WO2007072110A1 - Enhancement of carotenoids in plants - Google Patents
Enhancement of carotenoids in plants Download PDFInfo
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- WO2007072110A1 WO2007072110A1 PCT/IB2005/003995 IB2005003995W WO2007072110A1 WO 2007072110 A1 WO2007072110 A1 WO 2007072110A1 IB 2005003995 W IB2005003995 W IB 2005003995W WO 2007072110 A1 WO2007072110 A1 WO 2007072110A1
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- C—CHEMISTRY; METALLURGY
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
Definitions
- the present invention relates to a method of enhancing the content of carotenoids and other isoprenoids, preferably of lycopene and or ⁇ -carotene, in a plant, plant cell, callus, tissue, fruit, root or other part of a plant, and/or a method of increasing the height in a plant, to a plant, plant cell, callus, tissue, root or fruit produced by such method, to a method of obtaining carotenoids, preferably lycopene and/or ⁇ -carotene, to a nucleic acid construct and to the use of such nucleic acid construct.
- lycopene an acyclic isomer of ⁇ -carotene
- lycopene an acyclic isomer of ⁇ -carotene
- Lycopene's stability in serum and its superior quenching ability have made it an extensively studied compound in terms of its production and disease treatment. Its 11 conjugated and 2 non-conjugated double bonds give it its characteristic red colour (peak absorbance at 472-476 nm). Further of the possible 1056 isomers about 12 are found in nature with the linear all trans isomer being the most stable and the one found in plants. This compound is a polyunsaturated hydrocarbon, lipophylic, and sensitive to light, heat and oxidation (Faulks and Southon, 2005).
- Lycopene is abundant in several fruits and vegetables such as grapefruit, guava juice, watermelon and tomatoes. In tomatoes, the red pigmentation is largely accounted for by lycopene and high-lycopene fruits exhibit enhanced redness. In plants lycopene is found in the all trans- form and is poorly absorbed from the diet without cooking. Heating in the presence of oil has shown better extractability and certain tomato products such as juice, puree, paste and sauce have been found to have better, i.e. higher amounts of lycopene. Cooking has shown to isomerize lycopene to a czs-conf ⁇ guration that increases its bioavailability. Further in serum the preponderance of lycopene is in the cw-form. It has been estimated that 35 mg of lycopene is required on a daily basis and may not be currently part of the current intake of consumers (Faulks and Southon, 2005).
- ROS Reactive oxygen species
- ROS have been linked with a number of chronic diseases, due to the oxidative damage caused.
- antioxidants have been associated with protecting lipids, membranes, low- density lipoproteins, proteins and DNA from damage. More specifically, lycopene has been demonstrated to prevent cancer and cardiovascular diseases.
- lycopene by binding to lipophylic moieties, protects such complexes from oxidative damage.
- IPP is isomerized to dimethylallyl pyrophophate (DMAPP), an activated monomer that is eventually oligomerized into geranyl geranyl pyrophosphate, the precursor to C40 carotenoids and the first compound in the pathway, phytoene.
- DMAPP dimethylallyl pyrophophate
- Phytoene is desaturated to phytofluene by phytoene desaturase and further to ⁇ -carotene.
- Lycopene is next produced via neurosporene by ⁇ -carotene desaturase.
- the bacterial crtl (from Erwinia uredovord) converts phytoene to all trans-lycopene (Ye et al 2000, Bramley 2002). While beyond the scope of this work, it is to be noted that all trans- lycopene is further converted to ⁇ - and ⁇ -carotene of which ⁇ -carotene is a pro-vitamin A.
- hp-2 and dg were also identified from natural populations and linked to enhanced carotenoid levels (for review Levin). Drawing from these results, hp2 and dg were determined to be tomato homologs of the Arabidopsis DETl (DE-ETIOLATED 1). The constitutive silencing of this gene resulted in elevated levels of ⁇ -carotene and lycopene in tomato (Davuluri et al 2004). As with hp-2, these mutants had severe development defects in that, they were stunted, bushy and dwarf.
- Davuluri and coworker (2005) further, reduced the level of DETl using an RNA interference strategy that significantly increased the level of carotenoids, resulting in a doubling of lycopene and a 10-fold increase in ⁇ -carotene.
- over-expression of the blue light photoreceptor cry2 increased lycopene contents by decreasing the expression of lycopene ⁇ cyclases (Giliberto et al, 2004).
- the object of this invention is to provide means to enhance carotenoid levels in plants and/or plant parts.
- a further object of the present invention is to provide a method for producing enhanced levels of carotenoids, in particular lycopene, in plants and/or plant parts.
- Yet a further object of the present invention is to provide a plant or plant part having enhanced levels of carotenoids, in particular lycopene.
- the objects of the present invention are solved by a method of enhancing the content of carotenoids and other isoprenoids, preferably of Iycopene and or ⁇ -carotene, in a plant, plant cell, callus, tissue, fruit, root or other part of a plant, and/or increasing the height in a plant, said method comprising:
- impairment of mitochondrial function preferably impairment of mitochondrial complex I, II, III and /or IV, more preferably mitochondrial complex I in said plant, plant cell, callus, tissue, fruit, root or other part of a plant.
- said impairment occurs using a modified protein component of mitochondrial complex I of said plant cell, preferably a protein component of mitochondrial complex I that is the translation product of an unedited coding sequence.
- said impairment occurs by transforming said plant cell with a nucleic acid construct, preferably a DNA-construct, or by transforming said plant cell with a nucleic acid construct via Agrobacterium species - mediated transformation, preferably Agrobacterium tumefaciens, or by viral transfection using a suitable plant virus such as Tobacco Mosaic Virus, or by protoplast transformation.
- Agrobacterium species - mediated transformation preferably Agrobacterium tumefaciens
- viral transfection using a suitable plant virus such as Tobacco Mosaic Virus, or by protoplast transformation.
- said nucleic acid construct preferably said DNA-construct, or said Agrobacterium comprises, preferably in a binary vector, a nucleic acid encoding said modified protein component of said mitochondrial complex I.
- said modified protein component of mitochondrial complex I is a dysfunctional protein from another plant species than said plant cell or a dysfunctional protein of the same plant species as said plant cell.
- said modified protein component of mitochondrial complex I is a dysfunctional protein selected from the group comprising NAD 1, 2, 3, 4, 4L, 5, 6, 7, 9, nuclear mitochondrial proteins 76 Kda, 55 Kda, 28.5 Kda, 22 Kda and Acyl carrier protein.
- nucleic acid encoding said modified protein component is selected from the group comprising SEQ ID NO: 1 - 3
- said nucleic acid construct preferably said DNA-construct, or said Agrobacteriiim, preferably said Agrobacterium binary vector, additionally comprises a nucleic acid encoding a mitochondrial transit peptide, operably linked to said nucleic acid encoding said modified protein component of said mitochondrial complex I.
- nucleic acid construct preferably said DNA-construct, or said Agrobacterium, preferably said Agrobacterium binary vector, additionally comprises a promoter and a terminator, and said promoter and terminator are operably linked to said nucleic acid encoding said modified protein component of said mitochondrial complex I.
- nucleic acid construct preferably said DNA-construct, or said Agrobacterium, preferably said Agrobacterium binary vector
- said nucleic acid construct comprises said promoter and said terminator, said nucleic acid encoding mitochondrial transit peptide and said nucleic acid encoding said modified protein component of said mitochondrial complex I, all as defined before, all of them being operably linked.
- said impairment occurs by mutating said plant cell with respect to at least one of the components of said mitochondrial complex I in said plant cell.
- said mutating occurs by mutating said plant cell at random using a chemical and/or physical mutagenic agent being applied to at least one plant cell, preferably a plurality of plant cells of the same plant, said chemical mutagenic agent preferably being selected from the group comprising ethyl methane sulfonate and said physical mutagenic agent being selected from the group comprising fast neutron bombardment, X-ray, gamma ray and other mutagenic irradiation.
- the method after mutating, further comprises the additional step of screening for a modified protein component of mitochondrial complex I or other mitochondrial functions of said plant cell in said plant cell or plurality of plant cells.
- said impairment occurs by applying a chemical inhibitor of mitochondrial function, preferably a chemical inhibitor of mitochondrial complex I of said plant cell, to said plant cell, plant, callus, tissue, a part of said plant, said plant in its entirety, fruit, root and / or other plant organ.
- a chemical inhibitor of mitochondrial function preferably a chemical inhibitor of mitochondrial complex I of said plant cell
- said chemical inhibitor is selected from the group comprising rotenone, antimycin A, oxyfluorfen, violaxanthin, piericidin, piericidine A, pyrazoles, pyridaben, quinazolines, acetogenins, thiangazoles and fenaza.
- said impairment is an inhibition of said of mitochondrial complex I.
- said method further comprises the step of raising said plant cell, plant part, tissue, seed or organ having undergone the method of any of the foregoing claims, to produce a plant callus, tissue, plant, root and/or fruit.
- the objects of the present invention are solved by a plant cell, callus, tissue, plant, root or fruit produced by the method according to the present invention.
- the plant cell, callus, tissue, plant, root or fruit is/are derived from a plant origin selected from the group comprising solanaceous species, including tomato, pepper, capsicum, potato, petunia and or tobacco.
- the amount of carotenoid, preferably lycopene is enhanced to >10mg, preferably >15 mg, even more preferably >16 mg and most preferably >20 mg/lOOg fresh weight of plant cells, callus tissue, plant, root and/or fruit.
- the amount of carotenoid, preferably lycopene is enhanced by at least two-fold, preferably three-fold, in relation to a plant cell/callus/tissue/plant, root or fruit not having undergone the method according to the present invention.
- the objects of the present invention are solved by a method of obtaining carotenoids, preferably lycopene and /or ⁇ -carotene, comprising the steps:
- the objects of the present invention are also solved by a nucleic acid construct comprising a nucleic acid sequence encoding a modified protein component of mitochondrial complex I.
- said modified protein component of mitochondrial complex I is selected from the group comprising NAD 1, 2, 3, 4, 4L, 5, 6, 7 and 9 or other proteinaceous component of said complex.
- said modified protein component of mitochondrial complex I is from a species selected from the group comprising tomato, potato, tobacco, rice, maize, petunia, Arabidopsis, and or Lotus, Medicago, wheat and/or Sorghum.
- said modified protein component of mitochondrial complex I has a sequence selected from the group comprising SEQ ID NO: 4, 5, and 6.
- nucleic acid construct for enhancing the content of carotenoids and other isoprenoids, preferably of lycopene and /or ⁇ -carotene, in a plant cell, plant, callus, tissue, fruit, root or other part of said plant and/or for increasing the height in a plant.
- a “transgenic or transformed plant” refers to a plant which contains a recombinant polynucleotide introduced by transformation. Transformation means the introduction into a plant of a polynucleotide sequence in a manner so as to cause a stable integration of the nucleotide sequence or a transient expression of the sequence. This may be achieved by particle bombardment (biolistic), Agrobacterium-mediated (using a suitably developed plasmid vector), transfection with viral DNA or vectors, introduction of DNA by electroporation or lipofection. Plant transformation may be carried out on plant cells, pollen, on plant seeds, on plant protoplasts, or any other type of plant tissue, intact plant or plant part under sterile or non-sterile conditions.
- a transformed plant may refer to a whole plant, any plant part, plant cell, plant organ, plant tissue, seed, root, flower, fruit, root or shoot. It may also refer to the progeny thereof.
- a “vector” is a polynucleic acid construct, generated recombinantly, artificially or chemically, comprising nucleic acid elements that may encode genes, proteins, promoters, terminators and transit peptides. These segments will be operably linked so as to enable the expression of the gene encoded or the complete execution of the process encoded.
- the promoter region may include constitutive or tissue-specific, tissue-active, developmental stage-active/specific, or inducible promoters such as but not limited to the cauliflower mosaic virus 35S promoter, the cassava vein mosaic virus promoter or the maize ubiquitin promoter.
- a nucleotide sequence is "operably linked" when adjacent segments of DNA sequence are linked in such a manner so as to enable a cellular/biological function as encoded by the gene sequence.
- Carotenoids are a class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls) consisting of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the centre of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship.
- All carotenoids may be formally derived from the acyclic C40H56 structure, having a long central chain of conjugated double bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization or (iv) oxidation or any combination of these processes. Impairment
- Impairment refers to the partial or complete loss of function of the organelle, functional complex, enzyme or other functional entity.
- a complete loss of function as used herein is also sometimes referred to as inhibition.
- Mitochondria complexes refer to the four electron transport chain complexes of mitochondria called I, II, III and IV respectively, where complex I is NADH- dehydrogenase, Complex II is succinate dehydrogenase, Complex III is cytochrome c reductase and complex IV is cytochrome c oxidase. These are multipolypeptide complexes involved in ATP production by oxidation of NADH+H 4" and FADH2 to NAD+ and FAD respectively and water.
- This complexes comprises about 43 polypeptide chains including the mitochondrial encoded nadl, nad2, nad3, nad4, nad4Lm, nad5, nad ⁇ , nad7, nad9 and the nuclear encoded 76 Kda, 55 Kda, 28.5 Kda, 22 Kda and Acyl carrier protein.
- Modified protein component refers to a protein component that differs from the native functional protein in its amino acid sequence, structure or function and may be nonfunctional. It is to be noted that in plant mitochondria, the amino acid sequence of the native (functional) protein differs from the hypothetical translation production of the native gene sequence that encodes the protein due to one or more post-translational RNA editing events.
- Unedited refers to a gene sequence identical to the gene sequence present naturally in the mitochondrial genome. It is often different from the mRNA product that encodes the native (functional) protein due to post-transcriptional RNA editing whereby certain C ribonucleotides are modified to U ribonucleotides and rarely certain U ribonucleotides to C ribonucleotides.
- the "translation product of an unedited coding sequence” is a protein that would be expected to be produced if no editing events at a post- transcriptional level occurred.
- Transit peptide refers to an N-terminal presequence, which directs mitochondria-bound proteins encoded by the nucleus to the mitochondrion.
- the transit peptide is required for the transport of such proteins across the relevant membranes from their site of synthesis in the cytoplasm.
- Inhibitors of mitochondria include but are not limited to Rotenone, Antimycin A, Cyanide, malonate (succinate dehydrogenase inhibitor), 2,4-Dinitrophenol (DNP), Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), Oligomycin, oxyfluorfen, violaxanthin, piereicidin A, pyrazoles, pyridaben, quinazolines, acetogenins, thiangazoles, fenaza, thenoxyltrifluoroacetone, carfboxin, oxycarboxin, fenfuran, DDT, chlorproham, propanil, dinoseb, ioxynil, cyclodiene, paraquat, dinoseb, diafenthiuron, methomyl, Bongkrekic acid and hydramethyl ⁇ on.
- Agrobacterium refers to a bacterium of Agrobacterium species which is used to transform plants with gene(s) of interest using a suitable recombinant Agrobacterium binary vector.
- Agrobacterium binary vector refers to a recombinant DNA construct, which with other gene(s) of interest will be transformed into Agrobacterium cells. These cells in turn will be used for the transformation of plant cells, parts, seeds, or intact entire plants.
- the inventors have surprisingly found that enhanced levels of carotenoids may be achieved by impairing mitochondrial function in plants, by way of genetic engineering or other means.
- the inventors were able to produce surprisingly high levels of carotenoids in a plant, most notably lycopene, in comparison to a plant not having undergone the method according to the present invention (table 1).
- Similar levels of carotenoids may only be encountered in processed and artificially enriched foods such as ketchup or concentrated tomato paste (see also Table 2).
- said objective may be achieved by assembling a polynucleotide construct encoding a maize ubiquitin promoter (SEQ ID No 7), an Arabidopsis At-mRBPla mitochondrial targeting transit peptide (SEQ ID No 8-nucleotide sequence, SED ID No 9- polypeptide sequence), an unedited nad9 gene from rice mitochondria peptide (SEQ ID No 1 -nucleotide sequence, SED ID No 2- polypeptide sequence) and a Nopaline synthase (NOS) terminator.
- This assembly may be carried out so as to render the construct transcriptionally and translationally competent in plants, and additionally allow the protein product to be translocated to the mitochondrion.
- a plant cell/plant/plant part transformed by such or similar construct can be observed to show the aforementioned high levels of carotenoids, most notably lycopene and/or ⁇ -carotene.
- This invention thus relates to a method of developing high carotenoid plants, plant cell, tissues or plant parts (comprising leaves, stems, roots, fruits and flowers).
- This invention also relates to a method of developing plants with enhanced levels of lycopene and or/ ⁇ -carotene.
- This invention furthermore relates to a method of enhancing the levels of plant compounds derived from the isoprenoid pathway including but not limited to gibberellic acid, abscisic acid, pigments, sterols.
- Figure 1 shows: Plasmid pBS(SK-) construct used as a basic construct for transformation. Targeting sequence indicates the coding regions of At-mRBPla cloned in between Sac I and Xba I restriction sites to get pNGl.
- Figure 2 shows: Plasmid pBS(SK-) construct used as a basic construct for transformation. TS and unedited nad9 indicates the coding regions of At-mRBPla and edited nad9 gene cloned in between Xba I and BamHI restriction sites to get pNG3.
- Figure 3 shows: Plasmid pBS(SK-) construct used as a basic construct for transformation.
- the boxes named TP and unedited nad9 indicate the coding regions of At-mRBPla and unedited nad9 inserted in multiple cloning site of the pBS(SK-) vector.
- the chimeric genes are under the control of ubiquitin promoter and Nos terminator.
- Figure 4 shows: Plasmid pLAU ⁇ .hph encoding the hygromycin resistance gene (hph) driven by a Cassava vein Mosaic Virus promoter (CVMV) with a NOS terminator.
- hph hygromycin resistance gene driven by a Cassava vein Mosaic Virus promoter (CVMV) with a NOS terminator.
- CVMV Cassava vein Mosaic Virus promoter
- Figure 5 shows: The Standard curve for Lycopene used for determining the lycopene content in one embodiment of the present invention.
- Figure 6 shows: A typical chromatogram obtained by resolving a lycopene extract as described herein.
- SEQ ID NO 1 Rice (Oryza sativd) mitochondrial nad9 gene for NADH dehydrogenase subunit 9 (GenBank Accession number D50099 [RICMTNAD9]).
- SEQ ID NO 2 Potato (Solarium tuberosum) mitochondrial nad9 gene for NADH dehydrogenase subunit 9 (GenBank Accession number X79774 [STMINAD9]).
- SEQ ID NO 3 Tobacco (Nicotiana tabacum) mitochondrial nad9 gene for NADH dehydrogenase subunit 9 (GenBank Accession number YP 173479 [YP 173479]).
- SEQ ID NO 4 Rice (Oryza sativd) mitochondrial nad9 protein for NADH dehydrogenase subunit 9 (translation of SEQ ID NO 1). 0.1 % BSA
- Rice mitochondria (Approximately 75 ug) was resuspended in resuspension buffer and lysed with 1 A volumes of lysis buffer. After gentle mixing by inversion, phenol was added, followed by chloroform. Phenol chloroform extraction was carried out 3 times followed by chloroform extraction. DNA was precipitated from the aqueous phase with 2.5 volumes of ethanol and 1/1 Oth volumes of 3 M sodium acetate, by centrifugation at 13, 000 rpm for 20 minutes after a 30 minute incubation at —20 C. The DNA pellet was washed with 70 % ethanol, dried and dissolved in water.
- the mitochondrial targeting sequence (At-mRBPla, see SEQ ID No 8 for DNA and SEQ ID No 8 for DNA and SEQ ID No 8 for DNA and SEQ ID No 8 for DNA and SEQ ID No 8 for DNA
- the forward and the reverse primers have the Sad site and the Xbal site incorporated within them respectively and this facilitated the cloning of the PCR product into pBS
- Rice mitochondria (Approximately 75 ⁇ g) was resuspended in resuspension buffer and lysed with 1 A volumes of lysis buffer. After gentle mixing by inversion, phenol was added, followed by chloroform. Phenol chloroform extraction was carried out 3 times followed by chloroform extraction. DNA was precipitated from the aqueous phase with 2.5 volumes of ethanol and 1/10 th volumes of 3 M sodium acetate, by centrifugation at 13, 000 rpm for 20 minutes after a 30 minute incubation at -20 C. The DNA pellet was washed with 70 % ethanol, dried and dissolved in water.
- Unedited nad9 (SEQ ID NO 1 for DNA and 4 for peptide) is obtained by PCR from rice mitochondrial DNA. The following primer combination was used for the amplification:
- Reverse primer 5 ⁇ AAGGATCCGGGATTATCCGTCGCTACG y
- the forward primer has the Xba I site and the reverse primer has the BamHI site with which the unedited nad9 gene was cloned adjacent to the mitochondrial targeting sequence and the vector was named as pNG3 (fig 2).
- the unedited nad9 gene was excised out of pNG3 with Sad and BamHI and was ligated with the ubiquitin promoter upstream (SEQ ID NO 7) and the Nos terminator downstream.
- the resulting construct was called pNGl 1 (fig 3).
- Tomato ⁇ Lycopersicon esculentum Mill, variety S-22 (Arka Vikas) seeds were obtained from Indian Institute of Horticultural Research (IIHR), Hesarghatta, Bangalore. " The seeds were washed twice with double distilled autoclaved water. ⁇ The seeds were rinsed in 70% Ethanol for 2 minutes.
- the seeds are immersed in a 70% solution of commercial bleach for 30 minutes, in a shaker.
- the seeds are dried on autoclaved tissue papers.
- Steps 'd' and 'e' are carried out in the laminar hood to avoid contamination.
- the seeds are germinated in-vitro on half strength MS media.
- the 10-day old seedlings are uprooted the cotyledonary leaves are cut out and the center portion of the leaves are used for bombardments.
- About 40 such explants are placed at the center of a Petri plate containing osmoticum medium. After 4hrs incubation on this medium the calli were immediately subjected to microprojectile bombardment using the particle accelerator, PDS-1000/He.
- the size of gold particles used was between 1.5-3.0 ⁇ , 6mg of gold particles was weighed in a 0.5 ml eppendorf tube. lOO ⁇ l of autoclave double distilled water was added to the gold particles and vortexed for 30 seconds in a microfuge. It is centrifuged for 30 seconds. lOO ⁇ l of 100% ethanol was added and vortexed for a minute. This was centrifuged for 30seconds in a microfuge at 10000 rpm. The supernatant was pipetted out. The ethanol wash is repeated again. 100 ⁇ l of sterile distilled water was added to the pellet. It is vortexed and 50 ⁇ l of the suspension is transferred into another 0.5 ml tube. Each of these tubes contains 50 ⁇ l of gold suspension and was stored at room temperature or at 4 0 C until DNA coating was done.
- plasmid-containing gene of interest plasmid containing the unedited nad9 gene or edited nad9 or unedited nad9 in antisense version
- pLAU6hph the selectable marker hygromycin containing Plasmid, see Figure 4
- 20 ⁇ l of 0.1M spermidine Sigma, Aldrich
- 50 ⁇ l of 2.5M CaCl 2 was then added and mixed well. The mix was left at room temperature for 10 minutes. It was later centrifuged for 30 seconds in a microfuge. The supernatant is removed in the laminar hood.
- Pre-assemble and pre-sterilize the macrocarrier set in a macrocarrier holder prior to performing sample cell/tissue bombardments The macrocarrier was first immersed in 100% ethanol and then dried on autoclaved tissue paper. Then macrocarrier was left under UV light for surface sterilization for 10 minutes in the laminar hood.
- rupture disks Transfer selected rupture disks to individual Petri dishes for easier handling. Sterilize rupture disks by briefly dipping them in 70% isopropanol just prior to insertion in the Retaining Cap. Do not soak for more than a few seconds. Extensive soaking may delaminate the disks, resulting in premature rupture. All disks, with the exception of those rated at 450, 650 and 1,100 psi are laminated. Autoclaving is not recommended because of potential delamination.
- stopping screens Transfer selected stopping screens to individual Petri dishes for easier handling. Sterilization by autoclaving is recommended. Alternatively, these parts can be sterilized by soaking in 70% ethanol, followed by drying in a sterile environment. The stopping screens can be double autoclaved and reused.
- Coating of Microcarrier onto Macrocarrier The supernatant is removed from the DNA coated gold particles. Based on the number of plates to be bombarded, absolute ethanol is added to the DNA coated gold pellet - 10.0 ⁇ l is added for every macrocarrier to be coated, i.e. for every plate to be bombarded. The mixture is vortexed until a homogenous suspension is obtained and 10.0 ⁇ l is pipetted out and coated onto the center of the macrocarrier disc. The ethanol is allowed to dry away leaving the gold particles on the macrocarrier disc.
- the bombardment parameters for gap distance between rupture disk retaining cap and microcarrier assembly are selected and adjusted.
- the bombardment was carried out at 900-psi rupture pressure and at a distance of 9cm.
- the stopping screen is supported in proper position inside fixed nest of microcarrier launch assembly.
- the helium supply is adjusted to 200 psi in excess of the desired rupture pressure.
- Plug in power cord form main unit to electrical outlet.
- Target cells removed from chamber.
- the calli were transferred to tomato regeneration medium containing BAP (4.5 mg/L) and IBA (0.2 mg/L - selection media containing 10mg/L hygromycin) medium for selection & incubated at 250C in light for 15 days.
- the resistant calli are subculture every 15 days onto fresh media.
- the shoots are cut out and place on MS media containing BAP (4.5 mg/L), IBA (0.2 mg/L) and GA (1.0 mg/L), for elongation if only if the shoots have not elongated in the regeneration medium.
- the elongated shoots are shifted to MS media containing IAA (0.1 m/L) for rooting.
- the rooted plantlets are shifted to autoclaved distilled water for hardening for a couple of days and then into vermiculite. The plantlets are then shifted to red soil.
- Plantlets on soil are transferred to the greenhouse. Plants are raised under standard conditions for the variety and fruits harvested. Cross-fertilization may be carried out for experimental or breeding purposes. Genetic studies may also be carried out on plants in the greenhouse.
- the dried samples are redissolved in 1 ml of Sample solvent and 1:10 dilution was made from this stock. This diluted sample was passed through 0.22-micron filter before subjecting to HPLC analysis. Sample preparation should be carried out quickly to avoid loss of solvent.
- a binary solvent system of methanol : methylene chloride (95:5) was used to resolve the Lycopene from the samples over 15 minutes.
- the run time was reduced to 15 minutes with a flow rate of 1.5ml/min to accommodate more samples.
- the detection wavelength for lycopene is 476nm, which was quantitated using standard curve.
- Table 1 Levels of lycopene analysed including transgenic lines and commercial varieties.
- the levels of carotenoids, most notably lycopene, achieved by the present invention may be as high as 6.5 (26.9:4.1), and range from 2.1 (14.3:6.7) to 6.5, and include ratios such as 4.2 (17.4:4.1), 3.0 (16.7:5.5), and 5.7 (23.5:4.1).
- Giege P Sweetlove LJ et al (2003) Identification of mitochondrial proteini complexes in Arabidopsis using two-dimensional blue-native polyacrylamide gel electrophoresis. Plant MoI Biol Rep 21: 133-144 14. Giliberto L, Perrotta G et al (2004) Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol 137: 199-208
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (6)
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AU2005339391A AU2005339391A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
EP05850740A EP1963511A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
CA002638818A CA2638818A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
PCT/IB2005/003995 WO2007072110A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
AP2008004538A AP2008004538A0 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
CNA2005800525085A CN101360828A (en) | 2005-12-23 | 2005-12-23 | Reinforcement of carotenoid in plants |
Applications Claiming Priority (1)
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PCT/IB2005/003995 WO2007072110A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
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WO2007072110A1 true WO2007072110A1 (en) | 2007-06-28 |
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PCT/IB2005/003995 WO2007072110A1 (en) | 2005-12-23 | 2005-12-23 | Enhancement of carotenoids in plants |
Country Status (6)
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EP (1) | EP1963511A1 (en) |
CN (1) | CN101360828A (en) |
AP (1) | AP2008004538A0 (en) |
AU (1) | AU2005339391A1 (en) |
CA (1) | CA2638818A1 (en) |
WO (1) | WO2007072110A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013121329A (en) * | 2011-12-09 | 2013-06-20 | Sumitomo Rubber Ind Ltd | Method for producing isoprenoid, isoprenoid and callus |
WO2018234580A1 (en) * | 2017-06-23 | 2018-12-27 | Institut National De La Recherche Agronomique | Method for improving plant regeneration |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104686215B (en) * | 2015-02-10 | 2017-05-03 | 中国农业大学 | Method for increasing content of carotenoids in tomato fruits |
US10380915B2 (en) * | 2017-06-29 | 2019-08-13 | Stephen Sophorn Lim | Braille dot delivery system |
CN109655422A (en) * | 2018-10-30 | 2019-04-19 | 晨光生物科技集团股份有限公司 | A kind of detection method of lycopene content |
CN112890199B (en) * | 2021-02-27 | 2022-05-31 | 中国农业科学院油料作物研究所 | Composition for stably and efficiently regulating immunologic function and preparation and application thereof |
Citations (1)
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WO2001062889A2 (en) * | 2000-02-25 | 2001-08-30 | Grinenberger, Jean-Michel | A process for generating cytoplasmic male sterile line in rice and other crops by rna editing |
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2005
- 2005-12-23 CA CA002638818A patent/CA2638818A1/en not_active Abandoned
- 2005-12-23 WO PCT/IB2005/003995 patent/WO2007072110A1/en active Application Filing
- 2005-12-23 CN CNA2005800525085A patent/CN101360828A/en active Pending
- 2005-12-23 AU AU2005339391A patent/AU2005339391A1/en not_active Abandoned
- 2005-12-23 EP EP05850740A patent/EP1963511A1/en not_active Withdrawn
- 2005-12-23 AP AP2008004538A patent/AP2008004538A0/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001062889A2 (en) * | 2000-02-25 | 2001-08-30 | Grinenberger, Jean-Michel | A process for generating cytoplasmic male sterile line in rice and other crops by rna editing |
Non-Patent Citations (8)
Title |
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BRAMLEY PETER M: "Regulation of carotenoid formation during tomato fruit ripening and development", JOURNAL OF EXPERIMENTAL BOTANY, vol. 53, no. 377, October 2002 (2002-10-01), pages 2107 - 2113, XP002399322, ISSN: 0022-0957 * |
DATABASE EMBL [online] 16 June 1994 (1994-06-16), "S.tuberosum mitochondrion nad9 DNA", XP002399343, retrieved from EBI accession no. EM_PRO:X79774 Database accession no. X79774 * |
DATABASE EMBL [online] 22 February 2001 (2001-02-22), "Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, PAC clone:P0557A01.", XP002399342, retrieved from EBI accession no. EM_PRO:AP003280 Database accession no. AP003280 * |
DATABASE EMBL [online] 28 December 2004 (2004-12-28), "Nicotiana tabacum mitochondrial DNA, complete genome.", XP002399344, retrieved from EBI accession no. EM_PRO:BA000042 Database accession no. BA000042 * |
GROHMANN LUTZ ET AL: "Translation of nad9 mRNAs in mitochondria from Solanum tuberosum is restricted to completely edited transcripts", NUCLEIC ACIDS RESEARCH, vol. 22, no. 16, 1994, pages 3304 - 3311, ISSN: 0305-1048 * |
GUTIERRES S ET AL: "Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in nicotiana sylvestris mitochondrial deletion mutants", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 94, no. 7, April 1997 (1997-04-01), pages 3436 - 3441, XP002955476, ISSN: 0027-8424 * |
KARPOVA OLGA V ET AL: "A partially assembled complex I in NAD4-deficient mitochondria of maize", PLANT JOURNAL, vol. 17, no. 5, March 1999 (1999-03-01), pages 511 - 521, XP002399321, ISSN: 0960-7412 * |
LIU Y-S ET AL.: "There is more to tomato fruit colour than candidate carotenoid genes", PLANT BIOTECHNOLOGY JOURNAL, vol. 1, 2003, pages 195 - 207, XP002399323 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013121329A (en) * | 2011-12-09 | 2013-06-20 | Sumitomo Rubber Ind Ltd | Method for producing isoprenoid, isoprenoid and callus |
WO2018234580A1 (en) * | 2017-06-23 | 2018-12-27 | Institut National De La Recherche Agronomique | Method for improving plant regeneration |
Also Published As
Publication number | Publication date |
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AP2008004538A0 (en) | 2008-08-31 |
CA2638818A1 (en) | 2007-06-28 |
EP1963511A1 (en) | 2008-09-03 |
CN101360828A (en) | 2009-02-04 |
AU2005339391A1 (en) | 2007-06-28 |
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