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Transformation system in camelina sativa

Inactive Publication Date: 2004-02-12
UNICORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0193] "Agrobacterium tumefaciens" is a naturally occuring bacterium which when containing a circular Ti (Tumor inducing) plasmid is able to form crown gall disease in many species of dicotyledonous plants. Crown gall occurs when a wound is invaded by Agrobacterium. Agrobacterium tumefaciens natively has the ability to transfer a portion of its DNA called T-DNA, into the genome of the plant cells. In Agrobacterium-mediated transformation T-DNA is replaced with a foreign set of genes, thus, making the bacterium capable of transferring the foreign genes into the genome of the plant cell. "Agrobacterium tumefaciens" can be one of the three different strains of Agrobacterium used in the transformation of Camelina sativa or an equivalent strain suitable for the transformation. The strain LB4404 carries the plasmid pAL4404, the strain C58C1 carries the plasmid pGV3850 and the strain EHA105 carries the plasmid pTiBo542.
[0208] Gibberellins (GA for gibberellic acid) can stimulate extensive growth of intact plants, the transition from juvenile to adult growth, bolting of biennials, fruit formation, and germination of some cereal grains. More than 80 gibberellins have been isolated from various fungi and plants including GA.sub.3.

Problems solved by technology

Camelina sativa plants producing seed are grown in greenhouse conditions, since field grown plants produce seed contaminated with bacteria, which later prevents successful transformation with Agrobacterium.

Method used

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  • Transformation system in camelina sativa
  • Transformation system in camelina sativa
  • Transformation system in camelina sativa

Examples

Experimental program
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Effect test

example 1

[0253] Transformation Protocol for Camelina sativa cv. Calena with Agrobacterium tumefaciens Strain LBA4404 Harboring the Binary Plasmid pGPTV-HPT with uidA Intron Containing Gene.

[0254] The seeds of Camelina sativa plant grown in greenhouse were sterilized by immersing in 70% ethanol for 1 min and then treating for 10 min with Na-hypochlorite solution (3% active Cl.sup.-) with an addition of Tween-20 (1 drop per 100 ml). After sterilization the seeds were washed three times in sterile water and placed on solid Murashige and Skoog (MS) agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962) without sugars for germination. Sterilized seeds were germinated and grown 2-3 weeks on solid Murashige and Skoog (MS) medium without hormones (FIG. 1). Green leaves served as a source of explants for transformation procedure.

[0255] Agrobacterium tumefaciens strain LBA4404 carrying pGPTV-HPT-GUSint vector was grown overnight at 28.degree. C. with shaking in liquid YEB medium (Lichtens...

example 2

[0257] Transformation Protocol for Camelina sativa cv. Calinca with Agrobacterium tumefaciens Strain C58C1 pGV3850 Harboring the Binary Ti Vector with Kanamycin Selection.

[0258] Seeds were taken from greenhouse grown Camelina sativa cv. Calinca plants (no older than 4 months). Transformation efficiency increases from 66% to 100% if donor plants are grown in greenhouse.

[0259] 10 Days Before Excision of the Explants.

[0260] Seeds of Camelina sativa were sterilized and placed in vitro on Murashige and Skoog (MS) agar medium without sucrose and grown at temperatures of 25.degree. C. (day) and 18.degree. C. (night) as described in Example 1.

[0261] 1.sup.st Day.

[0262] A fresh colony of Agrobacterium tumefaciens strain C58C1pGV3850 carrying binary pGPTV-KAN vector (Becker et al., Plant Mol. Biol. 20:1195-1197, 1992) containing uidA-int gene under 35S promoter and selectable marker gene nptII, was inoculated in 3 ml of liquid YEB medium supplemented with 25 mg / l rifampicin (Rif) and 50 mg / l ...

example 3

[0276] Transformation Protocol for Camelina sativa cv. Calena with Agrobacterium tumefaciens Strain 4 C58C1 pGV3850 Harboring Cointegrative Ti DNA Without Selection of Transgenic Tissues.

[0277] Seeds were taken from greenhouse grown Camelina sativa cv. Calena plants (no older than 4 months). Transformation efficiency increases from 66% to 100% if donor plants are grown in greenhouse.

[0278] 10 Days Before Explants Excision.

[0279] Seeds were sterilized and placed in vitro on Murashige and Skoog (MS) medium without sucrose and grown at temperatures of 25.degree. C. (day) and 18.degree. C. (night) as described in Example 1.

[0280] 1.sup.st Day.

[0281] A fresh colony of C58C1pGV3850 with interned Ti DNA from pHTT-HPT (Elomaa et al., Bio / Technology 11:508-511, 1993) vector containing GUS gene under 35S promoter and hpt selectable marker was inoculated in 3 ml of liquid YEB supplemented with 25 mg / l rifampicin (Rif) and 100 mg / l spectinomycin (Spe) or streptomycin (Str). The bacteria were gr...

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Abstract

The present invention relates to plant biotechnology and specifically to a method for genetically transforming Camelina sativa with Agrobacterium-mediated transformation system. It comprises Camelina sativa for producing homologous and heterologous recombinant products including oil and protein products and assessing and screening the efficacy of plant transformation. Also disclosed are transgenic Camelina sativa plants, seeds as well as cells, cell-lines and tissue of Camelina sativa.

Description

[0001] The present invention relates to plant biotechnology and plant cell transformation. More particularly the invention relates to a method for genetically transforming Camelina sativa using Agrobacterium-mediated transformation of a plant tissue explant and subsequent regeneration of the transformed cells into whole Camelina sativa plants. It further relates to the use of an Agrobacterium-mediated transformation method of Camelina sativa for producing homologous or heterologous recombinant products including for example proteins, enzymes and oil products and for assessing and screening the properties, and effects of DNA sequences and recombinant DNA constructs in plants.[0002] Genetic transformation of plants allows the introduction of genes of any origin into the target species providing novel products for e.g. agricultural, horticultural, nutritional and chemical applications. Furthermore, transgenic plants provide more information about basic plant biology, gene function and ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84
CPCC12N15/8205
Inventor KUVSHINOV, VIKTORKANERVA, ANNEKOIVU, KIMMOPEHU, EIJAKUVSHINOVA, SVETLANA
Owner UNICORP
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