AU669980B2 - Transgenic plants - Google Patents

Description

1 141 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

Name of Applicant: THE UNIVERSITY OF WESTERN AUSTRALIA e~ r Actual Inventor(s): PIGEAIRE, Alix Anne Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

TRANSGENIC PLANTS Invention Title: ~s r o Details of Associated Provisional Application(s): No(s): Australian Patent Application No. PL8947/93 filed 21 May, 1993 The following statement is a full description of this invention, including the best method of performing it known to me/us:

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TRANSGENIC PLANTS The present invention relates generally to transgenic plants and methods of transforming same. More particularly, the present invention is directed to transgenic plants of the genus Lupinus. The present invention provides the means for generating genetically altered plants exhibiting a range of beneficial characteristics.

Bibliographic details of publications referred to herein are given at the end of the specification.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Agrobacterium tumefaciens induces gall formation in nature by transferring a piece of DNA (T-DNA) from its own plasmid into the plant genome. The T-DNA can be "disarmed" so that it will no longer cause tumorous growth but will still integrate into the plant chromosomal DNA. Genes conferring desired properties, for example pathogen or herbicide resistance, can be inserted into the disarmed T-DNA and thereby transferred to plants by co-cultivation of plant tissue with bacteria carrying the disarmed T-DNA. This method is critically dependent on an infection and co-cultivation method for transferring genes into plant cells, as well as an efficient regeneration system to recover plants from transformed cells.

Agrobacterium-mediated transformation systems have been developed for a number of dicotyledonous species. but no effective method for the genus Lupinus has yet been described. Lupins are an important crop in Australia, producing a high value, protein-rich grain. Lupins, therefore, represent an important cash crop with potential as a valuable source of animal and human food, world-wide. Furthermore, the use of lupins has the environmental advantage of restoring soil fertility, due to their leguminous nature.

9405 20.p:\oper\cjhupn.spe, I

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-2- There is a need, therefore, to develop an effective and efficient method for transforming Lupinus and other similar or related plants with genetic material so as to permit the generation of transgenic plants exhibiting a range of useful characteristics such as but not limited to resistance to one or more herbicides, resistance to plant diseases, improved protein quality of the seed and higher grain yield. There is a particular need to generate new modified species of Lupinus exhibiting such properties for domestic and foreign markets.

Accordingly, the present invention contemplates a method for transferring genetic material from cells of Agrobacterium to cells of a plant explant, said method comprising cocultivating a transformation-effective amount of Agrobacterium with a wounded form of said plant explant for a time and under conditions sufficient for transfer of genetic material to occur from Agrobacterium to the cells of said explant and then culturing the inoculated portion of said explant in a growth medium for a time and under conditions sufficient to allow cells to begin regeneration. In one embodiment, the growth medium is a selection medium. In another embodiment, the medium is a non-selection medium.

With regard to the latter embodiment, the present invention provides a method for transferring genetic material from cells of Agrobacterium to cells of a plant explant, said method comprising co-cultivating a transformation-effective amount of Agrobacterium 20 with a wounded form of said plant explant for a time and under conditions sufficient for transfer of genetic material to occur from Agrobacterium to the cells of said explant, culturing said co-cultured explant in a non-selection medium for a time and under conditions sufficient to allow cells to begin regeneration, wherein during culturing, one or more cells of said explant are contacted at least once with a selective agent.

The present invention further relates to a method for transferring genetic material from cells of Agrobacterium to cells of a plant explant and regenerating one or m'ore plants thereafter, said method comprising co-cultivating a transformation-effective amount of :Y Arobacterium with a wounded form of said plant explant for a time and under conditions sufficient for transfer of genetic material to occur from Agrobacterium to the ce, of said explant and then culturing the inoculated portion of said explant in a selection medium for a time and under conditions sufficient to allow cells to begin regeneration, 940520,p:\opcr\cjh,lupin.spe,2 contacting regenerating shoots with an effective amount of medium for a time and under conditions sufficient for shoots to grow and thereafter grafting regenerated shoots onto cotyledonary nodes of seedlings.

The present invention is more particularly directed to a method for transferring genetic material from cells of Agrobacterium to cells of a plant explant and regenerating one or more plants thereafter, said method comprising co-cultivating a transformation-effective amount of Agrobacterium with a wounded. form of said plant explant for a time and under conditions sufficient for transfer of genetic material to occur from Agrobacterium to the cells of said explant. culturing said co-cultured explant in a non-selection medium for a time and under conditions sufficient to allow cells to begin regeneration, wherein during culturing, one or more cells of said explant are contacted at least once with a selective agent and contacting regenerating shoots with an effective amount of medium for a time and under conditions sufficient for shoots to grow and thereafter grafting regenerated shoots onto cotyledonary nodes of seedlings.

Yet another aspect of the present invention contemplates a method for introducing genetic material containing non-Agrobacterium DNA into cells of a Lupinus explant said method comprising co-cultivating a transformation-effective amount ofAgrobacterium carrying S 20 said genetic material with a wounded form of said Lupinus explant and then culturing the inoculated portion of said explant in a selection medium for a time and under conditions sufficient to allow cells to begin regeneration and thereafter grafting the regenerated shoots onto cotyledonary nodes of seedlings.

This aspect of the present invention more particularly provides a method for introducing genetic material containing non-Agrobacterium DNA into cells of a Lupinus explant said method comprising co-cultivating a transformation-effective amount of Agrobacterium carrying said genetic material with a wounded form of said Lupinus explant, culturing said co-cultured explant in a non-selection medium until cells regenerate into shoots.

wherein during said culturing, one or more cells of said explant are contacted at least once with a selective agent and thereafter grafting the regenerated shoots onto cotyledonary nodes of seedlings.

40520.p:opchejh.luptn.spc3 -4- In these and other related aspects of the invention the preferred species of Agrobacterium is Agrobacterium tumefaciens. It is most preferably A. tumefaciens strain AGLO.

By "non-Agrobacterium DNA" is meant a genetic construct containing heterologous DNA of non-Agrobacterium origin but introduced into cells of Agrobacterium.

Preferably the selective agent is a herbicide having an active agent or ingredient se',cted from phosphinothricin. glyphosate, bromoxynil and chlorsulfuron.

In a most preferred aspect of the present invention, the selective agent is Basta® (trade mark registered in the name of Hoechst which is a commercial herbicide having phosphinothricin (otherwise known as glufosinate) as the active ingredient. Other suitable agents include Round-up® (trade mark registered in the name of Monsanto Co), Buctril® (trade mark registered in the name of Rhone Poulenc Agriculture Limited), and Glean® (trade mark registered in the name of DuPont de Nemours, e.i. Co), the active ingredients of which are glyphosate, bromoxynil and chlorsulfuron, respectively.

Preferably, the concentration of the Basta® solution added is between 1 mg/ml and mg/ml. In a more preferred embodiment the concentration is between 1 mg/ml and 3 e mg/ml. Cells of the said explant are generally contacted with discrete applications of the 20 selective agent from about 1 to about 50 times, more preferably from about 1 to about 30 times and still more preferably from about 1 to about 10 times, over the course of several weeks.

Preferably, the cells of the explant contacted by the selective agent constitute the tip of the explant.

Although the present invention extends to a method of transforming plants in general with genetic material, the preferred plants include leguminous plants such as soybean, peas, beans, clovers, alfalfa and leguminous trees and shrubs such as acacia. Particularly preferred plants are Lupinus plants and those having Lupinus plant-like properties such as but not limited to at least one or more of Lupinus angustifolius, Lupinus albus, Lupinus luteus. Lupinus cosentinii, Lupinus pilosis and Lupinus atlanticus. The most preferred 940520.p:%operejhupin.spc,4 D- I I L plant is Lupinus angustifolius.

In a preferred aspect of the present invention, explants are derived either from late immature seeds or from the shoot axis of germinating seeds. In a most preferred embodiment, the explants are derived from the shoot axis of germinating seeds. The explant may be i-,oculated withAgrobacterium immediately upon plating, or alternatively, may be pre-cultured for up to about 6 days prior to inoculation.

The methods of the present invention provide efficient and reliable means for generating transgenic plants and in particular transgenic Lupinus plants. The present invention also extends to the progeny and reproductive material (eg: seeds and seed material) of the transgenic plants.

In a preferred embodiment the transgenic plants of the present invention contain introduced genetic material which confers on the transgenic plant at least one of the following properties: resistance to one or more distinct or related herbicides, general or specific resistance to insect attack, general or specific resistance to disease-causing agents such as nematodes, viruses, fungi, bacteria and micoplasma, improved protein quality of the seed, higher grain yield, improvement of root nodule formation and function including 20 improved nitrogen fixation capacity, male sterile plants and proprietary tags (eg: colour change, specific amino acid sequence, or particular nucleotide sequence) to plants and their seeds.

There are various possible sources of genetic material which could be useful in achieving some or all of these ends. The following non-limiting examples may be usefully exploited. The use of the Cowpea trypsin inhibitor (CpTi) or Bacillus thuringiensis (BT) gene (Hoffman et al., 1992) for example, or other protease inhibitors (Gatehouse et al.) or Galanthus nivalis lectin (van Damme et al.) could confer insect resistance. The isopentenyl transferase (ipt) gene from Agrobacterium (Smigocki et al., 1993) may also be useful. This gene likewise has the potential to be useful in increasing yield (Atkins and Pigeaire. 1993), as does the 1-amino-cyclopropane-l-carboxylic acid (ACC) deaminase gene (Kende, 1993). Coat and movement protein genes from viruses when 940520p:opefr\tjhjupt

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-6transferred to plants may confer resistance in the plants to their viral source (Bol et al., 1993). Chitinase (US Patent No. 4,751,081; US Patent No. 4,940,840) and glucanase (European Patent Application No. 0 440 304) genes can be used to transfer bacterial and/or fungal resistance to plants, while nodule-specific "nodulin" genes could possibly be exploited to improve rates of nodulation.

In accordance with the methods of the present invention, the selective agent is applied directly to potentially-transformed cells of the plant explant and may be applied in any convenient and/or conventional manner, such as but not limited to, drop-wise by micropipette or other suitable applicator, use of a brush, or by spray including by use of an airbrush.

The present invention extends to transgenic plants regenerated from cells of an explant transformed with genetic material according to the methods contained, described and contemplated I erein, to their progeny and to reproductive material (eg: seeds or seed material) from such transgenic plants or from their progeny.

The present invention is further described by reference to the following non-limiting Figures and Examples.

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In the Figures: FIGURE 1 is a diagrammatic representation of the binary expression vector pCGP963 which contains the bar expression cassette consisting of the cauliflower mosaic virus promoter (35S), the bar coding region (bar) and the octopine synthase terminator (ocs).

LB left border; RB right border; Tc tetracycline resistance gene. Restriction enzyme sites are marked.

FIGURE 2 is a diagrammatic representation of the binary expression vector pCGP1258, which contains the same bar expression cassette as does pCGP963 and, in addition, the P-glucuronidase (GUS) expression cassette, consisting of the cauliflower mosaic virus promoter (35S), the GUS coding region and the nopaline synthase terminator (nos). LB 940S20,p:\apcr\ej h, Iupin.spc.6

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-7left border; RB right border; Tc tetracycline resistance gene. Restriction enzyme sites are marked.

FIGURE 3 shows an autoradiographic representation of a Southern hybridization of Unicrop T o plants transformed with pCGP963. Genomic DNA was digested with the enzyme combination BamHI/XhoI and probed with the bar gene coding region. The negative control is non-transformed Unicrop. The arrow indicates the 700bp band expected in transformed plants.

FIGURES 4A and 4B show an autoradiographic representation of a Southern hybridization of second generation T 1 plants. Genomic DNA was extracted from 45 T 1 plants (1-45) representing the total progeny of one primary Unicrop transformant, and from a non-transformed Unicrop control. The DNA was digested with the enzyme combination BamHI/XhoI which releases the 700bp bar gene coding region. The bar gene coding region from pCGP963 was used as probe. Arrows mark the 700bp product expected in the transformed plants.

FIGURE 5 shows seed progeny of transgenic lupin plants which have maintained resistance to the herbicide. Selected leaves were treated with a 1 mg/ml solution of 20 Basta® herbicide, the active ingredient of which is phosphinothricin, otherwise known as glufosinate. Arrows indicate the treated leaves. Non-transgenic control seedling: treated leaf dies within 3 days, and Leaf of transgenic seedling painted with Basta® solution shows resistance to the herbicide solution. Photos were taken 5 days after treatment.

FIGURE 6 is a cross-section of a leaflet from a cv. Unicrop T o shoot transformed with pCGP1258/AGLO, stained in the GUS histochemical assay. The blue staining of all the tissue in the cross-section indicates the presence and expression of the GUS gene in all tissues of the transformed leaflet.

940520, p:\opcr\cjh.Iupinspe,7 EXAMPLE 1 Materials Phosphothricin-ammonium (50% technical grade) was obtained from Hoechst Australia SLimited, 606 St Kilda Road, Melbourne, Australia.

The Escherichia coli K12 strains used were JM101 (Janisch-Perron et al., 1985) and (Hanahan, 1983 and BRL, 1986).

The disarmed Agrobacterium tumefaciens strain AGLO (Lazo et al., 1991) was obtained from Dr R Ludwig, Department of Biology, University of California, Santa Cruz, USA.

The Bradford reagent was obtained from Biorad.

Plants were grown in specialised growth rooms with a 12 hr day length at a light intensity of 10,000 lux minimum and a temperature of 22 to 26 0

C.

EXAMPLE 2 Construction of pCGP 963 20 The binary vector pCGP963 was derived from pGA492 (An, 1986). Between the T-DNA borders it carries a single functional gene cassette bounded by a unique PstI site and a unique EcoRI site. The PstI-EcoRI insert was derived from pSLJ 2011 (Jones et al., 1992) and contains the bar coding region encoding the phosphinothricin acetyltransferase from Streptomyces hygroscopicus controlled by the CaMV 35S promoter and an ocs (octopine synthase) terminator.

o*o EXAMPLE 3 Construction of pCGP 1258 The binary vector pCGP1258 was derived from pCGP963 by the addition of a marker gene cassette, 35S-GUS-nos, adjacent to the right border. The 2.5kb fragment originated from an EcoRI/HindlII/Scal digest of pJB 1 (obtained from Dr J.P Bodeau, Department of Biological Sciences, Stanford University, California, USA) which was 940520.p:\opcr\cjh.,upin,spc.8

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-9subcloned into pBluescript. Digestion with PstI released the 2.5 kb cassette which was then sub-cloned into the PstI site of pCGP963 to yield pCGP1258.

EXAMPLE 4 Transformation of E. coli and A. tumefaciens Transformation of the Escherichia coli K12 strains JM101 and DH5c with vectors pCGP963 and pCGP1258, respectively, was performed according to standard procedures (Sambrook et al., 1989). Escherichia coli JM101 and DH5a cells were made competent by the methods of Sambrook et al., (1989) and Inoue et al., (1990), respectively.

The vectors pCGP963 and pCGP1258 were introduced into the Agrobacterium tume/aciens strain AGLO by adding 5 Ag of plasmid DNA to 100 Al of competent Agrobacterium tumefaciens cells prepared by inoculating a 50 ml MG/L (Garfinkel and Nester, 1980) culture and growing for 16 h with shaking at 28 0 C. The cells were then pelleted and resuspended in 0.5 ml of 85% 100 mM CaCl 2 /15% glycerol. The DNA-Agrobacterium mixture was frozen by incubation in liquid N 2 for 2 min and then allowed to thaw by incubation at 37 0 C for 5 min. The DNA/bacterial mixture was then placed on ice for a further 10 min. The cells were then mixed with 1 ml of MG/L media.

S: incubated for 4 h at 28 0 C, centrifuged to pellet cells and plated on solid MG/L selection S 20 media.

Transformants carrying pCGP963 or pCGP 1258 were selected under tetracycline selection at 50 gg/ml. The presence of the plasmid was confirmed by restriction mapping of DNA isolated from the tetracycline-resistant transformants.

EXAMPLE Transformation of Lupinus angustifolius a. Plant Material Seeds of Lupinus angustifolius (cv Unicrop, Merrit and 85L:482) were obtained from the Western Australian Department of Agriculture, South Perth, Western Australia (Dr Wallace Cowling). The seeds were sterilized with 2.5% sodium hypochlorite (with Triton X100) for 12 min. then with 70% ethanol for 1.5 min and then rinsed three 94052O.p;\o pr\ejh.Iupin.spe.9 L_ 10 times with sterile water. To induce germination, the seeds were put into petri dishes on moistened filter paper for 24 hours. Whole shoot axes were excised from germinating seeds. The two pair of leaves present in the plumule were removed. The apical dome and primordia of the third pair of leaves were damaged by wounding with a 30 gauge Sneedle. The shoot axis explants were plated upright on Murashige and Skoog's (1962) medium (MS) supplemented with B5 vitamins, 5 mg/1 benzylaminopurine (BAP), mg/1 ac-napthalene acetic acid (NAA) and solidified with 0.25% gelrite (pH 5.7).

The concentrations of BAP and NAA may be increased two-fold without reducing success.

b. Co-cultivation of Agrobacterium and Lupinus tissue A4grohacterium tumefaciens strain AGLO (Lazo et al., 1991), containing either of the binary vectors pCGP963 or pCGP1258, was maintained in a liquid culture of MG/L with Ag/ml tetracycline and 15% glycerol stored at -80 0 C. An aliquot was grown overnight at 28°C in liquid MG/L broth with 50 jg/ml tetracycline, and diluted to 5 x 108 cells/ml next day before inoculation. A drop of Agrobacterium was placed on top of the shoot apex on each explant. The explant may be inoculated immediately upon plating or alternatively, may be pre-cultured for up to about 6 days prior to inoculation. The :explants were co-cultivated with Agrobacterium for 2 days.

S c. Recovery of transgenic Lupinus plants After co-cultivation, a drop of 2 mg/ml Basta® (commercial herbicide, the active ingredient of which is phosphinothricin, otherwise known as glufosinate) was applied to the tip of the explant and then sucked back into the tip of the pipette so as to leave a wet film over the shoot apex. Alternatively, an air brush can be used to spray the top of the explant with the Basta® solution. In addition, the concentration of the solution may be varied between about I mg/ml and about ,1 mg/ml. In most cases tested a concentration of between about 1 mg/ml and about 3 A, na gave an optimal effect.

The explants were transferred to MS medium supplemented with B5 vitamins, mg/l BAP, 0.5 mg/1 NAA 150 mg/l timentin, solidified with 0.25% gelrite (pH Again, the concentrations of BAP and NAA are not critical; I mg/I BAP, and 0.1 940520.p\opcrkejh,Iupin.sp.,

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11mg/l NAA was also effective. After 3 weeks, the shoots and shoot clumps from the explants were transferred to MS medium supplemented with 0.1 mg/1 BAP, 0.01 mg/I NAA, 150 mg/1 timentin and 20 mg/l phosphinothricin.

Alternatively, the explants may be treated with Basta® several times over the first 3 week period, following which individual shoots are removed and transferred directly to MS medium supplemented with 0.1 mg/1 BAP, 0.01 mg/l NAA, 150 mg/I timentin and mg/l phosphinothricin.

Individual green shoots were then repeatedly transferred to fresh plates of MS medium supplemented with 0.05 mg/1 BAP, 0.005 mg/I NAA, 150 mg/1 timentin and 20 mg/1 phosphinothricin, at two-week intervals until they started micropropagating and were large enough to be taken out of tissue culture 8-10 transfers). Shoots were grafted onto the cotyledonary node of 10-14 day old seedlings.

A "leaf' assay, involving painting the tissue culture leaflets with phosphinothricin at 2g/l, the GUS histochemical assay (Jefferson, 1987) and/or Southern blot analysis (see Figure was used to confirm the transgenic nature of the T o plant material in tissue culture.

Using the above method, or minor variations as indicated or as would be apparent to the skilled technician, phosphinothricin-resistant lupin shoots were repeatedly obtained, with a frequency of from 0% to at least about 6% and, in some circumstances, from 0.6% to at least about 2%.

Leaves from cv Unicrop T o shoot, transformed with pCGP1258/AGLO, were stained in the GUS assay. A cross-section of a leaflet may be seen in Figure 6. The blue staining indicates the presence and expression of the GUS gene n all tissues of the transformed leaflet.

While the above methods have been used to transform three cutivars of Lupinus angustifolius. comparable methods may be used for the transformation of other Lupinus or Lupinus-related species such as: Lupinus albus, Lupinus luteus; Lupinus cosentinii; '14052O,p:\opcr\ch.lupin spc,I I I- L LL I- L__ 12 Lupinus pilosis and Lupinus atlanticus.

EXAMPLE 6 Analysis of Second Generation (T 1 The grafted plants were grown in the glasshouse until they set seeds and these matured, which took about 5 months from the time of grafting. Approximately 40-50 seeds per plant were generated when the plants were grown over the winter months, the normal cropping season for lupins. During the summer months about 1-5 seeds were produced per plant.

The T, seedlings were grown in pots in the glasshouse. After their first pair of leaves unfolded, one leaflet of each seedling was challenged with a solution of Basta® at lg/1.

Leaves of normal, untransformed plants die within 3 days of the challenge, whereas leaves of transformed seedlings demonstrated resistance to the herbicide and were still 15 viable after 5 days (Figure Leaf material was also collected from the T 1 seedlings for DNA extraction, Southern blotting and phosphinothricin-acetyltransferase assays. The second generation T 1 plants inherited the transgene, as can be seen on Figures 4A and 4B.

S: EXAMPLE 7 Southern Analysis a. Preparation of genomic DNA DNA was extracted by grinding tissue in the presence of liquid N 2 in a mortar and pestle and adding Iml of extraction buffer (0.14 M sorbitol, 0.22 M Tris-HCl [pH8.0], 0.022 M EDTA. 0.8 M NaCl. 0.8% CTAB, 1% N-laurylsarcosine) heated at 65 0

C.

Chloroform (200tl) was added and the mixture incubated at 65 0 C for 15 min. Following centrifugation, the supernatant was phenol-chloroform extracted and then added to an equal volume of isopropanol, inverting to mix. This mixture was centrifuged and the pellet washed with 95% ethanol, re-centrifuged and washed with 70% ethanol.

The pellet was vacuum-dried and resuspended in 3 0p TE buffer (pH )940520,p:opcr\cjh,lupin.spe, 12 13 b. Southern blots Lupin genomic DNA was digested for 16 h with 6 units//g of appropriate restriction enzyme and electrophoresed through a 0.7% agarose gel in a running buffer of TAE mM Tris-acetate, 50 mM EDTA). The gel was then washed in 0.5 M HCI for min. DNA was denatured in denaturing solution (1.5 M NaCl/0.5 M NaOH) for 40 min, neutralized in 0.5 M Tris-HCI (pH 1 M NaCl for 30 min, and then rinsed with 2 x SSC. The DNA was then transferred to a Hybond N (Amersham) filter in 20 x SSC, and the DNA was fixed to the filter by UV irradiation for 5 min.

c. 32 P-labelling of DNA probes The 700 bp bar coding region from pCGP963 was used as probe in the Southern analyses. DNA fragments (100 ng) were radioactively labelled with 50 .zCi of [c- 32

P]-

dCTP using an oligolabelling kit (Bresatec). Unincorporated [a- 32 P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine) column.

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Southern analysis of putative transgenic lupin plants with the bar gene probe confirmed the transgenic nature of the phosphinothricin-resistant shoots.

EXAMPLE 8 Phosphinothricin-acctyltransferase Enzyme Assay Phosphinothricin-acetyltransferase enzyme activity was assayed from crude extracts of leaf tissue from transformed tissue culture plants. Assays were performed according to the method of De Block et al. (1987), with the following modifications.

Approximately 100-150mg of fresh tissue was ground in 100-150 1 of extraction buffer and centrifuged at 4 0 C for 20 min. The protein content of the supernatent was quantified using Bradfords reagent (Bradford, 1976) and adjusted so as to use 25 and 50Ag of protein in the reactions. The extracts were incubated with phosphinothricin/extraction buffer/ 1 4 C acetyl Co-A mix for 2 h at 37 0 C and spotted onto silica gel 60 TLC plates (Merck). Chromotography was carried out in a solvent system using a ratio of 3:2 of propan-l-ol to ammonium hydroxide. Acetylated phosphinothricin can be visualized by autoradiography using Kodak XAR film after a one week exposure.

14O452Op.'operejh.upin.spc. 13 L I__ 14- Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

**ee 940520.p:\opcr\cjhjupin.sp, 1 4 ~esPI 15

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Claims (17)

17- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method for transferring genetic material from cells of Agrobacterium to cells of a Lupinus or Lupinus-related plant explant, said method comprising co-cultivating a transformation-effective amount of Agrobacterium with a wounded form of said plant explant for a time and under conditions sufficient for transfer of genetic material to occur from Agrobacterium to the cells of said explant, culturing said co-cultured explant or part or portion thereof in a growth medium for a time and under conditions sufficient to allow cells to begin regeneration to form shoots. 2. A method according to claim 1 wherein the growth medium is a selection medium. 3. A method according to claim 1 wherein the growth medium is a non-selection medium and wherein during culturing, one or more cells of said explant are contacted at least once with a selective agent. 4. A method according to any one of claims 1 to 3 wherein the said part or portion is that part or portion which has been inoculated with Agrobacterium 5. A method according to any one of claims 1 to 4 further comprising contacting the regenerating shoots with an effective amount of a medium for a time and under conditions sufficient for the shoots to grow and thereafter grafting regenerated shoots onto cotyledonary nodes of seedlings. S. 6. A method according to any one of claims 1 to 5 wherein the plant is a leguminous S* plant. 7. A method according to claim 6 wherein the leguminous plant is a species of Lupinus. 94O52Op:\opcr\cjh,Iupin.spe, 17 -i 18 8. A method according to claim 7 wherein the Lupinus species is selected from Lupinus angustifolius, Lupinus albus, Lupinus luteus, Lupinus cosentinii, Lupinuspilosus and Lupinus atlanticus. 9. A method according to claim 7 wherein the species of Lupinus is Lupinus ungustifolils. A method according to any one of claims 1 to 9 wherein the selective agent is a herbicide having an active ingredient selected from phosphinothricin, glufesinate- glyphosate, bromoxynil and chlorsulfuron. 11. A method according to claim 10 wherein the selective agent is a herbicide having phosphinothricin as active ingredient. 12. A method according to any one of the preceding claims whereIn th.. lant explants are derived from late immature seeds. 13. A method according to any one of claims 1 to 11 wherein the plant explants are derived from the shoot axis of germinating seeds. S: 14. A method according to claim 13 wherein the explant is inoculated with .grobacterium immediately upon plating. A method according to claim 13 wherein the explant is pre-cultured for up to about 6 days prior to inoculation. 16. A method according to any one of the preceding claims wherein the genetic material from Agrobacterium comprises material of non-Agrobacterium origin and is capable of conferring in a transgenic plant one or more of the following properties selected from resistance to a herbicide, resistance to insect attack, resistance to a pathogen, improved seed protein quality, higher grain yield, improved root nodule 940520,p:\opcr\ejh,lupin.spc, 18 -1_ 19 formation and improved nitrogen fixation capacity. 17. A method according to claim 16 wherein the pathogen is selected from nematodes, viruses, fungi, bacteria and mycoplasma.

18. A method for introducing genetic material containing non-Agrobacterium DNA into cells of a Lupinus explant said method comprising co-cultivating a transformation- effective amount of Agrobacterium carrying said genetic material with a wounded form of said Lupinus explant, culturing said co-cultured explant in a growth medium until cells regenerate into shoots.

19. A method according to claim 18 wherein the growth medium is a selection medium.

20. A method according to claim 18 wherein the growth medium is a non-selection medium and wherein during said culturing one or more cells of said explant are contacted at least once with a selective agent.

21. A method according to claim 18, 19 or 20 wherein the species of Lupinus is selected from Lupinus angustifolius, Lupinus albus, Lupinus luteus, Lupinus cosentinii, Lupinus pilosus and Lupinus atlanticus.

22. A method according to claim 21 wherein the species of Lupinus is Lupinus angustifolius.

23. A method according to any one of claims 18 to 22 wherein the selective agent is a herbicide having an active ingredient selected from phosphinothricin, -gkfes-inate- glyphosate, bromoxynil and chlorsulfuron.

24. A method according to claim 23 wherein the selective agent is a herbicide having phosphinothricin as active ingredient. 94OSO,p:opcr\ejh.lupin.spe. 19 I I g 20 A method according to any one of claims 18 to 24 wherein the plant explants are derived from late immature seeds.

26. A method according to any one of claims 18 to 24 wherein the plant explants are derived from the shoot axis of germinating seeds.

27. A method according to claim 26 wherein the explant is inoculated with Agrobacterium immediately upon plating.

28. A method according to claim 26 wherein the explant is pre-cultured for up to about 6 days prior to inoculation.

29. A method according to any one of claims 18 to 28 wherein the genetic material is capable of conferring in a transgenic plant a property selected from resistance to a herbicide, resistance to insect attack, resistance to a pathogen, improved seed protein :i quality, higher grain yield, improved root nodule formation and improved nitrogen fixation capacity. A method according to claim 29 wherein the pathogen is selected from nematodes, viruses, fungi, bacteria and mycoplasma.

31. A transgenic plant regenerated from cells of an explant transformed with genetic material according to the method of any one of the preceding claims or a progeny plant thereof.

32. Reproductive material of the transgenic plant of claim 31 or a progeny plant thereof.

33. Reproductive material according to claim 32 in the form of seeds. 940520,p:\opcr\cjh.lupin.spc,20 I_ -21

34. A method according to any one of claims 1 to 30 or a transgenic plant according to claim 31 or reproductive material according to claim 32 or 33, substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 20th day of May, 1994 THE UNIVERSITY OF WESTERN AUSTRALIA By Its Patent Attorneys DAVIES COLLISON CAVE ee o ee *11 *"1 )405.0,p \opcr\ejh.upaspc,21 -22 ABSTRACT The present invention relates generally to transgenic plants and methods of transforming same. More particularly, the present invention is directed to transgenic plants of the genus Lupinus. The present invention provides the means for generating genetically altered plants exhibiting a range of beneficial characteristics. e ee

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