KR20120043371A - Mass production method for developing transgenic plant by using somatic embryo(incuding somatic embryodenic callus) in rose sweet yellow - Google Patents

Abstract

PURPOSE: A method for producing a large amount of transgenic plants using somatic cell embryo of Sweet Yellow is provided. CONSTITUTION: A method for producing a large amount of transgenic plants using rose 'Sweet Yellow' somatic cell embryo and embryogenic callus comprises: a step of co-culturing somatic cell embryo GUS gene-transformed Agrobacterium tumefaciens and GFP gene-transformed Agrobacterium tumefaciens; a step of culturing the co-cultured somatic cell embryo or embryogenic callues in an embryo selection medium containing 250mg/L^-1 of cefotaxim; a step of culturing the somatic cell embryo or embryogenic callus in a medium containing cefotaxim solely or together with PPT(phosphinothricin); a step of culturing a cultured shoot in a medium; a step of culturing the shoot in a shoot growth medium; a step of culturing 1-1.5cm of shoots in a multi-shoot growth medium; and a step of culturing the multi-shoots in a root inducing medium.

Description

MASS PRODUCTION METHOD FOR DEVELOPING TRANSGENIC PLANT BY USING SOMATIC EMBRYO (INCUDING SOMATIC EMBRYODENIC CALLUS) IN ROSE SWEET YELLOW}

The present invention relates to an efficient gene transfer method capable of mass-producing Agrobacterium-mediated transformants using somatic embryos and embryogenic callus derived from in-flight plant roots of the rose variety 'Sweet Yellow'.

New varieties of roses are cultivated mainly by cross-breed or cross-breeding. In late 2006, Suntory Co., Ltd. developed blue rose varieties using transformation technology and commercialized them in major crops such as rice and soybeans. Transforming technology, which has been established as a technology, has recently been used as a breeding technique for breeding rose varieties, but there are no commercially available transgenic rose varieties.

The previously reported rose transformation technique uses Agrobacterium tumefaciens ( Agrobacterium tumefaciens) inserting a foreign target gene into a plasmid vector using a callus derived from the leaves of in-flight culture plants as a fragment of the gene transfer. Tumefaciens ) was attempted to transfer the plasmid vector into which the gene was inserted or by inserting the gene using a gene gun. However, the gene gun technique causes too many foreign genes to be copied and introduced into the transformant, causing complex foreign gene loci. At the same time, it showed a problem of causing unexpected gene silencing. In addition, the gene transfer efficiency was very low and the transgene expression rate in plants re-differentiated in the selection medium after metastasis was not only low, but also the expression of the transgene was reduced. Few reports have been confirmed in.

In addition, transformation by electrophoresis is used, which has the advantage that it can be easily performed without tissue culture while having a problem that it is difficult to obtain a stable transformant.

On the other hand, when the gene is used as a fragment of the gene transfer, the gene transfer to the rose using a somatic embryogenic callus, which is not only high in gene transfer efficiency but also highly expressed in the whole replanted plant in the selection medium after the gene transfer Few examples have been attempted. Agrobacterium tumefaciens -mediated transformation of somatic embryonic callus (transformer) is known to have a weak root system and not bloom new branches (Alexander Vainstein (2002) Breeding For Ornamentals: Classical and Molecular Approches) , Kluwer Academic 185--186). In addition, there are no research reports in which gene expression has been identified throughout rose plants.

In the present invention, which is a partial gene transfer to a target material for gene transfer, unlike the existing gene transfer technology, which has a high probability of generating a chimeric transformant, the present invention can be a gene transfer as a whole of the target material for gene transfer. Gene transfer technology using somatic embryogenic callus is commercialized in blue rose varieties developed by Japan's Suntory in collaboration with Florigene of Australia, and transformation technology is breeding technology for breeding rose varieties. When it was used as a blue color variety, it is possible to increase the market share of the rose by developing the rose varieties that have the possibility of creating infinite added value due to the scarcity, but are difficult to develop as the traditional breeding technology. It is expected that you can.

The present invention relates to the development of transformation technology in order to cultivate new varieties of roses using transformation technology. When used as a material for transformation, the gene transfer efficiency is high, but it has not been studied until now. The aim of this study was to develop a technique for transferring genes and for purifying greenhouses after rooting by growing plants obtained from selection medium after gene transfer.

The present invention is to induce somatic embryogenic callus from the in-roots of the rose cultivars 'Sweet Yellow' fostered by the National Institute of Horticultural & Herbal Science, using a somatic embryo selected from the proliferation process (including embryogenic callus) as an exogenous gene transfer material. When establishing the transformation technology, after infection with Agrobacterium tumefaciens inserting GUS (β-glucuronidase) and GFP (Green Fluorescent Protein) genes which are widely used as marker genes, they are co-cultured After introducing the gene, transfer to the embryonic embryo selection medium and confirm the introduction of the foreign genes, and then induce the new primordial cells from the somatic embryos (including embryogenic callus) to which the foreign genes have been transferred, and regenerate the shoots from the originals, Rooting and rooting after confirming the expression of transgenic genes in shoot growth and polycystic induction selection medium for sound ground growth It was screen.

The present invention is a method for obtaining a large amount of transformed plants using rose sweet yellow somatic embryos and embryogenic callus, wherein the GUS gene is transformed into somatic embryos or embryogenic callus induced or selected from the roots of in vitro plants of the rose variety 'Sweet Yellow'. Agrobacterium tumefaciens bacteria Titanium (Agrobacterium tumefaciens) and the GFP gene is transformed Agrobacterium tumefaciens (Agrobacterium tumefaciens ) , culturing the co-cultured somatic embryo or embryogenic callus in embryonic embryo selection medium supplemented with 250 mg L- ¹ cefotaxim, the cultured somatic embryo or embryogenic callus Cultured in embryonic selection medium or embryonic selection medium to which 250 mg L- ¹ cefotaxim alone or 250 mg L- ¹ cefotaxim and 2 mg L- ¹ PPT (phosphinothricin) were added. Step, the step of culturing in the embryonic selection medium or embryonic induction selection medium added with 250mg? L- ¹ cefotaxim and 2mg? L- ¹ PPT (phosphinothricin) Cultivating shoots cultured in the shoot-derived selection medium in the shoot growth medium added with 250mg? L- ¹ cefotaxim and 2mg? L- ¹ PPT (phosphinothricin), the shoot growth medium 1-1.5 cm god Cultivating candles in polycythemia induced selection medium added with 250 mg L- ¹ cefotaxim and 2 mg L- ¹ phosphinothricin (50 mg?) Culturing in a rooting induction selection medium to which L- ¹ cefotaxim has been added, and purifying the rooting seedlings cultured in the rooting induction selection medium.

At this time, the co-culture of 30 ~ 80μM acetosyringone (acetosyringone), 3mg? L- ¹ SH medium with 2,4-dichlorophenoxyacetic acid, 30 g? L ⁻¹ sucrose, 300 mg L ⁻¹ L-proline, and 2.5 g L ⁻¹ phytagel and Hildebrandt's basal salts medium (pH 5.2) at 25 ± 2 ° C under dark conditions for 3 days.

The co-cultured somatic embryo or embryogenic callus can be cultured at 4 ° C. for 7 days.

???? The selection medium the germination times is 0.1mg L ^-1 indole-3-butyric acid (IBA), 1mg L ^-1 6- benzylamino purine (BAP), 30g L ^-1 maltose (maltose), 4g L ^{- It} may be an SH medium to which ¹ agarose is added.

Also, the medium is baeseongsuk ^{1mg? L -1 2,4-D,} 1mg? L -1 abscisic acid ^{(ABA), 1mg? L -1} BAP, 0.3mg? L -1 gibberellin (GA _3), 30g? It may be an SH medium to which L ⁻¹ sucrose and 4 g · L ⁻¹ agarose are added.

In addition, the shoot-derived selection medium may be MS medium (Murashige and Skoog's basal salts medium) to which 0.3mg? L- ¹ BAP, 30g? L- ¹ sucrose and 8g? L- ¹ agar are added. have.

In addition, the shoot growth selection medium is 2mg? L- ¹ BAP, 0.1mg? L- ¹ α-naphthaleneacetic acid (NAA), 30g? L- ¹ sucrose (8g? L- ¹ agar) is added MS medium.

In addition, the second guided selection medium again is ^{1mg? L -1 BAP, 0.001mg?} L -1 IBA, 30g? L -1 sucrose (sucrose), and 8g? L ^-1 agar (agar) can be an MS medium containing have.

In addition, the rooting induction selection medium may be MS medium to which 0.3mg? L- ¹ NAA, 30g? L- ¹ sucrose and 8g? L- ¹ agar are added.

Through the present invention, by using the somatic embryo (including embryogenic callus) derived from the domestically grown variety 'Sweet Yellow', the transfer efficiency of GUS (β-glucuronidase) and GFP (Green Fluorescent Protein) genes was dramatically improved. We have developed a technology that enables the growth of plants obtained from selection media to be healthy and to purify greenhouse after rooting.

The efficient exogenous transfer technology of roses developed by the present invention is expected to contribute to the growth of rose varieties having characteristics that are difficult to be introduced by traditional breeding methods.

Figure 1 shows the process of gene transfer and transgenic plants to rose somatic embryos (including embryogenic callus).
FIG. 2 is a photograph of somatic embryogenic callus (a) of 'Sweet Yellow' growing in embryogenic and secondary embryogenic callus induction medium and somatic embryos (including embryogenic callus) (b) selected as transgenic materials.
Figure 3a shows the GUS gene (gene accession number: AF234316) nucleotide sequence inserted in the pCAMBIA3301 vector (red nucleotide sequence is the portion where the catalase intron is inserted).
3B is a cleavage map of the pCAMBIA3301 vector, showing the position of the GUS gene inserted into the pCAMBIA3301 vector.
Figure 4a shows the sGFP gene (gene accession number: U84737) nucleotide sequence inserted into the pMJ109 vector.
4B is a cleavage map of the pMJ109 vector, showing the position of the GFP gene inserted into the pMJ109 vector.
5 is a photograph (a) confirming gene transfer to a sample after Agrobacterium co-culture and a photograph (b) of a sample cultured in embryo germination induction medium.
6 is a photograph (a) in which a sample in which GUS gene transfer is confirmed is germinated, a photograph in which shoots are induced (b), a photograph of shoots (c), and a picture (d) in which polycythes are grown.
Figure 7 is a photograph of the transgene, GUS gene expression in shoots obtained from selection medium after GUS gene transfer.
Figure 8 is a photograph of the expression of the transgene, GFP gene in the shoots obtained from the selection medium after GFP gene transfer.
Figure 9 is a photograph rooted in shoots obtained after GUS (a) and GFP (b) gene transfer.

The present invention is a somatic embryo (including embryogenic callus) selected during the proliferation process after being derived from the in-roots of the rose variety 'Sweet Yellow', which was grown at the National Institute of Horticultural & Specialty Science as a gene transfer material as described in FIG. Agrobacterium tumefaciens ( Agrobacterium) After infection with tumefaciens ) , in order to establish the transformation technology of a specific crop through cancer culture, the gene after attempting the transfer of GUS (β-glucuronidase) and GFP (Green Fluorescent Protein) genes which are widely used as marker genes Induction of shoots from somatic embryos (including embryogenic callus), where the metastasis was confirmed, and re-differentiated shoots from the shoots, followed by shoot growth and polycythemia induction medium to grow into healthy plants. It consists of purifying greenhouse by rooting. The process is described in detail as follows.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited only by these examples.

Example 1 Efficient Gene Introduction into Somatic Embryos (including Somatic Embryogenic Callus)

Example 1-1 Gene Preparation

Somatic Embryogenesis and Secondary Somatic Embryogenesis after Callus Induction from Plant Roots of Sweet Yellow, a Rose Variety Fostered by National Institute of Horticultural & Herbal Medicine (3 mg? L ^-1 2,4-dichlorophenoxyacetic acid 2,4-dichlorophenoxyacetic acid (hereinafter referred to as "2,4-D"), 30 g L- ¹ sucrose, 300 mg L- ¹ L-proline and 2.5 g L- ¹ Pita Gel (phytagel) added to SH medium (Schenk and Hildebrandt's basal salts medium), hereinafter referred to as 'RCP medium' and incubated after subcultured every 3-4 weeks to 1.5 ~ 2cm diameter (Fig. 2 ( a)). In this process, as shown in FIG. 2 (b), round, smooth and white somatic embryos and surrounding embryonic callus (hereinafter referred to as “sample”) were selected and prepared as gene transfer materials. A detailed description thereof is described in Korean Patent Application No. 10-2008-0115875, the contents of which are incorporated by reference in their entirety.

Example 1-2 Bacterial Culture Inserting Genes

It is stored in a -70 freezer ℃ Agrobacterium tumefaciens (Agrobacterium tumefaciens) AgL1 the strain stock solution 1mL 50mg? L ^-1 of kanamycin (Kanamycin) and 50mg? L ^-1 of rifampicin (rifampicin) is added to the LB (Luria -Bertani) After the addition to the liquid medium was incubated for 1-2 days until the OD ₆₀₀ = 0.7 ~ 1.6 at a speed of 170 ~ 200rpm in a rotary incubator at 28 ℃.

In addition, Agrobacterium tumefaciens (Agrobacterium a tumefaciens) LBA4404 strain stock solution 1mL 50mg? L ^-1 of spectinomycin (spectinomycin) and 10mg? L ^-1 of tetracycline (tetracycline) in 28 ℃ was added to the addition of LB (Luria-Bertani) liquid medium Incubate for 1 to 2 days until the OD ₆₀₀ = 0.7 ~ 1.6 at a speed of 170 ~ 200rpm in a rotary incubator.

Agrobacterium tumefaciens (Agrobacterium tumefaciens ) AgL1 is transformed by inserting the pCAMBIA3301 vector represented by the cleavage map of FIG. 3B into which the GUS gene (FIG. 3A) is inserted. As shown in Figure 3b, Agrobacterium Te Solarium tyume used in the present invention Pacific Enschede (Agrobacterium tumefaciens ) AgL1 was transformed by pCAMBIA3301 vector whose expression of GUS (β-glucuronidase) gene and PPT (phosphinothricin) gene is controlled by CaMV35S promoter.

In addition, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) LBA4404 is transformed by inserting the pMJ109 vector represented by the cleavage map of FIG. 4B into which the GFP (Green Fluorescent Protein) gene (FIG. 4A) is inserted.

Example 1-3 Attempt of Gene Transfer Through Bacterial Infection and Culture of Samples

OD ₆₀₀ = 0.7 ~ 1.6 by the cultured Agrobacterium until about 7 minutes and centrifuged at a rate of 6,000rpm, Agrobacterium tumefaciens Gon ache the Agrobacterium precipitated lumps separated from the culture medium 50μM acetonitrile on a liquid medium ( agrobacterium suspension was mixed homogeneously in SH liquid medium to which acetosyringone) was added. The somatic embryos and embryonic callus prepared in Example 1-1 were infected with the Agrobacterium suspension for 30 minutes, and then, the sterilized filter paper was used to remove the Agrobacterium solution. Ag-bacterium-infected samples were transferred to RCP medium (pH 5.2) containing 30-80 μM acetosyringone (pH 5.2), and cultured for 3 days in a temperature condition of 25 ± 2 ° C. and dark for 3 days. .

Example 1-4 Identification of Transgene Transfer and Embryonic Induction

Somatic embryos and embryogenic callus co-cultured with Agrobacterium to transfer β-glucuronidase (GUS) and Green Fluorescent Protein (GFP) genes to SH liquid medium containing 250 mg L- ¹ cefotaxim (pH) 5.7), and then the washing solution on the sample was removed from the sterilized filter paper. After that, the sample is Embryo germination medium with 250 mg L- ¹ cefotaxim (0.1 mg L- ¹ indole-3-butyric acid, hereinafter referred to as "IBA"), 1 mg L- ¹ -Benzylaminopurine (6-benzylaminopurine, hereinafter referred to as 'BAP'), SH medium added with 30 g L- ¹ maltose, 4 g L- ¹ agarose, REG medium (See FIG. 5 (b)), and cultured for 7 days under a temperature condition of 4 ° C. Afterwards, the cells were transferred to bright culture conditions and the GUS transgenic samples were examined by GUS assay analysis according to Jefferson (1987). GUS gene transfer It can be seen that the GUS gene has been transferred to all the trial samples (see Fig. 5 (a)).

Example 2 Transformation from Somatic Embryos (Including Embryonic Callus) to which Genes were Transferred Plant acquisition

If the transfer of the GUS gene is confirmed or if the sample being cultured in the REG selection medium after the attempt to transfer the GFP gene is a somatic embryo, 250 mg L- ¹ cefotaxim alone or 250 mg L- ¹ cefotaxim And 2 mg? L- ¹ PPT (phosphinothricin) added to the REG medium, and if the sample in culture is embryogenic callus, 250 mg? L- ¹ cefotaxim alone or 250 mg? L- ¹ cell Taxim ( cefotaxim) and 2 mg-L- ¹ PPT (phosphinothricin) combined with embryonic medium (1 mg L- ¹ 2,4-D, 1 mg L- ¹ Abscisic acid, hereinafter referred to as "ABA") ), SH medium supplemented with 1 mg L- ¹ BAP, 0.3 mg L- ¹ Gibberellin (GA ₃ ), 30 g L- ¹ sucrose, and 4 g L- ¹ agarose (Hereinafter referred to as 'REM medium'), and after the green color (see FIG. 6 (a)), the culture medium was changed every three weeks until the emergence of shoots.

The shoots identified by the shoots were visually derived from the shoot-derived selection medium (250 mg? L- ¹ cefotaxim) and 2 mg? L- ¹ PPT (phosphinothricin) added to the shoot until they grew in the shape of shoots (0.3 mg? L). Transfer it to MS medium (Murashige and Skoog's basal salts medium) to which ^-1 BAP, 30 g L ^-1 sucrose and 8 g L ^-1 agar were added (see FIG. 6 (b)), Shoot growth medium (2 mg L- ¹ BAP, 0.1 mg L- ¹ α-) with 250 mg L- ¹ cefotaxim and 2 mg L- ¹ phosphinothricin added for robust growth of shoots Naphthalene acetic acid (α-naphtalene acetic acid; hereinafter referred to as "NAA"), 30g-L- ¹ sucrose, 8g-L- ¹ agar added MS medium) and was incubated (see Fig. 6 (c)). Then, polycythemia-induced selection medium (1mg? L- ¹ BAP, supplemented with 250mg? L- ¹ cefotaxim and 2mg? L- ¹ PPT (phosphothricin) to maintain the ground growth of the plant before rooting ^{0.001mg? L -1 IBA, 30g?} L -1 can croissants five (sucrose), 8g? L ^-1 transferred was incubated with the agar (agar), the addition of MS medium) (see Fig. 6 (d)).

Example 3 Confirmation of Introduction Gene Expression and Purification of Greenhouse from Selected Plants

After the transfer of the GUS gene was confirmed, a part of the plants growing in the polycythenic induction selection medium was analyzed by GUS assay, and as shown in Table 1 and FIG. 7, the expression of the GUS gene was confirmed in the entire assayed plant.

GUS gene expression rate of shoots obtained from selection medium after GUS gene transfer division Black shoots GUS gene expression rate (%) Primary 3 100 Secondary 5 100 system 8 100

In addition, after the GFP gene transfer attempt microscopic observation of the neomyocytes induced in the shoot-derived selection medium, as shown in Figure 8 it was confirmed the expression of the GFP gene, which is the introduction gene in the whole neomyocytes including the sample.

In this way, the expression of the transgene in the entire selection of plants can be said to be the result of the greatest advantage when using somatic embryos (including embryogenic callus) as a material for gene transfer.

? The expression of the introduced gene was confirmed 250mg L ^-1 plant cells Taksim (cefotaxim) is added inducing rooting selection medium ^{(0.3mg L -1 NAA, 30g L} -1 sucrose (sucrose), 8g L?? ? - ¹ agar (MS medium to which agar) was added (see FIG. 9), cultured, and transferred to a greenhouse after rooting to obtain a complete plant of transformed roses.

Claims (9)

As a method for obtaining a mass of transformed plants using rose sweet yellow somatic embryos and embryogenic callus,
The rose cultivar 'Yellow Room' derived from the roots of the meal object? A starting somatic embryos or embryonic callus, GUS gene transgenic Agrobacterium tumefaciens (Agrobacterium tumefaciens), and GFP gene transfection step of co-culturing with the conversion Agrobacterium tumefaciens (Agrobacterium tumefaciens),
Culturing the co-cultured somatic embryo or embryogenic callus in embryonic germ selection medium supplemented with 250 mg L- ¹ cefotaxim,
The cultured somatic embryo or embryogenic callus was added with 250 mg L- ¹ cefotaxim alone or with 250 mg L- ¹ cefotaxim and 2 mg L- ¹ PPT (phosphinothricin). Culturing in a selection medium or embryonic selection medium,
Cultivating the shoots in the embryonic selection medium or embryonic selection medium medium cultured in shoot-derived selection medium added with 250mg? L- ¹ cefotaxim and 2mg? L- ¹ PPT (phosphinothricin),
Culturing the shoots cultured in the shoot-derived selection medium in shoot growth medium added with 250 mg? L- ¹ cefotaxim and 2 mg? L- ¹ PPT (phosphinothricin),
Cultivating the shoots of 1 ~ 1.5 cm cultured in the shoot growth medium, in polycythemia induced selection medium added with 250 mg? L- ¹ cefotaxim and 2 mg? L- ¹ PPT (phosphinothricin),
Culturing the polycynic cultivated in the polycythenic induction selective medium in a rooting induction selective medium to which 50 mg? L- ¹ cefotaxim was added, and
Mass production method of the transformed plant using rose sweet yellow somatic embryo and embryogenic callus comprising the step of purifying the rooting seedlings cultured in the rooting induction selection medium. The method of claim 1,
The co-culture is 30 ~ 80μM acetosyringone (acetosyringone), 3mg ~ L ^-1 of SH medium supplemented with 2,4-dichlorophenoxyacetic acid, 30 g? L ⁻¹ sucrose, 300 mg L ⁻¹ L-proline, and 2.5 g? L ⁻¹ phytagel Mass harvesting method of transgenic plants using rose sweet yellow somatic embryos and embryogenic callus, which were carried out in Schenk and Hildebrandt's basal salts medium (pH 5.2) for 25 days at 25 ± 2 ° C and in the dark. The method of claim 1,
Method for obtaining a mass of transformed plants using rose sweet yellow somatic embryos and embryogenic callus to culture the co-cultured somatic embryo or embryogenic callus for 7 days at 4 ℃. The method of claim 1,
???? The selection medium the germination times is 0.1mg L ^-1 indole-3-butyric acid (IBA), 1mg L ^-1 6- benzylamino purine (BAP), 30g L ^-1 maltose (maltose), 4g L ^{- 1 A} method for obtaining a large amount of transformed plants using rose sweet yellow somatic embryos and embryogenic callus, which are SH medium to which agarose is added. The method of claim 1,
????? The baeseongsuk medium ^{1mg L -1 2,4-D, 1mg} L -1 abscisic acid ^{(ABA), 1mg L -1 BAP} , 0.3mg L -1 gibberellin (GA _3), 30g L ^{- 1} Mass harvesting method of transgenic plants using rose sweet yellow somatic embryos and embryogenic callus, which are SH medium to which sucrose and 4 g L ¹ agarose are added. The method of claim 1,
The shoot-derived selection medium is rose sweet yellow, which is MS medium (Murashige and Skoog's basal salts medium) to which 0.3 mg? L ⁻¹ BAP, 30 g? L ⁻¹ sucrose and 8 g? L ⁻¹ agar are added. Mass production method of transformed plants using somatic embryos and embryogenic callus. The method of claim 1,
The shoot growth medium was 2 mg? L- ¹ BAP, 0.1mg? L- ¹ α-naphthaleneacetic acid (NAA), 30g? L- ¹ sucrose, 8g? L- ¹ agar added MS Mass harvesting method of transformed plants using rose sweet yellow somatic embryo and embryogenic callus as a medium. The method of claim 1,
The polycythemia induced selection medium was rose sweet yellow, which is an MS medium to which ¹ mg L- ¹ BAP, 0.001 mg L- ¹ IBA, 30 g L- ¹ sucrose and 8 g L- ¹ agar were added. Mass production method of transformed plants using somatic embryos and embryogenic callus. The method of claim 1,
The rooting induction selection medium using rose sweet yellow somatic embryos and embryogenic callus, which is MS medium to which 0.3 mg L ^-1 NAA, 30 g L ^-1 sucrose and 8 g L ^-1 agar are added. Method of obtaining a transgenic plant mass.

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