WO2010034961A1 - Procédé d’obtention de plants d’hévéa brasiliensis - Google Patents

Procédé d’obtention de plants d’hévéa brasiliensis Download PDF

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Publication number
WO2010034961A1
WO2010034961A1 PCT/GB2008/003227 GB2008003227W WO2010034961A1 WO 2010034961 A1 WO2010034961 A1 WO 2010034961A1 GB 2008003227 W GB2008003227 W GB 2008003227W WO 2010034961 A1 WO2010034961 A1 WO 2010034961A1
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plants
haploid
plant
rubber
doubled
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PCT/GB2008/003227
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English (en)
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Brian Peter Forster
Stephen Peter Connor Nelson
Peter D. S. Caligari
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Sumatra Biosciences Pte Ltd
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Priority to PCT/GB2008/003227 priority Critical patent/WO2010034961A1/fr
Publication of WO2010034961A1 publication Critical patent/WO2010034961A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/04—Stems

Definitions

  • the present invention relates to a method of obtaining special forms of rubber plant (Hevea brasiliensis Muell. Arg.) useful in plant breeding, or in rubber production, or both: and to plants obtainable thereby.
  • Plants obtainable according to the invention include haploid rubber plants as well as triploids, tetraploids and plants of higher ploidy.
  • the method relates to obtaining doubled haploid rubber plants: in a further aspect, it relates to obtaining haploid rubber plants from which doubled haploid rubber plants can be obtained.
  • the invention further comprises a method of producing uniform F] hybrid rubber plants by crossing two different doubled haploids and the Fi hybrid rubber plants obtainable by the process.
  • 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.
  • the problem is exacerbated when the desired traits include yield and quality characteristics that can only be determined once the plants have reached maturity.
  • haploid plants derived from the gametic cells of parental individuals.
  • the chromosome complements of these haploids sometimes double spontaneously to produce homozygous doubled haploids (DHs); or they may be induced to double by treatment with certain chemicals, for example colchicine.
  • DHs homozygous doubled haploids
  • One way of producing doubled haploids is by the in vitro culture of anthers or microspores (androgenesis) or analogously from female gametic cells by culturing flower, ovary and ovule tissues (gynogenesis).
  • a second method of producing haploids and doubled haploids has been via wide-species crosses.
  • the resultant doubled haploid plants regardless of how they are derived, are instantly and completely homozygous. This means that on selling 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 Fi 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.
  • x the number present in gametic cells of a diploid organism.
  • Haploid sporophytes of higher plants can be distinguished from diploids in many ways. They are usually smaller (partly because of their smaller cell size). In general terms, cell volume in plants is positively correlated with ploidy level. Haploids are also usually sterile. Several methods for the provisional assignment of haploid 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 is also easily identified because the haploids are sterile. However, none of the phenotypic predictors of haploidy are absolutely reliable. Methods that directly measure genome size are far more reliable.
  • Haploids 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.
  • haploids An important use of haploids 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. For rubber, there are no spontaneous haploid plants reported. Rubber is a clonally propagated crop and as such breeding and planting procedures are geared to selecting uniform high performing plants. Potential off types are therefore eliminated before sowing. Rubber production via seed is subject to a pre-screen in a bounce test. Only normal seed, those that bounce, are selected for sowing. The abnormal, non-bouncing, seed are discarded at the beginning of the process and therefore a major source of atypical seedling phenotypes is lost at the onset, they are never germinated. In addition rapidly germinating seedlings are selected within 20 days of sowing and slow germinating seed (another source of off types) are discarded. The skilled person therefore would have no incentive to try to identify off types in rubber through phenotypic selection because of the high level of prejudice against such selection in normal rubber agriculture.
  • homozygous doubled haploid plants also have utility for the generation of Fi hybrid plants, produced from crosses made between selected homozygous male and female parents. These Fi 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 Fj hybrids allows the breeder to produce large quantities of uniform seed of a single genotype from homozygous parental lines. Fi 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.
  • Haploid 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.
  • 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 haploids exploit their capacity for the rapid generation of homozygous genotypes after chromosome doubling, with advantages including:
  • polyploid 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 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).
  • step (c) assessing the ploidy level of plants in the subset; (d) classifying plants in the subset as haploid, diploid or polyploid according to the results of step (c)
  • step (c) assessing the ploidy level of atypical plants will apply flow cytometry to assess ploidy levels in cells extracted from root, shoot, leaf or other plant tissue.
  • the step (e) of assessing the homozygosity of chosen plants uses molecular, biochemical or phenotypic markers.
  • Phenotypic markers that correlate with homozygosity may be for example, height, pigmentation, leaf size and shape, stem thickness, grassy leaves, leaflet internode length and number and rate of leaf production. It is convenient to assess homozygosity using multiple molecular markers.
  • a preferred technique is to use multiple molecular markers, for example between 2 and 40, which may be microsatellite markers (also known as simple sequence repeats, SSRs): or Sequenced Characterised Polymorphic Regions (SCARs) markers or Single Nucleotide Polymorphism (SNP) markers. It is convenient to employ co-dominant molecular markers, particularly microsatellite markers, although many other marker systems could also be applied, for example, protein profiling, isozymes, High Resolution Melt analysis or pyrosequencing.
  • stage (b) of the method It is preferred to carry out stage (b) of the method upon germinating seed or seedlings.
  • step (e) assessing homozygosity
  • step (c) assessing homozygosity
  • 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.
  • mixtureaploid 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.
  • One convenient application of the invention is to select candidate haploid seedlings from germinated seed after the plumule and radical have developed and once the first whorl of leaves has developed. Germination usually commences about 10 days from sowing. Cohorts of germinating rubber seedlings typically exhibit a fairly synchronous developmental pathway and reasonably homogenous phenotype (see Figure 1, seedling beds). Abnormal germinated seed may deviate from the characteristic phenotype in one of many ways ( Figure 2 and Figure 3 show examples of altered seedling and leaf morphology for haploid, diploid, triploid and tetraploid plants) 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 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; number of leaves per whorl.
  • Another way of carrying out the invention is to select among a population of nursery or field planted plants.
  • the atypical morphology or growth pattern used as the basis of selection is preferably one or more of: slower vegetative growth, reduced ratio of leaflet width to length, altered inter- whorl distance, angle of leaf to plant axis, leaf colour, branching pattern and precocious flowering.
  • 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 haploid, or doubled haploid, mixaploid, triploid, tetraploid or higher ploidy 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.
  • the step of further assessing the homozygosity of the chosen plants uses multiple molecular markers. More preferably, this step 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.
  • the population of plants comprises at least 500 particularly 750 - 2,500 individual plants.
  • 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 and would normally be rejected by commercial breeders and growers. Seeds are normally placed in a sand bed for germination.
  • Mixaploids 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 a later stage. Mixaploid plants may also indicate the presence of doubled haploids in a progeny, the progeny can then be screened intensively using molecular markers to select the doubled haploids.
  • Plants may also be obtained by application of an external agent to vegetative and generative meristems of haploid plants or clones derived from them; or by application of an external agent to floral tissues of the haploid or clones derived from it; or by selfing the haploid plant by exploiting the occasional spontaneously doubled chromosome number in male and female reproductive cells; or through any other spontaneous doubling event.
  • a method for producing a diploid Fi hybrid rubber plant which comprises crossing two distinct doubled haploid rubber plants obtainable by the method of the invention; and Fi hybrids so produced.
  • the rubber germplasm Hevea brasiliensis Muell. Arg. used in the following experiments was obtained in Indonesia where the initial stages of the procedure (seed collections and selection of material of atypical phenotype) were carried out.
  • the historic origin of the rubber tree is understood to be the Amazon rainforest, where it still grows wild: it is believed that the species was introduced from Brazil via Kew Gardens (England) to South-East Asia in the last half of the nineteenth century, since when it has been widely cultivated throughout that region. Examples
  • Figure 1 is a photograph of a seedling bed of rubber in which off-types may be observed
  • Figure 2 is a photograph of selected abnormal seedlings after germination: A: triploid; B: haploid; C: tetraploid; D: diploid;
  • Figure 3 is a photograph of leaves of some abnormal seedlings: A: triploid; B: haploid; C: tetraploid; D: diploid;
  • Figures 4A - 4F show flow cytometry output histograms: of diploid (Figure 4A-C) and haploid (Figure 4D-F) samples, respectively;
  • Figure 5 is a photograph of a rubber inflorescence carrying male and female flowers
  • Figure 6 is a photograph of a rubber inflorescence carrying male flowers at anthesis (pollen shedding);
  • Figure 7 is a photograph of the removal of a staminal column containing dehiscing anthers from a male flower
  • Figure 8 is a photograph of insertion of the excised stamina column into a female flower, thus making contact with the stigma and effecting pollination;
  • Figure 9 is a photograph of artificially pollinated female flowers, covered with cotton wool to prevent uncontrolled pollinations
  • Figure 10 is a photograph of the labelling of crosses
  • FIG 11 is a photograph of crossed flowers bagged for protection. Detailed Description of the Preferred Embodiments
  • a total of 44,742 rubber seed were collected and sown directly into outside sand beds from the period September 2007 - January 2008, i.e. during the seed fall season of North Sumatra.
  • the "bounce test” would typically be applied to such seed, to remove seed less likely to germinate, but the test was not used on these seeds.
  • Seed were planted so that Vi of each seed was embedded in the sand, at a density of approximately 1 ,200 seeds per square metre. The exposed parts were covered with sacking until seedling emergence had commenced. The seedbeds were shaded (Fig. 1). Seeds were watered daily. Germination began 7-10 days from sowing and continued for two to three months.
  • the cell nuclei were prepared from fresh plant material (leaves or roots), by chopping the tissue (about 0.5 cm square leaf tissue or about 1 cm length of root of about 20-25 mg) with a clean, sharp razor blade in 0.5 ml buffer, or more depending in amount of tissue chopped, in a plastic Petri dish.
  • the DNA buffer is based on Arumuganathan and Earle (1991) and consisted of:
  • DAPI a fluorescent dye that selectively binds to form a complex with double stranded DNA and gives a product that fluoresces at 465 nm wavelength
  • DAPI has specific DNA-binding properties, with preference for adenine-thymine (AT)-rich sequences.
  • AT adenine-thymine
  • a solution containing stained nuclei was passed through the flow cytometer.
  • a standard normal diploid rubber sample was used as a reference.
  • Dichroic mirrors TK 420 abd TK 560
  • Genome characterisation is used in two ways: firstly to identify homozygous diploids among off-types confirmed by flow cytometry to be diploid and secondly, if so desired, as a prescreen for homozygous off-types before flow cytometry.
  • the protocol applied to perform a molecular pre-screen of seedlings showing abnormal phenotypes to discard heterozygotes comprises the following stages.
  • microsatellite markers have been used:
  • 10 ml of PCR reaction mixture contained the following reagents; 1.0 ml of 10x PCR buffer (Bioline), 0.3 ml MgCl 2 (10 mM), 0.4 ml dNTPs (10 mM of each), 0.2 ml of each primer pair (10 mM), 1 -5 ng of DNA (extracted as above) and 1 U of Taq DNA polymerase (5 U mI-I Bioline).
  • TBE running buffer 0.089 M Tris base, 0.089 M boric acid,(pH 8.3) and 2 mM Na2EDTA
  • 2 % (w/v) agarose 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 is cooled to approximately 55°C prior to the addition of ethidium bromide (3.5 ⁇ l 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
  • masking tape an appropriate number and type of combs placed in position. Combs with 16 x 20 ⁇ l wells are most often employed.
  • the gel solution is carefully poured into the prepared tray and allowed to cool for at least 20 min. Combs and tape are then removed and the gel tray submerged into a tank 10 containing 1 x TBE buffer.
  • 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).
  • electrophoresis the blue dye migrates at approximately the same position as DNA fragments 200 bp in length.
  • 4 ⁇ l of 100 bp Gibco's ladder Gibco's ladder (Gibco Life Science BRL) are loaded together with the analysed samples. Electrophoresis of mid-gels (100 ml) is performed at 75 Volts in IX TBE buffer for approximately 2 h.
  • gels are removed from the rig and post-stained in 5 mg/1 aqueous ethidium bromide solution for 30 min, destained in distilled water for 20 min and then viewed under Ultra Violet Illumination using a UVP Bio-Doc-system. Images of the gels are captured by the UVP Bio-Doc system as Polaroid format and used for scoring.
  • PCR products generated by each microsatellite-genotype combination are evaluated for the presence of one or two distinct bands after fractionation by agarose gel electrophoresis (stages 1-3 above). Any genotype that yields two products (two bands) for any of the microsatellite loci is deemed to be heterozygous and so discarded as a possible doubled haploid plant.
  • DNA from 130 abnormal seedlings has been screened using microsatellite (SSR) markers: M 124 and M574.
  • SSR microsatellite
  • PCR products produced using these primers were subjected to capillary electrophoresis, and outputs analysed using gene mapper software. From this initial screen 69 abnormal seedlings were identified as homozygous for the two markers.
  • Haploid cells will sometimes undergo "spontaneous doubling" whereby failure of complete mitosis gives rise to a doubling of the chromosomes. If this occurs early in development, the seed, plantlet and plant derived is a doubled haploid. If no such doubling occurs then a haploid is obtained and in most circumstances such haploid plants are intrinsically infertile, in that the process of meiosis is unable to generate gametes capable of fertilisation.
  • the nucleus of an individual cell may occasionally fail to divide normally at mitosis and thus form a diploid cell that ultimately gives rise either to a diploid sector(s) that may encompass most or all of the main shoot axis or (if it occurs in the first embryonic division) a doubled haploid plant.
  • the selfed seed secured from such individuals will be completely homozygous and genetically identical to the parent. This process can occur during the formation of reproductive cells and in this case it is possible that fertile gametes (pollen or egg cells) may be produced. When both male and female gametes form on the same plant then successful fusion of gametes can take place and an embryo will develop.
  • Such an embryo will be a homozygous diploid, and will breed true in all future selfed generations; all its selfed progeny will be genetically identical.
  • seedlings are found to be mixaploid (carrying cells of different ploidy level) and may develop into one ploidy class.
  • a seedling carrying n and 2n cells may develop to be completely homozygous diploid.
  • they provide another route of obtaining doubled haploids that can be detected by monitoring their ploidy development using flow cytometry.
  • colchicine as the doubling agent, but other chemicals that induce chromosome doubling may also be used.
  • Various plants, plantlets and plant parts in situ or in vitro are treated with colchicine.
  • the stock colchicine solution is prepared in a fume hood to avoid inhalation of the colchicine powder.
  • the stock solution contains 1 g colchicine in 1 litre of water to which 20 ml DMSO (Dimethyl sulphoxide), 1 ml of a 10 ppm solution of GA3 (Gibberelin A3) and 10 drops of Tween 20 (surfactant) may also be added.
  • the stock solution is diluted to give working solutions of colchicine between 0.2 and 12 ppm.
  • the concentration used is inversely related to the length of exposure.
  • Colchicine is toxic and is handled in a designated area using protective clothing. Typically, treatments involve colchicine concentrations in the range of 2.5 - 12 ppm for 2 - 10 hours.
  • Seedlings detected as being haploid are washed clean with water and the bare rooted plants placed into a colchicine solution so that their roots and crowns are fully immersed. Treatment is done in the light at ambient temperatures for 2-10 hours. After treatment seedlings are rinsed in water for 15 minutes, potted into soiled- filled pots and placed into nurseries or greenhouses where they receive shading and misting. Chromosome doubling is monitored by flow cytometry analysis of leaves or roots emerging after treatment. Once doubled haploidy is confirmed, established plants are removed to harden off.
  • Rooted micropropagated plantlets can be treated with colchicine using the in vivo method described for seedlings above. Alternatively, treatment with colchicine may be carried out in vitro. In vitro methods involve either 1) placing a clonal haploid plantlet into a sterile culture medium containing colchicine for a suitable period, or 2) treating a shoot meristem in a solution of colchicine and growing on the treated meristem in vitro.
  • Floral organs Floral buds developing on haploid plants can be treated with colchicine to induce doubling and restore fertility to male and female gametic cells. Self pollination of such flowers will yield doubled haploids.
  • Fi hybrid is produced by crossing 2 different homozygous doubled haploid rubber plants.
  • the inflorescence of rubber contains separate male and female flowers (see Fig. 5). Crossing is done artificially by following the following procedure:
  • the F] seed are then sown. They may be cultivated and tapped for the production of latex, in conventional manner.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Physiology (AREA)
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  • Developmental Biology & Embryology (AREA)
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Abstract

L’invention concerne un procédé permettant d’obtenir des plants de caoutchouc possédant une ploïdie et un génotype particuliers et utiles pour la production de semences ainsi que pour la multiplication et l’amélioration des cultures, ledit procédé comprenant : (a) la fourniture d’une population de plants de caoutchouc; (b) la sélection à partir de ladite population d’un sous-ensemble de plants individuels possédant un phénotype atypique; (c) l’évaluation du contenu ADN des plants dans ledit sous-ensemble; (d) la classification des plants du sous-ensemble en tant qu’haploïdes, diploïdes ou polyploïdes en fonction des résultats de l’étape (c).
PCT/GB2008/003227 2008-09-23 2008-09-23 Procédé d’obtention de plants d’hévéa brasiliensis WO2010034961A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019002569A1 (fr) * 2017-06-30 2019-01-03 University Of Ljubljana Procédé pour sélectionner des plantes hybrides
CN112088774A (zh) * 2020-10-28 2020-12-18 中国热带农业科学院橡胶研究所 一种植物多倍体的诱导方法
CN116267594A (zh) * 2023-02-04 2023-06-23 云南省热带作物科学研究所 一种促进橡胶树2n配子形成的装置

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019002569A1 (fr) * 2017-06-30 2019-01-03 University Of Ljubljana Procédé pour sélectionner des plantes hybrides
CN112088774A (zh) * 2020-10-28 2020-12-18 中国热带农业科学院橡胶研究所 一种植物多倍体的诱导方法
CN116267594A (zh) * 2023-02-04 2023-06-23 云南省热带作物科学研究所 一种促进橡胶树2n配子形成的装置

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