WO2015099674A1 - Procédés de régénération et de transformation de canne à sucre - Google Patents

Procédés de régénération et de transformation de canne à sucre Download PDF

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WO2015099674A1
WO2015099674A1 PCT/US2013/077572 US2013077572W WO2015099674A1 WO 2015099674 A1 WO2015099674 A1 WO 2015099674A1 US 2013077572 W US2013077572 W US 2013077572W WO 2015099674 A1 WO2015099674 A1 WO 2015099674A1
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explants
days
medium
light
regeneration
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PCT/US2013/077572
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Charles Armstrong
David Duncan
Edson Luis Kemper
Silvia Balbão Filippi OLIVEIRA
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Monsanto Technology Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • 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.
  • sugarcane Sacharum spp., including S. officinarum and S. spontaneum
  • 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.
  • 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.
  • the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the nucleic acid molecule of interest is comprised within an expression cassette.
  • the expression cassette comprises a selectable marker gene.
  • 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.
  • regenerated shoots are produced from the transformed tissue or cell within about 20-50 days from the transformation of the cell.
  • a rooted plant may be produced from the regenerated shoots within about 27-60 days.
  • step c) is carried out without producing a callus from said tissue or cell thereof.
  • the sugarcane tissue that is transformed and/or regenerated comprises a transverse midrib explant, a transverse leaf whorl explant, or a midrib longitudinal explant.
  • the sugarcane tissue comprises a transverse leaf whorl explant.
  • 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.
  • a plant is regenerated in media comprising a cytokinin and an auxin.
  • the cytokinin is BAP or kinetin
  • the auxin is 2,4-D, NAA or IBA.
  • 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.
  • a regenerated plant is produced within about 30-60 days of the start of co- cultivating.
  • the plant is a monocot; in a particular embodiment the plant is sugarcane (Sacchcirum spp).
  • the light is provided by a Gro-lux® lamp; in certain embodiments the light has an irradiance of about 0.4- 30 ⁇ m "2 s "1 ; in particular embodiments the light has an
  • gradually increased light conditions are utilized during regeneration.
  • the gradually increased light conditions comprise: 0.4 ⁇ m “2 s “1 for about 4 days, followed by 2 ⁇ m “2 s “1 for about 4 days, followed by 10 ⁇ m “2 s _1 for about 4 days, followed by 30 ⁇ 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.
  • previously "recalcitrant" genotypes that have shown limited embryogenesis response and transformability via a callus-based approach may now be directly used as transformation targets.
  • 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.
  • DR direct transformation and regeneration systems
  • 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.
  • auxin pulse PULA1 medium
  • CC-03 medium co-culture with aux
  • 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®.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • explants such as from leaf whorl transverse segments may be prepared to minimize production of red pigment components.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • SYLVANIA Gro-lux® Standard and/or Wide Spectrum Lamps may be utilized.
  • 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.
  • 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.
  • CHS chalcone synthase
  • 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.
  • 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).
  • 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).
  • sugarcane cells e.g. Arencibia et al., Transgenic Res. 7:213- 222, 1998.
  • 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.
  • transformed cells are identified for further culturing and plant regeneration.
  • a selectable or screenable marker gene with a transformation vector.
  • 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.
  • 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
  • the tissue employed as target for Agrobacterium-mediated transformation may affect transformation and regeneration efficiency.
  • numerous genotypes of sugarcane were successfully regenerated.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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 ⁇ , 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 1 ⁇ 2 strength MS macro salts; 1 mg/L IBA; 4% sucrose; ImM isoleucine; 0.1 ⁇ 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.
  • 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.
  • 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 ⁇ m "2 s "1 or gradually increased light conditions, e.g., 0.4 ⁇ m “2 s “1 for 4 days, 2 ⁇ m “2 s “1 for 4 days, 10 ⁇ m “2 s “1 for 4 days and then 30 ⁇ 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 1 ⁇ 2 strength MS macro salts; 1 mg/L IB A; 4% sucrose; ImM isoleucine; 0.1 ⁇ 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.
  • 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.
  • 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 OD 6 60nm 0.8 comprising a construct conferring glyphosate tolerance.
  • 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 ⁇ 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.
  • agarose SIGMA agarose A6013 type I, low EEO
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ glyphosate; 100 ⁇ 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
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ 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 ⁇ m " 2 s “ 1 ; 4 days in 2 ⁇ m “ 2 s “ 1 ; 4 days in 10 ⁇ m " 2 s “ 1 ; and then 30 ⁇ m " 2 s " 1 for the remaining time of culture.
  • Rooting Positive events displaying growth and regeneration were moved onto rooting medium containing 1 ⁇ 2 strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IB A; 1 mM isoleucine; 0.1 ⁇ 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
  • 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 OD 6 60nm 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 ⁇ acetosyringone (3,5-dimethoxy-4- hydroxyacetophenone) and the density was adjusted to an OD 6 60nm 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).
  • Sugarcane top stalks were obtained from 9 1 ⁇ 2 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 N 6 -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 ⁇ 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.
  • 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.
  • NAA a-napthaleneacetic acid
  • 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.
  • 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 ⁇ 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
  • 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
  • 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 1 ⁇ 2 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.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ 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.
  • 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 ⁇ m "2 s "1 ; 4 days in 10 ⁇ m "2 s “1 ; and then 30 ⁇ 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).
  • 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.
  • 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
  • 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 63 ⁇ 4 to 11 months old plants grown in field conditions.
  • auxin e.g. CC-03 medium
  • auxin delay medium e.g. Delay-3 medium
  • regeneration medium e.g. Delay-2 medium
  • auxin containing medium e.g. PULA1 medium
  • auxin delay medium for 2 days and then moved to regeneration medium for the remaining time of the experiment.
  • auxin pulse e.g. PULA1 medium
  • auxin pulse e.g. CC-03 medium
  • regeneration medium e.g. Delay-2
  • 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 ⁇ 61 ⁇ 2 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.
  • Auxin pulse 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.
  • Co-culture & auxin pulse 12 inoculated explants were co-cultured on solid medium containing agarose (Agarose Sigma A6013 Type I, Low EEO) 5.5 g/L; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3 mg/L 2,4-D; ⁇ 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 100 ⁇ acetosyringone. The pH of the medium was adjusted to 5.2 with 1 M NaOH before autoclaving.
  • agarose Agarose Sigma A6013 Type I, Low EEO
  • 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;
  • 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 ⁇ 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 ⁇ m "2 s “1 ; 4 days in 2 ⁇ m “2 s “1 ; 4 days in 10 ⁇ m “2 s “1 ; and then 30 ⁇ 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).
  • the buffer (100 mM NaH 2 P0 4 .H 2 0; 0.5 mM K 4 Fe(CN) 6 .3H 2 0; 10 mM Na 2 EDTA.2H 2 0; 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.
  • 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.
  • 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.
  • Example 9 Direct Agrobacterium -media ted T-DNA delivery into leaf whorl explants of different sugarcane genotypes
  • 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 OD 6 60nm 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 ⁇ 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 ⁇ 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
  • Gro-lux® lamp Sylvania
  • 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 ⁇ 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 ⁇ m "2 s “1 ; 4 days at 2 ⁇ m “2 s “1 ; 4 days at 10 ⁇ m “2 s “1 ; and then 30 ⁇ m "2 s "1 for the remaining time of the experiment.
  • the tissue was subcultured to fresh medium every 14 days.
  • Rooting phase Positive events were moved onto rooting medium containing 1 ⁇ 2 strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IBA; lmM isoleucine; 0.1 ⁇ 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
  • 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 BiosystemsTM product, Life TechnologiesTM). 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.
  • 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 BiosystemsTM product, Life TechnologiesTM).
  • 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
  • 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.
  • genotypes have particular characteristics of plantlet development.
  • commercial genotype Line 1 of FIG. 8
  • 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.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each amino acid aromatic (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; and 0 ⁇ , 5 ⁇ , 10 ⁇ , 20 ⁇ , 33 ⁇ , 66 ⁇ or 100 ⁇ 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
  • selection phase I "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 ⁇ copper sulphate; 0.1 ⁇ of each amino acid aromatic (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 0 ⁇ , 5 ⁇ ,10 ⁇ , 20 ⁇ , 33 ⁇ , 66 ⁇ or 100 ⁇ glyphosate; 100 ⁇ 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
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 0 ⁇ , 5 ⁇ ,10 ⁇ , 20 ⁇ , 33 ⁇ , 66 ⁇ or 100 ⁇ glyphosate; 0.2% phytagel. The ⁇ 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 ⁇ m "2 s “1 ; 4 days in 2 ⁇ m “2 s “1 ; 4 days in 10 ⁇ m - " 2 s- “ 1 ; and then 30 ⁇ 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 1 ⁇ 2 strength MS salts and vitamins; 4.0% sucrose; 1.0 mg/L IBA; 1 mM isoleucine; 0.1 ⁇ 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 ⁇ 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.
  • 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.
  • 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.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ glyphosate; 100 ⁇ 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 ⁇ m "2 s "1 with light supplied by Gro-lux® lamps (Sylvania).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 OD 6 60nm of 0.8. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour.
  • 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.
  • CC-03 co-culture & auxin pulse medium
  • CC-02 co-culture & regeneration medium
  • 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 ⁇ 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 1.0 mg/L BAP; 0.1 mg/L NAA; 100 ⁇ 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.
  • MS salts MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ glyphosate; 100 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ 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.
  • 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.
  • no GFP -positive stable expression was observed after infection of explants after pre-culture (3 or 6 days) in all 6 S. spontaneum tested.
  • 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 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.
  • 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 ( ⁇ 84-003; CHITTAN; FIJI44; KHAM; and NG77-065) only in non-induced leaf whorl explants.
  • 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.
  • 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.
  • 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 ⁇ 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 ⁇ copper sulphate; 300 mg/L Timentim; MS vitamins; 100 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 100 ⁇ 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 ⁇ 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 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 0.2% phytagel.
  • the pH of both media was adjusted to 5.8 with 1 M NaOH before autoclaving.
  • 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 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ 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 ⁇ m - " 2 s- " 1 for 3 days; 2 ⁇ m - " 2 s- " 1 for 2 days and than 2 days at 10 ⁇ m - " 2 s- " 1.
  • the tissue was left 2 to 1 day at 0.4 ⁇ 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 ⁇ m "2 s "1 or 60 ⁇ 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 ⁇ 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.
  • 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 ⁇ m "2 s "1 ; 2 ⁇ nfV 1 ; 10 ⁇ m ' V 1 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).
  • 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 33 ⁇ concentration of glyphosate.
  • the transformed explants became oxidated in all treatments. This oxidation after agroinfection is a characteristic of the CV7231 genotype.
  • the explants under cool white seemed to be more oxidated than any of the other treatments.
  • 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.
  • 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.
  • 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 ⁇ m - " 2 s " 1 instead of 30 ⁇ m ' V 1 ) 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.
  • explants were kept under twilight conditions (0.4 ⁇ 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.
  • twilight conditions 0.4 ⁇ m " s "
  • UV light 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.
  • 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 ⁇
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 100 ⁇ 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 ⁇ 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.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 0.2% phytagel.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ 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 ⁇ 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 ⁇ m ' V 1 , even in the Gro-lux® treatment (as the standard protocol).
  • 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.
  • 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 ⁇ 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
  • 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.
  • 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 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 0.2% phytagel.
  • 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 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 0.2% phytagel.
  • 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (Tyrosine, Phenylalanine, Tryptophan); 300 mg/L Timentim; 33 ⁇ glyphosate; 0.2% phytagel; 10-5 M abscisic acid (ABA).
  • 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 ⁇ m "2 s "1 or 60 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • adding ABA to the maltose/glucose medium resulted in promotion of shoot formation in both control and selection explants.
  • 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.
  • DR direct transformation system
  • 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; 1 ⁇ 2 MS salts; MS vitamins; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 100 ⁇ 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.
  • agarose Sigma A6013 Type I, low EEO
  • 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 ⁇ 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.
  • PULA 1 Liq liquid medium
  • 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
  • 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 ⁇ 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.
  • the explants 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 ⁇ copper sulphate, until the end of the process.
  • 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 ⁇ copper sulphate, until the end of the process.
  • the tissue was left 2 to 1 days at 0.4 ⁇ 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 ⁇ m "2 s "1 or 60 ⁇ 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 ⁇ 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.
  • Bioreactors can be used for micropropogation of several crop types in liquid medium, including sugarcane.
  • liquid medium including sugarcane.
  • direct transformation of sugarcane in a liquid medium with or without a bioreactor system has not been reported.
  • transgenic sugarcane plants were successfully produced in liquid medium via direct Agrobacterium-mediated DNA delivery into non-induced leaf whorl explants.
  • 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.
  • 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 OD 6 60nm of 0.8. The explants remained in contact with the Agrobacterium solution for about 40 minutes to 1 hour.
  • 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 ⁇ copper sulphate; 300 mg/L Timentin; MS vitamins; 100 ⁇ acetosyringone; 0.2% phytagel and other plates were moved onto Delay-3 Liquid, with the same content but without the use of phytagel.
  • Delay-3 containing MS salts; 3.0% sucrose; 150 mg/L citric acid; 3.0 mg/L 2,4-D; 2 ⁇ copper sulphate; 300 mg/L Timentin; MS vitamins; 100 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ glyphosate; 100 ⁇ 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 ⁇ 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 ⁇ 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 ⁇ copper sulphate; 0.1 ⁇ of each aromatic amino acid (tyrosine, phenylalanine, tryptophan); 300 mg/L Timentin; 33 ⁇ glyphosate; 0.2% phytagel or SEL33 Liquid with the same content but without the use of phytagel, according to the treatments (Table 10).
  • 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 ⁇ m "2 s “1 for 3 days; 2 ⁇ m “2 s “1 for 2 days and than 2 days at 10 ⁇ m “2 s “1 . From day 14, the tissue was left 2 to 1 day at 0.4 ⁇ 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 ⁇ m "2 s i or 60 ⁇ m "2 s "1 ).
  • 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 ⁇ 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.
  • Table 11 shows phenolic compounds released into the regeneration and selection liquid medium.
  • Table 11 shows phenolic compounds released into the regeneration and selection liquid medium.
  • Putative events were also observed under standard conditions (T13 solid medium).
  • 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 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.
  • 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.

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Abstract

L'invention concerne des procédés de tranformation et de régénération améliorées de plantes monocotylédones telles que la canne à sucre, permettant d'obtenir une production rapide et efficace de plantes transformées. L'invention concerne également des procédés améliorés de préparation d'explants, de synchronisation d'infection, ainsi que des procédés de manipulation de qualité de lumière et d'éclairement énergétique et de culture d'explants. Les procédés selon l'invention permettent une régénération améliorées de nombreux génotypes de la canne à sucre et d'autres monocotylédones.
PCT/US2013/077572 2013-12-23 2013-12-23 Procédés de régénération et de transformation de canne à sucre WO2015099674A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109644873A (zh) * 2019-02-01 2019-04-19 云南省农业科学院甘蔗研究所 一种甘蔗热带种胚性细胞团诱导方法
US11180770B2 (en) 2017-03-07 2021-11-23 BASF Agricultural Solutions Seed US LLC HPPD variants and methods of use
US11371056B2 (en) 2017-03-07 2022-06-28 BASF Agricultural Solutions Seed US LLC HPPD variants and methods of use
CN114847115A (zh) * 2021-02-03 2022-08-05 广西大学 甘蔗黑穗病菌高通量快速简易接种方法及其应用
WO2023036984A1 (fr) 2021-09-13 2023-03-16 Plantibodies Organisme génétiquement modifié pour la production de protéines recombinées
CN117925690A (zh) * 2024-01-15 2024-04-26 云南省农业科学院甘蔗研究所 一种农杆菌介导割手密遗传转化的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030180952A1 (en) * 2000-04-28 2003-09-25 Sugar Research & Development Corporation Plant regeneration
US20110124107A1 (en) * 2008-07-23 2011-05-26 Temasek Life Sciences Laboratory Limited Method of high frequency regeneration of sorghum
US20130152232A1 (en) * 2010-06-24 2013-06-13 Synegenta Participatins AG Methods for Agrobacterium-Mediated Transformation of Sugar Cane
US20130255150A1 (en) * 2010-06-11 2013-10-03 Stanislaw Karpinski Method and apparatus for plant protection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030180952A1 (en) * 2000-04-28 2003-09-25 Sugar Research & Development Corporation Plant regeneration
US20110124107A1 (en) * 2008-07-23 2011-05-26 Temasek Life Sciences Laboratory Limited Method of high frequency regeneration of sorghum
US20130255150A1 (en) * 2010-06-11 2013-10-03 Stanislaw Karpinski Method and apparatus for plant protection
US20130152232A1 (en) * 2010-06-24 2013-06-13 Synegenta Participatins AG Methods for Agrobacterium-Mediated Transformation of Sugar Cane

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11180770B2 (en) 2017-03-07 2021-11-23 BASF Agricultural Solutions Seed US LLC HPPD variants and methods of use
US11371056B2 (en) 2017-03-07 2022-06-28 BASF Agricultural Solutions Seed US LLC HPPD variants and methods of use
CN109644873A (zh) * 2019-02-01 2019-04-19 云南省农业科学院甘蔗研究所 一种甘蔗热带种胚性细胞团诱导方法
CN109644873B (zh) * 2019-02-01 2021-11-09 云南省农业科学院甘蔗研究所 一种甘蔗热带种胚性细胞团诱导方法
CN114847115A (zh) * 2021-02-03 2022-08-05 广西大学 甘蔗黑穗病菌高通量快速简易接种方法及其应用
WO2023036984A1 (fr) 2021-09-13 2023-03-16 Plantibodies Organisme génétiquement modifié pour la production de protéines recombinées
CN117925690A (zh) * 2024-01-15 2024-04-26 云南省农业科学院甘蔗研究所 一种农杆菌介导割手密遗传转化的方法

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