AU727616B2 - Method for producing polyploid plants, and polyploid plants - Google Patents

Description

lhr (File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz Description Method for Producin Polyploid Plants, and Polvploid Plants The present invention concerns a method for producing polyploid plants in accordance with the preamble of claim 1 and a polyploid plant in accordance with claim 13.

As a rule, somatic cells of animals and of many plants have a double set of chromosomes. These organisms are referred to as being diploid.

Numerous culture plants such as oats, wheat or potatoes, however, present doubled or multiplied sets of chromosomes (polyploidy). Thus diploid germ cells may be created where reduction division does not take place in meiosis, with the fusion of these diploid germ cells then resulting, in tetraploid cells.

In particular, polyploid plants are frequently also encountered in ornamentals. Thus it is e.g. known of the ornamental plant Primula malacoides, the wild form of which originates in China, that following successful ploidisation and subsequent modification by conventional breeding techniques, the occurring varieties were beyond expectation.

In order to obtain polyploid plants, the development of mitosis in the anaphase has in the past been influenced by impairing the functionality of the spindle fibers through mitotic poisons. Thus e.g. colchicine is known to inhibit the migration of chromosomes to the cell poles, possibly resulting in the formation of tetraploid cells.

In plant breeding this kind of effect is frequently desired because polyploid nuclei having a larger size than diploid ones will bring about larger cells, tissue and organs of the plants in accordance with the nucleus-cytoplasm relation. Namely, it has been found that polyploid plants, in particular polyploid culture plants, often have a higher yield than the respective wild types of a plant species.

Seite 1 (j r I ^e [File:ANM\GA230382.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang GArditz As a rule, tetraploid forms can perform better, however in certain cases other ploidies may have an even higher yield.

This is true for several culture plants; thus, the hexaploid form of wheat or the triploid stage of some sugar beets, and in various apple trees.

In DE-PS-34 23 207, production of a tetraploid camomile of the camomile culture plant species Chamomilla recutita Rauschert, is described.

Starting out from a diploid camomile, tetraploidisation is performed in the prior art in accordance with DE-PS-34 23 207 by means of chemicals at temperatures between 00 and 35°C, by means of gamma rays, X-rays or UV-rays at temperatures between 0° and 35°C, by means of high temperatures from 330 to 50°C, by means of lower temperatures of 0° to by means of the decapitation-callus technique, or by anther culture.

In chemical polyploidisation, besides colchicine a variety of highly toxic compounds such as nicotine or mercury organic compounds are utilised.

Details of the mechanism in terms of molecular biology are as yet largely not understood.

Among the worldwide most significant culture plants in the agricultural range there are wheat, rice, maize/corn, cassava/manioc, millet/sorghum and the soybean.

The soybean was, and is, of particular importance for human nutrition' in the Far-East region. Since World War II, it has experienced particular appreciation in the U.S.A. and is nowadays one of the most important export goods. This is founded particularly in its high oil/protein content suited particularly for supplying the animal feed market as well as the foodstuffs market and the non-food area.

Seite 2 i h [File:ANM\GA2303B2.DOC] March 6,1998 j ll Tetraploid Lupine Dr. Wolfgang Garditz] l In the European region, cultivation of the soybean is gaining importance for human and animal nutrition in those climatic regions which are suited for growing corn and wine.

Inasmuch as the soybean is not compatible with the European climate, and owing to the circumstance that the soybean is a so-called short-day plant and cannot normally be cultivated on a large scale in Europe, the ever increasing demand for soybeans must be satisfied by imports. The major part of its demand is covered by the EU through imports from the U.S.A. and Brazil.

Here another plant, namely the sweet lupine, might contribute to providing relief. In the past, lupine species were cultivated as protein supplying plants, particularly Lupinus albus in France and Poland, L. luteus in Eastern Europe, and L. angustifolius in semi-arid regions. With the exception of the angustifoliate lupine in Western Australia, they attained limited economic importance. Novel lupine breeds were for the first time bred by the inventor of the present application and were even then named "Europe's soybean" by the press owing to the good harvest index of the topless types, and these satisfied the criteria of a largely economic culture plant: sweet, short-stalked, day-neutral, and limited growth breeds.

The following is a short survey of the biology of the white lupine.

Taxonomically speaking, L. albus belongs to the family of papilionaceous plants (Papilionaceae, Leguminosae).

The diploid form of the white lupine (Lupinus albus is an annual, large-grain leguminous plant. It stores considerable amounts of protein and oil in the seeds. In cultivation as a feed plant, it attains a high protein yield per surface unit when cut twice a year.

The growth height of the plant, depending on the specific line, is 41i between 50 cm for modern, day-neutral cereal lines and about 100 cm as a pure forage crop.

-Seite 3 Seite -3 [File:ANM\GA2303B2.DOC] March 6,1998 lU Tetraploid Lupine Dr. Wolfgang Grditz The thousand grain weight (TGW) is approx. 300 g for early-maturing, small-grain types and approx. 400 g for lines reaching maturity later.

Apart from the lupine species L. luteus and L. angustifolius, Lupinus albus was further developed into a culture plant at the end of the 1920's by selection of plants low in alkaloids.

The white lupine derives from the gene center of the Mediterranean area. Its cultivation in the eastern Mediterranean was known as early as since classical times. In contrast with the other old-world lupine species, wild forms of the white lupine constituting direct ancestors are not known.

Modification of the white lupine by breeding was only carried out sporadically during the past 50 years. Owing to strong variations in the yield performance it hardly attained any economic importance. Nowadays, however, breeds are available which have a better capacity of competing with comparable economic plants as highly nutritious food and fodder plants, but also as a source of replenishable resources.

Such breeds have a higher self-fertility and enable a shorter sequence in the cropping rotation. Apart from pure protein or oil breeds there exist combination types which, at 35% to. 37% of raw protein, store an approximate average of 12-14% of raw fat in the grain.

The total nutrient values of the lupine are at 815 higher than those of vegetable pea, bean and animal feed barley.

Culture-technical problems with cultivation usually do not exist any.

more at the present age.

The grains by themselves, as a 15-% to 20-% admixture to the staple fodder, are a protein source of high biological value. Other than in the soybean, trypsin-inhibiting substances do not exist. This property allows for.- direct utilisation in agricultural facilities by the producer without additional costs for toasting.

Seite 4 '"roFiIe:ANM\GA230382.DOC] March 6,1998 H Tetraploid Lupine Dr. Wolfgang Garditz When considered under ecological aspects, all lupines have a number of desirable properties. They are suited for loosening the narrow rotation sequences in cereal/root crop agriculture, they activate the soil life, contribute to soil recovery by reducing the infection pressure and have, besides the activation of potassium, a particularly high capacity of exploiting phosphates for nutrients.

Owing to the great depth of rooting and because they improve the buffer capacity of the soil in the root area, they have a better effect as a preparatory crop on light soils than the root crops.

As they do not constitute a strain on the environment in combination with suitable intermediate and follow-up crops, they are suited for concentrated cultivation as main crop plants. Locally adapted rhizobia strains which are known to live in symbiosis with lupines fix up to 360 kg of nitrogen per hectare and year. They thus cover 95% of the nitrogen demand of the plant. A slow, but steadily flowing source of nitrogen is then available for the following crop.

As a plant for replenishable resources, the white lupine suppliesvaluable fatty acids and a broad range of amino acids. In particular the range of amino acids in its composition very much resembles the one of the soybean (Glycine max.).

As regards the composition of the raw fat content (fatty acid methyl ester and fatty acids), the monounsaturated fatty acids predominate in the lupine over the polyunsaturated fatty acids. In the lipids of the soybean, the polyunsaturated fatty acids predominate.

In comparison with other plants that are rich in oil and protein, the diploid white lupine, together with the soybean, combines the highest total contents of raw fat and raw protein in the seed.

In practical application, difficulties and (constant) problems have in the past always been created by the extensive propagation culture of the p arial cross-fertilisers, particularly with respect to acceptable alkaloid AIVT 0- Seite 5 If, S [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine

HII

Dr. Wolfgang Garditz levels, stabilisation of desired oil contents and most recently susceptibility to some pathogenic fungi.

The true cause for these deficits must be seen in the much too narrow genetic base of an economic plant whose origins rely too heavily on the "Sengbusch material" of the 1930's.

Starting out from the above described prior art, it is therefore an object of the present invention to provide novel, improved polyploid plants, particularly lupine plants.

This object is attained in terms of method technology by the characterising features of of claim 1.

With respect to novel polypolid plant species, in particular lupine species, the object is attained by the characterising features of claim 13 and the propagation material according to claim 22.

The plants in accordance with the invention have a genome which does not contain any genes of foreign species -for ecological safety cannot interact with any other plant in terms of biological information -only includes recessive linkage groups with respect to bitter principles owing to the at least four physiologically active alleles is capable of summing the important performance factors ingredients, resistances, stress stabilities) as concerns the gene dosage effect following a corresponding modification by breeding, and -has sufficient genetic plasticity to enable the development of.

further economic plant species through the addition of peripheral genomes.

In accordance with the method of the invention, protoplasts are obtained from cells of diploid plants. Obtaining such protoplasts is performed by means of techniques known per se by enzymatically R/removing the cell wall of the starting plant cells, which essentially contains I[c ellulose. Following purification of the protoplasts these are fusioned and Seite 6a 4 i, [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz even-numbered multiple protoplasts, preferably twin protoplasts, are isolated, i.e. those protoplasts engendered by fusion of two protoplasts.

Subsequently, ovaries of a receiver plant which have the capacity of conceiving are opened, and the isolated multiple protoplasts are introduced into the embryo sack.

This requires a certain skill as the desired protoplasts are transplanted into the embryo sack of the receiver plant for example by means of a glass capillary under the binocular, in which process the egg cell complex is squeezed more or less strongly.

Following transfer of the protoplasts into the embryo sack of the receiver plant, the ovary is closed again.

The stigma of the receiver ovary is dusted with mature pollen (mentor pollen) of a plant of a foreign species in order to stimulate the metabolism within the ovary.

Subsequently the polyploid plant embryo is allowed to grow into agerminable seed and following maturing of the seed the seed is allowed to germinate, whereby a polyploid plant is obtained.

By the described method it is possible to obtain, for example a novel species of polyploid culture plants for which the Name "European lupine", "Lupinus europaeus" is herewith being proposed.

The plant species or lupine species in accordance with the invention are a reproductively isolated, autopolyploid oil and protein plant. The plants obtained in accordance with the invention satisfy all the criteria which are demanded by taxonomy for the term "species" in distinction from the term "variety".

For the purposes of the present invention, a species is understood in the following terms: Seite 7

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(File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz Species are groups of actually or potentially propagating natural populations which are reproductively isolated from other such groups, or the largest and most comprehensive propagation community of sexual and cross-fertilising individuals pertaining to a common gene pool (cf. Siewing, "Evolution", Fischer-Verlag, Stuttgart, 1982, p. 284).

Accordingly the polyploid plants, particularly lupines, in a preferred manner form tetraploid lupines, form a novel species in accordance with the present invention inasmuch as they cannot be fertilely cross-bred with the natural diploid plants and thus, in the sense of the above definition, are reproductively isolated from other groups of plants.

Moreover, for example the tetraploide lupine species according to the invention has a tetraploid (4n) genome including approx. 100 chromosomes. A free gene flow exists only within this genome, and recombination of the genes is only possible within this gene pool.

In particular for the lupine of the invention the inventor proposes, as was mentioned above, the species name "European lupine", "Lupinus europaeus".

Other than a species, individuals which differ in at least one, but the same feature from other individuals of their species form a variety (of animals and plants) of this species.

A more precise definition of the variety term can be found in Art. 1 vi) of the International Convention for the Protection of New Varieties of Plants (UPOV) in the version of March 19, 1991 revised in Geneva.

Accordingly, a variety is: A plant grouping within a single botanical taxon of the lowest known rank, which can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, distinguished from any other plant grouping by the S expression of at least one of the said characteristics, and I A, 1 [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz considered as a unit with regard to its suitability for being propagated unchanged.

As the above discussion of the species term shows, however, the polyploid plants in accordance with the invention, particularly tetraploid lupines, are not covered by the variety term which they transcend by taxonomical definition, but form a novel species.

In particular, conventional lupine species have a diploid (2n) genome including e.g. approximately 50 chromosomes in varieties of Lupinus albus.

A fertile, recombinative gene exchange between species having 2n genomes and species having 4n genomes is not possible in a natural way.

The novel plants, particularly lupine plants, present the following features: Physiological features: Better growth, a substantially larger assimilation surface than plants having the starting genome, higher biomass productivity, hybrid enzymes increase the ingredient substances such as oil and raw protein content.

Moreover in accordance with previous observations, the polyploid plants in accordance with claim 13 present a higher resistance against pathogens.

Ecological features: The polyploid plants, particularly polyploid lupines, present a higher stress stability against temperature, aridity and salt content of the groundand have as we also know of other polyploid plant species the ability of settling new habitats. Thus the proportion of polyploids among other florae in central Europe is approx. 50%, however more than 76% in Spitzbergen.

In addition, the polyploid plants of the present invention offer a realistic genetic basis for the development of a perennial lupine.

Genetic features: [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz Owing to the polyploidy, there exists thanks to the higher-thanaverage number of chromosomes a possibility of considerably expanding variability to theoretically more than, 3 50 genotypes. When considering a natural mutation rate and upon induction of chromosome structure mutations, the potential of possible genotypes certainly cannot be exhausted in the foreseeable future. A high phenotypical and physiological variation may be expected in a few generations' time.

In addition, valuable breeding material for developing interspecific hybrids as well as for establishing addition and substitution lines is available through the polyploid plants, particularly polyploid lupines, of the present invention.

Being partially and facultatively cross-fertilising, this genome in lupines guarantees high stability but also plasticity of the population's gene pool.

There is moreover a lasting heterosis effect due to the high heterozygosity which theoretically amounts to 99.7%. Homozygous lines can hardly be developed by conventional methods. This would hold much work in store for biotechnology reproduction techniques would moreover have to be developed from gametes and for molecular biology (physical and physiological genetic maps and genetic probes would have to be developed for identifying homozygote genomes).

Moreover the genome of the polyploid plants of the present invention may serve as a starting genome for developing novel allopolyploid plants.

It is conceivable to add a suitable foreign genome to the tetraploid europaeus genome in analogy to the creation of the club wheat in eras of cultural history.

Thus a tetra-wheat of the Emmer series spontaneously added a diploid Aegilops genome. The present-day hexaploid genomes of wheat, the world i economy plant, have been developed from the latter.

Seite 10 0-1 [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz Pathogenic populations have hitherto not developed from the new genome. In this sense it would be a worthwhile task to keep an eye on phytosanitary prophylaxis.

In addition maximum ecological safety is provided for insertion of transgenic linkage groups inasmuch as reproductive isolation exists and wild forms of the polyploid plants, particularly lupines, of the invention do not exist.

The phenomenon of genes of foreign species "turning rampant", which was ascertained in Denmark for transgenic rapeseed'genomes, could never occur in the postulated transgenic genomes of L. europaeus.

In particular in the case of lupines the genome can be utilised entirely or partially as a genetic component if tetraploid albus genomes were to be developed subsequently from other diploid ones.

In the same way the entire biomass, the grain yield and all of the secondary metabolic products such as amino acids, vitamins, enzymes, etc. may be employed for most variegated purposes in terms ofbiochemistry and nutritional physiology because the oils of the lupines of the present invention may also be utilised as starting materials for the conversion technology. There is also the possibility of utilising the polyploid plants according to the invention, particularly lupines, as replenishable resources.

It is conceivable to aspire the same width of industrial use as in the case of the soy bean until recently in more than 20,000 product types and to learn from the experiences gained there.

In the case of lupines, particularly tetraploid lupines in accordance with the present invention, it is also sensible to settle colonies of bumblebees in the vicinity of the lupine fields inasmuch as it was found that bumblebees visit the novel lupines at the same frequency as the old lupine species to thereby intensify fructification. In this context, reference is made to the dissertation by Wolfgang D. Garditz: "Die Bedeutung der ,^RA fieimischen Hummeln and Bienen (Apoideae) for die Ertragsstruktur der \i Seite 11- 4 [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz WeilSen Lupine (Lupinus albus Dissertation am Lehrstuhl for Biogeographie, Abteilung Agrar6kologie der Universitat Bayreuth [The importance of native bumblebees and honeybees (Apoideae) for the yield structure of the white lupine (Lupinus albus dissertation at the Faculty for Biogeography, Dept. for Agricultural Ecology of Bayreuth University,] 1989). This dissertation is herewith fully incorporated by way of reference.

Obtaining cells from lupines in accordance with claim 2, particularly cells from Lupinus albus in a preferred manner from the C17/8 line, has the advantage that a lupine culture plant is available which hitherto did not exist in nature and which meets the taxonomical criteria of a species when starting out from these lupine cells.

Use of the lupine line C17/8 moreover has the advantage that it is publicly available in the gene bank at Gatersleben (Institut fOr Pflanzengenetik; Genbank, Corrensstr. 3, 06466 Gatersleben, Federal Republic of Germany) and has already been described in literature, for example in Wolfgang D. Garditz: "Die Bedeutung der heimischen Hummeln und Bienen (Apoideae) fir die Ertragsstruktur der WeiBen Lupine (Lupinus albus (Dissertation am Lehrstuhl fCr Biogeographie, Abteilung Agrar6kologie of Bayreuth University, 1989) and is therefore available to the public.

The method according to claim 3 has the advantage that, when tetraploid lupines are produced, it was found that they can be stabilised genetically upon suitable processing. By the presently described method it is, however, also possible to develop higher ploidies (cf. Figure 5) which might, depending on the circumstances, develop higher yields and an inestimable variability.

The use of apical meristems in accordance with claim 4 has the advantage that apical meristems are easily accessible for the implementation of the described method and that plants can moreover be regeneriated from genetically omnipotent and, virus-free cells.

Seite 12 I i. 1 0-, [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz The measuresof claim 5 have the advantage that a routine technique for removal of the cell walls and for obtaining the protoplasts may be employed without major modification.

Using density gradients of Ficoll and/or cane sugar for isolating the protoplasts in accordance with claim 6 has the advantage that in this way an etablished technique of separation and purification is available.

Performing protoplast fusion by means of a polyethylene glycol in accordance with claim 7, particularly one having a molecular mass of approx. 1550 Da, has the advantage that hereby a safe technique for the fusion of single protoplasts is available and that the fusion medium is inexpensive and commercially available.

The measures of claim 8, namely splitting and enriching fusioned protoplasts into twin and multiple protoplasts by centrifugation over a density gradient, particularly a Ficoll and/or cane sugar density gradient, has the advantage that hereby a customary technique is available for isolating the desired fusion products, the twin protoplasts.

In accordance with claim 9 it is preferred to introduce twin protoplasts into the embryo sack as this results in the tetraploid plants which are generally preferred.

In accordance with claim 10, the cut on the treated ovary is closed up with lanolin, wherein the lanolin preferably includes an admixture of a broad range antibiotic and/or antimycotic. This has the advantage of largely avoding infections of the injured plant to increase the chances of success of the plant surgery.

This equally applies to the measures of claim 11.

After approx. 24 to 48 hours all non-transplanted ovaries of the inflorescence are removed in accordance with claim 12, which has the advantage that only the transplanted ovaries receive optimum support by 4s the plant, a fact that equally improves the chances of success of the -described method.

Seite 13- A [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz Claims 13 to 18 concern the polyploid plants obtainable in accordance with the described method, particularly lupines, and claims 19 to 21 concern the propagating material, particularly the seed grains and meristem cells of the polyploid plant according to the invention, particularly of the polyploid lupine.

Further advantages and features of the present invention are described by way of embodiments and by referring to the drawing, wherein: Fig. 1 shows a photograph of the central 3rd) leaflet, or pinna, of the fifth foliage leaf on the main sprout of the following lupine species (the order proceeding from the left to the right): L. angustifolius angustifoliate lupin L. europaeus tetraploid lupine of the invention L. albus white lupin size comparison: matchstick; Fig. 2 shows a photograph of the last foliage leaf of the vegetative stage of (clockwise, from the top left): L. angustifolius, tetraploid lupine according to the invention Lupinus albus Lupinus mutabilis Lupinus angustifolius size comparison: matchstick; Fig. 3. shows photographs of the blossoms of (from left to right): L. angustifolius tetraploid lupine of the invention L. albus, size comparison: coin; [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang GArditz Fig. 4 shows a photograph of the third foliage leaf of the tetraploid lupine of the invention on the main sprout, size comparison: matchstick; Fig. 5 shows a photograph of a leaf of an octoploid lupine in accordance with the invention having a particular morphology, size comparison: fly (Sarcophaga carnaria) Fig. 6 shows a histogram explaining the determination of the ploidies of a sample carried out on a lupine in accordance with the invention; Fig. 7 shows a histogram for determination of the ploidies of a sample of a lupine in accordance with the invention; Fig. 8 shows a histogram for determination of the ploidies of another sample of a lupine according to the invention; and Fig. 9 shows a histogram presumably demonstrating octoploid lupines.

Example In the present example the obtension of a tetraploid lupine of the invention is described.

Several fully turgescent apical meristems of plants in the vegetativephase are prepared as donator genomes from Lupinus albus line C17/8 (described, in Wolfgang D. Garditz: "Die Bedeutung der heimischen Hummeln and Bienen (Apoideae) fir die Ertragsstruktur der Weilen Lupine (Lupinus albus Dissertation am Lehrstuhl for Biogeographie, Abteilung Agrar6kologie der Universitat Bayreuth, 1989, not reserved), the tissue is dissolved enzymatically by pectinases and/or cellulases having concentrations of approx. and the remaining cell walls are removed by a cellulase having a concentration of approx. 3% in osmotic medium 1.

SSeite- [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Grditz For obtaining the osmotic medium 1, initially a pollen extract, hereinafter abbreviated as PE 2 0 is produced in the following manner: The contents of the pollen basket of a ground bumblebee (Bombus terrestris) visiting a blossoming lupine inflorescence are removed, sterilised, the pollen grains are allowed to germinate in a germination solution cane sugar, 100 ppm H 3 B0 3 300 ppm Ca(N0 3 2 homogenised, supplemented with 4 parts of Gamborg B5 plant medium (macrosalts and microsalts) [detailed information on the germination solution is described, in Simson, C. J. and Stanford, J. "Induction of gametic selection in situ by stylar application of selective agents", in: Biotechnology and Ecology of Pollen; Springer, New York, 1986, pp. 107-112] and the homogenate is clarified in the centrifuge. The supernatant is subjected to steril filtration by using prefilters above a membrane filter.

Osmotic medium 1 is a solution containing 0.6M mannitol and

PE

2 0 pH 5.8 in water.

The obtained lupine protoplasts are isolated on a Ficoll density gradient and fusioned with the aid of polyethylene glycol (PEG 1550).

The fusion products are transferred from the PEG solution into osmotic medium 2.

Osmotic medium 2 contains 0.5M mannitol, 0.3ppm Gibberellin A 3 in

PE

2 0 pH 6.2.

The protoplast fusion products in osmotic medium 2 are equally.

separated in a density gradient, in an exemplary case of sucrose/Percoll, separated into twin and multiple protoplasts and enriched.

The desired band is removed from the centrifuge tube and should essentially contain twin protoplasts in order to obtain tetraploid lupines.

Initially the desired fraction is then transferred into a micro-reactor, in the exemplary case a microslide or a "suspended drop", photographed and Seite 16 [File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Grditz (visually) inspected as to vitality. The employed nutrient solution is fresh osmotic medium 2.

Subsequently ovaries of the receiver plant which are capable of conceiving in the exemplary case lupines of the Lupinus albus var. C17/8 line which are kept in in vitro culture are opened aseptically by means of a horizontal cut, occurring interstitial fluid is removed with a swab impregnated with an antioxidant, and the wound area is kept humid with MES buffer (morpholinoethanesulfonic acid) in PE 2 0 By means of a glass capillary viable protoplasts are then transplanted directly and as accurately as possible into the embryo sack of a receiver ovary under the binocular microscope using a micro-mechanism. Hereby osmotic medium 2 is jointly transferred, and the egg cell complex is squeezed more or less strongly.

Following closing of the ovary, the suture is protected with lanolin having an admixture of a broad range antibiotic, in the exemplary case totocilline.

Subsequently the stigma of the receiver ovary is dusted excessively with mature pollen grains (mentor pollen) of a foreign plant. This measure has the purpose of activating the metabolism in the ovary by means of the developing pollen tubes. In this phase the transplant does, however, have a spatial and temporal headstart on the mentor pollen as it is already located inside the embryo sack and can therefore develop in contrast to the pollen grains which are offset in time and distance.

The treated inflorescence is finally wrapped in air-permeable foil as aprotection against water loss and secondary infections. After 24 to 48 hours all non-transplanted ovaries of the inflorescence are removed.

All work steps are performed under clean-room conditions. The nutrient solutions are subjected to sterile filtration by membrane filter.

Pollen grains and apical meristems are sterilised with sodium hypochloride.

RA All reagents used are "analytical grade".

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[File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz The starting genomes originate from Lupinus albus var. C17/8 as described, in Wolfgang D. Garditz: "Die Bedeutung der heimischen Hummeln and Bienen (Apoideae) fur die Ertragsstruktur der Weilen Lupine (Lupinus albus Dissertation am Lehrstuhl fir Biogeographie, Abteilung Agrar6kologie der Universitat Bayreuth, 1989.

In an exemplary case, the symbiotic bacterium is an adapted genotype isolated from C17/8, with the symbiotic bacteria being rhizobia in the known manner.

Culture technique for the rhizobia was performed in accordance with known processes recently also summarised in Somasegaran, P.: "Handbook for Rhizobia" Springer, New York 1994 Maturing of the seeds in a case of successful transplantation takes place within 140 to 170 days.

The matured seeds of the polyploid lupines were germinated under the usual conditions.

Figure 1 shows a photograph of the central, i.e. the third pinna of the fifth foliage leaf on the main sprout of the tetraploid lupine according to the invention in comparison with the lupine species Lupinus angustifolius and Lubinus albus on the one hand, and in size comparison with a matchstick on the other hand.

The pinna shown in the center of Figure 1 has approx. four times the size of the leaf of a same-age comparative plant of L. albus.

Figure 2 shows a photograph of the last foliage leaf of the vegetative phase of the lupine according to the invention in tetraploid embodiment in comparison with the known species L. angustifolius, L. albus, L. mutabilis and in size comparison with a matchstick.

As can be seen in the lower right-hand half of Figure 2, even large- Teaved forms e.g. of Lupinus mutabilis which were grown under identical Seite -18- Sft (File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang GArditz conditions are at least smaller by half than the tetraploid lupine of the invention.

The blossoms of the tetraploid version of the lupine according to the invention are also clearly larger than those of the comparative species L.

angustifolius and L. albus in accordance with Figure 3.

Figure 4 shows a photograph of the third foliage leaf of the tetraploid lupine according to the invention on the main sprout in size comparison with a matchstick.

Figure 5 shows a photograph of a leaf of an octoploid lupine according to the invention having a particular morphology which dramatically differs from the tetraploid lupine of the invention and from the prior art lupines.

In order to convey the size, a fly, i.e. Sarcophaga carnaria, was included in the photograph.

Figures 1 to 5 show impressively that the entire plant mass of the novel lupine species is many times higher than in the prior art lupine species.

Radicle tissue and lamina tissue were subjected to examination of ploidy by flow cytometry. The ploidies were determined by Partec GmbH of Munster.

Measurement of ploidy was performed in accordance with A. M. M.

de Laat, W. G6hde and M. J. D. C. Vogelsang: "Determination of Ploidy of- Single Plants and Plant Populations by Flow Cytometry", Plant Breeding 99, 303-307 (1987) and I. Ulrich and W. Ulrich: "High-resolution flow cytometry of nuclear DNA in higher plants", Protoplasma (1991) 165: 212- 215. The fluorescence dye used was DAPI (4',6-diamidino-2-phenylindole).

As the result of ploidy measurements, so-called histograms are obtained wherein the ordinate indicates the number of nuclei per channel, ST whereas the abscisse indicates the fluorescense intensities which are Seite 19 Seite-19-

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[File:ANM\GA2303B2.DOCl March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz proportional to the relative DNA contents of the measured nuclei, expressed in a channel number.

Figure 6 shows a like histogram in which 4 peaks are recognisable.

Peak 1 corresponds to haploid G1 nuclei, peak 2 corresponds to haploid G2 nuclei and diploid G1 nuclei, peak 3 corresponds to diploid G2 nuclei and tetraploid G1 nuclei, and peak 4 corresponds to tetraploid G2 nuclei and octoploid G1 nuclei.

On the extreme left side in Figure 6 the histograms show nucleic fragments brought about by preparation. The frequency of occurrence of the single populations corresponds to the areas under the single peaks.

Figures 7 and 8 each show such a histogram of samples 3 and 5 of the lupines of the invention.

In the resulting histograms in accordance with Figures 7 and 8 it was found that tetraploid lupines were obtained, a fact which can be derived from the peak at channel 200.

Moreover these histograms also provide indications that lupines having higher ploidies were obtained, a fact which can be derived from the small peaks channel 300 and from the leaf in accordance with Figure Figure 9 shows another histogram of a lupine sample yielding the result that octoploid lupines are also obtained by means of the method of the invention. This can be taken from the fact that, apart from the peak around channel 200 corresponding to tetraploid G2 nuclei and octoploid G1 nuclei, one more distinct peak can clearly be perceived beyond channel 300.

As is shown in Figure 5, octoploid lupines have an entirely different morphology in comparison with tetraploid lupine species or even with the prior art lupine species.

The polyploid lupines of the invention represent novel culture plant s pecies which, being sweet lupines, are a suitable food plant for human Seite- S [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz and animal nourishment and which have the genetic potential for equalling the soybean in terms of quality and for distinctly topping it in yield per surface unit.

The sweet lupines of the prior art as a rule still contain bitter principles, i.e. alkaloids, in the order of 0.02 to with a bitter principles content in the order of more than approx. 0.02% being unsuitable for human or animal nutrition.

The polyploid lupines according to the invention, particularly tetraploid lupines, were cultivated in a secluded facility which was not open to the public and have regularly been subjected to examination as to their alkaloid content for more than the past seven years; in accordance with the method of Plarre, W. et al. (1975) "Verbesserte Methodik zur qualitativen bis halbquantitativen Alkaloidbestimmung bei Lupinen" [Improved methodology for the qualitative to semi-quantitative determination of alkaloids in lupines], periodical: Pflanzenzichtung [Plant Breeding] 74, 89 to 96, alkaloids were not found.

Parallel sensory tests of the lupine in accordance with the inventionalso yielded a negative result with respect to bitter principles.

It has moreover been found that the lupine of the invention, in accordance with the observations made until the present, is probably resistant against the fungus Colletotrichum spec. which presumably was introduced from Chile, secondarily, however, also from eastern European populations, and causes a lupine disease known under the name of antracnose. Despite experimentally performed contacts, antracnose did not occur.

The physiological performance of the polyploid plants of the invention, particularly lupines, preferably of the novel tetraploid lupine species, resides in its higher biomass and in the oil and protein content which is expected to be higher than in known lupine species, and in the concurrent absence of bitter principles.

Seite 21 Y ~Seite 21 0- 1~ S [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr. Wolfgang Garditz

HII~

SIn small-scale field cultivation of the lupine species according to the invention it was moreover found that ground bumblebee colonies, particularly the variety Bombus magnus flavoscutellaris which are settled in the vicinity of the lupine fields, pollinate the novel lupines and can increase the yield of the tetraploid lupine of the invention. For details, reference is again made to Wolfgang D. G&rditz: "Die Bedeutung der heimischen Hummeln and Bienen (Apoideae) fir die Ertragsstruktur der Weil3en Lupine (Lupinus albus Dissertation am Lehrstuhl for Biogeographie, Abteilung Agrar6kologie der Universitat Bayreuth, 1989, which is herewith fully incorporated by way of reference.

Table 1 shows an analysis of amino acids for lines a, P, y of a tetraploid lupine according to the invention.

Table 1 Analysis of amino acids for 3 lines of a tetraploid lupine in accordance with the invention Line Line Line Amino acids Alanine 1.83 1.54 1.92 Glycine 2.31 2.00 2.13 Valine 2.66 2.11 2.39 Threonine 1.77 1.56 1.57 Serine 2.20 1.90 1.75 Leucine 4.59 4.00 3.80 Isoleucine 2.70 2.11 2.12 Proline 1.98 1.83 1.69 Methionine 0.14 0.12 0.19 Aspartic acid 2.16 1.80 2.05 Phenylalanine 1.40 1.36 1.17 Glutamic acid 6.41 5.59 5.25 Seite 22 [File:ANM\GA2303B2. DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang G~rditz *Lysine 1.66 1.59 1.47 Tyro sine 1.49 1.43 1.16 Arginine 8.55 6.19 3.45 Seite 23

Claims (24)

1. A method for obtaining a polyploid plant, characterised in that protoplasts are produced from cells of diploid plants, the protoplasts are fusioned, and multiple protoplasts are isolated; ovaries of a receiver plant which are capable of conceiving are opened, the isolated multiple protoplasts are introduced into the embryo sack; the ovary is closed again; the stigma of the receiver ovary is dusted with mature pollen of a plant of a foreign species; the polyploid plant embryo is allowed to mature into a germinable seed; and the seed is allowed to germinate into a polyploid plant.

2. The method according to claim 1, characterised in that cells from lupines, in particular cells from Lupinus albus preferably line C17/8, are used.

3. The method according to claim 1 or 2, characterised in that tetraploid lupines are obtained. ^-Seite -24 S[File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Grditz____

4. The method according to one of claims 1 to 3, characterised in that apical meristem tissue is used for obtaining the protoplasts.

5. The method according to claim 4, characterised in that the apical meristem tissue is dissolved enzymatically and the cell walls of the meristem cells are removed by means of a cellulase in order to obtain protoplasts.

6. The method according to one of claims 1 to characterised in that the protoplasts are isolated by centrifuging over a density gradient, in particular a Ficoll and/or cane sugar density gradient.

7. The method according to one of claims 1 to 6, characterised in that the protoplasts are fusioned by using a polyethylene glycol, particularly one having a molecular mass of approx. 1550 Da.

8. The method according to one of claims 1 to 7, characterised in that fusioned protoplasts are separated into twin and multiple protoplasts by centrifuging over a density gradient, in particular a Ficoll and/or cane sugar density gradient, and preferably enriched.

9. The method according to one of claims 1 to 8, characterised in that preferably twin protoplasts are introduced into the embryo sack. The method according to one of claims 1 to 9, characterised in that the cut on the treated ovary is closed with lanolin, with the lanolin preferably having an admixture of a broad range antibiotic and/or antimycotic. [File:ANM\GA2303B2.DOC] March 6, 1998 Tetraploid Lupine Dr.

Wolfgang GdrditzI,, 11i m

11. The method according to one of claims 1 to characterised in that the treated inflorescence is wrapped in air-permeable foil as a protection against water loss and secondary infections.

12. The method according to one of claims 1 to 11, characterised in that all non-transplanted ovaries of the inflorescence are removed after approx. 24 to 48 hours.

13. A polyploid plant, characterised in that it is obtainable by a method in accordance with at least one of claims 1 to 12.

14. The polyploid plant according to claim 13, characterised in that it is a tetraploid lupine.

The polyploid plant according to claim 13 or 14, characterised in that it is obtainable from Lupinus albus preferably line C17/8.

16. The polyploid plant according to claim 13 or 14, characterised in that it attains at least twice the leaf size of the parent plant.

17. The polyploid plant according to one of claims 14 to 16, characterised in that it can be propagated by seeds and/or by cell culture.

18. The polyploid plant according to one of claims 13 to 17, characterised in that it is substantially free of alkaloids. Seite 26 (File:ANM\GA2303B2.DOC] March 6,1998 Tetraploid Lupine Dr. Wolfgang Garditz

19. The polyploid plant according to one of claims 13 to 17, characterised in that, being a tetraploid lupine, it has amino acid and oil spectra similar to those of the white lupine.

The polyploid plant according to one of claims 13 to 19, characterised in that, being a tetraploid lupine, fertile cross-breeding with any other plant is not possible.

21. The polyploid plant according to one of claims 13 to characterised in that, being a tetraploid lupine, it has a DNA-content twice that of diploid species, with the DNA being present particularly in approx. 100 chromosomes.

22. Propagating material of a polyploid plant in accordance with one of claims 13 to 21.

23. The propagating material according to claim 22, characterised in that it is propagating material, in particular seed grains, of polyploid lupines, in particular of tetraploid lupines.

24. The propagating material according to claim 22 or 23, characterised in that it is meristem cells, in particular apical meristem cells, of tetraploid lupines.