WO2015099674A1 - Sugarcane regeneration and transformation methods - Google Patents

Abstract

The present invention provides methods for improved transformation and regeneration of monocot plants such as sugarcane, leading to rapid and efficient production of transformed plants. Improved methods for explant preparation, timing of infection, as well as for manipulation of light quality and irradiance, and explant culture, are provided. The provided methods allow for enhanced regeneration of numerous genotypes of sugarcane, and other monocots.

Description

SUGARCANE REGENERATION AND TRANSFORMATION METHODS

FIELD OF THE INVENTION

The present invention generally relates to plant transformation and tissue culture methods. More specifically, the invention relates to methods for efficient regeneration of sugarcane (Saccharum spp., including S. officinarum and S. spontaneum) cells, as well as production of transformed sugarcane tissue using direct Agrobacterium-mQdiatQd DNA delivery into non-precultured leaf whorl explants.

BACKGROUND OF THE INVENTION

Sugarcane is an important food and energy crop which is typically clonally multiplied by stalk planting in furrows. Higher, but still limiting, multiplication ratios are achieved by micropropagation using shoot meristem explants. Rapid regeneration of plants directly from explants presents an effective strategy to avoid or substantially reduce somaclonal variation as it minimizes culture duration and eliminates or minimizes callus formation in culture, since use of callus based systems often causes genetic instability. Improved methods for regeneration of plants also allow for development of efficient genetic transformation systems, leading to crop plants, such as sugarcane, with enhanced agronomic characteristics. However, reliable methods for rapid, high-frequency plant regeneration are limited in monocots, especially for Poaceae. Thus, improved methods for genetic transformation and regeneration of such plants are needed. SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of transforming sugarcane tissue or a cell thereof, comprising: a) inoculating sugarcane tissue or a cell thereof with Agrobacterium comprising a nucleic acid molecule of interest; and b) co-cultivating the Agrobacterium- inoculated sugarcane tissue or a cell thereof to produce a transformed sugarcane tissue or cell thereof comprising the nucleic acid molecule of interest. In certain embodiments the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes. In some embodiments, the nucleic acid molecule of interest is comprised within an expression cassette. In certain embodiments the expression cassette comprises a selectable marker gene. Thus, certain embodiments of the method may comprise culturing the co-cultivated sugarcane tissue or cell thereof in the presence of a selection agent to select the transformed sugarcane tissue or cell thereof. The method may further be defined as comprising the step of: c) regenerating a transgenic sugarcane plant from said tissue or cell thereof, wherein the transgenic plant comprises the nucleic acid molecule of interest. In some embodiments regenerated shoots are produced from the transformed tissue or cell within about 20-50 days from the transformation of the cell. In these or other embodiments, a rooted plant may be produced from the regenerated shoots within about 27-60 days. In particular embodiments step c) is carried out without producing a callus from said tissue or cell thereof.

In certain embodiments, the sugarcane tissue that is transformed and/or regenerated comprises a transverse midrib explant, a transverse leaf whorl explant, or a midrib longitudinal explant. In particular embodiments the sugarcane tissue comprises a transverse leaf whorl explant. In other embodiments of the invention, an explant is inoculated with Agrobacterium shortly or immediately after the explant is prepared, such as by partial or complete excision or starting plant tissue.

In other embodiments, a plant is regenerated in media comprising a cytokinin and an auxin. In some embodiments the cytokinin is BAP or kinetin, while the auxin is 2,4-D, NAA or IBA. Thus, in some embodiments the media comprises about 0.1 mg/L to about 1.0 mg/L BAP.

Another aspect of the invention provides a method of transforming plant tissue comprising: a) inoculating plant tissue or a cell thereof with Agrobacterium comprising a nucleotide sequence of interest; b) co-cultivating the Agrobacterium-moculated plant tissue or a cell thereof to produce a transformed plant tissue or cell thereof; and c) regenerating a transformed plant from the transformed tissue or cell thereof; wherein the plant is regenerated without an intervening callus phase of growth, and wherein regeneration is performed by growth of plant cells in light comprising enhanced red wavelength and reduced blue wavelength. Thus, in some embodiments a regenerated plant is produced within about 30-60 days of the start of co- cultivating. In one embodiment the plant is a monocot; in a particular embodiment the plant is sugarcane (Sacchcirum spp).

In some embodiments the light is provided by a Gro-lux® lamp; in certain embodiments the light has an irradiance of about 0.4- 30 μΕ m^"2 s^"1; in particular embodiments the light has an

-2 -1

irradiance of about 30 μΕ m^" s^" .

In other embodiments of the method, gradually increased light conditions are utilized during regeneration. Thus, in certain embodiments, the gradually increased light conditions comprise: 0.4 μΕ m^"2 s^"1 for about 4 days, followed by 2 μΕ m^"2 s^"1 for about 4 days, followed by 10 μΕ m^"2 s^_1for about 4 days, followed by 30 μΕ m^"2 s^"1 until a regenerated plant is obtained.

The system of the present invention provides a rapid, yet less labor-intensive, efficient generation of transgenic sugarcane plants from non-induced leaf whorl explants. Thus, previously "recalcitrant" genotypes that have shown limited embryogenesis response and transformability via a callus-based approach may now be directly used as transformation targets.

DESCRIPTION OF FIGURES FIG. 1: Box plot indicating the mean and the quartiles of number of shoots regenerated per plate when treated by light provided by different sources. A trend was seen to obtain more shoots per plate of explants maintained under Gro-lux®, compared to cool white fluorescent.

FIG. 2: Box plot indicating the mean and the quartiles of number of shoots regenerated per plate as per light and BAP medium composition treatment.

FIG. 3 : Box plot indicating the mean and the quartiles of number of shoots regenerated per plate as function of the light provided by different light sources. A 2 fold increase in the number of shoots regenerated under the light provided by Gro-lux® lamps, compared to the standard light provided by cool white fluorescent lamp, was seen. This increase in the number of shoots is statically significant at 95% confidence level.

FIG. 4: Transient GFP expression in sugarcane leaf whorl explants exposed to two tested centrifugation treatments, and with or without acetosyringone in the co-culture medium. Higher speed centrifugation (650g) and addition of acetosyringone have a beneficial impact on the number of foci expressing GFP, which reaches 12% on a per explant piece basis. These data represent the total number of pieces of explant expressing GFP/total number of pieces of explant x 100.

FIG. 5: Schematic representation of four different infection timings.

FIG. 6: GUS transient days after infection on the different days of preculture (infection timing). Around 55% GUS transient expression was seen 3 days after infection for non-induced leaf whorl explants. However, no GUS activity was observed on induced treatments, that is, on the explant submitted to pre-culture with auxin pulse. FIG. 7: GFP transient expression (%) 7 days after infection. About 90% of non- precultured transverse leaf whorl explants showed GFP transient expression. However, for infection after auxin pulse (3, 5 or 7 days of preculture) only a low level of GFP transient expression was seen.

FIG. 8: Regenerable structures expressing GFP were seen by 15 days after infection in all 5 genotypes tested, following infection of non-precultured leaf whorl explants via Agrobacterium-mediated delivery.

FIG. 9: Comparison of quality (low copy number and no backbone) events, between the previous callus-based methods and the present direct transformation and regeneration systems ("DR"). A higher frequency of events with low copy (1 - 2 copies) was observed with the DR system when compared with the previous callus system. From the low copy events, the backbone assay resulted in similar proportion of events lacking vector backbone sequence.

FIG. 10: Schematic representation of three different infection timings for S. spontaneum and S. officinarium: (1) infection of non-induced leaf whorl explants, followed by co-culture with auxin pulse (CC-03 medium) for 3 days, followed by auxin pulse and selection (SELA 33) for 4 days and removal to regeneration and selection medium (SEL 33) for the remaining time of the experiment; (2) infection after 3 days on the auxin pulse (PULA1 medium) followed by co- culture with auxin pulse (CC-03 medium) for 3 days, followed by auxin pulse and selection (SELA 33) for 1 day and removal to regeneration medium and selection medium (SEL 33) for the remaining time of the experiment; (3) infection after 6 days on the auxin pulse (PULA1 medium), followed by co-culture with auxin pulse (CC-03 medium) for 1 day and co-culture with regeneration (CC-02 medium) for 2 days, and removal to regeneration and selection medium (SEL 33) for the remaining time of the experiment.

FIG. 11: GFP transient expression (%) 7 days after infection in 6 different genotypes of Saccharum spontaneum. Infection of non-precultured leaf whorl explants demonstrated acceptable GFP transient expression. In contrast, the infection of explants after auxin pulse (3 or 6 days of preculture) showed a little or no GFP transient expression.

FIG. 12: GFP transient expression (%) 7 days after infection in 6 different genotypes of Saccharum spontaneum. About 57 to 83% of non-precultured leaf whorl explants showed GFP transient expression. However, for infection after auxin pulse (3 or 6 days of pre-culture) only a low level of GFP transient expression was seen.

FIG. 13: Transient expression of GFP in regenerable structures from three LED treatments compared to the explants under Gro-lux®. Regenerable structures expressing GFP were obtained in all treatments, 21 days after infection. The graphic shows the number of embryos expressing GFP in each treatment. The LED 1 : 1 (Red:Blue) treatment group yielded 28 transformed embryos, in comparison to the 2 embryos expressing GFP under Gro-lux®. The LED 2: 1 and 4: 1 (Red:Blue) treatments presented at least twice the number of embryos expressing GFP than the embryos under Gro-lux®.

FIG. 14: Transient expression of GFP in regenerable structures for four LED treatments compared to the explants under Gro-lux®. The graphic shows the number of embryos expressing GFP in each treatment. Under different LED conditions, an increase of at least 2 or 2.5 fold was observed in terms of somatic embryo expressing GFP when compared with Gro- lux® as standard protocol.

DETAILED DESCRIPTION

The present invention provides improved methods and compositions relating to plant tissue culture, including transformation and regeneration of plants such as sugarcane, other Poaceae, and other monocots, among other plants. Methods are provided to obtain high frequency rapid regeneration of plants such as sugarcane by manipulating the light spectrum provided to cultured explants. Thus, transformation of plants such as sugarcane by co- cultivation with a bacterial strain comprising a nucleotide molecule of interest, may be coupled with efficient, rapid, and genotype-independent methods for regeneration, allowing for production of transgenic plants.

Among other variables, light quality and intensity plays a major role in plant development, and stress during tissue culture may lead to the appearance of red or brown pigmentation of cultured cells, which correlates with delayed or reduced regeneration of plant tissue. As described in the Examples below, use of Gro-lux® lamps, LED, or other light sources which are enriched in the red spectrum in comparison to cool white fluorescent lamps, were found to reduce the amount of such pigment formation and to allow for an increase of 2 fold or more in the number of plantlets regenerated, for instance by a direct sugarcane regeneration system without an intervening callus phase of growth. The resulting plantlets also display higher vigor, and develop roots faster than conventionally grown regenerating plantlets. The result unexpectedly contrasts with results obtained by Weiss and Jaffe (PI. Physiol. 22: 171 - 176, 1969) and Seibert (In Vitro 8:435, 1973), who found that the critical portion of the light spectrum for shoot induction in tobacco was the blue region. Further, the present invention demonstrates for the first time that use of a light source with enhanced emission in red wavelengths is beneficial for high efficiency shoot regeneration and rooting, such as in sugarcane. In certain embodiments of the invention, a gradual increase in light intensity may also be employed to further improve regeneration efficiency and vigor (e.g. growth and rooting rate) of resulting plants. Exemplary parameters for such gradual light intensity increase include,

-2 -1 -2 -1 -2 -1

for instance, 4 days in 2 μΕ m^" s^" ; 4 days in 10 μΕ m^" s^" and then 30 μΕ m^" s^" for the rest of time of the explants in culture; or 3 days in 0.4 μΕ m^"2 s^"1; 3 days in 2 μΕ m^"2 s^"1; 9 days in 10 μΕ m^"2 s^"1; 4 days in 20 μΕ m^"2 s^"1; and then 30 μΕ m^"2 s^"1 for the remaining time of culture. Such lighting parameters may also be varied as needed. Explant Preparation

Separately or in conjunction with manipulating the light source utilized during tissue culture, improved methods for explant preparation are also provided. For instance, midrib transverse segments, midrib longitudinal segments, or transverse leaf whorl segments of, for instance, sugarcane tissue may be prepared as disclosed to allow for efficient transformation and regeneration, as well as multiplication, of tissue.

In certain embodiments, explants such as from leaf whorl transverse segments may be prepared to minimize production of red pigment components. Further, use of a wide spectrum light source such as a Gro-lux® lamp, relatively enriched in red spectrum wavelength, promotes shoot formation, and is suitable for the regeneration process. The use of properly prepared explants along with control of light intensity and quality during regeneration steps allows for a highly efficient regeneration system. Thus, minimizing formation of dark red pigments on the surface of the original explant at the regeneration step is beneficial. Enhancements in explant preparation, use of thermotherapy, fungicide application on the buds that will originate the top stalks, and use of antioxidants can contribute to reduce browning and improve regeneration as well. Shoots regenerated under a wide spectrum lamp displayed an impressively superior vigor. Regeneration

In further embodiments, a direct regeneration system may be utilized, to avoid or minimize the length of time spent in a callus phase of growth. Rapid regeneration of plants directly from explants presents an effective strategy to avoid or substantially reduce somaclonal variation, as it minimizes culture duration and eliminates or minimizes callus formation in culture. Such conditions also permit the advantage of regeneration of non-chimeric events. Thus, cytokinin and auxin may be used as described, as well as an "auxin pulse." However, reliable methods for rapid, high-frequency direct plant regeneration are limited in monocots, especially for Poaceae (Lakshmanan, In Vitro Cell Devel. Biol. 42:201-205, 2006). In general, the use of callus based systems often cause genetic instability and lead to somaclonal variation (Larkin and Scowcroft, TAG 60: 197-214, 1981; Lourens and Martin, Plant Cell Tiss. Org. Cult. 89:49-54, 1987; Zucchi et al. Genetics & Mol. Biol. 25:91-96, 2002; Gill et al, Plant Cell Tiss. Org. Cult. 84:227-231, 2006). Thus a lengthy intervening callus phase of growth is often not desirable during micropropagation, or when genetic transformation is attempted. Sugarcane callus cultures show a considerable variation from cell to cell and among differentiated plantlets (Sengar et al., Plant Sciences Feed 1 : 101-11 1, 2011). Several factors such as explant source, time of culture, number of subcultures, applied phytohormone type and amount, genotype, medium composition, level of ploidy and genetic mosaicism are capable of inducing in vitro variability (Silvarolla, J. Brazil Assoc. Adv. Sci. 44:329-335, 1992; Snyman et al. In Vitro Cell Devel. Biol. Plant 47:234-249, 2011).

Minimizing the production of, and period of time in which tissue undergoes, callus phase growth may also be beneficial for reducing, for instance, somaclonal variation. Thus, the present invention allows for rapid and efficient production of superior quality plants by utilizing tissue culture methods in which little or no callus growth occurs. A tissue culture regime which avoids callus growth, allowing for direct regeneration via embryogenesis and/or organogenesis, may also be employed.

Direct plant regeneration, for instance in sugarcane, occurs by two major routes: direct organogenesis, when the explants are exposed to at least one cytokinin and an auxin (Grisham and Bourg, J. Amer. Assoc. Sugarcane Technol. 9:97-102, 1989; Burner and Grisham, Crop Sci. 35:875-880, 1995; Gill et al. 2006, ibid; Lakshmanan et al. 2006, ibid); or direct regeneration through somatic embryogenesis, when the explants are exposed to potent auxins, like 2,4-D (Heinz and Mee, Crop Sci. 9:346-348, 1969; Nadar et al, Crop Sci. 18:210-216, 1978; Ho and Vasil, Protoplasma 118: 169-180, 1983; Lee, Plant Cell Tiss. Org. Cult. 10:47-55, 1987; Snyman et al., Acta Hort. 560, 2001; Franklin et al., Plant Growth 50: 11 1-119, 2006; Behera and Sahoo, Nature and Science 7: 1-10, 2009). Irrespective of the direct regeneration pathway, direct regeneration can eliminate or substantially reduce the occurrence of somaclonal variation in sugarcane (Burner and Grisham, Crop Sci. 35:875-880,1995; Lakshmanan et al. 2006, ibid). Normally, a direct regeneration pathway without callus formation is more common in dicots, although there are reports of direct regeneration in a few monocots, including sugarcane. In Saccharum, direct regeneration can be achieved from leaf tissue (Irvine and Benda, Plant Cell Tiss. Org. Cult. 5: 101-106, 1985; Grisham and Bourg, J. Amer. Assoc. Sugarcane Technol. 9:97- 102, 1989; Gambley et al., Plant Cell Rep. 12:343-346, 1993) from cell suspension (Aftab and Iqbal, Plant Cell. Tiss. Org. Cult. 56: 155-162, 1999), from immature inflorescence tissues (Desai et al., Curr. Sci. 87:764-768, 2004), immature leaf thin cell layers (Lakshmanan et al., 2006, ibid), immature leaf roll (Snyman et al., 2001 , ibid), immature leaf disc explants with or without pre-emergent inflorescence (Snyman et al., 2006, ibid), leaf segments (Gill et al., 2006, ibid) and leaf midrib explants (Franklin et al., 2006, ibid).

In all the basic work done for direct regeneration, including attempts to employ direct plant regeneration for bulk up purposes or for genetically modification of sugarcane, a relatively low frequency of plant regeneration is typically obtained in a limited number of genotypes. The present disclosure provides improved methods for direct regeneration, including novel methods of explant preparation and methods of using light provided by Gro-lux® lamps with an enhanced wavelength emission in the red parts of the spectrum (SYLVANIA Technical Information Bulletin Light and Plants. Standard and Wide Spectrum SYLVANIA Gro-lux® Fluorescent Lamps). Gro-lux® lamps provide a spectrum enriched in the red wavelengths and relatively poor in blue wavelengths, compared to cool white fluorescent lamps.

Optionally, gradually increasing lighting conditions may be employed as disclosed. Moreover, a genotype-independent multiplication system for the successful rapid regeneration of a large number of, for instance, sugarcane genotypes or of other monocots, can be the basis for the rapid and efficient production of genetically transformed plants. The disclosed methods may also be applied, for instance, to sugarcane clonal micropropagation and/or used as a regeneration system for rapidly obtaining genetically transformed sugarcane events with superior agronomic quality.

Due to the light wavelength spectrum provided by wide spectrum light sources, for instance by Gro-lux® or LED lamps, in comparison to cool white fluorescent lamps, the inventors were able to produce more shoots per plate, which are in turn of an unexpectedly superior vigor. Plants need light not only for photosynthesis but also need other light spectrum which are sensed for morphogenesis. The light requirements for photomorphogenesis are in the near-ultraviolet (300-380 nm), blue (430-490 nm), red (640-700 nm), and far-red (700-760 nm) regions of the light spectrum (Hart J.W., "Light and Plant Growth " Unwin Hyman, London, 1988). Light sources such as Gro-lux® lamps provide an increased proportion of light wavelength in the red spectrum and a lower proportion of light at blue spectrum (Technical Information Bulletin, Standard and Wide Spectrum SYLVANIA Gro-lux® Fluorescent Lamps). Thus, SYLVANIA Gro-lux® Standard and/or Wide Spectrum Lamps, or physiologically similar light sources, may be utilized. By "enhanced red wavelength and reduced blue wavelength" or "relatively enriched in red spectrum wavelength" is meant use of a light source which more closely mimics the spectral characteristics of daylight, particularly at wavelengths in the ranges of 300-380 nm, 430-490 nm, 640-700 nm, and 700-760 nm, as compared with a typical cool white fluorescent light source.

Among the different red components that a cell can produce, the most important are the anthocyanins, which are in turn induced by blue light spectrum. Blue light has been shown to regulate the expression of a number of genes (Kaufman, PI. Physiol. 102:333-337, 1993; Short and Briggs, Ann. Rev. Physiol. Plant Mol. Biol. 45: 143-171, 1994) including the gene encoding chalcone synthase (CHS) catalyzing the first committed step in the flavonoid biosynthetic pathway leading to anthocyanin (Batchauer et al., pp. 559-599 in Kendrick, RE and Kronenberg, GHM, eds. "Photomorphogenesis in Plants, 2^nd Ed." Kluwer, Dordrecht, 1994; Feinbaum et al., MGG 226:449-456, 1991). This is consistent with the reduced level of red compounds found when explants were grown under Gro-lux® lamps, since the light provided by the cool white fluorescent comprises relatively more blue light and is relatively deficient in red spectrum (Technical Information Bulletin, Standard and Wide Spectrum SYLVANIA Gro-lux® Fluorescent Lamps). Light-emitting diode (LED) light sources may also be employed. Light source (wavelength and intensity) may further interact with provided cytokinin levels in influencing regeneration and vigor (Su and Howel, PI. Physiol. 108: 1423-1430, 1995; Nemhauser and Chory, pp. 1-12 in: Somerville C, Meyerowitz E, eds. "The Arabidopsis Book" American Society of Plant Biologists 2002.

The effect of light provided by cool white fluorescent lamps, to quickly induce the formation of dark/red pigments on the explants tested, may be due to a response to light stress and/or due to this light source to provide a relative enriched blue spectrum light. Blue light is one of the most effective wavelengths regulating anthocyanin biosynthesis, and the cryptochrome acts as the blue light photoreceptor for this response (Ahmad et al. Plant J. 8:653- 658, 1995; Meng and Wang, J. Hortsci Biotech. 79: 131-137, 2004; Wang and Wang, J. Trop. Subtrop. Bot. 12:252-256, 2004). Chen et al. (J. Integr. Plant Biol. 48:420-425, 2006) showed that blue light strongly induced anthocyanin accumulation in WT seedlings of Arabidopsis thaliana, and that the anthocyanin amount in seedlings treated with blue light was 12- fold greater than that in the seedlings raised in darkness.

Anthocyanins generally accumulate in tissues exposed to high irradiance; anthocyanin accumulation requires light and generally coincides with periods of high irradiance and increased potential for photo-oxidative damage. The anthocyanin accumulation functions in photoprotection, and light exposure is a prerequisite for significant anthocyanin synthesis in vegetative tissues in response to both environmental (Franceschi & Grimes, PNAS 88:6745- 6749, 1991 ; Krol et al., Can. J. Bot. 73: 11 19-1 127, 1995) and developmental factors (Mancinelli, pp. 640-666 in Shropshire Jr W, Mohr H, eds. Photo morphogenesis. Berlin, Germany: Springer- Verlag, 1983). Generally, induction of anthocyanin synthesis requires high light intensities and anthocyanin levels in plants and in individual leaves varies in relation to the light exposure levels (Mancinelli, ibid). Iida et al., (Plant J. 24: 191-203, 2000) described a gene apparently involved in acclimation to visible light stress. This gene was rapidly induced in proportion to intensity and duration of irradiation stress. Over-expression of the gene resulted in constitutive high-light tolerance, anthocyanin accumulation and adaptive phenotypic changes, such as thicker leaves, usually associated with acclimation to high light, suggesting that anthocyanin accumulation is part of the general plant response to light stress.

Sugarcane Transformation

Agrobacterium-mediated gene transfer is a widely applicable system for introducing genes into plant cells, including sugarcane cells (e.g. Arencibia et al., Transgenic Res. 7:213- 222, 1998). However, although in the past decade considerable progress has been made in understanding and manipulating cell biological approaches regarding Agrobacterium-mediated transformation for genetic manipulation of sugarcane, provision of an efficient genotype flexible transformation protocol has remained elusive, because, for instance, cell death from Agrobacterium-mdweed hypersensitive reactions on cultured sugarcane tissues typically results in no or very low transformation from recalcitrant genotypes.

In the present methods, after effecting delivery of exogenous DNA to recipient cells, transformed cells are identified for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may employ a selectable or screenable marker gene with a transformation vector. In this case, one would then typically assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait. To express the foreign nucleic acid molecules encoding a gene of interest ("GOI") in sugarcane plants, the GOI is preferably linked in an expression cassette with regulatory DNA sequences which ensure transcription in plant cells. These include, for instance, a promoter, an intron, and/or a terminator. In principle, any promoter which is active in sugarcane plants/plant cells is suitable for the expression. The promoter may be chosen such that expression of a GOI occurs constitutively, or only in a particular tissue, at a particular timepoint in the plant's development, or at a point in time which is determined by external factors. The promoter can be homologous or heterologous, both with regard to the plant and with regard to the foreign nucleic acid molecule.

The tissue (e.g. as defined by age, by explant type, or by genotype, among other variables) employed as target for Agrobacterium-mediated transformation may affect transformation and regeneration efficiency. As disclosed herein, numerous genotypes of sugarcane were successfully regenerated.

The wide applicability of the transformation protocol was demonstrated on diverse sugarcane genotypes, including the Sacchcirum officinarium and Saccharum spontaneum species. Commercial hybrids are principally derived from S. officinarium and S. spontaneum.

Thus, the invention provides a simple, rapid and less labor-intensive system for sugarcane transformation. The method comprises: a) introduction of a nucleotide sequence of interest directly on non-precultured leaf whorl explants via Agrobacterium-mediated delivery; b) co- culture of said Agrobacterium-mocxAated explants on a gelled medium promoting a "gentle desiccation" process, wherein such medium contains auxin at a level sufficient to start the direct embryogenesis process; c) culturing the explants in at least a second and third culture medium that support selection and regeneration of transgenic sugarcane plants. The regenerated transformed sugarcane plants may be produced, for instance, within about 5 to 15 weeks, within about 8 to 13 weeks, or within about 10 to 13 weeks of inoculating the explants with Agrobacterium, depending upon on the genotype, or any subrange of the above.

"Polarity" as used here in is defined as the original (e.g. in planta) direction of meristem growth. While transformation is observed regardless of polarity, the transformed shoots preferably grow from the periphery of leaf whorl explants independently of the direction of meristem growth (e.g. non apical side). Further, co-cultivation on a gelled agent medium (such as agarose), is useful during early stages of glyphosate selection and for tissue survival. Co- cultivation on the gelled medium containing 2,4-D may enhance transformation as well as start the initial regeneration pathway thru somatic embryogenesis. Light intensity and quality during the selection and regeneration steps are important for high transformation efficiency. In particular, Gro-lux® lamps (or lamps with similar emission characteristics), gradually increasing light intensity, and use of LED light sources have been found to improve the number of transformants ("events"). Importantly, this system provides a rapid, yet less labor-intensive, efficient methods for obtaining transgenic sugarcane plants from non-induced leaf whorl explants and thereby avoids or substantially reduces the likelihood of somaclonal variation, as it eliminates intermediate callus formation in culture, and minimizes total in vitro culture duration.

In the present invention, multiple exemplary, distinct, economically relevant, and previously recalcitrant sugarcane genotypes were successfully transformed, thus overcoming a major hurdle in genetic manipulation of sugarcane. Most protocols in the literature suffer from either low transformability and/or genotype specificity. Thus, importantly, previously "recalcitrant" genotypes that have shown limited embryogenesis response and transformability via a callus-based approach may now be directly used as transformation targets. Methods of this invention can thus be applied to different sugarcane genotypes and represent a considerable improvement in the transformation art for this monocot. Sugarcane has been successfully transformed using either direct or indirect somatic embryogenesis routes (Bower and Birch, Plant J. 2:409-416, 1992; Falco et al. Plant Cell Rep 19: 1188-1 194, 2000; Snyman et al. Plant Cell Rep. 25: 1016-1023, 2006; Van Der Vyver, Sugar Tech 12:21-25, 2010; Kalunke et al., Sugar Tech 11 : 355-359, 2009). However, information on Agrobacterium-medi&ted sugarcane transformation is limited, with some reports of successful transformation and plant regeneration using either embryogenic callus or axillary bud as explants (reviewed by Brumbley et al., Sugarcane. In: Kole C, Hall TC (eds) Compendium of transgenic crop plants: transgenic sugar, tuber and fiber crops. Blackwell, Oxford, pp 1-58, 2008). Arencibia et al. {Transgenic Res 7:213-222, 1998) focused on type and age of sugarcane explants while Enriquez-Obregon et al. (Planta 206:20-27, 1998) targeted pre-conditioned meristematic sections to produce transgenic plants. Such methods required additional steps apart from callus production and inoculation for successful transformation. Previously published protocols suffer from one or the other constraints such as no or low transformability and genotype specificity ( e.g. Elliott et al., Aust J Plant Physiol 25:739-743, 1998; Kalunke et al., 2009, ibid; Taparia et al., In Vitro Cell.Dev.Biol. - Plant 48: 15-22, 2012; WO 201 1/163292 Al ; WO 02/37951 Al).

Thus, this is the first time that GFP stable expression was achieved in regenerable structures for 5 different commercial genotypes, for S. officinarium and S. spontaneum, as well as stable events for 4 different genotypes, through Agrobacterium infection of non-induced leaf whorl explants. The only tested genotype for which stably transformed plants were not produced was line 4 of FIG. 8. However, this was apparently due to media contamination, since approximately 90% of explants for this line were lost to contamination. However, GFP stable expression in regenerable structures in this genotype was seen as well. A transformation frequency (TF%) of about 1.6% to 4% for Line 3 of FIG. 8; 2.8% for line 1 of FIG. 8; and about 2.8% for line 2 of FIG. 8 was observed. For reference variety SP803280, a TF of 1.2-3.5% was observed in several experiments. Surprisingly, the transformation system coupled with direct regeneration yielded an increased proportion of events with low transgene copy number (1-2 copies), and no vector backbone sequences, as compared with the number of "quality" events produced with a callus-based regeneration system.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.

All references cited herein are hereby expressly incorporated herein by reference. EXAMPLES Example 1: Explant Preparation

A. Midrib transverse segment preparation.

Explant preparation: Sugarcane top stalks were obtained from 6.5 month old field grown plants of reference variety SP803280. Outer whorls of mature leaves were removed until a spindle of 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each time. After removing the outer two to three leaves, a cylinder was obtained with a diameter of around 0.5 cm. Immediately above the auricle, the outer two to three leaves were removed until the midrib became visible above the auricle region. The cylinders were then chopped transversely to obtain isolated midrib transversal segment. Globular structures were identified on the top of the midrib original surface after the explants were in auxin pulse medium for 8 days in dark conditions, with midribs showing dark red pigment development on the globular structures and greening of the recognizable parts of the midrib original explant, by about 3 days after the explants were transferred to light conditions at 30 μΕ in regeneration medium. Direct regeneration of shoots from midrib explants was observed, with shoot initial direct regeneration from midrib segments occurring by around 20 days after transferring the explants to regeneration medium in regular light conditions. Shoot regeneration continued and, by around 35 days after transferring the explants to regeneration medium in regular light conditions, a high frequency of direct leaf regeneration was observed for this explant type.

Auxin pulse: Midrib transverse sections were inoculated aseptically on MS medium (Murashige & Skoog, Physiol. Plant. 15:473-497, 1962) containing 2.5% (w/v) sucrose; 3 mg/L 2,4-D; 1% coconut water; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated under dark conditions at 26±2°C for 8 days. Each culture plate contained 50 ml medium and was inoculated with 12 explants. The plates were evaluated every other day for contamination and morphogenesis development assessment.

Regeneration: To induce shoot formation, the explants were transferred to MS medium containing 3.0% (w/v) sucrose; 1.0 mg/L BAP; 0.1 mg/L NAA; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

Light conditions: the explants were maintained on regeneration medium under a 16 h

-2 -1

photoperiod at 26±2°C and incubated at 30 μΕ m^" s^" supplied by cool white fluorescent lamps (Phillips).

Formation of globular structures on the original surface of the midrib explants was observed by 8 days after the introduction onto auxin pulse medium. However, as soon as 3 days after the explants were transferred from the auxin pulse to the regeneration medium in regular light conditions (30 μΕ cool white), globular structures were observed that turned dark red to brown, while midrib original explant tissue turned green. This demonstrated growth stress due to the irradiance employed on the explants. Initial formation of green sectors and initial shoot regeneration in between the dark pigmented areas was observed by about 20 days after transferring the explants to regeneration conditions. There is a negative correlation between dark red pigmented areas and the ability of such areas to regenerate into shoots. Regenerated shoots were seem by 35 days after transferring explants to regeneration conditions. A significant portion of the observed regenerated tissue comprised leaves being regenerated directly from the original surface of the midrib. Overall, few shoots were observed being formed per explant, associated with the high level of dark red pigment development that occurred as soon as the explants were transferred to regeneration medium in regular light conditions. The observation that regular light intensity, like 30 μΕ, quickly induced the appearance of dark red pigments in explants in regeneration medium indicated that controlling light intensity and quality might decrease pigment appearance, leading to an increased number of regenerated shoots per midrib explant.

B. Midrib longitudinal segment explant preparation.

Explant preparation: Sugarcane top stalks were obtained from 6.5 old field grown plants of reference variety SP803280. Outer leaves were removed to obtain a spindle of 20 cm length and 1.5 cm diameter. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three whorls of leaves, a cylinder was obtained with a diameter of around 0.5 cm. Immediately above the auricle, the outer two to three leaves were removed until the midrib became visible above the auricle region. Midrib longitudinal segments were obtained by removing the leaf blade and these were used as an explant. The cylinders were chopped longitudinally to obtain 6 segments with around 2.0 cm long.

Midrib segments from different leaf positions were utilized. The "leaf +3" explant represents midrib segments from the innermost leaf and the "leaf +1" represents the outermost leaf, with "leaf +2" in between. No developed midrib was identified inner to the midrib of the leaf +3 position tissue. Midrib longitudinal explants were sliced into six pieces (segment positions 1-6), with position 1 proximal to the auricle. Midrib longitudinal segments explants of about 2.0 cm in length were placed in contact with medium, alternating the explant upside up and upside down position. After 8 days on auxin pulse medium, the leaf + 2 segments showed pronounced browning at cut ends of segments 4 thru 6 and globular structures are the cut ends of segments 1 thru 3. In general, milder browning was observed in midrib segments of "leaf + 2" than "leaf + 1" or "leaf + 3". Globular structures formed along the longitudinal midrib explant segments just at the contact edge of the explant with the medium and at the cut ends, when the explants were introduced upside up or upside down. Direct regeneration of shoots on the midrib longitudinal segments was seen by around 20-30 days in regeneration medium. Relatively poor regeneration was seen after 30 days on regeneration medium for most explants. Intact regions (without globular regenerating structures) of the original longitudinal segments were also observed, indicating relatively poor regeneration.

Auxin pulse: Midrib longitudinal sectioned explants were inoculated aseptically on MS medium containing a combination of 3.0% (w/v) sucrose; 3 mg/L 2,4-D; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated under dark conditions at 26±2°C for 8 days. Each culture plate contained 50 ml medium and was inoculated with 6 explants. The plates were evaluated every other day for contamination and morphogenesis development assessment.

Regeneration medium: To induce shoot formation, the culture was transferred to MS medium containing 3.0% (w/v) sucrose; l .Omg/L BAP or 0.1 mg/L BAP; 0.1 mg/L NAA; without or with 150 mg/L citric acid; MS salts with vitamins; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

Light conditions: Explants were maintained on regeneration medium under a 16 h photoperiod at 26±2°C in gradually increased light intensity supplied by cool white fluorescent lamps (Phillips): 3 days in 0.4 μΕ m^"2 s^"1; 3 days in 2 μΕ m^"2 s^"1; 9 days in 10 μΕ m^"2 s^"1; 4 days in

20 μΕ m -^"2 s-^"1 ; and 30 μΕ m -^"2 s-^"1 for the remaining time of the experiment.

Certain explant preparation variables were examined, including position of midrib (midrib from leaf +3 versus leaf +2); addition (or not) of citric acid (150 mg/L) in regeneration medium; and 2 levels of BAP in regeneration medium (0.1 mg/L versus 1.0 mg/L). Possible polarity effects on midrib longitudinal segments were also examined, e.g., introducing the explants upside up and upside down to the tissue culture medium. Position 1 is proximal to the auricle.

Pronounced browning was observed on explants prepared from leaf +2 compared with leaf +3, as well as slower or reduced regeneration response, or even no globular structures being formed from explants prepared from such leaf (data not shown). Globular structures were visible by 5 days after application of the auxin pulse. Such structures were formed along the midrib segment in both positions, e.g., upside up and upside down. These globular structures were formed on the explant just when it came in contact with the medium and at explant ends.

Segments 4, 5 and 6 showed consistently more browning in comparison to segments 1 , 2 and 3, possibly as these explants are more differentiated and or have cellular components that resulted in browning of such explants, mainly at explant ends. Associated with this browning, globular structures were not observed in most segments of positions 4, 5 and 6, and no shoots regenerated once explants were put in regeneration medium. Also, regarding the light regime, segments 4 thru 6 produced more red pigments. The addition of citric acid in regeneration medium contributed to reduced oxidation/browning at explant ends inoculated into medium containing such antioxidant (data not shown).

The explants started to show a consistent regeneration after 30 days in regeneration medium. The regeneration efficiency observed was relatively poor, since few shoots were formed per explant. The results demonstrated that leaf +3 segments produced explants less subject to browning and that segments 1, 2 and 3 yielded more regenerated shoots from the cut ends. Explants of positions 4 thru 6 did not typically show as good regeneration potential associated with browning at the cut ends. It was also demonstrated that explants can be inoculated upside up or upside down, with no apparent interference to the regeneration potential. In this experiment data were clearly obtained indicating that light regime is important for avoiding extensive red pigment accumulation, which in turn is negatively correlated with regeneration on the explant tested. Overall, preparation of midrib longitudinal segments is relatively laborious and yields a relatively smaller number of shoots compared to other methods of explant preparation.

C. Transverse leaf whorl segment explant preparation.

Explant preparation: Sugarcane top stalks were obtained from 7 month old field grown plants of reference variety SP803280. Outer whorls of mature leaves were removed until a spindle of about 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three whorls of leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick. Cylinders of young top stalks were cut and removed just below the shoot tip.

Auxin pulse: Transverse thin slices were inoculated aseptically on MS medium containing a combination of 3.0% (w/v) sucrose; 3 mg/L 2,4-D; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. Cultures were incubated under dark conditions at 26±2°C for until 8 days. Each culture plate containing 50 ml medium was inoculated with 12 thin slices of explants. The plates were evaluated every other day for contamination and morphogenesis development assessment. Direct regeneration of shoots from transverse leaf whorl section explants was observed after about 8 days in auxin pulse media, with a rough surface due to appearance of globular structures at explant surface.

Regeneration medium: To induce shoot formation, the culture was transferred to MS medium containing 3.0% (w/v) sucrose; 1.0 mg/L BAP or 0.1 mg/L BAP ; 0.1 mg/L NAA; without or with 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

Light conditions: Explants were maintained on regeneration medium under a 16 h photoperiod at 26±2°C in gradually increased light intensity, supplied by cool white fluorescent lamps (Phillips): 4 days in 2 μΕ m^"2 s^"1; 4 days in 10 μΕ m^"2 s^"1 and then 30 μΕ m^"2 s^"1 the rest of time of the explants in culture.

Transverse leaf whorl section explants formed shoots by 26 days after initial inoculation. Again, an apparent negative correlation of red pigmented areas with regeneration of shoots was seen, although the formation of dark red pigments was lower with this explant compared to the isolated midrib transverse and longitidinal segments. This may be due to a nursery effect, e.g., isolated explants produce more dark red pigments than when explants are joined, as in the transverse leaf whorl section shown here. This explant was formed by several leaves, each containing an immature leaf blade and some leaves containing immature leaf midrib sections. Thus, in one embodiment, it may be beneficial that explants such as transverse leaf whorl sections be maintained intact during preparation, at cutting and initial manipulation, since in this configuration the subsequent production of dark red pigments is reduced, and thus a better yield of regenerated shoots is obtained, even if gradually increased light is not applied to the explants. Additionally, if a direct regeneration protocol is employed after transformation by Agrobacterium, downstream manipulation of the cut explant to infect (co-cultivate) the tissue may separate the different immature leaf segments that make up a transverse leaf whorl section, making gradually increased light conditions useful for achieving good regeneration of shoots.

The transverse leaf whorl explant, which is less labor intensive for obtaining explant material, allowed for relatively more shoots per explant plate than the other two treated explant types tested, e.g., midrib transverse and midrib longitudinal segments. Also, this explant is more amenable to automation of explant preparation, since simple cuts of initial cylinders suffice to prepare the explants.

Example 2: Influence Of Medium Composition And Light Source On Shoot Regeneration

Efficiency And Vigor

To investigate the interaction between light spectrum and cytokinin levels on regeneration efficiency and vigor of regenerated plantlets, a factorial study with 3 different levels of BAP (0.1 mg/L; 0.5 mg/L, 1.0 mg/1) was designed; all with addition of 0.1 mg/L NAA in regeneration medium phase. The light spectrum quality was also tested by exposing the explants to either cool white fluorescent lamp (Philips) or Gro-lux® lamp (Sylvania). Gro-lux® lamps provide a spectrum enriched in the red wavelengths and relatively poor in blue wavelengths, compared to cool fluorescent lamps. Both light treatments were applied through gradually increasing light intensity: 0.4 μΕ m -^"2 s -^"1 for 4 days, 2 μΕ m -^"2 s -^"1 for 4 days, 10 μΕ m -^"2 s -^"1 for 4 days and then 30 μΕ m^"2 s^"1 for the rest of time the explants were cultured. This study was conducted by 3 different operators, to observe possible effects in the first phase of the process, e.g., quality aspect when cutting the explant. Sugarcane top stalks were obtained from 9.6 month old field grown plants of reference variety SP803280. Outer leaves were removed to yield a spindle of 20 cm length and 1.5 cm diameter. In a laminar flowhood, the spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three leaves, a cylinder with a diameter of around 0.5 cm was obtained and then chopped transversely into thin slices (0.5 to 1.0 mm thick).

Culture conditions were as follows: twelve transversely-sliced leaf whorl explants were introduced on culture dishes containing 50 ml of MS medium, 3.0% (w/v) sucrose; 3 mg/L 2,4- D; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. Cultures were incubated under dark conditions at 26±2°C for 8 days. To induce shoot formation, the culture was transferred to medium without 2,4-D in medium containing MS salts; 3.0% (w/v) sucrose; l .Omg/L BAP, 0.5 mg/L BAP or 0.1 mg/L BAP; 0.1 mg/L NAA; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated in gradually increased light conditions, described above provided by cool white florescent light or Gro-lux® lamps. The photoperiod was 16/8 h light/dark at 26±2°C. Regenerated shoots were transferred to rooting medium containing ½ strength MS macro salts; 1 mg/L IBA; 4% sucrose; ImM isoleucine; 0.1 μΜ of each of the aromatic amino acids (tyrosine, phenylalanine, tryptophan), pH 5.8 and 0.16% phytagel. Plates were evaluated every other day for contamination and morphogenesis development assessment.

Regeneration was observed for all treatments. Some plates were seen with more than

400 shoots formed. The observed data was stratified according to each treatment to identify treatment effects.

When the data was stratified by operators, a statistical difference at 90% confidence level was seen, whereby plates manipulated by operator 3 yielded fewer shoots than plates manipulated by operators 1 and 2. The aim of this portion of the analysis was to test possible effects caused by individuals when preparing the explants; thus explant preparation standardization by automation could lead to more consistent results, including an increase in the yield of shoots regenerated.

The analysis was also stratified by light source, and use of Gro-lux® lamps displayed a trend to yield more shoots per plate than when light was provided by cool white fluorescent lamps (FIG. 1). Additionally, an unexpected increase in regenerated plantlet vigor was observed by about 45 days after explants were transferred to regeneration conditions, if the explants were regenerated under light provided by Gro-lux® lamps in comparison with light provided by white fluorescent lamps. This is apparently due to the wider light wavelength spectrum provided by Gro-lux® lamps in comparison to cool white fluorescent lamps. Plants need light not only for photosynthesis but also need other portions of the light spectrum which are sensed for morphogenesis.

Analysis was also performed by medium composition. Due to variability introduced by different operators, a statistically significant difference among the different regeneration media tested was not observed, although a possible interaction between medium composition and light quality was seen. The number of regenerated shoots per plate was consistent among the media containing different concentrations of BAP when the explants were treated with fluorescent cool light lamps. However, an increase in shoot regeneration as the BAP concentration increased from 0.1 to 1.0 mg/L when the explants were regenerated under light provided by Gro-lux® lamps was also observed (FIG. 2). The treatment of 1.0 mg/1 BAP and Gro-Lux yield more regenerated plantlets, indicating a interaction of BAP and light, or plant growth regulators and light conditions that should be adapted to each genotype. This may be due to the different light wavelengths provided to the culture, as well as to the observed decrease of red compounds being formed when explants are regenerated under light provided by Gro-lux® lamps or possibly by a photomorphogenic effect due to BAP levels when the explants are subjected to red light wavelengths provided by the Gro-lux® lamps. The direct regeneration system, was shown to be rapid and efficient, with shoots forming by around 50 days from initiation of culture, which may then be transferred to rooting medium and then, around 6-10 days later, the plantlets can be transferred to soil. The total time from introduction to plant-in-soil was about 60 days.

Example 3: Influence Of Light Spectrum Quality And Intensity On The Number Of

Regenerated Shoots

Explant preparation: Sugarcane top stalks were obtained from 7 month old field grown plants of reference variety SP803280. Outer mature leaves were removed to yield a spindle of 20 cm length and 1.5 cm diameter. In a laminar flow hood, the spindles were surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three leaves, a cylinder was obtained with a diameter of around 0.5 cm; these cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm, as previously discussed.

Culture medium and incubation conditions: Leaf whorl material was aseptically cut transversally and 12 explants introduced onto culture dishes containing 50 ml of MS medium, 3.0% (w/v) sucrose; 3 mg/L 2,4-D ; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated under dark conditions at 26±2°C for 8 days. To induce shoot formation, the culture was transferred to MS medium containing 3.0% (w/v) sucrose; 0.1 mg/L BAP; 0.1 mg/L NAA; 150 mg/L citric acid; MS salts with vitamins and 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated directly under 30 μΕ m^"2 s^"1 or gradually increased light conditions, e.g., 0.4 μΕ m^"2 s^"1 for 4 days, 2 μΕ m^"2 s^"1 for 4 days, 10 μΕ m^"2 s^"1 for 4 days and then 30 μΕ m^"2 s^"1 the remaining time of the culture with light provided either by cool white florescent light or Gro-lux® lamps. The photoperiod was 16/8 h light/dark at 26±2°C. Regenerated shoots were transferred to rooting medium contain ½ strength MS macro salts; 1 mg/L IB A; 4% sucrose; ImM isoleucine; 0.1 μΜ or each of the aromatic amino acids mix (tyrosine, phenylalanine, tryptophan), pH 5,8 and 0.16% phytagel). Plates were evaluated every other day for contamination and morphogenesis development assessment.

Regeneration of shoots varied somewhat in relation to the light treatment employed, e.g., different light quality provided by two different lamps, as well as gradually increased light and direct full light after auxin pulse treatment. Light provided by cool white fluorescent lamps induced a higher level of dark red pigments, probably anthocyanin. Representative plates of explants which were regenerated under light provided by cool white fluorescent lamps yielded more explants with sectors darkly pigmented in red, combined with fewer green regenerated shoots, When shoots did regenerate, they were developmentally delayed as compared to plates with the shoots being regenerated under Gro-lux®. Again, fewer shoots regenerated on plates treated by light provided by cool white fluorescent lamps. At 17 and 28 days in regeneration, a higher density of initial regenerating shoots was seen for explants treated with light provided by Gro-lux® lamps, than with fluorescent lamps. Thus, use of the wide spectrum lamp promoted a higher yield of regenerated plants of superior vigor and shortened the duration of the regeneration process. After 51 days on regeneration medium, the number of shoots formed and the vigor of regenerated plantlets was measured, under light provided either by Gro-lux® or cool white florescent lamps. Consistent with previous examples, a 2 fold increase in the number of shoots was seen from explants in plates regenerated under Gro-lux® conditions, when comparing use of cool white fluorescent (FIG. 3). Statistical significance at 95% was observed; the study clearly indicates that light spectrum allows for a high frequency of shoot formation and vigor in the direct regeneration system. Formation of dark red pigments, which occur when the explants are treated with regular light provided by cool white fluorescent lamps, was reduced under Gro-lux® conditions.

Next, a regeneration study after the auxin pulse was conducted, exposing explants directly to 30 μΕ light or to fade in light conditions, with the light being provided either by cool white fluorescent lamps or Gro-lux®. More plantlets were observed when the explants were submitted directly to 30 μΕ, irrespectively to the light source. In this study no interaction of light source and lighting intensity was seen.

The beneficial influence of light provided by Gro-lux® lamp on rooting efficiency and rooting timing was also studied. 30 plantlets regenerated under light provided by cool white fluorescent lamp and 30 plantlets regenerated under light provided by Gro-lux® lamp were transferred to rooting medium. 15 plantlets from each treatment were randomly put under light provided by cool white fluorescent lamp or Gro-lux® lamp. The general efficiency after 8 days is statistically the same, close to or 100% rooting efficiency, demonstrating the excellent regeneration obtained by this system. The speed of rooting, however, was different between the treatments. Over 50% of shoots rooted as soon as 5 days when shoots were regenerated under light provided by Gro-lux® lamps and maintained under such light in rooting step (Table 1). The shoots regenerated under light provided by cool white fluorescent lamps and maintained under light provided by Gro-lux® at rooting did not root as quickly as the shoots maintained all the time in light provided by Gro-lux® lamps, but by day 6 the rooting of this treatment also rose to same level of shoots regenerated and rooted under light provided by Gro-lux® lamps (Table 1). Table 1. Impact of light provided either by cool white fluorescent or Gro-lux® lamps on rooting of plantlets regenerated under different light provided by either light source.

Rooting under light provided by Gro-lux® lamp is normal. The results show that, similarly to shoot induction and development, rooting speed is stimulated by the light provided by Gro-lux® lamps. LED light sources may further enhance the efficiency of this process.

Example 4: Comparison Of Regeneration Response Of 21 Elite Sugarcane Clones

Previous examples defined explant preparation methods, lighting conditions to maximize regeneration of each kind of explant, and identified wide spectrum light, such as provided by Gro-lux® lamps, to maximize the number of regenerated shoots. Use of such a light source to allow for rapid rooting on such regenerated shoots was also demonstrated. Next, this system was tested on 21 different elite sugarcane clones, to demonstrate its value for the multiplication of superior varieties of sugarcane. Sugarcane reference variety SP803280 was used as control.

Sugarcane top stalks were obtained from about 7 to 9 month old field grown plants of the reference variety SP803280, and 21 tested elite sugarcane clones. Each study compared 1 1 of the clones to reference variety SP803280. Transverse leaf whorl segments were obtained and cultured essentially as described above.

Regeneration of shoots was consistently observed in 15 out of the 21 clones tested, and at least 200 plantlets per plate were obtained for most of the elite genotypes tested. The number of regenerated shoots per plate in each variety showed an acceptable level of variation as expected when working with unrelated genotypes. This result clearly indicated that the direct regeneration system developed for sugarcane is variety flexible, and that most genotypes regenerated shoots at comparable or higher levels than the reference variety. Only 3 out of 21 tested sugarcane clones did not demonstrate shoot formation and regeneration and other 3 showed a regeneration of less than 200 plantlets per plate. Enhancements in explant preparation, use of thermotherapy, fungicide on the buds that will originate the top stalks, and use of antioxidants can contribute to reduce browning and improve culture response. Also different light intensities and qualities will likely improve regeneration, for instance with varieties where extensive browning was observed.

Example 5: Production of Transformed Sugarcane Plants

In an initial study, transverse leaf whorl section explants from sugarcane were prepared as described above, and batches of 12 explants were co-cultivated with Agrobacterium by adding to 20 ml of Agrobacterium inoculum grown to OD₆60nm 0.8 comprising a construct conferring glyphosate tolerance. During the inoculation procedure, the explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C and then placed under 700 mmHg vacuum for 20 min, with pump on for 10 minutes, and then 10 min with the pump off.

Co-culture phase & auxin pulse 1: After inoculation, most of the Agrobacterium suspension was removed from the explants by briefly blotting onto sterile Whatman filter paper prior to placing onto co-culture plates. The explants were co-cultured on solid medium containing agarose (SIGMA agarose A6013 type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape (12.5mm x 10mm; 3M, St. Paul, MN) and cultures were co-cultured in the dark conditions at 22±l°C for 3 days.

Selection I & auxin pulse 2: At the end of the co-culture period the plates were moved onto selection I & auxin pulse 2 medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape (12.5mm x 10mm) and placed under a 16 h

-2 -1

photoperiod at 26±2°C for 4 days in 0.4 μΕ m^" s^" with light supplied by Gro-lux® lamps (Sylvania).

Selection II & regeneration: Plates from selection I & auxin pulse 2 were then moved onto selection 2 & regeneration media, containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape (12.5mm x 10mm) and placed under a 16 h photoperiod at 26±2°C under gradually increasing light conditions supplied by Gro-lux® lamps (Sylvania): 4 days in 0.4 μΕ m^" 2 s^" 1 ; 4 days in 2 μΕ m^" 2 s^" 1 ; 4 days in 10 μΕ m^" 2 s^"1 ; and then 30 μΕ m^" 2 s^"1 for the remaining time of culture.

Rooting: Positive events displaying growth and regeneration were moved onto rooting medium containing ½ strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IB A; 1 mM isoleucine; 0.1 μΜ of each aromatic amino acid (tyr, phe, trp); 0.16% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The events were moved into jars and placed

-2 -1

under a 16 h photoperiod at 26±2°C in 30 μΕ m^" s^" supplied by Gro-lux® lamps (Sylvania). The plantlets were kept under these conditions for 6 to 10 days, typically 10 days, and then transferred to greenhouse conditions.

Example 6: Agrobacterium Inoculation Following Auxin Pulse, with Comparison of Solid

Supports During Co-Culture

Plasmid vectors were constructed using standard molecular biological techniques known to one of ordinary skill in the art. The plant transformation vector comprises nucleic acid sequences including T-DNA border sequences (right border, RB; left border, LB) to promote the transfer of nucleic acid molecules into the plant genome; replication elements; a selectable marker gene (CP4 conferring glyphosate tolerance); and a reporter gene (encoding GFP). A disarmed Agrobacterium strain (ABI) harboring the binary vector was used.

Pre-inoculum: A \ .0\iL loopful of an Agrobacterium glycerol stock was streaked out onto an LB plate. The LB plate was inverted, and the Agrobacterium allowed to grow for 72 hours in a 28°C incubator. After this time, the pre-inoculum was initiated by taking uniformly growing colonies and dispersing into 25 mL of LB liquid medium containing 40 mg/L kanamycin; 62 mg/L spectinomycin and 25 mg/L chloramphenicol. These were allowed to grow at 28°C in the dark for 18-22 hours with shaking (150 rpm on a gyratory shaker).

Inoculum: The cells were collected and resuspended in 80 ml volume of LB liquid using the same antibiotics as the pre-inoculum., These cultures were incubated at 28 °C in the dark for an additional 4 hours with shaking (150 rpm), after which they had achieved an OD₆60nm of about 0.8 to 1.2 Pre-Induction of Agrobacterium: The inoculum was centrifuged at 20°C at 4500 rpm for 25 min. The pellet was then re-suspended in AB minimal ("ABmin") medium containing 40 mg/L kanamycin, 31 mg/L spectinomycin, and 200 μΜ acetosyringone (3,5-dimethoxy-4- hydroxyacetophenone) and the density was adjusted to an OD₆60nm of 0.4. The Agrobacterium cells were grown for 14-18 hrs at 28°C with shaking (150 rpm on a gyratory shaker). After overnight induction, a pre-spin OD was taken (optional) to determine if the culture had grown in the ABmin media (0.8 to 1.2 OD_660nm desired). Aliquots of the induced Agrobacterium were spun for 20 min at 4500 rpm at 20°C. After that, the induction medium supernatant was poured off, and the Agrobacterium pellet resuspended in 25 mL of wash medium (MS Basal reduced to ½ strength containing 200 μΜ acetosyringone). The resuspended Agrobacterium cells were centrifuged at 20°C, 4500 rpm, for 25 min. The supernatant was removed and the pellet resuspended in the final volume of MS Basal reduced to ½ strength containing 200μΜ acetosyringone. The final concentration of Agrobacterium cells was an OD₆60nm of 0.8 to 0.9. The prepared Agrobacterium suspension was placed at 4°C in the dark until ready to use within 1-5 days.

Sugarcane top stalks were obtained from 9 ½ month old plants grown in field conditions, of reference variety SP803280. Outer mature leaves were removed to yield a spindle of 20 cm in length and 1.5 cm diameter. Spindles were surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick. This thickness was chosen for reliable somatic embryogenesis initiation.

Culture conditions sufficient for induction of somatic embryogenesis are known to those skilled in the art. For example, suitable media for establishment of somatic embryogenesis are described in Taparia et al. (In Vitro Cell. Dev. Biol. Plant 48: 15-22, 2012; Kalunke et al. (Sugar Tech. 1 1 : 355-359, 2009); Irvine and Benda, (Plant Cell Tiss. Org. Cult. 5: 101-106, 1985); Grisham and Bourg (J. Amer. Soc. Sugarcane Technol. 9:97-102, 1989). The culture medium comprises a Murashige and Skoog (MS) formulation which is commercially available and known to those skilled in the art.

The employed cytokinin may be N⁶-benzyladenine (BAP), at a concentration of 0.1-1.0 mg/L, such as 1.0 mg/L. Additional components of the medium may include citric acid and copper sulphate, for instance at a concentration of 100-200 mg/L (e.g. 150 mg/L), and 1-3 mM, respectively, such as 2 μΜ CuSO/t.

Auxin is used for an initial pulse, such as 2,4 dichlorophenoxyacetic acid (2,4-D) at a concentration of 2-3 mg/L. For the regeneration phase of culture, the auxin which was employed was a-napthaleneacetic acid (NAA) at a concentration of 0.1-0.5 mg/L, such as 0.1 mg/L. For the auxin pulse step, transverse thin leaf whorl slices were inoculated aseptically on MS medium containing a combination of 3.0% (w/v) sucrose; 3 mg/L 2,4-D; 150 mg/L citric acid; MS salts, MS vitamins and 0.2% phytagel; pH adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape and were incubated under dark conditions at 27±2°C for 8 days. 12 thin slices of explant tissue were placed on each culture plate containing 50 ml medium. The plates were evaluated every other day for contamination and morphogenesis development assessment.

Infection was for around 20 min to 1 hour, and procedures to aid with Agrobacterium infection, such as sonication, vacuum infiltration, and centrifugation were studied. Studied physical conditions during the infection process were: (1) sonication of A. tumefaciens into explants for 5 min at 45 kHz at room temperature; (2) vacuum infiltration of Agrobacterium solution into explants under 700 mmHg for 20 min, with pump on for 10 minutes, and pump off for 10 minutes, at room temperature; (3) centrifugation of explants with Agrobacterium solution at 290 g for 20 min at 4°C; (4) incubation at room temperature for 25 min with Agrobacterium solution. 12 explants prepared as above were added into 20 ml of the Agrobacterium inoculum. The ratio between the Agrobacterium and leaf whorl explant was about 20 ml: 12 leaf whorl explants. An Agrobacterium density of 0.8 at OD₆60nm was used. The explants remained in contact with the Agrobacterium solution for about 20 minutes to 1.0 hours, typically for 1 hour.

After the infection period, most of the Agrobacterium suspension was removed from the explants by briefly blotting onto sterile Whatman filter paper prior to placing onto co-culture plates. Part of the explants were co-cultured on solid medium containing agarose (SIGMA A6013 type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The explants were moved onto sterile Whatman filter paper under 300 μΐ Milli-Q water sterilized. Both conditions were used to provide a "desiccating environment". The plates were sealed with micropore tape 12.5mm x 10mm and placed into the dark at 22±1°C for 3 days. Transient GFP expression was scored 3 and 10 days after infection.

At the end of the co-culture period the explants were subjected to a "delay" culture period (without selection), and moved onto MS salts medium containing 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 300mg/L Timentin; MS vitamins; 0.2% phytagel; pH 5.8 for a 10 day delay period. Plates were again sealed with micropore^® tape

-2 -1 and placed under a 16 h photoperiod at 27±2°C under very low light conditions (0.4 μΕ m^" s^" ) supplied by cool white fluorescent lamps (Phillips). The light intensity LUX meters illuminance was measured by Luxmeters on the lux scale and converted in μΕ m^"2 s^"1.

After the delay period, experimental treatment plates were transferred onto glyphosate selection/regeneration medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ amino acid aromatic mix (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel, pH 5.8. Control plates were moved onto regeneration medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentin; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

For both selection and regeneration, the plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 27±2°C with gradually increasing light intensity supplied by

-2 -1 -2 -1 cool white fluorescent lamps (Phillips): 4 days in 2 μΕ m^" s^" ; 4 days in 10 μΕ m^" s^" ; and then 30 μΕ m^"2 s^"1 for the remaining time of the experiment. Tissue was subcultured to fresh medium every 14 days. The transformation frequency was determined by the number of explants transiently expressing GFP as a proportion (%) of the total number of explants in the plate (% GFP). The formation of globular structures on the original surface of the leaf whorl explants 8 days after placement onto the auxin pulse medium. After the auxin pulse, the explants were submitted to infection with Agrobacterium solution under conditions described above (sonication and/or vacuum and/or centrifugation) and then were subjected to co-culture on filter paper or solid medium with agarose. A "gentle desiccation" assisted regeneration and decreased browning. When co-cultured under filter paper conditions, the explant tended to over-dry, causing a severe browning and loss of regenerability. However, explants survived very well after co-culture on agarose. A transient transformation rate of about <1% was observed. Example 7: Modification of Infection Procedures

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used. Preparation of Agrobacterium cultures was as described in Example 6. Sugarcane top stalks were obtained from 8 ½ months old plants of reference variety SP803280, grown under field conditions, and transverse leaf whorl explants were prepared as described. Auxin pulse conditions were as described in Example 6.

For infection, centrifugation at 290 g or 650 g was tested. 12 explants prepared according to Example 3 were added to 20 ml of the Agrobacterium inoculum. About 20 ml of Agrobacterium prepared as described above was used per 12 leaf whorl explants. An Agrobacterium cell density of 0.8 at OD₆60nm was used. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour, typically 1 hour. For inoculation all treatments were sonicated for 5 min at 45 kHz and then centrifuged (at 290 g or 650 g) for 20 min at 4°C and then placed under 700mm Hg vacuum for 20 min, with pump on for 10 minutes, and pump off for 10 minutes.

For co-culture, the addition (or not) of 100 μΜ acetosyringone to the agarose-solidified medium was tested. After inoculation, most of the Agrobacterium suspension was removed from the explants by briefly blotting on sterile Whatman filter paper prior to placing onto co-culture plates. Explants were co-cultured on solid medium containing agarose (SIGMA A6013 type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; with or without ΙΟΟμΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. Plates were sealed with micropore^® tape and the cultures were placed in the dark at 22±1°C for 3 days. Transient GFP expression was scored 11 days after the infection. At the end of the co-culture period explants were moved onto MS salts medium containing 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 300mg/L Timentin; MS vitamins; 0.2% phytagel; pH 5.8 for a ten day delay period. Plates were again sealed with micropore^® tape and placed under a 16 h photoperiod at 27±2°C under very low light conditions (0.4 μΕ m^"2 s^"1) supplied by cool white fluorescent lamps (Phillips).

After the delay period, tissue from some of the plates were transferred onto glyphosate selection/regeneration medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. Tissue from the remainder of the plates was moved onto regeneration medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentin; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

For both selection and regeneration the plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 27±2°C in gradually increasing light intensity supplied by cool white fluorescent lamps (Phillips): 4 days in 2 μΕ m^"2 s^"1; 4 days in 10 μΕ m^"2 s^"1; and then 30 μΕ m^"2 s^"1 for the remaining time of the experiment. The tissues were subcultured to fresh medium every 14 days. The transformation frequency was determined as the number of explants expressing GFP as a proportion (%) of the total number of explants in the plate (% GFP).

Centrifugation at 650 g as well as the addition of acetosyringone had a beneficial impact on GFP expression, which reached over 12% of infection on a per explant basis. Overall, the percent of explants expressing GFP ranged from about 5-12% (FIG. 4).

Although the level of transient GFP expression increased from less than 1% to around

12% as compared to the previous study, stable transformation of the reference variety SP803280 was not seen. The may be because infection efficiency was not high enough and/or that infection occurred after the auxin pulse step, wherein initial stages of somatic embryo development were already occurring. GFP expression in the initial shoot (or leaf) was thus tracked and later stage stereoscope views (at about 11 days post infection) under light and GFP filters indicated that the transformed explant tissues were chimeric. Thus transformation needs to be performed in a timely manner to ensure that chimeric shoots are not formed, or at least that their formation is minimized and also to increase infection. The study also shows that the infection procedure tested (sonication, vacuum and centrifugation parameters) did not interfere with formation and development of shoots.

Example 8: Direct Agrobacterium-Mediated T-DNA Delivery Into Leaf Whorl Explants

Agrobacterium-mediated T-DNA deliver into leaf whorl explants was employed, followed by subjecting the explants to culture conditions leading to direct embryogenesis. The method comprised: a) infection of non-cultured leaf whorl explants via Agrobacterium-mediated delivery; b) co-culture of said Agrobacterium-mocxAated leaf whorl explants on a solidified medium containing an auxin level sufficient to start the direct embryogenesis process; c) culturing the explants in at least a second and third culture medium that supports shoot formation, regeneration and selection of a regenerated transgenic sugarcane plant. These transformation experiments used Agrobacterium infection of non-precultured (i.e., non-cultured) or non-induced leaf whorl explants.

As used herein, the term "non-precultured" or "non-cultured" or "non-induced" encompasses the leaf whorl explant that has not gone through any induction medium, being infected immediately after being chopped transversely into thin slices

As used herein, the term "gentle desiccation" means to submit the leaf whorl explant to weak desiccation process by being maintained for 3 days on medium containing agarose as a gelling agent. All the leaf whorl explants were obtained from sugarcane top stalks obtained from 6¾ to 11 months old plants grown in field conditions.

A. Comparing direct Agrobacterium infection with infection after auxin pulse

Four different Agrobacterium infection timings were tested for leaf whorl explants, in order to increase the transient expression of reference variety SP803280. The experimental scheme for testing four distinct times of infection is illustrated in FIG. 5. These four timings were:

(1) infection of non-precultured leaf whorl explant, followed by co-culture with medium containing auxin (e.g. CC-03 medium) for 3 days, followed by transfer to auxin delay medium (e.g. Delay-3 medium) for 5 days and then moved to regeneration medium (e.g. Delay-2 medium) without auxin for the remaining time of the experiment;

(2) infection after 3 days of culture on auxin containing medium (e.g. PULA1 medium), followed by auxin delay medium for 2 days and then moved to regeneration medium for the remaining time of the experiment.

(3) infection after 5 days on the auxin pulse (e.g. PULA1 medium); followed by co-culture with auxin pulse (e.g. CC-03 medium) for 3 days, and then moved to regeneration medium (e.g. Delay-2) for the remaining time of the experiment.

(4) infection after 7 days on the auxin pulse followed by co-culture with BAP (e.g. CC-02 medium) for 3 days, and then moved to regeneration medium (e.g. Delay-2 medium) for the remaining time of the experiment. A binary plasmid vector comprising the CP4 gene which confers tolerance to glyphosate, as well as reporter genes encoding GUS and GFP was used. Agrobacterium cultures were prepared as described above. Transverse leaf whorl explants were prepared as described above. Sugarcane top stalks were obtained from ~6½ month old plants of reference variety SP803280 grown under field conditions.

Infection of non-introduced leaf whorl explants: As shown in FIG. 5, for approach 1 infection was done prior to introducing the leaf whorl explant into culture. That is, the explant had not gone been subjected to any induction medium, but rather was infected immediately after being chopped transversely into thin slices. 12 explants were added into 20 ml of the Agrobacterium inoculum. The Agrobacterium was at an OD_660nm of 0.8 for this sugarcane transformation. The explants remained in contact with the Agrobacterium solution for about 40 to 60 minutes. During the Agrobacterium inoculation procedure all explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C, and then placed under 700 mmHg vacuum for 20 min (with the pump on for 10 minutes, followed by 10 min with the pump off.

Infection after Auxin pulse: As shown in FIG. 5, for approaches 2, 3 and 4 the infection was done after an auxin pulse at three different timings (3, 5 and 7 days, respectively).

Auxin pulse (PUIA1): 12 explants prepared were placed onto MS salts medium containing a combination of 3.0% (w/v) sucrose; 3 mg/L 2,4-D; 150 mg/L citric acid; MS vitamins; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated under dark conditions at 27±2°C for 3, 5 or 7 days.

Co-culture phase: For the co-culture phase, Agrobacterium-moculated leaf whorl explants were placed onto a gelled medium containing an auxin level sufficient to start the direct embryogenesis process. This step also provided "gentle desiccation" conditions.

After the infection period, most of the Agrobacterium suspension was removed from the explants by blotting briefly on sterile Whatman filter paper prior to placing onto co-culture plates. The explants were then moved onto co-culture & auxin pulse medium (CC-03) or onto co-culture & regeneration (CC-02), according to the scheme shown in FIG. 5.

Co-culture & auxin pulse (CC-03): 12 inoculated explants were co-cultured on solid medium containing agarose (Agarose Sigma A6013 Type I, Low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3 mg/L 2,4-D; ΙΟΟμΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. Co-culture & regeneration (CC-02): The 12 inoculated explants were co-cultured on solid medium containing agarose (Agarose Sigma A6013 Type I, Low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving.

All the plates were sealed with micropore^® tape, and the cultures were co-cultured in the dark at 22±1°C for 3 days. Each culture plate contained 50 ml medium. The plates were evaluated every other day for contamination and morphogenesis.

Delay Phase: At the end of the co-culture period the explants were moved onto Delay-3 or Delay-2, as shown in FIG. 5.

Delay-3: The explants were transferred to medium containing MS salts; 3.0% sucrose;

150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 300 mg/L Timentin; MS vitamins; 0.2% phytagel; pH 5.8 for a delay period.

Delay-2: The explants were transferred to medium containing MS salts; 3.0% sucrose; 150 mg/L citric acid; 1 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 300 mg/L Timentin; MS vitamins; 0.2% phytagel; pH 5.8 for a delay period.

The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 27±2°C under gradually increasing light intensity supplied by cool white fluorescent lamps (Phillips): 4 days in 0.4 μΕ m^"2 s^"1; 4 days in 2 μΕ m^"2 s^"1; 4 days in 10 μΕ m^"2 s^"1; and then 30 μΕ m^"2 s^"1 for the remaining time of the experiment. The explants were subcultured to fresh medium every 14 days.

The transformation frequency was determined by the number of GUS-positive explants as a proportion (%) of the total number of explants in the plate (% GUS) after 3 days of co- culture, as shown in FIG. 5 (arrow).

Gus analysis: The buffer (100 mM NaH₂P0₄.H₂0; 0.5 mM K₄Fe(CN)₆.3H₂0; 10 mM Na₂EDTA.2H₂0; 0, 1 % Triton X- 100 - pH 7.0) was prepared previously and stored at -20°C. On the day of analysis the X-Gluc was weighed on an analytical balance and diluted in DMSO (0.05g X-Gluc l\ mL of DMSO). 0.5 g X-Gluc / 1 L Buffer was used; thus, 0.5 g X-Gluc was diluted in 10 mL of DMSO and this dilution was added in 1 L of Buffer. The samples from each plate was collected and put into the falcon tubes containing 10 mL of Gus Buffer (Buffer plus X- Glue). The tubes was opened and put into the dissector under 700 mmHg vacuum for 15 minutes. After that, the tubes were incubate at 37°C overnight. The following day, samples were analyzed in a Leica stereomicroscopic (MZFL III).

A significant increase in transient expression by GFP and GUS analyses was observed when using non-precultured leaf whorl explants (FIG. 6), with up to 55% of explants demonstrating reporter gene expression. For all the infection timing treatments, histochemical GUS analysis was carried out on the leaf whorl explants 3 days after inoculation with Agrobacterium. Transient expression, as observed by GUS activity, was seen only for treatment #1, e.g., infection of non-precultured leaf whorl explants. High GFP transient expression at non- induced leaf whorl explants reaching around 90% of GFP transient expression. A GFP-positive and non-chimeric globular structure was observed 15 days after infection from treatment #1 , e.g., non-induced leaf whorl explants. This supports stable transformation. On the other hand, for different infection timings, such as 3, 5 or 7 days of preculture with an auxin pulse, only a low GFP transient expression for the reference variety SP803280 was observed (FIG. 7). Improved transformation efficiency was obtained by: 1) co-culture under "gentle desiccation" by maintaining the leaf whorl explants for 3 days on medium containing agarose as a gelling agent; 2) co-culture in the presence of 2,4-D, to start the somatic embryogenesis pathway immediately after infection, since such earlier transformation may contribute to reduced occurrence of chimeras. Further, cell death due to Agrobacterium-induced hypersensitive reactions on the cultured sugarcane tissue, which typically results in no transformation or very low transformation frequency on recalcitrant genotypes was rarely observed. Shoot formation and plantlet development was seen by 42 days after infection of non-introduced leaf whorl explants via Agrobacterium-mediated delivery.

Example 9: Direct Agrobacterium -media ted T-DNA delivery into leaf whorl explants of different sugarcane genotypes

Cell death due to Agrobacterium-induced hypersensitive reactions on cultured sugarcane tissues often results in no transformation or very low transformation frequency on the recalcitrant genotypes. Thus, one embodiment of the present invention comprises a novel method to achieve plant cell trans formability for different recalcitrant elite sugarcane genotypes.

Certain sugarcane genotypes respond poorly to somatic embryo formation and or embryogenic callus development. Such "recalcitrant" genotypes have transformation frequencies at or near zero when sugarcane methods described in the literature are attempted. Also, for some genotypes, embryogenic callus formation from leaf whorl explants can be obtained, but the embryogenic callus is difficult to maintain in culture. Consequently, transformation frequencies at or near zero are not uncommon when previously known methods are attempted. In this example transformation and regeneration of different sugarcane genotypes using direct Agrobacterium-mediated T-DNA delivery into non-introduced leaf whorl explants was evaluated.

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used. Transverse leaf whorl explants were prepared as described above. 12 prepared explants were added to 20 ml of the Agrobacterium inoculum. An Agrobacterium density of 0.8 at OD₆60nm was used. The explants remained in contact with the Agrobacterium solution for about 40-60 minutes. During the Agrobacterium inoculation procedure, all explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C and then placed under 700 mmHg vacuum for 20 min (10 min with the pump on and 10 min with the pump off).

Co-culture phase After inoculation, most of the Agrobacterium suspension was removed from the explants by blotting onto sterile Whatman filter paper prior to placing onto co-culture plates. The explants were co-cultured on solid medium containing agarose (Agarose Sigma A6013 Type I, Low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape and cultures were co-cultured in the dark at 22±1°C for 3 days.

Delay Phase At the end of the co-culture period the explants were moved onto Delay-3 medium comprising MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 300 mg/L Timentin; 0.2% phytagel; pH 5,8, for 5 days delay period. The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 26±2°C under

-2 -1

very low light conditions (0.4 μΕ m^" s^" ) supplied by Gro-lux® lamp (Sylvania). Gro-lux® lamps provide a spectrum more enriched in the red wavelengths and relatively more poor on blue wavelengths, compared to cool white fluorescent lamps.

Regeneration phase In order to monitor regeneration of the leaf whorl explants, part of the plates were moved from Delay-3 onto Delay-2 medium containing MS salts; MS vitamins; 3.0%) sucrose; 150 mg/L citric acid; 1 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 300 mg/L Timentin; 0.2% phytagel; pH 5.8. The plates were sealed with micropore tape and placed under a 16 h photoperiod at 26±2°C in light of gradually increasing intensity supplied by Gro- lux® lamps (Sylvania): 4 days at 0.4 μΕ m^"2 s^"1; 4 days at 2 μΕ m^"2 s^"1; 4 days at 10 μΕ m^"2 s^"1; and then 30 μΕ m^"2 s^"1 for the remaining time of the experiment. The tissue was subcultured to fresh medium every 14 days.

Selection phase In order to obtain stable events, part of the plates were transferred from Delay-3 directly onto selection medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 26±2°C in light of gradually increasing intensity supplied by Gro-lux® lamps (Sylvania): 4 days in 0.4 μΕ m^"2 s^"

1 ; 4 days i ·n 2 μΕ m^" 2 s^" 1 ; 4 days i ·n 10 μΕ m^" 2 s^" 1 ; and then 30 μΕ m^" 2 s^"1 for the remaining time of the experiment. Tissue was subcultured to fresh medium every 14 days. Transient GFP expression was monitored throughout the experiment.

Rooting phase Positive events were moved onto rooting medium containing ½ strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IBA; lmM isoleucine; 0.1 μΜ of each aromatic amino acid (tyr, phe, trp); 0.16% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The events were move into jars and placed under a 16 h photoperiod

-2 -1

at 26±2°C in 30 μΕ m^" s^" supplied by Gro-lux® lamps (Sylvania). The plantlets were kept under these conditions for 6 to 10 days, typically for 10 days, and then transferred to greenhouse conditions.

Molecular analysis: Copy number PCR assay: genomic DNA was isolated from leaf tissue of plants at least 3-weeks-old in a greenhouse using the ChargeSwitcli gDNA Plant Kit (Invitrogen Life Technologies Company, Carlsbad, California, USA). The isolated gDNA was used for real-time PCR analyses. Detection was by TaqMan^® system using Gene Expression Master Mix (Applied Biosystems™ product, Life Technologies™). Reactions were performed using Applied Biosystems Real-Time PCR System, with PCR conditions of 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Copy number determination is based on a multiplex PCR reaction in which both target (cp4- epsps) and a sugarcane endogenous gene are amplified in a single well from transgenic sugarcane genomic DNA. The endogenous gene is used as a reference for copy number analysis. The plants used as positive control were validated by Southern blot. 3 plants with 1 copy, 2 plants with 2 copies, 1 plant with 3 copies and 1 plant with 4 copies were obtained. DNA isolated from a conventional plant, PCR reaction mix (no DNA template inserted) and blank of DNA isolation (no vegetal material) were used as negative controls.

Backbone analysis: The end-point PCR for backbone analysis utilized the same isolated gDNA used for copy number assay. Detection was via the TaqMan^® system using Gene Expression Master Mix (Applied Biosystems™ product, Life Technologies™). The backbone assay is based on a multiplex PCR reaction in which both target (aadA or oriV) and sugarcane endogenous gene are amplified in a single well from transgenic sugarcane genomic DNA. An endogenous gene was used as internal control for the PCR reaction. The aadA marker gene and the ori V (replication origin V) were used to verify the presence or absence of the backbone, if it has been inserted into the plant genome. Reactions were performed using Applied Biosystems Real-Time PCR System with PCR Conditions of 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A positive plant (with backbone inserted on its genome), a PCR reaction mix (no DNA template inserted), a blank of DNA isolation (no vegetal material) and a conventional plant were used as controls. High levels of transient GFP expression levels were observed in all five genotypes tested (reference variety SP803280; and four elite clones). Around 40-85% of explants showed GFP transient expression 3 days after infection of non-induced leaf whorl explants. GFP expression was seen in vascular and epidermal cells.

After the explants were transferred to a selection medium with 33μΜ glyphosate, regenerable solid structures expressing GFP were seen by 15 days after infection in all 5 genotypes tested (FIG. 8). About 35 to 55 days after infection and under glyphosate selection pressure, GFP positive somatic embryos and regenerating plantlets were seen. Bright field visualizations were carried out. Inhibition of growth of untransformed somatic sugarcane embryo was seen. Surprisingly, most of regenerated plantlets showed solid GFP expression, indicating that the plants were non-chimeric. Thus infection of the cut leaf whorl explants, without pre-culture, allowed for this result. Differences among the genotypes in terms of transgenic plantlet regeneration timing were seen. As expected the genotypes have particular characteristics of plantlet development. In this example, commercial genotype (Line 1 of FIG. 8) showed later development as compared with other sugarcane genotypes. Transgenic plantlets were then acclimated in substrate to promote growth into mature transgenic plants under greenhouse conditions. Rooting was observed by 6-10 days after transfer to rooting medium. By about 70-80 days after infection, plantlets were moved to soil.

For the sugarcane lines of FIG. 8, transformation frequencies (TF%) of about 1.6% to 4% for Line 3, and 2.8% for lines 1 and 2 were observed. For the reference variety SP80328018 a TF of about 1.2- 3.5% was seen in different experiments (Table 2). We also observed that 9 out of the 10 initial events obtained using this protocol showed low copy number and no vector backbone sequences.

Table 2: Observed TF's from 4 different genotypes (Line numbers as in FIG.

When the event frequency with low copy number and no backbone was compared between a callus based transformation system and direct transformation and regeneration system ("DR"), a tendency for an increased proportion of quality events, lacking such extraneous sequences, was seen for the DR system (FIG. 9).

It was demonstrated in this example that methods are useful for recovery of transgenic sugarcane events, and that the events obtained are non-chimeric and low copy number.

Example 10: Glyphosate Selection Curve For Reference Variety SP803280

The effect of different glyphosate levels and timing on regeneration and on positive event selection for reference variety SP803280 was studied. Seven different glyphosate concentrations were tested (0 μΜ, 5 μΜ ,10 μΜ, 20 μΜ, 33 μΜ, 66 μΜ, 100 μΜ). Also, 2 different timings of starting glyphosate selection were tested: Approach A utilized starting of glyphosate selection after 5 days in delay phase; approach B started glyphosate immediately after the co-culture phase.

In these studies, a binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used in conjunction with transverse leaf whorl explants. Preparation of Agrobacterium inoculum was as described above. Centrifugation was performed at 650 g, followed by sonication and application vacuum as described above. Co-culture utilized ΙΟΟμΜ acetosyringone.

Approach A: Glyphosate selection started after 5 days in delay medium

At the end of the co-culture period part of the plates were moved onto Delay-3 medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 300mg/L Timentin; 0.2% phytagel; pH 5.8, for a 5-day delay period. The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 26±2°C under very low

-2 -1

light conditions (0.4 μΕ m^" s^" ) supplied by Gro-lux® lamps (Sylvania). At around 62 days post infection, plates from delay phase were then moved onto selection medium under 7 different glyphosate concentrations. The medium contained MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each amino acid aromatic (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; and 0 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 33 μΜ, 66 μΜ or 100 μΜ glyphosate; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at

26±2°C gradually increasing light intensity supplied by Gro-lux® lamps (Sylvania): 4 days in

0.4 μΕ m^" 2 s^" 1 ; 4 days in 2 μΕ m^" 2 s^"1 ; 4 days in 10 μΕ m^" 2 s^"1 ; and then 30 μΕ m^" 2 s^"1 for the remaining time of the experiment. The tissue was subcultured to fresh medium every 14 days.

Approach B: Starting glyphosate selection immediately after co-culture phase

At the end of the co-culture period part of the plates were moved onto selection medium

("selection phase I" for 5 days) under 7 different glyphosate concentration containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4D; 2 μΜ copper sulphate; 0.1 μΜ of each amino acid aromatic (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 0 μΜ, 5 μΜ ,10 μΜ, 20 μΜ, 33 μΜ, 66 μΜ or 100 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore tape and placed under a 16 h photoperiod at 26±2°C for 5

-2 -1

days in 0.4 μΕ m^" s^" with light supplied by Gro-lux® lamps (Sylvania).

Plates from selection phase I were then moved onto selection medium under 7 different glyphosate concentrations. The medium contained MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 0 μΜ, 5 μΜ ,10 μΜ, 20 μΜ, 33 μΜ, 66 μΜ or 100 μΜ glyphosate; 0.2% phytagel. The ρΗ of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 26±2°C under gradually increasing light conditions supplied by Gro-lux® lamps (Sylvania): 4 days in 0.4 μΕ m^"2 s^"1; 4 days in 2 μΕ m^"2 s^"1; 4 days in 10 μΕ m -^"2 s-^"1 ; and then 30 μΕ m -^"2 s-^"1 for the remaining time of the experiment. The tissue was subcultured to fresh medium every 14 days. Transient GFP expression was monitored throughout the experiment.

Rooting phase. Positive events were moved onto rooting medium containing ½ strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IBA; 1 mM isoleucine; 0.1 μΜ of each amino acid (tyr, phe, trp); 0.16% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. Events were moved into jars and placed under a 16 h photoperiod at 26±2°C in 30 μΕ m^"2 s^"1 supplied by Gro-lux® lamps (Sylvania). The plantlets were kept under these conditions for 6 to 10 days, typically 10 days, and then transferred to greenhouse conditions.

An effect of glyphosate on shoot formation and regeneration for reference variety

SP803280 was observed. The best selection was in the range of 33 to 66 uM glyphosate. Starting glyphosate selection immediately after the co-culture phase, that is, without delay phase, can minimize the occurrence of chimeric plants. At low glyphosate selection pressure non- transformed cells overgrow the transformed cells. Such overgrowth promotes the regeneration of chimeric events.

These results demonstrated that direct transformation protocol is less labor intensive, by reducing the number of plates generated or background growth of tissue under glyphosate pressure when compared to a callus-based system. A tendency of glyphosate to act quickly in tissue was also seen, thus reducing the regeneration of plates under 33 to 66 μΜ glyphosate selective conditions. Co-cultivation of Agrobacterium -inoculated leaf whorls on a gelled medium {e.g. Agarose Sigma A6013 Type I, Low EEO at 5.5 g/L) allowed for effective early selection and tissue survival under glyphosate selection. If tissue is subjected to less gentle methods, the tissues may not survive such initial selection.

Gradually increasing light intensity and the quality light supplied by Gro-lux® lamps was useful for effective glyphosate selection and the health and regeneration of transgenic tissues. The Shikimic acid pathway is activated by light (McCue and Conn, Plant Physiol., 94:507-510, 1990) and such light, for instance as provided by Gro-lux® lamps also stimulates plantlet regeneration and vigor, as discussed above.

Example 11: Demonstration of a Direct Somatic Embryogenesis Pathway for

Transformation via Agrobacterium -Mediated Transgene Delivery

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used for transformation and regeneration of sugarcane transverse leaf whorl explants. Explant preparation, Agrobacterium preparation, infection, and co-cultivation, was essentially as described above. At the end of the co-culture period the plates were moved onto selection medium containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving.

The plates were sealed with micropore^® tape and placed under a 16 h photoperiod at 26±2°C for 4 days in 0.4 μΕ m^"2 s^"1 with light supplied by Gro-lux® lamps (Sylvania).

Histological analysis. Explant samples were collected at zero, four and seven days after infection, after which the samples were subjected to GUS assays. All samples were fixed in paraformaldehyde (4%, w/v). Fixed tissues were slowly dehydrated at room temperature in a series of ethanol (35-100%), followed by infiltration at 4°C overnight, in ethanol: infiltration medium (Historesin, Leica) (1 : 1). Infiltration was completed with infiltration medium (100%) for 24 h or until the samples appeared translucent and sank to the bottom of the flask. Polymerization was done at room temperature for 24 to 48 h. Serial sections (5 μΜ) were prepared in a rotary microtome (Leica, RM2255) with a tungsten-carbide knife, with the sections floated on water drops, and dried on a hot plate (40°C). Some explants were stained with acid fuchsin (0.05%).

Histological analysis of the explants at 4 days demonstrated the ability to use the described methods to transform single cells. Histological analysis of early globular somatic embryo stage and somatic embryos also demonstrated that initial steps of the direct regeneration are thru direct somatic embryogenesis. The studies therefore demonstrated why whole transformed plants rather than chimeric plants were recovered in the method.

Example 12: Transformation of Recalcitrant Sugarcane

An Agrobacterium-mediated transformation protocol of the present invention was used to transform recalcitrant sugarcane genotypes that have shown limited embryogenesis response and transformability via a callus-based approach. The non-cultured direct transformation system of the present invention was used to transform leaf whorl explants from Saccharum spontaneum and Saccharum officinarium, which have been bred to produce the modern varieties of sugarcane. As used herein, the term "non- cultured" or "non-induced" encompasses the leaf whorl explant that has not gone through any induction medium, being infected immediately after being chopped transversely into thin slices.

High transient expression by GFP analysis and stable structure expressing GFP were observed when non-cultured leaf whorl explants were inoculated with Agrobacterium. In contrast, inoculation of explants after induction resulted in little or no transient transformation and no GFP -positive somatic embryos. This further demonstrates the efficiency of the non- cultured direct transformation system.

A. Transformation of Leaf Whorl Explants of Saccharum spontaneum

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used to transform leaf whorl explants using standard molecular biological techniques known to one of ordinary skill in the art, as described above.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from 12 month-old plants of six Saccharum spontaneum genotypes: S. Kanashiroi; IN84-058; US56-016-01 ; SES323; NEPAL; MANDALAY. Outer mature leaves were removed until a spindle of 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick.

Infection: Three different Agrobacterium infection timings were tested for leaf whorl explants. The overall experimental scheme is shown in FIG. 10.

Infection of non-cultured leaf whorl explants: As shown in FIG. 10, in approach 1 , the infection was done prior to introducing the leaf whorl explant into culture. That is, the explant had not been subjected to any induction medium, but rather was infected immediately after being chopped transversely into thin slices.

Infection of pre-cultured explants (after auxin pulse): As shown in FIG. 10, for approaches 2 and 3, the infection was done after an auxin pulse at two different timings (3 and 6 days, respectively).

Auxin pulse phase: 18 explants were placed onto MS salts medium containing a combination of 3.0% (w/v) sucrose; 3 mg/L 2,4-D; 150 mg/L citric acid; MS vitamins; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The cultures were incubated under dark conditions at 27±2°C for 3 or 6 days as shown in FIG. 10.

Infection (inoculation culture): 18 explants were added into 20 ml of the Agrobacterium inoculum. A disarmed Agrobacterium strain (ABI) harboring a binary vector was used for this experiment. All of the explants obtained from the top stalks were transformed. The ratio between the Agrobacterium and leaf whorl explants was about 20 ml: 18 leaf whorl explants. Agrobacterium was used at OD₆60nm of 0.8. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour. During the inoculation procedure, all the explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C and then placed under 700 mmHg vacuum for 20 min (10 min with the pump on and 10 min with the pump off).

Co-Culture phase: After the infection period, most of the Agrobacterium suspension was removed from the explants by blotting briefly on sterile Whatman filter paper prior to placing onto co-culture plates. The explants were then moved onto a co-culture & auxin pulse medium (CC-03) followed or not onto co-culture & regeneration medium (CC-02), according to the schematic shown in FIG. 10.

Co-culture & auxin pulse: The 18 explants were co-cultured on solid medium containing agarose (Sigma A6013 Type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and the cultures were co-cultured in the dark at 22±1°C. Each culture plate contained 50 ml medium.

Co-culture & regeneration: The 18 explants were co-cultured on solid medium containing agarose (Sigma A6013 Type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and the cultures were co-cultured in the dark at 22±1°C. Each culture plate contained 50 ml medium.

Selection phase: At the end of the co-culture period the explants were moved onto SELA 33 or SEL 33, according to the schematic shown in FIG. 10.

Auxin & selection: MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm. Each culture plate contained 50 ml medium.

Regeneration & selection: MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm. Each culture plate contained 50 ml medium.

Light conditions: After the co-culture phase, the plates were placed under a 16 h photoperiod at 26±2°C under gradually increasing light conditions supplied by Gro-lux® lamps (Sylvania): 4 days in 0.4 μΕ m -^"2 s -^"1 ; 4 days in 2 μΕ m -^"2 s -^"1 ; 4 days in 10 μΕ m -^"2 s -^"1 ; and then 30 μΕ m -^"2 s-^"1 for the remaining time of the experiment.

All six of the S. spontaneum genotypes showed typical explant behavior at infection for non-cultured and pre-cultured leaf whorls. Transient expression levels of GFP in explants were analyzed seven days after infection. Little or no transient expression was observed in explants infected after pre-culture. In contrast, Agrobacterium infection of non-cultured leaf whorl explants (i.e., non-induced) resulted in high transient expression levels by GFP analysis in all the 6 different S. spontaneum genotypes (FIG. 11).

These results demonstrate the efficiency of direct transformation in non-cultured explants. Similar results were obtained for reference variety SP803280, described above, which showed little or no transient GFP expression in explants receiving 3, 5, or 7 days of pre-culture compared with high transient GFP expression in non-cultured leaf whorl explants.

After the explants were transferred to a selection medium with 33μΜ glyphosate, regenerable solid structures expressing GFP were observed 14 days after infection. Regenerable structures expressing GFP were obtained 14 days after infection in 2 out of 6 S. spontaneum genotypes tested (IN84-058; US 56-016-01) only after infection of non-precultured leaf whorl explants via Agrobacterium-mediated delivery. In contrast, no GFP -positive stable expression was observed after infection of explants after pre-culture (3 or 6 days) in all 6 S. spontaneum tested.

B. Transformation of Leaf Whorl Explants of Saccharum officinarium

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used to transform leaf whorl explants using standard molecular biological techniques known to one of ordinary skill in the art, as described above.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from 12 month-old plants of six Saccharum officinarium genotypes: IN84-003; CHITTAN; FIJI44; KHAM; ANOMAN; NG77-065. Outer mature leaves were removed until a spindle of 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick.

Infection: Infection was carried out as for Saccharum spontaneum explants, described above.

Co-Culture phase: Co-culture was carried out as for Saccharum spontaneum explants, as described. Selection phase: Selection was carried out as for Saccharum spontaneum explants, as described. Light conditions: Light conditions were the same as for Saccharum spontaneum explants, as described.

All six of the S. officinarium genotypes showed the typical explant behavior for non- cultured and pre-cultured leaf whorls. Transient expression levels of GFP in explants were analyzed seven days after infection. No transient expression was observed in explants exposed to six days of pre-culture, while little or no transient expression was observed in explants exposed to three days of pre-culture. In contrast, Agrobacterium infection of non-cultured leaf whorl explants (i.e., non-induced) resulted in high transient expression levels by GFP analysis in all the 6 different S. officinarium genotypes (FIG. 12).

After the explants were transferred to a selection medium with 33μΜ glyphosate, regenerable solid structures expressing GFP were observed 14 days after infection. Regenerable structures expressing GFP were observed in 5 out of 6 S. officinarium genotypes tested (ΓΝ84-003; CHITTAN; FIJI44; KHAM; and NG77-065) only in non-induced leaf whorl explants. However, no GFP- positive stable expression was observed for infection of explants after pre-culture (3 or 6 days) in any of the 6 S. officinarium tested. (Table 3). Similar numbers of regenerable structures expressing GFP were observed for several modern sugarcane varieties when non-cultured explants were infected with Agrobacterium as described above.

Table 3. Regenerable structures expressing GFP in S. officinarium genotypes after non- cultured explants were infected with Agrobacterium as described.

These results demonstrate for the first time that high transient expression of a heterologous gene, and stable structures expressing the heterologous gene can be achieved in Saccharum spontaneum and Saccharum officinarium by Agrobacterium infection of non- cultured leaf whorl explants. Example 13: Effects of LED Light on Direct Transformation of Recalcitrant Sugarcane

Genotype CV7231

Among other variables, light quality and intensity plays a major role in plant development. Stress during tissue culture and transformation may lead to the appearance of red or brown pigmentation of cultured cells, which correlates with delayed or reduced regeneration of plant tissue as well as low transformation frequency. Moreover, conventional cool white fluorescent light sources emit light with deficiencies in the red and far red wavelengths that are essential to the photomorphogenesis process. This example demonstrates that different LED lighting conditions can be used to mitigate the tissue browning, oxidation, and phenolic release in recalcitrant sugarcane commercial genotypes such as CV7231 and CV0470. In addition it is demonstrated that the direct transformation system described herein can result in improved somatic embryo formation. Therefore, this system is useful in mitigating tissue browning or oxidation, improving somatic embryo formation, and increasing the transformation frequency in sugarcane recalcitrant genotypes as well as in other crops, such as sorghum, soybean, wheat and corn.

The effect of different LED lighting conditions (varying combinations of spectrums and light intensity) on direct transformation of the commercial genotype CV7231 was studied in an effort to improve explant performance, for example by mitigating oxidation or phenolic compounds commonly observed in this and other recalcitrant genotypes. Control plantlets and transformed explants of the same genotype were grown under cool white and Gro-lux® lamps for comparison. The spectrums tested were: deep red (660 nm), blue (450 nm) and far red (740 nm), delivered by LED.

Plasmid vector: A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used to transform leaf whorl explants.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from eight month three-day old plants of the commercial genotype CV7231 grown in field conditions. Outer whorls of mature leaves were removed until a spindle of 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three whorls of leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick.

Infection (inoculation culture): 18 explants were added into 20 ml of the Agrobacterium inoculum. A disarmed Agrobacterium strain (ABI) harboring a binary vector was used for this experiment. Pre-inoculum, a 1.0\iL loopful of an Agrobacterium glycerol stock was streaked. All of the explants obtained from the top stalks were transformed. The ratio between the Agrobacterium and leaf whorl explants was about 20 ml: 18 leaf whorl explants. Agrobacterium was used at OD_660nm of 0.8. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour. During the inoculation procedure, all the explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C and then placed under 700 mmHg vacuum for 20 min (10 min with the pump on and 10 min with the pump off).

Co-Culture phase: After the infection period, most of the Agrobacterium suspension was removed from the explants by blotting briefly on sterile Whatman filter paper prior to placing onto co-culture plates. The explants were co-cultured on solid medium.

Co-culture medium (CC03): agarose (Sigma A6013 Type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and the cultures were co-cultured in the dark at 22±1°C for 3 days.

Auxin pulse phase: At the end of the co-culture period, the explants of some plates were moved onto the regeneration medium DELAY-03 containing MS salts; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 300 mg/L Timentim; MS vitamins; 100 μΜ acetosyringone; 0.2% phytagel. The other plates were transferred onto the selection medium SELA33 containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C for 4 days at a light intensity of 0.4 μΕ m -^"2 s -^"1 supplied by Gro-lux® lamps (Sylvania) (treatment under Gro-lux®), cool white lamps (Phillips) (treatment under cool white), or LED, according to each LED treatment (see Table 4 below). Regeneration & selection: After the auxin pulse, tissues from Delay-03 plates were transferred onto the regeneration medium DelayOl containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentim; 0.2% phytagel; 2 μΜ copper sulphate. The explants from SELA33 medium were moved onto the glyphosate selection medium SEL33 containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel. The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving. Twenty-one days after infection, the tissues were moved onto medium with a lower BAP concentration of 0.1 mg/L until the end of the process: DELAY01-01BAP containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 0.1 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentim; 0.2% phytagel; 2 μΜ copper sulphate; and SEL33-01B containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 0.1 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel. The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving.

Subcultures and light conditions: All plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C in a light source supplied by Gro-lux® lamps (Sylvania), cool white lamps, or LED, according to each treatment (see treatments in Table 4). The tissues were subcultured to fresh medium every 7 days. At the first regeneration/selection phase (7 days after infection), the plates from all treatments were put under 0.4 μΕ m -^"2 s-^"1 for 3 days; 2 μΕ m -^"2 s-^"1 for 2 days and than 2 days at 10 μΕ m -^"2 s-^"1. Starting at day 14, the tissue was left 2 to 1 day at 0.4 μΕ m^"2 s^"1 after each subculture (2 days: 5 subcultures, from day 14 until day 35; 1 day: from the 35th day to the remaining time of the process) and then was placed under total light condition (30 μΕ m^"2 s^"1 or 60 μΕ m^"2 s^"1).

Rooting phase: Control plantlets were moved into flasks containing the rooting medium containing MS macro salts; 4.0% sucrose; 1.0 mg/L IBA; 1 mM isoleucine; 0.1 μΜ of each amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The rooting flasks were placed under a 16 h photoperiod at 26±2°C at the same light conditions of each treatment as the regeneration phase (Table 4), supplied by Gro-lux®, cool white or LED lamps. The plantlets were kept under those conditions for approximately 10 days and then transferred to greenhouse. Five treatment conditions were tested, each with a different ratio of deep red and blue after the auxin pulse phase. 50 days after infection, some regenerated plantlets from treatments 3 and 4 were put into far red (treatment 5). The light quantity (%) and intensity (μΕ m-2s-l) of each spectrum are presented below.

Table 4. LED conditions tested in the direct transformation process with the CV7231 genotype.

Regeneration of control plantlets: In this example, the control explants were infected using Agrobacterium and then were put in the regeneration medium, without glyphosate. All treatments (using cool white, Gro-lux® and LED) were put under the same light conditions as the standard protocol, i.e. , passing through gradative light conditions (0.4 μΕ m^"2 s^"1; 2 μΕ nfV¹; 10 μΕ m^'V¹ and then total light intensity) in defined phases. Twenty-eight days after infection plants exposed to all treatments except the cool white treatment began developing shoots. Plants exposed to the cool white treatment developed only few shoots after 35 days. Regeneration was observed for all LED conditions tested (see conditions in Table 4). Fifty-six days after infection, the far red spectrum was added for some plantlets developed from treatments 3 and 4, in a ratio of 4: 1 :5 (Red:Blue:Far red), at a total light intensity of 60 μΕ m^'V¹. Seventy-seven days after infection. All treatments regenerated normal plantlets, showing that plantlets can be obtained using different light sources in the direct regeneration system.

When the far red spectrum was added to deep red and blue for plantlets from treatments 3 and 4 (4: 1 condition), 56 days after infection, shoot elongation of the plantlets was clearly observed when compared to the plantlets that grew under cool white and Gro-lux® lamps. Furthermore, plantlets under far red were more elongated than the plantlets that grew under the others LED conditions. Elongation of shoots was observed under the combination 4: 1 :5 (Red:Blue:Far red) compared to plantlets regenerated under cool white and Gro-lux® lamps. The plantlets under far red conditions also showed more elongated shoots than the plantlets grown under the other LED conditions. It was observed that the plantlets that elongated under far red showed thinner shoots.

Although far red light promoted shoot elongation, it was observed that the plantlets became thinner than the plantlets grown under cool white, Gro-lux®, and other LED conditions. Based on these results, it may also be advantageous to add far red light over the selection plates in earlier direct regeneration phases to elongate the initial shoots that arise from transformed explants, which in general take a long time to develop in plantlets, and then transfer the explants to other conditions, for example 4: 1 or 2: 1 (Red:Blue).

It was also observed that LED promoted root formation (Table 5) when compared to rooting time of control plantlets under cool white and Gro-lux® lamps. This was observed in two LED conditions: 4: 1 (Red:Blue) and 4: 1 :5 (Red:Blue:Far red), both at 60 μΕ m^'V¹. Rooting was evaluated 4, 7 and 10 days after transferring plantlets to the rooting medium. Table 5 shows that both LED conditions anticipated root formation of some plantlets in only 4 days in the rooting medium. With 7 days at the rooting medium it was observed that a higher percentage of plantlets under Gro-lux® formed roots, faster than plantlets under cool white and LED, showing that besides LED anticipated root formation, Gro-lux® lamps were more consistent to develop roots in a period of 7 days. After 10 days in the rooting medium, almost 100% of the plantlets formed roots.

Table 5. Rooting formation under different light conditions (cool white, Gro-lux® and

Selection: In this experiment, explants were put under selection using the same light conditions as the control explants shown above. The explants were transformed using Agrobacterium as described and the transformed explants were put in a selection medium with a 33μΜ concentration of glyphosate. The transformed explants became oxidated in all treatments. This oxidation after agroinfection is a characteristic of the CV7231 genotype. However, the explants under cool white seemed to be more oxidated than any of the other treatments. Also, in terms of somatic embryo formation, it was observed that the explants under cool white showed the least ability to form embryos, followed by the explants under Gro-lux® lamps. On the other hand, the explants under different LED conditions showed an improved ability to form embryos compared with the other treatments. Furthermore, 42 days after infection, all LED conditions led to earlier embryo maturation, and consequently regeneration events were observed earlier.

Explant oxidation was observed in all treatments, especially in the explants under cool white light source. All the LED treatments (1 : 1, 2: 1 and 4: 1 at 30 μΕ m -^"2 s-^"1 ; 4: 1 at 60 μΕ m -^"2 s-^"1 ) formed more somatic embryos than the explants under cool white and Gro-lux® lamps at 28 days after infection. Furthermore, 42 days after infection it was clear that the LED promoted event formation as evidenced by formation of green shoots by 42 days after infection. Fifty-six days after infection, all treatments regenerated green shoots, except the cool white treatment which did not develop shoots during the whole process.

These data further demonstrate the influence of lighting quality on the regeneration of transformed sugarcane explants. In particular, Gro-lux® lamps and gradually increasing light intensity have been found to be important. Moreover, the present disclosure shows that under different LED conditions, somatic embryo formation can be increased and regeneration events will occur faster using the direct transformation system of the present invention. These results are particularly significant for recalcitrant sugarcane genotypes in order to improve the embryogenesis response. Thus, importantly, previously "recalcitrant" genotypes that have shown limited embryogenesis response may now be directly used as transformation targets.

A decrease in the phenolic compounds released into the medium was observed in all plate treated with LED as compared to the medium of plates under cool white and Gro-lux® lamps.

Elongated plantlets were obtained for recalcitrant sugarcane genotypes when the far red spectrum was added to blue and deep red using LED as the light source. Moreover, improved rooting was observed under two LED conditions which used a higher light intensity (60 μΕ m -^"2 s^{" 1} instead of 30 μΕ m^'V¹) and a Red:Blue ratio of 4: 1 , demonstrating that the deep red spectrum and higher total light intensity can be positive to roots formation on the sugarcane crop. Regarding the transformed explants that were put under selection medium, it was observed that the explants exposed LED developed more somatic embryos than the explants under cool white and Gro-lux®. In addition, all the LED treatments promoted event formation. The quantity of phenolic compounds released in the medium, evaluated by the medium color, was lower in all plants exposed to LED than the plates exposed to cool white and Gro-lux® light sources. These results suggest that a reduction of phenolic compounds in the medium can improve the embryo development formation and conversion into whole plants. Taken together the data shows that transformation frequency can be increased in the recalcitrant sugarcane genotypes.

Example 14: Effects of LED Light on Transformed Explants of Recalcitrant Sugarcane

Genotype CV7231 Under Selection Pressure

In further experiments, a LED light source was used soon after the co-culture phase for explants of the commercial genotype CV7231. In the standard direct transformation protocol,

-2 -1

explants were kept under twilight conditions (0.4 μΕ m^" s^" ) for 4 days after co-culture phase using Gro-lux® as source of light, in order to mitigate the tissue stress due to light intensity. However, using the identified high quality spectrum for photomorphogenesis using LED lighting, potentially damaging ultra violet (UV) light can be avoided, and it is possible to use a higher intensity light soon after the co-culture phase when compared to a Gro-lux® light source. Moreover, the present experiment demonstrates that explants may be exposed to total light intensity directly following direct transformation, in contrast with the standard direct transformation protocol disclosed herein, which includes exposure to twilight conditions (0.4 μΕ

-2 -1

m^" s^" ). The spectrums tested in this experiment, delivered by LED, were deep red (660 nm) and blue (450 nm) in different ratios.

In this experiment, the plasmid vector used, the leaf whorl explant preparation, the infection procedure and the characteristics of the co-culture phase were as described above.

Auxin pulse phase: At the end of the co-culture period, all transformed explants were moved to the selection medium SELA33 containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore^® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C for 4 days at three LED light conditions with the fourth treatment, at 0.4 μΕ m^"2 s^"1 supplied by Gro-lux® lamps (Sylvania), according to the standard protocol.

Table 6 shows LED conditions tested at the auxin pulse phase. The use of LED lighting was tested after the co-culture phase, in three different conditions. At the auxin pulse phase, the total light intensity of each treatment was established based on the minimum of the software capacity of the deep red and blue spectrum in each ratio.

Table 6. LED conditions tested at the auxin ulse phase.

Regeneration/selection phase: After the auxin pulse phase, the tissues from SELA33 medium were moved onto the selection medium SEL33 containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L AA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel. 21 days after infection, the tissues were moved onto the selection medium SEL33-01B containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 0.1 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel. The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving.

Subcultures and light conditions: All plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C in light intensities supplied by LED (3 treatments) or Gro-lux® lamps (1 treatment) (see treatments in Table 7). The tissues were subcultured to fresh medium every 7 days. After each subculture, all plates were put directly under total light intensity of 30 μΕ m -^"2 s -^"1 , wi ·thout passing through a twilight condition.

Rooting phase: The plantlets were moved into flasks containing the rooting medium (see formulation above). The rooting flasks were placed under a 16 h photoperiod at 26±2°C at the same light conditions of each treatment as the selection phase (see Table 7) supplied by LED or Gro-lux® lamps. The plantlets were kept under those conditions for approximately 10 days and then transferred to greenhouse. For all treatments, the total light intensity used was 30 μΕ m^'V¹, even in the Gro-lux® treatment (as the standard protocol).

Table 7. LED conditions at selection and rooting phases performed with the CV7231 genotype.

Twenty-one days after infection, regenerable stable structures expressing GFP were observed in all treatments. Transformation occurred for the variety CV7231 even when the explants were put under LED immediately after the co-culture phase. The highest level of regenerable stable structures was observed in the 1 : 1 (Red:Blue) treatment, which had 28 GFP- positive somatic embryos. In comparison, just 2 transformed embryos were observed in the Gro- lux® treatment, at the same direct transformation phase. The LED treatments 2: 1 and 4: 1 showed, at least twice the number of embryos expressing GFP as the Gro-lux® treatment, suggesting that LED had a positive effect on the transformation level of this variety. Compared to explants under Gro-lux® lamps, a considerable reduction in explant oxidation was observed when LED was used as the light source. Moreover, higher light conditions with LED at the auxin pulse phase did not result in damage to the explant tissue. This contrasts with the standard protocol, which involves lower light twilight conditions in this phase. To the contrary, the use of LED light at the auxin pulse phase seemed to improve the material quality. Furthermore, we observed that the explants under LED showed more embryo formation than the explants under Gro-lux®.

In addition, a reduction of phenolic compound release on the medium in plates was consistently observed under LED as in the previous example. A reduction of phenolic compounds released into the plates under all LED treatments was observed when compared to cool white and Gro-lux® treatments.

Under LED light at a ratio of 2: 1 (Red:Blue), at a light intensity of 30 μΕ m^"2 s^"1, events developed 40 days after infection. In general, the emergence of events for the genotype CV7231 frequently occurs approximately 60 days after infection. Thus, LED light seemed to accelerate the event formation on this genotype. These data show for the first time that events can be obtained about 40 days after infection using the direct transformation protocol with this genotype. Using a Gro-lux® light source under the same conditions resulted in the emergence of events 56 days after infection. Therefore, in this case, the LED light source accelerated event formation by approximately 16 days.

These results for genotype CV7231 demonstrate that the use of a LED light source improved the direct transformation process, accelerating event formation by 16 days. Moreover, a reduction of explant oxidation and a reduction of phenolic compounds release into the medium was observed. An interesting result was that LED, even in different spectrums, promoted embryo formation at a higher level than the embryo formation under Gro-lux® lamps, increasing the probability to form transformed events, which reduces the time necessary to obtain useable plants. Another interesting point was that LED could be applied directly following the auxin pulse phase (4 days after infection), when plants are kept under twilight conditions according to the standard protocol. However, the addition of LED in this phase did not cause damage to the material, and they did not became oxidated even at higher light intensity. Regarding the regenerable stable structures expressing GFP, we observed a higher transformation level under

-2 -1

specific LED conditions (1 : 1 at 30 μΕ m^" s^" ). Example 15: Effects of LED Light on Direct Transformation of Recalcitrant Sugarcane

Genotype CV0470

Transformed leaf whorl explants from recalcitrant sugarcane genotype CV0470 were exposed to Gro-lux® and LED light sources in this study. The ratio of deep red and blue spectrums of the LED light treatments was changed through the initial phases of the process. LED was used starting after the auxin pulse phase (4 days after infection). The spectrums tested, delivered by LED, were deep red (660 nm) and blue (450 nm).

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used to transform leaf whorl explants using standard molecular biological techniques known to one of ordinary skill in the art, as described above.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from eight month twelve day-old plants of the commercial genotype CV0470. The leaf whorl explant preparation was as described.

Co-culture phase: After the infection period the Agrobacterium suspension was removed from the explants by passing the liquid through a sieve to separate the explants from the inoculum. The base of the sieve was briefly dried in a sterile Whatman filter paper. The separated explants were then transferred to a sterile Whatman filter paper, blotting them briefly on prior to being placed onto co-culture plates. The explants were co-cultured on CC03 medium containing agarose, as described.

Auxin pulse phase: At the end of the co-culture period the explants of some plates were moved onto the regeneration medium DELAY-03. The other plates were transferred to the selection medium SELA33. Both medium compositions are as described. The plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C for 4 days at a light intensity of 0.4 μΕ m^"2 s^"1 supplied by Gro-lux® (Gro-lux® treatments) or LED, according to each LED treatment (Table 8). Two LED treatment conditions were tested after the co-culture phase. At the auxin pulse phase, the total light intensity of each treatment was established based on the minimum of the software capacity of the deep red and blue spectrum in each ratio. Treatments 1, 3 and 4 were put under the same conditions at this phase. Table 8. LED conditions at the auxin pulse phase tested in the direct transformation process with the CV0470 genotype. Blue Total light

Ratio Percentage of each Deep red intensity

Treatment mV) intensity (μΕ intensity - Auxin

(Red:Blue) spectrum (%) (μΕ mV) pulse phase

1, 3 and 4 1 : 1 50% Red: 50% Blue 4,5 4,5 9 μΕ m^"V

2 2: 1 67% Red: 33% Blue 8,7 4,3 13 μΕ m^'V¹

Regeneration/selection phase: After the auxin pulse, tissues from Delay-03 plates were transferred onto the regeneration medium DelayOl. The explants from SELA33 medium were moved onto the glyphosate selection medium SEL33. Both media compositions are as described. Twenty-one days after infection, the tissues were moved onto medium with a lower BAP concentration of O. lmg/L until the end of the process: DELAY01 -01 BAP and SEL33-01B media, with a lower BAP concentration of O.lmg/L until the end of the process, as described. For the maltose plus glucose test, four media were used: DELA Y01 2M1 G: MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentim; 0.2% phytagel; 2 μΜ copper sulphate.

SEL33 2M1G: MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel.

DELAY01 BAP 2M1G: MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 0.1 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentim; 0.2% phytagel; 2 μΜ copper sulphate.

SEL33_0,1B_2M1G: MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 0.1 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel.

The pH of all media was adjusted to 5.8 with 1 M NaOH before autoclaving.

In the abscisic acid (ABA) test, the following two media were used on days 7 through 14 after infection: DELA Y01_2M1 G_[-5]ABA : MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentim; 0.2% phytagel; 2 μΜ copper sulphate; 10-5 M abscisic acid (ABA).

SEL33_2M1 G - 5] ABA : MS salts; MS vitamins; 2.0% maltose and 1.0% glucose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 μΜ glyphosate; 0.2% phytagel; 10-5 M abscisic acid (ABA).

The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving.

Subcultures and light conditions: All plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C in a light source supplied by Gro-lux® lamps (Sylvania) or LED according to each treatment (see treatments in Table 9). The tissues were subcultured to fresh medium every 14 days. After each subculture, all LED treatments were left 1 day at the twilight condition and the next day were put under total light intensity of 30 μΕ m^"2 s^"1 or 60 μΕ m^"2 s^"1, according to each treatment (Table 9). The Gro-lux® treatment was conducted with the light conditions as the standard protocol. For treatment 1 , the light quality was changed through the initial direct transformation phases. After 21 days, treatments 1 and 2 changed the Red:Blue ratio to 4: 1 , to increasing the quantity of the deep red spectrum over the explants. Table 9. LED conditions tested on the genotype CV0470.

After the auxin pulse phase, the oxidation of the explants was very similar in all treatments. Also, this genotype does not release a high amount of phenolic compounds in the medium, in contrast to genotype CV7231. Because of this, the subcultures were done every 14 days.

Twenty-one days after infection, it was observed that the explants under Gro-lux® had a higher level of anthocyanin accumulation when compared with the explants under all LED treatments. It was also observed that there were differences between the LED treatments related to the accumulation of this pigment. LED treatment that contained less quantity of the blue spectrum showed less anthocyanin accumulation over the somatic embryos than the LED treatments which had more blue quantity.

Regenerable structures expressing GFP were observed in all treatments 21 days after infection. However, under different LED conditions at least a 2 or 2,5 fold increase was observed in terms of somatic embryos expressing GFP. This result demonstrates an acceptable stable transformation based on the somatic embryo expressing GFP level for this recalcitrant sugarcane genotype CV0470 when compared with other non-recalcitrant genotypes.

Thirty-five days after infection, less oxidation was observed in the explants under LED conditions. Also, the explants under such conditions showed more green shoot formation, and less mucilage and anthocyanin accumulation. These factors may improve the transformation frequency in such recalcitrant sugarcane genotype.

Under Gro-lux®, the explants became oxidated, with high anthocyanin accumulation and formation of mucilage in the most of the explants (Left). In contrast, explants under LED (for example treatment 1 passing through three different LED conditions during the initial phases: 1 : 1 → 2: 1 → 4: 1 , at 30 μΕ m^"2 s^"1) were less oxidated (right). The explants did not show anthocyanin accumulation like the explants under Gro-lux®. Less mucilage formation was observed in LED treatments. For both conditions sucrose 3% was used as a carbohydrate source.

Tests were also conducted to determine whether carbohydrate source as well as LED conditions could promote a better morphogenesis response on recalcitrant genotype CV0470. First, only the LED effect was tested by keeping 3% sucrose as carbohydrate source. Gro-lux® and sucrose as the carbohydrate source were also tested according to the standard protocol.

The synergistic effect of LED with maltose 2% and glucose 1% added to the selection and regeneration medium was also tested. The maltose and glucose was used 21 days after the infection, being maintained in all the remaining phases. It was observed that the treatment under LED and also in a medium supplied with maltose and glucose instead of sucrose promoted a better explants behavior than the other treatments. Reduced oxidation of the explants and formation of green shoots structures as early as 35 days after infection was observed.

The effect of abscisic acid (ABA) on this genotype was tested in medium supplemented with maltose 2% and glucose 1% instead of sucrose 3% together with the use the LED 4: 1 (Red: Blue) conditions. The ABA effect alone was not tested, because of the known negative effect of sucrose in this CV0470 genotype under Gro-lux® conditions. The LED 4: 1 (Red:Blue) and sucrose 3% was used as reference protocol for this CV0470 genotype.

ABA at 10^~5 M was added to the SEL33 medium or in the DelayOl (see above). This medium was used starting on day 7 and lasting through day 14 of the direct sugarcane transformation process.

Early shoot formation was observed in explants treated for 7 days with ABA under regeneration medium without glyphosate. Furthermore, explants treated with the modified carbohydrate source plus ABA and under LED conditions seemed to develop fewer white shoots than the explants only under LED or Gro-lux® conditions or treated with sucrose and without ABA.

For explants under selection pressure, ABA treatment also promoted shoot formation. Although the improved shoot formation was not seen in all explants, we observed more green structures in the ABA treatment when compared to the explants treated with LED light but with sucrose as carbohydrate source.

In terms of oxidation, the results obtained for the genotype CV0470 were similar to those obtained for genotype CV7231. The use of LED reduced oxidation in both genotypes at specific times of the process. For CV0470, a reduction of anthocyanin accumulation in the somatic embryos was observed when the total blue intensity over the explants was reduced. This may be explained by the influence of blue light on anthocyanin biosynthesis. More somatic embryo development and shoot formation was also observed when LED was used. Changing the carbohydrate source, from sucrose to maltose and glucose resulted in improvement of the explants behavior in terms of decreased oxidation and increased green shoot formation. Furthermore, adding ABA to the maltose/glucose medium resulted in promotion of shoot formation in both control and selection explants.

These LED effect as well as a modified carbohydrate source resulted in improved embryogenesis in recalcitrant sugarcane genotypes. Thus, previously "recalcitrant" genotypes that have shown limited embryogenesis response may now be directly used as transformation targets.

Example 16: Use of Liquid Medium in the Direct Regeneration System

The liquid media have increased nutrient availability and uptake by the explants due to greater ease of absorption of nutrients and growth regulators and also greater contact between the medium and the explant, unlike most solid media, which provides only basal contact. The use of liquid media also allows greater dilution of exudates originating from the explant, thus avoiding the accumulation of phenolic compounds. Therefore, the direct transformation system (DR) is useful in automated processes utilizing liquid media, which can result in a reduction of the cost of labor.

This study tested the use of felt or acrylic blanket only in direct regeneration system using the genotype CV6984. Three conditions were tested using the felt as a support for the explants in liquid culture medium: acrylic blanket, felt, or felt and filter paper.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from eight month- old plants of the commercial genotype CV6984 grown in field conditions. Outer whorls of mature leaves were removed until a spindle of 20 cm length and 1.5 cm diameter was obtained. Spindles were then surface sterilized twice using ethanol (70%) for 5 min each. After removing the outer two to three whorls of leaves, a cylinder with a diameter of around 0.5 cm was obtained. The cylinders were chopped transversely into thin slices of 0.5 to 1.0 mm thick.

Co-culture phase: Eighteen leaf whorls were placed into co-culture medium. The explants were co-cultured on solid medium containing agarose (Sigma A6013 Type I, low EEO) 5.5 g/L; ½ MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 μΜ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and the cultures were co-cultured in the dark at 22±1 °C for 3 days.

Auxin pulse phase: At the end of the co-culture period the explants were moved onto plates containing liquid medium (PULA 1 Liq) MS salts; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were sealed with micropore® tape 12,5mm x 10mm and placed under a 16 h photoperiod at 26±2°C for 4 days at a light intensity of 0.4 μΕ m^"2 s^"1 supplied by Gro-lux® lamps (Sylvania). Regeneration/selection phase: After the auxin pulse, the liquid medium was removed from the plates and the same volume was added according. For the regeneration phase, liquid medium (Delay- 1 Liq) was used containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentin; 2 μΜ copper sulphate. The pH of the media was adjusted to 5.8 with 1 M NaOH before autoclaving. Twenty-one days after infection, the tissues were moved into a medium (DELAYOl BAP Liq LIMS#4085) with a lower BAP concentration. Forty-two days after infection, the explants were moved into a medium (Reg W/H Liquid) without hormones containing MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid 300mg/L Timentin; 2 μΜ copper sulphate, until the end of the process.

Subcultures and light conditions: All plates were placed under a 16 hr photoperiod at 26±2°C in a gradative light intensity supplied by Gro-lux® lamps (Sylvania). The tissues were subcultured to fresh medium every 7 days. At the first regeneration phase (7 days after infection), the plates were put under 0.4 μΕ m^"2 s^"1 for 3 days; 2 μΕ m^"2 s^"1 for 2 days and than 2 days under 10 μΕ m^"2 s^"1. Starting at day 14, the tissue was left 2 to 1 days at 0.4 μΕ m^"2 s^"1 after each subculture (2 days: 5 subcultures, from day 14 until day 35; 1 day: from the 35th day to the remaining time of the process) and than was put under total light condition (30 μΕ m^"2 s^"1 or 60 μΕ m^"2 s^"1).

Rooting phase: The plantlets were moved into flasks containing a rooting medium containing MS; 4.0% sucrose; 1.0 mg/L IB A; 1 mM isoleucine; 0.1 μΜ of each amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 0.2% phytagel. The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The rooting flasks were placed under a 16 h photoperiod at 26±2°, supplied by Gro-lux®. The plantlets were kept under those conditions for approximately 10 days and then transferred to greenhouse.

Explants were regenerated 14 days after inoculation, 42 days after inoculation, and 55 days after inoculation. These results demonstrate that that sugarcane plantlets can be produced by the direct regeneration system using liquid medium. The type of support used determined the best liquid medium conditions for use with the direct regeneration system.

Example 17: Direct Transformation Using Liquid Medium

Bioreactors can be used for micropropogation of several crop types in liquid medium, including sugarcane. However, direct transformation of sugarcane in a liquid medium with or without a bioreactor system has not been reported. In this study, transgenic sugarcane plants were successfully produced in liquid medium via direct Agrobacterium-mediated DNA delivery into non-induced leaf whorl explants.

A. Direct Sugarcane Regeneration Process Using the Bioreactor RITA® Four treatments were tested based on frequency and duration that the explants stayed in contact with liquid medium: treatment 1 - 1 minute each 3 hours (lmin/3hrs); treatment 2 - 1 minute each 6 hours (lmin/6hrs); treatment 3 - 1 minute each 12 hours (lmin/12hrs); treatment 4 - 1 minute each 24 hours (lmin/24hrs).

In this experiment, the leaf whorl explant preparation, the co-culture medium, auxin pulse, regeneration and rooting medium were conducted as described. All explants were kept for 3 days into co-culture medium and them were transferred to Bioreactor RITA® system.

Seven days after inoculation, the treatments with the cycles lmin/3hrs and lmin/6hrs showed the highest oxidation rates of the explants. Although more explant oxidation was observed in the 1 min/3hrs cycle conditions, subsequently the material presented the best behavior.

Fourteen days after inoculation, the presence of globular structures were observed in treatments with lmin/3hrs, lmin/6hrs and lmin/12h. In this phase, the formation and development of globular structures was observed in the bioreactor RITA® conditions at lmin/3hrs, lmin/6hrs and lmin/12hrs, except with the lmin/24hrs conditions.

Twenty-one days after inoculation, lmin/3hrs and lmin/6hrs treatments showed more developed structures than the treatment lmin/12hrs. In the lmin/24hrs treatment, a delay in globular structure formation was observed.

Twenty-eight days after inoculation, and In the lmin/3hrs treatment and lmin/6hrs treatment (not shown) explants were more developed than the lmin/12hrs treatment. After the lmin/24hrs treatment, the explants still were forming globular structures and were not developed in shoots.

Sixty- five days after inoculation it was observed that the cycle conditions lmin/3hrs showed the best sugarcane regeneration and multiplication on the direct regeneration system.

Plantlets from the treatments lmin/3hrs and lmin/6hrs were transferred for rooting medium (MENRY). Plantlets exposed to the lmin/12hrs and lmin/24hrs cycles were not available for the rooting process. Direct sugarcane regeneration using Bioreactor RITA® produced good results using the 1 min/3 hrs and 1 min/ 6hrs cycle conditions.

Example 18: Direct Transformation of Sugarcane Genotype CV6984 using Liquid Medium

(Bioreactor RITA®)

In this study, different RITA® conditions were tested using the genotype CV6984 in the direct transformation system. The treatments were based on frequency and duration that the explants stayed in contact with liquid medium, 1 minute per 3 hour time period (lmin/3hrs), 1 minute per 6 hour time period (lmin/6hrs), 1 minute per 12 hour time period (lmin/12hrs) and 1 minute per 24 hour time period (lmin/24hrs). Twelve conditions were tested, starting with the explants in the Bioreactor RITA® during different phases and varying the frequency that the explants stayed in contact with the liquid medium. For all the treatments, after infection the explants were maintained for 3 days in co-culture phase, and then were transferred or not to the Bioreactor according to the phase listed below.

Table 10. Conditions tested in the direct transformation process with the CV6984 genotype.

A binary plasmid vector conferring tolerance to glyphosate, as well as a reporter gene which encodes GFP was used to transform leaf whorl explants using standard molecular biological techniques known to one of ordinary skill in the art, as described above.

Leaf whorl explant preparation: Sugarcane top stalks were obtained from 8 month, 8 day-old plants grown in field conditions of the genotype CV6984 and prepared as described. Infection of non-cultured leaf whorl explants: 18 explants were added into 20 ml of the Agrobacterium inoculum. All explants obtained from the top stalks were transformed. The ratio between the Agrobacterium and leaf whorl explants was about 20 ml: 18 leaf whorl explants. Agrobacterium was used at OD₆60nm of 0.8. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour. During the inoculation procedure, all the explants were sonicated for 5 min at 45 kHz, centrifuged at 650 g for 20 min at 4°C and then placed under 700 mmHg vacuum for 20 min (10 min with the pump on and 10 min with the pump off).

Co-culture phase: After the infection period the Agrobacterium suspension was removed from the explants by blotting them briefly on sterile the filter paper prior to being placed onto co- culture medium according to Example 15.

Auxin pulse phase: At the end of the co-culture period the explants of some plates were moved into a regeneration solid medium (Delay-3) containing MS salts; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 300 mg/L Timentin; MS vitamins; 100 μΜ acetosyringone; 0.2% phytagel and other plates were moved onto Delay-3 Liquid, with the same content but without the use of phytagel.

Regeneration and selection medium: The other plates were transferred onto a selection solid medium (SELA33) containing: MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 100 μΜ acetosyringone; 0.2% phytagel or a selection liquid medium (SELA33 Liquid) with the same content but without the use of phytagel. The pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving. The plates were placed under a 16 h photoperiod at 26±2°C for 4 days at a light intensity of 0.4 μΕ m -^"2 s -^"1.

Regeneration only medium: After the auxin pulse phase, explants from Delay-3 solid medium or Delay-3 Liquid medium were transferred onto regeneration medium Delay- 1 solid containing: MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 300mg/L Timentin; 2 μΜ copper sulphate 0.2% phytagel or Delay- 1 Liquid with the same content but without the use of phytagel. 21 days after inoculation, the tissues were moved onto a medium with a lower BAP concentration until the end of the process, with phytagel (DELAY01 - 01BAP) or without phytagel (DELAY01 _B AP Liq) . Regeneration and selection medium: The explants from SELA33 solid medium or SELA33 Liquid were moved onto glyphosate selection SEL33 solid containing: MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; l .Omg/L BAP; 0.1 mg/L NAA; 2 μΜ copper sulphate; 0.1 μΜ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 μΜ glyphosate; 0.2% phytagel or SEL33 Liquid with the same content but without the use of phytagel, according to the treatments (Table 10).

Twenty-one days after inoculation, the tissues were moved onto a medium with a lower BAP concentration until the end of the process, with phytagel (SEL33-01B) or without phytagel (SEL33-01B Liq). The pH of media was adjusted to 5.8 with 1 M NaOH before autoclaving.

Subcultures and light conditions: At the first regeneration/selection phase (7 days after infection), the plates from all treatments were put under 0.4 μΕ m^"2 s^"1 for 3 days; 2 μΕ m^"2 s^"1 for 2 days and than 2 days at 10 μΕ m^"2 s^"1. From day 14, the tissue was left 2 to 1 day at 0.4 μΕ m^"2 s^"1 after each subculture (2 days: 5 subcultures, from day 14 until day 35; 1 day: from the 35^th day to the remaining time of the process) and was then put under total light condition (30 μΕ m^"2 s i or 60 μΕ m^"2 s^"1). All the treatments from RITA® were placed under a 16 h photoperiod at 26±2°C in a gradative light intensity supplied by Gro-lux® lamps (Sylvania). At the first regeneration/selection phase (7 days after infection), all treatments were put under 10 μΕ m^"2 s^"1 for 7 days. From day 14, the tissue was left under total light conditions (30 μΕ m^"2 s i or 60 μΕ m-V).

Rooting phase: The positive events were moved into flasks containing the solid rooting medium containing: MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IBA; ImM isoleucine; 0.1 μΜ of each amino acid (tyr, phe, trp); 300 mg/L Timetin; 0.2% phytagel). The pH of the medium was adjusted to 5.8 with 1 M NaOH before autoclaving. The rooting flasks were placed under a 16 h photoperiod at 26±2°C). All the plantlets were kept under these conditions for approximately 10-15 days and then transferred to greenhouse.

Twenty-one days after infection, regenerable stable structures were observed in all treatments. In this phase all of the treatments showed results better or similar than standard condition (T13 solid medium) for the genotype tested.

Table 11 shows phenolic compounds released into the regeneration and selection liquid medium. In this phase transformed events were observed in the immersion cycle time of treatment T2; T5; T6 and T12 and putative event T7. Putative events were also observed under standard conditions (T13 solid medium). In treatments with more cycle immersion, like lmin/3hrs or 1 min/6hrs, more phenolic compounds were released into the medium, possibly due to more frequent washing in these cycles. A decrease in pH in these cycles when compared with lmin/12hrs and 1 min/24hrs was also observed. The 1 min/3hrs the immersion cycle can therefore be used to obtain earlier events and move to a longer cycle, for example 1 min/12hrs. All the treatments were exposed to the same subculture conditions.

Table 11. Phenolic compounds released during the direct transformation process with the CV6984 genotype for the twelve conditions tested and shown in Table 10.

Table 12 shows the transformation frequency of genotype CV-6984 after direct regeneration using liquid medium. Transformed events on the lmin/3hrs and lmin/6hrs conditions were found to be ready for rooting phase around 77 days after inoculation. Table 12. Transformation frequency (TF%) on the genotype CV-6984. Events 70 days after inoculation and under selection medium.

Seventy-seven days after inoculation an estimate of the number of plantlets per RITA® in regeneration medium was made. Table 13 shows the estimated amount of plantlets per Bioreactor RITA® in the direct regeneration system. Induction of somatic embryos from leaf whorl explants yielded a large number of plantlets on the liquid medium. An average of 105 plantlets per explant was recorded for genotype CV-6984 on the direct regeneration medium.

Table 13. Estimated amount of plantlets per Bioreactor RITA® using the direct regeneration system.

These data demonstrate the successful production of transgenic sugarcane plants using a liquid medium (Bioreactor RITA® system) with direct Agrobacterium-mediated DNA delivery into non-induced leaf whorl explants. Whole plantlets can be obtained using the direct regeneration system without the necessity of transferring the shoots to rooting medium one. Induction of somatic embryos from leaf whorl explants yielded a large number of plantlets using the liquid medium. An average of 105 plantlets per explant was recorded for genotype CV-6984 on the direct regeneration medium.

Claims

WHAT IS CLAIMED IS:

Claim 1. A method of transforming sugarcane tissue or a cell thereof, comprising:

a) inoculating sugarcane tissue or a cell thereof with Agrobacterium sp. comprising a nucleic acid molecule of interest; and

b) co-cultivating the Agrobacterium-moculatQd sugarcane tissue or a cell thereof to produce a transformed sugarcane tissue or cell thereof comprising the nucleic acid molecule of interest.

Claim 2. The method of claim 1 , wherein the Agrobacterium is Agrobacterium tumefaciens (Rhizobium radiobacter) or Agrobacterium rhizogenes.

Claim 3. The method of claim 1 , wherein the nucleic acid molecule of interest is comprised within an expression cassette.

Claim 4. The method of claim 3, wherein the expression cassette comprises a selectable marker gene.

Claim 5. The method of claim 4, further comprising culturing the co-cultivated sugarcane tissue or cell thereof in the presence of a selection agent to select the transformed sugarcane tissue or cell thereof.

Claim 6. The method of claim 1 , further defined as comprising the step of

c) regenerating a transgenic sugarcane plant from said tissue or cell thereof, wherein the transgenic plant comprises the nucleic acid molecule of interest.

Claim 7. The method of claim 6, wherein regenerated shoots are produced from the transformed tissue or cell within about 20-50 days.

Claim 8. The method of claim 7, wherein a rooted plant is produced from the regenerated shoots within about 27-60 days.

Claim 9. The method of claim 6, wherein step c) is carried out without producing a callus from said tissue or cell thereof.

Claim 10. The method of claim 1 wherein the sugarcane tissue comprises a transverse midrib explant, a transverse leaf whorl explant, or a midrib longitudinal explant.

Claim 11. The method of claim 10, wherein the sugarcane tissue comprises a transverse leaf whorl explant.

Claim 12. The method of claim 1 , wherein the plant is regenerated in media comprising a cytokinin and an auxin.

Claim 13. The method of claim 12, wherein the cytokinin is BAP or kinetin.

Claim 14. The method of claim 12, wherein the auxin is 2,4-D, NAA or IBA.

Claim 15. The method of claim 12, wherein the media comprises about 0.1 mg/L to about 1.0 mg/L BAP.

Claim 16. A method of transforming plant tissue comprising:

a) inoculating plant tissue or a cell thereof with Agrobacterium sp. comprising a nucleotide sequence of interest;

b) co-cultivating the Agrobacterium-moculated plant tissue or a cell thereof to produce a transformed plant tissue or cell thereof; and

c) regenerating a transformed plant from the transformed tissue or cell thereof;

wherein the plant is regenerated without an intervening callus phase of growth, and wherein regeneration is performed by growth of plant cells in light comprising enhanced red wavelength and reduced blue wavelength.

Claim 17. The method of claim 16, wherein a regenerated plant is produced within about 30- 60 days of the start of co-cultivating.

Claim 18. The method of claim 16, wherein the plant is a monocot.

Claim 19. The method of claim 18, wherein the monocot is sugarcane (Saccharum officinarum or Saccharum officinarium).

Claim 20. The method of claim 16, wherein the light is provided by a Grow-lux® lamp.

Claim 21. The method of claim 16, wherein the light has an irradiance of about 0.4- 30 μΕ/m s.

Claim 22. The method of claim 16, wherein the light has an irradiance of about 30 μΕ/ηι²8.

Claim 23. The method of claim 21, wherein gradually increased light conditions are utilized during regeneration.

Claim 24. The method of claim 23, where in the gradually increased light conditions comprise: 0.4 μΕ/ηΛ for about 4 days, followed by 2 μΕ/ηΛ for about 4 days, followed by 10

2 2

μΕ/m s for about 4 days, followed by 30 μΕ/m s until a regenerated plant is obtained.

Claim 25. The method of claim 16, wherein the light is provided by a light-emitting diode (LED).

Claim 26. The method of claim 16, wherein the enhanced red wavelength comprises far red and deep red wavelengths.

Claim 27. The method of claim 26, wherein the deep red wavelength light has a wavelength of about 660nm, and the far red wavelength light has a wavelength of about 770nm.

Claim 28. The method of claim 16, wherein the plant is regenerated in in liquid medium.

Claim 29. The method of claim 16, wherein the plant is regenerated in medium comprising maltose and glucose.