HK1161032A - Polyploid castor plants, compositions derived therefrom and uses of same - Google Patents

Description

Polyploid castor plant, compositions derived therefrom and uses thereof

Related application

This application claims priority from U.S. provisional patent application 60/996,214 filed on 6/11/2007.

The contents of all of the above documents are incorporated by reference herein, if set forth at all.

Field and background of the invention

The present invention, in certain embodiments thereof, relates to polyploid castor plants, more particularly, but not exclusively, to compositions derived therefrom and uses thereof.

Castor (Ricinus communis L.) is an important crop of euphorbiaceae. It is a mono-type species of the genus ricinus and is of considerable economic value due to its oil-rich seeds that produce castor oil, a strategically important oil with many industrial uses. Its origin is in the southwest mediterranean and west africa, but is now common in many parts of the world.

Castor beans contain up to 40-50% oil of a unique composition. Chemically, castor is a triglyceride (ester) of fatty acids. The fatty acid content of up to 90% of the oil is ricinoleic acid (12-hydroxyoleic acid), an 18-carbonic acid with a double bond in the 9-10 position and a hydroxyl group on the 12 carbons. Castor oil ranges in color from colorless to pale yellow or pale green. Other characteristics include relatively high viscosity, non-drying, weak but characteristic odor, slightly pungent taste and aftertaste (what is aftertaste.

Many derivatives can be produced, having a chemical composition similar to petroleum-based oils. Blown Castor oil (blow reactor oil) is a derivative with a higher viscosity and specific gravity than natural Castor oil. These properties are induced by bubbling air through it at elevated temperatures. Its main use is as a plasticizer for inks, paints and adhesives. Hydrogenated Castor Oil (HCO) or castor wax is an insoluble, hard, brittle wax. It is produced by adding hydrogen in the presence of a nickel catalyst. It is used for coatings and greases mainly when resistance to moisture, oil and other petrochemicals is required.

As a result, castor oil and products derived therefrom are used in many industrial products, including bio-based lubricants, fuels, paints and coatings, plastics, antifungal compounds, and cosmetics. The world market for castor oil is approximately 7 billion to 5 million dollars per year.

One problem in recent years has been the instability of castor oil supply. Major suppliers, india, china and brazil, have experienced production difficulties in recent years.

Castor oil is a candidate feedstock for the emerging biodiesel industry, but currently it is too expensive to compete with petroleum-based diesel. Increasing castor production worldwide can make castor biodiesel more competitive in the future.

Autotetraploids have been produced using colchicine, and haploids have been reported in addition, but in nature, castor exists mainly in diploid form (Moshkin and doryadinka, 1986).

Summary of The Invention

According to an aspect of some embodiments of the present invention there is provided a polyploid castor plant, having at least as great fertility as a diploid castor plant isogenic to said polyploid castor plant when grown under similar conditions.

According to some embodiments of the invention, the fertility is determined by at least one of:

number of seeds per plant;

analyzing the reproductive capacity of gametes; and

carmine acetate was stained.

According to certain embodiments of the invention, the plant exhibits genomic stability for at least 5 passages.

According to some embodiments of the invention, the plant has seed yield at least similar to that of a diploid castor plant.

According to some embodiments of the invention, the plant has a greater leaf surface area than a diploid castor plant.

According to some embodiments of the invention, the plant has a larger stomatal surface compared to a diploid castor plant.

According to some embodiments of the invention, the plant is a tetraploid.

According to some embodiments of the invention, the plant has an oil yield at least similar to that of a diploid castor plant.

According to certain embodiments of the invention, the plant is capable of cross-breeding with a diploid plant.

According to some embodiments of the invention, the plant is an autopolyploid.

According to some embodiments of the invention, the plant is an inbred.

According to an aspect of some embodiments of the invention there is provided a castor plant deposited according to budapest treaty at NCIMB ltd, having accession number NCIMB 41593 castor B2-20-4N.

According to an aspect of some embodiments of the invention, there is provided a plant part of a castor plant.

According to an aspect of some embodiments of the invention, there is provided castor oil produced from the plant or plant part.

According to an aspect of some embodiments of the invention, there is provided castor meal produced from the plant or plant part.

According to some embodiments of the invention, the plant part is a seed.

According to an aspect of some embodiments of the present invention, there is provided an isolated regenerable cell of a castor plant.

According to certain embodiments of the invention, the cell exhibits genomic stability for at least 5 passages in culture.

According to certain embodiments of the invention, the cell is from a meristem, pollen, leaf, root tip, anther, pistil, flower, seed or stem.

According to an aspect of some embodiments of the invention, there is provided a tissue culture comprising the regenerable cells.

According to an aspect of some embodiments of the present invention there is provided a method of producing castor seed comprising self-breeding or cross-breeding the plant.

According to an aspect of some embodiments of the present invention there is provided a method of producing castor oil, the method comprising:

(a) harvesting seeds of a castor plant or plant part; and

(b) processing said seed to produce castor oil.

According to an aspect of some embodiments of the present invention there is provided a method of producing a polyploid castor seed, the method comprising contacting a castor seed with a G2/M cell cycle inhibitor under a magnetic field to produce the polyploid castor seed.

According to certain embodiments of the invention, the G2/M cell cycle inhibitor comprises a microtubule polymerization inhibitor.

According to certain embodiments of the invention, the microtubule polymerization inhibitor is selected from the group consisting of colchicine, nocodazole, oryzaline, trifluraline and vinblastine sulphate.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and/or materials similar or equivalent to those described herein can be used in the practice and/or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be necessarily limiting.

Brief description of the drawings

Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description taken with the drawings make it apparent to those skilled in the art how the embodiments of the invention may be practiced.

In the drawings:

figure 1 is a picture of a polyploid castor plant produced in accordance with the teachings of the present invention;

fig. 2A-B are images of a photosynthesis monitoring system used to evaluate photosynthesis efficiency according to some embodiments of the present invention.

Figures 3A-B are images showing a diploid castor plant (3A) and a tetraploid castor plant (3B). Leaf Chamber (LC) is given as reference for leaf size. Note that the leaves of diploid plants are much smaller than those of tetraploids;

figure 4 is a photomicrograph image of the stomata taken with a computerized microscope. Upper, tetraploid plants. Bottom diploid plants (examples of stomata are circled with white lines).

FIGS. 5A-B are images of the major and secondary veins of tetraploid (A) and diploid (B) plants.

Figure 6 is a photograph showing castor seeds of chinese variety (left), brazil variety (middle) and tetraploid (right) produced by applying the teachings of the present invention to brazil variety.

Description of embodiments of the invention

The present invention, in certain embodiments thereof, relates to castor plants, and more particularly, but not exclusively, to polyploid castor plants and methods of making and using the same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced and carried out in various ways.

Castor (Ricinus communis l., euphorbiaceae) is produced using this versatile agricultural chemical (chemurgical) raw material needed to supply castor oil to hundreds of products. Forty to forty-five million tons per year of castor oil and derivatives are imported into the united states to meet increasing demand (Roetheli et al 1991).

To meet these needs, the inventors identified conditions for genome multiplication in castor plant seeds. The genomically multiplied castor seeds produced according to the present teachings provide progeny plants characterized by as high yield (e.g., seed yield, oil yield) and fertility as their isogenic diploid castor ancestors. This is in sharp contrast to previous reports of tetraploid castor plants that exhibit low productivity compared to diploids (see work by Efremov 1972, Evstaf' eva Fedorenko 1972, reported in Moshkin and Doryandinka, 1986).

As illustrated hereinafter and in the examples section that follows, tetraploid castor plants produced according to the present teachings are qualified for both yield and fertility. The parameters tested included pore area, number of pores per unit area, photographs of the pores made in statistical analysis scale, typical vein size, and chemical analysis of seed oil. The average size of the diploid castor plant stomata is 150 mu²32, tetraploid is 221. mu²96 is added. The difference was significant at the 0.001 level. The number of stomata for tetraploid and diploid are 54 and 105 stomata per 1mm, respectively². Based on these findings, and the larger size of the tetraploid plants, it was suggested that the tetraploid plants exhibited higher photosynthetic efficiency per unit area than the diploid plants. According to the polyploid phenotype, the size tetraploids of the major and secondary petiole veins are larger than those of diploid plants. The seed number per plant was similar in tetraploid plants as in isogenic diploid castor plants. Seed size (i.e., length, width, and fresh weight) was greater in tetraploids than in diploid plants, indicating even higher yield in tetraploids than in diploid plants. The volumetric oil content is at least similar to that of an isogenic diploid plant. All of these indicate the advantages of polyploid plants over wild-type castor plants.

Thus, according to an aspect of some embodiments of the present invention, there is provided a polyploid castor plant, having at least as much fertility as a diploid castor plant isogenic to said polyploid castor plant when grown under similar conditions.

As used herein, the term "castor plant" also referred to as "castor oil plant" or "castor (Ricinus communis)" refers to a plant species of the Euphorbiaceae family (Euphorbiaceae).

The castor plant of certain embodiments of the invention refers to the whole plant or a part thereof, processed or unprocessed (e.g., seed, oil, dried tissue, meal, clumps, etc.), a regenerable tissue culture, or cells isolated therefrom.

As used herein, the term "polyploid" refers to a plant having three or more sets of chromosomes (e.g., 3N, 4N, 5N, 6N or more). According to certain embodiments of this aspect of the invention, the polyploid plant is an autopolyploid.

As used herein, the term "diploid" refers to a typical castor plant having two sets (2N) of chromosomes, wherein each set comprises 20 chromosomes. As used herein, a diploid castor plant is isogenic to a multiplied polyploid plant, i.e., the two sets of chromosomes contain alleles that are substantially identical at all positions. Diploid plants may be naturally occurring, modified to produce or bred to produce the product.

As used herein, the term "reproductive" refers to the ability to reproduce sexually. Fertility can be analyzed using methods well known in the art. The following parameters can be analyzed to determine fertility: the number of seeds; gamete fertility can be measured, for example, by pollen germination on a sucrose substrate; and alternatively or additionally, carmine acetate is dyed, whereby the fertile pollen is dyed.

According to certain embodiments of the invention, a mature polyploid castor plant has at least about the same number of seeds (+/-10%) as its isogenic diploid progenitor grown under the same conditions; alternatively or additionally, the polyploid plant has at least 90% of fertile pollen stained with carmine acetate; and alternatively or additionally, at least 90% of the seeds germinate on sucrose.

Comparative analyses performed to characterize the traits (e.g., fertility, yield, biomass and vigor) of the polyploid plants of the present invention are generally performed in comparison to its isogenic progenitors (hereinafter, "diploid plants") when both are at the same developmental stage and both are grown under similar growth conditions.

Thus, according to certain embodiments of the invention, the polyploid plant has a greater leaf surface area than the diploid castor plant. In an exemplary embodiment, the leaf area is 30% to 100% greater than that of a diploid plant and the leaf thickness is at least 1.5-2.5 times that of a diploid plant.

According to some embodiments of the invention, the polyploid plant has a larger stomatal surface than the diploid castor plant. In an exemplary embodiment, said stomatal surface area is at least 1.5-2.5 times that of said diploid plant.

According to certain embodiments of the invention, the polyploid plant is capable of cross-breeding with a diploid plant.

According to certain embodiments of the invention, the polyploid plant is stable for at least 4, 5, 7, 9 or 10 generations.

According to certain embodiments of the present invention, the polyploid plant has a seed yield (as determined by at least one of seed number, seed size and volumetric oil content) at least similar to that of the growing diploid castor bean. According to a further embodiment of the present invention, said seed yield exceeds that of said diploid plant by at least about 1, 1.5, 1.75, 2, 2.5, 3 or 5 fold.

As used herein, the term "stable" refers to chromosomes and the number of chromosome copies that remain constant over generations, while the plant does not exhibit a substantial decrease in at least one of the following parameters: yield, fertility, biomass, vigor.

Polyploid plants of the present invention may be produced using an improved method of colchicination as described below.

The seeds were germinated in distilled water at 25 ℃ for 12 hours. Thereafter, the seeds were soaked for 20 hours in a doubling solution comprising: 0.5% colchicine, 0.5% DMSO, 0.03% Tritonx 100. Finally, the seeds are washed and sown on a suitable germination bed.

Additionally or alternatively, the polyploid castor plant of the present invention may be produced using colchicine or any other cell cycle inhibitor (e.g., a G2/M phase inhibitor, e.g., a microtubule assembly inhibitor, e.g., colchicine, vinblastine, nocodazole, oryzaline, and trifluraline), wherein the targeting agent is a magnetic field for targeted delivery of the inhibitor to chromatin filaments.

Specific embodiments of such methods are provided below. Notably, measurements were made to maintain the indicated pH values for each phase (e.g., with HCL or NaOH).

Stage one-3 hours:

seeds were incubated in a solution of vinblastine sulfate (0.1% v/v) containing 0.5% DMSO titrated to pH 5.6 in a petri dish in the dark at a temperature of 26 ℃. The pH conditions were monitored to maintain a constant pH value (5.6) throughout this phase. The vessel was placed in a 1300 gauss magnetic field with the magnets placed 10.5cm apart from each other.

Stage two-3 hours:

the seeds were incubated in the above solution at 4 ℃ under daylight conditions and the pH was titrated to 6.

Three-6 hours

The seeds were incubated at 20 ℃ under daylight conditions and the pH was titrated to 5.4.

Stage four-12 hours

The seeds were incubated at 26 ℃ under daylight conditions and the pH was titrated to 6. The magnetic field was removed and nocodazole was added to a concentration of 5 m/ml.

Stage five-12 hours:

the seeds were incubated under constant temperature conditions (26 ℃) in the sunlight.

The seeds were washed thoroughly in water to raise the pH to 7. Thereafter, the seeds were sown in a suitable growth bed under long daylight conditions (16 hours) at 26 ℃.

Using these teachings, the present inventors were able to produce polyploid castor plants, such as the polyploid castor plant deposited under the budapest treaty at NCIMB ltd and having the accession number NCIMB 41593 castor B2-20-4N.

Once established, the castor plant of the present invention can be propagated sexually or asexually, for example, by using tissue culture techniques.

As used herein, the term "tissue culture" refers to plant cells or plant parts from which castor plants can be produced, including plant protoplasts, plant calli, plant pieces, and plant cells, which are intact in plants, or parts of plants, such as seeds, leaves, stems, pollen, roots, root tips, anthers, ovules, petals, flowers, embryos, fibers, and cotton capsules.

According to certain embodiments of the invention, the cultured cells exhibit genomic stability for at least 4, 5, 7, 9, or 10 passages in culture.

Techniques for producing plant tissue cultures and regenerating plants from tissue cultures are well known in the art. For example, such techniques are Vasil., 1984, Cell Culture and social cells genetics of Plants, Vol I, II, III, Laboratory Procedures and theoretical applications, Academic Press, New York; green et al, 1987 Plant tissue and Cell Culture, Academic Press, New York; methods for Plant Molecular Biology, Academic Press; gelvin et al, 1990, Plant Molecular Biology Manual, Kluwer academic publishers; evans et al, 1983, Handbook of Plant Cell Culture, MacMillian Publishing Company, New York; and Klee et al, 1987.Ann.Rev.of Plant Phys.38: 467486.

the tissue culture may be produced from cells or protoplasts of a tissue selected from the group consisting of seeds, leaves, stems, pollen, roots, root tips, anthers, ovules, petals, flowers, embryos, fibers, and cotton capsules.

It is to be understood that the plants of the present invention may also be used in plant breeding (i.e., self-breeding or cross-breeding) with other castor plants to produce new plants or plant lines that exhibit at least some of the characteristics of the castor plants of the present invention.

Plants produced with any of these crosses with other plants can be utilized in pedigree breeding, transformation and/or backcrossing to produce other cultivars that exhibit the characteristics of the castor plant of the invention and any other desired traits. Screening techniques employing molecular or biochemical manipulations, well known in the art, can be used to ensure that the important commercial features sought remain in each breeding generation.

The goal of backcrossing is to alter or replace individual traits or characteristics in the recurrent parent lines. To achieve this, a single gene of the recurrent parent line is replaced or supplemented with the desired gene from the non-recurrent line, while substantially all other desired genes are maintained, thus maintaining the desired physiological and morphological configuration of the original line. The selection of a particular non-recurrent parent will depend on the purpose of the backcrossing. One of the main objectives is to add certain commercially desirable, agronomically important traits to plants. The exact backcrossing protocol will depend on the characteristics or traits to be altered or added to determine the appropriate test protocol. When the characteristic to be transferred is a dominant allele, although the backcrossing method is simplified, a recessive allele may also be transferred. In this case, testing of the introduced children may be required to determine whether the desired feature was successfully transferred. Likewise, the transgene may be introduced into the plant using any of a variety of established transformation methods known to those skilled in the art, such as: gressel, 1985.biotechnological Conferring Herbicide Resistance in Crops: the Present reagents, In: molecular force Function of the plant Genome, L van Vloten-dotting, (ed.), plenum Press, New York; huftner, S.L., et al., 1992, reviewing overhead of genetic Modified Plants, Bio/Technology; klee, H., et al, 1989, planta genes Vectors and Genetic Transformation: plant Transformation System based on the use of Agrobacterium tumefaciens, Cell Culture and genomic cells Genetics of Plants; and Koncz, C., et al, 1986, molecular General Genetics.

It is to be understood that the castor plant of the present invention can be genetically modified, for example, to introduce traits of interest, for example, improved oil composition and enhanced resistance to stress (e.g., biotic or abiotic). Non-limiting examples of nucleic acid sequences and castor transformation methods useful for altering the oil content of a castor plant, and nucleic acid constructs useful therefor, are described in U.S. patent No. 6,974,893, which is incorporated herein by reference in its entirety.

According to some embodiments of the invention, the fatty acid composition of the multiplied castor is about the same as that of a diploid castor plant, however the levels of different components may vary.

Thus, the present invention provides novel castor plants and cultivars and seeds and tissue cultures for their production.

The castor plant produced according to the present teachings can be further processed to produce castor plant products, which are commonly used in many industrial applications, including bio-based lubricants, waxes, paints and coatings, plastics, antifungal compounds, and cosmetics.

Thus, according to one method of the present invention, there is provided a method of producing castor oil, the method comprising: harvesting seed of the above-described castor plant or plant part; processing said seed to produce said castor oil.

The following is a non-limiting description of seed collection and processing.

The castor fruit is harvested when fully ripe and the leaves are dry, about 95-180 days depending on the cultivar. Planting and harvesting can be performed by manual methods or completely mechanized. Harvesting should begin before the rainy season in tropical areas, but in arid areas it is best to harvest when all fruits are ripe. Cutting or breaking the spike-like inflorescences, and peeling and collecting the capsules. Unless the capsules are dry, they must be spread apart to dry quickly. Sun drying, lyophilizing or by using deciduous agent. Harvesting machines may be used, such as modified wheat headers (headers) which shake the capsules from the plants by shaking them on their base. A relative humidity of 45% or less is required for efficient operation of the mechanical harvester. Some varieties with unbreakable pericarp are threshed by a common grain thresher at the rotating speed of a drum of 400-800r.p.m. After harvesting, the seeds must be removed from the capsules or pods, usually with a huller if the capsules are dry. The percentage of seeds to pods averages 65-75 depending on the maturity of the seeds at harvest.

Extraction of oil from castor seeds is performed in a manner similar to most other oil-containing seeds. The seeds were cleaned, cooked and dried prior to extraction. Cooking is performed to coagulate the protein (necessary for efficient extraction), and release the oil for efficient pressing.

The first stage of oil extraction is pre-pressing, using a high pressure continuous screw press, called an expeller (expeller). The extracted oil is filtered and the material removed from the oil is returned to the flow path with fresh material. The material eventually discarded from the press, called cake, contains 8 to 10 percent oil. It is crushed into a coarse powder and subjected to solvent extraction with hexane or heptane.

Once the oil is extracted from the seeds, impurities must be removed from the oil. The oil is a substantially pure triglyceride containing almost 90% of glyceryl ricinoleate. This is the ricinoleic acid triglyceride required to produce high quality castor oil.

The step of refining the crude oil comprises:

precipitation and degumming of the oil-is performed to remove the aqueous phase from the lipids, and to remove the phospholipids from the oil.

Bleach-bleaching results in the removal of colored materials, phospholipids and oxidation products.

Neutralization-the neutralization step is necessary to remove free fatty acids from the oil. This can be done in one of two ways: (a) alkali (chemical) or (b) steam removal (physical). Alkaline/chemical method: caustic soda (alkali) is mixed in the appropriate amount and the aqueous solution is removed, leaving a neutral oil. Steam removal: under vacuum to remove moisture, free fatty acids, odor bodies and other impurities from the oil. When conducted under vacuum conditions, the oil can be maintained at a low temperature, maintaining its chemical structure by not subjecting it to temperatures at which undesirable dehydration reactions may occur.

Deodorization of oils-deodorization allows the removal of odors from oils.

Many derivatives can be produced from castor oil. Some of these derivatives have chemical compositions similar to petroleum-based oils.

Castor seed residue, also known as castor powder-castor powder is the residue obtained from castor cubes by a solvent extraction process. It is one of the most common natural fertilizers. This fertilizer increases the fertility of the soil without causing any damage or deterioration. It is rich in three elements, namely nitrogen, phosphorus and potassium, that are critical and conducive to proper growth of the crop. It also has trace amounts of nutrients such as manganese, zinc and copper, thus making it a balanced fertilizer. In addition, it helps to neutralize the harmful effects of chemical fertilizers. In addition to their contribution to nutrients, they have many agricultural benefits, which synthetic fertilizers or pesticides cannot provide. They provide slow and stable nutrition, stimulation, protection from soil nematodes and insects, improve yield and product quality, such as taste, flavor, amino acid composition, etc.

After pressing of the castor beans a compressed mass is obtained. The solvent-extracted mass, although rich in protein, cannot be used as cattle feed due to its toxicity. However, it can be used as a fertilizer. The protein content of castor seed meal varies between 21-48% depending on the degree of dehulling. It has an ideal amino acid profile with moderately high cystine, methionine and isoleucine. Its anti-nutritional substances, ricin (ricin), ricin (ricine) and allergen, however, limit its use in poultry feed, even at very low inclusion levels.

Hydrogenated Castor Oil (HCO), also known as castor wax, is a hard, brittle, insoluble wax. It is produced by adding hydrogen in the presence of a nickel catalyst. It is used for coatings and greases mainly when resistance to moisture, oil and other petrochemicals is required.

HCO is produced by hydrogenation of castor oil with a nickel catalyst. Its white flakes are extremely insoluble and water resistant. The main use is in the manufacture of greases, and in paper coatings for food packaging.

Hydrogenated oils are also utilized in the manufacture of waxes, polishes, carbon paper, candles and crayons.

12 Hydroxystearic acid (12HSA) -12 HSA is a beige solid fatty acid used to make lithium and calcium based lubricating greases. When reacted with an ester, 12HSA provides a hard finish for the automotive and small appliance industries.

Methyl 12HSA (methyl 12 hydroxystearic acid, methyl 12 hydroxystearate) -methyl 12HSA was formed by direct esterification of 12HSA with methanol. It is usually sold in liquid form and is widely used in continuous grease processing. It has a lower melting point than 12HSA and is therefore easier to handle in liquid state. Greases made with methyl 12HSA can be configured with higher drop points, they experience less gel shrinkage (bleading) and improved oxidation stability.

Blown castor oil-blown castor oil is a castor oil derivative that has a higher viscosity and specific gravity than natural castor oil. These properties are induced by bubbling air through it at elevated temperatures. Blown castor oil has use in plasticizers for inks, paints and adhesives.

COLM, urethane grade — COLM (low moisture castor oil) is a refined grade of castor oil for specialized applications requiring minimal moisture. Typical applications include urethane coatings, adhesives, and inks. COLM also has application in urethane blowing and urethane molding.

The dehydrated oil is an excellent drying agent comparable to tung oil and is used in paints and varnishes.

It is expected that during the prosecution of the patent maturing from this application many relevant derivatives will be developed and the scope of the term derivative is intended to include all such new technologies a priori.

As used herein, the term "about" means. + -. 10%.

The terms "comprising," including, "and" having, "and their conjugates mean" including, but not limited to. This term encompasses the terms "consisting of and" consisting essentially of.

The term "consisting essentially of means that the composition or method may include other ingredients and/or steps, but only if the other ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the present invention may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range from 1 to 6 should be considered to specifically disclose sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, this is meant to include any number (fractional or integer) recited within the indicated range. The terms "range" between "a first stated number and a second stated number, and" range from "the first stated number" to "the second stated number, used interchangeably herein, are meant to include the first and second stated values and all fractional and integer numbers therebetween.

As used herein, the term "method" refers to manners, means, techniques and steps for accomplishing a given task including, but not limited to, those manners, means, techniques and steps known to, or readily developed from known manners, means, techniques and steps by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless no such element of the described embodiments is inoperative.

Various embodiments and aspects of the present invention described hereinabove and claimed in the claims section below may find experimental support in the following examples.

Examples

Reference is now made to the following examples, which together with the above descriptions, illustrate certain embodiments of the invention in a non-limiting manner.

Example 1

Production of polyploid castor plant

Castor plants from different genetic backgrounds were collected from many regions of israel (uppergalile, Lower galile, Coastal plane and newev) and from different regions of the world (southern brazil, northern brazil, argentina, paraguay, northern china and central china). The genomes of these plants were multiplied by Mutation-free genome Multiplication (MFGM). The multiplication in this experiment was performed using any of the protocols described above.

All sources underwent a process of focused mutagenesis (focused mutagenesis) to extend pre-existing genetic variability and to localize male sterility in different backgrounds.

Plants that underwent the process of genome multiplication were planted in the field and their level of ploidy was checked using FACS (fluorescence activated cell sorting).

Briefly, nuclei were released from 2cm x 2cm leaf tissue by immersion in chopping buffer for 30 seconds.

The chopping buffer consists of 4.575 g MgCl per 500ml distilled water₂2.095 grams MOPS, 4.4 grams sodium citrate, 1 gram DTT, 1.65 grams (10 drops) Triton x 100. The chopping is performed in one direction with a blade. The minced tissue was transferred to petri dishes and placed on ice.

The samples were filtered (20 mesh) prior to use. Nuclear samples (2cc-6cc) were transferred to FACS tubes and 15. mu.l Propidium Iodide (PI) was added to each sample. After 15 minutes, the samples were analyzed in a flow cytometer equipped with a Cyonics argon laser (488nm) operating at 15 mW.

Fluorescence above 635nm was gated and the results are shown as a single parameter histogram of the number of nuclei in each of the 1024 channels. The control is fixed to the channel 300.

Polyploid plants were subjected to pollen fertility tests and microscopic examination of pollen grains by germination on a sucrose bed (as explained above, carmine acetate was used to stain the fertile pollen).

All plants that were phenotypically, and in terms of pollen grain appearance and structure, determined to be unharmed, and whose fertility level was not lower than that of the control plants underwent self-pollination (FIG. 1).

Progeny of plants multiplied by the plant genome were examined for stability and gamete fertility. The plant is selected to serve as a parent in the production of the hybrid.

The F2 population underwent genomic multiplication using MFGM technology and the ploidy levels of all plants were examined using FACS.

Plants tested to achieve genome doubling and also being fertile and interesting from a plant breeding perspective were tested to determine their level of pollen fertility and then self-pollinated.

Plants tested to achieve genomic doubling and male sterile serve as parents for hybrids, pollinated by the selected genomically multiplied plant.

Example 2

Characterization of stomata and seed markers for multiplied castor oil plant (Ricinus comurnis) genotypes, and their CO-targeting₂Effects of intake, transpiration and yield.

Materials and Experimental procedures

The study area was: the study was conducted in a typical mediterranean climate with a long-term annual average temperature of 18-20 deg.C (-8-10 deg.C for 1 month, 30-35 deg.C for 8 months), 600-700mm rainfall in winter (11-5 months), 1500-2000mm evaporation, incident PAR 300-350MJ m^-2. Maximum incident PAR occurs at 8 months (410-^-2Moon cake^-1) The minimum value is 12 months (1140-160MJ m)^-2Moon cake^-1)。

Surface soils (0-30cm) with different proportions of sand, silt and clay components in the study area were mainly fine grained soils, exhibiting a narrow range of variation for field water capacity and permanent wilting point, which correspond to-0.03 MPa and-1.5 MPa in matric potential, respectively. The dry bulk density of the soil is 1.22 and 1.35g cm^-3In the meantime. The soil has no salinity problem, and the total soluble salt content is less than 0.1 percent. The soil at the study site had a slightly alkaline pH of 7.5 to 7.7 and was determined to be deficient in soil organic matter (1.18 to 2.37%).

Genetic methods-castor bean polyploid line treated with "MFGM" technology was compared in fertility and yield to its isogenic diploid line. For this purpose, individual plants from each line were harvested and the capsule number and yield components of each plant were collected.

Photosynthesis and transpiration measurements: parts of leaves from both genotypes were inserted under a microscope Nikon TI inverted microscope equipped with a 20X super flur objective and manipulated with NIS components software. 30-50 pores were labeled in each treatment, their areas were automatically measured and statistically analyzed.

Measurement of net photosynthetic Rate (P)_N) Transpiration rate (E)_T) Water Use Efficiency (WUE), Photosynthetically Active Radiation (PAR), air temperature (T), Relative Humidity (RH) and atmospheric CO₂Concentration (C)_atm) The coupling kinetics of (2).

To measure various environmental and plant parameters, a PTM-48M photosynthesis monitor (fig. 2A) was used.

The system comprises: four self-clamping leaf chambers LC-4A (FIG. 2B) that are closed continuously for two minutes for monitoring leaf CO₂Exchange of infrared CO₂An analyzer and a built-in data logger. The monitor also has eleven inputs for other sensors. Other sensors used in the current experiments include: the PIR-1 photosynthesis radiation sensor ATH-2 air temperature and humidity sensor, photosynthesis and transpiration data from the four leaf chambers and data from other sensors are automatically recorded continuously every 30 minutes.

Diploid and tetraploid plants in the experiment are shown in figure 3. The Leaf Compartment (LC) is given as a reference for the size of the leaf. Note that the leaves of diploid plants are much smaller than those of tetraploid plants.

Fatty acid analysis-oils were transesterified by reaction with a solution of potassium hydroxide in methanol, tested by gas liquid chromatography (ISO 5509-.

Germination assay-seeds were grown on a sucrose bed (2% sucrose and 2mM H) at 26 deg.C₃BO₃) And incubated for 12 hours. Thereafter germination was evaluated.

Carmine acetate staining-pollen was stained with a 1% carmine acetate solution for 15 minutes and then observed under a microscope.

Statistical analysis-pore analysis was performed using NIS software. It includes t-tests for frequency distribution, standard deviation, and significance. The frequency distribution of the pores was close to normal, and the number of pores per unit area and the t-test result of the area of the pores were significant at the 0.001 level. The mean of the two leaf chambers and the two chambers of each diploid plant were used to measure the multiplied castor oil plant genotype. Ambient CO₂The standard deviation of the output values was calculated using four probe measurements. Preliminary tests showed that the coefficient of variation for photosynthesis and transpiration were both 0.13.

It is expected that polyploid plants produced according to the present teachings exhibit similar or even higher photosynthesis as compared to diploid plants, as evidenced by transpiration and photosynthesis analysis.

Pore area and density-figure 4 clearly shows that the size of tetraploid pores is larger than that of diploids, whereas the number (density) of diploid pores is larger. The statistics of both photographs are summarized in table 1 below. The first 4 lines (1-4) outline the physics of the image. Row 5 indicates that the stomatal density diploid is approximately 6-fold greater than the tetraploid.

TABLE 1 physical Properties of stomata in leaves of tetraploid and diploid plants

	Item	Unit of	Tetraploid	Diploid body
					1	Number of pixels in x-axis	An	1,392.0	1,392.0
2	Number of pixels on y-axis	An	1,040.0	1,040.0
					3	Length of pixel	μ	0.3	0.3
4	Area of pixel	μ2	0.1	0.1
					5	Average number of air holes per slide	An	8.0	47.0
6	Average area of each air hole	μ2	221.5	150.0
					7	Total area of air holes per slide	μ2	1,772.0	7,050.0
8	Area of slide	μ2	148,242.4	148,242.4
					9	Area of slide	mm2	0.1	0.1

10	Number of pores/mm2	An	54.0	317.0
					11	Per mm2Area of air hole	μ2	11,953.4	47,557.2

Row 6 shows that the area of a single tetraploid stomata is about 50% larger than that of a diploid. However, the specific stomatal area of the diploid (11 rows) is about 4 times that of the tetraploid (p ═ 0.001).

FIGS. 5A-B are images of the veins of diploid (B) and tetraploid (A) castor plants. The measured veins of both plants were taken from the fifth secondary vein above the middle of the leaf. The diameter of the major tetraploid veins is about 1500 μ, while the diameter of the equivalent diploid veins is only half. Similar observations can be found in the secondary veins. It was found to be 500 μ in tetraploids and less than 300 μ in diploids.

Seed properties-phenotypic representation of multiplied castor seeds is shown in figure 6. Seed length, width and fresh weight were significantly greater in tetraploids than in diploid genotypes (table 2, below). The differences within each group were negligible.

TABLE 2 comparison of seed size and fresh weight between Castor diploid (2n) and tetraploid (4n) genotypes^a

Genotype(s)	Obsr.	Length, cm (s.d)	Width, cm (s.d)	Weight, g (s.d)
					B2-74-1-22nB2-20(11)-114nB2-20(12)-74nB2(20)98-12nB2(20)52-12nB2(20)202nB2(20)292n	2117713233244	1.38(0.06) bc1.46(0.05) a1.48(0.035) a1.37(0.04) bc1.36(0.04) c1.39(0.04) b1.39(0.07) b	0.83(0.03) c0.985(0.03) a1.01(0.026) a0.85(0.016) b0.85(0.03) b0.83(0.03) c0.85(0.04) b	0.34(0.06) c0.48(0.04) a0.49(0.07) a0.36(0.08) bc0.38(0.035)b0.33(0.08) c0.34(0.07) c
Average		1.396	0.867	0.367
					FPr＞F		9.70.0001	730.0001	14.80.0001

^aANOVA and Tukey Range test (α ═ 0.05)

Fertility and yield data: literature data on polyploid castor seeds show a sharp decrease in polyploid yield and fertility when compared to isogenic diploids or hybrids (Moshkin, v.a. and a.g. dvoryadinka. castor genetics. in case. ed.v.a.moshkin. americand publish.co., New delhi.1986. pp.93-102).

The results of data acquisition for the 2N and 4N isogenic lines are shown in table 3 below.

TABLE 3 number of capsules and seed weight per plant in tetraploid (4n) and diploid (2n) isogenic lines

2N series	Capsule quantity	Total seed yield (gram)	4N series	Capsule quantity	Total seed yield (gram)
						18-22(43)	551	220	B2(20)1	553	287
18-24(44)	530	213	B2(20)2	542	280
						18-28(45)	542	217	B2(20)3	546	283
18-30(46)	548	220	B2(20)4	543	280
						18-31(47)	572	229	B2(20)5	570	296
18-35(48)	546	218	B2(20)6	548	284
						18-36(49)	554	219	B2(20)7	553	287
18-41(50)	538	214	B2(20)8	540	280
						18-42(51)	541	218	B2(20)9	554	289
18-46(52)	540	215	B2(20)10	547	284
						18-47(53)	547	218	B2(20)11	555	288
18-49(54)	543	215	B2(20)12	529	274
						18-54(55)	551	220	B2(20)13	537	279
18-57(56)	546	218	B2(20)14	541	281

Thus, it can be concluded that the fertility of the first (and subsequent) generation polyploid plants is not reduced compared to the diploid plants. However, clear significant differences in yield levels (by pairwise analysis) were exhibited for 4N plants compared to 2N isogenic plants. The difference is about a 30% increase in seed yield of polyploid plants compared to isogenic diploid plants, primarily due to the greater seed weight of the polyploids.

Seed fatty acid distribution: the major castor seed fatty acids are oleic, ricinoleic and linoleic (table 4 below). The results presented show that the difference in acid content between the tetraploid and diploid lines is no greater than between plants within each line. This strongly suggests that doubling of the source plant does not result in serious mutations that may affect the basic function of the plant.

FIG. 4 fatty acid profiles of Castor diploid (2n) and tetraploid (4n) lines

Tying code	B2(807)	B2(11)20	I3(4)B	I3(3)35	EXPR20
						Ploidy level	2N	4N	2N	4N	4N
Generation of generation	F5	F5	F5	F5	F1
						C 8:0	-	0.01	-	0.01	-
C10:0	-	-	-	0.01	-
						C 12:0	-	-	-	-	-
C 14:0	-	0.01	0.01	0.02	-
						C 14:1	-	-	-	-	-
C 15:0	0.02	0.02	0.01	0.01	-
						C 16:0	1.46	1.68	1.52	1.70	1.85
C 16:1	0.01	0.02	0.01	0.02	0.02
						C 17:0	0.03	0.06	0.06	0.06	0.03
C 17:1	0.02	0.15	0.13	0.15	0.02
						C 18:0	1.09	1.19	1.68	1.46	1.44
C18: 1 (oleic acid)	4.78	4.39	4.91	5.90	5.63
						C18: 1OH (ricinoleic acid)	83.56	83.94	83.75	83.37	82.90
C18: 2 (linoleic acid)	7.02	6.68	5.93	5.58	6.54
						C 18:3	0.68	0.64	0.59	0.56	0.60

C 19:1	0.02	0.01	0.02	0.01	0.02
						C 20:0	0.05	0.04	0.07	0.07	0.06
C 20:1	0.56	0.46	0.47	0.46	0.48
						C20:2	0.10	0.10	0.10	0.09	0.10
C 20:3	0.02	-	-	-	-
						C 20:4	0.02	-	0.07	-	0.02
C 20:5n3	-	-	-	-	-
						C 21:0	-	-	-	-	-
C22:0	0.03	0.01	0.01	0.01	-
						C 22:1	-	-	0.02	-	0.01
C 22:2	0.10	0.09	0.06	0.04	0.08
						C22:3	-	-	0.02	-	-
C 22:5n3	-	-	-	-	-
						C 22:6n3	-	-	-	-	-
C23:0	-	-	-	-	-
						C24:0	0.02	0.02	0.02	0.02	0.02
C24:1	-	-	-	-	-
Sum of	99.59	99.51	99.46	99.53	99.82

Volumetric oil content-table 5 below presents the oil content in 3N plants. The results show that 3N plants have similar oil content as 2N plants.

TABLE 5

1	Par-21	42.40	Control	2N
					2	B1-19	41.00	Control	2N

3	CH-2	41.70	Control	2N
					4	C-181	44.10	Control	2N
6	EXPR 10	47.40		2N
					7	EXPR 11			2N
8	EXPR 12			2N
					9	EXPR 13	47.10		2N
10	EXPR 14	42.80		2N
					11	EXPR 15	41.80		2N
12	EXPR 16			2N
					13	EXPR 17			2N
14	EXPR 18	43.10		2N
					15	EXPR 19	44.50		2N
16	EXPR26	46.00		3N
					17	EXPR27	44.10		3N
18	EXPR29	42.70		3N
					19	EXPR 31			3N
20	EXPR33			3N
					21	EXPR 39			2N
22	EXPR40	44.70		2N
					23	EXPR44	48.10		2N
24	EXPR46	47.60		2N
					25	EXPR 50	43.80		3N
26	EXPR 55	44.70		2N
					27	EXPR 60			3N

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of all references in this application shall not be construed as an admission that such references are available as prior art to the present invention. For the use of section headings, they should not be considered as necessarily limiting.

Reference to the literature

(other references are cited throughout the application)

1.Dhawan，O.P.(1996)Enhancing the productivity of secondary metabolites viainduced polyploidy：a review.Euphytica87(2)

2.Moshkin，V.A.and A.G.Dvoryadinka.Castor Genetics.In Castor.Ed.V.A.Moshkin.Amerind Publ.Co.，New Delhi.1986.pp.93-102.

3.Nagl，W.(1984)The fluorophenylalanine sensitive and resistant tobacco cell lines，TX1 and TX 4 1.DNA contents，chromosome numbers，nuclear ultrastructures，andeffects of spermidine.PROTOPLASMA 122

4.D_ARREN J.O_BBARD，S_TEPHEN A.H_ARRIS，R_ICHARD J.A.B_UGGS，J_OHN R.P_ANNELL(2006)HYBRIDIZATION，POLYPLOIDY，AND THE EVOLUTIONOF SEXUAL SYSTEMS IN MERCURIALIS(EUPHORBIACEAE)Evolution 60(9)，1801-1815.doi：10.1111/j.0014-3820.2006.tb00524.x

5.M.P.Timko，A.C.Vasconcelos(1981)Ptiotosynthetic activity and chloroplastmembrane polypeptides in euploid cells of Ricinus Physiologia Plantarum 52(2)，191-196.

6.Comparative study of chlorophyll content in diploid and tetraploid black wattleMathura et al.Forestry.2006；79：381-388

Claims (25)

1. A polyploid castor plant, having fertility at least as great as that of a diploid castor plant isogenic to said polyploid castor plant when grown under similar conditions.

2. The plant of claim 1, wherein said fertility is determined by at least one of:

number of seeds per plant;

analyzing the reproductive capacity of gametes; and

carmine acetate was stained.

3. The plant of claim 1, exhibiting genomic stability for at least 5 passages.

4. The plant of claim 1, having at least similar seed yield as said diploid castor plant.

5. The plant of claim 1, having at least similar oil yield as said diploid castor plant.

6. The plant of claim 1, having greater leaf surface area than said diploid castor plant.

7. The plant of claim 1, having a larger stomatal surface than said diploid castor plant.

8. The plant of claim 1, which is a tetraploid.

9. The plant of claim 1, capable of cross-breeding with a diploid plant.

10. The plant of claim 1, being an autopolyploid.

11. The plant of claim 1, being an inbred.

12. A castor plant deposited under the budapest treaty at NCIMB ltd and having accession number NCIMB 41593 castor B2-20-4N.

13. A plant part of the castor plant of any one of claims 1-12.

14. Castor oil produced from the plant or plant part of any one of claims 1-13.

15. Castor meal produced from the plant or plant part of any one of claims 1-13.

16. The plant part of claim 13, which is a seed.

17. An isolated regenerable cell of the castor plant of any of claims 1-12.

18. The cell of claim 16, exhibiting genomic stability for at least 5 passages in culture.

19. The cell of claim 17, which is from a meristem, pollen, leaf, root tip, anther, pistil, flower, seed or stem.

20. A tissue culture comprising the regenerable cells of claim 17 or 19.

21. A method of producing castor seed comprising self-breeding or cross-breeding the plant of any one of claims 1-12.

22. A method of producing castor oil, the method comprising:

(a) harvesting seed of the castor plant or plant part of any of claims 1-13; and

(b) processing said seed to produce castor oil.

23. A method of producing a polyploid castor seed, the method comprising contacting a castor seed with a G2/M cell cycle inhibitor under a magnetic field, thereby producing a polyploid castor seed.

24. The method of claim 23, wherein said G2/M cell cycle inhibitor comprises a microtubule polymerization inhibitor.

25. The method of claim 24, wherein said microtubule polymerization inhibitor is selected from the group consisting of colchicine, nocodazole, oryzaline, trifluraline and vinblastine sulfate.