WO2000021358A1 - Hybrid alfalfa (medicago sativa) - Google Patents

Abstract

Molecular and morphological data shows that sexual hybridization of Medicago laciniata M. sativa and Medicago laciniata Medicago truncatula was successful by using pre-flower opening emasculation, controlled pollination and pod/ovule/embryo culture techniques. The techniques are time consuming and inefficient at this point in time and result in a limited transfer of genetic material from the pollen donor. Further optimization of the techniques would likely improve efficiency. Recovered F1 hybrids consistently show morphological characteristics intermediate between the parents but characters are skewed toward the maternal parent. The morphological observations are supported by molecular evidence which shows that the majority of DNA in the hybrids in maternal in nature. The recovered F1 plants retained the plant nodulation specificity factor from M. laciniata and produced adequate amount of seed. This is the first report of a successful hybridization of these species. It is recommended that the F1 seed be tested in a plant breeding program for segregation of desirable agronomic characteristics, including nodulation specificity and crossing ability with alfalfa (M. sativa) at the diploid and tetraploid level. Crossing of hybrids with M. sativa may be difficult due to the predominance of M. laciniata genetic material. Further cycles of hybridization using the protocols described here may be necessary to increase the M. sativa germplasm component of the population to the point that it can be handled with conventional breeding approaches.

Description

HYBRID ALFALFA {MEDICAGO SATIVA)

CROSS-REFERENCE TO RELATED APPLICATIONS This Application is related to but does not claim priority from U.S. Provisional Application titled, "Polynucleotide Fragment of S. meliloti USD A 1170 Containing Nodulation Efficiency Factor, filed October 13, 1998, (Attorney Docket No. 018756-000100) which is herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not Applicable.

BACKGROUND OF THE INVENTION

Alfalfa (Medicago sativa) originated near Iran, but related forms and species are found as wild plants scattered over central Asia and into Siberia. Alfalfa is now found worldwide as a cultivated hay crop. Its value as feed for horses and other animals is well documented, having been lauded by the Romans as early as 490 B.C. It has the highest feeding value of all commonly grown hay crops and is often used in combination with com silage in livestock rations to take advantage of the protein and energy content of the two feeds. With careful management, alfalfa can be used successfully as a pasture crop, with stands lasting over 5 years.

In spite of the qualities listed above, there is room for improvement. In the quest for better production, beneficial traits of many cultivated plants, including alfalfa, have been bred out. Creating hybrids between the commercially valuable plant and a wild relative which exhibits the beneficial trait is one mechanism of reintroducing the trait into the commercially important line. However, in addition to reintroducing a lost trait into a plant, hybrid breeding can also introduce into a commercial plant a beneficial trait never before seen in the plant.

Such a beneficial trait in alfalfa, as well as other legumes, is the establishment of a specific symbiotic relationship with highly-efficient, nitrogen- fixing bacteria. Many commercial legumes, even though inoculated with efficient nitrogen-fixing bacteria upon sowing, are outcompeted by bacteria resident in the soil which form a majority of the nodules on the legume roots. This can lead to lower production yields for the farmer, since the indigenous bacteria are often less efficient at fixing nitrogen than the inoculant strain. M. laciniata, a wild relative of alfalfa, originated in the Middle East. It is nodulated efficiently and specifically by S. meliloti strain USD A 1170. On the other hand,

M. sativa is nodulated by USDA strain 1170 but the bacteria, which reside in the root nodules, do not fix nitrogen. Creating a hybrid in which the nodulation specificity gene from M. laciniata is transferred to M. sativa will result in an alfalfa hybrid with high nodulating specificity for S. meliloti USD A 1170. An obstacle to creating the hybrid described above, as well as other alfalfa hybrids, is reproductive incompatibility between species of Medicago. The ability to cross alfalfa outside of the commercially-cultivated Medicago sativa-falcata complex has been successful only in a few cases and the hybridization of M. sativa (subgenus Medicago, section Falcago, subsection Falcatae) and M. laciniata (subgenus Spirocarpos, section Leptospirae) has not been reported (Arcioni, et al., Somatic Hybridization and Embryo '

Rescue for the Introduction of Wild Germplasm in BIOTECHNOLOGY AND THE IMPROVEMENT OF FORAGE LEGUMES, McKersie and Brown, eds, CAB International (1997); McCoy & Smith, Theoretical and Applied Genetics 71:772 (1986); McCoy, Canadian J. of Genetics and Cytology 27:238 (1985); and Quiros & Bauchan, The genus Medicago and the origin of the Medicago sativa complex, in ALFALFA AND ALFALFA IMPROVEMENT, Hanson, et al,

(eds), American Society of Agronomy, Madison, WI (1988)). In fact, the only successful hybridization outside of the subgenus Medicago, was a cross between M. sativa and M. scutellata (subgenus Spirocarpos, section Rotatae). However, this hybrid was both male and female sterile (Sangduen, et al., Can. J. Genet. Cytol. 24:361 (1982). Thus, there is a need for a mechanism that can be used to transfer desirable traits from reproductively incompatible Medicago parents to hybrid progeny. This invention meets this and other needs.

BRIEF SUMMARY OF THE INVENTION One embodiment of this invention provides for a fertile hybrid Medicago plant resulting from sexually crossing, through embryo rescue, a reproductively incompatible Medicago plant and Medicago sativa. The two parental plants are from different subgenera of the genus Medicago. In a preferred aspect of this embodiment, the Medicago parent is effectively nodulated by one strain of Sinorhizobium which does not effectively nodulate Medicago sativa. In another aspect of this embodiment, the reproductively incompatible Medicago plant is Medicago laciniata. In a preferred aspect, the Medicago laciniata plant is land race 307. In another aspect of this embodiment, the Medicago sativa is Medicago sativa cultivar Regen S. In yet another aspect of this embodiment, the hybrid is diploid. And in yet another aspect, the strain of Sinorhizobium is Sinorhizobium meliloti. In a preferred aspect, the Sinorhizobium meliloti comprises a nodulation specificity factor from strain USDA 1170. Another embodiment of this invention provides for seeds produced by a hybrid resulting from sexually crossing, through embryo rescue, a reproductively incompatible Medicago plant and Medicago sativa. In a further aspect of this embodiment, the seed is treated with colchicine.

Yet another embodiment of this invention provides for a hybrid plant produced by backcrossing a hybrid plant resulting from sexually crossing, through embryo rescue, a reproductively incompatible Medicago plant and Medicago sativa with Medicago sativa. Also encompassed in this embodiment are the progeny of the backcrossed hybrid. In yet another aspect, the backcrossed hybrid is multiploid, preferably tetraploid.

In still another embodiment of this invention, a method of making a fertile hybrid Medicago plant is provided for comprising the steps of fertilizing a gamete from a first Medicago plant with a gamete from a second, reproductively incompatible Medicago plant, thereby forming an embryo; removal of the developing pod from the plant at a time period of 0-168 hours after fertilization, preferably 72 hours after fertilization; contacting the pod with nutrients supplied in an in vitro medium for 0-14 days, preferably 7 days; isolating developing ovules from the pod and culturing the isolated ovules in an in vitro environment and a nutrient medium for 14 days; removing the embryo from the ovule and contacting the embryo with nutrients supplied in an in vitro environment; and culturing in the in vitro environment a Medicago plant from the embryo. In a further embodiment, a flower comprising anthers and pistil from one plant is isolated, preferably in medium in an in vitro environment. In a preferred aspect, the flower is from Medicago laciniata.

In a preferred aspect of this embodiment, the anthers are removed from the flower, preferably by hand emasculation and between about 36 and about 50 hours prior to the flower opening. After the anthers are removed, pollen from the second Medicago plant is deposited onto the pistil of said flower. Preferably, the second Medicago plant is Medicago sativa.

In yet another aspect of this embodiment, fertilization of the gametes takes place in an in vitro environment with pollen from Medicago sativa and the female gamete from Medicago laciniata. After fertilization takes place, the embryo is dissected from the ovule to allow nutrients from the media to contact the embryo, and preferably removed from the ovule between about 65 and about 80 hours after said fertilizing of the gamete.

In another embodiment, a plant produced by the above embodiment is provided and preferably comprises a plant nodulation specificity factor (PSF). In yet another embodiment, a method for making a hybrid Medicago plant resulting from a cross of Medicago laciniata and Medicago sativa is provided comprising the steps of isolating a flower comprising anthers and pistil from a Medicago laciniata; removing the anthers from the flower; depositing pollen from a Medicago sativa plant onto the pistil of the flower; removing a embryo from an ovule of the flower; and culturing in an in vitro environment the hybrid Medicago plant from the embryo.

DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton, et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICΉONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., Rieger, R., et α/.(eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The term "cross" in the context of this invention refers to the fusion of gametes by pollination to produce seed. A "sexual cross" is pollination of one plant by another. "Selfmg" is the production of seed by self-pollinization, i.e., pollen and ovule are from the same plant. The term "backcrossing" refers to crossing a F_j hybrid plant to one of its parents. Typically, backcrossing is used to transfer genes which confer a simply inherited, highly heritable trait into an inbred line. The inbred line is termed the recurrent parent. The source of the desired trait is the donor parent. After the donor and the recurrent parents have been sexually crossed, F₍ hybrid plants which possess the desired trait of the donor parent are selected and repeatedly crossed (i.e., backcrossed) to the recurrent parent, inbred, or a another desirable line. For example, in the invention as exemplified herein, the donor parent is effectively nodulated by Sinorhizobium meliloti strain USD A 1170, i.e., Medicago laciniata. The recurrent parent is the commercially desirable Medicago sativa. After the initial cross between these two parents, Ε_{ hybrids which are effectively nodulated by strain USD A 1170 are selected and back-crossed with Medicago sativa to confer the desired, commercially important, phenotype. The end result is a hybrid which possesses the phenotype of M. sativa but is effectively nodulated by S. meliloti USDA strain 1170. The term "depositing" refers to the physical contacting of pollen and stigma.

The pollen can be from another plant, i.e., a sexual cross, or the pollen can be from the same plant, i.e., selfing.

The term "emasculation" refers to the removal of the anthers from the flowers of this invention. Emasculation can be any technique which completely removes the anthers without contacting the pollen contained therein with the stigma of the flower. Preferred ^' emasculation is by vacuum or by hand. Most preferred is hand emasculation.

The term "flower" refers to the reproductive structure of angiosperms, including Medicago. A complete flower includes the calyx, corolla, at least one stamen and at least one carpel which may contain one or more ovaries. The Medicago flowers of this invention also contain "anthers." Anthers are the pollen-bearing portion of a stamen. The carpel of Medicago flowers is centrally located. The carpel typically comprises one or more ovules contained within an ovary, style and stigma. After pollen is deposited on the stigma, the pollen germinates and forms a pollen tube. The micro gametophyte (sperm) in the pollen travels through the pollen tube to the ovule where fertilization takes place. The term "pollen" refers to mature microspores containing a mature or immature microgametophyte.

The phrase "hybrid plants" refers to progeny of a sexual cross between genetically different individuals. The phrase "inbred plants" refers to plants derived from a cross between genetically related plants. The phrase "diploid plants" refers to plants that have two sets of chromosomes, one from each parent. The phrase "tetraploid plants" refers to plants that have four sets of chromosomes, two sets from each parent. Tetraploidy can result from imperfect mitosis wherein chromosomes replicate but the cell does not divide. Tetraploidy often results from temporarily contacting the seed of a diploid plant with "colchicine." Colchicine inhibits the formation of the spindle apparatus in mitosis. Thus, after undergoing one round of mitosis in the presence of colchicine, the chromosomes of each cell are doubled.

The term "immobilization" refers to physically stabilizing a flower of the disclosed invention. Immobilization of a flower either can be on the plant or in an in vitro environment.

The phrase "in vitro environment" refers to an environment removed from a plant. In the context of this invention, unless indicated otherwise, in vitro refers to plant tissue culture. Plant tissue culture techniques are well known in the art and can be found in many practice guides and textbooks including, Dodds & Roberts, EXPERIMENTS IN PLANT TISSUE CULTURE, 3RD ED., Cambridge University Press (1995); and Pollarnd, ed., METHODS

IN MOLECULAR BIOLOGY: PLANT CELL AND TISSUE CULTURE TECHNIQUES, VOL. 6, Humana

Press (1990).

The term "isolating" in the context of this invention refers to physically removing possible contaminating plants or organs of plants from a Medicago flower. Typically this is done by removing the flower or parts of the flower from the plant and - incubating the flower or parts of the flower in an in vitro environment.

The phrase "Medicago plant" refers to a plant of the genus Medicago. "M. laciniata " is a wild relative of alfalfa. It is found naturally in the Middle East and Northern

Africa. Individual lines of M. laciniata are referred to as "land races." A preferred land race of this invention is "307." "M. sativa " for purposes of this invention is a parent and recurrent parent of this invention. M. sativa also refers to plants of the M. sativa-falcata complex. The complex contains diploid and tetraploid forms of M. sativa, M. falcata and M. glutinosa. There appears to be no barrier to crossing within the plants of complex at the same ploidy level. The complex encompasses all of the perennial forms cultivated for forage. Throughout this disclosure, M. sativa refers to the diploid plant and "alfalfa" represents the tetraploid commercially important plant. M. sativa strains are referred to as

"cultivars" A preferred cultivar is "Regen S."

The terms "nodulation" or "nodulated," for purposes of this disclosure, refers to the ability of a Sinorhizobium to nodulate and reside in the roots of legumes. The term "effective nitrogen fixation" refers to the ability of a Sinorhizobium to not only reside in the roots of legumes but to fix nitrogen once residing there. Strains of Sinorhizobium preferentially nodulate different species of legumes. Specific nodulation between a host legume and Sinorhizobium is the result of the complimentary genetic factors present in the host legume and the Sinorhizobium.

The term "parents" refers to the sources of the gametes which are fused and develop into the hybrid plants of this invention. Typically, the parents are the pollen donor and the ovule donor which are crossed to make the hybrid plant of this invention. Parents also refer to F, parents of a hybrid plant of this invention (the F₂ plants). Finally, parents refer to a recurrent parent which is backcrossed to hybrid plants of this invention to produce another hybrid plant of this invention.

The term "progeny" refers to the descendants of a particular plant (self-cross) or pair of plants (crossed or backcrossed). The descendants can be of the F,, the F₂, or any subsequent generation.

The phrase "reproductively incompatible" refers to a post-zygote formation mechanism which prevents hybrid embryo development. The Medicago plants of this invention are reproductively incompatible. One hypothesis for reproductive incompatibility is that after fertilization, endosperm fails to develop. Without endosperm to feed the developing embryo, the embryo dies. To overcome the incompatibility, "embryo rescue" is utilized. Embryo rescue refers to a technique wherein an embryo is grown on medium in an in vitro environment. The in vitro environment provides the nutrients and other physical requirements necessary for development of the embryo. To allow the nutrients present in the in vitro environment to contact the embryo, in some cases, the ovule surrounding the embryo must be removed or otherwise dissected away from it. Alternatively, the ovule can be made permeable to the nutrients present in the in vitro environment.

The term "Sinorhizobium" refers to a genus of bacteria which, in a symbiotic relationship with the host legume, resides in root nodules of the host legume and fixes atmospheric nitrogen. The nitrogen is then used by the legume. As mentioned above, different strains of Sinorhizobia nodulate different species of legumes. "S. meliloti" is a species of Sinorhizobia that nodulates legumes of the genus Medicago. "Strain USDA 1170" is a strain of Sinorhizobium meliloti which nodulates and fixes nitrogen, i.e., effectively fixes nitrogen, on M. laciniata. USDA 1170 also elicits nodules on M. sativa and resides in the nodules, but the resident bacteria do not fix nitrogen.

The term "seed" refers to a structure formed by the maturation of the ovule of seed plants following fertilization. In Medicago, seeds are typically in the form of "pods." Within the pod are "ovules." Ovules comprise integuments, nucellus, which forms the endosperm after a second fertilization, and an egg cell or gamete, which when fertilized becomes first a zygote and then an "embryo." An embryo is a sporophytic plant before the start of germination. "Fertilization" refers to the fusion of male and female gametes.

The terms "subgenus" and subgenera" refer to groupings of Medicago spp. in the genus Medicago. Typically, the genus is divided into subgenera. Each subgenus is divided into Sections. Section Falcago, of the subgenus Medicago, is further divided into Subsections, including Falcatae, which includes the species M. sativa. See Table 1 for a list of subgenera and sections within the genus Medicago.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for hybrid Medicago plants and methods for creating them. One embodiment of this invention is a hybrid alfalfa plant. In a preferred embodiment, the hybrid is derived from two reproductively incompatible parents. This hybrid is possible because of another embodiment of this invention; a novel embryo rescue technique. The techniques described herein can be used to cross a variety of reproductively incompatible members of the genus Medicago.

The ability to cross Medicago species would permit the introduction of genes of potential agronomic importance into commercially grown alfalfa. An example is the nodulation specific factor from M. laciniata as described in the given examples below. One of skill, upon review of this disclosure will realize other useful applications of the technology described herein.

I. PARENTS OF THE MEDIC A GO HYBRID OF THIS INVENTION

The genus Medicago, contains over 60 species which are grouped by subgenera and sections. See, Table 1. Some of the species, particularly of the subgenus

Medicago and section Falcago interbreed. However, across subgenus lines, Medicago are reproductively incompatible. The preferred parents for the Medicago hybrid plants of this invention cross subgenus lines and are selected from the group consisting of M. sativa, M. truncatula, and M. laciniata. In a most preferred embodiment, the parents of the hybrid plants of this invention are selected from the group consisting of M. sativa, cultivar Regen S; truncatula, cultivar J.E. 103, and laciniata, land race 307. Table 1: Medicago spp. (from Quiros & Bauchan, The genus Medicago and the origin of the Medicago sativa complex, in ALFALFA AND ALFALFA IMPROVEMENT, Hanson, et al , (eds), American Society of Agronomy, Madison, WI (1988))

Subgenera

Lupularia Orbtcularia Medicago Spirocarpos

M lupuhna Section Carstiensae Section Falcago Section Rotatae M secund ora M carstiensis Subsection Falcatae M rotata Section Platycarpae M sativa M blancheana

M platycarpa M glomerata M noeana

M ruthemca M prosttata M shepardu Section Orbiculares Subsection Rupestres M rugosa

M orbiculans M rhodopea M scutellata Section Hymenocarpos M saxatilhs Section Pachyspirae

M radiata M rupestris M soleirolu Section Heymanae M cancellata M tornata

M heymana Subsection Daghnestanicae M littorahs Section Cretaceae M daghestanica M truncatula

M cretacea M pironae M ngiduia Subsection Papύlosae M murex

M dzhawakhetica M lesnsu

M pap losa M constricta Section Arborea M turbinata

M marina M dohata Section Suffruticosae Section Leptospirae

M sujfruticosa M sauvagei

M hybrida M laciniata

M minima

M praecox

M coronata

M polymorpha

M arabica

M lanigera

M dtsciformis

M tenoreana Section Intertextae M intertexta M ciharis M muricoleptis M granadensis

M sativa, cultivar Regen S is a highly regenerable perennial species of Medicago It is a member of the subgenus Medicago, section Falcago and the subsection Falcatae This places it withm the M sativa-falcata complex Members of the complex, M sativa, M falcata and M glutinosa, intercross with each other and share the same karyotypes M sativa has lavender to violet flowers and a coiled pod For a more thorough description of the members of the M sativa-falcata complex, see Quiros & Bauchan, The Genus Medicago and the Origin of the Medicago sativa Complex, ALFALFA AND ALFALFA IMPROVEMENT, Hanson, et al (eds ), American Society of Agronomy, Inc , Madison, WI (1988)

M laciniata is a member of the subgenus Spirocarpos and the section Leptospirae It is an annual wild species grown m a natural state in a Mediterranean region It has small yellow flowers, an apical coil spmy pod with lateral veins on the coil face _joining as shoulders at πght angles to the elevated dorsal suture The preferred strains ofM laciniata mature withm 75 days after seeding m pots (seed to seed) M laciniata, as well as

M sativa, is diploid with 8 sets of chromosomes (for a total chromosome number of 16)

A. Maintenance of Diploid Medicago Parents Plant in a Greenhouse

The parent plants of this invention can be established in any environment suitable for growth of Medicago but preferably, to maintain a controlled climate, in a greenhouse If pests are a particular problem, the plants can be grown in growth chambers Because insecticides affect the flowering and pod development of Medicago, the use of msecticidal agents is not preferred The preferred conditions in a growth chamber are a temperature of 25°C and a 16 hour photopeπod The plants can be grown m any suitable container and m any soil mixture that supports Medicago, however m a preferred embodiment, the plants are grown m 5-7" fiber pots m a sandy soil of 1 1 1 soil sand peat The plants should be watered twice daily by misting and fertilized weekly with a commercial mix of 7 7 7 Nitrogen:Phosphorus:Potassium. The plant lines are maintained by repeated cuttings and replanting in a greenhouse.

B. Crossing Techniques. Upon review of this disclosure, those of skill will realize many different techniques for crossing the parent plants of this invention can be used. These techniques include in vitro fusion of male and female gametes, pollination by insect or bird vectors, pollination by manual tripping and physical transfer of pollen, and pollination in the open field. However, in a preferred embodiment, artificial pollination is used. Artificial pollination consists of at least two steps: (1) prevention of self- pollination; and (2) depositing pollen from the male parent onto the female stigma. In nature, the stigma is mostly inaccessible to pollen prior to tripping (the physical unfolding of the petals of the flower). Typically, in nature, bees and other insects trip flowers by landing on the keel petals of the Medicago flowers, however, some annuals, such as M. laciniata, are self-tripping. Once tripped, the restraint of the keel petals on the pistil is reduced. This causes the stigma to slap onto the standard petal and rupture the stigmatic cuticle. Pollen then can access the stigma and germinate. For a complete discussion of flower morphology and the tripping mechanism, see Viands et al, Pollination Control: Mechanical and Sterility, ALFALFA AND ALFALFA IMPROVEMENT, Hanson, et al. (eds.), American Society of Agronomy, Inc., Madison, WI (1988).

To prevent self-tripping and self-pollination, pollen from the maternal flower should be removed or otherwise prevented from fertilizing the egg cells within the ovule. In Medicago, pollen matures before the flower is tripped. Therefore, the pollen should be removed from the flower prior to the flower's opening. When both the male and female parents are at the flowering stage, the maternal buds are selected for emasculation. Emasculation is the removal of the anthers from the maternal flower to prevent self-pollination. Preferably, unopened buds are chosen. In a more preferred embodiment, the buds are about 35 to about 60 hours prior to opening. In a most preferred embodiment, the maternal buds are about 48 hours prior to opening. To allow for the selection of viable pollen, the flower of the male donor should just be opening.

Before manually tripping the maternal flower, the flower should be immobilized. Immobilization stabilizes the flower through the subsequent manipulations as well as protects the flower from adventitious pollination, e.g., selfing. Immobilization can be done while the flower is still on the plant in an greenhouse. However, it is preferable to remove the flower from the plant and immobilize it in an in vitro environment. This is done by securing the flower in a clamp, preferably metal, which has been lined with plastic foam or another coating to protect the flower. After the maternal flower has been immobilized, the flower is tripped, emasculated and pollinated. To avoid contacting the stigma with the standard petal which would cause the stigmatic cuticle to rupture, thus making the stigma more accessible to pollen from the maternal plant, the standard petal should be removed. The flower is tripped by gently squeezing it at the base. The anthers are then removed by emasculation. In a preferred embodiment, the anthers of the maternal flower are removed before pollination. Any method useful for removing the anthers can be used. However, hand and vacuum emasculation are preferred. Hand emasculation is the removal by forceps of the anthers, leaving the filaments behind. Vacuum emasculation is the removal of the anthers by pulling them up into the pipette under a slight vacuum source. To ensure that all of the pollen has been removed, the flower should be examined under a low-powered binocular magnifier.

The maternal flower should be pollinated immediately after emasculation. In a preferred embodiment, pollen from the male parent is placed directly on the stigma of the female parent. Depositing pollen can be done by any suitable means, including but not limited to, dotting with a sterile cotton swab, brushing the stigma with an anther of the male parent or painting the pollen on the stigma with a fine paintbrush.

After pollination, a solution of a plant growth regulator, preferably gibberellic acid, may be applied to the peduncle of the flower to ensure the flower remains attached to the ovule and pod formation is initiated. Preferably, pods are removed 72 hours after flower opening.

C. Hybrid Embryo Rescue

In Medicago, reproductive incompatibility is due to a variety of causes. After self-pollination, in some species, pollen tubes fail to develop or fail to reach the ovary. In other species, ovules are aborted or seeds are not formed. In interspecific crosses, reproductive incompatibility is often due to ineffective pollen germination, slow pollen tube growth rate, and failure of endosperm development. Pollen grains from angiosperm contain two microgametophytes. After a pollen tube forms, the two sperm travel to the ovule. The first sperm fertilizes the egg cell. The second fuses with the polar nuclei to form the endosperm (Stanford, et al, Cytology and Evolution of the Medicago sativa-falcata Complex, ALFALFA SCIENCE AND TECHNOLOGY, Hanson (ed), American Society of Agronomy, Madison, WI (1972)). If the second fertilization does not take place, the endosperm fails to develop. The embryo does not receive the nutrients from the endosperm and fails.

To support other Medicago hybrid embryos, ovule-embryo culture (see Arcioni et al, 1997; McCoy, 1985; McCoy and Smith, 1988), has been used to overcome post-zygote function incompatibility. However, these techniques have not been successful in all Medicago hybrids, particularly between crosses of annual and perineal species, e.g., M. sativa and M. laciniata.

In this invention, because previously published methods of embryo rescue have been proven to be unsuccessful, a novel embryo rescue technique is utilized to maintain a hybrid embryo. By removing and culturing an immature pod soon after fertilization, the abortion of the embryo at the late heart stage is avoided (see, McCoy and Smith) Typically, between 24 and 96 hours after fertilization, preferably between 64 and 80 hours after fertilization, and most preferably 72 hours after fertilization, the developing pods are removed from the plant and surface sterilized. The most preferred sterilization technique is rinsing the pods in 70% ethanol for approximately 5 minutes. After sterilization, the pods are transferred onto growth regulator- free medium in vitro.

5 -cm Petri dishes with 10 mL of medium are preferred for pod development, but one of skill will recognize that other sterile containers and amounts of medium may be used. Any growth regulator-free medium is acceptable, but the preferred medium is modified SH medium (Schenk & Hildebrandt, Canadian J. of Botany 50:199 (1972)). The pods are preferably cultured at about 25°C in a sterile environment.

After a suitable time in culture, preferably one week, the ovules are aseptically dissected from the pods and the viable ovules transferred to ovule medium (Table 1). So nutrients are made available to the embryo, the ovule is made permeable to the culture medium. A preferred method is removal of the embryo from the ovule. However, any method that allows the embryo to come into contact with the culture medium yet allows the embryo to grow and form roots and shoots may be utilized. The embryos are transferred to any container with medium, 5 -cm Petri dishes with 10 mL of medium being preferred. The preferred medium consists of the salts and nutrients as shown in Table 2, however, any suitable plant cell culture medium can be used.

Table 2. Composition of the medium used for the pod/ovule culture.¹

Macro elements Micro elements

(mg/L) (mg/L)

KNO₃ 2500 MnS0₄-4H₂O 10

MgS0₄ 400 H₃BO₃ 5

CaCl₂ 200 ZnS0₄-7H₂O 1

KH₂P0₄ 355 KI 1

(NH₄)₂SO₄ 991 CuS0₄-5H₂0 0.2 CoCl₂-6H₂O 0.1

Organic NaMo₂O₄-2H₂O 0.1

FeSO₄-7H₂O 15

Inositol 1000 Na-.EDTA 20

Nicotinic Acid 5

ThiamineΗCl 5 Gelrite 2000

Pyridoxine' HCl 0.5

Proline 11000 adjust to pH 5.8

Maltose 30000

After the embryos have begun to develop, typically two weeks after placing them on the ovule medium, the plantlets are transferred to growth boxes. "Growth boxes" refers to clear, sterile, polycarbonate or polypropylene boxes. Typically, such boxes have membrane-covered vents to allow gas exchange with the ambient atmosphere but prevent microbial contamination. The vents can be either in the sealable lid or drilled into a sidewall of the box. Preferred growth boxes are the Magenta Box™ brand from Sigma Chemical Co.

1 Based on Piccirilh & Arciom, New interspecific hybrids in the genus Medicago through in vitro culture of fertilized ovules, in ANGIOSPERM POLLEN AND OVULES, Ottaviano (ed), Springer- Verlag (1991). (St. Louis, MO). The growth conditions of the plantlets in the growth boxes are preferably kept the same as the embryo growth conditions but with proline-free medium.

After an adequate root system has developed, the developing plantlets are transferred to soil in a growth box, preferably a sterile sandy soil mix.

II. EMBRYOGENESIS AND EMBRYO CULTURE

In a most preferred embodiment, somatic embryogenesis is used to propagate the hybrid plants of this invention. Somatic embryogenesis is the direct or indirect production of embryos from cells, tissues and organs of plants (see McKersie & Brown, Somatic embryogenesis and artificial seeds, BIOTECHNOLOGY AND THE IMPROVEMENT OF

FORAGE LEGUMES, McKersie & Brown (eds), CAB International, Wallingford, U.K. (1997)).

Indirect somatic embryogenesis is characterized by growth of a callus and the formation of embryos on the surface of the callus. Direct somatic embryogenesis is the formation of an asexual embryo from a single cell or group of cells on an explant tissue without an intervening callus phase. Because abnormal plants tend to be derived from a callus, direct somatic embryogenesis is preferred.

Some species of Medicago, e.g., M. laciniata, are highly embryogenic and can be induced to form somatic embryos in tissue culture. Therefore, in a preferred embodiment, the F, hybrids are induced to form somatic embryos so that a plurality of plants can be grown from one hybrid embryo.

Induction of embryogenesis is as follows. After the cotyledon stage of development has been achieved, the embryos are transferred into fresh medium. A preferred medium is a MS-based medium as shown in Table 3. After the hybrid somatic embryos initiate root and shoot development, typically within two weeks, the plantlets are transferred to root- and shoot-development medium (e.g., Murashige and Skoog Shoot Multiplication

Medium). Table 3. Composition of modified MS medium used for late-stage embryo development.² --_____

Macro elements Micro elements (mg/L) (mg/L)

MgS0₄-7H₂O 92.5 H₃B0₄ 1.6

MnSO₄-H₂O 4.2 ZnS0₄-7H₂0 2.1

CaCl₂-2H₂O 110.0 CuS0₄-5H₂O 0.006

NH₄NO₃ 412.5 KI 0.22

KNO₃ 475 CoCl₂.-6H₂0 0.006

KH₂PO₄ 42.5 NaMo₄-2H₂0 0.063

FE(III)EDTA 212.5

Organic

Nicotinic acid 0.5 Gelrite 2000

PyridoxineΗCl 0.5

ThiamineΗCl 1.0 adjust to pH (5.8)

Myo-inositol 100

Sucrose 30,000

Those of skill in the art will realize that different acclimation procedures can be used to obtain ¥_l plantlets of hybrid crosses. In a preferred embodiment, cuttings, as well as somatic embryos of various crosses, after developing a root and shoot, are transferred to medium, e.g., the medium shown in Table 3, for establishment of plantlets. After the plantlets develop an adequate root system, typically 4-5 weeks, the plantlets are taken out from the medium and transplanted into soil. A sandy soil is preferred. The plantlets and soil can be placed in growth boxes or into fiber pots. If pots are used, they should be covered with plastic bags, e.g., polyethylene, to maintain humidity and placed in a growth chamber. Water should be supplied daily, preferably by misting.

After the plantlets initiate their shoot growth, the established F, plants are transferred to larger pots for full recovery. The entire acclimation process typically takes approximately 4-6 weeks from the time of removal from the tissue culture medium to soil

2. Based on Murashige & Skoog, Physiologia Plantarum 15:473 (1962). establishment, although more or less time may be required depending on the environment, e.g., culture medium, temperature, soil mix, watering, etc.

For further development of the hybrid plants of this invention and characterization of the hybrids as well as the parents, the Medicago parents and the recovered F, hybrids, once established in soil, are regenerated through cuttings. The plants are preferably kept in a greenhouse and, more preferably, in a mist bed.

III. MULTIPLOIDY

Like many plants raised for food, commercially grown alfalfa is multiploid. Typically, it is tetraploid but up to octoploid lines are sometimes seen. Multiploidy is desirable in that beneficial genes are overproduced. This leads to larger flowers, pods and leaves, as well as higher nutrient value.

The basic genomic number of the genus Medicago is x = 8. Multiploidy in Medicago results either from the fusion of unreduced gametes (diploid) or the treatment of the embryo with an inhibitor of spindle formation, i.e., colchicine.

In a preferred embodiment, polyploid hybrids of this invention are created by fusion of gametes. Most typically, polyploid progeny are produced by sexually crossing the diploid hybrids of this invention with polyploid alfalfa plants. In the Medicago sativa- falcata complex, it has been found that tetraploid progeny is found if the diploid parent is the female. Either triploid or tetraploid progeny result if the diploid parent is the male (see,

Stanford, et al, "Cytology and Evolution of the Medicago sativa-falcata Complex," ALFALFA SCIENCE AND TECHNOLOGY, Hanson (ed.), American Society of Agronomy, Madison WI (1992)). In another embodiment, polyploidy results from the fusion of nonreduced diploid gametes. In another embodiment, colchicine is used to generate polyploid hybrid plants. Optimum concentration and exposure of seeds to colchicine requires some experimentation since different plants react differently to the compound. Therefore, it is necessary to determine a dose response curve with the embryos of the hybrids of this invention before proceeding to large scale treatment. To ensure contact with the embryo, it usually is necessary to remove the ovules from the pods. It may also be necessary to remove the embryos from the ovules. The embryos are then contacted with an optimal concentration of colchicine for an optimal exposure time. Since embryos, at different stages of development, react differently with colchicine, embryos at different stages of development should be tested. Typically, meristematic stages are responsive. The dose response curve should encompass colchicine concentrations from 0.001% to 1.0%o and exposure times from 1 hour to 5 days.

The desired dose response is multiploidy in the plants. After the embryos have germinated and root and shoot development has begun, cells from the plants are removed and a karyotyping is done to determine the level of ploidy (see, infra, for a description of karyotyping). Alternatively, as a screening assay, the chloroplasts in the plant cells, particularly the guard cells of the epidermis, can be counted.

IV. CHARACTERIZATION OF HYBRIDS

The hybrid plants of this invention can be characterized either genotypically or phenotypically. Genotypic analysis is the determination of the presence or absence of particular genetic material. For purposes of this invention, the genetic material is conferred upon a hybrid plant of this invention by either one of its parents. Therefore, for comparison to the hybrid offspring, the parents of the hybrid plants of this invention also undergo genotypic analysis. The parents of the hybrid plant being analyzed are either the original reproductively incompatible parents of a F, hybrid, sexually crossed F, hybrids, or any other progeny of an original cross of reproductively incompatible parents. For example, the parents, in a backcross, will be the donor, or hybrid parent and the recurrent, or inbred, parent. One of skill will recognize that genotypic analysis also can be performed to determine the presence or absence of particular genetic material conferced upon a plant by other means, e.g., recombinantly.

Phenotypic analysis is the determination of the presence or absence of a phenotypic trait. A phenotypic trait is a physical characteristic of a plant determined by the genetic material of the plant in concert with environmental factors. Phenotypic traits can either be simple, e.g.,Mendelian, or complex, e.g., quantitative. Mendelian traits are those conferred upon the hybrid plant by dominant genes. One skilled in the art will realize that resistance to disease and nematode pests is conditions usually by one or a few genes with varying degrees of dominance. For example, resistance to bacterial wilt caused by Corynebacterium insidiosum is caused by a single dominant tetrasomic gene coded for resistance. This resistance gene was reported to have originated from a single regional (Turkestan) genetic source. In the case of summer black stem, caused by Cercospora medicaginis, controlled crosses show that both dominance and recessive genes appear to influence the expression of resistance to this disease. Resistance to root-knot nematode (Meloidogyne spp.) has been shown to be determined by a single dominant gene that is inherited tetrasomically in some clones but due to duplicate dominant genes in other clones.

A quantitative phenotypic trait is one wherein the physical characteristic of the F, hybrid is intermediate between the physical trait of the two parents. For example, M. laciniata has small, highly serrated leaves. M. sativa has larger, less serrated leaves. The F, hybrid produced by the cross of these two Medicago species has an intermediate leaf size with fewer serrations than the M. laciniata parent but the serrations are deeper than those of the M. sativa parent. Leaf size and degree of serration, are therefore, quantitative traits.

A. DNA Analysis

1. DNA Fingerprinting

In general, "DNA fingerprinting" is a broad term used to designate methods for assessing sequence differences in DNA isolated from various sources. Typically, DNA fingerprinting is used to analyze and compare DNA from different species of organisms. Tn a preferred embodiment of this invention, DNA fingerprinting is used to assess the relationship of individuals, particularly parents and progeny.

DNA sequence differences detected by fingerprinting are referred to as DNA polymorphisms. The presence of a DNA polymorphism in an organism's DNA can serve to indicate the genetic origin of such an organism and serve as a characteristic genetic marker of that organism. Such polymorphisms can result from insertion, deletion, and/or mutation events in the genome.

Many methods are known in the art for DNA fingerprinting. The restriction fragment length polymorphism (RFLP) technique employs restriction enzyme digestion of DNA, followed by size separation of the digested DNA by gel electrophoresis, and hybridization of the size-separated DNA with a specific polynucleotide fragment (or "polynucleotide probe"). As used herein, a "probe" is a biochemical labeled with a radioactive isotope or tagged in other ways for ease in identification. A probe is used to identify a specific region of DNA, a gene, a gene product or a protein. Thus a "polynucleotide probe" is a nucleic acid molecule that can be used to identify complementary nucleic acid sequences. The sequence of the polynucleotide probe may or may not be known. Differences in the size of the restriction fragments to which the polynucleotide probe binds reflect sequence differences in DNA samples, or DNA polymorphisms. See Tanksley, Biotechnology 7:257-264 (1988). Thus, a "polymorphic DNA fragment," is a DNA fragment which has a unique size and sequence, and is either present in other DNA samples with another unique size or is not present in other DNA samples. Other fingerprinting methods generate DNA fragments for fingerprint analysis using polymerase chain reaction (PCR) amplification of specific DNA sequences. See, e.g. Williams, Nucl. Acids Res. 18:6531-6353 (1990) (random amplified polymorphic DNA (RAPD) technique), Heath, Nucl. Acids Res. 21:5782-5785 (simple sequence repeat (SSR) technique), and PCT application WO 93/06239 (amplified fragment length polymorphism (AFLP) technique). See also U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202 (discussing PCR amplification techniques).

All of these PCR-based fingerprinting methods result in the generation of a large number of reproducible DNA fragments of specific size and sequence that can be separated according to size, typically by gel electrophoresis. Visualization of the size-separated fragments is effected either by direct visualization with a fluorescent dye, by hybridization with a labeled polynucleotide probe, or by labeling the amplification products during PCR (radioactively or fluorescently) followed by detection of the labeled products in the gel.

2. Making and Using Markers for Detection of Polymorphic Nucleic

Acids

Although DNA sequences which code for necessary proteins are well conserved across a species, there are regions of DNA which are non-coding or code for portions of proteins which do not have critical functions and therefore, absolute conservation of nucleic acid sequence is not strongly selected. The major causes of genetic variability are addition, deletion or point mutations, recombination and transposable elements within the genome of individuals in a plant population.

Point mutations are typically the result of inaccuracy in DNA replication. During meiosis in the creation of germ cells or in mitosis to create clones, DNA polymerase "switches" bases, either transitionally (i.e., a purine for a purine and a pyrimidine for a pyrimidine) or transversionally (i.e., purine to pyrimidine and vice versa). The base switch is maintained if the exonuclease function of DNA polymerase does not correct the mismatch. At germination, or the next cell division (in clonal cells), the DNA strand with the point mutation becomes the template for a complementary strand and the base switch is incorporated into the genome.

Transposable elements are sequences of DNA which have the ability to move or to jump to new locations within a genome. Several examples of transposons are known in the art (see, e.g., Freeling M., Ann. Rev. Plant. Physiol 35:277-298 (1984); Haring, et al,

Plant Mol. Biol 16:449-469 (1991); and Walbot, Ann. Rev. Plant Mol Biol 43:49-82 (1992)).

One of skill can generate probe nucleic acids for detecting markers, including probes which are PCR primers, allele-specific probes, RAPD probes and the like for the detection of polymorphic nucleotides at the loci disclosed herein, as well as the genetically linked sequences discussed below. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Vol. 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al, MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.) Vol. 1-3, Cold Spring

Harbor Laboratory, Cold Spring Harbor Press, NY (1989), (Sambrook); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (through and including the 1997 Supplement) (Ausubel). A catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., THE ATCC CATALOGUE OF BACTERIA AND BACTERIOPHAGE, Gherna et al. (eds) (1992) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Lewin, GENES V, Oxford University Press Inc., NY (1995) (Lewin); and Watson et -z/.,RECOMBiNANT DNA, 2ND ED., Scientific American Books, NY (1992).

Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the Sigma Chemical Company (Saint Louis, MO); New England Biolabs (Beverly, MA); R&D systems (Minneapolis, MN); Pharmacia LKB Biotechnology (Piscataway, NJ); CLONTECH Laboratories, Inc. (Palo Alto, CA); ChemGenes Corp.,

(Waltham MA) Aldrich Chemical Company (Milwaukee, WI); Glen Research, Inc. (Sterling, VA); GIBCO BRL Life Technologies, Inc. (Gaithersberg, MD); Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland); Invitrogen (San Diego, CA); Perkin Elmer (Foster City, CA); and Stratagene; as well as many other commercial sources known to one of skill.

The nucleic acid compositions of this invention, whether DNA, RNA, cDNA, genomic DNA, or analogs thereof, or a hybrid of these molecules, are isolated from biological sources or synthesized in vitro. The nucleic acids of the invention are present in plants, whole cells, cell lysates or in partially purified or substantially pure form.

In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well as U.S. Patent No. 4,683,202; PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; Kwoh et al, Proc. Nat 'lAcad. Sci. USA 86:1173 (1989); GuateUi et al, Proc.

Nat 'lAcad. Sci. USA 87:1874 (1990); Lomell et al, J. Clin. Chem. 35:1826 (1989); Landegren et al, Science 241:1077-1080 (1988); Nan Brunt, Biotechnology 8:291-294 (1990); Wu & Wallace, Gene 4:560 (1989); Barringer et al,Gene 89:117 (1990), and Sooknanan & Malek, Biotechnology 13: 563-564 (1995). Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al, U.S. Pat. No. 5,426,039. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra.

Ohgonucleotides for use as probes, e.g., in in vitro amplification methods, for use as gene probes, or as inhibitor components (e.g., ribozymes) are typically synthesized chemically according to the solid phase phosphorarnidite triester method described by Beaucage & Caruthers, Tetrahedron Letts., 22(20):1859-1862 (1981), e.g., using an automated synthesizer, as described in Needham-VanDevanter et al, Nucl. Acids Res., 12:6159-6168 (1984). Ohgonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of ohgonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Regnier, J. Chrom. 255:137-149 (1983). The sequence of the synthetic ohgonucleotides can be verified using the chemical degradation method of Maxam & Gilbert, Methods in Enzymology 65:499-560 (1980). 3. Labeling and Detecting Probes

A probe for use in an in situ detection procedure, an in vitro amplification procedure (PCR, LCR, etc.), hybridization techniques (allele-specific hybridization, in situ analysis, Southern analysis, northern analysis, etc.) or any other detection procedure herein can be labeled with any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include spectral labels such as fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, dixogenin, biotin, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³³S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g., horse-radish peroxidase, alkaline phosphatase etc.) and other labels known to those skilled in the art.

In general, a detector which monitors a probe- target nucleic acid hybridization is adapted to the particular label which is used. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill.

Because incorporation of radiolabeled nucleotides into nucleic acids is straightforward, this detection represents a preferred labeling strategy. Exemplary technologies for incorporating radiolabels include end-labeling with a kinase or phosphatase enzyme, nick translation, incorporation of radio-active nucleotides with a polymerase and many other well known strategies.

Fluorescent labels are also preferred labels, having the advantage of requiring fewer precautions in handling. Prefened labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labeling. Fluorescent moieties, which are incorporated into the labels of the invention, are generally are known, including but not limited to, Texas red, rhodamine and fluorescein. Individual fluorescent compounds which have functionalities for linking to an element desirably detected in an apparatus or assay of the invention, or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthydrol. Many fluorescent tags are commercially available from SIGMA chemical company (St. Louis, MO), Molecular Probes, R&D systems

(Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GLBCO BRL Life Technologies, Inc. (Gaithersberg, MD), Fluka Chemica- Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, CA) as well as other commercial sources known to one of skill.

4. Hybridization Strategies

In a preferred aspect, a labeled nucleic acid probe is specifically hybridized to nucleic acid from a biological sample and the label is detected, thereby determining that the marker nucleic acid is present in the sample. For example, a marker can be detected by Southern analysis, northern analysis, in situ analysis, or the like. Similarly, a marker comprising a polymorphic nucleic acid can be detected by allele-specific hybridization of a probe to the region of the marker comprising the polymorphic nucleic acid (as described below).

Two single-stranded nucleic acids "hybridize" when they form a double- stranded duplex. The region of double-strandedness can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single stranded nucleic acid, or the region of double-strandedness can include a subsequence of each nucleic acid. "Stringent hybridization conditions" in the context of nucleic acid hybridization are sequence dependent and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), id. Generally, stringent conditions are selected to be about 5 ° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50%) of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the T_m point for a particular probe. Sometimes the term "T_d" is used to define the temperature at which at least half of the probe dissociates from a perfectly matched target nucleic acid. In any case, a variety of estimation techniques for estimating the T_m or T_d are available, and generally described in Tijssen, id. Typically, G-C base pairs in a duplex are estimated to contribute about 3°C to the T_m, while a-T base pairs are estimated to contribute about 2°C, up to a theoretical maximum of about 80-100°C. However, more sophisticated models of T_M and T_d are available and appropriate in which G- C stacking interactions, solvent effects, the desired assay temperature and the like are taken into account. In one example, PCR primers were designed to have a dissociation temperature (T_j) of approximately 60 °C, using the formula: T_d = (((((3 x #GC) + (2 x #AT)) x 37) - 562) / #bp) - 5; where #GC, #AT, and #bp are the number of guanine-cytosine base pairs, the number of adenine-thymine base pairs, and the number of total base pairs, respectively, involved in the annealing of the primer to the template DNA.

An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg/mL heparin at 42 °C, with the hybridization being carried out overnight. An example of stringent wash conditions for a Southern blot of such nucleic acids is a 0.2x SSC wash at 65 °C for 15 minutes (see, Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40 °C for 15 minutes.

In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. For highly specific hybridization strategies such as allele-specific hybridization, an allele-specific probe is usually hybridized to a marker nucleic acid (e.g.; a genomic nucleic acid, an amplicon, or the like) comprising a polymorphic nucleotide under highly stringent conditions.

5. RAPD Analysis In a most preferred embodiment, RAPD analysis is used to analyze the genotypic information of the hybrids of this invention. RAPD analysis detects recombination events at the genome level and is based on the detection of specific but randomly generated fragments of DNA. Because it does not require labeled nucleic acid probes or hybridization as described above, it can be quickly performed. In a prefened embodiment, 5 different sets of primers (RAPD primer series 406, 417, 420, 427, and 429, available from the Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, CANADA) are used to generate amplified fragments of polymorphic DNA.

Briefly, the genomic DNA is isolated from the parents and the hybrid progeny. Each genomic DNA sample is amplified with sets of primers. The amplification products are then electrophoresed in an agarose gel to generate DNA profiles. Agarose electrophoresis of DNA is well known in the field. If desired, the PCR primers can be labeled with radioisotopes or fluorophores for better detection of low concentrations of DNA. However, usually enough DNA fragments are generated in the amplification step so that labeling is not necessary and the DNA profile can be visualized by staining the gel with Ethidium Bromide, again, a standard procedure.

If the profile in the lane with the maternal DNA is identical to the profile in the lane with the progeny's DNA, then no exchange of DNA has occurred and the progeny is a result of a self fertilization. If the DNA profile of the progeny is different in any way from the DNA profile of the maternal parent, then rearrangement of the genome has occuned. If a band corresponding to a band in the DNA profile of the paternal parent is present, then the rearrangement in the DNA of the progeny is due to the incorporation of new DNA (genes) from the pollen donor.

This type of data provides strong proof there has been some transfer of DNA (genes) from the pollen donor to the maternal flower and DNA from the pollen donor has been incorporated into some of the Fj plants; that is, the F_! plants are hybrid and contain DNA material from both parents.

B. Morphological and Quantitative Trait Study

The general appearance of progeny is also used to characterize the hybrids of this invention. Morphological traits of leaflet hairiness, flower morphology, pod shape and hairiness, growth and regeneration pattern are monitored throughout the generations of progeny. The quantitative traits of number of ovule/seed per pods, pod length (mm), pod width (mm), central leaflet length and breadth (mm) and number of serrations on the central leaflet length (mm) of all the maturing plants of each crosses are also monitored.

This quantitative trait data is significant for the identification of the heterotic expression or depression in the progeny. For confidence, at least twenty observations of each quantitative trait of the female parent, the hybrid progeny and the male parent are subjected to an analysis of variance. The results of each plant for various traits with a least significant difference (LSD) at 5% and the coefficient of variation are then determined and tabulated.

In addition to heterosis expression and depression in the progeny, differences in quantitative traits can indicate recombination at the genetic level. For example, in crosses between M. laciniata and M. sativa, the pod length (mm) and number of seeds per pod were reduced in the F, in all three plants measured, and pod width (mm) was increased significantly when compared with the female M. laciniata, indicating recombination had occurred.

In addition to gross morphology, cytology of the parents and progeny can be compared to characterize the hybrid progeny. Cytological analysis includes but is not limited to, karyotyping to determine chromosome number, chromosome painting to determine the presence of parentally derived chromosomes in the hybrid progeny and in situ hybridization for, e.g., isozyme analysis.

Karyotyping is well known in the art. Staining methods developed over the last 30 years permit unambiguous identification of each individual chromosome. To stain, the dividing cells are removed from the plant. The cells may be cultured in vitro if desired.

Colchicine can be added to the culture to stop mitosis at metaphase and the cells collected and spread on a microscope slide. Alternatively, the cells can be collected and then fixed on a microscope with 3:1 ethanol: acetic acid to stop mitosis. The cells on the microscope slide, are stained and photographed. Giemsa staining can be used to visualize the chromosomes, although more typically, the chromosomes are stained with fluorescent dyes. The chromosomes are then paired, depending on their size and banding pattern. The number of chromosomes is the chromosome number. In diploid Medicago somatic cells, the number of chromosomes is 16, or 8N. In alfalfa, it is possible and commercially desirable for plants to be at the tetraploid level (32 chromosomes). However, up to 64 chromosomes, the octoploid level, has been observed and may be even more desirable. Multiploidy in the progeny is accomplished as described above.

In addition to the older technique of karyotyping metaphase chromosomes and interphase nuclei, fluorescence in situ hybridization (FISH) is an important technique for visualizing DΝA sequences. The method is now in routine use in research laboratories for gene localization studies. For example, FISH is used to map genes to specific chromosome regions or to order clones along chromosomes to create. In addition, FISH can also be used to compare parental and progeny chromosomes.

A class of FISH probes termed "chromosome paints" are available. This type of probe is very useful for determining chromosome structure, as they more or less uniformly hybridize to the entire length of a given chromosome. Paints are used to determine chromosome complements of a cell, structural abnormalities such as translocations, and to identify the parental origin of marker chromosomes. Numerous methods are available to label DNA probes for use in FISH, including indirect methods whereby a hapten such as biotin or digoxigenin is incorporated into DNA using enzymatic reactions. Following hybridization to a metaphase chromosome spread or interphase nuclei, a fluorescent label is attached to the hybrid through the use of immunological methods. More recently, fluorescent dyes have been directly incorporated into probes and detected without the use of an intermediate step. Standard FISH dyes include fluorescein, rhodamine, Texas Red and Cascade Blue. Multiprobe FISH analysis can be accomplished by labeling different probes with different haptens or fluorescent dyes.

V. SELECTIVE BREEDING OF HYBRIDS

A. Genetic Mapping of a Hybrid Genome

After the initial cross, it is beneficial in selecting subsequent generations to select progeny which contain genetic material which confers a specific beneficial trait. Before selection can begin, however, a genetic map of the desirable genome should be made. Genetic mapping is done by finding polymorphic markers that are genetically linked to each other (in linkage groups) or linked to genes or QTL affecting phenotypic traits of interest. The alignment of markers into linkage groups is useful as a reference for future use of the markers and for accurately positioning genes or QTL relative to the markers. Many of these QTL's have multiple sub-loci and haplotypes across the sub-loci. Each haplotype provides a different allele composition within a locus, thereby expanding the utility of these marker loci to more Medicago mapping studies than possible with only two alleles per locus.

B. Allele-Specific Hybridization (ASH)

One especially prefened technique for screening progeny for particular sequences conferred upon it by its parents is allele-specific hybridization, or "ASH." This technology is based on the stable annealing of a short, single-stranded oligonucleotide probe to a single-stranded target nucleic acid only when base pairing is completely complementary. The hybridization can then be detected from a radioactive or non-radioactive label on the probe (methods of labeling probes and other nucleic acids are set forth in detail below). ASH markers are polymorphic when their base composition at one or a few nucleotide positions in a segment of DNA is different among different genotypes. For each polymorphism, two or more different ASH probes are designed to have identical DNA sequences except at the polymorphic nucleotide(s). Each probe will have exact homology with one allele sequence so that the complement of probes can distinguish all the alternative allele sequences. Each probe is hybridized against the target DNA. With appropriate probe design and stringency conditions, a single-base mismatch between the probe and target DNA will prevent hybridization and the unbound probe will wash away. In this manner, only one of the alternative probes will hybridize to a target sample that is homozygous or homogeneous for an allele (an allele is defined by the DNA homology between the probe and target). Samples that are heterozygous or heterogeneous for two alleles will hybridize to both of two alternative probes. Having a probe for each allele allows the polymorphism to be genetically co-dominant which is useful in determining zygosity. In addition, a co-dominant ASH system is useful when hybridization does not occur for either one of two alternative probes, so that control experiments can be directed towards verifying insufficient target DNA or the occurrence of a new allele.

ASH markers are used as dominant markers where the presence or absence of only one allele is determined from hybridization or lack of hybridization by only one probe. The alternative allele may be infened from the lack of hybridization. Heterogeneous target nucleic acids (i.e., chromosomal DNA from a multiallelic plant) are detected by monitoring simultaneous hybridization of two or more probes comprising different polymorphic nucleotides to a genomic nucleic acid.

An ASH probe is designed to form a stable duplex with a nucleic acid target only when base pairing is completely complementary. One or more base-pair mismatches between the probe and target prevents stable hybridization. This holds true for numerous variations of the process. The probe and target molecules are optionally either RNA or denatured DNA; the target molecule(s) is/are any length of nucleotides beyond the sequence that is complementary to the probe; the probe is designed to hybridize with either strand of a DNA target; the probe ranges in size to conform to variously stringent hybridization conditions, etc.

The polymerase chain reaction (PCR) (see, e.g., Mullis & Faloona, Methods Enzymol 155:335-350 (1987) and references supra) allows the target sequence for ASH to be amplified from low concentrations of nucleic acid in relatively small volumes (Koenraadt & Jones, Phytopatholog 82:1354-1358 (1992); Iitia et al, BioTechniques 17:566-571 (1994)). Otherwise, the target sequence from genomic DNA is digested with a restriction endonuclease and size separated by gel electrophoresis. Hybridizations typically occur with the target sequence bound to the surface of a membrane or, as described in U.S. Patent 5,468,613, the ASH probe sequence may be bound to a membrane.

Utilizing nucleotide alleles and polymorphisms, ASH data can be obtained by amplifying nucleic acid fragments from genomic DNA using PCR, transferring the target DNA to a membrane in a dot-blot format, hybridizing a labeled oligonucleotide probe to the target, and observing the hybridization dots by autoradiography.

In one variant, ASH technologies are adapted to solid phase anays for the rapid and specific detection of multiple polymorphic nucleotides. Typically, an ASH probe is linked to a solid support and a target nucleic acid (e.g., a genomic nucleic acid) is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore. Where the target is labeled, hybridization is detected by detecting bound fluorescence. Where the probe is labeled, hybridization is typically detected by quenching of the label. Where both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies, labels, and the like, particularly for fluorescent based applications are described, supra.

In one embodiment, an array of ASH probes are synthesized on a solid support. Using chip masking technologies and photoprotective chemistry it is possible to generate ordered anays of nucleic acid probes. These arrays, which are known, e.g., as "DNA chips," or as very large scale immobilized polymer arrays ("VLSIPS™" anays) can include millions of defined probe regions on a substrate having an area of about ' ^cm2 to several cm².

The construction and use of solid phase nucleic acid arrays to detect target nucleic acids is well described in the literature. See, Fodor et al, Science 251:767- 777 (1991); Sheldon et al, Clin. Chem. 39(4):718-719 (1993); Kozal et al, Nature Medicine

2(7): 753-759 (1996) and U.S. Pat. No. 5,571,639. See also, PCT/US95/16155 (WO 96/17958). In brief, a combinatorial strategy allows for the synthesis of anays containing a large number of probes using a minimal number of synthetic steps. For instance, it is possible to synthesize and attach all possible DNA 8mer oligonucleotides (4^s, or 65,536 possible combinations) using only 32 chemical synthetic steps. In general, VLSIPS™ procedures provide a method of producing 4ⁿ different oligonucleotide probes on an anay using only 4n synthetic steps. Light-directed combinatorial synthesis of oligonucleotide anays on a glass surface is performed with automated phosphoramidite chemistry and chip masking techniques similar to photoresist technologies in the computer chip industry. Typically, a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group. Photolysis through a photolithographic mask is used selectively to expose functional groups which are then ready to react with incoming 5'-photoprotected nucleoside phosphoramidites. The phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group). Thus, the .phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents. Monitoring of hybridization of target nucleic acids to the array is typically performed with fluorescence microscopes or laser scanning microscopes.

In addition to being able to design, build and use probe anays using available techniques, one of skill is also able to order custom-made anays and anay-reading devices from manufacturers specializing in anay manufacture. For example, Affymetrix Corp. in Santa Clara CA manufactures DNA VLSIP™ anays. It will be appreciated that probe design is influenced by the intended application. For example, where several allele-specific probe-target interactions are to be detected in a single assay, e.g., on a single DNA chip, it is desirable to have similar melting temperatures for all of the probes. Accordingly, the length of the probes are adjusted so that the melting temperatures for all of the probes on the array are closely similar (it will be appreciated that different lengths for different probes may be needed to achieve a particular

T_m where different probes have different GC contents). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further adjust probe construction.

C. Marker Assisted Selection

After genes or a QTL and a marker or markers are mapped together and found to be in linkage disequilibrium, it is possible to use those markers to select for the desired alleles of those genes or QTL - a process called marker-assisted selection (MAS). In brief, a nucleic acid conesponding to the marker nucleic acid is detected in a biological sample from a plant to be selected. This detection can take the form of hybridization of a probe nucleic acid to a marker, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker or the like. A variety of procedures for detecting markers are described herein. After the presence (or absence) of a particular marker in the biological sample is verified, the plant is selected, i.e., used to make progeny plants by selective breeding.

Another use of MAS in plant and animal breeding is to assist the recovery of the recunent parent genotype by backcross breeding. MAS for the recunent-parent genotype can be combined with MAS for the desired genetic material using these markers.

Accordingly, it is possible to use the markers to introduce QTLs into Medicago plants having an otherwise desirable genetic background using the markers of the invention for selection of the QTL and for selection of the otherwise desirable background.

Any of the cloning or amplification strategies described above are useful for creating contiguous sequences (contigs) of overlapping clones, thereby providing overlapping nucleic acids which show the physical relationship at the molecular level for genetically linked nucleic acids. A common example of this strategy is found in whole organism sequencing projects, in which overlapping clones are sequenced to provide the entire sequence of a chromosome. In this procedure, a library of the organism's cDNA or genomic DNA is made according to standard procedures described, e.g., in the references above. Individual clones are isolated and sequenced, and overlapping sequence information is ordered to provide the sequence of the organism. See also, Fleischmann et al, Science 269:496-512 (1995) describing whole genome random sequencing and assembly of the complete Haemophilus influenzae genome; Fraser et al, Science 270:397-403 (1995) describing whole genome random sequencing and assembly of the complete Mycoplasma genitalium genome and Bult et al, Science 273:1058-1073 (1996) describing whole genome random sequencing and assembly of the complete Methanococcus jannaschii genome. Hagiwara & Curtis, Nucl. Acids Research 24(12):2460-2461 (1996) developed a "long distance sequencer" PCR protocol for generating overlapping nucleic acids from very large clones to facilitate sequencing, and methods of amplifying and tagging the overlapping nucleic acids into suitable sequencing templates. The methods can be used in conjunction with shotgun sequencing techniques to improve the efficiency of shotgun methods typically used in whole organism sequencing projects. As applied to the present invention, the techniques are useful for identifying and sequencing genomic nucleic acids genetically linked to the loci described.

VI. EXAMPLES A. Example 1: Crossing Medicago Plants

The details of the crossing program for development of F₁ hybrid among M. sativa x M. laciniata and M. laciniata x M. truncatula species at diploid level are presented in Table 3. The crossing experiments indicated that, after repeated cycles of emasculation and pollination, there is reciprocal differences in these_. species. There was no pod formation if M. sativa or M. truncatula were used as the female parent. There was some pod formation over a 10-day period and plant recovery if M. laciniata was used as the female parent (see Table 4). After laciniata flower opening, the pods developed very quickly. Fusion of the gametes was found to occur about 48 hrs prior to M. laciniata flower opening.

Repeated experiments demonstrated that pods at 7-10 days of development had ovules and embryos at a stage of development where they could be excised and cultured in vitro.

Table 4. Details of the crossing program attempted for the development of F„ hybrids among M. sativa¹ x M. laciniata² and M. laciniata x M. truncatula³ diploids.

F_j Cross No. of flowers No. of pods No. of ovules emasculated and recovered in the recovered in the _pollinated medium medium

M. laciniata x M. sativa

307 x Regen S 285 2 5

308 x Regen S 126 2 0

598 x Regen S 131 0 0

M. sativa x M. laciniata

Regen S x 307 28 0 0

Regen S x 308 12 0 0

Regen S x 598 53 4 0

M. laciniata x M. truncatula

307 x J.E. 103 61 1 0 F_j Cross No. of flowers No. of pods No. of ovules emasculated and recovered in the recovered in the pollinated medium medium

307 x J.E. 129 14 0 0

308 x J.E. 103 37 0 0

308 x J.E. 129 21 1 4

598 x J.E. 103 48 1 0

598 x J.E. 129 21 0 0

M. truncatula x M. laciniata

J.E. 103 x 307 10 0 0

J.E. 129 x 307 10 0 0

J.E. 103 308 6 0 0

J.E. 129 x 308 11 0 0

J.E. 103 x 598 10 0 0

J.E. 129 x 598 38 0 0

Total 922 11 9

B. Example 2: Embryo Rescue

For successful crossing of Medicago, it was necessary to utilize a new technique of embryo rescue. Seventy-two hours after fertilization, the developing Medicago pods were surface sterilized by rinsing in 70% ethanol for 5 min. and subsequently washed in sterile distilled water for 10 min. After surface sterilization, the pods were transfened into 5-cm Petri dishes containing 10 mL of a growth regulator-free medium (Table 2). Cultures were incubated under fluorescent light (25°C, 21.2 μmol s^"1 m^"2, 16-hour photoperiod).

After a week in culture, the ovules were dissected away from the pods under aseptic conditions using a scalpel and forceps and the viable embryos contained in the ovules were transfened into 5-cm Petri dishes containing 10 mL of ovule medium and maintained on the same medium under the above-reported conditions. Two weeks later, the growing embryos that had emerged from the ovules were transfened into Magenta™ boxes containing same medium but without proline. Alternatively, developing embryos were dissected from ovules after about one week and isolated embryos grown on the same medium. After developing embryos formed an adequate root system, they were transfened into a sandy soil mix in doubled Magenta™ boxes under aseptic conditions. This process resulted in the recovery of two Fj plantlets of two crosses; namely, 307 x Regen S, and 308 x JE 103 (see Table 4).

C. Example 3: Somatic Embryogenesis and Embryo Culture While recovering the F, plantlets during multiplication in root- and shoot-development medium (Table 5), as outlined in the above section, extensive callus formation occurred on one of the cuttings of the 307 x Regen S hybrid. The large number of somatic embryos confirmed earlier observations that M. laciniata embryonic material was highly embryogenic and could be induced to form somatic embryos in tissue culture. When the somatic embryos reached the cotyledon stage after two weeks of growth in tissue culture they were transfened onto modified MS medium (Table 3) in 5-cm Petri dishes having 10 to 15 embryos each and containing 10 mL of medium. These embryos initiated root and shoot development within two weeks. At the two-leaf stage, they were transferred to root- and shoot-development medium in Magenta™ boxes. The details of recovered plantlets through the embryo development procedure are presented in Table -6.

Table 5. The status of the cross/Tj hybrid material at different growth stages.

Plantlets in Plant established Plants at culture in soil maturity Cross/Hybrid Plantlets

307 X REGEN S 402-2 1 - 2 1

307 X REGEN S 402-3 36 2 6 4

307 X REGEN S 402-5 - - 2 2

308 X J.E. 103-2 - - 1 308 X J.E. 103-4 34 8 5 5

It is evident that embryos from the crosses 307 x Regen S and 308 x J.E. 103 F, and recovered plantlets grew very slowly. Somatic embryos of cross 307 x Regen S, after developing a root and shoot, were established in MS medium in Magenta™ Boxes. Table 6. Comparison of F. plants and their parents for quantitative traits recorded at the maturity stage.

Centra; I Centra I No. of No. of leaflet leaflet serrations Pod Pod ovule/

Plant cross/parent No. length width on central length width seed per

(mm) (mm) leaflet (mm) (mm) pod

1. P, 308 (M. laciniata) 10.16 5.10 17.00 6.75 5.17 9.45

F, 308 x J.E. 103 11.70 5.85 16.00 7.65 5.37 11.50

P₂ J.E. 103 12.22 6.70 13.20 - - -

LSD at 5% 1.01 0.47 0.94 0.55 0.61 0.91

C.V. (%) 13.90 12.60 9.59 10.89 16.29 12.29

2. P, 307 (M. laciniata) 7.75 4.32 14.15 6.80 4.10 8.00

F, 307 x R 402-3 9.57 5.65 16.80 5.75 4.52 8.65

P, Regen S 402 18.85 6.33 9.15 - - -

LSD at 5% 1.17 1.28 0.64 0.38 0.38 0.54

C.V. (%) 14.62 18.62 15.40 12.60 12.60 9.25

3. P, 307 7.40 3.91 13.46 7.03 3.76 8.20

F, 307 x R 402-3-1 11.40 6.03 17.80 6.16 4.60 7.26

P₂ R 402 18.66 6.66 9.33 - - -

LSD at 5% 1.34 0.72 1.49 0.60 0.36 0.49

C.V. (%) 14.39 17.54 14.80 10.61 9.93 6.47

4. P, 307 7.40 3.93 13.46 7.03 3.76 8.20

F, 307 x R 402-3-2 8.90 5.40 11.26 5.06 4.16 6.93

P, R 402 18.66 6.66 9.33 - - -

LSD at 5% 0.87 0.50 1.04 0.63 0.28 0.93

C.V. (%) 10.10 12.73 12.34 11.99 8.39 14.76

J.E. 103 = Medicago truncatula; R 402 = Regen S = Medicago sativa

D. Example 4: Development of Hybrid Medicago Plants

The 307 x Regen S and the 308 x J.E. 103 hybrid cuttings, as well as somatic embryos of various crosses, after developing a root and shoot, were transferred to modified MS medium (Table 3) in Magenta™ boxes for establishment of plantlets. These plantlets developed a good root system in 4-5 weeks. At that time, they were taken out from the medium and washed with water to clean the root system thoroughly and transplanted into sandy soil in double Magenta™ boxes or put into fibre pots with the same soil. If pots were used, they were covered with polyethylene bags to maintain humidity. All of the plantlets were placed into a growth chamber at 20 °C with fluorescent light (21.2 μmol s^"! m^"2) and a 16-h photoperiod. Pots were supplied with water every morning using a spraying bottle. After 2-3 weeks, the polyethylene bags were removed for 6-8 hours during the day-time period for a period of one week. After the plants initiated their shoot growth, the polyethylene bags were removed. The established Fj cross plants were transfened to larger pot for full recovery.

This procedure took approximately 4-6 weeks from the time of removal from the tissue culture medium to soil establishment. The recovery rate ranged from 45 to 50%.

E. Example 5: Sinorhizobium meliloti Inoculum Strain Specificity

The recovered plants of Medicago crosses and F, hybrids established in soil were regenerated through cuttings in a mist bed for further root/shoot development in order to test for Sinorhizobium meliloti inoculum strain specificity. Ten cuttings of each plant cross were tested against Sinorhizobium meliloti USDA1170 and PRC 1520 strains.

F. Example 5: Genomic Analysis of the Hybrid Plants

The recovered F, plants were found to show evidence of recombination at the genome level by Random Amplified Polymorphic DNA (RAPD) analyses which has been shown to be effective for identifying Medicago genotypes (Osborn, et al, BIOTECHNOLOGY AND THE IMPROVEMENT OF FORAGE LEGUMES, McKersie & Brown (eds.), CAB International, Wallingford, UK (1997)). The RAPD technique for hybrid confirmation was initiated with plants recovered from all the crosses (see Table 4) during the months of March and April, 1996. In addition, DNA extraction of eight plants of various F,'s established in soil were completed for RAPD analysis.

Briefly, the results on DNA analysis were inconclusive; two tests were positive showing the presence of sativa DNA in the F, and, one test, the latest, was negative suggesting that there was no M. sativa DNA in the F_r

G. Example 6: Morphological and Quantitative Characters Study

The general appearance of F, plants was intermediate of that of the parental plants. Morphological traits of leaflet hairiness, flower morphology, pod shape and hairiness, growth and regeneration pattern were monitored. The quantitative traits of number of ovule/seed per pods, pod length (mm), pod width (mm), central leaflet length and breadth

(mm) and number of senations on the central leaflet length (mm) of all the maturing plants of each crosses were recorded with the use of a microscope. This data is significant for the identification of the heterotic expression or depression in the F_t plants. Twenty observations of each quantitative trait of P₁ (the female parent), Fj and P₂ (the male parent) were subjected to an Analysis of Variance. The results of each plant for various traits with a Least Significant Difference (LSD) at 5% and the coefficient of variation are presented in Table 5. In general, the F, of cross 308 x J.E. 103 showed significant differences and appeared to be intermediate in central leaflet length and width, and number of serration on central leaflet compared with both parents. It was also observed that a significant increase in pod length, pod width and number of seed per pod compared with P, (see Table 6) occuned. This indicates high heterotic expression in the F,.

Considering the data of cross 307 x R 4_.02 for central leaflet length and width, the F_j plants were observed to be intermediate and appeared to be more toward P₂ when compared with both parents, whereas the number of serrations on the central leaflet was more toward P_j, indicating a significant heterosis. The pod length (mm) and number of seeds per pod were reduced in the F, in all three plants measured, and pod width (mm) was increased significantly when compared with the female parent, indicating some recombination has occuned (see Table 6).

There is the possibility that some of the significant variation could be the result of somaclonal variation induced by the tissue culture; however, this is unlikely to have caused the variation in the measured traits to be intermediate between the two parents.

H. Example 7: Cytology of Diploid Parents and F_t Plants

Root tip and flower bud samples were collected for cytological investigation and determination of the chromosome level (diploid vs tetraploid) of the donor parents. The cytological work indicated that all the M. laciniata lines tested (307, 308 and 598) have as a basic chromosome number n=8, the same as M. sativa. Potential hybrids were recovered from crosses between M. laciniata 307 x M. sativa Regen S and M. laciniata 308 x M. truncatula J. E. 103 at the diploid (2n = 2x = 16) level (Table 5 and 6).

I. Example 8: Nodulation Tests

Specificity of nodulation between the host Medicago plant and Sinorhizobium is the result of an interaction between the host plant and the bacterium. These symbiotic partners carry complimentary genetic factors that permit them to recognize each other and to permit nodulation. In effect, the specificity of nodulation is comparable to a lock and key system with the bacterium having the key (the plant nodulation specificity factor gene) to the lock possessed by the host Medicago plant (the bacterial nodulation specificity factor gene). The recovered Medicago cross/Fj plants established in soil were regenerated through cuttings in a mist bed for further root/shoot development in order to test for Sinorhizobium meliloti inoculum strain specificity. Ten cuttings of each F, were tested against Sinorhizobium meliloti USDA 1170 and 1520 strains. None of the cuttings developed nodules when inoculated with the 1521 strain but all plants from all six crosses inoculated with strain 1170 developed nodules on at least some cuttings. This indicates the F, plants contained the plant nodulation specific factor, gene from M. laciniata. The results are summarized in Table 7.

Table 7. Phenotype screening for nodulation on cuttings from F_j plants. Ten cuttings of each plant were rooted and then transferred to silica sand, inoculated with S. meliloti and grown in a growth chamber for 5 weeks.

It is understood that the example and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WHAT TS CLAIMED IS: 1. A fertile hybrid Medicago plant resulting from crossing a first Medicago plant and a second Medicago plant, wherein said second Medicago plant is Medicago sativa, and wherein the first and second Medicago plants are from different subgenera and reproductively incompatible, and said crossing is through embryo rescue.

2. The hybrid Medicago plant of claim 1 , resulting from crossing the first Medicago plant which is effectively nodulated by a strain of Sinorhizobium and the second Medicago plant which is not effectively nodulated by the strain of Sinorhizobium.

3. The hybrid plant of claim 2, wherein the first Medicago plant is a Medicago laciniata plant.

4. The hybrid plant of claim 3, wherein the Medicago laciniata plant is land race 307.

5. The hybrid plant of claim 2, wherein the second Medicago plant is Medicago sativa cultivar Regen S.

6. The hybrid plant of claim 2, wherein said strain of Sinorhizobium is a Sinorhizobium meliloti.

7. The hybrid plant of claim 6, wherein said Sinorhizobium meliloti comprises a bacterial specificity nodulation factor gene.

8. The hybrid plant of claim 7, wherein the nodulation gene is from S. meliloti strain USDA 1170.

9. The hybrid plant of claim 1 , whereby the plant is diploid.

10. Seed produced by the hybrid plant of claim 1.

11. The seed of claim 10, further comprising treatment with colchicine.

12. A hybrid plant produced by backcrossing the hybrid plant of claim 1 , to a plant of Medicago sativa.

13. The progeny of the hybrid plant of claim 12.

14. The hybrid plant of claim 13, wherein said plant is tetraploid.

15. A method of making a fertile hybrid Medicago plant, said method comprising: (a) fertilizing a gamete from a first Medicago plant with a gamete from a second, reproductively incompatible Medicago plant, wherein the first Medicago plant and the second Medicago plants are from different subgenera, thereby forming an embryo; (b) contacting the embryo with nutrients supplied in an in vitro environment; and (e) culturing in the in vitro environment said Medicago plant from the embryo.

16. The method of claim 15, further comprising isolating a flower comprising anthers and pistil from the first plant.

17. The method of claim 16, wherein said flower is immobilized in medium in an in vitro environment.

18. The method of claim 16, wherein said flower is from a Medicago laciniata plant.

19. The method of claim 16, further comprising removing the anthers from said flower.

20. The method of claim 19, wherein removal of anthers is by hand emasculation.

21. The method of claim 19, wherein the anthers are removed between about 24 and about 72 hours prior to the flower opening.

22. The method of claim 16, further comprising depositing pollen from the second Medicago plant onto the pistil of said flower.

23. The method of claim 22, wherein the pollen is from a Medicago sativa plant.

24. The method of claim 15, wherein said fertilizing is in an in vitro environment.

25. The method of claim 15, wherein the first Medicago plant is Medicago laciniata.

26. The method of claim 15, wherein the first Medicago plant is Medicago truncatula.

27. The method of claim 15, wherein the second Medicago plant is Medicago sativa.

28. The method of claim 15, wherein said contacting comprises dissecting an embryo from the ovule.

29. The method of claim 28, further comprising removing the embryo from the ovule.

30. The method of claim 15, wherein said contacting occurs between about 48 and about 168 hours after said fertilizing of the gamete.

31. A hybrid plant obtained by the method of claim 15.

32. The hybrid plant of claim 31 , wherein said plant comprises a plant specific nodulation factor for Sinorhizobium meliloti.

33. A method of making a hybrid Medicago plant resulting from a cross of Medicago laciniata and Medicago sativa, said method comprising:

(a) isolating a flower comprising anthers and pistil from a

Medicago laciniata;

(b) removing the anthers from said flower prior to flower opening;

(c) depositing pollen from a. Medicago sativa plant onto the pistil of said flower;

(d) removing a developing pod from said flower;

(e) removing an ovule from said pod;

(f) culturing said ovule from said pod in an in vitro environment so that sufficient nutrients are present to support an embryo within said ovule;

(g) removing an embryo from said ovule of said flower; and (e) culturing in an in vitro environment said hybrid Medicago plant from said embryo.