WO2016054236A1 - In situ embryo rescue and recovery of non-genetically modified hybrids from wide crosses - Google Patents

Abstract

A method is disclosed of producing a hybrid plant, and the method includes the steps of obtaining an immature embryo in a developing ovule or caryopsis obtained from a wide cross between two parental plants, wherein at least one parental plant comprises a selectable marker, culturing the embryo without removing the maternal tissue in a medium comprising a selection agent, expanding the resulting callus, and regenerating a whole hybrid plant.

Description

IN SITU EMBRYO RESCUE AND RECOVERY OF

NON-GENETICALLY MODIFIED HYBRIDS FROM WIDE CROSSES

PRIORITY

The present application claims priority to U.S. Provisional Patent Application Ser. No. 62/057414 filed September 30, 2014, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

The present invention relates generally to the fields of plant genetics and plant breeding, and relates in particular to the production of hybrid plants.

The use of biotechnology approaches, and in particular non-GMO approaches for genetic improvement of plants for agricultural purposes generally are increasingly important. As population growth increases worldwide, now exceeding 7 billion with projected increases to over 9 billion in the next three decades, the need to improve food production and availability has been considered by some to be a moral imperative. In addition, as the population increases and the global environment, economy and food security issues increase the need for improved crop resources as struggles for remaining assets increase and availability of arable land and water are increasingly challenged.

Methods for increasing agricultural outputs are therefore greatly needed for all agricultural crops. The use of advanced genetic tools, including transgenics, genomics, bioinformatics, hybrid plant development, advanced tissue culture, and marker assisted breeding for the improvement of crop plants will be required to realize the full economic and environmental benefits of modern agriculture. The use of genetic modification for the improvement of all crop species will be required for the application of some target traits comprising water use efficiency, pest-, drought-, cold- and salt-tolerance, nutrient use efficiency, biopolymers and organic plastics, altered cell wall composition and improved processing and end-use characteristics, some of which may render plants competitive with their wild relatives.

Heterosis can cause dramatic improvements in various aspects of plant performance such as seed yield and size, floral number and size, first year biomass yield, second year biomass yield and other agronomic traits by recombination of genetic variation through intraspecific or interspecific hybrid production (Martinez -Reyna and Vogel 2008, Troyer 2006, Brummer 1999). To produce hybrid cultivars, breeders have not relied on non- additive genetic variance except where the hybrids can be vegetatively propagated (Burton, 1989; Vogel, 2000; Vogel and Burson, 2004, Martinez-Reyna and Vogel 2008). Heterosis, also known as hybrid vigor, must be addressed on a trait by trait basis, and is defined by Lamkey and Edwards (1999) as "the positive difference between the hybrid and the mean of the two parents". The phenomenon of hybrid vigor is best known as observed in maize breeding and the term heterosis was first coined by Shull (1952) regarding hybrid maize. Heterosis and identification of heterotic groups has played an essential role in maize becoming the highest tonnage crop worldwide in 2001(Birchler et al. 2003, Troyer 2006).

In a number of cross-fertilized crops the generation of Fl hybrids are of great economic importance, but their application to self-fertilized crops is not widely adapted. The main reason for this restriction is that in the self-fertilized crops is the high cost of seed production. Therefore exploitation of heterosis is limited to only certain crops. For example, the high value of Fl hybrid tomato seed in the United States justifies the high production costs and sells for over ten times the cost of self-pollinated seed. Hand pollination, and hand emasculation is required for Fl hybrid seed production in many crops and drives up the costs making commercialization prohibitive. Commercial production of Fl hybrid seed is not widely practiced owing to the difficulty of hand emasculating, hand transfer of pollen, and low numbers of seeds produced per pollination. However, such crosses demonstrate the utility and benefits of Fl crosses, even though these are within a given self-incompatible species.

Intervarietal, interspecific and intergeneric, or more distantly related crosses collectively are referred to as 'wide crosses'. Breeding of wide crosses are most often prevented through either pre- and post- fertilization incompatibility mechanisms. For example, Martinez-Reyna and Vogel (2002) demonstrated a pre-fertilization system similar to the S-Z incompatibility system previously characterized in the Poaceae family (Hayman 1956; Lundqvist 1956) exists in Panicum sp. (Martinez-Reyna and Vogel 2002) and has been described in many other plants. Incompatibility through this system involves the interactions of two multiallelic gene loci, S and Z. If the same alleles are present at these loci in both the pollen grain and the style of the female pollen recipient, fertilization is inhibited (de

Nettancourt 1997). The S-Z incompatibility system has been observed in many

monocotyledoneous including Secale cereale (Lundqvist 1954), Festuca pratensis (Lundqvist 1955), Phalaris coerulescens (Hayman 1956), Hordeum bulbosum, Dactylis aschersoniana (Lundqvist 1965), Briza media (Murray 1974), Lolium perenne (Cornish, Hayward et al. 1979), and Lolium multiforum (Fearon, Hayward et al. 1983). However, these pre fertilization self-incompatibility systems have been shown to be ineffective or non-existent in wide crosses, instead post-fertilization incompatibility is the main obstacle to successful seed development.

Post-fertilization incompatibility was also observed in interploid crosses within switchgrass (Martinez-Reyna and Vogel 2002). In the studies of Martinez-Reyna and Vogel, tetraploid (4n) x octaploid (8n) crosses yielded small, shriveled seed while the reciprocal octaploid (8n) x tetraploid (4n) crosses produced small seed with floury endosperm.

Caryopses from the 4n X 8n crosses developed abnormally and formed an aqueous endosperm at about 15 days post-pollination. Roughly 20 days after pollination, as these seeds ripened, the endosperm began to shrivel and was completely shriveled 16 days later. The 8n X 4n crosses yielded caryopses that developed sluggishly in comparison to control crosses but had solid endosperm at 15 days post-pollination. At 30 days post-pollination these seeds were about one-third the size of those obtained from control crosses and had developed a floury endosperm quite unlike the seeds from the reciprocal cross. These results indicate that fertilization and embryo development does occur following pollination but an abortive process is induced some days later (20-36 days after pollination). Results from this study were similar to those obtained by earlier studies addressing crossability of octaploid and tetraploid switchgrass cultivars (Taliaferro, Vogel et al. 1999).

In 1947, Brink and Cooper proposed the idea that endosperm degeneration was the chief mechanism behind the failure of interspecific and intraspecific interploid crosses in many plants which exhibit post-fertilization incompatibility (Brink and Cooper 1947). The endosperm is important in proper seed development through physiological and genetic relationships to the developing embryo (Johnston, Nijs et al. 1980). Johnston et al. proposed that normal endosperm development relies on achieving a 2: 1 maternal to paternal ratio of whole genomes in the endosperm (Johnston, Nijs et al. 1980). They termed this ratio as the Endosperm Balance Number (EBN) and further went on to suggest that any ratio that deviates from this EBN will cause abnormal endosperm development and will often result in seed abortion before the embryo can fully develop (Shen, Gmitter et al. 2011). In wide crosses which are not prevented by pre-fertilization incompatibility the technique of embryo rescue overcomes seed abortion that occurs through abnormal endosperm development by surgically excising the immature embryo and germinating or culturing it on artificial media, independent of the endosperm.

Since its inception, plant embryo culture has become a highly valued tool for plant breeding and is widely used for a diverse range of applications including further understanding requirements for development of the embryo, shortening the breeding cycle, overcoming seed dormancy, testing the viability of seeds, providing explants for

micropropagation, and rescuing immature embryos from incompatible crosses (Bridgen 1994). The successful in vitro germination of an excised mature embryo was first

demonstrated in 1904 by Hannig using embryos excised aseptically from two cruciferous plants and a medium containing basic mineral salts and sugar (Bridgen 1994). The first reported rescue and culture of interspecific hybrid embryos was performed by Laibach using hybrid Linum embryos (Mehetre and Aher 2004). Laibach emphasized the importance of this technique for recovering interspecific hybrids observing that the embryos would need to be excised and rescued prior abortion(Sharma, Kaur et al. 1996). Additional studies have validated his theory and technique in Lillium (Asano and Myodo 1977; Asano 1978), and Brassica (Quazi 1988). In 1933 Tukey opened the door to the potential of embryo rescue to aid in the development of fruit crops through his work with sweet cherry (Tukey 1933).

Since then, his medium and procedure have been adopted by many and applied to other crops like cucurbits (Whitaker and Davis 1962), peaches (Blake 1939). Embryo rescue has been utilized to recover many intergeneric and interspecific crosses in fruits; grape (Parks, Wakana et al. 2002), citrus (Shen, Gmitter et al. 2011), banana (Vuylsteke 2000), strawberry (Sayegh and Hennerty 1989), kiwi (Mu, Wang et al. 1990), vegetables; tomato (Smith 1944), potato (Chavez, Brown et al. 1988), legumes; Phaseolus (Andrade-Aguilar and Jackson 1988), Vigna (Fatokun and Singh 1987), Trifolium (Williams 1978), and Brassica (Matsuzawa 1978; Quazi 1988). In cereals intergeneric and interspecific hybrid plant production relied on embryo rescue to produce novel hybrids of rice (Oryza) (Butany 1958; Jena and Khush 1989), Hordeum (Morrison, Hannah et al. 1959), barley x rye (Fedak 1986), wheat x barley (Koba, Handa et al. 1991), wheat x rye (Oettler 1984), oat x maize (Rines and Dahleen 1990), wheat x maize (Laurie, O'Donoughue et al. 1990), Lolium x Festuca (Morgan and Thomas 1991), Hordeum x Elymus (Lu and Von Bothmer 1990), and Elymus x Triticum (Lu and Von Bothmer 1991). Additional work has been done in numerous species to optimize technique and cultural requirements, such as media components, temperature, light, and time of culture, and the literature has been reviewed extensively (Bridgen 1994; Sharma, Kaur et al. 1996; Mehetre and Aher 2004; Reed 2005).

Embryo rescue is defined as a tool frequently used in plant breeding to recover an immature embryo arising usually from an interploid hybrid cross by excising and culturing the embryo in vitro (Monnier 1990) and subsequently culturing the embryo to a whole plant (fertile or infertile). Typical the post-excision embryo is germinated directly on an appropriate medium. In some species it may not be technically feasible to surgically excise embryos out of fertilized ovules and in these cases the whole ovule or entire ovary can be cultured. This technique has been proven in tobacco (Reed and Collins 1978), impatiens (Arisumi 1980), cotton (Stewart 1981; Mehetre and Aher 2004), Brassica spp. (Bajaj, Mahajan et al. 1986), Brassica x Raphanus (Takeshita, Kato et al. 1980), Lilium (Van Tuyl, Van de Sande et al. 1990), Raphanus (Tang and Williams 1988), Pelargonium (Bentvelsen, Stemkens et al. 1990), Ornithogalum (Niederwieser, Van de Venter et al. 1990), Helianthus (Espinasse, Volin et al. 1991), Vitis (Fernandez, Clark et al. 1991), Hydrangea (Reed 2000), and more recently Stenotaphrum (Genovesi, Jessup et al. 2009). This technique is encumbered because it is tedious and time consuming resulting in low yields and inefficient recovery of rare wide crosses. In addition, effects of the maternal tissue (especially the ovular wall) may be deleterious to embryo rescue, further contributing to low yields.

The resultant plants or tissue cultured explants from outcomes generated from conventional embryo rescue techniques may be treated with compounds (such as colchicines) that result in chromosomal doubling or loss, to restore fertility so that these outcomes can be incorporated into normal breeding programs. This technique is also encumbered because it is tedious and time consuming resulting in low yields and inefficient recovery of rare wide crosses to conventional breeding programs where fertile plants are desirable. Therefore, while wide crosses have proven as valuable to breeding hybrids, the method of conventional embryo rescue is encumbered for a variety of reasons, which limit its application to certain plants and breeding schemes.

There remains a need therefore, for recovery of wide crosses resulting in both intraspecific and interspecific hybrid plants of switchgrass and related species.

SUMMARY

The present invention relates to methods involving the use of advanced tissue culture in conjunction with transgenic plants for embryo rescue from rare wide crosses that result in the recovery of progeny from wide inter- and intra- varietal specific and generic hybrid plants. This novel method is coined here as in situ embryo rescue since it does not involve surgical removal of the embryo according to traditional procedures. The present invention relates to the production of hybrid plants using transgenic bridge intermediates and methods for production of non-GMO hybrids and use thereof. This invention teaches methods to use biotechnology approaches to develop methods for rapid recovery of hybrids with improved traits that are non-GMO for commercialization.

In accordance with an embodiment, the invention provides a method of producing a hybrid plant, and the method includes the steps of obtaining an immature embryo in a developing ovule or caryopsis obtained from a wide cross between two parental plants, wherein at least one parental plant comprises a selectable marker, culturing the embryo without removing the maternal tissue in a medium comprising a selection agent, expanding the resulting callus, and regenerating a whole hybrid plant. In accordance with an embodiment, the parental plants are flowering plants

(angiosperms). The flowering plant may be a monocot or a dicot, and the monocot may be an Alismatidae, a Commelinidae, an Arecidae, or a Liliidae. The dicot may be a Magnoliidae, a Hamamelidae, a Caryophyllidae, a Dilleniidae, a Rosidae, or an Asteridae. The method does not comprise removing the embryo from the maternal tissue. The selectable marker of the parent plant and the selection agent in the medium may be matched. The selectable marker of the parent plant may be a resistance gene, and the resistance gene confers antibiotic or herbicide resistance. The maternal plant may comprise a selectable marker. The paternal plant may comprise a selectable marker. Both parental plants may comprise a selectable marker.

In accordance with further embodiments, the selection agent in the medium is an antibiotic or a herbicide. The selection agent in the medium may be used at a killing concentration for a wild-type or non-transgenic plants or tissues. The embryo may be rescued from abortion when cultured in the maternal tissue. The medium may further comprise a mutagen or chromosome doubling agent. The chromosome doubling agent may be used if progeny is infertile. At least one of the parental plant may further comprise a desired trait, and the desired trait may be carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

In further embodiments, the wide cross is an inter- or intra-varietial cross, or the wide cross is an inter- or intra- specific cross, or the wide cross is an inter- or intra-generic cross. The embryo or caryopsis may be obtained 1-45 days post pollination of the plants. The hybrid plant may be sexually crossed and/or vegetatively propagated.

In accordance with further embodiments, the invention provides a method of producing a non-transgenic plant comprising a desired trait. The method includes obtaining an immature embryo in a developing ovule or caryopsis obtained from a wide cross between two parental plants, wherein at least one parental plant comprises a selectable marker and at least one parental plant comprises a desired trait, culturing the embryo without removing the maternal tissue in a medium comprising a selection agent, expanding the resulting callus, regenerating the callus into a whole Fl plant comprising the selectable marker and the desired trait, selecting fertile Fl plant progeny to obtain viable Fl seeds, germinating the Fl seeds to produce fertile plants comprising the selectable marker and the desired trait, backcrossing or or outcrossing the Fl plants with a non-transgenic or wild-type parental plant, obtaining F2 plants from germinated seeds obtained from the backcross or outcross, contacting the F2 plants with a selection agent, and selecting a non-transgenic F2 plant not comprising the selection marker, thereby producing a non-transgenic plant comprising a desired trait.

In accordance with certain embodiments, the parental plants are flowering plants (angiosperms), the flowering plant is a monocot or a dicot, and where the monocot is an Alismatidae, a Commelinidae, an Arecidae, or a Liliidae, and where the dicot is a

Magnoliidae, a Hamamelidae, a Caryophyllidae, a Dilleniidae, a Rosidae, or an Asteridae. In certain embodiments, the method does not comprise removing the embryo from the maternal tissue, and the selectable marker of the parent plant and the selection agent in the medium are matched. The selectable marker of the parent plant may be a resistance gene, and the resistance gene confers antibiotic or herbicide resistance. The maternal plant may comprise a selectable marker, and the paternal plant may comprise a selectable marker. Both parental plants may compris a selectable marker. The selection agent in the medium may be an antibiotic or a herbicide, and the selection agent in the medium may be used at a killing concentration for a wild-type or non-transgenic plants or tissues. The embryo may be rescued from abortion when cultured in the maternal tissue, and the medium may further comprise a mutagen or chromosome doubling agent, wherein the chromosome doubling agent is used if progeny is infertile.

The desired trait may be carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof. The wide cross may be an inter- or intra-varietial cross, an inter- or intra- specific cross, or an inter- or intra-generic cross. The embryo or caryopsis may be obtained 1-45 days post pollination of the plants, and the hybrid plant may be sexually crossed and/or vegetatively propagated. In certain embodiments, the invention provides a hybrid bridge intermediate plant, embryos, caryopsis, seeds or progeny thereof obtained by the above methods.

In accordance with a further embodiment, the invention provides a plant breeding program to confer non-transgenic plant traits including obtaining the hybrid bridge intermediate plant of claim 48 or viable progeny thereof, backcrossing or outcrossing the hybrid bridge intermediate plant or progeny thereof with a non-transgenic or wild-type parental plant, obtaining progeny from the backcross or outcross, contacting the backcross or outcross progeny with a selection agent, selecting the backcross or outcross progeny that does not comprise the selection marker, wherein the backcross or outcross progeny comprises the desired trait, and cultivating the selected non-transgenic progeny. In certain embodiments, the plant breeding program further includes conventionally breeding the non-transgenic progeny, or additional trait selection, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

The invention also provides in certain embodiments, plant progeny, embryos, caryopsis, or seeds obtained from the above plant breeding program. The invention also provides seeds obtained from the cultivated non-transgenic progeny produced by the above breeding program, as well as plant progeny, embryos, caryopsis, or seeds obtained from the hybrid plant obtained from the above methods, and plant progeny, embryos, caryopsis, or seeds obtained from the Fl, F2, or resulting non-transgenic plant obtained from the above methods. In certain embodiments, the invention provides a field comprising the hybrid plant produced by the above methods, and a field comprising the Fl, F2, or resulting non- transgenic plant obtained from the above methods, as well as a field comprising the cultivated hybrid bridge intermediate plant or non-transgenic progeny produced by the above breeding program. In certain embodiments, the field comprises 10, 100, 1,000, or 10,000 plants.

In accordance with certain embodiments, the invention provides a plant system that includes: i) plant progeny of the hybrid plant produced by the above methods, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant. In certain embodiments, the invention provides a plant system including: i) plant progeny of the Fl, F2, or resulting non-transgenic plant obtained from the above methods, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant. In accordance with further embodiments, the invention provides a plant system including: i) plant progeny of the hybrid bridge intermediate plant or non-transgenic progeny obtained from the above plant breeding program, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant. In certain embodiments, the invention provides a plant breeding platform comprising the above hybrid bridge intermediate plant, seeds, embryos or progeny thereof comprising a desired trait, wherein the hybrid bridge intermediate plant or progeny thereof is backcrossed or outcrossed with a non-transgenic or wild-type parental plant and the backcrossed or outcrossed progeny is selected to obtain non- transgenic progeny plants comprising a desired trait.

In accordance with a further embodiment, the invention provides the above plant breeding platform, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description will be further understood with reference to the

accompanying drawings in which:

FIGURES 1 A - II show seed stratification and germination for eight varieties of switchgrass (Panicum virgatum L. ) and 'Atlantic' Coastal Panicgrass (Panicum amarum Ell. var. amarulum); FIGURE 2 shows illustrations of various callus morphotypes in switchgrass cv 'Alamo' cultures. (1) embryogenic (Type I) organized, compact embryogenic structures with an epidermal surface cell layer, light yellow (2) embryogenic (Type II), friable callus masses with white somatic embryoids, (3) organogenic (root forming) compact callus forming roots with root hairs and non-regenerable to shoots and (5) mucilaginous;

FIGURES 3 A and 3B show kill curve data on increasing amount of bialaphos

(FIGURE 3 A) and hygromycin (FIGURE 3B) for cv 'Alamo' shows adequate selection for bialaphos is at 3 mg- 1-1 and 300 mg- 1-1 hygromycin;

FIGURES 4A - 4C show carbenicillin effect on callus growth (FIGURE 4A), cefotaxime effect on callus growth (FIGURE 4B), and timentin effect on callus growth (FIGURE 4C);

FIGURE 5 shows an expression of pJLU13 26 days after co -cultivation and on selection shows a non-uniform distribution of GFP expression;

FIGURE 6 shows transformation results after 70 days post-transfection and 71 days hygromycin selection on 100 pieces of each of the five basic morphotypes consisting of (1) embryogenic (Type I), (2) embryogenic (Type II), (3) organogenic (root forming), (4) nondifferentiated, and (5) mucilaginous, using the 35S:hph selectable marker cassette in the EHA105 Agrobacterium strain;

FIGURE 7 shows the identification of embryogenic stem cells in switchgrass calli under selection. GFP imaging of a single hygromycin resistant colony is shown sequentially over 45 day period;

FIGURES 8A and 8B show expression of the GFP reporter gene in switchgrass (cv a€~Alamoa€™) in a single representative stable transgenic event using brightfield

microscopy (left side of FIGURE 8 A), and 470nm UV GFP microscopy (right side of FIGURE 8A), as well as the same transgenic event 7 days on regeneration media (Figure 8B); FIGURES 9A and 9B show regeneration of transgenic calli with expression of the GFP reporter gene. Transgenic events grow normally and have normal chlorophyll development. Autofluorescence of chlorophyll masks expression. While chlorophyll autoflorescence may mask the detection of GFP under these conditions, subsequent molecular analysis demonstrates that these plants are stable transgenic events. All plants are clonal as determined by Southern blot analyses without escapes.

FIGURE 10 shows Southern blot hybridization of Ncol-digested genomic DNA extracted from cv 'Alamo' switchgrass pHG018 primary trans formants with DIG-oxigenin labeled bar probe;

FIGURE 11 shows Southern blot hybridization of Ncol-digested genomic DNA extracted from cv 'Alamo' switchgrass primary transformant # 52-7 and 10 Tl individuals with DIG-oxigenin labeled bar probe;

FIGURE 12 shows various stages of ovule and embryo development suitable for in situ embryo rescue from Panicum viragtum cv Alamo in panels E - K;

FIGURE 13 shows a diagrammatic view of a strategy for recovery of wide crosses via in situ embryo rescue using embryogenic callus induction medium with herbicide selection;

FIGURES 14A and 14B show isolated ovules explanted from wide crosses onto media optimized for embryogenic callus initiation (FIGURE 14 A) and on selection for bar show generation of embryogenic callus (FIGURE 14B); and

FIGURES 15A - 15G shows isolated ovules from wild type Panicum virgatum cv Cave in Rock (CIR) plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycin (FIGURE 15A and FIGURE 15B), as well as callus on selection expresses the GFP and is cable of regeneration to plants (FIGURE 15C and FIGURE 15D), and isolated ovules from wild type Panicum amarum cv ACP plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycinm as well as callus on selection expresses the GFP (FIGURE 15E) and is cable of regeneration to plants (FIGURE 15F and FIGURE 15G).

DETAILED DESCRIPTION

Disclosed herein are methods for recovery of incompatible crosses through in situ tissue culture of immature embryos carrying a selectable marker gene and plant regeneration of the resulting hybrid crosses, and methods for in situ embryo rescue as used for producing genetically modified plants for recovery of non-genetically modified hybrids from plant crosses.

The outcome of both surgical removal embryo rescue and immature ovule or caryposis culture techniques is usually a single plantlet. In many wide cross embryo rescue crosses, the recovered Fl plants, especially between crosses involving different ploidy levels, the outcome is most often sterile. This sterile outcome may in some cases be desirable, especially where such plants can be vegetatively propagated by cuttings or grafting techniques, producing 'seedless' plants. Many types of seedless plants that are the result of triploidy or other increased ploidy levels have been commercially produced. However in some other cases it may be desirable for hybrid outcomes to be entered into a normal breeding program.

Through the application of a selectable marker, such as herbicide resistance, it would possible to selectively culture a fertilized embryo into embryogenic callus as an intermediate away from maternal tissue of the ovule. If the selectable marker were a transgene, present in the genome as a either a hemizygous (TO) homzygous (selfed T1,T2,...) of heterzygous (outcrossed Tl,T2,...the embryogenic callus could then be proliferated and regenerated under selection to produce clonal herbicide resistant wide-hybrid individual plantlets. After crossing of the putative hybrid with a wild-type parent, it would be possible to select for sensitive individuals and recover non-GMO hybrid individuals with potentially desirable hybrid characteristics.

The derivation of wide crosses can result in important breeding stocks. Plant breeding is a process that has a long history since antiquity for the selection of wild plants from for agricultural purposes. In fact most plants used by humans today are the long result of plant breeding and domestication efforts, including corn rice and wheat. Corn was invented by humans probably not longer than 10,000 yr BP in central Mexico, derived from its wild relative teosinte. In fact, most of the plants used by people today, common in our grocery stores, including the vegetable, fruit, nut and cereal crops would not have existed without human intervention. Many would cease to exist if humans suddenly ceased to exist to stop agricultural practices, such as growing corn.

Conventional plant breeding is the science that utilizes intentional crosses between individuals with different genetic constitutions with intentional trait selection followed by subsequent crosses. The conventional breeding process produces new hybrids varieties and indeed new species with desirable traits. These new plant types are the result of sexual recombination of genes mostly accomplished during meiosis. However additions, duplications, deletions, insertions and rearrangements of chromosomal sets, fragments and individual genes as well as non-coding DNAs also can play a significant role. These results can occur between different lines and varieties, species, genera, families or even more distant relatives. Humans have exploited this capacity for useful traits that are selected.

Thus wide crosses have been used as a method in plant breeding for decades and proven to be a useful method for transferring novel genetic materials and traits for new cultivar development. The recovery of wide crosses may require embryo rescue and the current invention uses in situ embryo rescue for recovery of any cross, including wide crosses. In the present invention, the use and recovery of wide crosses by culture of immature caryopsis from crosses with a transgenic parent to generate valuable bridge intermediates is exemplified in switchgrass.

In switchgrass cultivars, Martinez -Reyna and Vogel have analyzed incompatibility systems (Martinez-Reyna and Vogel, 2002; 2008; Martinez-Reyna et al. 2001), which provides a sound basis for the present invention. Wide crosses can be used to create new alloploids by combining genetic sets within the switchgrass gene pool and related species or genera (Martinez-Reyna and Vogel 2002; 2008). The work of Martinez-Reyna and Vogel (2002) demonstrates that caryopses can be recovered from wide crosses, even between varieties with various ploidy levels (the figures in their paper illustrate this point). It is also shown that in many cases these do not develop viable seed. Historically many products of wide crosses require tedious and inefficient embryo rescue to recover plants, and in addition, many of these will result in sterile hybrids. The genomes of sterile hybrids can sometimes be doubled using colchicine to create new fertile alloploids. Rooney et al. (2010), have developed the methods and have applied this technique to sorghum with an interest in bio fuels traits. The work of Martinez-Reyna and Vogel (2002) then provides and important basis for the present invention. Kausch and Dellaporta (unpublished) have explored using similar stage caryopses from wild type plants as explants for embryogenic callus initiation. In those experiments it would be impossible to know if callus was derived from maternal tissue, or from the products of fertilization. By using an herbicide resistant selectable marker (bar) however, this is easily discerned.

The present invention provides the methodology and techniques for simple recovery of wide crosses resulting in both intraspecific and interspecific hybrid plants of switchgrass and related species by combining novel applications of transgenics, selection for embryo specific herbicide resistance, tissue culture and classical breeding techniques. The techniques aim to use transgenic herbicide resistance as a selectable marker in switchgrass and related species for recovery of rare intra-specific and inter-specific hybrids and crossing out the transgene in the subsequent backcrossed generation.

Therefore, in its simplest application, a line of transgenic switchgrass with a dominant herbicide -resistance selectable marker gene may serve as the paternal parent in the proposed intra-specific and inter-specific crosses. Transgenic herbicide resistant switchgrass (cv Alamo) plants that have been developed in preliminary studies (Deresienski, 2011) were used to pollinate wild-type individuals of alternate switchgrass varieties or Panicum species. By isolating entire flowering switchgrass plants in crosses within individual pollen cages, as opposed to bagging inflorescences, the chances of recovering hybrid plantlets is increased dramatically. A simple herbicide treatment of seedlings from the maternal wild-type plant verifies the hybrid nature of the offspring. The transgenic traits can then be selected against in the F2 population to recover herbicide sensitive hybrids that are essentially non-GMO. These hybrids can be verified as non-GMO using our genomics and sequencing approaches and thus can be rapidly introduced to the commercial market without the costly and time consuming process of deregulation.

An opportunity addressed herein is to provide the technology that will rapidly accelerate new cultivar development that are non-GMO resulting in varieties with improved biofuels traits that may be introduced into the market without the process of deregulation. In addition, the use of a linked transgenic male sterility trait with herbicide resistance used in conjunction with advanced tissue culture embryo rescue techniques (Nelson et al. 2012) will be exploited to force recovery of rare wide crosses. When the herbicide resistance marker is linked to a dominant male sterility trait and used as the maternal parent, this will serve as: 1) an ample filter to facilitate forcing and recovery of rare wide cross progeny; but more importantly, (2) when pollinated by wild type pollen, the immature caryopses can be used as explants onto embryogenic callus initiation medium containing bialaphos for herbicide resistance selection, whereby only callus derived from a fertilized embryo will be recovered. The embryogenic callus, derived from a wide cross can then easily be regenerated to whole plants and further characterized. A benefit of this wide cross recovery method is that it will dramatically increase the numbers of wide crosses that can be recovered and the numbers of clones of each wide cross.

The technique of embryo rescue overcomes seed abortion that occurs through abnormal development caused by various incompatibility mechanisms. Conventional embryo rescue techniques are accomplished by surgical excision of the immature embryo resulting from incompatible plant crosses and germinating or culturing the developing embryo on artificial media, independent of the endosperm. These techniques are tedious, time consuming and have low yields especially with low fertilization frequencies. The present invention overcomes the problems of conventional embryo rescue by utilizing a transgenic selectable marker in the paternal parent and culturing the immature embryo in situ in the developing ovule on embryogenic culture initiation media which includes the selective agent. This method is coined here as in situ embryo rescue since it does not involve surgical removal of the embryo according to traditional procedures. The resulting embryogenic culture can be regenerated to whole plants. The ability to combine genomes within and between distant plant taxonomic groups including varieties, species, genera and more distant relatives offers the opportunity to rapidly accelerate the development of new varieties, cultivars, species, and crops. The utilization of these novel techniques and the exploitation of the recovery of fertile or infertile progeny from wide crosses increases genetic diversity and can be used to introduce novel genetic and useful traits. This process is advantageous since the outcomes can include genetically modified organisms (GMO) traits when appropriate, or these can be segregated to produce plants that do not contain transgenes and hence, are non-GMO.

The need for the world to increase its efforts on sustainable food and energy production and the role that advanced genetics will play are well understood and widely known. Rapid genetic improvement of the most domesticated crop plants is desirable for current and projected global agricultural needs. Addressing these needs is anticipated by current genomics, bioinformatics, association genetics, marker assisted breeding,

conventional genetics and other non-GMO approaches. While transgenics offer access to traits outside the conventional breeding pool they are time consuming, costly, and involve unresolved issues regarding public acceptance, governmental deregulation and commercial release. This invention involves the novel use of transgenic herbicide resistance in

conjunction with a new embryo rescue technique from wide crosses for recovery of bridge intermediates. A bridge intermediate refers to a genetic conduit for incorporation of new genes and conferred traits into new hybrids. The bridge intermediate provides a mechanism for importing many new genes and large amounts of genetic material that cannot be otherwise moved through common conventional breeding program materials. In addition, the creation of these intermediates provides new de novo genetic material that arises from these wide varietal, species or genera crosses which would not be possible using traditional plant breeding techniques.

These wide cross hybrids are then used as bridge intermediates in backcrosses or outcrosses to remove the transgene facilitating recovery of non-GMO hybrids that can be rapidly introduced to the agricultural market without costly R&D and deregulation of GM development. Using this novel system the problem of new varietal development can be addressed by the creation of new hybrid plants which include novel traits, allows for advanced trait selection using genomic assisted breeding technologies, and the exploitation of heterosis. Hybrid plants incorporate new genetic material in a breeding program that can result in dramatic improvements in various aspects of plant performance such as yield, including, but not limited to: fruit, biomass, grain, root or tuber and seed yield; plant size, color, or texture; plant growth rate; floral timing; floral numbers and size; secondary metabolite production and yields; first year and, second year biomass yield in bioenergy crops; root mass; water use efficiency; insect and pest tolerance, avoidance, or protection; drought, cold, and salt-tolerance; more efficient use of nutrients and, many other important agronomic traits. New traits are introduced by recombination of genetic variation through intra- or inter- varietal, specific or generic hybrid bridge intermediates and subsequent production through conventional breeding.

The specific examples of this invention teach the methods for high scale production of hybrid plants through wide crosses, and recovery of bridge intermediates in crop species. This invention introduces a new embryo rescue technique that relies on selection of rare crosses via tissue culture of embryogenic callus cultures and subsequent plant regeneration, rather than traditional embryo rescue techniques which rely on direct surgical removal of an immature embryo and direct germination. The practice of this technique is characterized by the absence of this procedure in global literature of agricultural breeding programs. It is also deemed useful since it results in novel germplasm and recovery of previously non-existing varieties and new species that could be created by other existing methods. It is also widely applicable across many crop species.

During the practice of this invention new transgenic lines are generated for improved efficiency of hybrid recovery on a case-by-case and species-by-species basis by using transgenic herbicide or antibiotic resistance which may be linked with male or female sterility and advanced tissue culture approaches. Through these efforts one may create new plant hybrids. Transgenic traits can be selected against in the F2 population to recover hybrids that are essentially non-GMO. In addition, crosses that result in sterile hybrids can be used as vegetatively propagated transgenic (GM) crops with 100% gene confinement. The anticipated results for the extension of this method to other angiosperms has direct and short and long term commercial application. The invention creates new varieties that will be superior in the marketplace, virtually replacing existing varieties.

This invention is specifically about the recovery of embryogenic or otherwise regenerable callus from wide crosses in flowering plants. To accomplish this task transgenic plants carrying a selectable marker is used as one of the parents to recover immature embryos in the developing ovule or caryopsis. This embryo is rescued from ensuing abortion that typically occurs as a post-fertilization barrier to fertile seed set in wide crosses by the tissue culture of the immature embryo in situ (i.e. without removal from maternal tissues). Selection of embryogenic or regenerable callus occurs in killing concentrations for the agent specified by the selectable marker resistance. Embryogenic or regenerable callus can then be cultured and expanded during subsequent growth and regenerated to whole plants, each of which is clonally and genetically equivalent. Growth conditions as well as media adjustments, such as inclusion of mutagens or chromosome doubling agents such as colchicines, can be included for desired effects prior to or during plant regeneration. The recovered plants are effectively hybrid bridge intermediates that can be used for the production of non- genetically modified hybrids via backcrossing or outcrossing to wild type plants followed by conventional breeding and trait selection.

The fact that wide crosses occur in nature is visible in many extant species of plants and animals. The frequency however, of fertile progeny from wide crosses in nature is low but exploitation of such events would be very useful for crop breeding purposes. The introgression of genes from wide crosses will increase genetic diversity and allow trait introduction that does not include transgenes, which will shorten the breeding and commercialization processes. This patent teaches methods for the establishment of an efficient breeding platform for agricultural improvement of members of Angiosperms comprising members of the Monocotyledonea, and the Dicotyledoneae.

Switchgrass and its related species are well known as bioenergy crops. There are global economic, political, US national security and environmental pressures to increase renewable biofuel production and utilization, to offset gasoline and diesel fuel use, especially in the liquid fuel transportation sector.

In one embodiment, one of the monocot parental types are transgenic members of the Poacea, such as switchgrass (Panicum virgatum L. cv Alamo). The second parental type is also a member of the Poacea, such as but not limited to, Andropogon sp., Panicum, sp., Pennisetum sp., Zea sp., Saccharum sp., Miscanthus sp., a Saccharum sp. x Miscanthus sp. hybrids, Erianthus sp., Tripsicum sp., or Zea X Tripiscum sp. hybrids, In general the procedure begins with a transgenic monocot parent with a transgenic selectable marker (typically, but not limited to a selectable marker conferring resistance to an antibiotic or herbicide that can be used for recovery of primary transgenics) as a maternal or paternal parent. In some crosses each parent may be independent transgenic events, containing the same or different selectable markers. The transgenic parent is used in wide crosses, defined as inter- and intra-varietial, inter- and intra-specific as well as inter-generic crosses. Recovery of putative wide crosses is accomplished and ovules or caryopses are isolated for tissue culture in the presence of the selective agent at killing levels for wild type plants or tissues specified by the transgenic selectable marker. The tissue culture media is intended to encourage emebryogenic or otherwise regenerable callus.

In a first embodiment, for example, transgenic herbicide resistant Panicum virgatum L. (cv Alamo or cv Kanlow) may be used in an intra-specific cross with non-transgenic wild type Panicum virgatum L. (cv Alamo or cv Kanlow). Note that directionality (maternal X paternal) is not relevant to the practice of hybrid plant recovery. Developing caryopses may be isolated from 1-45 days post pollination and plated on callus induction media containing concentrations of the selective agent that result in death or noticeable growth reduction of non-transgenic cells. The resulting Fl callus is expanded or regenerated to whole plants. The resulting whole plant Fl progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedling are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrids can be selected for desirable phenotypes contributed by either parent; including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus).

Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal

development. Hybrids can be sexually crossed and/or vegetatively propagated. In a second embodiment, serving as another example of the usefulness of this procedure, transgenic herbicide resistant Panicum virgatum L. cv Alamo may be used in difficult to recover wide crosses and using the in situ embryo rescue technique to produce viable herbicide resistant Fl seedlings to produce healthy plants to identify and define progeny useful for production of fertile hybrids. In this example, using a self-compatible intra-specific cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non-transgenic Panicum virgatum L. cv Kanlow (4x); where (x) is the basal number of chromosomes, and in Panicum sp. x=9, therefore, 4x refers to a tetraploid where 2n=4x=36 chromosomes, and 8x refers to an octaploid where 2n= 8x = 72 chromosomes. Note that in this example directionality (maternal X paternal) also does not matter to the practice of hybrid plant recovery. The Fl progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.

In a third embodiment, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in to recover rare intra- or inter-specific crosses between self-incompatible parents to identify and define progeny useful for production of fertile hybrids. In this example using a self-incompatible cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non-transgenic Panicum virgatum L. cv Cave-In-Rock (8x). Note that directionality (maternal X paternal) here may in some case play a role to the practice of hybrid plant recovery. The Fl progeny from the wide crosses may be infertile, thus using the embryo rescue technique to produce viable herbicide resistant Fl seedlings to produce healthy plants. In some cases chromosome doubling may be required by incorporating colchicine or other such agents in the embryo rescue medium to recover fertile Fl plants. If plants are infertile the outcome will be transgenic and can be vegetatively increased via clonal propagation for other purposes. Fertile Fl plants can be used in backcrosses to wild type non-transgenic Panicum virgatum L. hybrids. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.

In this embodiment, this invention can generate a series of intra- and inter-specific wide crosses as a breeding platform. The breeding platform will utilize transgenic male and female sterile lines from a reference switchgrass variety, Panicum virgatum L. cv. Alamo and herbicide selection for recovery of wide intra- and inter- specific Fl crosses by embryo rescue. Fl hybrids can be backcrossed to the reference Alamo cultivar to segregate away the transgene to generate a non-GMO BC 1 mapping population. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.

Phenotypic analysis is conducted on the non-GMO population in regionally selected field plots and phenotypic data is statistically correlated to genetic variation. Variation is assessed using genome -resequencing technologies and this data, along with phenotypic information is used to establish a computational and statistical pipeline to identify, map and introgress variation associated with biomass and other bioenergy traits.

Finally, This invention teaches the wide applicability of these techniques with application to selections of tomato (Solanum lycopersicum cvs) crosses. In this case the breeding and selection of hybrids is accelerated by the production of a transgenic intermediate followed by embryo rescue and backcrossing the transgene away from the hybrid background. Trait selection may be by direct phenotypic selection or by using genomic assisted breeding. In this example, Solanum lycopersicum cv Buffalo is transformed with a selectable marker gene (e.g., bar) and crossed with Solanum lycopersicum cv

Geronimo. Male and/or female sterility transgenes may also be used in combination with a selectable marker for recovery and selection of crosses. After 1-30 days post pollination ovules are excised from the wild type Solanum lycopersicum cv Geronimo plants and placed on media containing the selection agent (e.g., bialaphos). The resulting embryogenic callus is recovered and regenerated to whole hybrid Fl plants. These plants are backcrossed to either wild type cv Geronimo or wild type cv Buffalo. The resulting F2BC1 population is screen for herbicide resistance and sensitive plants are phenotypically selected. The phenotype of the F2BC1 population may be screened for traits defined by genomic markers (e.g., taste).

Demonstration of the usefulness of this technique for hybrid tomato selection extends this procedure to include dicot plant breeding.

This fundamental approach will take advantage of current transgenic, genetic and genomic work already in place for the development of hybrid selected cultivars. The utility of this invention is to develop new commercially available cultivars with improved-relevant characteristics.

Perquisite to the embodiments in this invention, transgenic plants were created using a herbicide resistance selectable marker bar, as one example and used to establish a basic protocol. Optimization of transgenic embryogenic callus (Plant Material, Explant

Sterilization, Callus Induction and Plant Regeneration).

Nine commercially available cultivars of switchgrass (Panicum virgatum L.); cvs 'Alamo', 'Blackwell', 'Carthage', 'Cave-in-Rock', 'Kanlow', 'Shawnee', 'Shelter', 'Southlow', 'Sunburst' and 'Atlantic' Coastal Panicgrass (Panicum amarum Ell. var.

amarulum) were kindly donated by Ernst Conservation Seeds (Meadville, PA, USA) for use in this study. All seed was harvested from field-grown plants grown during the previous season. Embryogenic calli were generated (Somleva et al. 2002) from mature caryopses subjected to a brief 70% ethanol rinse (2 min , 25 °C) followed by shaking in 25% sodium hypochlorite plus 100 μΐ Tween-20 for 15 min. After rinsing three times with sterile distilled water, caryopses were imbibed under constant agitation for 16 to 20 hrs in 20 ml of medium containing 2% (v/v) Plant Preservative Mixture (PPM, PhytoTechnology Laboratories, Shawnee Mission, KS, USA) and IX Murashige and Skoog (MS) salts (Murashige and Skoog 1962). Sterilized caryopses were then maintained for 0,2,3,4,5,6, and 8 weeks in darkness at 4°C on callus induction medium comprised of IX MS salts and vitamins

(PhytoTechnology Laboratories, Shawnee Mission, KS, USA), 30 g-1-1 D-Maltose, 5 μΜ 6- BAP, 22.5 μΜ 2,4-D, 0.8% (w/v) Plant Cell Culture Agar (Sigma-Aldrich, Inc., St. Louis, MO, USA), pH 5.7, Test cultures were also supplemented with 2%> (v/v) PPM. Plates containing cold-treated caryopses were removed from refrigeration at the time points indicated above and transferred to incubation at 27 °C in darkness. After 4 weeks, germinated seeds were transferred to fresh callus induction media and embryogenic calli were subsequently identified and sub-cultured every 14 days for use in transformation experiments. Embryogenic cell lines were established and multiplied. After 3 months, approximately 500 mg fresh weight callus from individual lines were placed on two distinct regeneration media, plant regeneration media I (Somleva et al. 2002) and regeneration media II (Nelson et al. current study). Regeneration media II consists of MS medium supplemented with 1.4uM gibberellic acid (GA3) and 5uM 6-benzylaminopurine (6-BAP). Callus was placed in the dark at 28°C for 1 week followed by incubation at 28oC in a 16-h photoperiod. Two weeks after callus was placed under lights plantlets were counted. Agrobacterium Preparation and Genetic Transformation. A. tumefaciens LBA4404 (pSBl) cultures harboring pOsUbi-bar or p35S-bar for transformation were initiated by streaking from glycerol stocks on plates containing 10 μg·ml-l rifampicin, 10 μg·ml-l tetracycline, and 50 μg·ml-l spectinomycin.

A single colony was then streaked onto solid YEP medium containing identical selective antibiotics and grown at 20 or 28 °C for 48 to 72 hrs as described elsewhere (Frame et al. 2002, Ishida et al. 2007, Vega et al. 2008). Approximately 2 loopfuls of Agrobacterium cultures were transferred to 20 ml infection medium containing IX MS salts and vitamins, 30 g-1-1 D-maltose (pH 5.7) supplemented with 200 μΜ acetosyringone. The culture was shaken at 100 rpm for approximately 4-5 hours at 28°C with an OD600 of 0.6-0.8, and was then used in transformation experiments. For transformation experiments using A.

tumefaciens EHA105 (pJLU13: p35S-hph), single colonies were selected from plates containing 10 μg·ml-l rifampicin and 30 μg·ml-l kanamycin, transferred to 100 ml YEP medium amended with identical antibiotics, and shaken at 225 rpm at 27 °C until OD600 reached 0.6 (approximately 24 hrs). Cultures were centrifuged at 3220 xg and the resulting pellets were re-suspended in the infection medium described above to an OD600 = 0.8.

Embryogenic type II calli were broken into approximately 2 x 2 mm pieces, sub- cultured on callus induction medium supplemented with 200μΜ acetosyringone, and maintained in darkness at 27°C. The following day individual calli were inoculated with approximately 10-15 μΐ of the A. tumefaciens cultures indicated above via micropipetting. After 15 min, excess A. tumefaciens suspension was carefully removed and the plates were incubated at 27°C in darkness for 3 days. Following co -cultivation, inoculated calli were moved onto callus induction medium containing 150 mg-1-1 timentin and incubated at 27°C for 7 days in the absence of selection.

The selection of bialaphos and hygromycin resistant calli and regeneration of transgenic plants was as follows. Transformation experiments using the p35S-bar: (rice) pOsUbi-bar vectors were compared using two target explants in cvs 'Alamo' and 'Kanlow'. Individual somatic embryos (0.5 mm) were isolated using a dissecting microscope according to Somleva et al. (2002) and larger (2-3 mm) embryogenic Type II calli were isolated for transformation. After 7 days, calli inoculated with the Agrobacterium strain LBA4404 (pSBl) harboring either p35S-bar or (rice) pOsUbi-bar were moved to callus induction medium amended with 3 mg-1-1 bialaphos, and incubated at 27°C for 10-12 weeks with subculture onto fresh medium every 14 days. Similarly, after 7 days, calli inoculated with Agrobacterium strain EHA105 harboring the construct pJLU13: p35S-hph were moved to callus induction medium amended with 300 mg-1-1 hygromycin and incubated at 27°C for 10-12 weeks with subculture onto fresh medium every 14 days. After 10-12 weeks, resistant calli were transferred to either media specified by Somleva et al (2002) or revised to contain IX MS salts and vitamins, 30 g-1-1 D-maltose, 3 mg-1-1 6-BAP, 0.48 mg-1-1 GA3) containing either 3 mg-1-1 bialaphos or 300 mg-1-1 hygromycin (as appropriate). Both were incubated in the dark at 27°C for 7 days. Embryogenic callus from cv. Callus were placed onto the respective regeneration media and incubated in the dark for one week, followed by incubation under lights. Regenerates were counted following one week under lights.

Following analysis of cv. Alamo regeneration, the remaining cultivars of switchgrass and Atlantic Coastal Panicgrass were evaluated for regenerability using the two media that gave the most obust regeneration for cv. Alamo. Plates were placed under fluorescent lights (40 μΜ m-2 s-1 ) on 16h light/8h dark cycles and calli were subcultured to fresh media after 14 days. Vigorously growing shoots were then transferred to Stage II Regeneration (Stage I medium sans 6-BAP and GA3) medium. Shoot formation per plate was counted following one week light incubation. Plantlets were transferred to rooting medium consisting of MS media supplemented with 30g/L Maltose. Plantlets were then moved into soil (Metromix 550, Sun Gro Horticulture, Bellevue, WA, USA) in 1" peat pots kept under identical growth conditions. Healthy plantlets were transferred to 12" pots and grown to maturity in the greenhouse under natural light supplemented with high-pressure sodium halide growth lamps on a 16h light/8h dark cycle. After 3 months of growth, plants were fertilized with 200 ppm Peterson's 20- 20-20 fertilizer (Scotts-Sierra Horticultural Product Company, Marysville, OH, USA).

The resulting plants from experiments using either p35S-bar or (rice) pOsUbi-bar were assayed for resistance to the herbicide Finale. At least 2 healthy leaves were chosen from each plantlet and swabbed with 3% (v/v) Finale. The leaves of two wild-type cv 'Alamo' plants were also swabbed. After 7 days the leaves of Finale sensitive plants were dried out, necrotic and dead and resembled the wild-type response to Finale. The leaves of Finale resistant plants remained healthy, green and actively growing. The herbicide resistance assay was repeated again approximately 3 weeks later. Finally, entire plants were sprayed with Finale at the concentration indicated above.

Seed inoculation, germination and callus induction was optimized Seed that was used for inoculation, germination and callus induction in this study was harvested from field grown plants the previous year. Germination frequencies varied widely. See FIGURES 1 A - II, which show seed stratification and germination for eight varieties of switchgrass

(Panicum virgatum L. ) and 'Atlantic' Coastal Panicgrass (Panicum amarum Ell. var.

amarulum). In particular, FIGURE 1 A shows Alamo seed stratification germination data, FIGURE IB shows Blackwell seed stratification germination data, FIGURE 1C shows cave- in-rock seed stratification germination data, FIGURE ID shows Atlantic C. panicgrass seed stratification germination data, FIGURE IE shows Kanlow seed stratification germination data, FIGURE IF shows Shawnee seed stratification germination data, FIGURE 1G shows Southlow see stratification germination data, FIGURE 1H shows Shelter seed stratification germination data, and FIGURE II shows Sunburst seed stratification germination data. PMP is also beneficial for prevention of contamination and is an improvement from previous methods for generating embryogenic callus. Stratification is beneficial for some cultivars.

The occurrence of unidentified endogenous fungal contaminants and/or endophytes were frequently observed associated only with calli derived from mature caryopses that had been surface sterilized as described and not the tissue culture medium. The growth of the fungi often led to callus culture contamination resulting in the growth and proliferation of black fungal hyphae. This contamination could be controlled via frequent subculture of non- contaminated embryogenic callus but rarely completely eliminated. Therefore we investigated the use of stratification to improve germination coupled with PPM treatment to improve germination frequencies and control fungal contamination. Stratification is a beneficial improvement for some cultivars. Surface sterilized mature caryopses were plated onto callus induction media supplemented with 0-4% (v/v) PPM and placed at 4°C in the dark for 0, 2, 3, 4, 5, 6, and 8 weeks. Following 1 week incubation at 28°C and subsequently, germinated caryopses were counted at each time point. Seed stratification either had no effect or improved germination. PPM treatment at 2% (v/v) eliminated all occurrence of fungal contamination. It is shown that seed stratification requirements vary across the genotypes tested, which either had no effect or improved germination frequencies and recovery of stable callus induction. The use of PPM treatment in the stratification medium dramatically decreased the occurrence of endophyte contamination and increased successful callus induction and subsequent maintenance.

Embryogenic Callus Culture was also Optimized. Switchgrass callus induction media have a profound effect on the efficiency of callus formation, transformation efficiency and plant regeneration of switchgrass. For this part of the analysis only the frequency of formation of embryogenic Type II callus was determined. Frequencies of embryogenic Type II callus production on callus induction media were counted using a dissecting microscope and determined for nine switchgrass cultivars and Atlantic Coastal Panicgrass on the Somleva et al (2002) media (TABLE 1).

TABLE 1. Embryogenic Type II callus induction varies widely across cultivars on the Somleva et al. (2002) medium.

TABLE 1

TABLE 1 shows that the embryogenic Type II callus induction varied widely across cultivars on the Somleva et al. (2002) medium and has been improved for the present invention. Embryogenic Type II callus from cv's. Alamo and Kanlow produced at the highest frequencies (5.39% and 6.85%, respectively) which are over ten fold higher than any switchgrass cultivars tested. Atlantic Coastal Panicgrass produced Type II callus at a frequency of 5.75 %. Embryogenic Type II callus from cv. Alamo was tested for

regenerability using two different regeneration media (TABLE 2).

TABLE 2. Plantlet regeneration is increased across all cultivars tested except non- responders in comparison to Somleva et al. (2002) regeneration medium. Regeeer&fioii

eiiia I Regeneration

S itciigrass

Soislff^" 3 aL Media 11

cultfvar (cv)

(2008) current study

and ACP

Average # Average

Regenerates Regenerates

'Alamo' 2.33 40.5

'Aiiaalis:^;

Coastal 15.11 79.7

^£Blackweir 0 a½

'Carthage' 0 0

'Cave-m-Rock' 6.1 1 63.55

' anlow' 10.69 47.69

'Shawn e^* 0.5? 4.14

'Shelter^* 0 0

30.63 247.2 1

'Sanbt&st' 1.33 12.5

TABLE 2

Embryogenic Type II calli were placed onto regeneration media and incubated in the dark for one week, followed by incubation under lights (161/8d). Regenerates were counted following one week under light incubation. The remaining cultivars were evaluated for regenerability using the two regeneration media as shown in TABLE 2. Regenerated plants were grown to soil and exhibited a high degree of phenotypic variation. These media modifications show large increases in all regenerable varieties with modest or no improvements for previously non-regenerable varieties. Southlow, Kanlow and Cave -in- Rock show the largest percent improvements. TABLE 2 shows that plantlet regeneration is increased across all cultivars tested except non-responders in comparison to Somleva et al. (2002) regeneration medium and this is applied to the present invention for the recovery of plantlets from in situ embryo rescue.

Callus Morphotype Identification-Importance of visual section of embryogenic callus

Callus cultures were induced from mature caryopses of all nine switchgrass cultivars and Atlantic coastal Panicgrass and subcultured at 14 d intervals on callus induction medium. The initial calli in all of these cultivars generally consisted of heterogenous cell masses, comprised of five basic morphotypes: (1) embryogenic (Type I) (2) embryogenic (Type II) (3) organogenic (root forming) (4) nondifferentiated, and (5) mucilaginous (FIGURE 2 ). In particular, FIGURE 2 shows various callus morphotypes in switchgrass cv 'Alamo' cultures. (1) embryogenic (Type I) organized, compact embryogenic structures with an epidermal surface cell layer, light yellow (2) embryogenic (Type II), friable callus masses with white somatic embryoids, (3) organogenic (root forming) compact callus forming roots with root hairs and non-regenerable to shoots and (5) mucilaginous. Embryogenic callus visual identification and selection is important to the practice of present invention. Bar = 0.5 mm.

These various morphotypes are formed on callus induction medium as heterogenous callus masses with two or more types per callus, yet only rarely do they appear all together on any single induced piece of callus. The individual morphotypes can be isolated by visual selection using a dissecting microscope , maintained for three to five subculture passages by routine subculture (14 d) and visual selection before reversion to heterogeneous cultures usually comprised again of several morphotypes. All callus morphotypes can each be generated from a single seed and all morphotypes occur on the same callus induction medium with various degrees of heterogeneity indicative of inherent variation in the seed stocks with response to these media. Only embryogenic calli (Type I and Type II) were regenerable on regeneration medium and result in production of both shoot and root axes during

regeneration. Embryogenic Type I callus is compact (non-friable) and is covered by a smooth differentiated epidermal layer observable under a dissecting microscope. Embryogenic Type II callus is friable without a distinct epidermal layer and produces early stage radially symmetric somatic embryos. Only Type II embryogenic callus results in recoverable transformation, selection, and regeneration of fertile transgenic plants using current selection protocols. Embryogenic Type II callus must be sub-cultured every 14 d and visually selected using a dissecting microscope to maintain this phenotype. Organogenic root- forming callus is characterized by a proliferation of root primordia that form observable root hairs and do not produce shoots on regeneration medium. However, this callus is capable of re-establishment of heterogeneous callus during maintained subculture. Nondifferentiated callus consists of large vacuolated cells (observed with brightfield and Nomarski optics; not shown) is not organized as tissue or organs, and does not produce either shoots or roots on regeneration medium. Mucilaginous callus is characterized by loose, slow-growing cell masses suspended in thick elastic mucilage. These cultures do not produce organs or plantlets on regeneration medium yet can be sustained on callus maintenance medium, and will at a low frequency revert to a heterogeneous callus. Embryogenic visual selection (FIGURE 2) is important to the practice of the present invention.

Optimization of transformation parameters was as follows. Kill curve data was generated on increasing concentrations of bialaphos or hygromycin to determine optimum amounts required for selection of transgenic embryogenic Type II calli. Embryogenic Type II calli from cv 'Alamo' were placed onto increasing concentrations of bialaphos (0-10 mg-1-1) and hygromycin (0-300 mg-1-1). Fresh weight measurements (g) were taken after 14 d and change in fresh weight was calculated. In particular, FIGURES 3A and 3B show kill curve data on increasing amount of bialaphos (FIGURE 3 A) and hygromycin (FIGURE 3B) for cv 'Alamo' shows adequate selection for bialaphos is at 3 mg-1-1 and 300 mg-1-1 hygromycin. Killing levels of selective agent are required for practice of the invention. The control Type II callus increased four- fold in fresh weight gain during these trials. While 1 mg-1-1 bialaphos reduced growth over 50%, an increase to 3 mg-1-1 sufficiently halted fresh weight gain.

Similarly, addition of 50-200 mg-1-1 hygromycin reduced growth to over 50% fresh weight gain, whereas addition of 300 mg-1-1 halted growth. Hence the levels chosen for tight selection of transgenics were 3 mg-1-1 bialaphos and 300 mg-1-1 hygromycin.

Antibiotics typically used to remove Agrobacterium after cocultivation were tested for their effects on growth of embryogenic Type II callus (cv 'Alamo') including cefotaxime (0- 300 mg-1-1 carbenicillin (0-300 mg-1-1), and timentin (0-200 mg-1-1). Change in fresh callus weight was calculated and used to determine the effects on callus growth after 14 days incubation. The control Type II callus increased 1.5-2.0-fold in fresh weight gain during these trials. FIGURES 4A - 4C show carbenicillin effect on callus growth (FIGURE 4A), cefotaxime effect on callus growth (FIGURE 4B), and timentin effect on callus growth (FIGURE 4C). FIGURES 4A - 4C show antibiotic effects on callus growth at increasing concentrations show that both cefotaxime and timentin can have positive effects on growth, while carbenicillin is benign Cefotaxime had the most positive effect on growth at a concentration of 50 mg-1-1.

Antibiotics are used to remove Agrobacterium from primary transgenic (TO) cultures. These requirements only apply to recovery of primary (TO) transformants required for crosses. They are not normally required for subsequent steps in the in situ embryo rescue procedure as practiced by the present invention, but may be useful for de-contamination of re-introduced cultures where necessary. All kill curve data has been verified against various types of explants including immature embryos, ovules and caryopses.

Transformation Frequency Studies- Transient expression analysis was as follows. The binary vector pJLU13 contains an enhanced green fluorescent protein (GFP) reporter gene cassette described previously (Lu et al. 2008). Expression of GFP was analyzed in calli one week after co-cultivation with EHA105 (pJLU13: p35S-hph). FIGURE 5 shows an expression of pJLU13 26 days after co-cultivation and on selection shows a non-uniform distribution of GFP expression. Positive expression is seen associated with embryogenic Type II (middle six arrows) but not in embryogenic Type I (top two arrows), mucilaginous (bottom two arrows) and nondifferentiated callus morphotypes. Embryogenic cultures derived from in situ recovered embryos are most useful for practice of the present invention.

GFP positive foci were then again observed by comparison of brightfield and UV light imaging 26 days after co-cultivation and on selection revealing differentially expressed GFP associated with the various callus morphotypes. Cells expressing GFP appear primarily in embryogenic Type II callus but rarely in embryogenic Type I, nondifferentiated, or mucilaginous callus morphotypes. The numbers of cells expressing GFP in embryogenic Type II callus tissues varies from tens to hundreds of individual cells. Callus health as observed by discolored sectors was affected by co -cultivation and was most commonly associated with embryogenic Type I sectors. Attempts to observe single GFP positive cells transition to stable colonies were not successful. Auto-fluorescence was observed as dull yellow sectors primarily associated with some, but not all, embryogenic type I callus. This further demonstrates that the correct identification of embryogenic callus by visual identification and selection (FIGURE 2) is important to the practice of the present invention.

The transformation frequency and optimization studies (selection of stable transformants) was as follows. Selection of resistant colonies occurs over a 6-8 week period for both hygromycin and bialaphos selection. Efficiency of transformation was calculated as the number of recovered calli and also as the percent of calli maintained on selection medium capable of regeneration to plantlets (TABLE 3). The results show recovery of stable transformants on both hygromycin and bialaphos selection. Φ d % piaaifcss per

Agro- strain

3:s:S.&2j- LBA44&4 3C'S IS3 34J.3% 32/lS.S7%

Absaa ¾S:, ISA44iJ- SOS 12¾ί*ΧΘ0% 34/1 33%

Akac 35S-.i LBA44C ! 13® 3:5

Ahiao 3:s:S.¾si- LBA44t> 1 «5 34/2.$ 1 % L2;^'l.¾3%

OsUbi-.&sr LBA440 577 2¾S £!3% 2¾5.S3¾

LBA 0 sss 4&7.S2% 43/7.31%

Aiss-a* LBA44t> -rOO m.5 6/1.50%

QsUbr.bsr LBA440 35-2.58% sm

LBA4404 .m:

LBA44 171 .Qi%

35S:½* EKAK Mil l 5$%

A-siae. }:-^<y.h≠- EH ; &~ see i i. %

35S;½* EK li S :07% 2¾4.¾¾

EHA l0> I TO 45/«Φ%

TABLE 3

TABLE 3. Switchgrass transformation efficiency data using either bialaphos or hygromycin selection.

Transformation experiments using EHA105 (pJLU13: p35S-hph) and selection with hygromycin were conducted and used to compare transformation frequencies in three switchgrass cultivars (cvs 'Alamo', 'Kanlow', and 'Blackwell'). These cultivars were chosen on the basis of their tissue culture response. Transformation efficiency experiments were conducted to compare cvs 'Alamo', 'Kanlow', and 'Blackwell'. In cv 'Alamo', inoculating 398 embryogenic Type II calli resulted in recovery of 74 independent resistant calli with 46 (62.2%) yielding transgenic plantlets. In a second transformation experiment,, inoculating 100 embryogenic Type II calli resulted in recovery of 13 independent resistant calli with 10 (77%) regenerating to transgenic plantlets (TABLE 3). The same procedure used on embryogenic Type II callus of cvs 'Kanlow' and 'Blackwell' resulted in recovery of 30 and 85 independent resistant calli; 29 and 45 calli yielded transgenic plantlets (respectively; TABLE 3). Transgenic TO switchgrass plants showed a low level of escapes but were otherwise similar to the bialaphos-resistant transgenics.

In TO generation experiments transformation frequencies routinely resulted in transformed colonies of resistant cells with transformation frequencies ranging from 1.5% - 40.0%). All of these transgenic events were subsequently verified by Southern blot analysis with no escapes. Every plantlet (i.e., clone) derived from a particular resistant callus showed consistent hybridization patterns in the DNA blot analyses, indicative of single cell origin. FIGURE 6 shows transfection results after 70 days post-transfection and 71 days with hygromycin selection on 100 pieces of each of the five basic morphotypes consisting of (1) embryogenic (Type I), (2) embryogenic (Type II), (3) organogenic (root forming), (4) nondifferentiated, and (5) mucilaginous, using the 35S:hph selectable marker cassette in the EHA105 Agrobacterium strain. This data shows that embryogenic calli are preferentially recovered by the medium used in the in situ embryo rescue procedure.

Four transformation efficiency experiments were conducted using p35S-bar (TABLE 3). All of these experiments were conducted using the LBA4404 (pSBl) Agrobacterium strain and embryogenic Type II callus of the cv 'Alamo' cultivar. In one experiment, inoculating 300 embryogenic Type II calli resulted in recovery of 120 independent resistant calli with 46 (38.3%) regenerating to transgenic plantlets. In another transformation efficiency experiment using the same cv 'Alamo' cultures and the same Agrobacterium strain, inoculating 1165 embryogenic Type II calli resulted in recovery of only 34 independent resistant calli; however, 32 (94.1%) were regenerable. Transformation efficiencies were also conducted using (rice) pOsUbi-bar in LBA4404 (pSBl). One experiment using 588 embryogenic Type II calli of cv 'Alamo' resulted in recovery of 46 independent resistant calli with 42 (91.3%) regenerating to transgenic plantlets. In another experiment, inoculation of 400 calli resulted in recovery of only 6 independent resistant calli with 5 (83.3%) yielding transgenic plantlets. The same procedure using the identical selectable marker cassette and Agrobacterium strain was also used to evaluate the cv

'Kanlow' cultivar. In one experiment inoculation of 559 embryogenic Type II calli resulted in recovery of 7 independent resistant calli with 3 (42.8%) regenerating to transgenic plantlets; and, in another experiment, inoculation of 171 calli resulted in recovery of only 9 independent resistant calli with 5 (55.5%) regenerating to transgenic plantlets. All transgenic TO switchgrass plants were grown in soil in 10 inch pots and flowered in the greenhouse. All plants were morphologically normal with respect to leaf, root, shoot and flower development in comparison to wild type non-trans genie plants.

Identification of embryogenic stem cells in switchgrass calli was as follows. The five basic morphotypes consisting of (1) embryogenic (Type I), (2) embryogenic (Type II), (3) organogenic (root forming), (4) nondifferentiated, and (5) mucilaginous, (as shown in FIGURE 2) of cv 'Alamo' were grown in isolation and used to compare transformation efficiency using the 35S:hph selectable marker cassette and the same EHA105

Agrobacterium strain as in the previous experiments. Visual selection of embryogenic callus is important to the practice of this invention. Kill curve data (FIGURE 3 ) on increasing amount of bialaphos and hygromycin for cv 'Alamo' shows adequate selection for bialaphos is at 3 mg- 1-1 and 300 mg- 1-1 hygromycin. Killing levels of selective agent with recovery of embryogenic calli are required for practice of the invention. Antibiotics are used to remove Agrobacterium from primary transgenic (TO) cultures. Antibiotic effects on callus growth at increasing concentrations show that both cefotaxime and timentin can have positive effects on growth, while carbenicillin is benign. These requirements only apply to recovery of primary (TO) trans formants required for crosses. They are not normally required for subsequent steps in the in situ embryo rescue procedure as practiced by the present invention, but may be useful for de-contamination of re-introduced cultures where necessary.

One hundred calli of each morphotype described in FIGURE 2 were transfected using the identical procedures. Only embryogenic Type II callus resulted high levels of transient expression in useful numbers of stable resistant colonies. Only embryogenic Type II derived colonies were regenerable to plantlets. FIGURE 5 shows expression of pJLU 13 16 days after co-cultivation shows a non-uniform distribution of GFP expression. Positive expression is seen associated with embryogenic Type II but not in embryogenic Type I, mucilaginous or nondifferentiated callus morphotypes. Embryogenic cultures derived from in situ recovered embryos are most useful for practice of the present invention.

Further indications of the importance of embryogenic cell cultures is revealed by transformation results after 70 days post-transfection and 71 days hygromycin selection on 100 pieces of each of the five basic using the 35S:hph selectable marker cassette in the EHA105 Agrobacterium strain. In particular, FIGURE 6 shows transformation results after 70 days post-transfection and 71 days hygromycin selection on 100 pieces of each of the five basic morphotypes consisting of (1) embryogenic (Type I), (2) embryogenic (Type II), (3) organogenic (root forming), (4) nondifferentiated, and (5) mucilaginous, using the 35S:hph selectable marker cassette in the EHA105 Agrobacterium strain. This data shows that embryogenic calli are preferentially recovered by the medium used in the in situ embryo rescue procedure.

Another indication of the importance of embryogenic cell cultures for the present invention is revealed by the identification of embryogenic stem cells in switchgrass calli under selection. In particular, FIGURE 7 shows the identification of embryogenic stem cells in switchgrass calli under selection. GFP imaging of a single hygromycin resistant colony is shown sequentially over 45 day period. Doubling time is approximately 5 days. By day 70 intense green foci (arrows) appear as cytoplasmically dense cells of somatic embryos characteristic of embryogenic Type II callus. These results demonstrate that embryogenic callus can be selected from nontransgenic cells in the presence of killing levels of the selective agent as applied during in situ embryo rescue. All maternal non-transgenic tissue is eliminated allowing only the proliferation of cells resulting from the growth from the fertilization event (i.e. from the contribution of the transgene from the GMO parent). The most intense inflorescence is associated with embryogenic Type II call even though these colonies are typically heterogenous for various morphotypes. GFP imaging of single hygromycin resistant colonies were observed and recorded over a 45 day period starting 35 days after co-cultivation and after 31 days of hygromycin selection. By day 70,

cytoplasmically dense cells of somatic embryos characteristic of embryogenic Type II callus appear as intense GFP positive green foci and are clearly visible. The approximate doubling time based on these images is approximately 5 days. Identification of embryogenic stem cells in switchgrass calli under selection demonstrates the basis for selection during in situ embryo rescue. These results demonstrate that embryogenic callus can be selected from nontransgenic cells in the presence of killing levels of the selective agent as applied during in situ embryo rescue. All maternal non-transgenic tissue is eliminated allowing only the proliferation of cells resulting from the growth from the fertilization event (i.e., from the contribution of the transgene from the GMO parent).

Stable transgenic colonies expressing GFP were observed to determine the nature of embryogenic stem cells that are useful for transformation. Resistant colonies were observed after 100 days on selection revealing non-uniform GFP expression patterns . Expression of the GFP reporter gene in switchgrass (cv 'Alamo') in a single representative stable transgenic event was observed using brightfield and 470nm UV GFP microscopy. Micrographs of GFP expression in a stable transgenic calli prior to regeneration reveal intense GFP fluorescence associated with meristematic and somatic embryogenic tissues. No calli observable as GFP positive embryogenic Type II colonies were recovered from the other morphotypes. While the callus eventually re-establishes as a heterogenous tissue mass, from Type II, GFP is most intense in cytoplasmically dense cells consistent with meristematic cells and therefore, embryogenic Type II cells are clearly visualized. This demonstrates co-expression in transformed callus recovered from a Tl seed, a necessary prerequisite for recovery of embryogenic callus via in situ rescued embryos.

Micrographs of GFP expression in a stable transgenic callus prior to regeneration is throughout the callus. Most intense GFP fluorescence is associated with meristematic and somatic embryogenic tissues. FIGURES 8A and 8B show expression of the GFP reporter gene in switchgrass (cv a€~Alamoa€™) in a single representative stable transgenic event using brightfield microscopy (left side of FIGURE 8 A) and 470nm UV GFP microscopy (right side of FIGURE 8A). These micrographs (labelled A, B) of GFP expression were in a stable transgenic callus prior to regeneration. Note intense GFP fluorescence associated with meristematic and somatic embryogenic tissues. The same transgenic event (as labelled A,B) 7 days on regeneration media are shown in FIGURE 8B (as labelled C,D) shows these cells are regeneration competant, consistent with their embryogenic phenotype. Morphological changes in development noted as well as early leaf development from germinated embryos are visible even prior to exposure to the light regime during plantlet regeneration. These results show that under these selection criteria a callus selected from in situ embryo rescue will uniformly be transgenic and regenerable to intact plants.

FIGURES 9A and 9B show regeneration of transgenic calli with expression of the GFP reporter gene. Transgenic events grow normally and have normal chlorophyll development. Autofluorescence of chlorophyll masks expression. All plants are clonal as determined by Southern blot analyses without escapes. FIGURES 9A and 9B shows that the expression of the GFP reporter gene in switchgrass (cv 'Alamo') in a single representative stable transgenic event is consistent in a selected colony and can be regenerated to whole plants expressing the selectable marker and reporter genes. These tissues support the regeneration of stably transgenic plantlets that also express the selectable marker, as determined by their resistance, and the expression of the GFP reporter. This data supports that callus derived from in situ embryo rescue that will develop embryogenic callus that is also capable of plant regeneration. Also apparent are actively growing tissues with other cell types (Type I callus) while vacuolated cells appear less intense (as observed in FIGURE 9B). Morphological changes as early leaf development ensues from germinated embryos is clearly observable with these colonies giving rise to plantlets which all uniformly express GFP before becoming eventually masked by chlorophyll autoflorescence. While chlorophyll autoflorescence may mask the detection of GFP under these conditions, subsequent molecular analysis demonstrates that these plants are stable transgenic events.

Southern blot analyses were performed to determine the number and structure of T- DNA insertion(s) carried by 30 independent transgenic events, a representative subset of the total independent and regenerable bialaphos resistant calli. Eighteen of 30 (60%) contained a single T-DNA insertion. In particular, FIGURE 10 shows Southern blot hybridization of Ncol-digested genomic DNA extracted from cv 'Alamo' switchgrass pHG018 primary transformants with DIG-oxigenin labeled bar probe. EcoRI-digested bar cassette was included as positive hybridization control followed by 12 independent transgenic events and finally an Ncol-digested genomic DNA sample of wild-type cv 'Alamo' DNA. These data demonstrate that plantlets derived from embryogenic calli under these conditions are all clonal as determined by Southern blot analyses without escapes and supports the use of these approaches for in situ embryo rescue for transgenic cells derived from crosses. This data supports the contention that stable integration of transgenes are inherited as Tl plants that can be used for donor plants during in situ embryo rescue. The other events analyzed revealed multiple insertions ranging from 2 to 10+. All hybridization signals represent T- DNA/genomic DNA junction fragments, and all were larger than the minimum possible size of 2.1 kb. The positive hybridization control revealed a single hybridized fragment at its expected size (4 kb) and the negative control wild-type cv 'Alamo' DNA showed no hybridization at all. All plantlets regenerated from an individual bialaphos-resistant callus generated the identical number and size of hybridizing fragments in the DNA blot analysis, indicating that the plantlets were ultimately derived from a single-cell T-DNA integration event.

Further DNA samples were taken from mature plants and processed for PCR and/or Southern blot analysis. TO plants were backcrossed to wild-type parents in pollen cages in growth chambers. Recovered seed was germinated, grown to seedlings, and sprayed with 3% Finale. The putative Tl seedlings were grown to maturity and verified by Southern blot analysis as the progeny of the TO crosses.

In particular, FIGURE 1 1 shows Southern blot hybridization of Ncol-digested genomic DNA extracted from cv 'Alamo' switchgrass primary transformant # 52-7 and 10 Tl individuals with DIG-oxigenin labeled bar probe. Included in blot is EcoRI-digested bar cassette included as positive hybridization control, followed by TO parent #52-7, five individuals from cross where wild-type cv 'Alamo' served as pistillate parent and five individuals from reciprocal cross where primary transformant #52-7 served as the pistillate parent. All herbicide resistant Tl plantlets contained an identical fragment to their TO transgenic parent. No contamination from an outside source of transgenic pollen was observed. This data verifies that the T-DNA was stably integrated into the host plant genome and was inherited through germline cells to Tl offspring. Stable transmission of the T-DNA insertion through both microspore and megaspore cells were verified. This data supports the contention that stable integration of transgenes are inherited as Tl plants that can be used for donor plants during in situ embryo rescue.

Utilization of embryo rescue for recovery of wide cross progeny. The development of the immature caryopses in wide crosses and control wild type plants were evaluated according to Martinez -Renya and Vogel (2002). Martinez-Renya and Vogel (2002) show the results of several wide crosses yield devloping caryopses which abort via post fertilization incompatibility. These results at 2-30 days post pollination form the basis for generation of embryogenic callus and/or embryo rescue. Following microscopy and documentation intact caryopses and dissected immature embryos from these stages of development (i.e. 2-30 days post pollination) were removed and placed on embryogenic callus induction medium.

Caryopses and immature embryos were removed and placed on media designed for conventional embryo rescue as controls. From crosses made with transgenic plants containing a selectable marker (either bar or hyg), embryogenic callus initiation is accomplished on selection (3mg/L bialaphos for bar and 100 mg/L hygromycin for hyg). Only callus resulting from wide crosses is developed and that these events could be proliferated and regenerated to whole plants by well established protocols (Somleva et al. 2002. These regenerated plants were treated as those described above recovered from seed.

The intent then, was to use genomic information data to develop a genotyping platform that can be applied to future characterization of wide cross analysis as well genome and trait association studies, trait mapping, wide crosses and marker-assisted breeding. This is an example of the type of information that is useful for the development of new hybrids and their analysis subsequent to selection by in situ embryo rescue. This same type of information is useful for the genotyping of vegetable crops, such as tomato, and recovery of new hybrids with improved characteristics, such as hybrid tastes. In the tomato example, Solanum lycopersicum cv Buffalo is transformed with a selectable marker gene (e.g., bar) and crossed with Solanum lycopersicum cv Geronimo. The resulting F2BC1 population is screened for herbicide resistance and sensitive plants are phenotypically selected. The phenotype of the F2BC1 population may be screened for traits defined by genomic markers (e.g., taste). The tomato sequencing/genotyping platform provides a broader genomic function for trait identification, association genetics, marker- assisted breeding, and introgression of genetic material though crosses into hybrid selected tomato germplasm. Hybrids can be selected and identified for desirable phenotypes contributed by either parent; including taste and texture traits, sugar and solids content, carpel number and development, photosynthetic efficiency, enhanced fruit set yield acre, reduction of perception of nearest neighboring plant, higher value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, input requirements and utilizations, such as ertilizers and pesticides, characteristics, vine size, leaf phenotypes (including size, color, length width and angle), root mass and depth, seed set, inflorescence number, plant height and width, floral development; as well as biotic and abiotic stresse resistance including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus).

SPECIFIC EXAMPLES

In a first example, a herbicide selection procedure was used to select Fl embryogenic callus and plants were regenerated. Thus, a routine and high through-put method for switchgrass transformation was tested and optimized for embryogenic callus production as a perquisite to the practice of the current invention. The transformation systems was used to introduce molecular constructs designed to test expression of transgenes and deliver these to wide crosses recipients which could be recovered via in situ embryo rescue. The switchgrass transformation sequence typically begins with mature seed to generate embryogenic callus, however, the present invention teaches that embryogenic calli can also be produced from immature embryos both excised from the developing immature caryopsis or/and left in situ. During routine transformation, embryogenic callus initiation occurs from mature caryopses followed by transfection of embryogenic callus with Agrobacterium carrying vectors;

selection of transformants for herbicide resistance; and transgenic TO plant regeneration . Also, embryogenic callus can be derive from Tl transformed seeds carrying a selectable marker gene and a reporter. The same embryogenic callus induction media optimized for those conditions is deployed for induction of callus from immature caryopses from in situ embryo rescue. Various stages of ovule and embryo development are suitable for in situ embryo rescue from Panicum viragtum cv Alamo. FIGURE 12 shows various stages of ovule and embryo development suitable for in situ embryo rescue from Panicum viragtum cv Alamo in panels E - K. In particular, panel E shows an ovule approximately 4 dpf, panel F shows an ovule approximately 6 dpf, pane G shows an ovule approximately 9 dpf, panel H shows an ovule approximately 12 dpf, panel I shows an immature excised embryo, panel J shows an immature embryo within spikelet, and panel K shows a nearly mature excised embryo.

As shown ovules and embryos can be isolated and explanted to a suitable medium; where (E) Ovule approximately 4 days after flowering (dpf) (F) Ovule approximately 6 dpf (G) Ovule approximately 9 dpf (H) Ovule approximately 12 dpf (I) Immature excised embryo (J) Immature embryo within spikelet and, (K) Nearly mature excised embryo, are all amenable to in situ embryo rescue. Generation of hundreds of transgenic switchgrass plants and evaluation of their Tl and T2 progeny showed efficacy of the transformation protocol.

A strategy for recovery of wide crosses via in situ embryo rescue using embryogenic callus induction medium with herbicide selection is shown in FIGURE 13. This diagrammatic scheme illustrates, as an example, the recovery of wide inter-specific crosses using herbicide selection as a marker, however, this same or similar scheme also applies to wide inter-varietal, inter-specific, inter-generic and distant relative crosses. In this example, genetically modified (GMO) Panicum virgatum L. cv Alamo switchgrass (4x), (at upper left) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialaphos and 3% Finale or Liberty) and is used as the paternal pollen donor in a wide cross. These plants may be hemizygous TO, or contain at least one copy of the transgene in T1,T2, or .... generations. The maternal pollen recipient is wild -type Panicum virgatum cv Cave-In- Rock (CIR) (8x, at middle upper right) which in non-genetically modified (non-GMO) and hence herbicide sensitive to bialaphos and 3% Finale or Liberty. Pollinations may be most conveniently accomplished in pollen cages using one several clones of an event herbicide sensitive as a pollen donor and a single wild type plant as pollen recipient. After pollination immature caryopses are harvested only from the wild type maternal parent and plated onto

embryogenic caluus induction medium (center, left). Some of the caryopses form

embryogenic calli. Seedlings are regenerated from the calli in the presence of bialaphos for selection of the paternally inherited bar gene. Regenerated seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +) and herbicide sensitive (bar-) populations (middle lower right). At floral maturity the (bar+) Hbl Herbicide

Resistant Alamo X CIR hybrid plant (s) are scored for fertility and if fertile (I. fertile) used preferably as paternal pollen donor (s) in a backcross to either wild -type Panicum virgatum cv Alamo switchgrass (4x) non-GMO herbicide sensitive plants (lower center) or CIR wild type plants. The resultant seed from each population is recovered and germinated. The resultant seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +) and herbicide sensitive (bar-) and populations (left). The non-GMO hybrid plants contain Alamo X CIR X Alamo (12x or lower, blue, lower left) or Alamo X CIR X CIR genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers. Desirable plants may enter into population block breeding plots, and using genomic assisted breeding and mass selection can enter subsequent commercial development. These plants, can also serve as bridge intermediates to cross with other compatible or incompatible parents. If sterile (lower left; II. sterile) seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for verification of resulting resistant plants after 21 days to reveal herbicide resistant (bar +) populations (lower right). These plants have a robust gene confinement phenotype for deregulation of transgenic traits in hybrid backgrounds. These GMO hybrid plants contain Alamo X CIR (lower left) ,with 12x or lower genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers. Desirable plants may enter into population block breeding plots, and using genomic assisted breeding and mass selection can enter subsequent commercial development.

Three commercially available cultivars of switchgrass (Panicum virgatum L.); cvs Alamo, Kanlow, Southlow, and Atlantic Coastal Panicgrass (Panicum amarum Ell. var. amarulum) are all routinely transformable. This protocol exploits this expertise to generate the transgenics used in this invention. Approximately 50 independent randomly inserted events per construct were created and regenerated to 3-5 plantlets per clone and grown to maturity in the TO generation. TO plants with reporter genes and sterility constructs were analyzed for their phenotypes in the TO generation, whereas, all fertile trangenics, including reporter constructs, are backcrossed to wild type plants to recover Tl plants and seed for further use in wide cross recovery and analysis. Molecular analysis is by routine PCR and standard Southern blot analysis. In addition a genomics platform has been established to follow transgene introgression in subsequent crosses. Analysis of the transgenic outcomes and wide crosses include both phenotypic and molecular investigations. First, reporter constructs (GUS and GFP) driven by the same promoters as the ablation constructs (SL) are analyzed in TO and Tl plants using microscopy to verify tissue specificity and the absence of ectopic expression. Verification of intact inserts was conducted by, Southern blot analysis, RT-PCR and sequencing.

TABLE 4. List of seventeen public switchgrass (Panicum virgatum L) cultivars and their corresponding State of Origin, and Plant Form (i.e. Upland or Lowland). Corresponding sources shown in superscript. This type of information serves as an example for the present invention how a database can be generated to follow the hybridization of new cultivars or plants after in situ embryo rescue. USDA Soil Conservation Service Agricultural Handbook No. 170. Grass Varieties in the United States. 2 Plant Patent Application. USDA- NRCS.Release documents from Brooksville, FL Plant Materials Center. 4 Personal communication to Calvin Ernst. USDA NRCS Bismarck, ND Switchgrass Biomass Trials in North Dakota, South Dakota, and Minnesota. USDA-NRCS, Cape May Plant Materials Center "High Tide Switchgrass" release brochure. USDA-NRCS, Rose Lak Plant Materials Center "Southlow Switchgrass" release brochure.

TABLE 4

Isolated ovules from wide crosses where the parental pollen donor carried the selectable marker gene (bar) were explanted to media optimized for embryogenic callus initiation and containing killing levels of bialaphos. Two to fourteen days after plating ovules show development of embryogenic callus. FIGURES 14A and 14B show isolated ovules explanted from wide crosses onto media optimized for embryogenic callus initiation (FIGURE 14A) and on selection for bar show generation of embryogenic callus (FIGURE 14B). This callus must be derived from the fertilization product from the cross inherited from the parental transgenic plant and hence could not be derived from the maternal wild type tissue. In addition, if this callus derived from the endosperm it would not have the appearance of embryogenic callus. A wide self incompatible inter-varietial cross was recovered by in sit embryo rescue. Isolated ovules from wild type Panicum virgatum cv Cave in Rock (CIR) plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycin.

FIGURES 15A - 15G show isolated ovules from wild type Panicum virgatum cv Cave in Rock (CIR) plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycin (FIGURE 15A and FIGURE 15B), as well as callus on selection expresses the GFP and is cable of regeneration to plants (FIGURE 15C and FIGURE 15D), and isolated ovules from wild type Panicum amarum cv ACP plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycinm as well as callus on selection expresses the GFP (FIGURE 15E) and is cable of regeneration to plants (FIGURE 15F and FIGURE 15G).. Callus on selection expresses the GFP and is cable of regeneration to plants. In an example of a wide interspecific cross recovered by in situ embryo rescue, isolated ovules from wild type Panicum amarum cv ACP plant that had been fertilized with a hyg+/GFP+ transgenic Panicum virgatum cv Alamo paternal pollen donor and plated to embryogenic callus induction media containing killing levels of hygromycin.

Target genes for <$ and 9 sterility expression cassettes is show in TABLE 5. Target cells Reference

Gene

eveloping anthers Sorenseti er al. 2003

MS

developing Robinson-Beers et eL

ELI raegaspores 1992

developing, anthers von Malefc et eL 2002

BE2

developing IVA

Al megaspores

developing Marian ei sL 2905

Al

developing amber? Wiison ef eL 2001

SI

developing Ro iisoii-Beers ef aL

INI isiegas ores 1992

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Pollen Hague et ai, 2013

M 13

TABLE 5

TABLE 5. Target genes for $ and 9 sterility expression cassettes, promoters from these genes may be operably linked to cytoxic genes, including barnase or RNAi, or comparable technology to direct cell specific ablation leading to the developmental disruption of male or female floral structures. A detailed meta-analysis of known male- and female- specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation. These genes have been used to create "expression cassettes" using SLIC technology, a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. These genes or their orthologues, could also be the target for ZFN or TAL modifications to direct sterility functions. Promoters from these genes may be operably linked to cytoxic genes, including barnase or RNAi, or comparable technology to direct cell specific ablation leading to the developmental disruption of male or female floral structures. A detailed meta-analysis of known male- and female-specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation. These genes have been used to create "expression cassettes" using SLIC technology, a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. These genes or their orthologues, could also be the target for ZFN or TAL modifications to direct sterility functions. Concurrently, analysis of the sterility of male and female lines was also by microscopy but with the addition of the results of controlled crosses conducted in growth chambers. Male and female sterility lines are useful to enhance or force wide crosses increasing the probablity of recovery a hybrid. Since switchgrass is an obligate outcrosser, this system cleanly identified sterile lines and was also be used for the recovery of wide crosses through the exploitation of the herbicide selectable marker. This data was used to determine the degree of sterility per event, aware of position effects, to identify adequate expressers for the introduced constructs. IKI staining for pollen fertility was used on male and pollen specific ablations, but reliance on seed set and linked resistance in controlled crosses is required to evaluate female sterility. Wide crosses were conducted with these transgenic lines then in both directions as described in the embodiments.

This is an important fact used in selection of GMO and non-GMO populations in the F2 and subsequent generations recovered from wide crosses recovered by in situ embryo rescue. These switchgrass transgenics show that herbicide selection and "paint assays" correlate and most importantly that they could be used as paternal donors to wild type cv Alamo in preliminary wide cross experiments. These results also underscore the importance of a functional male sterility system for switchgrass. Such a system has not been previously described for switchgrass. This shows that co-transformation of a male sterility gene co- integrates with the selectable marker gene that have utility for in situ embryo rescue with applications for 'forcing' of wide crosses and/or recovery of wide crosses, hybrid plant formation or where hybridization may be impeded (i.e. tomato and other vegetable crops) is important.

Following a cross using a GMO parent (TO, Tl, or other) as the pollen donor (paternal) ovules are excised 1-30 post fertilization and plated either to (1) a 'resting' phase on embryogenic induction medium (TABLE 2) without selection for 1-30 days then transferred to the same medium containing a killing level of selection agent, or (2) directly to embryogenic induction medium (TABLE 2) containing a killing level of selection agent. Typically only embryogenic callus resulting from the cross is recovered with consistency. These calli are increased via tissue culture in the presence of the selective agent and regenerated as previously described.

This method permits the selection of hybrids without direct surgical removal of the immature embryo. A putative wide cross hybrid is isolated as a developing caryopsis (dl - 40 post-pollination) and plated on embryogenic callus induction medium. Alternatively, and as a control, conventional embryo rescue can be performed by direct surgical isolation of the embryo, followed by induction on embryogenic callus medium. In both of these cases, the callus intermediated can be regenerated to produce one to several hybrid plantlets that are genetically identical clones. Thirdly, conventional embryo rescue can be performed also by direct surgical isolation of the embryo and directly germinated to yield a single hybrid plant. In this second embodiment, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in difficult to recover intra-specific crosses to identify and define progeny useful for production of fertile hybrids. In this example using a self-compatible intra-specific cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non- transgenic Panicum virgatum L. cv Kanlow (4x). Note that directionality (maternal X paternal) here also does not matter to the practice of hybrid plant recovery. The Fl progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.

In a second example, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in to recover rare intra- or inter-specific crosses between self-incompatible parents to identify and define progeny useful for production of fertile hybrids (FIGURE 13). These plants may be hemizygous TO, or contain at least one copy of the transgene in T1,T2, or .... generations. This scheme illustrates, as an example, the recovery of wide inter-specific crosses using herbicide selection as a marker, however, this same or similar scheme also applies to wide intra- and inter-varietal, intra-and inter-specific, inter-generic and distant relative crosses. In this example, genetically modified

(GMO) Panicum virgatum L. cv Alamo switchgrass (4x) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialphos and 3% Finale or Liberty) and is used as the paternal pollen donor in a wide cross. These plants may be hemizygous TO, or contain at least one copy of the transgene in T1,T2, or .... generations. The maternal pollen recipient is wild -type Panicum virgatum cv Cave-In-Rock (CIR) (8x) which in non-genetically modified (non-GMO) and hence (shown in dark green) herbicide sensitive to bialaphos and 3% Finale or Liberty. Pollinations may be most conveniently accomplished in pollen cages using one several clones of an event herbicide sensitive as a pollen donor and a single wild type plant as pollen recipient. After pollination immature caryopses are harvested only from the wild type maternal parent and plated onto embryogenic caluus induction medium (center, left).

Caryopses can be selected from various stages of development, as shown by in the accompanying micrographs of isolated caryopses from various wide crosses in switchgrass, from the figures of Martinez -Reyna and Vogel (2002) as a guide. Some of the caryopses form embryogenic calli. In this embodiment chromosome 'doubling' treatments can be applied at this stage.

While the treatments may not result in precise chromosomal doubling, they can, in some cases restore fertility. Treatments may include but are not limited to colchicines and/or temperature shocks. Seedlings are regenerated from the calli in the presence of bialaphos for selection of the paternally inherited bar gene. Regenerated seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +, red) and herbicide sensitive (bar-) populations. Seedlings may also be analyzed for chromosomal number, content and/or integrity. At floral maturity the (bar+) Hbl Herbicide Resistant Alamo X CIR hybrid plant (s) are scored for fertility and if fertile used preferably as paternal pollen donor (s) in a backcross to either wild -type Panicum virgatum cv Alamo switchgrass (4x) non- GMO herbicide sensitive plants or CIR wild type plants. These plants, can also serve as bridge intermediates to cross with other compatible or incompatible parents. The resultant seed from each population is recovered and germinated. The resultant seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +) and herbicide sensitive (bar-) and populations. The non-GMO hybrid plants contain Alamo X CIR X Alamo (12x or lower) or Alamo X CIR X CIR genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers.

Desirable plants may enter into population block breeding plots, and using genomic assisted breeding and mass selection can enter subsequent commercial development. If sterile seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for verification of resulting resistant plants after 21 days to reveal herbicide resistant (bar +) populations. These plants have a robust gene confinement phenotype for deregulation of transgenic traits in hybrid backgrounds. These GMO hybrid plants contain Alamo X CIR ,with 12x or lower genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers. Desirable plants may enter into population block breeding plots, and using genomic assisted breeding and mass selection can enter subsequent commercial development.

This second example teaches, as an example, the recovery of wide inter- varietal crosses using herbicide selection as a marker, however, this same or similar scheme also applies to wide inter-varietal, inter-specific, inter-generic and distant relative crosses as a method to enhance recovery of fertile bridge intermediates. In this example using a self- incompatible cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non-transgenic Panicum virgatum L. cv Cave-In-Rock (8x). This example shows that various stages of developing caryopses can be chosen for in situ embryo rescue. In addition, this embodiment also shows that chromosome doubling agents (such as colchicine) or techniques (such as temperature shocks) can be applied to the developed embryogenic calli to enhance the recovery of fertile plants for the generation of non-GMO population. But as in the previous embodiment, this protocol shows the generation of sterile hybrids that may also present utility. Note that directionality (maternal X paternal) here may in some case play a role to the practice of hybrid plant recovery. The Fl progeny from the wide crosses may be infertile, thus using an embryo rescue technique to produce viable herbicide resistant Tl seedlings to produce healthy plants. In some cases chromosome doubling may be required by incorporating colchicine or other such agents in the embryo rescue medium to recover fertile Tl plants.

If plants are infertile the outcome will be transgenic and can be vegetatively increased for other purposes. Fertile Tl plants can be used in backcrosses to wild type non-transgenic Panicum virgatum L. hybrids. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.

FIGURE 13 shows a general strategy utilizing isolated caryopses from self- compatible crosses compared against the stages of caryopsis developed described by

Martinez -Reyna and Vogel (2002) When caryopses from self-incompatible crosses where one of the parents is transgenic carrying the herbicide selectable marker, and plated onto embryogenic initiation medium (Somleva et al. 2002; Nelson et al. 2012) embryogenic callus is isolated which is phenotypically distinct from calli forming from the endosperm. Although endosperm derived callus may form from the outcome of some crosses in some cases, it is not embryogenic and will not regenerate plantlets. Herbicide resistant embryogenic callus can be regenerated to plants. In some cases it may be desirable to treat calli with a chromosome doubling agent, such as colchicine, to derive a fertile Tl population. In the cases when fertile Tl plants are derived these will enter a strategy for deriving the non-GMO population via backcrossing to the wild type to segregate the transgene. The paint assay can then be used to identify the sensitive and resistant BC1 hybrid populations. These plants can be used as bridge intermediates in subsequent breeding crosses. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment. Selected individuals are characterized against the genomic platform and entered into a block population breeding strategy for varietial increases.

In the third embodiment, a series of intra- and inter- varietal, intra- and inter-specific or intra- and inter-generic wide crosses in switchgrass and related species are generated. These can be used directly in breeding programs or used as bridge intermediates to generate new cultivars and hybrids. This breeding platform utilizes transgenic male and female sterile lines from a reference genotype. In the present embodiment, the reference genotype is a switchgrass variety, such as Panicum virgatum L. cv. Alamo that is transgenic and either male or female sterile and linked to herbicide resistance. The sterility characteristic is used to 'force' rare wide crosses and herbicide selection is used for recovery of wide Tl (or Fl) crosses by embryo rescue intermediates. Tl(or Fl) hybrids can be backcrossed to the reference genotype, in this embodiment, the cv Alamo cultivar to segregate away the transgene to generate a non-GMO BC 1 mapping population.

One improvement on hybrid recovery in this embodiment is this method to 'force' outcrossing between parental lines. In this embodiment the generation of exclusively staminate and pistillate lines of Alamo are made specifically for this purpose. A detailed meta-analysis of known male- and female-specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation (TABLE 5). These genes have been used to create "expression cassettes" using SLIC technology, a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. These genes could also be the target for ZFN or TAL modifications to direct sterility functions. Alamo was chosen as the reference for several reasons including its ability to transform with Agrobacterium (FIGURE 2 ) and extensive genomic resources developed for the genome mapping component of this invention. It is referred to as the "reference" genome in this embodiment. Transformation with these cassettes has been conducted using reporter gene expression (GUS and GFP) as well as male and female specific cell ablation phenotypes and evaluated in mature TO florets. The observed reporter and ablation phenotypes demonstrate exclusively staminate (female sterile) or pistillate (male sterile) and are dependent on the appropriate expression cassette in the transgenic.

Alternatively, and similarly, male sterility lines can be used to for recovery of rare wide crosses. One target for male-sterility is the tapetum, the innermost layer of the anther wall that surrounds the pollen sac, which is needed for pollen development. A variety of anther and tapetum- specific genes have been identified that are involved in normal pollen development in many plant species, including maize (Hanson et al. 1989), rice (Zou et al. 1994), tomato Twell et al. 1989, Brassica campestri, (Theerakulpisut et al. 1991) and Arabidopsis thaliana (Xu et al. 1995). Selective ablation of tapetal cells by cell-specific expression of nuclear genes encoding cytotoxic molecules or an antisense gene essential for pollen development (Xu 1995, Goetz et al. 2001,) blocks pollen development, giving rise to stable male sterility. To induce male sterility in the turfgrass, Agrostis stoloniferia L

(creeping bentgrass )the 1.2-kb rice rts gene regulatory fragment, tap (Luo et al. 2003) was fused with two different genes. One was the antisense of rice rts gene that is predominantly expressed in tapetum cells during meiosis. Another gene was the Bacillus amyloliquefaciens ribonuclease gene, barnase, which ablates tapetal cells by destruction of RNA. Both approaches have been shown to be effective in various plant species. Therefore by supplying wild type pollen as a donor male sterile maternal lines such as these can provide important breeding tools and perhaps function as a filter for forcing generation and recovery of rare wide crosses. The use of Zm msl (for microspore abortion) zml 3 (for pollen sterility) can also be used for this purpose. These genes could also be the target for ZFN or TAL modifications to direct sterility functions. Transgenic recovered by both and can be subjected to colchicine treatments to recover homozygous lines to be used for future breeding and cultivar development. Most importantly the use of the randomly inserted maternal herbicide resistance in male sterile plants to select for wide crosses can be efficiently embryo rescued by plating immature caryopses on embryogenic callus induction medium and selecting for resistant calli. These calli must be derived as products from wide crosses and will be regenerated to whole plants by routine methods. A subset of this callus will be treated with colchcine to double the chromosome number to recover fertile allopolyploids. The resultant hybrids (if fertile) can then be backcrossed and selected for herbicide sensitivity rendering a non-GMO hybrid. This is a novel method which greatly accelerates new germplasm development. However, it has not escaped attention that a sterile transgenic hybrid with biofuels specific transgenic traits would be an attractive outcome to the solution of the gene confinement problem. This invention can also use traditional embryo rescue tissue culture techniques as a backup and control for comparative purposes, as described in the previous three embodiments. The scientific rationale for this invention therefore, has a long history involved with generating wide crosses in plants and hybrid plant development.

In this embodiment, a series of intra- and inter-specific wide crosses in switchgrass and related species is generated. Another important strategy in this inventive design is to physically link herbicide resistance (HR1 and HR2; i.e. bar or glyphosate) with male- and female-sterility transgenes, respectively. The breeding platform utilizes transgenic male and female sterile lines from a reference switchgrass variety, Panicum virgatum L. cv. Alamo and herbicide selection for recovery of wide intra- and inter- specific Fl crosses by embryo rescue. Fl hybrids can be backcrossed to the reference Alamo cultivar to segregate away the transgene to generate a non-GMO BC 1 mapping population. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment. Phenotypic analysis is conducted on the non-GMO population in regionally selected field plots and phenotypic data is statistically correlated to genetic variation. Variation is assessed using genome -resequencing technologies and this data, along with phenotypic information is used to establish a computational and statistical pipeline to identify, map and introgress variation associated with biomass and other bioenergy traits. This permits a single herbicide for single sex sterility in parental lines and progeny. A schematic for transgene cassette design may also be provided to generate male and female sterile lines under different selectable markers, designated Hbl and Hb2. Note that double herbicide selection can be used for complete sterility in Fls and that this accomplishes a separate and useful different objective, (i.e. namely gene confinement and trait stacking for GMO plant populations). Any other trait gene of interest (GOI) or series of GOIs, can be combined through this strategy into said bridge intermediate. Physical linkage of herbicide resistance (HR1 and HR2) with male- and female-sterility transgenes, respectively. This permits a single herbicide for single sex sterility in parental lines and progeny. Note that double herbicide selection can be used for complete sterility in Fls and that this accomplishes a separate and useful different objective. Two lines are created that, when crossed, would give rise to a fully sterile individual.

Male and female lines are created through the application of the promoters and/or the coding sequences described in TABLE 5. Male sterile lines (top, line A-Male Sterility) are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as RNAi. These genes or their orthologues, could be the target for ZFN or TAL modifications to direct sterility functions. Female sterile lines (bottom , line B-Female Sterility) are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as R Ai. These genes or their orthologues, could also be the target for ZFN or TAL modifications to direct sterility functions.

The final transgene contains the target promoter translationally fused or operably linked to a selected CDS or open reading frame (ORF) and 3' non-translated region (3'-UTR) with compatible 5' and 3 ' ends which are readily cloned into the LIC-adapted T-DNA vector. The SLIC-LIC method is highly scalable and permits construction of many independent versions of promoter elements fused to reporter CDS, such as GUS and GFP, as well as cell ablation genes (barnase) or RNAi. Transgenic cv. Alamo (sequenced reference line) have been generated for male and female test vectors (10-20 independent single gene insertion events per vector) and have been analyzed molecularly for single-copy insertions and phenotypically for reporter gene expression and floral phenotypes characterized in our greenhouses. Single copy insertions have been detected using a Taqman qPCR assay, to detect low copy insertions (1-2 copies), followed by genomic Southerns for verification.

Physical linkage of herbicide resistance (HR1 and HR2) may be used with male- and female-sterility transgenes can be used for creation of bridge intermediate hybrid breeding populations. Physical linkage of herbicide resistance (HR1 and HR2) with male- and female- sterility transgenes, respectively, which can also be used to create total sterile outcomes. This permits a single herbicide for single sex sterility in parental lines and progeny. Note that double herbicide selection can be used for complete sterility in Tl (Fl)s and that this accomplishes a gene confinement strategy. Two lines are created that, when crossed, would give rise to a fully sterile individual.

In this strategy, as shown in this example, single-copy transgenics are backcrossed to wild type cv Alamo reference plants to test for stability and inheritance of the transgene phenotype. Stable single copy lines are used in conjunction with embryo rescue to create inter- varietial, inter-specific and inter-generic hybrids of switchgrass and related species. The breeding platform for efficient wide-cross production produces important bridge intermediates. Success at using a dominant herbicide marker to create inter-specific hybrids in switchgrass form the basis of establishing an efficient breeding platform. This embodiment teaches a greatly improved efficiency of hybrid production as well as the rescue of hybrid embryos by incorporating staminate and pistillate lines and herbicide selection into this program. The basic design is to use the pistillate reference plants as pollen recipient with a wide variety of cultivars and species. [Note: reciprocal crosses, using the staminate reference plant are also possible.] In closed pollen cage experiments parental types are set up in pairwise combinations. Seed set is monitored and collected for subsequent analysis, using this novel technique, this intermixing to produce developing caryopses (Fl progeny) on the pistillate plants that are the result of pollen flow from the staminate plants but not vice versa.

Note that in most cases of wide crosses, Tl (Fl) sterility, caused by embryo- endosperm incompatibility, is common and this may require the use of embryo rescue techniques, as described in the previous embodiments, to recover Fl progeny or reciprocal Fl crosses to avoid incompatibility. Recovery of rare wide cross progeny can be forced.

Immature, isolated caryopses can be excised and grown in vitro to recover plantlets. In rare wide cross cases, it may be necessary to generate embryogenic callus that will be regenerated to whole plants.

All recovered Tl (Fl) hybrid plants are grown in the greenhouse and characterized molecularly. For instance in one direction of the cross, initially one can use a female cytoplasmic (chloroplast) marker and a male nuclear marker (trans gene) to detect hybrids. A more detailed phenotypic and genomic analysis can follow in the BC 1 population. Hybrids are then examined for fertility and seed set in backcrosses to wild-type Panicum virgatum cv Alamo reference plants. For instance, since the Fl hybrids will retain the pistillate phenotype when selected for herbicide resistance, these Fl will be mated to wild-type reference plants in cage experiments to recover BC 1 population.

The problem often seen with sterility of the Fl hybrids in wide crosses is not necessarily a disadvantage for biomass perennial crops that are grown vegetatively, however. In fact, this may be an advantage in allocating resources to biomass production in the field. Nevertheless, a breeding program may wish to introgress traits from one line into the genome of another, a process of recurrent backcrossing. In cases where Fl sterility is observed, this embodiment addresses this issue by creating alloploid lines to attempt to restore fertility in the Fl . Basically, immature caryopses of the Fl plants can be used as to initiate embryogenic callus in a medium containing herbicide for selection and simultaneously treated with colchicine and regenerated to recover alloploid plants, which in some cases will restore fertility. If fertility is not recovered in the Fl hybrid the new alloploid sterile hybrids that will be increased as vegetative plugs for stand establishment. This method for creation of new sterile alloploid hybrids may serve as a platform vehicle for inclusion of additional stacked transgenes as a GMO product that will be sterile solving the problem of gene confinement as a stable mechanism for control of transgene escape.

Tissue samples from hybrid plants are collected for genomic studies and the non- transgenic (non-GMO) BC 1 population can then transferred outdoors for field trial analysis where they can will be vegetatively propagated and entered into block breeding increases. A set of clones are grown in several locations for regional selection and extensively

characterized for biomass production and additional selected biofuels and agronomic traits such as above ground biomass, leaf number, inflorescence height and number, crown size per year and seed set. Characterized individuals can then be included in a downstream breeding process using genomic assisted breeding. Hybrids can be selected for desirable phenotypes contributed by either parent;

including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus). Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development. Hybrids can be sexually crossed and/or vegtetatively propagated.

In the last embodiment, in situ hybridization is extended to dicot plants, exemplified here in the tomato. The wide applicability of this technique with application to selections of wide crosses in tomato (Solanum lycopersicum cvs), In this case the breeding and selection of hybrids is accelerated by the production of a transgenic intermediate followed by embryo rescue and backcrossing the transgene away from the hybrid background. Trait selection may be by direct phenotypic selection or by using genomic assisted breeding. In this example, Solanum lycopersicum cv Buffalo is transformed with a selectable marker gene (i.e. bar) and crossed with Solanum lycopersicum cv Geronimo. Male and/or female sterility transgenes may also be used in combination with a selectable marker for recovery and selection of crosses. The tomato variety Solanum lycopersicum cv Buffalo is known for superior taste characteristics of significant market value and Solanum lycopersicum cv Geronimo has certain production characteristic of value. Flowers of wild type Solanum lycopersicum cv Buffalo at anthesis with full developed anthers and fertile pollen may be provided. An inflorescence of wild type Solanum lycopersicum cv Buffalo withimmature fruits may also be provided at a stage suitable for in situ embryo rescue. A vegetative leaf from a mature wild type Solanum lycopersicum cv Buffalo may be provided with characteristic shape and deep green color (mature flower is shown at the lower left). The Hybrid tomato variety Solanum lycopersicum cv Buffalo X Solanum lycopersicum cv Geronimo is recovered by in situ embryo rescue. Their combined hybrid characteristics forms a new variety of commercial value. The ability to perform in situ embryo rescue for recovery of the hybrid saves valuable breeding time and effort. After 1-30 days post pollination ovules are excised from the wild type Solanum lycopersicum cv Geronimo plants and placed on media containing the selection agent (i.e. bialaphos). The resulting embryogenic callus is recovered and regenerated to whole hybrid Fl plants. These plants are backcrossed to either wild type cv Geronimo or wild type cv Buffalo. The resulting F2BC1 population is screen for herbicide resistance and sensitive plants are phenotypically selected. The phenotype of the F2BC 1 population may be screened for traits defined by genomic markers (i.e. taste). Demonstration of the usefulness of this technique for hybrid tomato selection extends this procedure to include dicot plant breeding.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 is a nucleic acid sequence of a corn ovule-specific gene SEQ ID NO: 2 is a nucleic acid sequence of a corn female inflorescence developmentally-specifically expressed gene .

SEQ ID NO: 3 is a nucleic acid sequence of a corn tapetum-specific gene

SEQ ID NO: 4 is a nucleic acid sequence of a corn pollen-specific gene

Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "a transgenic plant" includes one or a plurality of such plants, and reference to "the floral-specific promoter" includes reference to one or more floral-specific promoters or their homologues and equivalents thereof known to those skilled in the art, and so forth.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

Allele: One of the different forms of a gene that can exist at a single locus

Anther-specific gene: A gene sequence that is primarily expressed in the anther, relative to expression in other plant tissues. Includes any anther-specific gene whose malfunction or functional deletion results in male-sterility. Examples include, but are not limited to: anther-specific gene from tobacco (GenBank Accession Nos. AF376772- AF376774), and Osg4B and Osg6B (GenBank Accession Nos. D21159 and 21160). Anther-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in anther tissue relative to the other tissues of the plant. Examples include, but are not limited to: anther-specific gene promoter from tobacco (GenBank Accession Nos. AF376772-AF376774), and the promoters of Osg4B and Osg6B (GenBank Accession Nos. D21159 and D21160).

Asexual: A plant lacking floral structures such that it is incapable of participating either as a male or female parent in sexual reproduction and propagates vegetatively.

Bridge intermediate: refers to a genetic bridge for importing genes into hybrids providing a mechanism for importing any new genes not found in common breeding program materials, and any de novo genetic material that arises from these wide varietal, species or genera crosses using traditional plant breeding techniques.

Comprises: A term that means "including." For example, "comprising A or B" means including A or B, or both A and B, unless clearly indicated otherwise.

Deletion: The removal of a sequence of a nucleic acid, for example DNA, the regions on either side being joined together.

Desirable trait: A characteristic which is beneficial to a plant, such as a commercially desirable, agronomically important trait. Examples include, but are not limited to: resistance to insects and other pests and disease-causing agents (such as viral, bacterial, fungal, and nematode agents); tolerance or resistance to herbicides; enhanced stability; increased yield or shelf-life; environmental tolerances (such as tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress, or oxidative stress); male sterility; and nutritional

enhancements (such as starch quantity and quality; oil quantity and quality; protein quality and quantity; amino acid composition; and the like). On one example, a desirable trait is selected for through conventional breeding. In another example, a desirable trait is obtained by transfecting the plant with a transgene(s) encoding one or more genes that confer the desirable trait to the plant.

Egg: In seed plants an egg is an ovum (plural; ova, from t ovum meaning egg or egg cell) is a haploid female reproductive cell or gamete.

Floral deficient: A plant that is lacking, or is functionally deficient in, one or several parts of the male or female structures contained within a single flower or inflorescence effectively resulting in either male or female sterility.

Floral-specific gene: gene sequence that is primarily expressed in floral tissue or during the transition from a vegetative to floral meristem, such as the tapetum, anther, ovule, style, or stigma, relative to the other tissues of the plant. Includes any floral-specific gene whose malfunction or functional deletion results in sterility of the plant either directly or by preventing fertilization of gametes through floral deficiencies.

Floral-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in floral tissues or during the transition from vegetative to floral meristem relative to the other tissues of the plant. Examples include, but are not limited to: meristem transition-specific promoters, floral meristem-specific promoters, anther-specific promoters, pollen-specific promoters, tapetum-specific promoters, ovule-specific promoters, megasporocyte-specific promoters, megasporangium-specific promoter-0, integument- specific promoters, stigma-specific promoters, and style-specific promoters. In one example, floral-specific promoters include an embryo-specific promoter or a late embryo-specific promoter, such as the late embryo specific promoter of DNH 1 or the HVA1 promoter, the GLBl promoter from corn, and any of the Zein promoters (Z27). In another example, floral- specific promoters include the FLO/LFY promoter from switchgrass. The determination of whether a sequence operates to confer floral specific expression in a particular system (taking into account the plant species into which the construct is being introduced, the level of expression required, etc.), is preformed using known methods, such as operably linking the promoter to a marker gene (e.g. GUS, and GFP), introducing such constructs into plants and then determining the level of expression of the marker gene in floral and other plant tissues. Sub-regions which confer only or predominantly floral expression, are considered to contain the necessary elements to confer floral specific expression.

Functionally equivalent: Nucleic acid sequence alterations in a vector that yield the same results described herein. Such sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, and insertions. For example, in a nucleic acid including a barnase sequence that is cytotoxic, a functionally equivalent barnase sequence may differ from the exact barnase sequences disclosed herein, but maintains its cytotoxic activity. Methods for determining such activity are disclosed herein.

Genetic markers: Alleles used as experimental probes to keep track of an individual, a tissue, a cell, a nucleus, a chromosome, or a gene.

Gene of interest: (GOI) Any gene, or combination of functional nucleic acid sequences (such as comprised in plant expression cassettes such as with a promoter, coding sequence and termination sequence) in plants that may result in a desired phenotype.

Genotype: The allelic composition of a cell—either of the entire cell or, more commonly, for a certain gene or a set of genes of an individual.

Hybrid plant: An individual plant produced by crossing two parents of different genotypes or germplasm backgrounds.

In situ (/in ^'sitju:) is a Latin phrase which translates literally to 'In position'. It is used in many different contexts, but here is used referring to the in place context of the embryo within a fertilized plant. Intergeneric (literally between/among genera) describes relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of separate genus thereby contrasting with interspecific.

Interspecific (literally between/among species) describes relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of separate species thereby contrasting with intraspecific.

Intervarietal (literally between varieties, or cultivars) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of a single variety, thereby contrasting with interspecific

Intraspecific (literally within species) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues within individuals of a single species, thereby contrasting with interspecific

Intravarietal (literally within varieties, or cultivars) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues within individuals of a single variety, thereby contrasting with interspecific.

Isolated: An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides

Locus: The place on a chromosome where a gene is located. Molecular genetics: The study of the molecular processes underlying gene structure and function.

Mutagens: Include physical mutagens, such as, e.g. ionizing radiations (e.g. X-rays, gamma rays and alpha particles), as well as DNA reactive chemicals, such as, DNA adducts, deaminating agents, alkylating agents, intercalating agents, metals, biological agents, e.g. transposons and viruses.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide: A linear polynucleotide (such as DNA or RNA) sequence of at least 9-350 nucleotides, for example at least 15, 18, 24, 25, 27, 30, 50, 100 or even 200 nucleotides long.

ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Ovule: In seed plants, the ovule is the structure that gives rise to and contains the female reproductive cells. It consists of three parts: The integument(s) forming its outer layer(s), the nucellus (or megasporangium), and the megaspore-derived female gametophyte (or megagametophyte) in its center. The megagametophyte (also called embryo sac in flowering plants) produces an egg cell (or several egg cells in some groups) for. After fertilization, the ovule develops into a seed.

Peptide: A chain of amino acids of which is at least 4 amino acids in length. In one example, a peptide is from about 4 to about 30 amino acids in length, for example about 8 to about 25 amino acids in length, such as from about 9 to about 15 amino acids in length, for example about 9-10 amino acids in length.

Perennial: A plant which grows to floral maturity for three seasons or more. Whereas annual plants sprout from seeds, grow, flower, set seed and senesce in one growing season, perennial plants persist for several growing seasons. The persistent seasonal flowering of perennial plants may also, but not necessarily, include light and temperature requirements that result in vernalization. Examples include, but are not limited to: certain grasses, such as members of the Poacea, such as switchgrass (Panicum virgatum L. cv Alamo). Andropogon sp., Panicum, sp., Pennisetum sp., Zea sp., Saccharum sp., Miscanthus sp., a Saccharum sp. x Miscanthus sp. hybrids, Erianthus sp., Tripsicum sp., or Zea X Tripiscum sp. hybrids, also including species of turfgrass, forage grass or various ornamental grasses; trees, including poplar, willow, eucalyptus, Paulownia and also trees broadly known such as fruit and nut, and crop trees (for example bananas and papayas), forest and ornamental trees, rubber plants, and shrubs; grapes; roses.

Plant: Any kind of plant. In specific embodiments, the "plant" is a flowering plant or Angiosperm. In some embodiments, the Angiosperm is a Dicotyledon (or Dicot). In some embodiments, the Angiosperm is a Monocotyledon (or Monocot). In some embodiments, the Dicot is a plant selected from Magnoliidae, Hamamelidae, Caryophyllidae, Dilleniidae, Rosidae, or Asteridae. In some embodiments, the Monocot is a plant selected from

Alismatidae, Commelinidae, Arecidae, or Liliidae. In some embodiments, the plant is a row crop or field crop. Row crops and field crops include, without limitation, grains, including small grains (e.g. corn/maize, (buck)wheat, millet, oats, rye, beans, sorghum, rice, barley), commercial crops (e.g. sugar beets, cotton, sunflowers, Kenaf, tobacco, soybeans, canola, (oilseed) rape, sugar cane, cassava), vegetables (e.g. cabbage, tomatoes, cucumbers, beets, squash, carrots), and forage crops (e.g. perennial grasses, annual grasses, perennial legumes, annual legumes, alfalfa, root crops, feed cabbage, potatoes). In some embodiments, the plant is a nut plant, e.g. peanut or almond. In some embodiments, the plant is a fruit plant, e.g. orange, apple, cherry, grape, watermelon, papaya, banana, or plum.

Plant breeding: The application of genetic analysis to development of plant lines better suited for human purposes

Pollen- specific gene: A DNA sequence that directs a higher level of transcription of an associated gene in microspores and/or pollen (i.e., after meiosis) relative to the other tissues of the plant. Examples include, but are not limited to: pollen-specific promoters LAT52, LAT56, and LAT59 from tomato (GenBank Accession Nos. BG642507, X56487 and X56488), rice pollen specific gene promoter PSI (GenBank Accession No. Z16402), and pollen specific promoter from corn (GenBank Accession No. BD 136635 and BD 136636).

Pollen-specific promoter: A gene sequence that is primarily expressed in pollen relative to the other cells of the plant. Includes any pollen-specific gene whose malfunction or functional deletion results in male-sterility. Examples include, but are not limited to: LAT52, LAT56, and LAT59 from tomato (GenBank Accession Nos. BG642507, X56487 and X56488), PSI (GenBank Accession No. Z16402), and pollen specific gene from corn

(GenBank Accession No. BD136635 and BD136636).

Promoter: An array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included .

Specific, non-limiting examples of promoters that can be used to practice the disclosed methods include, but are not limited to, a floral-specific promoter, constitutive promoters, as well as inducible promoters for example a heat shock promoter, a

glucocorticoid promoter, and a chemically inducible promoter. Promoters produced by recombinant DNA or synthetic techniques may also be used. A polynucleotide encoding a protein can be inserted into an expression vector that contains a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host. In one example, an expression vector contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Probe: Defined nucleic acid segment that can be used to identify specific molecules bearing the complementary DNA or RNA sequence, usually through autoradiography, chemiluminescence or color detection.

RFLP: Refers to restriction fragment length polymorphism that is a specific DNA sequence revealed as a band of particular molecular weight size on a Southern blot probed with a radiolabeled RFLP probe and is considered to be an allele of a gene.

Southern blot: Transfer of electrophoretically separated fragments of DNA from the gel to an absorbent surface such as paper or a membrane which is then immersed in a solution containing a labeled probe that will bind to homologous DNA sequences.

Selectable marker: A nucleic acid sequence that confers a selectable phenotype, such as in plant cells, that facilitates identification of cells containing the nucleic acid sequence. Transgenic plants expressing a selectable marker can be screened for transmission of the gene(s) of interest. Examples include, but are not limited to: genes that confer resistance to toxic chemicals (e.g. ampicillin, spectinomycin, streptomycin, kanamycin, geneticin, hygromycin, glyphosate or tetracycline resistance, as well as bar and pat genes which confer herbicide resistance), complement a nutritional deficiency (e.g., uracil, histidine, leucine), or impart a visually distinguishing characteristic (e.g., color changes or fluorescence, such as 13-gal).

Tapetum-specific gene: A gene sequence that is primarily expressed in the tapetum relative to the other tissues of the plant. Includes any tapetum cell-specific gene whose malfunction results in male-sterility. Examples include, but are not limited to: TA29 and TA13, pca55, pEl and pT72, Bcpl from Brassica and Arabidopsis (GenBank Accession Nos. X68209 and X68211), A9 from Brassicaceae (GenBank Accession No. A26204), and TAZl, a tapetum-specific zinc finger gene from petunia (GenBank Accession No. AB063169).

Tapetum-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in tapetal tissue relative to the other tissues of the plant. Tapetum is nutritive tissue required for pollen development. Examples include, but are not limited to the promoters associated with the genes listed under tapetum-specific genes.

Tissue culture: is a collection of techniques used to maintain or grow cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture, specifically, refers to a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition.

Transduced and transformed: A virus or vector "transduces" or transfects" a cell when it transfers nucleic acid into the cell. A cell is "transformed" by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation, lipofection, Agrobacterium- mediated transfer, direct DNA uptake, and microprojectile bombardment.

Transgene: An exogenous nucleic acid sequence. In one example, a transgene is a gene sequence, for example a sequence that encodes a cytotoxic polypeptide. In yet another example, the transgene is an antisense nucleotide, wherein expression of the antisense nucleotide inhibits expression of a target nucleic acid sequence. A transgene can contain native regulatory sequences operably linked to the transgene (e.g. the wild-type promoter, found operably linked to the gene in a wild-type cell). Alternatively, a heterologous promoter can be operably linked to the transgene.

Transgenic Cell: Transformed cells that contain a transgene, which may or may not be native to the cell.

Vector: A nucleic acid molecule as introduced into a cell, thereby producing a transformed cell. A vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. Examples include, but are not limited to a plasmid, cosmid, bacteriophage, or virus that carries exogenous DNA into a cell. A vector can also include one or more cytotoxic genes, antisense molecules, and/or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express the nucleic acids and/or proteins encoded by the vector. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a, liposome, protein coating or the like.

Wild type: refers to a reference and it can mean an organism, set of genes, gene or nucleotide sequence. For purposes herein the wild type refers to the parents of hybrid progeny.

In some embodiments, plants, including parental plants and hybrid plants, as well as progeny, seeds and embryos thereof, comprise one or more selectable marker and/or reporter. In some embodiments, the selectable markers and/or reporters are utilized to regulate the expression of a transgene or to allow for selection of plants or plant tissues comprising (or not comprising) the selectable marker and/or reporter. Selectable marker genes include, but are not limited to nptll (Neomycin phosphotransferase II), hpt (Hpt (hygromycin

phosphotransferase), hph or aphlV-Hygromycin (Hyg) B resistance), acc3, aadA, bar (Bar- Phosphinothricin (PPT) resistance), and pat. Recovery of a transgenic plant can be facilitated by selection of putative transformants on a medium containing a (matched) selection agent, such as antibiotic (nptll (neomycin), hpt (hygromycin B), acc3, aadA), antimetabolite (dhfr), herbicide (bar, pat), or similar, such as, e.g., kanamycin and other aminoglycoside antibiotics. Other selectable marker genes include pmi, codA, aux2, tms2, dhlA, CYP105A CYP105A, and cue. Other selectable marker genes include isopentyl transferases, histidine kinase homologue, and hairy root-inducing genes. A selectable marker will protect the organism from a selection agent that would normally kill it or prevent its growth. In some

embodiments, the killing concentration for a plant or plant tissue not containing the selectable marker is used in the methods described herein. The killing concentration for specific selectable markers and corresponding selection agents (in a medium) can be determined without difficulty by using standard methods well known in the art. In some embodiments, reporter genes, such as, e.g., cat, lacZ, GUS (beta-glucuronidase), uidA, luc (luciferase), gfp (green fluorescent protein), are utilized, either alone or in combination with a selectable marker. Reporters allow to distinguish transformed and non-transformed plants. (See, e.g. A. Ziemienowicz, "Plant selectable markers and reporter genes", Acta Physiologiae Plantarum, 23:3, 363-374, 2001; B. Miki et al. "Selectable Marker Genes in Transgenic Plants - Applications, Alternatives and Biosafety", Journal of Biotechnology, 107:3, 193-232, 2004; P. Hare et al. "Excision of Selectable Marker Genes from Transgenic Plants", Nature

Biotechnology, 20:6, 575-580, 2002; Goldstein et al. "A Review - Human Safety and Genetically Modified Plants - A Review of Antibiotic Resistance Markers and Future Transformation Selection Technologies. Journal of Applied Microbiology, 99, 7-23, 2005).

In some embodiments, the methods described herein may be used to confer a desired trait of one plant on another plant. The traits include, e.g., carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof. In some embodiments, the desired trait that is to be conferred is not present in the plant variety, genus or species. In some embodiments, the trait to be conferred is present, but is enhanced upon conferral. An enhancement may be in the order of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%), 800%), 900%), 1000%) or more when the resulting plant (e.g. hybrid plant) is compared to a comparable parent plant. For example, if the trait of "biomass" is enhanced, the biomass of the resulting plant or progeny thereof is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more after conferral of the trait when compared to a comparable parental plant without the trait. The increase may be measured in gram or kilogram of harvested biomass. In another example, if the trait of "fertilizer use" is enhanced, the fertilizer use required to bring about equal growth of the resulting plant or progeny thereof is decreased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%), 60%), 70%), 80%), or 90% or more after conferral of the trait when compared to a comparable parental plant without the trait. A decrease in fertilizer growth may be measured in gram or kilogram of less fertilizer used (e.g. per sq ft (or m²) or sq mi (or km²) of planted crop). In another example, if the trait of "cold or freeze tolerance" is enhanced, the temperature tolerance range of the resulting plant or progeny may be increased by, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, such that, e.g. the resulting plant or progeny tolerates lower temperatures, such as 0.5°C, 1°C, 2°C, 3°C, 4°C, or 5°C lower, or such that the resulting plant or progeny tolerates longer freezing periods, e.g. lh, 2h, 6h, 12h, 24h, 36, 48h, or 72h of below freezing temperatures when compared to a comparable parental plant without the trait. The increased tolerance may be measured, e.g. by a corresponding reduction in crop loss (e.g. for a period of freezing or cold snap) or by a corresponding increase in, e.g. biomass, flowering, fruit development, etc. because of increased cold tolerance.

Generally, a characteristic mentioned herein as it pertains to an individual plant or plant tissue (e.g. embryos, calli, cells, leaves, stems, seeds) is absolute, e.g. 100%, such as a fertile or infertile plant, a transgenic plant, a non-transgenic plant, etc. It is to be understood, however, that with respect to a population of plants or plant tissues (e.g. embryos, calli, cells, leaves, stems, seeds), e.g. 10, 100, 1,000, 10,000, 100,000 or more plants or plant tissues, a characteristic may not be absolute. In some embodiments, e.g. through random events, the characteristic may be lost in individual members of the population, such that, e.g., only about 99.99%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, or less than 85% of the population exhibit the characteristic.

In accordance with various embodiments, therefore, the invention provides a method for embryo rescue to recover a hybrid plant for breeding purposes using at least one transgenic plant as a parent. In certain embodiments, the method uses a transgenic plant containing one or more selectable markers, and produces a hybrid plant. In certain embodiments, the method produces a hybrid plant from intra-varietial parents. In certain embodiments, the method produces a hybrid plant from inter-varietial parents or from intra- specific parents or from inter-specific parents or from intra-generic parents or from inter- generic parents.

In certain embodiments, the method produces a fertile Fl hybrid embryo, seed or plant, such as a fertile FI hybrid plant that is used to backcross to its non-transgenic compatible parent. In certain embodiments, the method produces a fertile hybrid plant used to outcross to a non-transgenic compatible parent. In certain embodiments, the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers. In certain embodiments, such as a mature hybrid, and the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

In certain embodiments, the hybrid F2 individuals are all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses. In certain embodiments, the subsequent crosses involve inter- or intra- varietal, specific or generic parents. In certain embodiments, the F3 hybrid progeny are used in subsequent crosses. In certain embodiments, the method produces an infertile Fl hybrid embryo, seed or hybrid plant, such as a Fl hybrid embryo, seed or plant that is recovered to produce a fertile Fl embryo, seed or hybrid plant [using embryo rescue and chromosomal doubling using colchicine for example].

In certain embodiments, the method produces a fertile F2 hybrid embryo, seed or hybrid plan that is used to backcross to its non-transgenic parent. In certain embodiments, the method produces a fertile F2 hybrid embryo, seed or plant used to outcross to a non- transgenic parent. In certain embodiments, the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers. In certain embodiments, the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome. In certain embodiments, the hybrid F2 individuals are all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses. In certain embodiments, the subsequent crosses involve inter- or intra- varietal, specific or generic parents. In certain embodiments, the F3 hybrid progeny are used in subsequent crosses.

In certain embodiments, the method produces an infertile Fl hybrid embryo, seed or plant is vegetatively propagated as a sterile population [for gene confinement purposes], such as a fertile FI hybrid plant used to outcross to non-transgenic incompatible inter- or intra- varietal, specific or generic parents. In certain embodiments, the method produces a fertile FI hybrid plant used to outcross to non-transgenic incompatible inter- or intra- varietal, specific or generic parents. In certain embodiments, the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers. In certain embodiments, the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

In certain embodiments, the hybrid F2 individuals are all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses. In certain embodiments, the subsequent crosses involve inter- or intra- varietal, specific or generic parents. In certain embodiments, the F3 hybrid progeny are used in subsequent crosses. In certain embodiments, the method produces a F2 hybrid embryo, seed or plant is recovered to produce a fertile embryo, seed or hybrid plant [using embryo rescue and chromosomal doubling using colchicine for example]. In certain embodiments, the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals are all sequences of the transgene cassette have segregated from the genome. In certain embodiments, the hybrid F2 individuals are all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses.

In certain embodiments, the subsequent crosses involve inter- or intra- varietal, specific or generic parents. In certain embodiments, the F3 hybrid progeny are used in subsequent crosses. In certain embodiments, the method produces an infertile Fl hybrid embryo, seed or plant that is vegetatively propagated as a sterile population [for gene confinement purposes] .

Those skilled in the art will appreciate that numerous modifications and variations may be made to the above disclosed embodiments without departing from the spirit and scope of the present invention.

What is claimed is:

Claims

1. A method of producing a hybrid plant, the method comprising:

obtaining an immature embryo in a developing ovule or caryopsis obtained from a wide cross between two parental plants, wherein at least one parental plant comprises a selectable marker,

culturing the embryo without removing the maternal tissue in a medium comprising a selection agent,

expanding the resulting callus, and

regenerating a whole hybrid plant.

2. The method of claim 1, wherein the parental plants are flowering plants

(angiosperms).

3. The method of claim 2, wherein the flowering plant is a monocot or a dicot.

4. The method of claim 3, wherein the monocot is an Alismatidae, a Commelinidae, an Arecidae, or a Liliidae.

5. The method of claim 3, wherein the dicot is a Magnoliidae, a Hamamelidae, a

Caryophyllidae, a Dilleniidae, a Rosidae, or an Asteridae.

6. The method of any one of the preceding claims, wherein the method does not

comprise removing the embryo from the maternal tissue.

7. The method of any one of the preceding claims, wherein the selectable marker of the parent plant and the selection agent in the medium are matched.

8. The method of claim 7, wherein the selectable marker of the parent plant is a

resistance gene.

9. The method of claim 8, wherein the resistance gene confers antibiotic or herbicide resistance.

10. The method of any one of claims 1-9, wherein the maternal plant comprises a selectable marker.

11. The method of any one of claims 1-9, wherein the paternal plant comprises a

selectable marker.

12. The method of any one of claims 1-9, wherein both parental plants comprise a

selectable marker.

13. The method of claim 7, wherein the selection agent in the medium is an antibiotic or a herbicide.

14. The method of claim 13, wherein the selection agent in the medium is used at a killing concentration for a wild-type or non-transgenic plants or tissues.

15. The method of any one of the preceding claims, wherein the embryo is rescued from abortion when cultured in the maternal tissue.

16. The method of any one of the preceding claims, wherein the medium further

comprises a mutagen or chromosome doubling agent.

17. The method of claim 16, wherein the chromosome doubling agent is used if progeny is infertile.

18. The method of any one of the preceding claims, wherein at least one of the parental plant further comprises a desired trait.

19. The method of claim 18, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

20. The method of any one of claims 1-19, wherein the wide cross is an inter- or intra- varietial cross.

21. The method of any one of claims 1-19, wherein the wide cross is an inter- or intra- specific cross.

22. The method of any one of claims 1-19, wherein the wide cross is an inter- or intra- generic cross.

23. The method of any one of the preceding claims, wherein the embryo or caryopsis is obtained 1-45 days post pollination of the plants.

24. The method of any one of the preceding claims, wherein the hybrid plant is sexually crossed and/or vegetatively propagated.

25. A method of producing a non-transgenic plant comprising a desired trait, the method comprising:

obtaining an immature embryo in a developing ovule or caryopsis obtained from a wide cross between two parental plants, wherein at least one parental plant comprises a selectable marker and at least one parental plant comprises a desired trait, culturing the embryo without removing the maternal tissue in a medium comprising a selection agent,

expanding the resulting callus,

regenerating the callus into a whole Fl plant comprising the selectable marker and the desired trait,

selecting fertile Fl plant progeny to obtain viable Fl seeds,

germinating the Fl seeds to produce fertile plants comprising the selectable marker and the desired trait, backcrossing or or outcrossing the Fl plants with a non-trans genie or wild- type parental plant,

obtaining F2 plants from germinated seeds obtained from the backcross or outcross,

contacting the F2 plants with a selection agent, and

selecting a non-transgenic F2 plant not comprising the selection marker, thereby producing a non-transgenic plant comprising a desired trait.

26. The method of claim 25, wherein the parental plants are flowering plants

(angiosperms).

27. The method of claim 26, wherein the flowering plant is a monocot or a dicot.

28. The method of claim 27, wherein the monocot is an Alismatidae, a Commelinidae, an Arecidae, or a Liliidae.

29. The method of claim 27, wherein the dicot is a Magnoliidae, a Hamamelidae, a

Caryophyllidae, a Dilleniidae, a Rosidae, or an Asteridae.

30. The method of any one of claims 25-29, wherein the method does not comprise

removing the embryo from the maternal tissue.

31. T The method of any one of claims 25-30, wherein the selectable marker of the parent plant and the selection agent in the medium are matched.

32. The method of claim 31, wherein the selectable marker of the parent plant is a

resistance gene.

33. The method of claim 32, wherein the resistance gene confers antibiotic or herbicide resistance.

34. The method of any one of claims 25-33, wherein the maternal plant comprises a

selectable marker.

35. The method of any one of claims 25-33, wherein the paternal plant comprises a selectable marker.

36. The method of any one of claims 25-33, wherein both parental plants comprise a selectable marker.

37. The method of claim 31, wherein the selection agent in the medium is an antibiotic or a herbicide.

38. The method of claim 37, wherein the selection agent in the medium is used at a killing concentration for a wild-type or non-transgenic plants or tissues.

39. The method of any one of claims 25-38, wherein the embryo is rescued from abortion when cultured in the maternal tissue.

40. The method of any one of claims 25-39, wherein the medium further comprises a mutagen or chromosome doubling agent.

41. The method of claim 40, wherein the chromosome doubling agent is used if progeny is infertile.

42. The method of any one of claims 25-41, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photo synthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

43. The method of any one of claims 25-42, wherein the wide cross is an inter- or intra- varietial cross.

44. The method of any one of claims 25-42, wherein the wide cross is an inter- or intra- specific cross.

45. The method of any one of claims 25-42, wherein the wide cross is an inter- or intra- generic cross.

46. The method of any one of claims 25-45, wherein the embryo or caryopsis is obtained 1-45 days post pollination of the plants.

47. The method of any one of claims 25-46, wherein the hybrid plant is sexually crossed and/or vegetatively propagated.

48. A hybrid bridge intermediate plant, embryos, caryopsis, seeds or progeny thereof obtained by the method of any one of claims 1-24 or 25-47.

49. A plant breeding program to confer non-transgenic plant traits comprising:

obtaining the hybrid bridge intermediate plant of claim 48 or viable progeny thereof, backcrossing or outcrossing the hybrid bridge intermediate plant or progeny thereof with a non-transgenic or wild-type parental plant,

obtaining progeny from the backcross or outcross,

contacting the backcross or outcross progeny with a selection agent, selecting the backcross or outcross progeny that does not comprise the selection marker, wherein the backcross or outcross progeny comprises the desired trait, and

cultivating the selected non-transgenic progeny.

50. The plant breeding program of claim 49, further comprising conventionally breeding the non-transgenic progeny.

51. The plant breeding program of claim 49, further comprising additional trait selection.

52. The plant breeding program of any one of claims 49-51, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photosynthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.

53. Plant progeny, embryos, caryopsis, or seeds obtained from the plant breeding program of any one of claims 49-52.

54. Seeds obtained from the cultivated non-transgenic progeny produced by the breeding program of any one of claims 49-52.

55. Plant progeny, embryos, caryopsis, or seeds obtained from the hybrid plant obtained from the method of any one of claims 1-24.

56. Plant progeny, embryos, caryopsis, or seeds obtained from the Fl, F2, or resulting non-transgenic plant obtained from the method of any one of claims 25-47.

57. A field comprising the hybrid plant produced by the method of any one of claims 1- 24.

58. A field comprising the Fl, F2, or resulting non-transgenic plant obtained from the method of any one of claims 25-47.

59. A field comprising the cultivated hybrid bridge intermediate plant or non-transgenic progeny produced by the breeding program of claim 49-52.

60. The field of any one of claims 57-59, wherein the field comprises 10, 100, 1,000, or 10,000 plants.

61. A plant system comprising: i) plant progeny of the hybrid plant produced by the method of any one of claims 1-24, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant.

62. A plant system comprising: i) plant progeny of the Fl, F2, or resulting non-transgenic plant obtained from the method of any one of claims 25-47, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant.

63. A plant system comprising: i) plant progeny of the hybrid bridge intermediate plant or non-transgenic progeny obtained from the plant breeding program of claim 49-52, ii) soil in which the plant progeny resides, and iii) a container holding the soil and the plant.

64. A plant breeding platform comprising the hybrid bridge intermediate plant of claim 48, seeds, embryos or progeny thereof comprising a desired trait, wherein the hybrid bridge intermediate plant or progeny thereof is backcrossed or outcrossed with a non- transgenic or wild-type parental plant and the backcrossed or outcrossed progeny is selected to obtain non-transgenic progeny plants comprising a desired trait.

65. The plant breeding platform of claim 64, wherein the desired trait is carbon allocation in root or shoot mass, cellulose content, lignin content, sugar content, photo synthetic efficiency, biomass yield, perception of nearest neighboring plant or tiller, photomorphogenic response, photoperiod, floral sterility, regulated dormancy, fertilizer use, pesticide use, stratification, crown size, leaf phenotype, root mass and depth, tillering, stand development, seed set, inflorescence number, height and width, floral development, water use efficiency, cold and freeze tolerance, pest resistance, or any combination thereof.