WO1997026363A2 - Method of inducing nitrogen fixation in plants - Google Patents
Method of inducing nitrogen fixation in plants Download PDFInfo
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- WO1997026363A2 WO1997026363A2 PCT/GB1997/000120 GB9700120W WO9726363A2 WO 1997026363 A2 WO1997026363 A2 WO 1997026363A2 GB 9700120 W GB9700120 W GB 9700120W WO 9726363 A2 WO9726363 A2 WO 9726363A2
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- 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
- A01H17/00—Symbiotic or parasitic combinations including one or more new plants, e.g. mycorrhiza
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the invention relates to plant biotechnology and particularly to me stimulation of nitrogen-fixation in non-leguminous plants by interaction with nitrogen-fixing bacteria.
- Legumes such as clover
- non-legumes do not naturally form such nodules. It would be desirable to enable commercially important non-leguminous cereal crops such as wheat, rice, maize, barley, millet, sorghum and rice; tomatoes and other horticulturally important crops, and especially oilseed rape (Brassica napus and other Brassica species) to fix nitrogen as this would offer the possibility of reducing the amount of nitrogenous fertilizers used at present.
- Nodulation of legumes by the nitrogen-fixing bacteria rhizobia is generally considered to be controlled by rhizobia nodulation (nod) genes. These nod genes encode enzymes involved in the syndiesis of Nod factors that induce morphological changes in legume roots.
- the four nod genes A,B,C and D are u e minimum number of genes required for the formation of such extracellular metabolites which induce the root hair curling response in legumes. It has been demonstrated that the expression of some of the rhizobia symbiotic genes wim the associated production of rhizobia Nod factors, can induce changes in the growth of rice root hairs comparable to the curling and distortion observed in root hair of legumes during interaction wi ⁇ i rhizobia.
- Some naturally occurring rhizobia induce nitrogen-fixing root nodules on a wide range of legumes.
- strain NGR234 nodulates many legumes, including species in the genera Arachis, Glycine, Lablab and Vigna. Additionally, this wide host range strain NGR234 induces effective root nodules on the non-legume Parasponia. This unique association has prompted a genetic analysis of the nitrogen fixing symbiosis between Parasponia and rhizobia. The infection of Parasponia by rhizobia requires the nod regulatory gene, together with host-specific nodulation (hsri) genes.
- nodulation of Parasponia requires the co ⁇ ordinated expression of nodA,B and C genes together with rhizobia nsn genes under the regulation of the nodD product.
- This knowledge enabled uie construction of a new rhizobium strain (ANU536) which possessed multiple copies of the nodD gene on a high copy number vector.
- This strain was able to induce nodule-like strucmres on the roots of rice seedlings at a low frequency of 0.1-0.25%.
- These nodule-like strucmres were small, white and contained membrane encapsulated bacteria; one of these strucmres was observed to have an internal strucmre resembling that found within legume root nodules.
- nitrogen fixation activity has not been detected.
- R Bradyrhizobium
- S Sinorhizobium
- A Azorhizobium
- R. meliloti Rmnod-1 identified as a Nacyl-tri-N-acetyl-/°-l,4-D- glucosamine tetrasaccharide bearing a sulphate group on carbon 6 of the reducing sugar moiety.
- nodbl D2,D3 Transcriptional regulator required for the expression of the other nod genes.
- nodE Host range reported sequence similarity to the ⁇ MJ gene of f. coli, condensing enzyme of fatty acid synthase. Membrane protein.
- nodF Host range reported sequence similarity to acyl carrier protein.
- NodF carries 4'-phosphopantetheine as a prosthetic group.
- At least rwo different forms of ⁇ odF can be isolated from a ⁇ odFe- overproducing strain of R. leguminosarum bv . viciae.
- nodG Host range reported sequence similarity to dehydrogenases.
- nodti Host range many modify factor produced by NodABC. may function as a sulphate transferase for the synthesis of ⁇ odRm-I. nodi Reported sequence similarity to ATP binding transport proteins.
- nodi Membrane protein may act in conjunction with nod .
- nodL Host range reported sequence similarity to acetyltransferase.
- nodM Host range reported sequence similarity to amidotransferase.
- nodO Function unknown, encoded protein is exported. Reported to bind calcium. nod? Function unknown, gene hybridizes to E. coli genomic D ⁇ A.
- nodQ Host range reported sequence similarity to GTP binding proteins, EF-tu and EF-1. May modify factor produced by NodAB .
- Host range. nodS Function unknown. MW 23 kDa.
- nodT Host range. Function unknown.
- nodV Function unknown. MW 62 kDa. nodV Reported only in B. japonicum.
- nodW Reported only in B. japonicum Important for host range, sequence similarity to family of transcriptional regulatory proteins.
- Expression of nod genes may be regulated in legumes by inducers such as the flavones or flavonones exuded by legume roots.
- the present invention aims to provide a method for inducing nitrogen fixation in non-legume plants.
- a method of inducing nitrogen fixation in a non-leguminous plant by inoculating the plant with a nitrogen- fixing bacterium; wherein the bacterium is exposed to a nod-factor inducing agent.
- inoculating is intended to embrace any means by which the plant and bacterium are brought into nitrogen-fixing interaction.
- the inventor has found that exposure of die bacterium to a nod-factor inducing agent in the kannod of the invention induces the bacterium to produce Nod factors which stimulate the entry of the bacterium into the non-leguminous plant root system and nitrogen fixation.
- the invention provides a non-leguminous plant in a nitrogen fixing interaction with a bacterium obtainable by the method of the invention.
- nitrogen-fixing interaction is intended to include any plant/bacterial interaction in which atmospheric nitrogen is made available to the plant as a nutrient.
- the interaction may, for example, result in uie formation of root or stem nodules containing the bacterium, or, by bacterial invasion of lateral roots, in the formation of short, thickened lateral roots (STLRs) which are very similar in appearance to the normal short lateral roots of the plant.
- STLRs thickened lateral roots
- t e bacterium for use in the method of the invention is oxygen tolerant.
- oxygen tolerant we include any bacterium which is able to fix atmospheric nitrogen in the presence of at least 0.1% oxygen.
- suitably tolerant rhizobia bacteria comprise stem-nodulating rhizobia including Azorhizobium species such as A caulinodans (eg. strain ORS571 ATCC deposit No. 43989) and Bradyrhizobium species such as B. ORS310, which normally nodulates stems of Aeschynomene indicia (Van Rhiju and Vanderleyden (1995) J. Microbiol Rev. 59: 124-142).
- the nitrogenase of A caulinodans is tolerant of up to approximately 3% oxygen and the nitrogenase of B.
- ORS310 is tolerant of up to approximately 0.5% oxygen.
- Oxygen tolerant bacteria are preferred because, unlike leguminous plants, non-leguminous plants do not normally contain molecules such as leghaemoglobin which can take up free oxygen to minimize inhibition of nitrogen fixation activity due to inactivation of nitrogenase.
- Rhizobia which namrally infect legumes (especially temperate legumes) via an infection thread in uie root hairs such as Rhizobium leguminosarum, R. meliloti, R. loti, R. phaseoli, R. japonicum, R. lupini and R. t ⁇ folii.
- Rhizobia used in theicaiods of he invention are hose which disturb the epidermis themselves or which can infect the plant by "crack- entry", that is, take advantage of a dismrbance of the epidermis (for example resulting from physical injury or the emergence of an organ such as a lateral root) and in either case thereby form a nodule.
- Such Rhizobia include those which namrally form nodules on uie roots of Parasponia species and those which infect the roots (including lateral roots) or other tropical plants (especially legumes), such as Aeschynomenes spp. (for example A.
- Rhizobia also include those which induce nodules on the stems of Sesbania, Aeschynomenes, and Neptunia species. Rhizobia which infect Parasponia and cause nodulation include:
- the Parasponia-wfecting Rhizobium strains CP283 and 501 are particular examples of effective Rhizobia.
- the nod-factor inducing agents useful in the present invention comprise any agent which can induce nod-factors in the bacterium used to inoculate the non-leguminous plant.
- the inducing agents are obtained from plant exudates.
- Particularly preferred inducing agents are flavonoids. Dakora, F.D. (1995) Aust. J. Plant Physiol 22, 87-99 provides a review of plant flavonoids.
- Flavonoids are naturally occurring plant products which originate from the phenylpropanoid pathway (Table A). Each molecule has a C 15 skeleton formed from condensation of three malonate units with a phenylalanine- derived -C 3 precursor. Their distribution is widespread in higher plants.
- Flavonoid structures namely flavones, flavanones, isoflavones and chalcones (Table A), induce transcription of nodulation (nod) genes in Rhizobium cells as the first step towards root nodule formation and symbiotic N 2 fixation.
- Anthocyanidins such as delphinidin, petunidin and malvidin are also able to transcribe nod genes in Rhizobium legumino ⁇ sarum biovar phaseoli.
- the molecules identified are released from seed coats during imbibition and from root exudates of sterile young seedlings. This pool of active molecules is increased in exudate by the release of additional nod-gene inducers when rhizobial cells are present on infectible roots.
- Useful flavonoids can be provided as exudate from the seedlings of plants, particularly legumes which normally have nodules on their roots containing the nitrogen fixing bacterium used to inoculate the non- leguminous plant.
- Flavonoids can be purified from the exudate of seedlings using conventional methods such as those described by Messens et al (1991 , Mol. Plant-Microbe Interactions, vol.4, No.3, pp.262-267). They extracted 7,4'-Dihydroxyflavanone (liquiritigenin) as the major nod-factor inducing component of exudate obtained from seedlings of the tropical legume Sesbania rostrata which normally includes nitrogen-fixing nodules on its roots and stems containing the bacterium Azorhizobium caulinodans strain ORS571.
- O her flavonoids which were shown by Messens et al to imitate the inducing effect of liquiritigenin were 5,7,4'- trihydroxyflavanone (naringenin) and 4,2',4'-trihydroxychalcone (isoliquiritigenin).
- the method of the invention comprises inoculating a non-leguminous plant with Azorhizobium wherein uie bacterium is exposed to one or more nod-factor inducing flavonoids selected from naringenin, liquiritigenin and isoliquiritigenin.
- the bacterium is Azorhizobium caulinodans.
- Non-flavonoid nod-factor inducing agents for use in the invention may include betaines, preferably trigonelline and stachydrine. The latter two betaines were identified by Phillips et al (1992) Plant Physiol 99: 1526-
- the inventors have also identified di-iodo 4-hydroxybenzoic acid (Pfaltz Bauer Inc) as a nod-factor inducer in Azorhizobia inoculated wheat seedlings and the related 4-hydroxybenzoic acid as a nod-factor inducer in Rhizobium NGR234.
- the preferred amount of the agent for use in uie me ⁇ iod of the invention to induce nod- factor production in a particular bacterium and allow it to form a nitrogen- fixing interaction with a particular non-leguminous plant can be determined using routine tests involving nothing more than trial and error.
- the bacterium is exposed to an inducing agent solution having a molarity of from 1 x IO "6 to 5 x 10 "4 and especially from 2 x IO "5 to 1 x 10
- seedlings of the non-leguminous plants are inoculated with uie nitrogen-fixing bacterium in the presence of the nod factor inducing agent shortly after germination.
- wheat seedlings may be inoculated approximately 3 to 6 days after germination.
- the bacterium can be pretreated with the inducing agent prior to inoculation of the plant seedlings.
- the seeds are sterilised prior to germination and grown under sterile conditions prior to inoculation.
- such exposure is achieved by degrading the plant cell wall at the apices of the root hairs enzymatically.
- the bacterium is preferably exposed to the nod-factor inducing agent during or prior to this step.
- the invention provides a composition comprising a non- leguminous plant seed in combination with a nitrogen-fixing bacterium which is exposed to a nod-factor inducing agent such as a flavonoid.
- non-leguminous plant seeds are mixed with a plant growth medium such as peat in combination whh a bacterial inoculum and a nod-factor inducer
- a plant growth medium such as peat in combination whh a bacterial inoculum and a nod-factor inducer
- the growth medium is treated with a solution of the inducer.
- uie bacterium can be pre-treated by incubation with the inducer.
- the composition comprises sterile plant growth medium, such as
- the composition comprises a non-legume seed or a somatic non-legume plant embryo enclosed within a synthetic coating comprising a growth medium.
- a non-legume seed or a somatic non-legume plant embryo enclosed within a synthetic coating comprising a growth medium.
- Such coated seeds are well known, wi ⁇ i alginate beads being the most widely studied coating material.
- the coating comprises a pharmaceutical type capsule as described by Dupuis et al (1994) Bio/Technology Vol.12 April pg.385-389 the disclosure of which is incorporated herein by reference.
- the capsule body acted as a strong water soluble hull, covered on its inner surface by a watertight film composed of polyvinyl chloride (PVC), polyvinyl acetate (PVA) and bentone as a thickener, to control the nutrient supply and the subsequent development of the somatic embryo. A germination medium and the somatic embryo were then placed in die capsule.
- PVC polyvinyl chloride
- PVA polyvinyl acetate
- bentone as a thickener
- a bacterial inoculum is added to the growth medium.
- the nod-factor inducing agent can also be included in die growth medium, but it is preferably used to induce nod-factor expression in the bacterium before the bacterium is added to uie growth medium.
- the invention provides a growth medium for a non- leguminous plant comprising a bacterium capable of fixing nitrogen; wherein the bacterium is exposed to a nod-factor inducing agent.
- the medium can be prepared simply by inoculating a known medium with a bacterium which has been pretreated with an inducing agent.
- uie invention provides a method of inducing nitrogen fixation in a non-leguminous plant comprising providing a non-leguminous plant or reproduction material of said plant including heterologous genetic material capable of expression to produce a nod-factor inducing agent and inoculating the plant with a nitrogen-fixing bacterium.
- Another somewhat similar procedure is to utilise biolistics, the so-called particle gun bombardment method, involving the high velocity microprojectile delivery of me gene(s) into plant cells (without having to remove their cell walls to produce protoplasts) which are capable of regenerating shoots and, ultimately, fertile plants.
- the bacterium Agrobacterium tumefaciens can also be used to deliver genes into cells of the major non-legume crops from which transgenic plants can be regenerated using standard tissue culture procedures.
- the gene or genes to be inserted for the required flavonoid syndiesis and secretion in die root system or target crops will be introduced into the non-legume crops using a plasmid construct containing a suitable promoter, terminator and uie gene of interest.
- the pathway of flavonoid synthesis is well established
- This specific "flavonoid synthesis" gene can be produced from legumes by standard molecular procedures involving isolation of messenger RNA and reverse transcription of cDNA followed by introduction into the target non-legume crop plant by delivery into isolated protoplasts, or by incorporating into totipotent cells through biolistics or the use of Agrobacterium tumefaciens as the gene vector.
- the cDNA can be tailored with a suitable promoter (preferably a root-specific promoter) and termination sequence. Putative transgenic non-legume crops would be evaluated at the molecular level for the presence of the "flavonoid synthesis" transgene. Their required flavonoid synthesis capability can be demonstrated using the common acetylene reduction assay for nitrogen fixation (Witty J.F. [1979] Soil. Biol. Biochem. 11 : 209-210).
- expression of a nod-factor inducing agent by a non- leguminous plant can be provided by blocking a step in a biosynuietic pathway of the plant whereby an intermediate capable of inducing nod- factors accumulates so that the bacterium can be exposed thereto.
- an intermediate capable of inducing nod- factors accumulates so that the bacterium can be exposed thereto.
- the conversion of the intermediate naringenin (a nod-factor inducing agent) to Dihydrokaempferol by the enzyme Flavanone-3'- hydroxylate can be blocked by a variety of techniques in which expression of the gene encoding the enzyme is suppressed. Suitable suppression methods are described by Courtney-Gutterson et al (1994) Bio/Technology vol.12, pg.268-271 and the disclosure of that document is incorporated herein by reference.
- Suitable methods for making transgenic plants capable of expressing flavonoid biosynthetic pathway genes are described in WO90/11682 (DNA Plant Technology Corporation) die disclosure of which is incorporated herein by reference. Also described are methods for suppressing the expression of flavonoid biosynthetic genes.
- Figure 1 shows: a) Formation of short thickened lateral roots (STLRs; arrowed) on a 2 Id-old wheat seedling inoculated mt Az. caulinodans in die presence of 10 ⁇ M naringenin. b) Root of a seedling inoculated wititi Az. caulinodans, without naringenin, which lacks STLRs. c) Longitudinal section of a STLR from (a) showing crack entry invasion (arrowed), widi azorhizobia penetrating between cells at the base of the merging lateral root. d) The region arrowed in (c) showing azorhizobia between
- Figure 2 shows die gene sequence of die chalcone synthase of Petunia hybrida (Koes et al, 1989, Gene 81, 245-257);
- Az caulinodans IRBG314, nod- V44 Nod A::Tn5 and ORS 571 (ATCC deposit No. 43989) ntf " 57004 (nif04-Tn5) were maintained on YEM medium (Vincent J M. Int. Biological Programme Handbook 15, Oxford. Blackwell Scientific Publications (1970). After 3 d ays (d) of culture in TGYE liquid medium (Ladha J K, et al. Appl. & Environ. Microbiol. 55:454-460 (1989)) die rhizobia were used for inoculation.
- Nitrogenase activity was dete ⁇ nined by the acetylene reduction assay using 10% acetylene with 90% air in tubes closed with subaseals (Witty J F. Soil Biol. Biochem. 11:209-210 (1979).
- the number of short diickened lateral roots (STLRs) was determined by direct measurement of the number of lateral roots which were more than 0.5 mm wide at the base and up to 2 mm in length.
- Nano moles STLRs Nano moles STLRs Nano moles STLRs Nano moles STLRs Molarity ethylene/ ethylene/ ethylene/ pla.it/24h plant/24h plant/24h
- Nitrogenase activity was determined using the acetylene reduction assay at various times post inoculation. Uninoculated seedlings, with and without naringenin, and seedlings inoculated with lz. caulinodans nif ⁇ and Az caulinodans nod , widi and without naringenin, failed to produce ethylene or STLRs either at 14d, 2 Id or 28d. Thirty replicates were assayed per treatment. All seedlings inoculated with Az. caulinodans in the presence of naringenin showed some positive ethylene production and die formation of STLRs.
- Roots were fixed in 2.0% (v/v) glutaraldehyde for 24h at 4°C, followed by 1.0% (w/v) osmium tetroxide (2h, 4°C). Fixatives were prepared in 0.1M sodium phosphate buffer, pH 7.0. Specimens were dehydrated uirough 10% (v/v) ethanol to absolute ethanol (30 min each) and embedded in LR White medium grade resin (The London Resin Co. , Basingstoke, UK) (Davey M R et al. J. Exp. Bot. 44:863-867 (1993). For light microscopy (Fig.
- narmgenin stimulates nitrogen fixation in wheat when otiier rhizobia, such as Bradyrhizobium ORS310 which normally nodulates stems of Aeschynomene indica are used for inoculation (data not shown). This suggests that narmgenin may be a generally active nod gene inducing signal for various rhizobia interacting with non- legumes, leading to nitrogen fixation.
- otiier rhizobia such as Bradyrhizobium ORS310 which normally nodulates stems of Aeschynomene indica are used for inoculation.
- Table 1 The fact that wheat seedlings inoculated with the nod- strain of Az.
- caulinodans which is capable of nitrogen fixation in the free-living state in the presence of up to 3% oxygen, failed to show nitrogenase activity with and without naringenin, as did the wild-type Az.
- caulinodans used for inoculation wiuiout naringenin (Table 1), also indicates diat nitrogenase activity is arising from rhizobia within die root system of the wheat seedlings. Moreover, the nitrogen fixation activity of surface azorhizobia would be completely inhibited by die concentration of oxygen
- Rice seeds are surface sterilised in 30% (v/v) Domestos solution and germinated on nitrogen-free agar at 28 °C in die dark.
- This medium (Fahraeus) has the following composition:- [CaCl 2 (0.1 g), MgSO 4 .7H 2 0 (0.12 g), KH 2 PO 4 (0.1 g), Na 2 HPO 4 .2H 2 O (0.15 g), Fe citrate (0.005 g), traces of: Mn,Cu,Zn,B,Mo] per litre widi 0.8% agar, pH 6.5-7.0.
- Exponential phase Rhizobia are cultivated by centrifugation and resuspension in fresh yeast extract-mannitol solution (0.5 ml) having the composition [K 2 HPO 4 (0.5 g), MgSO 4 .7H 2 0 (0.2 g), NaCl (0.1 g), mannitol (10.0 g), yeast extract (Difco)(0.4 g)] per litre, pH 6.8-7.0.
- this preparation is mixed with 1 ml of polyethylene glycol solution of composition [Polyethylene glycol M.W. 6000 (20.0 g), CaCl 2 .2H 2 0 (0.15 g)] per 100 ml [Difco is a Registered TM].
- the enzyme-treated roots are immersed in die polyetiiylene glycol widi Rhizobia for 5 minutes followed by two washings widi mannitol solution [Mannitol (9.0 g) per 100 ml, pH 6.5-7.0].
- the seedlings are transferred to nitrogen-free agar in square Petri dishes which can be stacked vertically for optimum seedling growth. All seedlings survive the enzyme treatment altiiough die root growtii maybe impaired in some cases. Root samples are removed for light and electron microscopic examination.
- 24 fusion using eidier chemical or electrical procedures may be used to transfer nuclear genes (controlling for instance symbiotic associations) or cytoplasmic genes (conveying for instance male sterility) without impairment of the functional integrity of the plant.
- Exposed plasma membranes readily regenerate a new cell wall and root hairs with exposed plasma membranes, after fusion, may be stabilised in this way.
- protoplasts can be isolated from root hairs and used for somatic hybridisation by fusion with otiier isolated protoplasts.
- This first procedure involves fusion of protoplasts containing Rhizobia (diese protoplasts are isolated enzymatically from nodules of the legume) with the exposed plasma membrane of die non-legume root hair whereby the root hair of the non-legume will contain Rhizobia in its cytoplasm.
- the second procedure involves fusion of protoplasts (subprotoplasts) released from the tips of root hairs of enzymatically- treated root hairs of legumes with die exposed plasma membrane of the non-legume root hair; die hybrid root hair on the non-legume then behaves like a legume root hair and interacts with Rhizobia in the usual way that legumes do during their normal infection with Rhizobia.
- protoplasts subprotoplasts
- Rice seeds (or seeds of any other non-legume) are surface sterdised in 30% (v/v) Domestos and germinated on nitrogen-free agar (see Example 2) at 28° in the dark, and 2-day-old seedlings are incubated in isotonic enzyme mixmre for 5 minutes to expose the plasma membranes at the surface of their root hairs.
- Protoplasts are isolated from young nodules of the legume using the procedure described by Davey and Cocking, 1973, Namre, 224,
- Seedlings of the non-legume following the treatment as detaded in (a) are mixed witii nodule protoplasts (which contain Rhizobium) such that die root hairs are mixed widi these nodule protoplasts widi a ratio of approximately four nodule protoplasts to every root hair.
- the root hair system of the non-legume with associated isolated root nodule protoplasts is then incubated in autoclaved 30% w/v polyediylene glycol (PEG) M.W. 6000 containing 0.01
- Subprotoplasts are isolated from root hairs of the legume by treating root hairs of seedlings with the enzyme mixmre under conditions which cause extrusion of the protoplast from the root hairs.
- the subprotoplasts are collected by flotation.
- Rhizobia of die required legume specificity for infection of the non- legume to take place.
- compositions of non-legume seeds coated with a growth medium containing a bacterium exposed to a nod-factor inducing agent are produced.
- Somatic wheat embryos can be coated widi a pharmaceutical type capsule as described by Dupuis et al (1994) Bio/Technology, Vol.12 April 385- 389.
- Natrosol HHR 250 ® (Aqualon, France), Tylose ® (Hoechst, Germany), Methocel ® (SEPPIC) and Bentone SDI ® (Eurindis, France) were used as tiiickeners.
- the capsule was filled with 0.5ml of germination medium containing 6g/l of Phytagel ® mixed with an inoculum (approx. 0.2ml of a IO 9 ml "1 culture medium) of Az. caulinodans strain ORS571 which had been pretreated with a 10 ⁇ molar solution of the nod-factor inducing agent naringenin..
- a 0.5 to 1.5mm long torpedo shaped embryo was placed on the intemal medium without special orientation. As the embryoes converted witiiout arrest into plants, the capsules were placed directly in germination conditions.
- Capsule cap Different metiiods for closing the capsule can be used.
- a fdm cap was obtained by placing in die capsule opening 0.5ml of a liquid polymeric mixmre or oil, then subjecting it to air drying (polymers) or lower temperamres (oil).
- Suitable ods include jojoba oil (Sigma), rape seed oil (Robbe). Where cotton or rockwoll is used as a cap, fibers are then sprayed with silicon before being inserted by hand into the opening.
- transgenic non-legume capable of producing flavonoids for forming a nitrogen-fixing interaction with a bacterium.
- Flavonoid biosyntiiesis in plants is based on a series of common steps; the key enzyme involved is chalcone svntiiase which catalyses die syndiesis of tetrahydroxy chalcone from coumaryl-CoA and malonyl-CoA. Tetrahydroxy chalcone is readily converted to naringenin by chalcone isomerase of general occurrence in plants.
- a preferred example of commercially important transgenic non-legume crop is rice, which does not naturally syntiiesise the nod factor inducer naringenin.
- die rice is preferably manipulated genetically to produce naringenin by inserting die gene for chalcone synthase; the apparent inability of rice to produce this flavonoid namrally is probably due to die absence in rice of the chalcone syndiase, ChsA gene.
- a cDNA library was prepared in ⁇ gtlO using poly A+RNA isolated from flowers of the petunia (Ausubel et al., 1990 Current Protocols in Molecular Biology. Greene Publishing Associates, New York). Approximately 96,000 plaques of the library were screened for hybridisation to a full length CHS DNA clone of petunia. Hybridisation was carried out under low stringency conditions (2xSSC, 45 °C) resulting in die production of high background activity.
- Oligonucleotide mutagenesis was performed to introduce an Ncol site at the start codon and a BamHIsite just after the stop codon for the predicted amino acid sequence so that a perfect fusion could be obtained.
- the coding sequence was cloned into an expression cassette based on die 35S promoter, [The 35S promoter (from cauliflower mosaic virus) has been fully characterised and is standard in transgenic plant production (Ausubel et al., 1990)] and a 5' untranslated region of a chlorophylla/b binding protein gene of petunia (Cab22L) and die nopaline syntiiase termination region.
- This expression construct was introduced into the binary vector pAGS802, which contains a 35S-nptII-ocs construct to confer kanamycin resistance on plant tissue, a lacZ ⁇ region for ease of cloning and replication origins of ColEI and pVSP (Courtney-Gutterson et al., 1994 Bio/Technology 12: 268-271).
- the binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation.
- This procedure is designed to produce rice plant transgenic for chalcone synthase. No specific root promoter is required since a general expression of the gene in all parts of die plant is desirable to ensure production of naringenin and secretion from the plant (including the roots).
- Rice tissues (shoot apices, roots and calli derived from roots) were immersed in die bacterial suspension for several minutes and tiien transferred without rinsing on to 2N6-AS medium (all tissues except for shoot apices) or N6S3-AS medium (shoot apices), and incubated at 25 °C in darkness for 3 days. After the co-cultivation, the materials were rinsed thoroughly with 250 mg l "1 cefotaxime in sterile water and placed on 2N6-CH medium. Colonies of cells that had proliferated were plated on a regeneration medium, N6S3- CH, and incubated at 25 °C under continuous illumination (about 2000 lux).
- Regenerated plants were eventually transferred to soil in pots and grown to mamrity in a greenhouse as described by Yukoh Hiei et al., 1994.
- the regenerated rice transgenic plants (tested by Soutiiern and Northern analysis) containing and expressing die chalcone syntiiase gene were screened for secretion of naringenin using the stimulation of nitrogen fixation assay described in Example 1(a).
- OS Petunia hybrida Eukaryota;Plantae;Embryobionta; Magnoliophyta; Magnoliopsida;
- XX CC ChsA is die major expressed member of the genefamdy in various
- Exudate from seedlings or roots of plants such as the legume Sesbania rostrata can be subjected to standard purification procedures and die nod- factor inducing components separated and identified to prepare nod-factor inducing agents useful in die mediod of the invention.
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AU13946/97A AU1394697A (en) | 1996-01-19 | 1997-01-17 | Method of inducing nitrogen fixation in plants |
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GBGB9601110.1A GB9601110D0 (en) | 1996-01-19 | 1996-01-19 | Method of inducing nitrogen fixation in plants |
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Cited By (17)
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WO2000004778A1 (en) * | 1998-07-21 | 2000-02-03 | Bios Agriculture Inc. | Composition for accelerating seed germination and plant growth |
US6869773B2 (en) | 1999-12-01 | 2005-03-22 | Dsm Nutritional Products, Inc. | Process for the manufacture of carotenoids and biologically useful materials thereof |
EP1555321A1 (en) * | 2004-01-15 | 2005-07-20 | Institut National De La Recherche Agronomique | CCaMK involved in nodulation and endomycorrhization |
WO2005087005A1 (en) * | 2004-03-18 | 2005-09-22 | Agribiotics Inc. | Isoflavonoid compounds and use thereof |
US20140342904A1 (en) * | 2011-09-14 | 2014-11-20 | Novozymes Bioag A/S | Use of lipochito oligosaccharides and/or chito oligosaccharides in combination with phosphate solubilizing microorganisms to enhance plant growth |
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US11046905B2 (en) | 2018-05-03 | 2021-06-29 | Upm-Kymmene Corporation | Method for producing renewable fuels |
US20210204501A1 (en) * | 2018-05-24 | 2021-07-08 | The Regents Of The University Of California | Compositions and methods for increasing plant yield |
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WO2022015762A1 (en) * | 2020-07-13 | 2022-01-20 | The Regents Of The University Of California | Plant metabolite-mediated induction of biofilm formation in soil bacteria to increase biological nitrogen fixation and plant nitrogen assimilation |
CN114806989A (en) * | 2022-04-28 | 2022-07-29 | 中农创达(北京)环保科技有限公司 | Artificial nodule nitrogen-fixing body and construction and application thereof |
US11560340B2 (en) | 2011-09-08 | 2023-01-24 | Novozymes Bioag A/S | Seed treatment methods and compositions |
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1996
- 1996-01-19 GB GBGB9601110.1A patent/GB9601110D0/en active Pending
-
1997
- 1997-01-17 AU AU13946/97A patent/AU1394697A/en not_active Abandoned
- 1997-01-17 WO PCT/GB1997/000120 patent/WO1997026363A2/en active Application Filing
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Also Published As
Publication number | Publication date |
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GB9601110D0 (en) | 1996-03-20 |
WO1997026363A3 (en) | 1997-10-09 |
AU1394697A (en) | 1997-08-11 |
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