2 or double cross progeny for an increased frequency of apomictic seed set as compared to the parent plants.
In this embodiment, step (f) can optionally be repeated at least once to obtain advanced breeding generations, followed by screening the advanced generation plants for an increased frequency of apomictic seed set as compared to the parent plants.
The methods of the invention can also further comprise the step of doubling the chromosome number of the progeny to stabilize apomixis. The invention further is directed to apomictic or apomictic-enhanced plants produced according to the methods disclosed herein, and progeny thereof.
Preferably, the plant used in the methods of the invention is a rice, wheat, maize, barley, sorghum, millet, soybean, potato or cotton plant. In a preferred embodiment, the plant is sorghum.
In angiosperms, the nucellus and MMC begin to differentiate early in development of the funiculus and chalaza, and prior to integument differentiation. Differentiation of the integument(s) then initiates by periclinal divisions in the epidermis of the chalaza. The chalaza, funiculus and integument(s) remain largely undifferentiated during megasporogenesis and early embryo sac formation (Esau 1977). In Antennaria, zones of coordinated periclinal cell divisions in intercalary meristematic tissues located near the chalaza/funiculus and chalaza/integument junctions cause the ovule to curve and assume an anatropous form (see photomicrographs, left column). Technically, the integument does not grow around the developing gametophyte, as is often stated in the literature. Instead, the terminus of the integument remains close to the funiculus base, and elongation of intercalary-meristem-produced cells causes the chalaza and chalaza-attached meiocyte or gametophyte to recede deep into the ovarian cavity (locule).
The formation of anatropous ovules was observed to be a canalized process that occurred essentially identically for all Antennaria species evaluated. In this respect, the distance from the integument terminus to the funiculus origin was similar for all species studied and did not change appreciably during embryo sac development (
Anatropous ovule formation appears to be developmentally canalized across the Antennaria species studied. In contrast, much genetically-determined heterochronicity was observed among accessions (
The unadjusted lines in
Before the present methods of increasing the frequency of apomixis expression in angiosperms are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the instant specification will be limited only by the appended claims and equivalents thereof.
The publications and other reference materials referred to herein are added to describe the background of the invention and to provide additional detail regarding its practice. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventor is not entitled to antedate such disclosure by virtue of prior invention.
It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
In describing and claiming the instant specification, the following terminology will be used in accordance with the definitions set out below.
As used herein, “comprising,” “including,” “containing,” “characterized by,” and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps. “Comprising” is to be interpreted as including the more restrictive terms “consisting of” and “consisting essentially of.”
As used herein, “consisting of” and grammatical equivalents thereof exclude any element, step, or ingredient not specified in the claim.
As used herein, “consisting essentially of” and grammatical equivalents thereof limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic or characteristics of the claimed invention.
As used herein, “apomixis” and grammatical equivalents thereof refer to asexual reproduction of plants through seed. Primarily in older literature, the term apomixis has included reproduction through vegetative structures other than seeds. However, during the past 50 years, the term “apomixis” has increasingly been restricted in the literature to asexual seed formation, including gametophytic apomixis (apospory and diplospory) and forms of adventitious embryony that result in asexual seed formation (Asker and Jerling 1992). In the instant specification, the restricted definition of the term “apomixis” is used. Accordingly, apomictically-produced seeds of apomictic plants contain embryos that are generally genetic clones of the mother plant.
As used herein, “facultative apomict” and grammatical equivalents thereof refer to a solitary plant that reproduces both sexually and apomictically, i.e. one or more ovules of the plant may produce seed sexually and one or more ovules of the plant may produce seed apomictically. With few exceptions all angiospermous apomicts are considered to be facultative apomicts (Asker and Jerling 1992).
As used herein, “obligate apomict” and grammatical equivalents thereof refer to a solitary plant that reproduces only by apomixis. It is believed that few if any obligate plant apomicts exist in nature (Asker and Jerling 1992).
As used herein, “levels of apomixis” and grammatical equivalents thereof refer to the percentage of ovules of a plant that produce seed apomictically. Most of the ovules of a highly or strongly apomictic plant produce seed apomictically. Generally, more than 98% of the ovules of a near-obligate apomictic plant produce seed apomictically. Few ovules of a weakly apomictic plant produce seed apomictically.
As used herein, “genetically-unstable apomictic plant” and grammatical equivalents thereof refer to an apomictic plant in which the level of apomixis among sexually-derived progeny of said genetically-unstable apomictic plant is on average lower than said genetically-unstable apomictic plant. Facultative apomictic reproduction tends to be replaced by sexual reproduction or sterility after several sexually-derived generations starting from a genetically-unstable facultative apomict (WO 01/32001).
As used herein, “MMC” and grammatical equivalents thereof refer to the megaspore mother cell (megasporocyte) in the ovule of an angiospermous plant.
As used herein, “GDS components” and grammatical equivalents thereof refer to components of the germline development sequence. These components consist of MMC differentiation, megasporogenesis, embryo sac formation, fertilization, embryony, and endosperm formation.
As used herein, “elements of apomixis” and grammatical equivalents thereof include unreduced embryo sac formation (aposporous and/or diplosporous), parthenogenesis and/or adventitious embryony, and autonomous or pseudogamous endosperm formation.
Apomixis occurs naturally in only a few crops and in close relatives of only a few other crops. It occurs in cultivated species or closely-related wild species of sugar cane, citrus, apples, pears, mangos, blackberries, raspberries, walnuts, strawberries, sunflowers, beets, cucumbers and onions. Among forage and turf crops, it occurs in wild or cultivated species of Poa (Kentucky bluegrass), Brachiaria, Bouteloua, Elymus, Cenchrus, Eragrostis, Panicum, Pennisetum, and Paspalum (Carman 1997). The instant specification is directed toward inducing or enhancing the expression of apomixis in such crops as well as all other angiospermous crops that may not have close apomictic relatives. It should be appreciated that the instant specification has additional commercial applications, e.g. in such fields as horticulture, floriculture and forestry (particularly hardwoods).
Apomictic ovule development in angiosperms often begins with megasporogenesis being preempted by a precocious embryo sac formation that initiates from the MMC itself (diplospory) or from a nearby cell of the nucellus (tissue that surrounds the MMC) or rarely the integument (leafy structure that surrounds the nucellus, apospory,
Wobble in the intensity of signals that cause apomixis allows for the facultative expression of sexual reproduction in apomicts. Most if not all apomicts are facultative, which means a certain percentage of seeds produced by the apomict will form sexually, rather than apomictically, and this percentage is influenced by genetic and environmental factors (Asker and Jerling 1992). In a near-obligate apomict, the percentage of seeds per plant that form sexually may be less than 1%. In contrast, weak facultative apomicts may produce less than 1% of their seed apomictically.
Several types of apomixis have been described. Most of these were discovered in the early part of the last century. In Antennaria-type diplospory, signals for precocious embryo sac formation occur very early and cause the MMC, which normally undergoes megasporogenesis, to form a genetically-unreduced embryo sac with no trace of megasporogenesis having been initiated. In Taraxacum-type diplospory, signals for embryo sac formation are less precocious and disrupt megasporogenesis after the first meiotic division. Wobble in the onset time of embryo sac formation in apomictic Elymus rectisetus allows for sexual development, Taraxacum-type diplospory, Antennaria-type diplospory, and various forms that are intermediate between the Taraxacum and Antennaria-types (Crane and Carman 1987). In Hieracium-type apospory, cells affected by precocious and ectopic embryo-sac-inducing signals are located in the nucellus or rarely the integument(s). The affected nucellar or integumentary cell undergoes three rounds of endomitosis to produce a mature genetically-unreduced 8-nucleate embryo sac. In Panicum-type apospory, only two rounds of endomitosis occur resulting in a mature genetically-unreduced 4-nucleate embryo sac. Additional types of apomixis have been described and are reviewed by Asker and Jerling (1992).
Five models of inheritance for apomixis have been hypothesized during the past 100 years:
The chromosomal nonhomology model was championed by Ernst in the early part of the 20th century. It stated that apomixis is a function of chromosomal nonhomology, i.e. it is one of several cytogenetic anomalies caused by wide hybridization. Accordingly, apomixis is not controlled by genes, but is a consequence of divergence in chromosome structure. This hypothesis was abandoned shortly after its inception because apomixis was shown to occur in plants whose chromosomes were largely homologous. Later in the century, genetic studies suggested that apomixis is simply inherited, i.e. that it involves genes (Carman 1997).
The quantitative-mode-of-inheritance model was popular in the early to mid 20 h century. It was championed by Muntzing who believed apomixis resulted from a delicate balance of few to many recessive genes, and by Powers, who believed that recessive genes caused three major elements of apomixis: failure of megasporogenesis, apomictic embryo sac formation, and parthenogenesis. In the latter half of the 20th century, results from numerous genetic analyses of apomixis, involving many natural apomicts, strongly implied that apomixis is simply inherited. Hence, the quantitative inheritance hypotheses were largely abandoned (Asker and Jerling 1992).
From the 1960s to the present, most apomixis scientists have favored simple inheritance models, i.e. that one or two dominant genes confer apomixis. Until recently, this conclusion appeared well founded in that Mendelian analyses repeatedly produced simple inheritance segregation ratios, e.g. 1:1 ratios of apomictic to sexual progeny had often been produced in crosses made between sexual and apomictic plants (Asker and Jerling 1992; Savidan 2000, 2001; Sherwood 2001). Based on these findings, several well-funded R&D programs were initiated in the 1980s and 1990s that have focused on transferring the “apomixis gene(s)” from a wild naturally apomictic species to a related sexual crop species through introgressive plant breeding schemes (crossing and backcrossing). However, despite seemingly simple inheritance, years of effort have not resulted in the identification or isolation of the hypothesized “apomixis gene” nor in its transfer to sexual crops to produce commercially-viable crop plants. While apomictic hybrids between apomictic wild relatives and crop species have been readily produced, such hybrids and their backcross progenies have remained weedy or largely sterile (Spielman et al 2003). Furthermore, mapping studies are now indicating that if apomixis is controlled by one or a few apomixis genes, such genes are located in large chromosomal regions in which recombination is suppressed (Spielman et al 2003; Koltunow and Grossniklaus 2003). Consequently, many scientists today are considering the possibility that apomixis may be controlled by numerous genes and modifiers (Carman 1997; WO 98/33374; WO 01/32001; Grimanelli et al 2001; Grossniklaus et al 2001; Richards 2003; Spielman et al 2003; Koltunow and Grossniklaus 2003).
The epigenetic model suggests that apomixis is caused by heritable epigenetic changes in gene regulation. The epigenetic changes are caused by changes in DNA methylation, which may accompany structural changes in chromatin due to hybridization, chromosomal rearrangements and polyploidy. This model combines elements of the mutation and hybridization models. Epialleles are heritable, like mutations, and they can be induced by hybridization and polyploidization (Koltunow and Grossniklaus 2003). However, the epigenetic model does not explain the fact that hybridization and polyploidization have played major roles in the evolution and speciation of over 460 angiospermous families (Ramsey and Schemske 1998), yet over 75% of the genera that contain apomictic species belong to only three families (Carman 1997).
The RS model was developed by the inventor of the instant specification and states that apomixis is the product of multiple quantitatively-inherited traits that are genetically stabilized by structural heterozygosity (
The two previous patent applications, WO 98/33374 and WO 01/32001, and the new technology described in the instant specification represent major advances in the state-of-the-art for producing apomictic plants. These patent applications are unique from other published approaches to producing apomictic plants in that the inventor's procedures involve the manipulation of naturally occurring genetic variability for initiation times and durations of megasporogenesis, embryo sac formation, stigma exertion, fertilization, embryony and endosperm formation, all of which are normal sexual processes (
The instant specification and WO 98/33374 are similar to each other in that both involve methods of producing plants that express a higher frequency of apomixis than the starting plants. They fundamentally differ with regard to how megasporogenesis, embryo sac formation, fertilization, embryony and endosperm formation are uncoupled so as to permit apomixis to occur. In WO 98/33374, the methods largely rely on asynchrony of entire GDS sequences (
Before elucidating the specific processes of the instant specification, it is desirable to review current hypotheses concerning genes that regulate the various elements of apomixis. By comparing these hypotheses with the inventor's discoveries, it is clear that the inventor's processes materially advance the state-of-the-art with regard to the development of methods for predictably producing apomictic plants.
The elements of apomixis were clearly described in the first half of the 20th century and include disruption or abortion of megasporogenesis, aposporous or diplosporous embryo sac formation, parthenogenesis or adventitious embryony, and autonomous or pseudogamous endosperm formation (Gustafsson 1946, 1947a, 1947b). Clearly, the most popular school of thought during the past 40 years, concerning the genetic regulation of apomixis, is that a single mutation that causes aposporous or diplosporous embryo sac formation could pleiotropically cause parthenogenesis as well (Savidan 2000, 2001). However, many scientists, starting with several in the first half of the 20th century, have genetically uncoupled apomictic embryo sac formation from parthenogenesis, which suggests that these elements of apomixis are regulated by separate genes (Gustafsson 1947a; Spielman et al 2003). This understanding of an uncoupling of discrete developmental steps, which was first arrived at in the first half of the 20th century (Gustafsson 1947a), was later memorialized by Nogler, who extended the idea by describing apomictic embryo sac formation as an opening (or uncoupling) of the developmental bonds linking megasporogenesis with sexual embryo sac formation (Nogler 1984).
Most scientists who study apomixis today believe that the uncoupling and reshuffling events that cause apomixis are controlled in most cases by at least two apomixis genes (mutations of normal genes), which are generally tightly linked, or by epigenetic modifications to the regulation of wild-type genes (Spielman et al 2003; Koltunow and Grossniklaus 2003). In contrast, the inventor of the instant specification has demonstrated that apomixis is the result of competition between asynchronously-expressed developmental programs that are combined together either naturally or intentionally by hybridization. As taught in WO 98/33374, apomictic embryo sac formation may occur when genes that initiate sexual embryo sac formation (inherited from a plant wherein megasporogenesis, embryo sac formation, fertilization, embryony and endosperm formation occur early in ovule development) are expressed at about the same time as or earlier than genes that initiate megasporogenesis (inherited from a different plant wherein megasporogenesis, etc, occur relatively late in ovule development) (
It was taught in WO 01/32001 that sexually-derived segregants of a facultatively apomictic plant would express either the same level of apomixis as the parent plant (same percentage of ovules developing apomictically) or a lower level. For example, recombination involving heterozygous loci responsible for apomixis (asynchronously expressed female developmental sequences) could, if inherited, result in progeny wherein the critical loci are homozygous, which could cause a disruption of the developmental competition responsible for apomixis. In this respect, the uncoupling of megasporogenesis and fertilization were viewed only as a preemptive removal of these processes by competing signals (
The inventor recently discovered that genes controlling initiation times and durations of megasporogenesis are different from genes controlling initiation times and durations of embryo sac formation, etc (
The instant specification, in combination with WO 98/33374 and WO 01/32001, provide methods of producing genetically-stabilized apomictic plants from sexual or facultatively-apomictic plants, the latter being less apomictic than the apomictic plants produced. High frequency apomixis (near obligate expression) is important for many agricultural applications of apomixis. Near obligate expression assures crop uniformity (flowering date, plant height, yield variables, nutrition variables, etc), which is necessary for modern agricultural practices. Consequently, it is advantageous to provide methods that result in plants with an increased frequency of apomixis expression relative to the plants from which the improved lines were derived. It should be appreciated that these and other advantages of the present application (discussed below) represent major advancements in the state-of-the-art.
It is convenient to separate the processes of the instant specification into five categories: (a) compiling groups of lines that contain sufficient genetic variability to produce, through plant breeding or genetic engineering, plants in which the within-plant frequency of expression of apomixis (or one or more of its elements) is increased, (b) selfing, crossing or otherwise genetically-modifying said groups of lines in such a way as to produce plants from which more highly apomictic plants may be obtained, (c) producing subsequent-generation recombinant plants, (d) screening plants for apomixis, and (e) repeating certain steps to increase the frequency with which apomictically-enhanced plants are produced.
Compiling Groups of Lines from which Plants with Enhanced Levels of Apomixis May be Produced
One step of the methods involves selecting or producing sexual or facultatively-apomictic plants from which plants with an increased expression of apomixis can be produced. One process for obtaining said sexual or facultatively-apomictic plants is to follow the methods of WO 98/33374. Accordingly, the parent plants of each said sexual or facultatively-apomictic plant will have been delineated such that initiation of embryo sac formation in one parent occurs at about the same time as or before megasporogenesis in the other parent relative to developmental maturity of sporophytic ovule or ovary tissues (
Another step involves choosing said related plants so that they contribute genetic variability (different alleles) to the next generation of hybrids with regard to initiation times and durations of megasporogenesis, embryo sac formation, fertilization, embryo formation, or endosperm formation relative to sporophytic ovule or ovary tissues (
It should be appreciated that genetic variability for initiation times and durations of the various stages of female development in ovules is a new discovery of the inventor (
Genetically-Modifying Groups of Lines to Increase the Frequency in which Plants with Enhanced Levels of Apomixis are Produced
Another step of the methods involves either crossing or selfing said sexual or facultatively-apomictic hybrid plants or outcrossing one or more of them to a related plant of the same species, genus or family. It is anticipated that genes controlling timing of GDS stages will soon be cloned, and these could be used in an alternative approach involving transformation to modify GDS timing in appropriate ways so as to induce apomixis. For plant breeding, standard plant breeding procedures may be used to accomplish selfing, crossing or outcrossing such as are taught in Poehlman (1987), and for mapping, cloning and transformation, standard approaches well practiced in the industry may be used such as are taught in Weigel and Glazebrook (2002).
Producing Subsequent-Generation Recombinant Plants from which Plants with Enhanced Levels of Apomixis May be Selected
Other steps of the methods include sowing seed obtained from selfing, backcrossing, cross-hybridizing (e.g., full-sib or half-sib crossings, see Poehlman 1987), outcrossing or genetic engineering and growing the resulting second-generation or later generation plants.
Selecting Plants with Enhanced Levels of Apomixis
Additional steps of the methods include screening first, second or subsequent generations of plants for apomixis by (a) using cyto-embryological procedures (
Repeating Certain Steps to Increase the Frequency with which Apomictically-Enhanced Plants are Produced
Further steps of the methods include repeating one or more times prior steps to produce additional segregating generations from which plants with enhanced apomixis are derived and identified by conducting additional screening and progeny test procedures.
The present invention is described by reference to the following examples. These are offered by way of illustration only. They are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were used. It will be appreciated that the instant specification may be embodied in many specific forms without departing from its spirit or essential characteristics.
Apomixis was first described at the embryological level in Antennaria alpina (Juel 1900). Antennaria (x=14) are dioecious, herbaceous perennials and are usually stoloniferous. Morphology-based cladistic analyses of 32 sexual diploid species coupled with analyses of sequenced internal transcribed spacer regions of nuclear ribosomal DNA (ITS-1 & ITS-2) indicated that Antennaria is composed of six clades (Bayer 1990; Bayer et al 1996). Apomixis occurs only in the Catepes clade, which contains 17 of the 32 sexual Antennaria species and sexual and apomictic polyploids ranging from 4× to 12× (Bayer and Stebbins 1987; Bayer and Minish 1993). All members of this group are stoloniferous and sexually dimorphic. Five geographically-divergent complexes of interbreeding sexual and apomictic Antennaria species (agamic complexes), A. alpina (L.) Gaertn., A. howellii E. L. Greene, A. parlinii Fern., A. parvifolia Nutt., and A. rosea, have evolved from among the sexual members of the Catipes (Bayer 1987; Bayer et al 1996).
Antennaria sp, number of sites collected and latitudinal range.
A. alpina
A. aromatica
A. corymbosa
A. densifolia
A. friesiana alaskana
A. friesiana friesiana
A. friesiana neoalaskana
A. marginata
A. media
A. microphylla
A. monocephala
A. parvifolia
A. racemosa
A. rosea
A. rosulata
A. umbrinella
The center of diversity for the A. rosea agamic complex is the Rocky Mountains of North America, and its range is from New Mexico and southern California, north to Alaska and the Northwest Territories, and east through Alberta, Saskatchewan, the northern Great Lakes and with disjunct populations in Atlantic Canada (Bayer 1989a). It is generally tetraploid, although triploid and pentaploid plants have been found (Bayer and Stebbins 1987). It appears to have arisen by multiple hybridizations and introgression involving A. aromatica Evert, A. corymbosa E. Nelson, A. pulchella E. Greene, A. marginata E. Greene, A. microphylla Rydb., A. racemosa Hook., A. rosulata Rydb. and A. umbrinella Rydb. These are sexual species that primarily inhabit unglaciated portions of the western cordillera. Phenetic analyses indicated that A. aromatica, A. corymbosa, A. microphylla, A. pulchella/media, and A. umbrinella are the major sexual progenitors of the A. rosea complex. Only a few A. rosea clones displayed morphological characteristics that can be attributed to A. marginata or A. rosulata (Bayer 1990). Analyses of all pairwise comparisons of zymograms from 33 A. rosea populations indicated only slight divergence. The genetic identity (I) averaged 0.944 (range=0.802-0.997). Most of the A. rosea populations were most similar to populations of A. corymbosa, A. microphylla, A. pulchella/media and A. umbrinella. Fewer were similar to A. aromatica, A. marginata, and A. rosulata (Bayer 1989b).
In an extensive survey of biotic and abiotic factors, the sexual A. aromatica, A. corymbosa, A. marginata, A. media/pulchella, A. microphylla, A. racemosa, A. rosulata, and A. umbrinella occurred in distinct habitats but apomictic A. rosea occupied the center of the overall ordination and overlapped at least parts of the ordination space of all sexual species except A. aromatica and A. racemosa (Bayer et al 1991). This study further supported the hypothesis of a multiple hybrid origin for A. rosea. Many sites of A. rosea occupy similar habitats to their diploid sexual progenitors, and other sites occupy hybrid habitats that are intermediate between those of their sexual progenitors (Bayer et al 1991). Hence, there is good evidence from a variety of sources (morphologic, isozymic, ecological) to support the hypothesis of a multiple hybrid origin for the A. rosea agamic complex.
Sexual Antennaria were collected from their native habitats throughout the Rocky Mountain Cordillera (Table 1), grown in cultivation, embryologically analyzed for GDS variation (
There is evidence that low level facultative apomictic seed formation (up to 25%) has occurred in at least some Sorghum lines (Hanna et al 1970; Tang et al 1980; Schertz 1992; Bala Ravi 1993). To assess whether apomixis in these lines arose from hybridization, rather than fortuitous mutation, we tested the following null hypothesis: apomixis fails to arise in hybrids produced by crossing progenitors of known facultatively-apomictic sorghum lines. To our knowledge, such simple tests had not previously been conducted, i.e. conventional wisdom assumed that apomixis arose by mutation. Progenitors of two facultatively-apomictic Sorghum lines, ‘R473’ and ‘302’, were obtained. Progenitors of R473 are ‘IS 2942’ (a day neutral Kafir line) and ‘Aispuri’ (a short day Indian variety) (Tang et al 1980). Progenitors of 302 are ‘IS 3922’ and ‘Karad Local’ (Rana et al 1981). Additional lines totaling 20 S. bicolor, 14 tetraploid Sorghum×Almum, four tetraploid S. halapense, three S. arundinacium, three tetraploid S. australiense (2n=4x=20, i.e. n=5), and two wide hybrids of unknown parentage were chosen based on diversity of habitat or prior history of being involved in apomixis research. GDS-characterized lines involved in producing hybrids that were subsequently screened embryologically for apomixis are listed in Table 2.
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. bicolor
S. hybrid
S. halepense
S. hybrid
S. arundinaceum
S. bicolor
S. bicolor
S. bicolor
Pistils for cytological analysis were killed, fixed, cleared and observed using DIC microscopy as in Peel et al (1997a,b). Cytological data was obtained at the MMC, dyad, triad/tetrad, functional megaspore, 1-nucleate embryo sac, 2-nucleate embryo sac, 4-nucleate embryo sac, early 8-nucleate embryo sac, mature embryo sac, stigma exertion, and ripe seed stages. The following data was obtained for each ovule analyzed: meiotic or embryo sac development stage, pistil length and width, integument length and width, and meiocyte or embryo sac length and width. Tables 3-4 exemplify data sheets used to GDS-characterize Sorghum lines from the MMC to mature embryo sac stages (data from line SB1001.1 are shown). Additional data sheets were used to obtain cytological data for the stigma exertion and ripe seed stages. Plants from Table 2 were grown, embryologically analyzed for GDS variation (
Genetic segregation was extensive among the 21 F2s of two Sorghum bicolor parent lines (5.1, 7.1) for initiation times and durations of meiosis and embryo sac formation relative to the maturity level (length) of the inner integument (
The F2 progeny in
Sorghum bicolor Tag # PI253638 Plant # 1-1 variety Aispuri (tall)
The described steps and materials in the foregoing examples are to be considered in all respects only as illustrative and not restrictive, and the scope of the invention is indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
This application is a continuation of U.S. application Ser. No. 10/969,054 filed Oct. 21, 2004, claims the benefit of priority of U.S. Provisional Application No. 60/512,919, filed Oct. 22, 2003; and is also a continuation-in-part of U.S. application Ser. No. 10/772,243 filed Feb. 6, 2004, which is a continuation-in-part of U.S. application Ser. No. 09/744,614 filed Jan. 26, 2001, which is the U.S. National Stage of International Application No. PCT/US00/29905, filed Oct. 30, 2000, which claims priority to U.S. Application No. 60/162,626, filed Oct. 29, 1999; and is also a continuation-in-part of U.S. application Ser. No. 10/785,157 filed Feb. 25, 2004, which is a divisional of U.S. application Ser. No. 09/576,623 filed May 23, 2000, now U.S. Pat. No. 6,750,376, which is a continuation of U.S. application Ser. No. 09/018,875 filed Feb. 5, 1998, which claims the benefit of priority from U.S. Application No. 60/037,211 filed Feb. 5, 1997, the entire contents of each of which are expressly incorporated herein by reference thereto.
| Number | Date | Country | |
|---|---|---|---|
| 60512919 | Oct 2003 | US | |
| 60162626 | Oct 1999 | US | |
| 60037211 | Feb 1997 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09576623 | May 2000 | US |
| Child | 10785157 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10969054 | Oct 2004 | US |
| Child | 12416883 | US | |
| Parent | 09018875 | Feb 1998 | US |
| Child | 09576623 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10772243 | Feb 2004 | US |
| Child | 10969054 | US | |
| Parent | 09744614 | Jan 2001 | US |
| Child | 10772243 | US | |
| Parent | 10785157 | Feb 2004 | US |
| Child | 09744614 | US |
