WO2010034997A1 - Methods of obtaining plants and novel plants obtainable thereby - Google Patents

Abstract

The invention relates to a method of obtaining mixoploid plant material useful in plant breeding and commercial production, and to plants obtainable thereby, including mixoploid oil palm.

Description

Methods of obtaining plants and novel plants obtainable thereby

Field of invention

The present invention relates to a method of obtaining mixoploid plant material useful in plant breeding and commercial production, and to plants obtainable thereby. Plants obtainable according to the invention include mixoploid plants comprising haploid regions as well as diploid, triploid, tetraploid regions and regions of higher ploidy. Also included are plants obtainable from said regions.

Background of the invention - plant breeding

Although plant breeding programmes worldwide have made considerable progress in developing new cultivars with improved yield, quality, disease resistance and other useful traits, breeding is primarily a stochastic process. It typically involves generating and screening large numbers of individuals to identify rare types containing novel and desirable trait combinations. For this purpose very large numbers of progeny from crosses need to be screened over several seasons in order to select one or a few plants with the desired characteristics.

In a typical plant breeding programme, two plants (parental lines) are crossed: the resultant progeny are screened and one or more plants possessing the desired phenotype (combination of expressed traits) are identified and selected. The selected plant or plants may then be self-pollinated or crossed to yield a second-generation progeny population. This population may undergo another round of individual plant screening to select from it those lines that possess the desired traits originally introduced in the first generation. If, as is often the case, the phenotypic traits are derived from the combined effect of several genes, then the number of progeny plants that must be screened depends on the number of genetic differences between the two original parental lines. Thus, the greater the number of genetic differences the greater the number of plants that need to be grown and evaluated and the lower the probability of obtaining progeny with all the desired traits. The problem is exacerbated when the desired traits include yield and quality characteristics that can only be determined once the plants have reached maturity.

One possible solution to the problem of screening large numbers of individuals in a progeny that segregates for traits of interest depends on the ability to produce or identify haploid or double haploid plants derived from the gametic cells of parental individuals. The chromosome complements of haploids sometimes double spontaneously to produce homozygous doubled haploids (DHs). Mixoploids, which are plants which contain cells having different ploidies, can sometimes arise and may represent plants that are undergoing chromosome doubling so as to spontaneously produce doubled haploid tissues, organs, shoots, floral parts or plants. A phenomena which has been observed by the inventors is that the proportion of haploid:diploid cells in, for example, leaves of a mixoploid plant may change as the plant grows. As such mixoploid plants are valuable sources for generating homogenous doubled haploid plants in addition to producing doubled haploids, mixoploids have the potential to generate, haploid, triploid, tetraploid or other polyploids as well as chimeric and mixoploid plants which are valuable for breeding. Furthermore, haploids produced from mixoploid plants may be induced to double by treatment with certain chemicals, for example colchicine. The resultant doubled haploid plants, regardless of how they are derived, are instantly and completely homozygous. This means that on selfmg the plants breed true, i.e. their progeny are genetically identical to the parent doubled haploid plant, so clones can be generated and multiplied rapidly. Furthermore, when two such doubled haploids are crossed sexually the resultant hybrid is genetically invariant and heterozygous for all loci that differ between the two parents. With a sufficient stock of the two parental doubled haploids, the same F₁ hybrid can be produced repeatedly and reliably and grown commercially.

The ploidy level of somatic cells is defined as the number of genome sets of chromosomes that they contain. A genome set of chromosomes (also known as the base number, x) is most simply described as the number of heterologous chromosomes present in the nuclear genome and equals n, the number present in gametic cells of a diploid organism. For example, humans are a diploid species, having 2n=2x=46 chromosomes in their somatic cells and n=x=23 in their gametes (sperm and egg). When the ploidy level is greater than one, genetic analysis is made more difficult by the effects of dominance. When more than one copy of a gene is present, only one copy, the dominant one, may influence the phenotype: or else both copies can contribute to the expression of the phenotype (partial or no dominance). With dominance, the other copy of the gene (the recessive allele) is masked: as a consequence its presence cannot be determined by the observed phenotype. Haploid organisms contain the same number of chromosomes (n) in their somatic cells as the normal gametes of the species have. The term haploid sporophyte is generally used to designate sporophytes having the gametic chromosome number.

Mixoploid sporophytes of higher plants can be distinguished from diploids in many ways. They may be smaller, partly because they can contain smaller cell sizes in haploid regions or larger, due to the presence of cells of higher than normal ploidy. In general terms, cell volume in plants is positively correlated with ploidy level. Several methods for the provisional assignment of ploidy status to a plant exploit phenotypic characteristics. The most widely used of these methods is the measurement of stomatal guard cell length and chloroplast content in these cells. In some fast-growing annuals such as barley, haploidy, for example, is easily identified because the haploids are sterile. However, none of the phenotypic predictors of altered ploidy are absolutely reliable. Methods that directly measure genome size are far more reliable. These include direct measurement of the chromosome number using conventional chromosome counting techniques, and measurement of the DNA content using microdensitometry or more especially by flow cytometry (Coba de Ia Pena and Brown, 2001). It is also possible to exploit the absolute absence of heterozygosity in haploids and doubled haploids to detect such plants using various co-dominantly inherited molecular marker methods (e.g. Chani et al., 2000; Tang et al., 2006).

Haploids, which can be produced from mixoploids may have intrinsic value because of their overall reduction in size compared with diploids. Haploids also have value in allowing the isolation of mutants, which may be masked in a diploid, particularly where the mutant allele is either non-functional or recessive. Haploids also have value in transformation programmes. If haploids are transformed directly, then true-breeding doubled haploid transgenic plants can be produced in one step following chromosome doubling of transgenic haploids. A wide range of techniques for chromosome doubling are known (Kasha, 2005) and these techniques, or modifications of them, may be used in the present invention.

An important use of such plants derived from mixoploids is based on the fact that marked improvements in the economics of plant breeding can be achieved via doubled haploid production, since selection and other procedural efficiencies can be markedly improved through the provision of elite true-breeding (homozygous) lines (Nei, 1963). With doubled haploid production systems, homozygosity is achieved in one generation. Thus, the breeder can eliminate the numerous cycles of inbreeding that are usually necessary to achieve practical levels of homozygosity by conventional methods. Indeed, absolute homozygosity for all traits is not achievable by conventional breeding methods. Consequently, an efficient doubled haploid technology would enable breeders to reduce the time and the cost of cultivar development relative to conventional breeding practices.

Spontaneous haploids may occur in many species of plants, usually at very low frequencies. However, for some species, for example, rubber, there are no spontaneous haploid plants reported. For this reason, emphasis has turned to alternative means of generating haploids and doubled haploids.

Mixoploids arise at an even lower level than spontaneous haploids, occurring at a frequency of less than 0.5%.

As well as having value in their own right as potential new varieties, homozygous doubled haploid plants produced from mixoploids also have utility for the generation of Fi hybrid plants, produced from crosses made between selected homozygous male and female parents. These F₁ plants are also of value as cultivars as they may exhibit so-called hybrid vigour (heterosis), a characteristic often associated with dramatic increases in yield compared with either parent. Furthermore, the production of Fi hybrids allows the breeder to produce large quantities of uniform seed of a single genotype from homozygous parental lines. F₁ hybrids have many advantages over a genetically heterogeneous mix of genotypes because one can select single elite genotypes that possess various desirable characteristics, for example high yield. One may also achieve higher yields by selecting genotypes adapted to specific environments and to optimise agronomic and management practices. In many crops, the only way of producing a single genotype in commercial quantities has previously been by asexual cloning, using suckers, cuttings or grafts. Thus Fi hybrid production from doubled haploid parents is a rapid method of achieving distinctive, uniform and stable (DUS) crop varieties demanded by many national regulatory authorities. Haploid (H) plants (and doubled haploids - DH plants) express all their genetic information or, in other words, their genotype is completely displayed by their phenotype. Resistance to pest and diseases or unfavourable external factors (drought, salinity, heavy metal toxicity, temperature, light etc.) can thus be directly recognized and selected. Haploid plants allow the detection of mutants that are unable to pass through the embryonic phases of development. They also allow: 1) screening for both recessive and dominant mutants in the first generation after mutagenic treatment, 2) immediate fixation of mutant genotypes via doubled haploidy, 3) increased selection efficiency, and 4) applying in vitro selection methods at the haploid or doubled haploid level. For similar reasons haploid plant tissues make ideal vehicles for genetic transformation, to give genetically modified haploids that on doubling give homozygous diploids containing the introduced gene or genes.

The agricultural applications for mixoploids exploit their capacity for the rapid generation of homozygous genotypes after chromosome doubling of haploid regions, with advantages including:

• Reduced time for variety development;

• Homozygous recombinant lines can be developed in one generation instead of after numerous backcross generations;

• Selection for recessive traits in recombinant lines is more efficient because recessive alleles are not masked by the effects of dominant alleles; and

• Introduction of "alien" genes is speeded up by allowing homozygotes to be developed readily.

The crossing of two homozygous elite lines (such as can be produced by doubling haploids) can generate genetically uniform, highly heterozygous hybrid varieties, as is exemplified by the highly successful hybrid maize varieties first produced in the USA during the 1930s. There have been many efforts to reproduce the yield increase gained in hybrid maize varieties in other crops. Other examples include: F₁ hybrid varieties of sunflower, sugar beet and carrot which are now widely grown, and hybrid lines of oilseed rape (canola) and rice which are becoming increasingly available; more than half the rice grown in China is hybrid, with yields at least 20% higher than the non-hybrid equivalent. However, to date, there has been no corresponding progress in other important crop species, for example, oil palm and rubber. The lack of progress with hybrid cultivar production is principally because of the traditional cropping systems used in these species. In rubber this is based on grafted clonal material. F₁ hybrid production has not been an option as no homozygous, true breeding lines (doubled haploids or other inbred lines) have been available in rubber. In oil palm the breeding system of the crop precludes the simple production of inbred lines. Oil palm is essentially an outbreeding species, but unlike corn in which a male and a female flower are produced on the same plant at the same time, each oil palm plant produces either male or female flowers at any one time, and therefore a palm can only readily be self-pollinated by methods of controlled pollination using stored pollen. Progress in converting oil palm into a hybrid crop, and thereby exploiting the potential hybrid vigour, depends upon the development of a process for the reliable production of homozygous plants. To date, however, there is no published example of any haploid, or homozygous diploid oil palm plant. There has nevertheless been extensive breeding (Wahid et al., 2004), cell culture (Abdullah 2005; Abdullah et al., 2005; Rival and Parveez, 2005; Te-chato et al., 2005), and transformation studies (US Application 20030159175) geared towards the genetic improvement of the oil palm crop.

Several important crops are polyploids, for example the Cavandish banana is triploid, potato is tetraploid and bread wheat and oat are hexaploids. Ploidy levels of crops can be either natural or induced. Increasing the ploidy of a crop via mixoploids or other techniques can have several advantages. By simply increasing the copy number of the genome (from diploid to triploid, tetraploid or higher ploidy) the nucleus of the cell is enlarged proportionally. As a consequence cell size increases, which in turn increases tissue and organ size. The later often being harvestable products. Induced polyploidy to increase crop yields began in the 1930s and was successful in producing new varieties in vegetable crops such as Swedes (rutabaga). Odd ploidy levels such as triploids are meiotically unbalanced and often result in sterile, seedless fruits, as in banana. Sterility of fruits can lead to greater vegetative growth, as resources that would normally be used in seed development are diverted to vegetative tissues. In China, triploids in rubber have been reported to have increased latex yields by about 20% compared to a standard (FAO, 1995) and are therefore of commercial interest.

For the reasons discussed above, it is therefore an object of the present invention to provide a method of identifying and isolating mixoploids plants of various species, particularly palms, including oil palms, and rubber.

Summary of invention

In accordance with a first aspect of the current invention, there is provided a method for obtaining mixoploid plants useful for seed production, multiplication and crop improvement, said method comprising:

(i) providing a population of plants;

(ii) choosing from the population a subset of individual plants with atypical phenotype;

(iii) assessing the DNA content in cells extracted from the subset of plants; (iv) identifying plants in the subset that are mixoploid.

Mixoploids may represent plants that are undergoing chromosome doubling, i.e. they are developing spontaneously into double haploids. This can be monitored by repeating the ploidy analysis at different stages as the plant develops.

In a first preferred embodiment, the atypical phenotype is an atypical morphology or growth pattern.

Preferably the atypical morphology or growth pattern is a seed, seedling, plantlet or plant trait. In one embodiment this may be retarded or accelerated growth. More preferably, the atypical morphology is one or more of: radicle growth; radicle :plumule length ratio; radicle:plumule angle; colour of radical, plumule or leaf; seed shape or size during germination; radicle width:length ratio; plant height, stem morphology, petiole morphology, venation, distance between leaf whorls and the number of leaves per whorl , distance between leaf nodes, slower vegetative growth, altered leaf and stem shape and/or size, leaf angle, pigmentation, branching pattern, altered reproductive tissues and altered flowering time. The atypical phenotype of a germinated seed may also be the germination of two or more embryos from a single seed.

Preferably, the plants are germinated seeds or seedlings.

It will be understood that any suitable population of plants can be used in the methods of the current invention. Preferably, the population of plants comprises in vitro, nursery or field grown plants.

In preferred embodiments the plants are palms, or rubber plants. More preferably, the plants are oil palms. It will be apparent to the skilled person that the methods of the current invention are equally applicable to other plant species.

Preferably the step at assessing the ploidy level of atypical plants to identify mixoploids will apply flow cytometry to assess ploidy levels in cells extracted from root, shoot, leaf or other plant tissue.

According to a second aspect there is provided a method for producing haploid plants comprising:

(i) identifying mixoploid plants according to the method of the first aspect;

(ii) identifying a region of said mixoploid plant which comprise at least one haploid cell;

(iii) propagating plants and clones from said haploid region or at least one haploid cell.

Said haploid region or cell of step (ii) may be subjected to a chromosome doubling step to produce doubled haploid cells prior to step (iii) wherein said plants produced in step (iii) are doubled haploids. In a first preferred embodiment, the doubled haploid is obtained through spontaneous chromosome doubling; or by doubling the chromosome number by application of an external stimulus to the haploid region or plant in vivo or in vitro.

In a second preferred embodiment, the doubled haploid is obtained by the application of an external stimulus to at least one cell isolated from a haploid region or plant.

According to a third aspect of the present invention, there is provided a method for producing homozygous doubled haploid plants comprising: (i) identifying mixoploid plants according to the method of the first aspect; (ii) identifying regions of said mixoploid plant which comprise diploid cells; (iii) assessing the homozygosity of said diploid cells to identify doubled haploids; (iii) propagating plants and clones from said doubled haploid regions or cells.

According to a fourth aspect of the present invention, there is provided a method for producing triploid plants comprising:

(i) identifying mixoploid plants according to the method of the first aspect;

(ii) identifying regions of said mixoploid plant which comprise triploid cells;

(iii) assessing the homozygosity of said triploid cells; (iv) propagating plants and clones from said triploid regions or cells.

According to a fifth aspect of the present invention, there is provided a method producing tetraploid plants or plants of higher ploidy comprising:

(i) identifying mixoploid plants according to the method of the first aspect; (ii) identifying regions of said mixoploid plant which comprise tetraploid or higher ploidy cells;

(iii) assessing the homozygosity of said cells;

(iv) propagating plants and clones from said tetraploid or higher ploidy regions or cells. It will be understood that the following preferred features of the invention are applicable to any of the preceding aspects described above.

Preferably, the homozygosity screening step uses molecular or biochemical markers. More preferably multiple biochemical or molecular markers. A preferred technique is to use multiple molecular markers, for example between 2 and 40, these may be microsatellite markers (also known as simple sequence repeats, SSRs), or Sequenced Characterised Polymorphic Regions (SCARs) markers or Single Nucleotide Polymorphism (SNP) markers, or any other suitable polymorphic marker. It is convenient to employ co-dominant molecular or biochemical markers, particularly microsatellite markers, although many other marker systems could also be applied, for example, protein profiling, isozymes, High Resolution Melt analysis or pyrosequencing. Even more preferably, multiple molecular markers are used and the analysis is performed on a pooled sample of markers. A chosen plant is identified as highly homozygous if it is homozygous for each molecular marker used.

Preferably the step at assessing the ploidy level of atypical plants to identify mixoploids will apply flow cytometry to assess ploidy levels in cells extracted from root, shoot, leaf or other plant tissue.

The order in which the various possible stages of the method are carried out is not necessarily fixed. For example, an assessment of homozygosity may be carried out before an assessment of the ploidy levels. In this case, all plants in the set will be screened for homozygosity, not just the diploids: however, since the vast majority of plants in the set are diploid, this makes little difference in practice.

It will be understood that plant regions or cells are considered heterozygous unless they show only one allele per locus for each co-dominant marker.

It will be further apparent that the identification of mixoploid regions or cells in a progeny may indicate an increased likelihood of the presence of doubled haploids in that progeny. Said progeny can be intensively screened to identify completely double haploid plants or plants possessing doubled haploid regions or cells.

Furthermore, the identification of mixoploids in a progeny may indicate an increased likelihood of the parental crosses producing doubled haploids, crosses thus identified can be repeated and the progeny screened to obtain more doubled haploids.

Preferably the plants are palms or rubber plants. More preferably the plants are oil palms.

According to a sixth aspect of the present invention there are provided progeny plants produced from somatic or reproductive cells of a plant produced according to any of aspects two to five.

According to a still further aspect there are provided clones, pollen or ovules from plants obtained by a method according to any of aspects two to six.

According to another aspect there is provided a mixoploid or chimeric plant obtainable by the method of the first aspect.

Also provided is a haploid plant obtainable by the method of the second aspect.

A doubled haploid plant obtainable by the method of the second or third aspect.

A triploid plant obtainable by the method of the fourth aspect.

A tetraploid or higher polyploid plant obtainable by the method of the fifth aspect.

It will be understood that one or more of the plants produced by the methods of the second to fifth aspects can be subsequently used in breeding, multiplication or seed production. According to a further aspect there is provided a method comprising crossing two distinct doubled haploids produced by the methods of the current invention to produce a Fi hybrid.

Also provided is a F₁ hybrid produced by the method of the previous aspect.

In a further aspect, the present invention provides haploid, mixoploid and double haploid plants produced from tissue culture of mixoploid plant material.

One convenient application of the invention is to select candidate mixoploid seedlings from germinated seed after the plumule and radical have developed or, for example in the case of rubber, once the first whorl of leaves has developed. Cohorts of germinating seedlings typically exhibit a fairly synchronous developmental pathway and reasonably homogenous phenotype. Abnormal germinated seed may deviate from the characteristic phenotype in one of many ways which may include diverse atypical features of morphology or growth pattern. The atypical morphology or growth pattern may be reduced plant, organ and tissue growth and size, or germination of two embryos from a single seed (multiple seedlings). The atypical growth pattern may be accelerated or retarded growth. The atypical morphology or growth pattern may be one or more of atypical radicle growth; radicle :plumule length ratio; radicle :plumule angle; colour of radicle, plumule or leaf; seed shape or size during germination; altered radicle width:length ratio; altered plant height; stem or petiole morphology; venation; distance between leaf whorls or leaf nodes; number of leaves per whorl; slower vegetative growth, altered leaf and stem shape and size; leaf angle; pigmentation; branching pattern; altered reproductive tissues and/or altered flowering time; plant height and stature and morphological variation of one or more of these characteristics within a plant, for example, between leaves.

Another way of carrying out the invention is to select among a population of nursery or field planted plants. In this method, the atypical morphology or growth pattern used as the basis of selection is preferably one or more of: slower vegetative growth, plant height and stature, reduced ratio of leaflet width to length, altered inter- whorl distance, angle of leaf to plant axis, leaf colour, branching pattern, precocious flowering and morphological variation within a plant.

A preferred process is one in which the atypical phenotype by which plants are selected is chosen from atypical phenotypes shown from previous tests to correlate with mixoploid characters. This process progressively improves accuracy of the phenotypic screen as increasing numbers of off-types are observed, as traits are correlated with ploidy type and uninformative traits are discarded from consideration.

Preferably, the population of plants comprises at least 500 particularly 750 - 2,500 individual plants. By "providing germinated seeds or seedlings", we refer to any process whereby seeds sprout and seedlings begin to grow.

In the case of rubber, this includes any germination technique used by commercial and plant breeding seed production units. Seed tested may include seeds selected using the bounce test (Setyamidjaja, 1993) as well as those that don't bounce which would normally be rejected by commercial breeders and growers. Seeds are normally placed in a sand bed for germination.

In the case of oil palm, it includes both the germination techniques commonly used by commercial and plant breeding seed production units, the wet heat method and the dry heat method. The former method is now less used. The whole process may be shorter (95 days against 120 days for dry heat), but some germination will take place during the heating period and so a less uniform set of seedlings will be produced. Oil palm seed is dormant when it is harvested, and under natural conditions germinates sporadically over several years. The critical requirement to break dormancy is to maintain the seed at a raised temperature of 39-4O⁰C for up to 80 days.

Flow cytometry is used for assessing the genome content of plant or animal cells, and can be used to distinguish between haploid, diploid, triploid, tetraploids and polyploids as well as mixoploid material. By "flow cytometry" is meant methods for counting, examining, quantifying and sorting analyte suspended in a stream of fluid. It permits simultaneous analysis of two or more characteristics of single cells flowing through an optical or electronic detection apparatus. In this invention, flow cytometry may be applied to individuals exhibiting an abnormal phenotype. In the first instance flow cytometry will be used to divide the population into haploids, diploids, other higher ploidies and mixoploids.

Definitions

Following are definitions of words used in the specification and claims:

"plant" means any seed and any growing plants at any stage of development, for example germinated seeds, seedlings, nursery and mature plants, including plants in vitro culture.

"haploid" means any plant cell containing a single set of chromosomes, or any tissue or plant comprising such cells.

"diploid" means any plant cell containing two sets of chromosomes, or any tissue or plant comprising such cells

"triploid" means any plant cell containing three sets of chromosomes, or any tissue or plant comprising such cells.

"tetraploid" means any plant cell containing four sets of chromosomes, or any tissue or plant comprising such cells.

"polyploid" means any plant cell containing multiple sets of chromosomes, or any tissue or plant comprising such cells.

"mixoploid" means a plant containing cells of two or more different ploidies;

"heterozygous" characterises any cell containing two or more sets of chromosomes that are not all identical sets; or any tissue or plant composed of such cells. Material which is not heterozygous is either homozygous or haploid (containing only one set of chromosomes).

"plantlet" means any small plant which is not fully grown. [1] Origin of materials used

The oil palm germplasm (Elaeis guineensis Jacq) used in the following experiments was obtained in Indonesia (Sumatra) where the first stage of the procedures (selection of material of atypical phenotype) was carried out. The historic origin of the oil palm (Elaeis guineensis) is understood to be West Africa, where it has been cultivated for many years: the species was introduced from West Africa to the Pacific region in the first half of the last century, since when it has been widely cultivated throughout that region.

Examples

Identification and development of mixoploid oil palm plants

_L Seed processing

1 The mesocarp was mechanically removed from oil palm seeds and the seeds were air dried for 24 hours at ambient temperature and then for 24 hours in an air- conditioned room at 25 ⁰C to a seed moisture content of 15-18%. The seeds were then stored, usually for one to three months in an air-conditioned room (25 ⁰C) in plastic bags or trays (although it is possible to store seeds for up to one year in this way).

2 The seeds were soaked for three days to increase their moisture content to 18 - 20 % and then heat treated in plastic bags or trays for 40 to 60 days at 38 - 40 ⁰C.

3 After heating, the seeds were soaked for five days to raise their moisture content to >22% and then dried at ambient temperature for approximately four hours.

4 The seed were transferred to a germination room where under ambient temperatures germination usually starts after 7 to 10 days and continues for two to three months.

2. Morphological screen

There were two large-scale morphological screens of oil palm seedlings for morphological off-types. The first consisted of 10,900,000 germinated seeds, of which 3,854 were identified as being morphologically deviant (3,801) or twin-seeded (53), with the remaining individuals all being deemed 'normal'. Thus, in this instance 99.96% of seeds evaluated were classified as exhibiting a normal phenotype and 0.035% being aberrant. In the second screen, approximately 10,000,000 commercial seedlings were screened, together with approximately 1,000,000 seedlings taken from breeding experiments. This trial generated 5,704 morphological candidates, of which 5,601 were phenotypically abnormal and 103 were twin-seeded. In this screen, therefore, 99.95% of seedlings were classed as normal and 0.05% as aberrant prior to transfer to the nursery house.

3. Assessment of nuclear genome content by flow cytometry

Flow Cytometry

Individuals identified as morphologically abnormal were subjected to flow cytometry to establish their ploidy level using the following protocol.

Sample preparation for flow cytometry

The cell nuclei were isolated from fresh plant material (leaves or roots), by chopping the plant material (a few cm² /20-50 mg) with a sharp razor blade in an ice-cold buffer, in a plastic Petri dish. The DNA buffer (stored at 4 ⁰C) is based on: Arurnuganathan, K. and Earle, E.D. Estimation of Nuclear DNA Content of Plants by Flow Cytometry. Plant Molecular Biology Reporter, VoI 9(3) 1991, Pages 229-233.

5mM Hepes; 1OmM Magnesium sulphate heptahydrate; 5OmM Potasiuin chloride; 0.2% Triton X-100; 2% DTT (Dithiothreitol); 2mg/litre DAPI pH 8.

DAPI, a fluorescent dye that selectively binds to form a complex with double-stranded DNA and give a product that fluoresces at 465 nm, was introduced to the solution. DAPI has specific DNA-binding properties, with preference for adenine-thymine (AT)-rich sequences. After chopping, the buffer (ca. 2 ml.), containing cell constituents and large tissue remnants, is passed through a nylon filter of 40 micrometer mesh. This method will produce thousands of nuclei from a leaf piece of a few cm . The solution containing stained nuclei was passed through the flow cytometer. Controls are required of known ploidy (DNA content) as reference - for oil palm, tissue from diploid Tenera palms were used because the shell thickness must be heterozygous and therefore the palm cannot be haploid.

The fluorescence of the stained nuclei, passing through the focus of a light beam from a high-pressure mercury lamp, was measured by a photomultiplier and converted into voltage pulses.

These voltage pulses were electronically processed to yield integral and peak signals that can be processed by a computer. When the samples are run with the appropriate filter- settings for excitation and emission, DNA histograms can be produced.

Material

Flow cytometer: CyFlow ML (Partec GmbH, Otto Hahnstrasse 32, D-4400 Munster, Germany) with a high pressure mercury lamp, OSRAM HBO 100 long life. Objective: 40 x N. A. 0,8 air (Partec); Filter combination with DAPI: Heat protection filter KG-I; Excitation-filters: UG-land BG-38. Dichroic mirrors: TK 420 and TK 560. Emission- filter: GG 435. Software: Flomax version 2.4 d (Partec).

Flow cytometry of plants having an abnormal phenotype identified 8 plants which were composed of both haploid and diploid cells. Further analysis of these spontaneous mixoploids showed that these plants were composed of different ratios of haploid and diploid cells as shown in Table 1.

Table 1

The genome of the diploid regions of the plants identified as mixoploids were further assessed for homozygosity.

4. Screening of mixoploids for homozygosity

1. DNA extraction

2. Amplification of microsatellite markers by PCR

3. Separation of PCR products by agarose gel electrophoresis

4. Scoring of results to discard identify the homozygosity of the mixoploid plants

DNA extraction

Around 0.5 cm of the radicle (around 50 mg) was removed from the seedling and used to extract DNA using the Qiagen 96 DNeasy extraction kit according to the manufacturer's instructions as described below, although other systems for DNA extraction could also be used.

A. PREPARATION

1. For new kits, add 100% ethanol to AP3/E buffer and AW buffer

2. Set water bath to 65⁰C

3. Preheat AE and API buffer to 65⁰C

4. If API buffer has a cloudy appearance, heat to 65⁰C and shake until the solution becomes clear B. PROTOCOL

1. Add 50 mg plant material into each tube in two collection microtube racks. Retain the clear cover.

2. Add one tungsten carbide bead into each microtube.

3. Prepare the lysis solution: (400 μl APl+ 1 μl RNAse + 1 μl Reagent DX)/reaction plus 15% of each component.

4. Disrupt the sample using MM 300, 30 Hz for 1.5 minutes.

5. Pulse centrifuge to 3000 rpm.

6. Remove and discard caps, add 130 μl AP2 buffer into each collection microtube.

7. Close the microtubes with new caps. Place a clear cover (from step 1) over the 96 well plate. Shake the plate vigorously for 15s. Pulse centrifuge to 3000 rpm.

8. Incubate the racks for 10 min at -20°C.

9. Remove and discard the caps. Transfer 400 μl of each supernatant to new plate of collection microtubes (provided). Do not transfer pellet and floating particles. Hold the strips and use the lowest pipette speed. Recover the tungsten beads.

10. Add 1.5 volume (typically 600 μl) of AP3/E buffer.

11. Close the microtubes with new caps and mix vigorously.

12. Pulse centrifuge (3000rpm) to collect solution).

13. Place 96 well plates on top of S-Blocks provided.

14. Transfer ImI of sample into each well of the 96 well plate.

15. Seal with Airpore Tape sheet and centrifuge for 4 min at 6000 rpm.

16. Add 800 μl of Buffer AW to each sample.

17. Centrifuge for 15 min at 6000 rpm. 18. Add 100 μl of buffer AE to each sample and seal with new AirPore sheets.19.

Incubate for 1 min at room temperature (15-25⁰C).

20. Centrifuge for 2 min at 6000 rpm.

Amplification of microsatellite markers by PCR

The microsatellite markers shown in Table 2 were used.

Table 2

Reaction Mixtures

In all cases, 10 μl a PCR reaction mixture contained the following reagents; 1.0 μl of 10x PCR buffer (Bioline), 0.3 μl MgCl₂ (10 mM), 0.4 μl dNTPs (10 mM of each), 0.2 μl of each primer pair (10 μM), 1-5 ng of DNA (extracted as above) and 1 U of Taq DNA polymerase (5 U μl^"1 Bioline). PCR conditions

The following conditions were used for the Polymerase Chain Reaction (PCR) for all microsatellite markers: an initial 94⁰C denaturing step for 2 min followed by 35 cycles of; 94⁰C for 30 sec, 52⁰C for 30 sec and 72⁰C for 45 sec, with a final extension step of 72⁰C for 7 min.

Separation of PCR products by agarose gel electrophoresis

Agarose gel electrophoresis and ethidium bromide staining were routinely used to fractionate and visualise products generated by microsatellite PCR.

Reagents

TBE running buffer: 0.089 M Tris base, 0.089 M boric acid, (pH 8.3) and 2 mM Na₂EDTA

Loading buffer: 0.23% (w/v) bromophenol blue; 60 mM EDTA; 40% (w/v) sucrose

Ethidium bromide stain: 1% (w/v) ethidium bromide

Ladder 100 bp (Gibco Life Science BRL)

Gel preparation and loading

1.0 - 1.5 % (w/v) agarose (was prepared in 1 x TBE buffer and subjected to heating in a microwave (700W) for 2 x 1 min at full power to create a gel solution. The gel solution was cooled to approximately 55⁰C prior to the addition of ethidium bromide (3.5 μl per 100 ml gel). The ends of a suitable gel tray rig (midi-gel tray for 100 ml gels, maxi-gel tray for 250 ml gels) were sealed with masking tape and an appropriate number and type of combs placed in position. Combs with 16 x 20 μl wells were most often employed. The gel solution was carefully poured into the prepared tray and allowed to cool for at least 20 min. Combs and tape were then removed and the gel tray submerged into a tank containing 1 x TBE buffer.

Generally, 5 μl of sample were mixed with 2 μl of bromophenol blue buffer prior to loading. The loading buffer serves two functions: first, it increases the specific gravity of the sample thereby preventing diffusion of DNA from the top of the well into the surrounding buffer, and second, it indicates the progress of product as they migrate through the gel by electrophoresis (the blue dye migrates at approximately the same position as DNA fragments 200 bp in length). To estimate the size of the amplicons, 4μl of 100 bp Gibco's ladder (Gibco Life Science BRL) were loaded together with the analysed samples.

Electrophoresis of mid-gels (100 ml) was performed at 120 Volts in IX TBE buffer for approximately 1 h. Following electrophoresis, gels were removed from the rig and post- stained in 5 mg/1 aqueous ethidium bromide solution for 40 min, destained in distilled water for 2 min and then viewed under Ultra Violet Illumination using a UVP Bio-Doc- system. Images of the gels were captured by the UVP Bio-Doc system as jpeg format and used for scoring.

Scoring of results

PCR products generated by each microsatellite-genotype combination were evaluated for the presence of one or two distinct bands after fractionation by agarose gel electrophoresis (stages 1-3 above). Any genotype that yielded two products for any of the microsatellite loci was deemed to be heterozygous and so discarded as a possible candidate doubled haploid plant as shown in Table 3.

Table 3

Horn = homozygous; Het = heterozygous 5. Artificial production of mixoploids

Further work has been undertaken on haploid plants identified via flow cytometry. Haploid plants were treated with colchicine to induce development of mixoploid plants. Plants were treated as shown in the table and the next five leaves which developed assessed by flow cytometry to identify any diploid cells. As can be seen from Table 4, a number of plants became mixoploid. More interestingly, it can be seen that in a number of plants the percentage of diploid cells present in the leaves which developed after treatment with colchicine increased with time. This suggests that mixoploids may represent haploid plants which are undergoing chromosome doubling to produce doubled haploid tissues, organs or plants. In addition the floral tissues associated with leaves are expected to have the same % of diploid cells with doubled sectors bearing fertile flowers that can be selfed to produce* doubled haploid: seed.

Method of monitoring mixoploidy during plant development.

Both spontaneous and induced mixoploids can be monitored for changes in the percentage of cells of various ploidy levels. The method involves labelling each leaf as it is produced on the seedling, e.g. 1 -10 with one being the oldest leaf and ten being the latest fully expanded leaf. Leaf samples (a few cm² /20-50 mg) are removed from a leaf once it is fully expanded and analysed for ploidy as described above in 'Sample preparation for flow cytometry'. Data are then recorded for each leaf. The results shown in Table 4 show the effect of Colchicine treatment on the % diploid cells found in isolated haploid oil palm plants.

Mixoploid frequencies in oil palm progenies

Experiments conducted over a period of about a year have suggested that the frequency of mixoploids in abnormal oil palm seedlings is in the order of 1 in 10,000. About 170,000 abnormal oil palm seedlings were investigated: among these 17 mixoploids were identified.

The current work undertaken by the inventors suggests that mixoploid plants provide a valuable source of material for generating homozygous doubled haploid plants. Additionally, mixoploids are also potentially useful for generating haploid, triploid, tetraploid and higher ploidy plants. Furthermore, mixoploid plants may also themselves be of value in plant breeding.

Although the Example described above relates only to Oil Palm, it will be readily apparent that the techniques are equally applicable to other plant species where it may be desirable to identify mixoploid plants. The following references are incorporated by reference:

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Claims

Claims

1. A method for obtaining mixoploid plants useful for seed production, multiplication and crop improvement, said method comprising:

(i) providing a population of plants; (ii) choosing from the population a subset of individual plants with atypical phenotype;

(iii) assessing the DNA content in cells extracted from the subset of plants;

(iv) identifying plants in the subset that are mixoploid.

2. The method according to claim I₅ wherein the atypical phenotype is an atypical morphology or growth pattern.

3. The method according to claim 2, wherein the atypical morphology or growth pattern is a seed, seedling, plantlet or plant trait.

4. The method according to claim 2 or claim 3, wherein the atypical phenotype is an atypical morphology or growth pattern that can be detected in the seed, or during germination of seeds or seedling stages.

5. The method according to any one of claims 2 to 4, wherein the atypical morphology is one or more of slower or accelerated vegetative growth, altered leaf and/or stem shape, altered leaf angle, altered pigmentation, altered branching pattern, altered reproductive tissues, altered flowering time, altered shoot growth, altered root: shoot length ratio, altered rootshoot angle, altered colour of root, shoot or leaf, altered seed shape or size during germination, altered root width: length ratio, altered plant height, altered stem morphology, altered leaf morphology, altered venation, altered distance between leaf nodes and/or altered distance between leaf whorls.

6. The method according to claim 1 wherein the atypical phenotype is germination of two or more embryos from a single seed.

7. The method according to any preceding claim, wherein the plants are germinated seeds or seedlings.

8. The method according to any preceding claim, wherein the population of plants comprises in vitro, nursery or field grown plants.

9. The method according to any preceding claim, wherein the plants are palms, or rubber plants.

10. The method according to claim 9, wherein the plants are oil palms.

11. A method for producing haploid plants comprising: (i) identifying mixoploid plants according to the method of any one of claims

1 to 10;

(ii) identifying a region of said mixoploid plant which comprise at least one haploid cell;

(iii) propagating plants and clones from said haploid regions or at least one cell.

12. The method according to claim 11, wherein said haploid region or cell of step (ii) are subjected to a chromosome doubling step to produce doubled haploid cells prior to step (iii).

13. The method according to claim 12, wherein the doubled haploid is obtained through spontaneous chromosome doubling; or by doubling the chromosome number by application of an external stimulus to the. haploid region or plant in vivo or in vitro.

14. A method according to claim 12, wherein the doubled haploid is obtained by the application of an external stimulus to at least one cell isolated from a haploid region or plant.

15. A method for producing doubled haploid homozygous plants comprising:

(i) identifying mixoploid plants according to the method of any one of claims

1 to 10;

(ii) identifying regions of said mixoploid plant which comprise diploid cells;

(iii) assessing the homozygosity of said diploid cells to identify doubled haploids;

(iv) propagating plants and clones from said doubled haploid regions or cells.

16. A method for producing triploid plants comprising:

(i) identifying mixoploid plants according to the method of any one of claims

1 to 10; (ϋ) identifying regions of said mixoploid plant which comprise triploid cells;

(iii) assessing the homozygosity of said triploid cells; (iv) propagating plants and clones from said triploid regions or cells.

17. A method for producing tetraploid plants or plants of higher ploidy comprising:

(i) identifying mixoploid plants according to the method of any one of claims

1 to 10; (ii) identifying regions of said mixoploid plant which comprise tetraploid or higher ploidy cells;

(iii) assessing the homozygosity of said cells;

(iv) propagating plants and clones from said tetraploid or higher ploidy regions or cells.

18. The method according to any one of claims 15 to 17 wherein the homozygosity screening step uses molecular or biochemical markers.

19. The method according to any one of claims 15 to 18, wherein the homozygosity screening step uses multiple co-dominant molecular or biochemical markers.

20. The method according to claim 19, wherein the multiple co-dominant molecular or biochemical markers comprise at least one of microsatellite markers, sequenced characterised polymorphic region (SCARs) markers or single nucleotide polymorphisms (SNP) markers.

21. The method according to any one of claims 18 to 20, wherein the homozygosity screening step uses between 2 and 40 co-dominant markers.

22. The method according to any one of claims 15 to 21, wherein plant regions or cells are declared heterozygous unless they shows only one allele per locus for each co- dominant marker.

23. The method according to any one of claims 11 to 22, wherein the plants are palms or rubber plants.

24. The method according to claim 23, wherein the plants are oil palms.

25. Progeny plants produced from somatic or reproductive cells of a plant produced according to the method of any one of claims 11 to 24.

26. Clones, pollen or ovules from plants obtained by a method according to any one of claims 11 to 24 or from a plant of claim 25.

27. A mixoploid plant obtainable by the method of any one of claims 1 to 10.

28. A chimeric plant obtainable by the method of claims 1 to 10.

29. A haploid plant obtainable by the method of claim 11.

30. A doubled haploid plant obtainable by the method of any one of claims 12 to 15.

31. A triploid plant obtainable by the method of claim 16.

32. A tetraploid or higher polyploid plant obtainable by the methods of claim 17.

33. The method according to any one of claims 11 to 26, wherein one or more of the plants produced thereby are subsequently used in breeding, multiplication, seed production or clonal production.

34. A method according to claim 33, comprising crossing two distinct doubled haploids produce a F₁ hybrid.

35. An F i hybrid produced by the method of claim 34.

36. Haploid, mixoploid and double haploid plants produced from tissue culture of mixoploid plant material.