WO2001094602A2 - Procede de regeneration de plantes et utilisation de ce procede pour multiplier et/ou transformer des plantes - Google Patents

Procede de regeneration de plantes et utilisation de ce procede pour multiplier et/ou transformer des plantes Download PDF

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WO2001094602A2
WO2001094602A2 PCT/IT2001/000280 IT0100280W WO0194602A2 WO 2001094602 A2 WO2001094602 A2 WO 2001094602A2 IT 0100280 W IT0100280 W IT 0100280W WO 0194602 A2 WO0194602 A2 WO 0194602A2
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shoots
meristematic
platforms
plant
bulks
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PCT/IT2001/000280
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WO2001094602A3 (fr
Inventor
Oriano Navacchi
Bruno Mezzetti
Giuseppe Zuccherelli
Angelo Spena
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G.In.E.S.Tr.A. S.C. Ar.L.
Vitroplant Vivai Di Zuccherelli Giuseppe & C. S.S.
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Priority to AU74484/01A priority Critical patent/AU7448401A/en
Publication of WO2001094602A2 publication Critical patent/WO2001094602A2/fr
Publication of WO2001094602A3 publication Critical patent/WO2001094602A3/fr

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    • 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

  • the present invention relates to a method for the plant regeneration by induction of adventitious meristems in explants, as well as to applications thereof to multiply and transform plant species.
  • the invention relates to a method to regenerate by caulogenesis starting from meristematic bulks, in vitro induced in somatic tissue explants, by a breakage of the apical dominance in proliferating shoots and by a hyper-cytokininic stimulus, which determines an hyperplasic and hypertrophic development of the shoot base and then a cellular bulk with a complex cellular and tissue structure.
  • the cellular bulk is characterised by the predominant presence of cells with an high caulogenetic regenerative competence.
  • the method is applicable for vegetative propagation and multiplication as well as for the genetic manipulation (mediated or direct) of various plant species.
  • the method allows an efficient regeneration of shoots and therefore of plants, from vegetal tissues.
  • the method can be widely utilised during plant genetic multiplication and transformation.
  • the regeneration occurs from a "platform" tissue characterised by an high number of cells with high competence for the formation of adventitious meristems (Meristematic Platform).
  • the methodology is of great importance due to the possibility to increase the efficiency and/or to extend both in vitro multiplication and genetic transformation techniques to many species of agronomic interest. Therefore the method, increasing the efficiency of multiplication methods, in addition to its use for genetic transformation, allows to implement the application of the vegetal biotechnologies also to plants which are not easily manipulated, also providing cost reduction and standardisation of production techniques.
  • Background of the In vitro culturing techniques and regeneration The in vitro cultures consist of the growth of cells, tissues or organs, isolated from a parent plant on artificial substrates.
  • the conventional micropropagation technique is based on the proliferation of apical and axillary buds.
  • Direct or indirect differentiation of adventitious buds and/or somatic embryos from de-differentiated tissue allows possible alternatives but, due to the high genetic variability often induced thereby, it results in a difficult practical application in the propagation systems.
  • Meristematic tissues are undifferentiated tissues causing the growing process of the plant.
  • Meristematic cells are actively self-divided generating both other meristematic and differentiated cells.
  • Meristems can be derived directly from zygote or adult cells regressing to de- differentiated state; they are named "primary” (for example vegetative and root tips) and “secondary” (for example cambium, adventitious meristems and meristemoids) in the first and second case, respectively.
  • primary for example vegetative and root tips
  • secondary for example cambium, adventitious meristems and meristemoids
  • the more diffused method for the propagation is shoots culture.
  • the propagation occurs by the generation of axillary shoots, isolated and used as explants for subsequent sub-cultures.
  • As primary explants usually lateral 5-10 mm long apexes or buds are used.
  • meristem culture The culture of the terminal ends of the meristematic apexes is named meristem culture. This technique is usually used to remove viruses form the plants, often in combination with heat treatment. Explants consists of small portions (0,2-1 mm long) of apical meristems, with one or two leaf primordia. Nodus culture is a technique derived from shoot culture, more recently provided (Wang, 1977), wherein the stems are localised horizontally on solid substrate. The propagation, like in the shoot culture, occurs by axillary buds.
  • Step 0 Selection of the parent plant and preparation.
  • Step 1 Start of the culture.
  • Step 2 Multiplication.
  • Step 3 Elongation and induction of the root development.
  • Step 4 Adaptation.
  • step 3 to 4 The passage from step 3 to 4 is critical because the seedlings are adapted to a sterile environment characterised by high humidity, low light intensity and supported by external carbon source (for example sucrose). Therefore it is required to control, after the transplant in a yet sterile soil, the light and humidity conditions until the plants re-acquired the structural and functional autonomy. Adaptation yields depend on the type and quality of the material derived from the previous steps. Morphogenesis and Differentiation
  • organogenesis The generation process of a new organ from a vegetal tissue is named organogenesis or morphogenesis.
  • organs developing from somatic tissues are named adventitious.
  • the potential to produce organs or even a complete plant is owned not only by the meristematic but also by the somatic tissue cells.
  • somatic tissue cells the more specialised plant cells, as vascular tissue and sclerenchyma ones, do not modify their differentiated state.
  • the cell and tissue transition from a differentiated to undifferentiated state is named de-differentiation or callogenesis and it can be induced in vivo or in vitro following chemical, physical or biological stimuli.
  • Vegetal tissues can growth in an organised way, wherein the cells maintain a well defined structure or in an unorganised way (callus).
  • the organised tissue is constituted of differentiated cells (morphologically and functionally organised), while the unorganised tissue contains a minimal amount of differentiated cells.
  • Callus is an amorphous tissue generated when cells are divided disorderly. In vivo it can be induced in a plant from produced injury, in the presence of microorganism or stress conditions. During the formation of callus, the cellular metabolism passes from a differentiated and quiescent to a meristematic, not specialised and actively dividing state.
  • the in vitro induction of callus takes advantage of the same phenomenon which can be amplified or inhibited by the application of chemical and physical factors.
  • the in vitro production of callus is increased by carrying out further cuts on explants before their transfer onto induction substrates and by the combination of growth regulators added to the substrate the light and temperature conditions being adjusted. Three are the major factors inducing the callus: 1) explant type, 2) selection of the substrate and culturing conditions and 3) separation of the callus from the explant and culturing maintenance thereof (Constabel, 1984). Morphogenetic Processes
  • Competent cells can be induced in a determined state wherein they tend to follow a precise, genetically determined, development path; this path can go on cellular differentiation or morphogenesis (Christianson, 1987). The assumption of the determined state can take advantage of the presence of certain growth regulators.
  • somatic tissue both differentiated and undifferentiated, can lead to the formation of unipolar or bipolar structures.
  • caulogenesis and rhizogenesis which include the formation of adventitious shoots and roots, respectively; while somatic embryogenesis leads to the formation of somatic embryos having a bipolar development.
  • Morphogenesis can be direct or indirect, depending on its occurrence in the presence or absence of an undifferentiated proliferating tissue (Hicks, 1980). Most of studied vegetal species is able to generate adventitious roots and buds from explants of various plant tissues and organs, while the formation of adventitious embryos is very less usual. Caulogenesis and rhizogenesis
  • Direct caulogenesis consists of the formation of shoots from somatic tissue, while in the indirect one the shoots are formed from dedifferentiated tissue.
  • the direct caulogenesis about 48 hours following the transfer of a tissue explant onto the culture substrate initiate mitosis which can originate the meristems of the shoots.
  • meristems can generate primordia, whose disposition appears to be random, tendentiously equidistant. Absence of chimera indicates that the shoots are generated from an epidermis individual cell or few daughter cells, anyway deriving from a single cell (Broertjies and Van Harten, 1978; Broertjes and Keen, 1980).
  • the beginning of the shoot meristems includes the association and possible inclusion of cells situated below the epidermis.
  • Ability to form adventitious shoots is anyway dependent on plant genotype and often tissue type of the explant too.
  • Callus culture along with the subsequent seedling regeneration, can be used if the object of the culture is not the propagation but the induction of a new variability to be used for the genetic improvement.
  • Rhizogenesis is the process by which the roots are formed and, as caulogenesis, can occur directly, from somatic tissue or indirectly through an unorganised step. Firstly rhizogenesis proceeds, till to the primordium induction, in the same manner as caulogenesis. Successively primordia can be differentiated in shoots or roots, as a response to stimuli of different typei (Halperlin, 1069). High levels of auxin usually stimulate the rhizogenesis. Generally in vitro rhizogenesis occurs much more easily than caulogenesis.
  • Direct caulogenesis often is used for the propagation of various decorative and crop plants.
  • some decorative plants like Fresia (Hussey and Hargraves, 1974) and Pelargonium (Holdgate, 1977) were propagated by indirect formation of adventitious shoots.
  • Fresia Hussey and Hargraves, 1974
  • Pelargonium Hussey and Hargraves, 1974
  • this process involves a too high variability to maintain the commercial characteristics of the plant.
  • Somatic embryogenesis can occur directly or indirectly.
  • In vitro direct embryogenesis consists of the formation of somatic embryos on tissue explants without involvement of undifferentiated tissue.
  • In vitro direct embryogenesis principally was described in gametophyte and sporophyte tissues, in association with gametophyte or derived from fertilisation thereof (Smith and Krikorian). Practically it occurs only in cells suitable to produce embryos, named pre-embryogenically determined cells (PEDC) and the transfer onto the culture medium has only the function to increase such a process (Sharp et al., 1980; Evans et al., 1981a and Sharp and Evans, 1982).
  • PEDC pre-embryogenically determined cells
  • Somatic embryos can be formed indirectly when undifferentiated callus cells are induced to the embryogenetic determination (Sharp et al., 1980). These cells are named "induced embryogenetic determined cells” (IEDC). Although the embryos are difficult to be observed, they can be distinguished from adventitious shoots in that, differently from these, they are bipolar, comprising both an apical and a radical pole, an axis of the shoot and cotyledons, do not have vascular connections with adjacent parental tissues. Embryos (both zygotic and somatic) are in fact new individuals deriving from single cells and not having vascular connections with parental tissues (Haccius, 1978).
  • Somatic embryos can develop from a single cell, directly from somatic tissue, on callus or cellular suspensions (Backs-Husemann and Reinert, 1970; Nomura and Komamine, 1986a; Miura and Tabata, 1986) and from protoplasts (Miura and Tabata, 1986).
  • Embryogenetically determined cell can be subjected to a periclinal division forming a cytoplasm rich terminal cell, which will generate embryo, and a large and vacuolated basal cell which in turn can divide a few times generating a suspensor. If the basal cell divides according to anticlinal planes or randomly, can generate pro-embryonal cellular complexes or anomalous embryos with multi-serial suspensor (Trigiano et al., 1989).
  • Possibility to obtain effective differentiation protocols for the various iinteresting species is usually determined by the study and acknowledgement of the genotype response (plant, cell, tissue) to various factors possibly affecting the regulation of the various steps of the morphogenetic process (induction, determination and development). Usually in this process are involved the following factors: - growth regulators (phytohormones, stress inductors, oligosaccharides, etc.);
  • transgenic plants uses the transformation of vegetal cells from a tissue explant (protoplasts).
  • the production of transgenic plants depends essentially on the introduction frequency of the interest gene and the ability of the transformed cells to regenerate the whole plant, shoots or roots. Both the aspects can represent limiting factors for the fruit plants.
  • Indirect methods use microbial expression vectors. Gene transfer into the vegetal cell is facilitated by the naturally occrring gene transfer system of Agrobacterium tumefaciens (Gheysen et al., 1985; Fraley ef al., 1986; Lichtenstein and Fuller, 1987; Zambryski et al., 1989; Schuerman and Dandekar, 1991). Also DNA (Gronenborg and Matzeit, 1989) and RNA (French ef al., 1986) viruses were modified in order to insert an exogenous gene replacing part of the viral genome, thus generating a viral defective particle able to carry out gene transfer.
  • the Agrobacterium mediated transformation technique was the first tool for the transformation of many dicotyledonae. It is certainly preferred method for all the vegetal species for which T-DNA transfer is possible due to relative transfer easiness and precision in undamaged and able to regenerate explants explants. However many vegetal species, among which economically important cereals like rice, maize and wheat, are not easily transformed by conventional indirect method. Therefore alternative systems were provided wherein DNA insertion into the host cell occurs as free DNA followed by the random integration into the genome. Among these the biolistic method represent a mechanical method for the introduction of DNA into most of the vegetal species. It is advantageous in all the situations wherein the Agrobacterium mediated transformation and free transfer into protoplasts are not possible.
  • the technique is based on the acceleration of heavy particles with some micrometer diameter, principally gold or tungsten, which can penetrate through the cellular wall and plasmatic membrane of undamaged vegetal cells, thus delivering the genetic matter. Because the small holes through the wall and plasmalemma quickly self-close, the produced injuries are temporary and do not compromise irreversibly the cell integrity.
  • the first transgenic plant was soy-bean.
  • the most interesting result was the production of fertile transgenic maize plants (Fromm ef al., 1990; Gordon-Kamm ef al., 1990).
  • selection markers Many genes encoding for antibiotics or herbicides can be used as selection markers, usually required for an efficient production of transgenic plants.
  • Selection agents are different each other relating to the toxicity grade against the plant, which depends on the size and development step of the cell o tissues. In the presence of the selection agent indifferentiated cells respond differently to the explants of organs as leaves and cotyledons. In addition the susceptibility to the various agents depends on the species. Therefore for each transformation and regeneration protocol it is required to carry out the precise determination of the concentration of the selection agent which allows on the hand the growth and development of the transformed cells and on the other hand inhibits the proliferation of undifferentiated cells. Often herbicides are more toxic than antibiotics against vegetal tissues such that their use is carried out only successively, i.e.
  • Prior art techniques prove to have an effective application only for species having a cellular structure of the various somatic tissues characterised by high cellular competence for organogenesis and somatic embryogenesis processes.
  • Currently known and/or used regeneration protocols depend on the cellular competence of the interest plant genotype (Genus-Species-Variety) and tissue type (both meristematic and somatic: leaves, stem, stipule, etc.).
  • Many vegetal species having high agronomic interest show an extremely reduced competence to the regeneration of their various cellular typologies. This behaviour, mainly apparent in many woody species, often represents the determining factor in limiting the application of the in vitro multiplication technologies and gene manipulation, due to reduced efficiency and difficulty in obtaining the regeneration of interest gene modified plant, respectively.
  • the method of the invention allows to obtain the formation of tissues characterised by a major cellular component having high competence for the regeneration of adventitious buds.
  • the method allows to rationalise the production process of nurseries because it can be used both for the multiplication and transformation. Thus the costs for the production and implementation of genetic vegetal biotechnological innovations are reduced.
  • the present invention consists of method of in vitro culturing plant tissues that, by inducing and culturing meristematic cellular bulks, allows to produce cellular layers (platforms) characterised by an high regeneration efficiency, via caulogenesis, of new plants.
  • the basic principle of the method is the induction of growing cellular bulks with high regenerative competence from which, via sectioning, it is possible to obtain cellular layers (platforms), again with high regenerative competence.
  • the choice of the initial concentration of the growth regulators is defined on the base of the indications from individual species during the in vitro standard proliferation step (proliferation of axillary buds).
  • the chemical treatment consists of progressive increase of the cytokininis content in the substrate of the proliferating shoots, reaching in few sub-cultures (3-5) an effective concentration of the cytikininis at least three times higher than initial value in the proliferating substrate (1-3).
  • the mechanical treatment includes, at each transplant of the proliferating shoots, without removing the base, the removal of the apex of the individual shoots (topping) originating from axillary buds. This treatment promotes a strong growth of the base which more and more is characterised by an increasing presence of various meristematic centres.
  • parenchymatic cells (callus) to complete primary meristem (adventitious bud) comprising all the intermediates steps (nodules, meristematic primordium).
  • These cellular bulks therefore prove to be with high regeneration competence which can be maintained during successive sub-culture on substrates characterised by the cytokininis concentrations reached in the last step of the bulk induction.
  • the essential characteristic of the produced bulks is that from their fragmentation, following three or four culturing weeks, identical formations are obtained.
  • these meristematic bulks can be maintained in culture and multiplied by sectioning (about 2 mm thick homogenous sections) resulting in new meristematic platforms characterised by the same regenerative efficiency.
  • Effective induction and maintenance of meristematic bulks (MB) and high regenerative efficiency of new adventitious shoots of meristematic platforms represent an effective multiplication method resulting in simplified techniques for the in vitro manipulation of the vegetal matter (simple sectioning of the bulks) and the achievement of explants maintaining yet high regenerative efficiency.
  • the method is suitable to produce large amounts of these cellular bulks (both on liquid and solid substrates), easily to be sectioned and usable for the production and differentiation of new shoots and plants in great number.
  • formed meristematic bulks can be used for the production, via regular sectioning, of cellular platforms (about 2 cm thick and 1 cm long and wide sections) which maintain the characteristic of meristematic-vascularized bulks with high competence to determine the regeneration of new adventitious shoots (caulogenesis).
  • Cellular platforms obtained by the method of the invention are advantageously applied for the genetic transfer both by Agrobacterium tumefaciens mediation and direct techniques.
  • the combined effect of the injury and high frequency of regeneration competent cells promote high efficiency also for the transformation.
  • the infection is carried out according to the conventional PM infection method, preferably including also a treatment with acetosyringone and syringaldehyde (25 ⁇ l), which are organic compounds known to activate the expression of vir genes and, therefore, to mobilise the DNA of the binary plasmid.
  • the explants are dehydrated using 3MM sterile absorbent paper and transferred into Petri dishes containing the regeneration substrate characterised by hormonal concentration as defined for the PM regeneration.
  • the transformation occurs for an extremely reduced number of cells and the following selection step is extremely important to discriminate stable and homogeneous transformation events.
  • a protocol of continuous regeneration in selecting medium based on various passages of PM on substrates added with same concentrations of growth regulators and increasing doses of the selecting factor (kanamycin), from a minimum level of 25 mg/l to a final concentration (after 4 sub-cultures) from 50 to 100 mg/l, depending on the kanamycin sensitivity of the species, is started.
  • transgenic-not transgenic chimera the selective pressure exerted by kanamycin results in selective advantage of transgenic cells.
  • a moderate selection during proliferation and subsequent radication with kanamycin are preferred for easy rooting species. It is extremely important to proceed by a continuous regeneration over quite long periods and frequent sub- cultures. Continuous regeneration means to maintain, after the first regeneration, continuously the explants in regeneration and selection substrate. In fact it is believed that the initial regeneration events usually are related to already formed and therefore rarely transgenic meristems. In a following step, the vegetal matter being maintained in a regeneration- selection substrate, the probability to increase the frequency of transformed cells, whose subsequent differentiation promotes the regeneration and development of permanently and homogeneously genetically modified individuals, is higher.
  • FIG. 1 Southern Blot analysis on 0,7 % agarose gel in 0,5 X TBE of DNA of Defh9-iaaM transgenic, vine, cv "Thompson”. Column 1 , Hindlll digested clone 4 DNA. Column 2, Hindlll/EcoRI digested clone 4 DNA.
  • FIG. 235023 Southern Blot analysis of DNA of two DefH9-iaaM transgenic, vine, cv Silcora (l.G. 235023). Column 1 , Hindlll digested clone 29 DNA. Column 2, Hindlll/EcoRI digested clone 29 DNA. Column 3, Hindlll digested clone 35 DNA. Column 4, Hindlll/EcoRI digested clone 35 DNA. Column 5, Hindlll digested control DNA. Column 6, Hindlll/EcoRI digested control DNA. Vegetative Multiplication
  • the described method allows to produce the meristematic platforms with high regeneration capability, resulting in higher production efficiency of shoots than proliferating rate usually found during a conventional in vitro proliferation step from axillary buds and also than regeneration rate from somatic tissue (usually leaf and stalk).
  • Figure 1 shows Southern Blot analysis of Defh9-iaaM transgene transgenic cv "Thompson” vine DNA.
  • the probe used homologous to nptll gene, is of 544 bp, produced by PCR using following primers: 5'CAGAGTCCCGCTCAGAAGAACTCGTCA3' and
  • the plant contains 4 copies of T-DNA inserted into the genoma. Only one of four fragments, about 8,6, 7,5 and 2,8 kb, is cut by EcoRI, as deduced from the disappearance of the heaviest fragment by double digestion and concurrent detection of 2,4 Kb fragment. Because the distance from Hindlll site, present in T-DNA at nptll gene 5', to T-DNA right border (RB) is about 2 kb, EcoRI site is present in genomic DNA of the vine transgenic plant at 400 bp from the insertion site.
  • Figure 2 shows Southern Blot analysis of Defh9-iaaM transgene transgenic cv Silcora (l.G. 235023) two vine DNA.
  • the probe used homologous to iaaM gene encoding region, is of 589 bp, produced by PCR using following primers:
  • 5OCATGCTCTTTTCACCCGTATTAG3' Clone 29 contains 3 copies of T-DNA inserted into the genoma, as deduced from the presence of three bands of about 1 ,2, 3,5 and 6 kb, produced by Hindlll enzyme digestion and homologous to the probe obtained from Defh ⁇ -iaaM ⁇ ene p romoter. 35 plant contains on ' one T- DNA copy, as deduced from the detection of a single band of about 2,1 Kb. In both cases the Hinf ⁇ ! and EcoRI double digestion produce an about 0,8 Kb band.
  • Plant gene vectors and genetic transformation plant viruses as vectors. In: Schell J. Vasil J.K. (Ed.) Cell Culture and Somatic Cell Genetics of Plants. Vol 6. Pp. 69-100. Academic Press, New York.

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Abstract

L'invention concerne un procédé permettant de produire des agrégats cellulaires végétaux dotés d'une grande capacité de régénération, lequel procédé consiste en un traitement chimique et un traitement mécanique. L'invention concerne également les agrégats cellulaires végétaux ainsi obtenus. Le procédé de cette invention est utilisé de manière avantageuse pour produire des plates-formes méristématiques à haute capacité de régénération, pour la multiplication végétative d'une plante et l'obtention d'une plante modifiée génétiquement.
PCT/IT2001/000280 2000-06-05 2001-05-31 Procede de regeneration de plantes et utilisation de ce procede pour multiplier et/ou transformer des plantes WO2001094602A2 (fr)

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AU74484/01A AU7448401A (en) 2000-06-05 2001-05-31 Method to regenerate plants and uses thereof to multiply and/or transform plants

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IT2000RM000305A IT1317038B1 (it) 2000-06-05 2000-06-05 Metodo per la rigenerazione di piante e suoi usi per lamoltiplicazione e/o la trasformazione di piante.
ITRM2000A000305 2000-06-05

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Cited By (4)

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EP1571899A2 (fr) * 2002-12-06 2005-09-14 Del Monte Fresh Produce Company Transformation et regeneration organogenique
WO2011030083A1 (fr) 2009-09-11 2011-03-17 Imperial Innovations Limited Procédé
JP2017520277A (ja) * 2014-07-11 2017-07-27 メディカゴ インコーポレイテッド 植物におけるタンパク質生産の改変
CN116420621A (zh) * 2023-04-24 2023-07-14 玉林师范学院 一种促进墨兰根状茎芽分化方法

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EP1571899A4 (fr) * 2002-12-06 2006-08-30 Del Monte Fresh Produce Compan Transformation et regeneration organogenique
AU2003302775B2 (en) * 2002-12-06 2010-05-27 Del Monte Fresh Produce Company Organogenic transformation and regeneration
US8049067B2 (en) 2002-12-06 2011-11-01 Del Monte Fresh Produce Company Organogenic transformation and regeneration
WO2011030083A1 (fr) 2009-09-11 2011-03-17 Imperial Innovations Limited Procédé
JP2017520277A (ja) * 2014-07-11 2017-07-27 メディカゴ インコーポレイテッド 植物におけるタンパク質生産の改変
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US11959088B2 (en) 2014-07-11 2024-04-16 Aramis Biotechnologies Inc. Modifying protein production in plants
CN116420621A (zh) * 2023-04-24 2023-07-14 玉林师范学院 一种促进墨兰根状茎芽分化方法

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