CA2641868A1 - Method of selection and regeneration of transgenic brassica jxmcea on xylose - Google Patents

Abstract

The present invention describes an improved method of generation of Brassica transformants based on a method of selecting genetically transformed Brassica juncea explants based on their ability to utilize Xylose as a sole carbohydrate source. The said invention encompasses the process of Agrobacterium Mediated Transformation of the target host plant with the constructed vector for the expression of the enzyme Xylose Isomerase. Also disclosed is the method of selecting the putative transformants subsequent to transformation with the said vector to obtain a metabolic advantage of utilizing Xylose as a carbon source. The subject invention alleviates the disadvantage of the negative selection methods. Reported is a stable transformation efficiency of 34-40% using Xylose Isomerase for selection.

Description

DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS

THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:

NOTE POUR LE TOME / VOLUME NOTE:

METHOD OF SELECTION AND REGENERATION OF TRANSGENIC BRASSICA JUNCEA ON XYLOSE
FIELD OF INVENTION:

The present invention describes an improved method of generation of Brassica transformants based on a method of selecting genetically transformed Brassica juncea explants based on their ability to utilize Xylose as a sole carbohydrate source. The said invention encompasses the process of Agrobacterium Mediated Transformation of the target host plant with the constructed vector for the expression of the enzyme Xylose Isomerase. Also disclosed is the method of selecting the putative transformants subsequent to transformation with the said vector to obtain a metabolic advantage of utilizing Xylose as a carbon source. The subject invention alleviates the disadvantage of the negative selection methods. Reported is a stable transformation efficiency of 34-40%
using Xylose Isomerase for selection.

BACKGROUND OF INVENTION:

The production of transgenic plants often requires the use of a selection system that allows the regeneration and the growth of the successfully transformed cells.
When a nucleotide of interest (NOI) or Gene of Interest (GOI) has to be introduced into a population of cells by transformation only a certain number of cells are successfully transformed ie., only few cells receive the NOI or GOI. Since the transformed cells constitute a minor fraction of the treated cells, compared to the majority of cells which remain untransformed, the selection system has to enable efficient screening in selecting the successfully transformed cells such that the probability to recover transgenic plants in the presence of a selective agent is greater than in its absence, especially because the transformation rate is always low (10-3 to 10-6).

The selection process is undertaken by the introduction of a selectable marker gene with the gene of interest. The use of this kind of a selectable marker gene in the transformation process aims to a give a selective advantage to the transformed cells, allowing them to grow faster, better and to kill the non-transformed cells.

An ideal selectable marker gene should be capable to express in any cell or tissue and in great number of plant species. This expression should be easily distinguished from any endogenous activity in the plant tissue allowing to differentiate the phenotype of the transformed from the untransformed tissues. During the regeneration steps, the influence exerted by the dying non-transformed cells on the transformed cells should be minimal on the selective medium. In addition to the above an easy assay should be able to confirm 'the presence the marker (ACNFP, 1994) Selectable markers identified today can be differentiated into two types that enable transgenic plants or cells to be identified after transformation. They are positive and negative markers conferring a selective advantage or disadvantage respectively. Negative Selection kills the cells which do not contain the introduced DNA and includes antibiotic and herbicide based "selection. They allow the selection of transformed cells, or tissue explants by their ability to grow in the presence of an antibiotic or a herbicide.

So far, the most widely used selectable gene is the neomycin phosphotransferase II
(NPTII) gene (Fraley et al.1986) which confers resistance to the aminoglycoside.
antibiotics kanamycin, neomycin and G418 (Bevan et al. 1983). A number of other selective systems has been developed based on resistance to bleomycin (Hille et al. 1986) bromoxynil (Stalker et al. 1988), chloramphenicol (Fraley et al. 1983), 2, 4-dichlorophenoxyacetic acid (Streber and Willmitzer 1989), glyphosate (Shah et al. 1986), hygromycin (Waldron et al. 1985) or phosphinothricin (De Block et al. 1987).

Negative Selection methods, which rely on the use of antibiotics or herbicides, suffer from a number of disadvantages. Plant cells dying from antibiotic toxicity release growth inhibitors and toxins, which are thought to negatively, affect transformed cells and hinder their growth (Haldrup et al.1988a). The non-transformed cells are killed in the presence of the phyto-toxic product and in cases where a coherent tissue is used there is a risk that the transformed cells also die, due to the fact that the death of the non-transformed cells may cut off supply of the nutrients to the transformed cells or because of the damaged or dying non-transformed cells may excrete toxic compounds, thus limiting the transformation efficiency of the negative systems. In addition selection of cells or tissues using negative selection requires the precise timing of expression of the introduced genes in relation to the selection process. If the transgenic cells are treated with a toxic compound before the detoxifying gene is expressed or before enough gene products are produced to ameliorate the action of the toxic compound, both the transgenic and the non-transgenic cells are killed. If selection is delayed, the selection of the transgenic cells or tissues may be hindered by, for example, shoot or callus formation from non-transgenic cells or tissues, which form a barrier to the penetration of the compound used to select the transformed cells. They cause genome wide alterations in DNA
methylation (Schmitt et al. 1997). This non-reversible phenomenon leads to gene silencing and is thought to hinder both the selection of transgenic cells and the plant regeneration process.
Changes in methylation can be dosage dependant and lead to sequence mutation (Bardini et al.2003.) The presence of an antibiotic resistance gene in ingested plants is a matter of environmental concern with the major concern being the potential transfer of the genes conferring antibiotic resistance into gut microorganisms. Homology between sequences in an antibiotic resistance marker gene that is prokaryotic in origin and the recipient's DNA is more likely to be found in gut microorganisms, which are prokaryotic in origin.
The probability of integration and expression of a marker gene is therefore greater in gut microorganisms than in the gut epithelial cells. Rare transfer events can be amplified very rapidly under selective pressure. The health impact would be significant if a gene conferring resistance to a clinically important antibiotic was transferred and expressed in a pathogenic microorganism normally treated with that antibiotic. While such ecological concerns may lead to governmental restrictions on the use of antibiotic resistance genes in transgenic plants, and it is therefore desirable to develop new selection methods, which are independent of such genes. The European Union has enacted a ban on antibiotic resistance genes for the selection of transgenic plant cells effective at the end of 2004, and thus any future genetically enhanced plants and the food products sold in EU will have to contain alternative selectable markers. Even with respect to the usage of herbicide resistance genes as a selectable marker, there is a possibility that resistance might transfer to weedy relatives of crops via outcrossing (Rieger et a1.1999).

The above-mentioned concerns are overcome to a substantial extent, by the method of positive selection whose operating principle is converse to negative selection. In contrast, to negative selection, positive selection gives the transformed cells the ability to grow using a specific carbon, nitrogen or growth regulator as the selection agent (Joersbo and Okkels 1996; Bojsen et al. 1998; Haldrup et al 1998a). The transgenic cells acquire a gene which confer a metabolic advantage to those cells whilst non-transgenic cells are starved rather than killed.

The selection method employed in the invention uses the selection agent, which is the carbohydrate xylose, which are not metabolized by a number of plant species (Bojsen et al. 1994). By substituting the normally employed carbohydrate with one of these compounds, cells transformed with the gene encoding an enzyme capable of converting it to metabolizable isomer are favored in growth while the non-transgenic cells are starved.
Thereby giving the transformed cells a metabolic advantage. Xylose can be converted to xylulose by xylose isomerase which functions as a selectable marker in this system (Haldrup 1996). These marker genes for positive selection make possible the identification and the selection of genetically modified cells without injuries or death of the non-transformed population of cells (negative selection). The addition of a new compound in the culture medium as nutrient source during the regeneration process, allow the norinal growth and the differentiation of transformed cells, while non-transformed cells will not be capable of growth or generate denovo plants.

Disclosed in this invention is a method of generation of Brassica transgenics employing a selection method without usage of any of the commonly known antibiotic resistance genes or herbicide resistance genes. Described is the method of selecting and regenerating transformants of the species Brassica juncea, specifically exemplifying a positive selection method that involves conferring to the transformed tissue explants an ability to utilize certain carbon sources preferably Xylose and transformed explants can be selected by simply subjecting them to a medium containing the referred selection agent.

Prior art describes a positive selection method that has been developed using the xylose isomerase gene (xylA) isolated from Therrraoaerobaeterium thermosulfurogenes or from Streptomyces rubiginosus, as a selective marker gene (Haldrup et al., 1998a;).
Transgenic plants of potato, tobacco and tomato were successfully selected on xylose-containing media. The method of selection disclosed herein is lucid enough to distinguish from the prior art documents cited. Our invention specifically aims at the method of positive selection of the transformed plant explants that belong to the species Brassica juncea.
The selectable marker gene has been isolated from the organism Schizochytrium and has been suitably modified for its expression in the host plant after successful transformation.
PRIOR ART:

Our prior application filed describes a method of transformation and selecting genetically transformed Sunflower explants based on their ability to utilize Xylose as a sole carbon source.

US Patent Application 5,767,378 titled "Mannose or Xylose positive selection"
relates to the method of identifying or selecting from a population of eukaryotic cells cultivated on or in a medium containing at least one compound, cells which have a metabolic advantage as a result of being transformed. This invention provides a basis of specifically selecting transformed sugar beet or potato cells. The above referred prior art specifically claims the described protocol of positive selection in sugar beet and potato cells as against our invention that refers to the positive selection of Brassicajuncea per se. The protocols of transformation are unique with respect to every plant species.
Moreover, the methodology of transformation described in the subject invention is completely different in comparison to the prior art cited. Furthermore, no cited literature describes the method of transformation based on positive selection in Brassica juncea.

Haldrup et al., 1998 have established that the xylose isomerase gene from the organism Thermonanaerobacterium thermosulfurogenes allows the selection of transgenic plants using D-xylose as the selection agent (Plant Molecular Biology (1998), 37(2), 287-296.
The Xylose isomerase gene was transferred to the target plant by Agrobacterium mediated transformation. Unoptimized selection studies showed in that in potato and tomato, the xylose isomerase selection was more efficient than the kanamycin resistance selection, whereas in the tobacco plants the opposite effect was observed.

The method of selection disclosed herein is lucid enough to distinguish from the prior art documents cited. Described is an improved method of generation of Brassica transgenics employing a selection method without usage of any of the commonly known antibiotic or herbicide resistance genes. Also described is the method of selecting and regenerating transformants of the species Brassica juncea, specifically exemplifying a positive selection method that involves conferring to the transformed tissue explants an ability to utilize alternate carbon source preferably Xylose. Transformed explants can be selected by simply subjecting them to a medium containing the referred selection agent.
The selectable marker gene used in the invention has been isolated from the marine Organism Schizochytrium and further the gene was codon optimized for the expression of the gene in the host plant. 3-4 fold greater numbers of regenerating plantlets were observed using xylose isomerase XI as against the commonly used hpt hygromycin based method of selection. Using the aforementioned method of positive selection which alleviates the problems associated with the commonly used antibiotic resistance or the herbicide resistance selection markers and also reported is a stable transformation efficiency of 35-40%.

SUMMARY OF THE INVENTION:

The subject invention relates to an improved method of transforming the plant species Brassica juncea, and thereby selecting the transformants with a method of positive selection utilizing Xylose. According to the first aspect of the invention, there is provided a method of isolation of the total RNA and the synthesis of cDNA from the mRNA. The EST clones randomly picked from the primary cDNA library have been subjected to PCR
amplification thereby leading to the identification of cDNA clone of 1.5kb, which encodes a full length Xylose Isomerase (xylA) cDNA possessing an ORF of 148 1bp.

Further still, an additional aspect of the subject invention pertains to the polynucleotide molecule that encodes the protein having the biological activity of Xylose Isomerase.
Specifically, the aspect pertains to a polynucleotide as represented in the SEQ ID 3.
Also represented is the polypeptide encoded by the polynucleotide molecule of the subject invention.

A specific aspect of the invention describes the homology of Xylose. Isomerase obtained from Schizochytrium to that of Arabidopsis thaliana and its cloning into a pGEM-T
Easy.

According to a further aspect, the sequence of xylose isomerase is codon optimized by subjecting the clone to multi-site directed mutagenesis for substituting 9 nucleotides and a successful expression of the gene in the host plant.
In one preferred aspect, the codon-optimized gene is cloned into an expression vector, transformed into E.coli and experiments were conducted for proof of function to detect for the transcription and the translation of the xylose isomerase gene.
The xylose isomerase gene is then cloned into the pCAMBIA to be further transformed into the host plant Brassicajuncea.

In accordance with the highly preferred aspect of the invention, describes the Agrobacterium mediated gene transfer of the constructed vector comprising the selectable marker gene into the host plant explants under conditions suitable for infection thereby inducing the transformed cells with a positive effect that gives the cells a metabolic advantage over the non-transformed cells when cultured together on a suitable medium containing the selective agent.

According to the most significant aspect of the invention, described herein is a selection system for selecting atleast one genetically transformed cell from the population of cells from the medium, wherein at least one genetically transformed cell is transformed with the nucleotide sequence which encodes an expression product capable of converting a component or precursor thereof that is present in the medium. More specifically the selective component referred in the invention is Xylose. The term "cells" is intended to refer to any type of cells from which individual genetically traiisformed cells may be identified and isolated using the method of the invention.

The transformation efficiency reported from the disclosed method of transformation is 34-40% and the regeneration efficiency has been 3-4 fold greater than the known hygromycin based methods.

Brief Description of the Drawings:

FIG. 1. Complete Xylose isomerase domain in the 1.5kb sequence of SC-1 desaturase FIG. 2. Homology of the motif from SC-1 to the Xylose isomerase domain FIG. 3. Map of pGEX-XI. The Xylose Isomerase gene was cloned at EamHI and Xhol of pGEX.

FIG. 4. Induction of Xylose isomerase in E.coli. Amplification of transformed SEA
explants with XI primers. Lanes: I: Induced; U: Uninduced; 1,2,3: Different clones carrying the xylose isomerase gene; M: MW marker.

FIG. 5. A. Amplification of the ORF from the xylose Isomerase clone.
B: Restriction of the pCAMBIA-XI with Xho I. Note the release of the hpt gene from the Xho 1 site.

FIG. 6. Replacement of hpt with XI in pCAMBIA-CO-XI.

FIG. 7. Brassicajuncea cotyledonary petiole explants A) cultured on sucrose (35 gm/1) B) cultured on D-Xylose (30 gm/1) C) cultured on medium without any carbon source. ~
FIG. 8. Budding comparison of cotyledonary petiole explants transformed with pCAMBIA-CO-XI and pCAMBIA 1380 and cultured on Xylose (25g/1) + Sucrose (5gm/1) and Hygromycin (25mg/1) respectively after A) 3 weeks; B) 6 weeks of selection.
FIG. 9. Selection and regeneration of transgenic shoots using positive selection in explants transformed with pCAMBIA+XI construct.

FIG. 10. Amplification of transformed cotyledonary petiole explants with XI
primers.
lOOng of genomic DNA of the selected plantlets, carrying pCAMBIA1301-XI were amplified with XI primers Lane 1: d15 plasmid DNA; Lane2-6 and 8-13: DNA
samples from different explants; M: 1 kb ladder.

FIG. 11. Brassicajuncea var "Varuna" Xylose Isomerase positive Plant.

FIG. 12. Amplification of Genomic DNA of TI seeds of XI positive To Plant.
100 ng of Genomic DNA isolated from T, seeds was amplified using XI primers Lane 1: Marker (1Kb), Lane 2-7: Amplified of product (1.32 kb) of XI gene from the seeds of 6 separate pods, Lane 8: positive control, Lane 9: marker.
BRIEF DESCRIPTION OF SEQUENCE LISTING.

SEQ ID NO 1: Sequence of full length of Xylose isomerase transcript of SC 1 SEQ ID NO 2: Translated Protein sequence of Xylose Isomerase of SC 1 SEQ ID NO 3: Sequence of Xylose isomerase after optimization for expression in Plants SEQ ID NO 4: Sequence of full-length codon optimized Xylose isomerase transcript of SCI in frame in pCAMBIA vector.

DETAILED DESCRIPTION OF THE INVENTION:

The term "Selectable marker gene" refers to any nucleotide sequence that is preferably co-introduced with the gene of interest, wherein a selective advantage is conferred to a cell transformed with the said selectable marker gene.

The term "marker compound" or "selective agent" is the compound or nutrient which only the successfully transformed cells are able to metabolize thereby allowing the selection of the transformed cells over the non-transformed cells by virtue of transformation and expression of the selectable marker gene The term "selective advantage" as used herein includes the terms selective, metabolic and the physiological advantage and means the transformed cells are able to grow more quickly than the disadvantaged (non-transformed) cells, or are advantageously able to utilize substrates (such as nutrient precursors, etc.) which disadvantaged cells are not able to utilize.

The term "selecting" refers to the process of identifying and/or isolating the genetically transformed cells from the non-genetically transformed cells using the method of the present invention.

The term "genetically transformed" includes transformation using recombinant DNA
techniques.

The term "vector" includes expression vectors and transformation vectors.

Cloning of Xylose Isomerase from a Thraustochytrid strain SC-1.

Total RNA was isolated from three-day-old cultures of the Schizochytrium SC-1, a Thraustochytrid isolated from the backwaters of Goa. cDNA was synthesized from the mRNA using superscript Rnase (GibcoBRL). The cDNA was cloned into the Notl-Sall site of pSPORTI vector and transformed into E.coli DH10B.

The primary cDNA library consists of 2 x 106 clones, while the ainplified library has a titer of 2 x 1010 clones/ml. EST clones were randomly picked and inserts amplified with SP6 and T7 primers. The 5' ends of the 2000 cDNA clones were randomly selected and sequenced with T7 primers. 5' end sequencing of clones from the cDNA library of SC-1 led to the identification of cDNA clone of 1.5kb which encodes a full-length Xylose Isomerase (xylA) cDNA of 1511bp with a 5' UTR of 158bp and a 3' UTR of 30bp.
It has an ORF of 1481 bp.

The sequence of the CDS is given in the SEQ ID 1. It is the sequence of the full length Xylose Isomerase transcript of SC-1. The sequence translates into a protein of 440 amino acids. The amino acid sequence of the translated protein is represented in SEQ
ID 2. The sequence shows 74% homology to the Xylose isomerase of ANabidopsis thaliana.
It contains the complete Xylose isomerase domain and has been designated as the xylose isomerase of SC-1. Presence of the complete Xylose isomerase domain in the 1.5kb sequence of SC-1 desaturase has been represented in the FIG. NO 1.

FIG. NO.2 represents the homology of the motif from SC-1 to Xylose Isomerase domain.
EXAMPLE: 2 Codon optimization of the Xylose isomerase The xylose isomerase (xylA) sequence of SC-1 uses codons that are comparatively rarely used in plants; SC-1 predominantly utilizes CGC to code for Arginine where only 9% of the plant codons for Arg are CGC. Hence, the CGC codons were replaced with more frequently used codons for arginine. The clone was subjected to two rounds of multi-site directed mutagenesis for substituting 9 nucleotides prior to introduction into plants. The optimized sequence is represented in SEQ ID 3.

EXAMPLE: 3 Cloning of Xylose Isomerase into pGEX Expression vector To confirm that the codon optimized gene does transcribe and translate, the codon optimized SC-1 XyIA gene from pSPORTl, was amplified with the forward primer (5'GCGCGGATCCATGGGTGAATTCTTTC3') containing an BamHI site and the Reverse primer (5'GAAACTCGAGCTTGTCGATTAAGAAATGTATTGGTT3') containing an XhoI site. The amplified PCR product (1323) was digested with BamHI
and XhoI. pGEX-4T-3 is an expression vector used to express the proteins as fusion proteins with the 26-kDa glutathione S-transferase (GST under control of the tac promoter. pGEX-4T-3 was digested with BamHI and Xhol and the PCR product (restricted with BamHI and Xhol) cloned directionally between the two sites -the resultant clone was called pGEX-XI. Map of pGEX-XI has been represented in the FIG.NO. 3.

Expression of the Xylose isomerase fusion protein in E.coli The pGEX-XI carrying the Xylose Isomerase gene was transformed into E coli BL21.
The transformed cells were selected on LB media containing 100 g/ml of Ampicillin.
Colonies were picked up at random and were grown overnight in LB containing 100ug/ml of ampicillin. 1% of these overnight cultures were used as starter cultures for inoculating 5m1 of LB broth containing 100ug/ml of ampicillin. Cultures were incubated till O.D. 600 reached approximately 0.6-0.7. 2 ml each of these cultures was induced with 1mM IPTG for 3 hrs. Simultaneously, 2ml of the culture was pelleted down as control without induction. The cell pellets were resuspended in 100 1 sample buffer and 50ul loaded onto a 10% SDS-PAGE gel. The induction is shown in the FIG. NO 4.

A protein of 76KDa, the expected size of the GST-Xylose isomerase fusion protein, was observed in the induced cells of clones 1 and 2 while being absent from the uninduced cells. Thus the codon optimized xylose isomerase is in the right reading frame and is capable of being transcribed and translated.

Proof of function of Xylose Isomerase gene in E. coli The E.coli strain AB477 (proA2, his-4, aroCl, thi-1, lacYl, ga1K2, xyl-5,.mtl-l, lambda supE44) is Xylose Isomerase (XylA) deficient (Haldrup et al., 1998 & 2001).
The complementation of the Xylose Isomerase gene in the mutant will enable this strain to utilize and grow and thereby proving its function. E.coli strain AB477 was obtained from the E.coli Genetic Stock Center, USA. Xylose isomerase gene in pGEX-4T-3 was transformed into competent cells of E.coli strain AB477 which was plated onto MacConkey agar plates containing 1%(w/v) D-Xylose with ampicillin (100 g/ml).
Transformants harboring the XyIA gene were able to ferment D-Xylose and appear red on MacConkey agar plates containing xylose.

AB477 host cells, cells transformed with pGEX4T-3 and those transformed with pGEX-XI were plated onto MacConkey's media containing ampicillin (100 g/ml) as well as media containing Xylose (1% w/v) and ampicillin (100 g/ml) respectively. Red colonies were obtained with pGEX-XI in media containing xylose, while host cells and carrying pGEX4-T3 did not grow in the media. Thus, the codon optimised xylose isomerase gene isolated from SC-1 is translated and functional.

EXAMPLE: 4 Cloning of Xylose Isomerase into pCAMBIA

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

The ORF of the codon optimized Xylose Isomerase (XylA) gene in pSPORT was amplified with Forward primer (5' CTCTCTCGAGCAACCATGGGTGAATTCTTTCC
3') & the reverse primer (5'GAAACTCGAGCTTGTCGATTAAGAAATGTATTGGTT
3') containing the Xhol sites each. The amplified fragment was restricted with Xhol.
pCAMBIA 1301 was simultaneously restricted with XhoI to release the hpt gene and the vector purified from agarose gel was ligated to the amplified fragment.
Amplification of the ORF from the Xylose isomerase clone and the restriction of the pCAMBIA-XI
with Xhol is represented in the FIG.NO.5 The ligation mix was transformed into DH10B. Transformed colonies were picked up and the plasmid isolated from these colonies amplified with XI primers and sequenced with the same. Thus constructs carrying the XI gene in the right orientation and right frame were identified. The sequence of the gene, cloned in the right frame, is given in the SEQ ID.4 The FIG NO.6 represents the sequence of full-length codon optimized Xylose Isomerase transcript of SC-1 in frame in pCAMBIA vector and the replacement of hpt with XI in pCAMBIA-CO-XI. The hpt gene coding for hygromycin phospho transferase cloned between Xho-I sites of vector pCAMBIA1301 has been replaced by codon optimized Xylose isomerase in place of hygromycin and the resulting construct has been named pCAMBIA-XI.

Transformation of Brassica using xylose isomerase as positive selection marker It is known that when genetic material is to be introduced into a population of cells by transformation, only a certain number of the cells are successfully transformed.
Identification and selection of the transformed cells has traditionally been accomplished using negative selection, whereby transformed cells are capable of survival on media containing an agent, which they are able to degrade, while non-transformed cells are killed on the media. Hygromycin is the most commonly used antibiotic while the hygromycin phospho transferase gene (hpt) in the construct used for transformation provides resistance to the transformed cells grown in media containing the antibiotic.
Kanamycin and herbicide (Phosphinothricin) resistance are the other markers used for selection of brassica transformants.

These negative selection methods have certain disadvantages. While the non-transformed cells may die because of the presence of antibiotics in the growth medium, they release toxins into the medium, which are inhibitory and toxic to the transformed cells as well.
Moreover, the presence of an antibiotic resistance gene in ingested plants and microorganisms is a matter of concern for environmental groups and governmental authorities. In addition, selection of cells or tissues using negative selection requires precise timing of expression of the introduced genes in relation to the selection process. If the transgenic cells are treated with a toxic compound before the detoxifying gene is expressed or before enough gene products are produced to ameliorate the action of the toxic compound, both the transgenic and the non-transgenic cells are killed.
If selection is delayed, the selection of transgenic cells or tissues may be hindered by, for example, shoot or callus formation from non-transgenic cells or tissues, which forms a barrier to the penetration of the compound used to select the transformed cells.

The above disadvantages are overcome, to a substantial extent by the method of positive selection which makes it possible to selectively grow transformed cells without damaging or killing the non-transformed cells in the population and without introduction of antibiotic or herbicide resistance genes.

Many plant species do not have the innate ability to metabolise xylose and hence fail to thrive on media where xylose is the sole carbon source. Transformation of such species with constructs carrying xylose isomerase, as selection marker, would impart the transformed cells the ability to utilize xylose as the carbon source.

Transformation and selection in Brassicajuncea.
Plant Material:

For our study, we have used Brassica juncea vaf . `varuna' - A selection from Varanasi local, having plant height of 145-155 cm, erect and stout with moderate branching. Its leaves are medium in size, dark green, sparsely hairy on lower surface with purplish pigment at the base of leaves. Varuna is moderately resistant to Alternaria blight and Aphids. It is a medium duration variety (135-140 days) with oil content of 43%
(21%
linoleic acid) and yield of 20 -22 qtls/ha.

Seeds of Brassica juncea `varuna' were sterilised in 70% alcohol for 2 minutes and with 0.1% Mercuric Chloride for 5 minutes. The sterilized seeds were vigorously washed 4-5 times with sterile water, followed by drying by blotting. These seeds were then germinated on half strength hormone free Murashige and Skoog Medium (MS media) solidified with 0.8% agar in tissue culture bottles (30-40 seeds/bottle). The seedlings were grown for 2 days in dark and 3 days in light (16h light:8h dark) photoperiod at 25 C in BOD, until the cotyledons were fully expanded and hypocotyls were 4 to 5 cm long.
Different explants were tested for their regeneration efficiency. Among these, the cotyledonary petiole explants was found to have maximum potential for regeneration and were used for transformation.

Isolation of Cotyledonary petiole explant Five days old healthy seedlings were collected and the bottom portion of the seedlings were cut and removed. The cotyledonary petiole was isolated by giving a cut at the point of attachment of hypocotyl and the petiole stem just above the meristem avoiding the inclusion of leaf primordial tissue. It is important that the scalpel blade is sharp, as petioles isolated with a good `clean' cut surface (i.e. when the tissue is not torn) respond best. Two explants per seedling were obtained each having a petiole length of 3-5 mm.
Explants were collected and stored in co cultivation media with petiole dipped in the media until further use.

Screening for capability to utilize xylose as carbon source In order to determine if Brassicajuncea is capable of using xylose as the carbon source, explants were cultured on media containing sucrose and Xylose respectively.
100 cotyledonary petiole explants were isolated and cultured in medium containing MS
salts, 0.2mg/1 NAA, 2 mg/l BAP and 8 g/l Agar where the carbon source was a) 3.5%
sucrose; b) 3% Xylose; c) no carbon source for 3 weeks. The effect of the carbon source on the explant was observed at the end of 3 weeks. Represented in FIG. 7.

Explants cultured in media containing xylose as the carbon source bleach or brown and die within 2-3 weeks. The explants grown in the absence of carbon source in the medium looked pale and unhealthy and did not callus, while explants grown in media containing sucrose were healthy and well developed. Thus, the Brassicajuncea cotyledonary petiole explants do not appear to be able to use xylose as a carbon source.

Transformation of Brassica using pCAMBIA-XI
Transfection with Agrobacterium The cotyledonary petiole explants were isolated from the five days old seedlings as described earlier. Agrobacterium tumefaciens strain GV3 101 harboring the binary vector pCAMBIA 1301 carrying hpt gene (negative selection) and the strain carrying vector pCAMBIA- XI (positive selection) were used for comparison.

Bacterial activation: Agrobacterium GV3101 cells carrying the respective vectors were grown overnight in Luria-Bertani (LB) medium containing Sodium Chloride lOg/1, Tryptone lOg/1, Yeast extract 5g/l, Rifampicin (10 g/ml), Gentamycin (l0 g/ml) and Kanamycin (50 g/ml) and grown exponentially in 28 C shaker at 200 rpm for a period of 4-6 hrs to reach O.D600 = 1Ø The cells were centrifuged at 6000 rpm for 10 min at 4 C, washed and resuspended in MS liquid containing MS salts and 30g/1 sucrose to adjust the density of bacteria to A600 of 0.3-0.5.

Infection: Cotyledonary explants are picked up and the petiole tip dipped and held in the Agrobacterium suspension for 10-15 seconds and then immediately transferred to the co cultivation media with petiole dipped in the medium. The cotyledon should not be immersed in the suspension, before being transferred back to the co cultivation medium (2 cm deep petri-dishes are ideal for holding the bacterial suspension). The explants were co cultivated for 3 days at 28 C under dark. By the time explants are moved onto selection medium (72 hours after inoculation), petioles will have lengthened and thickened. It should then be possible to embed the petiole into the selection medium, with the cotyledonary lamella clear of the medium.

Selection and Regeneration:

Positive Selection and Regeneration After three days of co-cultivation, the explants were washed thrice thoroughly with MS
liquid containing MS salts, 30 g/l sucrose and Cefotaxime 250 mg/1 for 15 min.
These were blotted dry with sterile filter paper and then transferred to I selection medium containing MS salts, 0.2mg/1 NAA, 2mg/l BAP, 25g/1 D-Xylose, 5g/1 Sucrose, 250mg/1 Cefotaxime, 8g/l Agar, pH 5.6 and kept under photoperiod of 16hr light and 8hr dark condition for 4 weeks. These were then subcultured to Ilnd selection medium containing MS salts, 0.lmg/1 NAA, 3mg/1 BAP, 29g D-Xylose, lg/1 Sucrose, 250mg/1 Cefotaxime, 8 g/l Agar, pH 5.6 and retained for the another 3 weeks. During this period a number of shoot buds develop from the callus. After the selection period the shoots were subcultured onto 1/2 MS medium containing half strength MS salts, 15g/l sucrose, 250mg/l cefotaxime with 8g/I Agar, pH 5.6 for elongation. After 2 weeks the elongated shoots were transferred to Rooting medium consisting of half strength MS
salts, 0.01mg/l IBA, 30g/l sucrose, 150mg/l cefotaxime with 8g/1 Agar, pH 5.6. The rooted plants were hardened for 2 days in water before transferring to soil and vermiculite (1:2) mixture.
Negative Selection and regeneration After three days of co cultivation the explants were washed thrice thoroughly with liquid medium containing MS salts, 30g/1 sucrose and 250mg/1 Cefotaxime. The explants were transferred to selection medium containing MS salts, 0.1mg/1 NAA, 2mg/1 BAP, 25mg/i Hygromycin, 250mg/1 Cefotaxime, 30g/1 Sucrose, 8g/1 Agar, pH 5.6. After the selection period the shoots were subcultured onto 1/2 MS medium containing half strength MS
salts, 30g/1 sucrose, 250mg/l Cefotaxime and 8g/l Agar, pH 5.6 for 2 weeks.
The elongated shoots were transferred to Rooting medium containing 1/2 Strength MS
salts, 30g/1 sucrose, 150mg/1 cefotaxime and 8g/1 Agar, pH 5.6) and the rooted plants were subsequently transferred to soil.

Comparing Positive and negative selection Explants were transformed with pCAMBIA 1301 (with hpt) and pCAMBIA-XI (Xylose Isomerase replacing hpt) and selected on respective selection media containing different concentrations of hygromycin and xylose respectively. The explants surviving first selection were transferred for a second round of selection to the same medium for 21 days. Explants were subsequently transferred to the elongation media. After the second selection lOOng DNA from the leaves of the shootlets were amplified with hpt and XI
primers respectively under the following cycling conditions: 94 C for I min., 55 C for I
min. and 72 C for 1.3 min for 40 cycles.

The observations made are tabulated below:

A) Transformation of explants with pCAMBIA-1301. Selection on media containing Hygromycin Hygromycin No. of explants No. of PCR +ve Transgenic Conc. placed in plants obtained after 2 a m /1 selection selection B) Transformation of explants with pCAMBIA-XI. Selection on media containing Xylose Xylose No. of explants No. of PCR +ve Transgenic Cone. placed in plants obtained after 2 a (9/1) selection selection Table 1: Transformation of sunflower SEA explants with pCAMBIA-1301 and pCAMBIA-XI lOOng DNA extNacted fi om the leaves of the transformed plants obtained was subjected to PCR with gene specific prinaers for confirming the transgenic status of the plants.

There is a considerable difference in the number of transgenic regenerants obtained from positive selection compared to negative selection as seen in the table. The selection pressure on the explants in media containing hygromycin is found to be hindering the development of the transformed cells. Also, the buds put forth by explants growing on Hygromycin containing media are seen to be stunted and hydric. They do not grow to develop into plantlets and a very few survive on elongation media. The regeneration efficiency is very low even at low concentrations of hygromycin.

Xylose appears to have a lesser toxic effect on the explants than hygromycin.
The explants grown on media containing different concentrations of Xylose showed better budding efficiency compared to hygromycin selection. The number of transgenic plants obtained on selection with Xylose Isomerase was 3-4 times more than with Hygromycin selection.

Optimization to increasing the regeneration efficiency:

Haldrup et al. (2001) reported that the use of minimal quantity of sucrose or any other alternative carbon source in combination with xylose increases the selection and regeneration efficiency in positive selection in several crops.

In order to increase the regeneration efficiency of transformed explants, media optimization was carried out, wherein combinations of different concentrations of xylose and sucrose was used in the selection media siuch that the sucrose is just sufficient to allow survival but not growth of the untransformed cells, while transformed cells being able to utilize xylose would be able to grow and reproduce in the medium.

A total of 100 explants each were infected with pCAMBIA 1301-XI and placed in selection media containing MS salts, 0.2 mg/1 NAA, 2 mg/1 BAP, 250mg/1 cefotaxime and Agar 8g/1 pH5.6 containing various combinations of sucrose and xylose and grown for 4 weeks in light room conditions of 16h light: 8h dark, at 25 C
temperature and 60%
humidity. These were then subcultured to 2nd selection medium containing MS
salts, 0.2 mg/l NAA, 2 mg/1 BAP, 29g/1 Xylose, lg/1 sucrose, 250mg/1 cefotaxime and Agar 8g/1 pH5.6 and grown for a period of three weeks. The shootlets developed are transferred to elongation medium containing half the concentration of MS salts, 15g/l Sucrose, 250mg/i cefotaxime and Agar 8g/l pH5.6. The elongated shootlets are then transferred to the rooting medium containing 1/2 MS salts, 0.01mg/1 IBA, 30 g/l sucrose, 150mg/1 cefotaxime and Agar 8g/l pH5.6. The leaf samples from these plants are collected for the molecular analysis. The observations made are as follows:

No of No. of plants PCR +ve PCR ve Xylose Sucrose explants obtained conc.(g/1) cone. (g/1) placed in after 2 d Transgenic Plants selection selection plants (escapes) Table : Regeneration observed in the different combinations of Xylose and Sucrose.
lOOng DNA extracted f~om the leaves of the plants obtained was subjected to PCR with gene specific primers for determining the transgenic status of the plants.

It was observed that explants regenerated and grew better in media containing a combination of Xylose and sucrose. More number of shootlets could be obtained in media having 5 and 10 g/l sucrose with xylose (30g/l). However, with the increase in sucrose concentration, more escapes were obtained as seen by PCR results.
Thus, the use of 25g/l xylose and 5g/l sucrose in the first selection was found to be optimal for the positive selection.

Budding as observed in transformed cotyledonary petiole explants cultured on Xylose (25g/1) + Sucrose (5gm/1) and Hygromycin (25mg/1) after A) 3 weeks; B) 6 weeks of selection has been represented in FIG. 8.

Thus, the following protocol for the transformation and regeneration of Brassica using positive selection results in ca. 35% transformation efficiency in BNassica juncea `varuna' :

^ The seeds are sterilized in 70% alcohol for 2 minutes followed with 0.1%
Mercuric Chloride treatment for 5 minutes.
^ The sterilized seeds are vigorously washed 4-5 times with sterile water;
blot dried and placed in half MS media. They are grown for two days at 25 C in BOD and three days in light.
^ The cotyledonary petioles are excised avoiding the inclusion of any leaf primordial tissues.
^ 2 ml of the overnight grown agrobacterium culture GV3 101 carrying pCAMBIA
1301-XI is inoculated in 25 ml Luria-Bertani (LB) medium containing Rifampicin (10 g/ml), Gentamycin (10 g/ml), Kanamycin (50 g/ml) and grown exponentially at 28 C in shalcer at 200 rpm for a period of 4-6 hrs to reach O.D600 = 1Ø The cells were centrifuged at 6000 rpm for 10 min at 4 C and then washed and resuspended in liquid medium containing MS salts and 30g/l Sucrose to adjust the density of bacteria to A600 of 0.3-0.5.
^ The Petiole region of the cotyledonary petiole explants is infected by dipping in agro suspension for 10-15 seconds and is immediately placed into the cocultivation medium with petiole part pierced into the medium.

^ Co cultivated for three days on Co cultivation Media, containing MS salts, 0.2 mg/1 NAA, 2 mg/1 BAP, 15g/1 Sucrose, 8g/l Agar pH 5.6.
^ The explants are washed for 15 min in Washing Solution, containing MS salts, 30g/l Sucrose, 250mg/1 Cefotaxime pH 5.6 and blot dried.
^ Subcultured onto I Selection Media, containing MS salts, 25g/l xylose, 5gm/1 Sucrose, 0.2mg/l NAA, 2 mg/l BAP, 250mg/1 Cefotaxime, 8g/l Agar, pH 5.6 for 4 weeks in light room conditions of 16h light: 8h dark, at 25 C temperature and 60% humidity.
= Subcultured onto II Selection Media, containing MS salts, 29g/l xylose, 1 gm/1 Sucrose, 0.1mg/1 NAA, 3 mg/l BAP, 250mg/1 Cefotaxime, 8g/l Agar, pH 5.6 for 3 weeks in light room conditions of 16h light: 8h dark, at 25 C temperature and 60% humidity.
^ Subcultured onto Elongation Medium, containing half strength MS salts, 15g/l Sucrose, 250mg/i Cefotaxime, 8g/l Agar, pH5.6 for 1 week in light room conditions of 16h light: 8h dark, at 25 C temperature and 60% humidity ^ Subcultured onto Rooting Media, containing half strength MS salts, 0.01mg/1 IBA, 30g/l sucrose, 150mg/1 Cefotaxime and 8g/1 Agar with pH 5.6 in light room conditions of 16h light: 8h dark, at 25 C temperature and 60% humidity.
^ Finally the rooted plants are removed from the bottles and the roots are washed with water. These plants are hardened by placing in bottles with tap water for days followed by transferring into plastic cups containing Red soil (20%) and vermiculite (80%) mixture for 4 days in light room conditions. These are transferred to green house and maintained as such till it 'is completely acclimatized before transferring to pots containing red soil and manure.

Selection and regeneration of transgenic shoots using positive selection in explants transformed with pCAMBIA+XI construct has been represented in FIG. 9 Starting with 100 explants, 2-3 transgenic plants carrying the hpt gene are obtained on transformation with pCAMBIA-1301. However, starting with the same number of explants, 80-85 explants show budding; 40 of the explants develop into shootlets in elongation media and ca. 35 plants survive to grow into plants, which show normal flowering and seed set. The fully-grown To transgenic plants are healthy and phenotypically similar to control plants. They produce the same amount of seeds (Tt) that are healthy and weigh as much as untransformed seeds.

Molecular Analysis ofplants Leaves from To Plants obtained after transformation were screened for the selectable marker gene by amplification with 1 XI primers respectively. Represented in FIG. 11.
The different stages of the Xylose Isomerase positive Brassica juncea plants are shown in FIG. 10.

Xylose Isomerase - Forward Primer 5' CTCTCTCGAGCAACCATGGGTGAATTCTTTCC 3' Xylose Isomerase- Reverse Primer 5' GAAACTCGAGCTTGTCGATTAAGAAATGTATTGGTT-3' Table 14. Primers used for amplification of xylose isomerase genes.

All the transgenic plants show integration of the XI gene into their genome as seen by amplification of the XI gene in the seed samples of the To transgenic plants.
Represented in FIG 12.

Thus, we report successful transformation of Brassica using Xylose Isomerase as the selection marker. An efficiency of ca. 35 % is observed using the selection system using Xylose Isomerase (positive selection) compared to the transformation efficiency of 15-23 % reported using herbicides and antibiotics resistance genes (negative selection) around the world so far.

Thus, Brassica has been successfully transformed using positive selection. The use of xylose isomerase of SCI for positive selection is an efficient method for transformation of Brassica explants. This selection system is more efficient and results in larger number of transgenic plants than traditional antibiotic (Kanainycin, Hygromycin) and herbicide (phosphinothricin) based systems. Finally, it also fulfills the demand of alternative selective markers and avoids the risk and environmental concern involved with the use of antibiotic resistance genes in the development of genetically modified plants.

MEDIA COMPOSITION
Murashige and Skoog (MS) salts:
1.9g/l KNO3 1.65g/1 NH4NO3 370mg/1 MgSO4 170mg/1 KH2PO4 440mg/1 CaC12.2H20 15mg/1 MnSO4.7H20 8.6mg/1 ZnSO¾.7H20 6.2mg/1 H3B03 0.025mg/1 CuSO4.5H20 0.025mg/1 CoC1z 0.83mg/1 KI
0.025mg/1 NaZMoO4.2H20 36 mg/1 Na2EDTA
28mg/1 FeSO4 100mg/1 Myoinositol 0.1 mg/1 Nicotinic acid 1.0 mg/1 Thiamine HCl 0.1 mg/1 Pyridoxine HCl MS liquid MS salts 30 g/l Sucrose Half MS medium Half MS salts 30 g/1 Sucrose 8 g/l Agar Luria Bartani (LB broth) g/l Tryptone 5 g/l Yeast extract 10 g/l NaCI

PCR conditions for amplifying Brassica Genomic DNA.
DNA 100ng dNTP 200 M
Primer 0.25 M
MgC12 mM
lOX Buffer 2.51i1 (Bangalore Genei) Taq Polymerase lU
Total Volume 25 l Amplifying Conditions Followed.
94 C -3 min 94 C -1 min 55 C -1 min 40Cycles 72 C -1.3 min 72 C-10 min DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS

THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:

NOTE POUR LE TOME / VOLUME NOTE:

Claims (15)

1) A novel and improved method of generating Brassica juncea transformants based on a method of positive selection and regeneration comprising:

(a) construction of a recombinant vector incorporating the nucleic acid sequence separably linked to a regulatory sequence encoding the enzyme xylose Isomerase required for the metabolism of the selection agent xylose.
(b) Agrobacterium mediated method of transformation of the said vector into the plant cells or plant tissues under conditions optimal for infection.
(c) A step of selection of putative transformants from a population of genetically non-transformed plant cells or plant tissue on MS medium containing the selection agent of step (a).
(d) A step of regeneration of the selected plant cells or tissues.

2) A method according to claim 1, wherein the plant used belongs to the species Brassica juncea.

3) A method according to claim 1, wherein the nucleic acid sequence encoding the enzyme xylose isomerase is isolated from the organism Schizochytrium and is represented in the SEQ ID 1.

4) The nucleic acid sequence according to claim3, wherein atleast one of the nucleotide sequence is modified in that the expression of thus modified DNA
occurs in the host plant explant.

5) The nucleic acid sequence according to claim 4, wherein the modified sequence is represented in SEQ ID3.

6) The corresponding amino acid sequence of the expressed protein is represented in SEQ ID 2.

7) A method according to claim1, wherein the plant cells or tissues subjected to transformation is obtained from the cotyledonary petiole post germination of seeds of the host plant in MS medium.

8) A method according to claim1, wherein the plant cells/tissues prior to transformation with the vector construct are unable to utilize xylose as a sole source of carbon.

9) A method of transformation according to claim1, wherein the plant of the genus Brassica, transformed with the recombinant vector comprising the nucleic acid sequence encoding the enzyme xylose isomerase required for the metabolism of xylose, characterized in that, in the step of transformation a stagewise co-cultivation of explants is used which comprises:
(a) A step of preparing an explant by excision of cotyledonary petiole avoiding the inclusion of any primordial tissue.
(b) A step of infection of the said explant with the Agrobacterium carrying the said vector construct for 10 seconds and the subsequent transfer to the co-cultivation medium.
(c) A step of co-cultivation of the said explants for 3 days at 28°C, in dark conditions.

(d) A step of transferring the explants with the selection medium containing the selection agent.
(e) A step of subsequent transfer of the selected explants into the selection and elongation medium.
(f) A step further comprising the regeneration of the selected transformants.

10) A method according the preceding claim, characterized in that the confirmation of transformation of the said selectable marker gene in the regenerated plantlets is done using the known molecular techniques of screening.

11) A method according to any of the said preceeding claims, wherein the genetically transformed plant explants harboring the vector comprising the nucleotide sequence of claim3, the expression of which confers a metabolic advantage to the transformed explants over the non-transformed cells.

12) A method according to any of the said preceding claims, wherein the transformed cells are selected based on the said competitive metabolic advantage of utilizing the selection agent xylose as a carbohydrate source attributed to the expression of the enzyme by the nucleic acid of claim 3.

13) A method according to claim1, wherein the transformation efficiency obtained is greater than 35%

14) A method according to claim 1, wherein the transformation efficiency obtained is greater than 30%

15) A method according to claim 1, wherein the transformation efficiency obtained is greater than 25%