KR20080097993A - New Efficient Transformation Methods of Sunflower and Oilseeds Based on Positive Selection - Google Patents

Abstract

The present invention discloses a highly improved, reproducible, stable transformation and regeneration method that makes it possible to obtain 12-15% of transgenic plants. The present invention relates to a method for selecting genetically transformed sunflower explants based on the ability to use xylose as the only carbohydrate source. Also targeted with a vector comprising a gene encoding the enzyme xylose isomerase under functional combination of the nucleic acid sequence of the xylose isomerase gene, the vector construction for incorporation of the selection marker, and the heterologous regulatory sequence. A method of transforming a host plant by Agrobacterium mediated transformation is disclosed. Also disclosed is a method of selecting putative transformants with metabolic benefit using xylose as the only carbon source after transformation with the vector. Application of the positive selection method disclosed herein observed increased regeneration efficiency, better growth and survival. The present invention alleviates the disadvantages of negative selection methods, such as the potential environmental harm caused by unwanted removal of transformed cells and the dispersion of antibiotic and herbicide resistant genes.

Description

Novel efficient transformation method for sunflower and oil seeds based on poritive selection

Helianthus annuus ), or sunflower, is a difficult species that is very difficult to transform. We report a highly improved transformation method which results in a 2-3 fold increase in the number of transformants obtained compared to the cited prior art. The present invention relates to a method for selecting a genetically transformed sunflower explant based on the ability to use xylose as the only carbohydrate source. Encoding the enzyme xylose isomerase under control of heterologous regulatory sequences by nucleic acid sequences of xylose isomerase genes, vector construction for inclusion of selection marker genes, and Agrobacterium mediated transformation A method of transforming a target host plant with a vector comprising a gene is disclosed. Also disclosed is a method of selecting putative transformants with metabolic benefit using xylose as the only carbon source after transformation with the vector. By the positive selection method described herein, increased efficiency, better growth and survival of regeneration were observed. The present invention avoids the disadvantages of negative selection, such as the use of antibiotic or herbicide resistance genes.

Plant transformation is now a key research tool in plant biology and a practical tool for breed development: transformation of several plant species via Agrobacterium mediated transformation has become a common technique. Sunflower is a major crop used worldwide for the production of edible oils; Attempts to transform sunflower species have been underway for decades. However, sunflower is an unwieldy species that resists the transformation methods used to date. Attempts to transform sunflower species using Agrobacterium or biolistic methods or different selectable markers have resulted in very low transformation efficiency in the range of 0.6-6%.

When a population of plant cells is transformed, the selection of transformed cells is generally made using selection markers. A major technical challenge in plant transformation is the development of selection markers for the screening and selection of successfully transformed plants. This selection process is so important that the cost of screening the transformant often exceeds the cost of the transform itself. The selection of selectable marker genes, selection agents, their concentrations and timing of application is very important for the complete selection of transformed cells. On the other hand, regeneration should not be disturbed. Thus, a strict selection regime is desirable, but it should not interfere with the developmental potential of the transformant. The choice of conditions to harmonize the two needs appropriately is a technique in itself.

Selective markers identified to date can be distinguished into two types that allow transgenic plants or cells to be identified after transformation. They can be classified into positive markers and negative markers, respectively, which confer selective benefits or disadvantages. Negative selectable markers are markers that allow the selection of transformed cells, or tissue explants by their ability to grow in the presence of antibiotics or herbicides. In addition to the selection of transformants, such markers can be used to track the inheritance of foreign genes in a separate plant population.

This voice selection method has significant disadvantages. The most serious of the disadvantages are the non-transformed cells, where non-transformed cells are killed in the presence of phyto-toxic products and coherent tissue is used. The fact that their death blocks the supply of nutrients to the transformed cells or the fact that damaged or deceased non-transforming cells can secrete toxic compounds means that the transformed cells are also at risk of death. . Moreover, the presence of antibiotic resistance genes in the plants to be eaten is a concern. In addition, the selection of cells or tissues using negative selection requires precise timing of the expression of the introduced genes in connection with the selection process. Both transgenic and non-transforming cells die when the transformed cells are treated with the toxic compound before the detoxifying gene is expressed or before the gene product is produced in sufficient quantity to mitigate the action of the toxic compound. . If the selection is delayed, the selection of transformed cells or tissues shoots from non-transformed cells or tissues, for example, forming a barrier to penetration of the compound used to select the transformed cells. Or by callus formation.

The aforementioned disadvantages are overcome to a considerable extent by the method of positive selection, where the principle of operation is the opposite of negative selection. Rather than confer resistance to negative or toxic substances, positive selection involves giving the transformed cells a metabolic advantage or other competitive advantage over non-transformed cells. They identify and select genetically transformed cells without damaging or killing non-transformed cells in the population.

Sunflowers are important fat-seed crops and have been reported to be resistant to transformation and regeneration (Schrammeijer et al., Plant Cell Reports, 1990 9: 55-60). Schrammeijer et al. Selected transformants by their ability to grow on negative selection based agents, evaluating some potential problems with the regeneration of sunflower from various explants. Kanamycin and hygromycin are known to be detrimental to the organogeneric potential of sunflower, but are the most widely used selection agents for sunflower transformation (Everett et al., 1987; Muller et al., 2001).

A novel method of selecting transformants belonging to Helianthus annuus species, in particular, the step of conferring the transformed tissue explants with the ability to metabolize particular compounds, preferably xylose Methods for illustrating positive selection methods are disclosed herein; Transformed explants are selected by simply culturing them on a medium containing the aforementioned selection agent.

The selection methods disclosed herein relate to methods of screening and selecting transformed explants suitable for regeneration. Stable, accurate and high efficiency transformation and regeneration were achieved through the method of the present invention. The advantages of the techniques disclosed herein are clear enough to distinguish them from existing techniques.

Summary of the Invention

Sunflower is a major crop used worldwide for the production of edible oils. Attempts to transform sunflower species using Agrobacterium or bioolithic methods or different selectable markers resulted in very low transformation efficiency in the range of 0.6-6%. The present invention discloses a highly improved, reproducible and robust transformation and regeneration method that yields 12-15% of transgenic plants.

The present invention relates to a positive selection method that imparts the ability to metabolize xylose to transformed explants of Helianthus annuus species. One aspect of the invention relates to the construction of a vector construct comprising a selectable marker gene. Certain embodiments relate to the Agrobacterium-mediated method of transforming a constructed vector comprising said selectable marker gene into a host explant under conditions suitable for infection. Successfully transformed cells containing the gene of interest are induced with a positive effect that gives a selective metabolic advantage over non-transformed cells when incubated together in a suitable medium containing a selection agent. do. This selective benefit is attributed to the successful expression of the marker gene in the presence of the marker compound.

In another embodiment, the selective marker compound supplied to the population of transformed tissues is xylose that induces expression of the marker gene in the transformed explant.

Further aspects of the present invention also relate to polynucleotide molecules encoding proteins with biological activity of xylose isomerase. Specifically, the above aspect relates to the polynucleotide represented by SEQ ID NO: 3.

In addition, the transformation efficiency achieved with the disclosed method is 12-15%, and the regenerative efficiency has been demonstrated to be 2-3 times higher than the antibiotic (hygromycin) based method of negative selection.

Prior art:

U.S. Pat.No. 5,767,378, entitled "Mannose or Xylose positive selection," describes cells with metabolic benefits as a result of transformation in a eukaryotic population cultured in a medium comprising one or more compounds. To identify or select. The cells are transformed with nucleotide sequences or co-introduced nucleotide sequences that, upon expression, result in metabolism of compounds such as mannose or xylose.

Haldrup et al. (1998) reported that the xylose isomerase gene from Thermonan aerobacterium thermosulfurogenes can be used to select D-xylose as a selection agent in potato, tomato and tobacco species. It has been established that it enables the effective selection of transgenic plants (Plant Molecular Biology (1998), 37 (2), 287-296). The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. Studies on selection have shown that in potatoes and tomatoes, xylose isomerase selection is more efficient than kanamycin resistance selection, but the opposite effect is observed in tobacco plants.

A recent document titled "Positive Selection Marker Genes for Routine Plant Transformation" indicates that the selective marker genes used to date are antibiotic resistance genes or herbicide tolerance genes. It is mentioned. The document also describes a new class of marker genes that confer metabolic benefits to transformed cells relative to non-transformed cells.

Haldrup et al. (2001) disclose the selection of transformed plants based on the use of xylose isomerase as a selection marker (In Vitro Cellular & Developmental Biology: Plant (2001), 37 (2), 114-119.

The continuation part of U. S. Patent No. 5,767, 378 also includes introducing a desired nucleotide sequence or co-introduced nucleotide sequence into the cell's genome when the inert compound is supplied to the population of cells. In a method of selecting a genetically transformed cell from a population of cells, inducing a positive effect that gives the transformed cell a competitive advantage so that the transformed cell can be identified from a population of non-transformed cells. Novel glucuronide compounds, including cytokinin glucuronide compounds to be used.

Hou et al. Disclose the use and removal of selectable marker genes in transgenic plants. This document focuses on positive selection markers in transgenic plants using isopentenyl transferase gene, xylose isomerase gene, phosphomannose-isomerase gene and beta-glucuronidase gene. And to the use and removal of selectable marker genes in transgenic plants. Also disclosed are methods of removing marker genes using cotransformation, site-specific recombination systems, transposon-mediated reposition and homologous recombination.

The selection method disclosed herein is clear enough to be distinguished from the above cited prior art documents. The present invention specifically aims at a method of positive selection of transformed plant explants belonging to the plant species Helianthus annus. In addition, the selectable marker gene encoding the protein xylose isomerase was isolated from individual Schizochytrium and modified appropriately for expression in host plant species. It should be particularly noted that high transformation efficiency in sunflower plants has been reported rarely. More specifically, several issues have been noted for the regeneration of sunflower from various explant types (Schrammeijer et al., Plant Cell Reports, 1990 9: 55-60).

We report a transformation and regeneration efficiency of 12-15%, which is 2-3 times higher than the efficiencies achieved to date.

Detailed description of the invention

The term "selectable maker gene" is a nucleotide sequence that is preferably introduced with the gene of interest, which confers a selective advantage to cells transformed with the selectable marker gene. Nucleotide sequence.

The term “selective agent” is a compound or nutrient in an inactive form or in the form of a precursor, in the absence of which, for example, the expression of the selectable marker gene is present in a form which is substantially biologically inactive with respect to the subject cell, When a marker is expressed or transcribed, it is a compound or nutrient that will be hydrolyzed or activated or metabolized to provide selective benefits to the genetically transformed cell comprising the gene of interest and thereby select the cell.

As used herein, the term "selective advantage" includes the terms selective, metabolic and physiological benefits and wherein the transformed cells have adverse conditions (non-transformation). This means that one can grow faster than the cells, or advantageously use substrates (eg, nutrient precursors, etc.) that are not available to cells with adverse conditions.

Figure 1. Complete xylose isomerase domain at 1.5 kb sequence of SC-1 desaturase

Figure 2. Homology of the xylose isomerase domain of the domain from SC-1

Figure 3. Map of pGEX-XI. To study expression in E. coli, the xylose isomerase gene was cloned between the BamHI and XhoI sites.

Induction of xylose isomerase in E. coli. Amplification of Transformed SEA Explants by XI Primer. Lane: I: Induced; U: not induced; 1,2,3: different clones with xylose isomerase gene; M: molecular weight (MW) marker.

A. Amplification of ORF from xylose isomerase clone.

B: Restriction by XhoI of pCAMBIA-XI. Note the release of the hpt gene from the XhoI site.

Figure 6. Substitution of hpt by XI in pCAMBIA-CO-XI.

Figure 7. A) Sucrose (35 gm / l) B) D-xylose (30 gm / l) and C) Untransformed sunflower SEA explants cultured in medium without a carbon source. Three weeks later, the food was photographed.

8. Germination observed in transformed SEA explants cultured in A) hygromycin (25 mg / l) and B) xylose (15 g / l) + sucrose (5 mg / l) after two weeks of selection.

A) Primary selection medium. B) & C) Elongation Media D) SEA explants on Rooting Media and E) & F) Plantlets in the Hardening stage.

10. Amplification of transformed SEA explants with XI primers.

Brief description of sequence listing:

SEQ ID NO: 1 Full length sequence of xylose isomerase transcript of SC1

Translated protein sequence of xylose isomerase of SEQ ID NO: SC1

SEQ ID NO: 3: Sequence of xylose isomerase after optimization of expression in plants

SEQ ID NO: 4: Sequence of full-length codon optimized Xylose isomerase transcript of SC1 fused in frame in pCAMBIA vector.

Example : One

Trausuto Kyrid ( Thraustochytrid ) Variants SC - From 1 Xylose this Somerase Cloning .

Total RNA was isolated from a three-day culture of Schizochytrium SC-1, a troutstochytrid isolated from the drainage of Goa. CDNA was synthesized from mRNA using Superscript Rnase (GibcoBRL). The cDNA was cloned into the NotI - SalI site of the pSPORTl vector and transformed into E. coli DHlOB.

The primary cDNA library consisted of 2 × 10 6 clones and the amplified library had a titer of 2 × 10 10 clones / ml. EST clones were randomly selected and inserts were amplified with SP6 and T7 primers. The 5'-terminus of the 2000 cDNA clones was randomly selected and sequenced by T7 primers. The 5 'terminal sequencing of the clones from the cDNA library of SC-1 was 1.5 kb encoding 1511 bp full length xylose isomerase ( xylA ) cDNA with 158 bp 5' UTR and 30 bp 3 'UTR. This led to the identification of cDNA clones. The cDNA has an ORF of 1481 bp.

The sequence of the CDS is given by SEQ ID NO: 1. The sequence of CDS is the sequence of the full length xylose isomerase transcript of SC-1. The sequence is translated into a protein consisting of 440 amino acids. The amino acid sequence of the translated protein is represented by SEQ ID NO: 2. The sequence shows 74% homology to xylose isomerase from Arabidopsis thaliana . The sequence comprises the complete xylose isomerase domain and is represented by the xylose isomerase of SC-1. The presence of the complete xylose isomerase domain in the 1.5 kb sequence of the SC-1 desaturase is shown in FIG. 1.

2 shows homology to the xylose isomerase domain of motifs from SC-1.

Example : 2

Xylose Isomerase  Codon optimization

The xylose isomerase ( xylA ) sequence of SC-1 uses codons which are relatively rarely used in plants; SC-1 mainly uses CGC to encode arginine, but only 9% of plant codons for Arg are CGC. Thus, CGC codons were substituted with more frequently used codons for arginine. Two multi-site directed mutagenesis were performed on clones to replace 9 nucleotides prior to introduction into plants. The optimized sequence is represented by SEQ ID NO: 3.

Example 3

Xylose Isomerase pGEX  To expression vectors Cloning

In order to confirm that the codon optimized gene transcription and translation that, the forward primer for the codon optimized gene from the SC-1 XylA pSPORTl includes a BamHI site (GGATCC 5'GCGC ATGGGTGAATTCTTTC3 ') and a reverse primer having a XhoI site (5 'GAAA CTCGAG CTTGTCGATTAAGAAATGTATTGGTT3'). Amplified PCR product (1323) was treated with BamHI and XhoI. pGEX-4T-3 is an expression vector used to express proteins as fusion proteins with 26-kDa glutathione S-transferase (GST under the control of a tac promoter). pGEX-4T-3 was treated with BamHI and Xhol and PCR products (treated with BamHI and XhoI) were directionally cloned between the two sites-the resulting clone was named pGEX-XI. A map of pGEX-XI is shown in FIG. 3.

In E. coli Xylose Isomerase  Expression of Fusion Proteins

PGEX-XI with xylose isomerase gene was transformed into E. coli BL21. Transformed cells were selected on LB medium containing 100 g / ml of ampicillin. Random colonies were harvested and incubated overnight in LB medium containing 100 g / ml ampicillin. As above, 1% of the culture cultured overnight was used as a starter for inoculating 5 ml of LB culture solution containing 100 g / ml of ampicillin. Culture medium was O.D. Incubate until 600 reached about 0.6-0.7. 2 ml of each culture was induced with 1 mM IPTG for 3 hours. At the same time, 2 ml of culture was precipitated into pellets without induction as a control. Cell pellets were resuspended in 100ul sample buffer and 50ul loaded on a 10% SDS-PAGE gel. Induction is shown in FIG. 4.

In clones 1 and 2, a protein of 76 KDa, the expected size of the GST-xylose isomerase fusion protein, was observed, but not in uninduced cells. Thus, codon optimized xylose isomerase could be present in the correct reading frame and transcribed and translated.

In E. coli Xylose Isomerase  Evidence of the function of genes

E. coli strain AB477 (proA2, his-4, aroCl, thi-1, lacYl, galK2, xyl-5, mtl-1, lambda ^- supE44) is a xylose isomerase ( XyIA ) defective strain (Haldrup et at. , 1998 & 2001). Compelementation of the xylose isomerase gene in the mutant will allow this strain to grow using xylose, thereby demonstrating its function. E. coli strain AB477 was obtained from E. coli Genetic Stock Center, USA. E. coli strain AB477 plated on a MacConkey agar plate containing x% isomerase gene in pGEX-4T-3 with ampicillin (lOO μg / ml) containing l% (w / v) D-xylose The cells were transformed into competent cells. Transformants with the XyIA gene can ferment D-xylose and appear red on MacConkey agar plates containing xylose.

AB477 host cells, cells transformed with pGEX4T-3 and cells transformed with pGEX-XI were treated with MacConkey medium and xylose (1% w / v) and ampicillin (100) containing ampicillin (lOOµg / ml), respectively. And plated in medium containing [mu] g / ml). Red colonies with pGEX-XI were obtained in media containing xylose, but host cells and AB477 with pGEX4-T3 did not grow in the media. Thus, the codon optimized xylose isomerase gene isolated from SC-1 was translated and functioned.

Example : 4

Xylose Isomerase pCAMBIA By Cloning

pCAMBIA-1301 was derived from the pPZP vector. The vector comprises a hygromycin phospho-transferase (hpt) under the CaMV35S promoter and terminated by a CaMV35S polyA signal. The gene provides resistance to hygromycin so that the transformed cells can be selected in a hygromycin containing medium.

ORF of codon-optimized xylose isomerase (XylA) gene in pSPORT using forward primers (5 'CTCT CTCGAG CAACCATGGGTGAATTCTTTCC 3') & reverse primers (5'GAAA CTCGAG CTTGTCGATTAAGAAATGTATTGGTT 3 '), each containing an XhoI site Was amplified. The amplified fragment was treated with XhoI. pCAMBIA-1301 was simultaneously treated with XhoI to release the hpt gene and a vector purified from an agarose gel was ligated to the amplified fragment. Amplification of ORF from xylose isomerase clones and restriction by xhoI of pCAMBIA-XI is shown in FIG. 5.

The ligation mix was transformed with DHlOB. Transformed colonies were harvested and plasmids isolated from these colonies were amplified with XI primers and sequenced with the same primers. Thus, constructs with the XI gene were identified with the correct orientation and the correct frame. The sequence of the gene, cloned in the correct frame, is given by SEQ ID NO: 4.

FIG. 6 depicts the sequence of the full-length codon optimized xylose isomerase transcript of SC-1 embedded in the pCAMBIA vector in frame and the substitution by XI of hpt in pCAMBIA-CO-XI. The hpt gene encoding the cloned hygromycin phospho-transferase cloned between the XhoI sites of the vector pCAMBIA1301 was named pCAMBIA-XI, which was substituted by codon-optimized xylose isomerase instead of hygromycin.

Example : 5

Xylose Isomerase  Positive selection With marker  Transformation of sunflower used

If genetic material is to be introduced into a cell population by transformation, it is known that only some cells are successfully transformed. Identification and selection of transformed cells has traditionally been done by negative selection, and according to negative selection, transformed cells can survive in a medium containing an agent that they can degrade, but non-transformed cells Is killed in the medium. Hygromycin is the most commonly used antibiotic, and in the constructs used for transformation, the hygromycin phosphotransferase gene (hpt) provides resistance to transformed cells grown in the medium containing the antibiotic. .

These voice selection methods have some disadvantages. Non-transformed cells can die because of the presence of antibiotics in the growth medium, they can excrete toxins into the medium, which toxins are inhibitory and toxic to the transformed cells. Moreover, the presence of antibiotic resistance genes in the plants and microorganisms eaten is a concern for environmental groups and government authorities. In addition, the selection of cells or tissues using negative selection requires precise timing of the expression of the introduced genes in relation to the selection process. If transgenic cells are treated with toxic compounds before detoxifying genes are expressed or before sufficient gene product is produced to mitigate the action of toxic compounds, both transformed and non-transformed cells are Die. When selection is delayed, shoot or callus formation from non-transforming cells or tissues forms a barrier to penetration of the compound used to select, for example, the transformed cells. May be interrupted by

The aforementioned disadvantages are overcome to a considerable extent by the method of positive selection, which enables the selective growth of transformed cells without the introduction of antibiotic or herbicide resistance genes without damaging or killing non-transforming cells in the population. do.

Many plant species do not have the innate ability to metabolize xylose and thus do not proliferate in the medium where xylose is the only carbon source. Transformation of such species by constructs with xylose isomerase as the selection marker will give transformed cells the ability to use xylose as a carbon source.

Transformation and Selection in Sunflowers:

Plant material:

For this study, we used Helianthus annus CMS234B, the plant is a maintenance line for 234A, an inbred line widely used as a magnetic parent for the production of hybrid sunflowers in India. )to be. Helianthus Annus CMS234B is the parent of KBSH1 hybrid, a nationally-checked, short-lived variant (90-100 days) with an oil content of 42%, used as a maintenance system for all hybrid production. It is a parental line.

Seeds of Helianthus annus CMS 234B were peeled and sterilized for 2 minutes with 70% alcohol and for 4 minutes with 0.1% Mercury Chloride. The sterilized seeds were washed strongly 4-5 times with sterile water, followed by imbibition in water for 2 hours at 25 ° C. in BOD. Three different explants, shoot apical meristem, split embryonic axis and cotyledon, were tested for their regeneration efficiency. Among them, the split embryonic axis explants were identified as having the greatest potential for regeneration.

SEA ( Split embryonic axis ) Eating out  detach

After sterilization of the seeds, a thin, transparent papery translucent seed coat and a transparent drainage layer therein were carefully peeled off. A first incision was performed through the cotyledon parallel to the line to which two cotyledons were attached to incision the shoot meristem. If there is a primordial leave, it was removed by carefully cutting it from the tip of shoot apical meristem. The tissue including the root meristem was removed and the final incision was performed directly through the center of the shoot apical meristem. Explants were recovered on filter paper soaked.

As a carbon source Xylose  Screening of the ability to use

To determine whether sunflowers could use xylose as a carbon source, explants were cultured on regeneration media containing sucrose and xylose, respectively.

One hundred SEA explants were isolated to have a carbon source of a) 3.5% sucrose; b) 3% xylose; c) incubation for 3 weeks on MS medium (Murashige and Skoog Medium) containing 0.5 mg / l BAP, without carbon source. The effect of carbon source on explants was observed at the end of 3 weeks.

FIG. 7 shows A) sunflower SEA explants cultured in sucrose (35 gm / l), B) sunflower SEA explants cultured in D-xylose (30 gm / l), C) cultured on medium without carbon source Shows sunflower SEA explants. The explants were photographed after 3 weeks.

Explants cultured in a medium containing xylose died brown within 2-3 weeks. These explants looked similar to explants cultured in the absence of a carbon source in the medium. Thus, sunflower CMS234B SEA explants did not appear to be able to utilize xylose as a carbon source.

pCAMBIA - XI Transformation of Sunflowers

Transfection with Agrobacterium transfection )

Seeds were sterilized and SEA isolated as described above. For comparison, a strain comprising Agrobacterium tumefaciens strains GV3101 and pCAMBIA-XI comprising a binary vector pCAMBIA 1301 (with hpt gene) was used.

Agrobacterium cells from glycerol stock were incubated overnight in Luria-Bertani (LB) medium containing rifampicin (10 μg / ml), gentamycin (lOμg / ml) and kanamycin (50 μg / ml) and O.D600 Growth exponentially for 4-6 hours until reaching 1.0. The cells were washed by centrifugation at 6000 rpm for 5 minutes at 4 ° C. and resuspended in MS liquid to reach 1.0 D ₆₀₀ of 1.0. The excised explants were immediately transferred to a vacuum infiltration flask containing 25 ml of resuspended culture. Vacuum was applied for 15 minutes at 200 atm pressure and quickly released. The liquid was then completely removed from the culture and the explants were recovered on blotting paper. The explants were transferred to MS medium containing 0.5 mg / l BAP + 2 gm / l sucrose and left for 2 days at 26 ° C. for dark period.

Example : 6

Select and play

Positive selection and regeneration

After two days of co-cultivation, the grafts were thoroughly washed with Murashige and Skoog (1972) liquid medium containing 250 mg / l of Cefotaxime. The graft was then transferred to MS medium at pH 5.6 containing 0.5 mg / l BAP, 15 gm D-xylose, 5 gm / l sucrose, 250 mg / l Cytoxim, 8 gm / l agar and subjected to 16 hours of light conditions and Photoperiod with 8 hours of cancer conditions was maintained with two subculturings for 3 weeks. After the selection period, passages were made with Elonglation medium containing 1/2 Strength MS medium. After 2 weeks, elongated shoots were transferred to Rooting medium (1/2 MS medium containing 0.01 mg / l IBA). The rooted plants were hardened in water for 2 weeks before being transferred to the soil and vermiculite mixture.

Voice selection and playback:

After two days of co-culture, the grafts were thoroughly washed with MS medium containing 250 mg / l of Celltaxime. The explants were transferred to selection and regeneration medium (0.5 mg / l BAP, 15 mg / l hygromycin, 250 mg / l Celltaxim, MS medium containing 30 gm / l sucrose, 8 gm / l agar, pH 5.6). After the selection period, shoots were passaged for 2 weeks on kidney medium (1/2 Strength MS medium). Elongated shoots were transferred to rooting medium (1/2 MS medium containing 0.01 mg / l IBA) and rooted plants were subsequently transferred to the soil.

Comparison of positive and negative choices:

Explants were transformed with pCAMBIA 1301 (including hpt) and pCAMBIA-XI (substituting hpt with xylose isomerase) and selected on separate selection media containing different concentrations of hygromycin and xylose, respectively. . Grafts surviving the primary selection were transferred to the same medium for secondary selection and selected for 21 days. The grafts were then transferred to individual kidney media. Healthy shootlets were transferred to individual rooting media.

Observations are made in the following table:

A) pCAMBIA By -1301 Graft  Transformation. Hygromycin  Choice in Badges to Include

Concentration (mg / l) Number of infected explants Number of explants surviving primary selection (15 days) Number of explants surviving primary selection (21 days) Number of Explants Surviving in Secondary Choice (15 Days) PCR + ve number of explants Number of explants played with shootlets PCR + ve regenerant 30 100 45 22 12 12 One One 25 100 50 30 17 15 3 2 20 100 58 42 28 20 5 2 15 100 65 50 43 27 7 3

B) pCAMBIA - XI On by Eating out  Transformation. Xylose  Selection in medium containing

Concentration (mg / l) Number of infected explants Number of explants surviving primary selection (15 days) Number of explants surviving primary selection (21 days) Number of Explants Surviving in Secondary Choice (15 Days) PCR + ve number of explants Number of explants played with shootlets PCR + ve regenerant 30 100 50 38 20 18 5 5 25 100 65 55 40 28 7 6 20 100 72 65 57 30 12 7 15 100 80 70 60 28 20 10

Table 1: Transformation of Sunflower SEA Explants by pCAMBIA-1301 and pCAMBIA-XI

FIG. 8 shows germination observed in transformed SEA explants cultured in A) hygromycin (25 mg / l) and B) xylose (15 g / l) + sucrose (5 gm / l) after two weeks of selection. do. Hygromycin appears to have a stronger toxic effect on grafts than xylose. Eyes formed by explants growing in hygromycin media were stuted and wet. They did not grow well in the renal medium and only a few survived. Regeneration efficiency was very low even at low concentrations of hygromycin.

Better germination efficiency was observed on selected explants on xylose. Most explants formed normal shoot buds and these eyes survived the second choice better. As compared to 1-2 colonized dwarf shoots observed in hygromycin media, 2-3 healthy shoots per explant were observed in positive selection. Seedlings grown in D-xylose medium contained 90% green tissue as compared to seedlings grown in hygromycin. The graft also showed better survival and growth in kidney medium. The eyes were healthy and developed into young branches in the kidney medium.

We observed 2-3 times more number of regenerated seedlings using XI compared to using hpt for selection. Further improvement in regeneration efficiency was observed when 5 g sucrose and 15 g xylose were used in the selection medium after transfection. The following is a standardized protocol for the transformation and regeneration of sunflower with positive selection:

Peel the seeds of Helianthus Annus CMS 234B for 2 minutes with 70% alcohol, followed by 4 minutes with 0.1% mercuric chloride.

The sterilized seeds are washed 4-5 times strongly with sterile water, followed by infiltration in water for 2 hours at 25 ° C. in BOD.

Carefully remove cotyledon, radicle and leaf primordial to expose SEA (shoot apical meristem).

Infect SAG explants with Agrobacterium GV3101 culture containing AGT-D15-XI (OD600 = 1.0).

Perform vacuum infiltration for 15 minutes at 200 mbar.

Co-cultivation is carried out for 2 days in MS medium at pH 5.6 containing 100 mg / l myoinositol, 0.5 mg / l BAP, 2 g / l sucrose, 8 g / l agar.

The grafts are washed for 15 minutes in MS medium at pH 5.6 containing 35 g / l sucrose, 100 mg / l myoinitol, 250 mg / l Celltaxim, and blot dried.

Passage for 3 weeks in MS medium at pH 5.6 containing lOOmg / l myoinositol, 15 g / l xylose, 2 gm / l sucrose, 0.5 mg / l BAP, 250 mg / l Cytotaxime, 8 g / l agar .

Passage for 1 week in 1/2 MS medium at pH 5.6 containing 50 mg / l myoinositol, 15 g / l sucrose, 250 mg / l cetactaxi, 8 g / l agar.

Passage with 1/2 MS medium at pH 5.6 containing 50 mg / l Myoinositol, O.Olmg / l IBA, 250mg / l Celltaxim, 8g / l agar.

-Seedlings are finally transplanted into pots containing a mixture of red soil (10%) and vermiculite (90%).

9 shows seedlings in SEA grafts on primary selection medium, B) & C) SEA grafts on renal medium, D) SEA grafts on rooting medium, and E) & F) strengthening step.

Starting with 100 explants, transformation with pCAMBIA-1301 yielded 2-3 transgenic plants with the hpt gene. However, starting with the same number of explants, transformation with pCAMBIA-XI yielded 12-15 transgenic plants with the XI gene. The seedlings obtained through the selection of hygromycin were very few, and these seedlings were also very weak and did not grow into complete plants.

Example  7

Molecular  analysis

Seedlings obtained after transformation were screened for selectable markers by amplification with XI primers.

Table 2. Primers used for amplification of xylose isomerase genes.

Genomic DNA isolated from the transformed seedlings was amplified with the primers. Amplification conditions were as follows:

DNA lOOng

dNTP 200 μM

Primer 0.25 μM

MgCl ₂ mM

2.5 μl 1OX Buffer (Bangalore Genei)

Taq polymerase 1U

25 μl total volume

The cycling conditions used were as follows:

Applied amplification conditions.

10 shows amplification of transformed SEA explants with XI primers. 100 ng of genomic DNA of selected seedlings with pCAMBIA1301-XI was amplified with XI primers. Lanes 1-10: DNA samples from different explants; '+' Agrobacterium DNA; M: 1 kb ladder

Note: Amplification of nine explants using XI primers suggests that these samples have XI integrated into their genome.

About 90% of the selected explants showed integration of the XI genes into their genomes, as confirmed by amplification of the XI gene in the transformed explants.

Thus, we report a stable transformation efficiency of 12-15% using xylose isomerase for selection versus 0.2-6.2% transformation efficiency reported by other selection markers used to date worldwide. do.

Thus, we have demonstrated that the use of xylose isomerase of SC1 for positive selection is an efficient method for transformation of sunflower explants. This selection system was more efficient and produced a larger number of transgenic plants than traditional kanamycin and hygromycin based systems. The system also solves the problem of growing transformants from difficult crops, such as sunflowers, to successfully produce transformed sunflowers. Finally, the system also meets the needs of alternative selection markers in the development of genetically modified plants and avoids the use of antibiotic resistance markers.

                         SEQUENCE LISTING <110> Avestha Gengraine Technologies Pvt Ltd.        Gengraine Technologies Pvt.Ltd, Avestha        Morawala Patell, Villoo   <120> Positive selection method using Xylose Isomerase in Sunflower <130> 09-1-2005 <160> 7 <170> PatentIn version 3.3 <210> 1 <211> 1323 <212> DNA <213> Schizochytrium <220> <221> CDS (222) (1) .. (1323) <400> 1 atg ggt gaa ttc ttt ccc gag gtt ggc aag att gaa tac aag gga cca 48 Met Gly Glu Phe Phe Pro Glu Val Gly Lys Ile Glu Tyr Lys Gly Pro 1 5 10 15 ggc agc aac gat gtg ctt tcg tac agg tgg tat aac cct gat gaa gag 96 Gly Ser Asn Asp Val Leu Ser Tyr Arg Trp Tyr Asn Pro Asp Glu Glu             20 25 30 atc ctt gga aag aag atg aaa gac tgg ctt aag ttt tct gtc tgc ttt 144 Ile Leu Gly Lys Lys Met Lys Asp Trp Leu Lys Phe Ser Val Cys Phe         35 40 45 tgg cat act ttc cga ggc gtc ggc atg gac ccc ttt ggc aaa cct acg 192 Trp His Thr Phe Arg Gly Val Gly Met Asp Pro Phe Gly Lys Pro Thr     50 55 60 atc acc agc cgc ttc cag ggc gat gat gga tct gac tca gtc gaa aat 240 Ile Thr Ser Arg Phe Gln Gly Asp Asp Gly Ser Asp Ser Val Glu Asn 65 70 75 80 gcg ctc cgt cgc gta gac gcc gcc ttt gag cta ttt aca aag ctt ggc 288 Ala Leu Arg Arg Val Asp Ala Ala Phe Glu Leu Phe Thr Lys Leu Gly                 85 90 95 gtt gag tac tac agc ttt cat gat gtg gat gtt tct ccc gaa ggt gca 336 Val Glu Tyr Tyr Ser Phe His Asp Val Asp Val Ser Pro Glu Gly Ala             100 105 110 aca ctc aag gag aca aat gag aac ctt gac aag att acc gac cgc atg 384 Thr Leu Lys Glu Thr Asn Glu Asn Leu Asp Lys Ile Thr Asp Arg Met         115 120 125 cta gaa ctg caa aag aaa aca gga gtg aag ctt ttg tgg ggt aca gct 432 Leu Glu Leu Gln Lys Lys Thr Gly Val Lys Leu Leu Trp Gly Thr Ala     130 135 140 aac ctc ttt acc aac ccc cgc tac atg aac ggt ggc tcc acg aac ccg 480 Asn Leu Phe Thr Asn Pro Arg Tyr Met Asn Gly Gly Ser Thr Asn Pro 145 150 155 160 gac ccg aat gtc ttc att cga gct gct gca cag gtt aaa aag gct atc 528 Asp Pro Asn Val Phe Ile Arg Ala Ala Ala Gln Val Lys Lys Ala Ile                 165 170 175 gat gtt acg cac aag ctt ggt ggt caa ggt ttt gta ttc tgg ggt ggt 576 Asp Val Thr His Lys Leu Gly Gly Gln Gly Phe Val Phe Trp Gly Gly             180 185 190 cgc gaa ggc tac atg cac att ctc aac act gat gtt gtg cgt gag atg 624 Arg Glu Gly Tyr Met His Ile Leu Asn Thr Asp Val Val Arg Glu Met         195 200 205 aac cac tat gcc cag atg ctg aag atg gcg att gca tac aag aag aag 672 Asn His Tyr Ala Gln Met Leu Lys Met Ala Ile Ala Tyr Lys Lys Lys     210 215 220 atc ggc ttt gat ggt caa att ctt gtg gag cca aaa cct cgt gaa cca 720 Ile Gly Phe Asp Gly Gln Ile Leu Val Glu Pro Lys Pro Arg Glu Pro 225 230 235 240 atg aag cac caa tat gat tac gat gta caa acc gtg att ggg ttc ttg 768 Met Lys His Gln Tyr Asp Tyr Asp Val Gln Thr Val Ile Gly Phe Leu                 245 250 255 cga gag cat ggg ctt gaa aaa gaa gtc ctg ctg aat gtt gaa ccc aac 816 Arg Glu His Gly Leu Glu Lys Glu Val Leu Leu Asn Val Glu Pro Asn             260 265 270 cac acc cag ctt gcg ggc cac gaa ttt gag cat ggc ttt atc ttt gct 864 His Thr Gln Leu Ala Gly His Glu Phe Glu His Gly Phe Ile Phe Ala         275 280 285 gcc aaa ctt ggc atg ctt gga agc att gat gct aat acc ggt tct gag 912 Ala Lys Leu Gly Met Leu Gly Ser Ile Asp Ala Asn Thr Gly Ser Glu     290 295 300 agt ctt ggc tgg gac act gat gag ttc atc act gac cag act cat gcg 960 Ser Leu Gly Trp Asp Thr Asp Glu Phe Ile Thr Asp Gln Thr His Ala 305 310 315 320 act ctg ctg tgt cgc acg att att gag atg ggc ggt ttc aaa aaa ggt 1008 Thr Leu Leu Cys Arg Thr Ile Ile Glu Met Gly Gly Phe Lys Lys Gly                 325 330 335 ggc ctt aac ttt gat gcc aag gtg cga cgc gaa agt act gat cca gag 1056 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Glu Ser Thr Asp Pro Glu             340 345 350 gat ctc ttt atc gcg cac gtg gca tct atg gat gca ctt gct aaa ggt 1104 Asp Leu Phe Ile Ala His Val Ala Ser Met Asp Ala Leu Ala Lys Gly         355 360 365 ctt cgc aat gct gcc aaa ttg gtt gat gaa ggc cgt atg gct aag atg 1152 Leu Arg Asn Ala Ala Lys Leu Val Asp Glu Gly Arg Met Ala Lys Met     370 375 380 ctc gca gag cgc tac gct gga tgg gac tct gga cta ggt aag aga att 1200 Leu Ala Glu Arg Tyr Ala Gly Trp Asp Ser Gly Leu Gly Lys Arg Ile 385 390 395 400 gag gat ggc cag agt tcg ctt gac gag ctg gag gag cat gcg ctc cag 1248 Glu Asp Gly Gln Ser Ser Leu Asp Glu Leu Glu Glu His Ala Leu Gln                 405 410 415 aat gat gag gag ccc gct aag act tca gca aaa cag gag aag ttt att 1296 Asn Asp Glu Glu Pro Ala Lys Thr Ser Ala Lys Gln Glu Lys Phe Ile             420 425 430 gct gtt ctc aac caa tac att tct taa 1323 Ala Val Leu Asn Gln Tyr Ile Ser         435 440 <210> 2 <211> 440 <212> PRT <213> Schizochytrium <400> 2 Met Gly Glu Phe Phe Pro Glu Val Gly Lys Ile Glu Tyr Lys Gly Pro 1 5 10 15 Gly Ser Asn Asp Val Leu Ser Tyr Arg Trp Tyr Asn Pro Asp Glu Glu             20 25 30 Ile Leu Gly Lys Lys Met Lys Asp Trp Leu Lys Phe Ser Val Cys Phe         35 40 45 Trp His Thr Phe Arg Gly Val Gly Met Asp Pro Phe Gly Lys Pro Thr     50 55 60 Ile Thr Ser Arg Phe Gln Gly Asp Asp Gly Ser Asp Ser Val Glu Asn 65 70 75 80 Ala Leu Arg Arg Val Asp Ala Ala Phe Glu Leu Phe Thr Lys Leu Gly                 85 90 95 Val Glu Tyr Tyr Ser Phe His Asp Val Asp Val Ser Pro Glu Gly Ala             100 105 110 Thr Leu Lys Glu Thr Asn Glu Asn Leu Asp Lys Ile Thr Asp Arg Met         115 120 125 Leu Glu Leu Gln Lys Lys Thr Gly Val Lys Leu Leu Trp Gly Thr Ala     130 135 140 Asn Leu Phe Thr Asn Pro Arg Tyr Met Asn Gly Gly Ser Thr Asn Pro 145 150 155 160 Asp Pro Asn Val Phe Ile Arg Ala Ala Ala Gln Val Lys Lys Ala Ile                 165 170 175 Asp Val Thr His Lys Leu Gly Gly Gln Gly Phe Val Phe Trp Gly Gly             180 185 190 Arg Glu Gly Tyr Met His Ile Leu Asn Thr Asp Val Val Arg Glu Met         195 200 205 Asn His Tyr Ala Gln Met Leu Lys Met Ala Ile Ala Tyr Lys Lys Lys     210 215 220 Ile Gly Phe Asp Gly Gln Ile Leu Val Glu Pro Lys Pro Arg Glu Pro 225 230 235 240 Met Lys His Gln Tyr Asp Tyr Asp Val Gln Thr Val Ile Gly Phe Leu                 245 250 255 Arg Glu His Gly Leu Glu Lys Glu Val Leu Leu Asn Val Glu Pro Asn             260 265 270 His Thr Gln Leu Ala Gly His Glu Phe Glu His Gly Phe Ile Phe Ala         275 280 285 Ala Lys Leu Gly Met Leu Gly Ser Ile Asp Ala Asn Thr Gly Ser Glu     290 295 300 Ser Leu Gly Trp Asp Thr Asp Glu Phe Ile Thr Asp Gln Thr His Ala 305 310 315 320 Thr Leu Leu Cys Arg Thr Ile Ile Glu Met Gly Gly Phe Lys Lys Gly                 325 330 335 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Glu Ser Thr Asp Pro Glu             340 345 350 Asp Leu Phe Ile Ala His Val Ala Ser Met Asp Ala Leu Ala Lys Gly         355 360 365 Leu Arg Asn Ala Ala Lys Leu Val Asp Glu Gly Arg Met Ala Lys Met     370 375 380 Leu Ala Glu Arg Tyr Ala Gly Trp Asp Ser Gly Leu Gly Lys Arg Ile 385 390 395 400 Glu Asp Gly Gln Ser Ser Leu Asp Glu Leu Glu Glu His Ala Leu Gln                 405 410 415 Asn Asp Glu Glu Pro Ala Lys Thr Ser Ala Lys Gln Glu Lys Phe Ile             420 425 430 Ala Val Leu Asn Gln Tyr Ile Ser         435 440 <210> 3 <211> 1323 <212> DNA <213> Schizochytrium <220> <221> CDS (222) (1) .. (1323) <400> 3 atg ggt gaa ttc ttt ccc gag gtt ggc aag att gaa tac aag gga cca 48 Met Gly Glu Phe Phe Pro Glu Val Gly Lys Ile Glu Tyr Lys Gly Pro 1 5 10 15 ggc agc aac gat gtg ctt tcg tac agg tgg tat aac cct gat gaa gag 96 Gly Ser Asn Asp Val Leu Ser Tyr Arg Trp Tyr Asn Pro Asp Glu Glu             20 25 30 atc ctt gga aag aag atg aaa gac tgg ctt aag ttt tct gtc tgc ttt 144 Ile Leu Gly Lys Lys Met Lys Asp Trp Leu Lys Phe Ser Val Cys Phe         35 40 45 tgg cat act ttc cga ggc gtc ggc atg gac ccc ttt ggc aaa cct acg 192 Trp His Thr Phe Arg Gly Val Gly Met Asp Pro Phe Gly Lys Pro Thr     50 55 60 atc acc agc cgt ttc cag ggc gat gat gga tct gac tca gtc gaa aat 240 Ile Thr Ser Arg Phe Gln Gly Asp Asp Gly Ser Asp Ser Val Glu Asn 65 70 75 80 gcg ctc cgt cgt gta gac gcc gcc ttt gag cta ttt aca aag ctt ggc 288 Ala Leu Arg Arg Val Asp Ala Ala Phe Glu Leu Phe Thr Lys Leu Gly                 85 90 95 gtt gag tac tac agc ttt cat gat gtg gat gtt tct ccc gaa ggt gca 336 Val Glu Tyr Tyr Ser Phe His Asp Val Asp Val Ser Pro Glu Gly Ala             100 105 110 aca ctc aag gag aca aat gag aac ctt gac aag att acc gac cgt atg 384 Thr Leu Lys Glu Thr Asn Glu Asn Leu Asp Lys Ile Thr Asp Arg Met         115 120 125 cta gaa ctg caa aag aaa aca gga gtg aag ctt ttg tgg ggt aca gct 432 Leu Glu Leu Gln Lys Lys Thr Gly Val Lys Leu Leu Trp Gly Thr Ala     130 135 140 aac ctc ttt acc aac ccc cgt tac atg aac ggt ggc tcc acg aac ccg 480 Asn Leu Phe Thr Asn Pro Arg Tyr Met Asn Gly Gly Ser Thr Asn Pro 145 150 155 160 gac ccg aat gtc ttc att cga gct gct gca cag gtt aaa aag gct atc 528 Asp Pro Asn Val Phe Ile Arg Ala Ala Ala Gln Val Lys Lys Ala Ile                 165 170 175 gat gtt acg cac aag ctt ggt ggt caa ggt ttt gta ttc tgg ggt ggt 576 Asp Val Thr His Lys Leu Gly Gly Gln Gly Phe Val Phe Trp Gly Gly             180 185 190 cgt gaa ggc tac atg cac att ctc aac act gat gtt gtg cgt gag atg 624 Arg Glu Gly Tyr Met His Ile Leu Asn Thr Asp Val Val Arg Glu Met         195 200 205 aac cac tat gcc cag atg ctg aag atg gcg att gca tac aag aag aag 672 Asn His Tyr Ala Gln Met Leu Lys Met Ala Ile Ala Tyr Lys Lys Lys     210 215 220 atc ggc ttt gat ggt caa att ctt gtg gag cca aaa cct cgt gaa cca 720 Ile Gly Phe Asp Gly Gln Ile Leu Val Glu Pro Lys Pro Arg Glu Pro 225 230 235 240 atg aag cac caa tat gat tac gat gta caa acc gtg att ggg ttc ttg 768 Met Lys His Gln Tyr Asp Tyr Asp Val Gln Thr Val Ile Gly Phe Leu                 245 250 255 cga gag cat ggg ctt gaa aaa gaa gtc ctg ctg aat gtt gaa ccc aac 816 Arg Glu His Gly Leu Glu Lys Glu Val Leu Leu Asn Val Glu Pro Asn             260 265 270 cac acc cag ctt gcg ggc cac gaa ttt gag cat ggc ttt atc ttt gct 864 His Thr Gln Leu Ala Gly His Glu Phe Glu His Gly Phe Ile Phe Ala         275 280 285 gcc aaa ctt ggc atg ctt gga agc att gat gct aat acc ggt tct gag 912 Ala Lys Leu Gly Met Leu Gly Ser Ile Asp Ala Asn Thr Gly Ser Glu     290 295 300 agt ctt ggc tgg gac act gat gag ttc atc act gac cag act cat gcg 960 Ser Leu Gly Trp Asp Thr Asp Glu Phe Ile Thr Asp Gln Thr His Ala 305 310 315 320 act ctg ctg tgt cgt acg att att gag atg ggc ggt ttc aaa aaa ggt 1008 Thr Leu Leu Cys Arg Thr Ile Ile Glu Met Gly Gly Phe Lys Lys Gly                 325 330 335 ggc ctt aac ttt gat gcc aag gtg cga cgt gaa agt act gat cca gag 1056 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Glu Ser Thr Asp Pro Glu             340 345 350 gat ctc ttt atc gcg cac gtg gca tct atg gat gca ctt gct aaa ggt 1104 Asp Leu Phe Ile Ala His Val Ala Ser Met Asp Ala Leu Ala Lys Gly         355 360 365 ctt cgt aat gct gcc aaa ttg gtt gat gaa ggc cgt atg gct aag atg 1152 Leu Arg Asn Ala Ala Lys Leu Val Asp Glu Gly Arg Met Ala Lys Met     370 375 380 ctc gca gag cgt tac gct gga tgg gac tct gga cta ggt aag aga att 1200 Leu Ala Glu Arg Tyr Ala Gly Trp Asp Ser Gly Leu Gly Lys Arg Ile 385 390 395 400 gag gat ggc cag agt tcg ctt gac gag ctg gag gag cat gcg ctc cag 1248 Glu Asp Gly Gln Ser Ser Leu Asp Glu Leu Glu Glu His Ala Leu Gln                 405 410 415 aat gat gag gag ccc gct aag act tca gca aaa cag gag aag ttt att 1296 Asn Asp Glu Glu Pro Ala Lys Thr Ser Ala Lys Gln Glu Lys Phe Ile             420 425 430 gct gtt ctc aac caa tac att tct taa 1323 Ala Val Leu Asn Gln Tyr Ile Ser         435 440 <210> 4 <211> 1323 <212> DNA <213> Schizochytrium <220> <221> CDS (222) (1) .. (1323) <400> 4 atg ggt gaa ttc ttt ccc gag gtt ggc aag att gaa tac aag gga cca 48 Met Gly Glu Phe Phe Pro Glu Val Gly Lys Ile Glu Tyr Lys Gly Pro 1 5 10 15 ggc agc aac gat gtg ctt tcg tac agg tgg tat aac cct gat gaa gag 96 Gly Ser Asn Asp Val Leu Ser Tyr Arg Trp Tyr Asn Pro Asp Glu Glu             20 25 30 atc ctt gga aag aag atg aaa gac tgg ctt aag ttt tct gtc tgc ttt 144 Ile Leu Gly Lys Lys Met Lys Asp Trp Leu Lys Phe Ser Val Cys Phe         35 40 45 tgg cat act ttc cga ggc gtc ggc atg gac ccc ttt ggc aaa cct acg 192 Trp His Thr Phe Arg Gly Val Gly Met Asp Pro Phe Gly Lys Pro Thr     50 55 60 atc acc agc cgt ttc cag ggc gat gat gga tct gac tca gtc gaa aat 240 Ile Thr Ser Arg Phe Gln Gly Asp Asp Gly Ser Asp Ser Val Glu Asn 65 70 75 80 gcg ctc cgt cgt gta gac gcc gcc ttt gag cta ttt aca aag ctt ggc 288 Ala Leu Arg Arg Val Asp Ala Ala Phe Glu Leu Phe Thr Lys Leu Gly                 85 90 95 gtt gag tac tac agc ttt cat gat gtg gat gtt tct ccc gaa ggt gca 336 Val Glu Tyr Tyr Ser Phe His Asp Val Asp Val Ser Pro Glu Gly Ala             100 105 110 aca ctc aag gag aca aat gag aac ctt gac aag att acc gac cgt atg 384 Thr Leu Lys Glu Thr Asn Glu Asn Leu Asp Lys Ile Thr Asp Arg Met         115 120 125 cta gaa ctg caa aag aaa aca gga gtg aag ctt ttg tgg ggt aca gct 432 Leu Glu Leu Gln Lys Lys Thr Gly Val Lys Leu Leu Trp Gly Thr Ala     130 135 140 aac ctc ttt acc aac ccc cgt tac atg aac ggt ggc tcc acg aac ccg 480 Asn Leu Phe Thr Asn Pro Arg Tyr Met Asn Gly Gly Ser Thr Asn Pro 145 150 155 160 gac ccg aat gtc ttc att cga gct gct gca cag gtt aaa aag gct atc 528 Asp Pro Asn Val Phe Ile Arg Ala Ala Ala Gln Val Lys Lys Ala Ile                 165 170 175 gat gtt acg cac aag ctt ggt ggt caa ggt ttt gta ttc tgg ggt ggt 576 Asp Val Thr His Lys Leu Gly Gly Gln Gly Phe Val Phe Trp Gly Gly             180 185 190 cgt gaa ggc tac atg cac att ctc aac act gat gtt gtg cgt gag atg 624 Arg Glu Gly Tyr Met His Ile Leu Asn Thr Asp Val Val Arg Glu Met         195 200 205 aac cac tat gcc cag atg ctg aag atg gcg att gca tac aag aag aag 672 Asn His Tyr Ala Gln Met Leu Lys Met Ala Ile Ala Tyr Lys Lys Lys     210 215 220 atc ggc ttt gat ggt caa att ctt gtg gag cca aaa cct cgt gaa cca 720 Ile Gly Phe Asp Gly Gln Ile Leu Val Glu Pro Lys Pro Arg Glu Pro 225 230 235 240 atg aag cac caa tat gat tac gat gta caa acc gtg att ggg ttc ttg 768 Met Lys His Gln Tyr Asp Tyr Asp Val Gln Thr Val Ile Gly Phe Leu                 245 250 255 cga gag cat ggg ctt gaa aaa gaa gtc ctg ctg aat gtt gaa ccc aac 816 Arg Glu His Gly Leu Glu Lys Glu Val Leu Leu Asn Val Glu Pro Asn             260 265 270 cac acc cag ctt gcg ggc cac gaa ttt gag cat ggc ttt atc ttt gct 864 His Thr Gln Leu Ala Gly His Glu Phe Glu His Gly Phe Ile Phe Ala         275 280 285 gcc aaa ctt ggc atg ctt gga agc att gat gct aat acc ggt tct gag 912 Ala Lys Leu Gly Met Leu Gly Ser Ile Asp Ala Asn Thr Gly Ser Glu     290 295 300 agt ctt ggc tgg gac act gat gag ttc atc act gac cag act cat gcg 960 Ser Leu Gly Trp Asp Thr Asp Glu Phe Ile Thr Asp Gln Thr His Ala 305 310 315 320 act ctg ctg tgt cgt acg att att gag atg ggc ggt ttc aaa aaa ggt 1008 Thr Leu Leu Cys Arg Thr Ile Ile Glu Met Gly Gly Phe Lys Lys Gly                 325 330 335 ggc ctt aac ttt gat gcc aag gtg cga cgt gaa agt act gat cca gag 1056 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Glu Ser Thr Asp Pro Glu             340 345 350 gat ctc ttt atc gcg cac gtg gca tct atg gat gca ctt gct aaa ggt 1104 Asp Leu Phe Ile Ala His Val Ala Ser Met Asp Ala Leu Ala Lys Gly         355 360 365 ctt cgt aat gct gcc aaa ttg gtt gat gaa ggc cgt atg gct aag atg 1152 Leu Arg Asn Ala Ala Lys Leu Val Asp Glu Gly Arg Met Ala Lys Met     370 375 380 ctc gca gag cgt tac gct gga tgg gac tct gga cta ggt aag aga att 1200 Leu Ala Glu Arg Tyr Ala Gly Trp Asp Ser Gly Leu Gly Lys Arg Ile 385 390 395 400 gag gat ggc cag agt tcg ctt gac gag ctg gag gag cat gcg ctc cag 1248 Glu Asp Gly Gln Ser Ser Leu Asp Glu Leu Glu Glu His Ala Leu Gln                 405 410 415 aat gat gag gag ccc gct aag act tca gca aaa cag gag aag ttt att 1296 Asn Asp Glu Glu Pro Ala Lys Thr Ser Ala Lys Gln Glu Lys Phe Ile             420 425 430 gct gtt ctc aac caa tac att tct taa 1323 Ala Val Leu Asn Gln Tyr Ile Ser         435 440  

Claims (13)

a) constructing a recombinant vector encoding the enzyme xylose isomerase required for metabolism of the selective agent xylose and containing a nucleic acid sequence operably linked to a regulatory sequence, b) transforming the vector into a plant explant by an Agrobacterium mediated method under optimal infection conditions, c) selecting a putative transformant on MS medium containing the selection agent of step a), and d) genetically transformed fatty seeds, more specifically Helianthus , comprising regenerating said selected plant explants. annus ) A method for selecting and regenerating plant explants. The method of claim 1, wherein the species used is a species of Hellenanthus annus. The method of claim 1, wherein the nucleic acid sequence encoding the enzyme xylose isomerase is isolated from individual Schizochytrium, represented by SEQ ID NO: 1. The nucleic acid sequence according to claim 3, wherein at least one nucleotide sequence is modified and expression of said modified DNA occurs in a host plant explant. The nucleic acid sequence of claim 4, wherein the sequence is represented by SEQ ID NO: 3. 6. The corresponding amino acid sequence of the protein expressed according to claim 3, represented by SEQ ID NO: 2. The nucleic acid according to claim 2, wherein the regulatory sequence is a promoter sequence. 9. The method of claim 1, wherein the selection agent is xylose. 10. The plant of claim 1, wherein the genetically transformed plant explant has a vector comprising the nucleotide sequence of claim 5, wherein the expression of the sequence is a non-transformed cell in the transformed explant. Giving a metabolic advantage. The competitive metabolic benefit of claim 1 wherein said transformed plant explants and untransformed plant explants are capable of utilizing said selection agent as a carbohydrate source due to expression of the enzyme by the nucleic acid of claim 5. competitive metabolic advantage). The method of claim 1, wherein the transformation efficiency obtained is at least 10%. The method of claim 1, wherein the transformation efficiency obtained is at least 15%. The method according to any one of claims 1 to 12, wherein the regeneration efficiency is 2-3 times the efficiency of the voice selection method based on hygromycin based on Table 1 of the specification.

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