WO1999048355A1 - Procede de transformation - Google Patents

Procede de transformation Download PDF

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Publication number
WO1999048355A1
WO1999048355A1 PCT/AU1998/000195 AU9800195W WO9948355A1 WO 1999048355 A1 WO1999048355 A1 WO 1999048355A1 AU 9800195 W AU9800195 W AU 9800195W WO 9948355 A1 WO9948355 A1 WO 9948355A1
Authority
WO
WIPO (PCT)
Prior art keywords
transformation
pressure
tissue
vacuum
transformation method
Prior art date
Application number
PCT/AU1998/000195
Other languages
English (en)
Inventor
Robert Dixon Teasdale
Ekaterina Mouradova
Yumin Yang
Ding G. He
Cecilia O'dwyer
Original Assignee
Forbio Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forbio Limited filed Critical Forbio Limited
Priority to AU63864/98A priority Critical patent/AU6386498A/en
Priority to PCT/AU1998/000195 priority patent/WO1999048355A1/fr
Publication of WO1999048355A1 publication Critical patent/WO1999048355A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

Definitions

  • This invention relates to a method of transformation.
  • This invention has particular but not exclusive application to a method of
  • invention could be used in other applications such as whole plant or seed transformation or with other infective vector systems.
  • Eucalyptus has been considered a difficult genus for transformation, with the main obstacle the low regeneration potential of explants.
  • Agrobactenum mediated transformation is a commonly used method for transforming plants.
  • To inoculate explants the general practice is to incubate wounded explants with a suspension of Agrobactenum. Consequently, the transformation events occur mainly around the wound sites.
  • Arabidopsis thaliana Seeds have been imbided with Agrobactenum, and plants allowed to grow to maturity in soil. Transformed plants were selected out from the
  • Vacuum infiltration has been used as a means of agroinfection for introducing
  • transformation targets are somatic cells For example, in transformation of trees the targets are frequently the selected superior trees themselves, not the second generation from seeds, which are of different genotypes to the superior parent trees. Further, it may a substantial period of time for some plant species to reach maturity to enable selection of transformed seeds
  • the present invention aims to alleviate at least one of the foregoing
  • this invention in one aspect resides broadly in a method of transformation of plant including the steps of immersing plant tissue in a medium including an infective transformation vector, reducing the pressure on said tissue to -10 to -100 kPa gauge, maintaining said pressure for 10 to 60 minutes, and raising said pressure to
  • said transformation vector being selected to provide
  • this invention in one aspect resides in a method of transformation of eucalypts including the steps of
  • the eucalypt tissue may be selected from E grandis, E tereticornis or E camaldulensis
  • the eucalypt tissue may be cell culture, a whole plant, an explant or
  • the plant material be explants suitable for prolongation after transformation
  • the explants may be selected from shoots, cotyledons, hypocotyls, leaves, seedlings, or the me ⁇ stem
  • the infective transformation vector may be selected from any known suitable infective system to mediate transformation, such as a virus or bacterium
  • the vector is Agrobactenum
  • Agrobactenum is the most common type of bacterium used for bacterially mediated-transformation of plants
  • any other infective microorganism which may be proved to be effective in transformation of plant cells may be used
  • the medium may be any liquid medium used to support the bacterial suspension
  • the medium may include chemicals which assist
  • acetosy ⁇ ngone may be added to the medium
  • Agrobactenum is preferably established at a population in the range 1-5 x 10 8 cfu ml "1
  • the reduced pressure may be achieved in any vacuum chamber or desiccator
  • the vacuum vessel may be any vacuum vessel having pressure above atmospheric as is described hereinafter.
  • the duration of the exposure is in the range of 10 to 60 minutes, the longer periods being preferred for recalcitrant species of eucalypt
  • the duration of vacuum treatment is in the range of 15 to 20 minutes
  • the pressure may be raised to atmospheric pressure or above slowly or
  • the pressure is raised to atmospheric pressure or above very rapidly This may be achieved by opening the vacuum chamber while still under negative pressure Not being bound by theory, it is surmised that the vacuum (negative pressure) causes the air spaces between the cells in the plant tissue to
  • regeneration may be any method commonly used in the field tissue culture For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may be any method commonly used in the field tissue culture. For example, regeneration may
  • the multiple application of vacuum infiltration may also
  • the reduced pressure effect may be provided by cyclically applying vacuum to the tissue
  • this invention resides in a method of transforming plants including the steps of: immersing plant tissue in a medium including an infective transformation vector; reducing the pressure on said tissue;
  • the plant tissue may be cell culture, a whole plant, an explant or germ material.
  • the plant material be explants suitable for propagation after transformation.
  • the explant may be selected from shoots, cotyledons, hypocotyls,
  • the medium may include any infiltration media or bacterial suspensions typically
  • the pressure differential between the reduced pressure and the over-pressure may in a suitable range so as not to cause damage or excessive hyperhydricity to the
  • reduced pressure is preferably selected to avoid hyperhydricity of the tissue.
  • corresponding over pressure is preferably selected to provide sufficient pressure
  • differential between the reduced pressure and the over pressure to promote infiltration. 7 pressure may be in the range of 10 to 500 kPa
  • the plant material may be subjected
  • the pressure differential is preferably about 90 kPa or higher
  • pressure this may be achieved in any suitable vessel including a vacuum/pressure chamber or desiccator modified to allow the apparatus to function as a pressure vessel
  • the negative and positive pressure may be built by manual pulling and pushing of a piston of a syringe containing the plant material and medium
  • the piston may be pulled to a scale to create negative pressure or a vacuum
  • the pushing of the piston creates a positive pressure
  • the pulling and pushing may be performed repeatedly
  • the inoculated plant material may be co-cultivated and normal selection and regeneration procedures used to obtain a transgenic plant
  • this invention resides in a method of transforming plants
  • FIG 1 is the control (E grandis) seedlings, where the wounded explant FIG. 2 is the high frequency GUS expression in vacuum-infiltrated E. camaldulensis seedling as assayed 5-6 days after inoculation;
  • FIG. 3 is the high frequency GUS expression in vacuum-infiltrated E. grandis seedlings as assayed 5-6 days after inoculation;
  • FIG. 4 is the high frequency GUS expression in vacuum-infiltrated E. tereticornis shoot as assayed 5-6 days after inoculation;
  • FIG. 5 is the regeneration of multiple Gl/S-positive shoots from E. camaldulensis callus obtained from vacuum-infiltrated explants;
  • FIG. 6 is a GL/S-positive E. grandis shoot obtained from vacuum-infiltrated
  • FIG. 7 is a GL S-positive E. camaldulensis plantlet obtained from vacuum-
  • FIG. 8 is a transgenic E. grandis plant in soil obtained from vacuum-infiltrated
  • FIG. 9 is a southern blot analysis of transformed E. grandis lines EG66 and
  • the media used in this study were G22 and KG.
  • G22 medium was used in a modified 9 were omitted.
  • the phytohormone regimes in inductions medium were also different in using 1 ⁇ M BA, 0.03-.01 ⁇ M TDZ (Sigma) and 1 ⁇ M NAA for callus induction of E. grandis and E. tereticornis and 1 ⁇ M BA and 0.5 ⁇ M NAA for E. camaldulensis.
  • the differentiation medium was a G22 medium supplemented with 2-5 ⁇ M BA and 0.5 ⁇ M
  • NAA NAA.
  • Liquid medium used for selecting transformed shoots, was KG supplemented with 0.01 ⁇ M BA and antibiotics.
  • Clones are sub-cultured every 3-5 weeks on KG media [Laine E and David A:
  • the jars are kept at 23°C under dim light (5-10 ⁇ Mol m "2 ) with a 12-hour photo- period. Under such growth conditions, the shoot cultures are generally clusters
  • This particular protocol was optimised for use with Eucalyptus, but it is not restricted to said genus and is generally envisioned to be applicable to other plant genus/species, particularly those which lend themselves to clonal micro-propagation.
  • One aspect of this method is designed to clonally propagate large numbers of suitable plant starting material for use in transformation. In doing so, this methodology is
  • genotype independent as it is capable of generating as much starting material as
  • transformation e.g. stem or leaves. Transformation into the clonal plant starting
  • material may be by several transformation methods, including, but not limited to,
  • This vector carries a GUS gene interrupted by a plant intron. Consequently, GUS expression is exclusively from plant cells, and contamination from stained bacteria are
  • Agrobactenum were grown overnight in a liquid YEP medium at 28°C until the
  • OD 600 value was around 1.
  • the bacterium suspension was diluted to the desired density of 1-5x10 8 ml "1 (6-30 times) using a liquid KG medium.
  • Acetosyringone was
  • the plant materials were placed in an Agrobactenum suspension. The mixture was put into a desiccator, and vacuum infiltrated -95 kPa for 20 minutes in a BioHood. As the control, explants were immersed in an Agrobactenum bath for 20 minutes. After inoculation, the explants were blotted dry with sterile filter paper before transfer to an MG22 co- cultivation medium. The cultures were kept on the co-cultivation medium, in the dark for 2-5 days, depending on the degree of Agrobactenum overgrowth.
  • the time for vacuum treatment was chosen as 20 minutes for E.
  • Transgenic plants referring to Figs. 5-8, were obtained from explants vacuum infiltrated for 20 minutes under -95 kPa in a liquid containing 1-5x10 8 ml '1 Agrobactenum. The explants were co-cultivated in MG22 medium for 2-3 days before
  • the DNA samples were digested with Hind ⁇ , which released a 2.8 kb internal fragment of the T-DNA, containing the GUS gene sequence with 35S promoter and terminator sequences. A comparison of the intensities of the bands indicates that
  • EG66 and EG67 have multiple (three to four) copies of 35SGL SINT transgene, as
  • Eucalyptus has been considered a difficult genus for transformation, with the main obstacle the low regeneration potential of explants.
  • Methods in accordance with the foregoing embodiment results in an increase in regeneration capacity by vacuum infiltration, due to the minimisation of tissue damage by avoidance of wounding. It is
  • duration of the vacuum treatment will vary in relation to the type of plant material used
  • cotyledons and hypocotyls were then transferred to a selection/regeneration medium and subcultured as in routine transformation experiments.
  • One GL/S-positive shoot was obtained following 6 weeks' incubation on selection medium. The shoot was obtained from the first experiment, where 30 seedlings were used as starting material. Ten seedlings were assayed for transient expression and
  • the shoot was obtained from one of the remaining 20 hypocotyls.
  • vacuum infiltration is an effective way of infecting Eucalyptus explants
  • histochemical assays The procedure is highly time efficient and less damaging to the tissue, as wounding of cotyledon and leaf explants was not needed. Stably

Abstract

L'infiltration sous vide est une manière efficace d'infecter des explants d'Eucalyptus avec Agrobacterium. Il en résulte une haute fréquence d'expression tel que le montrent les analyses histochimiques GUS. Ledit procédé fait gagner beaucoup de temps et endommage moins les tissus végétaux du fait qu'il n'est pas nécessaire d'effectuer une lésion sur les explants sous forme de cotylédon ou de feuille. Des plantes à transformation stable ont pu être régénérées à partir d'explants infiltrés sous vide. L'analyse histochimique GUS et les analyses d'hybridation Southern ont confirmé cette transformation. Il a également été déterminé que l'application de vide peut être ajoutée à l'application ultérieure de pression pour créer un gradient efficace de pression de transformation.
PCT/AU1998/000195 1998-03-20 1998-03-20 Procede de transformation WO1999048355A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU63864/98A AU6386498A (en) 1998-03-20 1998-03-20 A method of transformation
PCT/AU1998/000195 WO1999048355A1 (fr) 1998-03-20 1998-03-20 Procede de transformation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/AU1998/000195 WO1999048355A1 (fr) 1998-03-20 1998-03-20 Procede de transformation

Publications (1)

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WO1999048355A1 true WO1999048355A1 (fr) 1999-09-30

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Application Number Title Priority Date Filing Date
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AU (1) AU6386498A (fr)
WO (1) WO1999048355A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005017169A1 (fr) 2003-08-13 2005-02-24 Japan Tobacco Inc. Procede visant a introduire un gene dans une plante
ES2299285A1 (es) * 2004-11-26 2008-05-16 Universidad De Vigo Procedimiento para transformar material vegetal procedente de arboles adultos.
US7960614B2 (en) 2003-06-06 2011-06-14 Arborgen, Llc Plant transformation and selection
WO2012007587A1 (fr) 2010-07-16 2012-01-19 Philip Morris Products S.A. Procédés de production de protéines dans des plantes
WO2012084962A1 (fr) * 2010-12-22 2012-06-28 Philip Morris Products S.A. Procédé et système pour l'infiltration sous vide de plantes
WO2012098119A3 (fr) * 2011-01-17 2012-10-18 Philip Morris Products S.A. Expression protéique chez des plantes
CN103068990B (zh) * 2010-07-16 2016-12-14 菲利普莫里斯生产公司 用于在植物中生产蛋白质的方法
CN109880847A (zh) * 2019-03-19 2019-06-14 兰州大学 一种高效的高山离子芥转基因植株的制备方法
CN115088621A (zh) * 2022-07-18 2022-09-23 南京林业大学 一种利用抽真空处理促进杨树外植体大量、快速分化成芽的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1807588A (en) * 1987-05-20 1988-12-21 Crop Genetics International Delivery of beneficial microorganisms to seeds and plants

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
AU1807588A (en) * 1987-05-20 1988-12-21 Crop Genetics International Delivery of beneficial microorganisms to seeds and plants

Non-Patent Citations (2)

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Title
C.R. ACAD. SCI. PARIS, (1993), Volume 316, by BECHTOLD N. et al., "In Planta Agrobacterium Mediated Gene Transfer by Infiltration of Adult Arabidopsis Thaliana Plants", pages 1194-1199. *
PLANT SCIENCE, (1989), Volume 63, by DALE P.J. et al., "Agroinfection of Wheat Innoculation of In Vitro Grown Seedlings and Embryos", pages 237-245. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7960614B2 (en) 2003-06-06 2011-06-14 Arborgen, Llc Plant transformation and selection
WO2005017169A1 (fr) 2003-08-13 2005-02-24 Japan Tobacco Inc. Procede visant a introduire un gene dans une plante
EP1662002A1 (fr) * 2003-08-13 2006-05-31 Japan Tobacco Inc. Procede visant a introduire un gene dans une plante
EP1662002A4 (fr) * 2003-08-13 2007-01-31 Japan Tobacco Inc Procede visant a introduire un gene dans une plante
US7812222B2 (en) 2003-08-13 2010-10-12 Japan Tobacco Inc. Method of transducing gene into plant material
ES2299285A1 (es) * 2004-11-26 2008-05-16 Universidad De Vigo Procedimiento para transformar material vegetal procedente de arboles adultos.
JP2013534430A (ja) * 2010-07-16 2013-09-05 フィリップ・モーリス・プロダクツ・ソシエテ・アノニム 植物においてタンパク質を産生する方法
CN106520520A (zh) * 2010-07-16 2017-03-22 菲利普莫里斯生产公司 用于在植物中生产蛋白质的方法
CN106520520B (zh) * 2010-07-16 2019-08-02 菲利普莫里斯生产公司 用于在植物中生产蛋白质的方法
CN103068990A (zh) * 2010-07-16 2013-04-24 菲利普莫里斯生产公司 用于在植物中生产蛋白质的方法
WO2012007587A1 (fr) 2010-07-16 2012-01-19 Philip Morris Products S.A. Procédés de production de protéines dans des plantes
US9512439B2 (en) 2010-07-16 2016-12-06 Philip Morris Products S.A. Methods for producing proteins in plants
CN103068990B (zh) * 2010-07-16 2016-12-14 菲利普莫里斯生产公司 用于在植物中生产蛋白质的方法
RU2662866C2 (ru) * 2010-07-16 2018-07-31 Филип Моррис Продактс С.А. Способы получения белков в растениях
WO2012084962A1 (fr) * 2010-12-22 2012-06-28 Philip Morris Products S.A. Procédé et système pour l'infiltration sous vide de plantes
WO2012098119A3 (fr) * 2011-01-17 2012-10-18 Philip Morris Products S.A. Expression protéique chez des plantes
US10472644B2 (en) 2011-01-17 2019-11-12 Philip Morris Products S.A. Protein expression in plants
CN109880847A (zh) * 2019-03-19 2019-06-14 兰州大学 一种高效的高山离子芥转基因植株的制备方法
CN109880847B (zh) * 2019-03-19 2023-10-13 兰州大学 一种高效的高山离子芥转基因植株的制备方法
CN115088621A (zh) * 2022-07-18 2022-09-23 南京林业大学 一种利用抽真空处理促进杨树外植体大量、快速分化成芽的方法
CN115088621B (zh) * 2022-07-18 2023-09-26 南京林业大学 一种利用抽真空处理促进杨树外植体大量、快速分化成芽的方法

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