WO2010058048A1 - Système d'androstérilité géno-cytoplasmique dans le blé - Google Patents

Système d'androstérilité géno-cytoplasmique dans le blé Download PDF

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Publication number
WO2010058048A1
WO2010058048A1 PCT/ES2009/070416 ES2009070416W WO2010058048A1 WO 2010058048 A1 WO2010058048 A1 WO 2010058048A1 ES 2009070416 W ES2009070416 W ES 2009070416W WO 2010058048 A1 WO2010058048 A1 WO 2010058048A1
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Prior art keywords
wheat
line
chromosome
restorative
chilense
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English (en)
Spanish (es)
Inventor
Antonio Pedro MARTÍN MUÑOZ
Francisco Barro Losada
Sergio Gustavo ATIENZA PEÑAS
Mª Carmen RAMÍREZ ALCANTARA
Azahara Carmen MARTÍN RAMÍREZ
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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Classifications

    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • 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
    • A01H5/10—Seeds
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4678—Triticum sp. [wheat]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • the present invention is within the field of biotechnology and, specifically within the plant genetic improvement. It refers to a plant cell with gene-cytoplasmic androesterility that incorporates fertility restorative genes from chromosomes of
  • Chilean Hordeum Said cells give rise to or are part of plants that form the restorative line, and integrate into their genetic material a fragment of the chromosome 1 H ch , a double monosomy for 6H ch and 6D, and / or the translocation 6H ch S / 6DL
  • MMV Plant Genetic Improvement
  • Hybrid varieties of this type are also called F1 hybrids and are, in addition to more productive (a high yield of the parts of the plant that can be marketed, such as fruits and the like), due to what is called “hybrid vigor", more homogeneous (uniformity in color, flavor, size and the like), resulting in a higher crop yield and a uniform quality of it.
  • the commercial hybrid is the product of the cross between two pure lines that have been selected for their ability to express heterosis.
  • the optimal combination has been the result of an expensive process and, as always in MGV, long, field evaluation of tens of thousands of crosses.
  • castration is not profitable to do it manually (mechanical castration or removal of anthers, also called emasculation).
  • the procedures used are: chemical castration (substances that eliminate or inactivate male gametes), genetics (nuclear) and gene-cytoplasmic. The latter is the one that has had the greatest commercial success.
  • CMS cytoplasmic male sterility
  • GMS male gene sterility
  • CMS cytoplasmic male sterility
  • chromosomal arms of group 4 the long arm of chromosome 4A (4AL) , the short arm of chromosome 4B (4BS) and the short arm of chromosome 4D (4DS), which carry the normal male fertility genes Ms - AI, Ms - B1 and Ms - D1, respectively, and the long arms of chromosomes from group 5: 5A, 5B and 5D (5AL, 5BL and 5DL, respectively), which carry the genes Ms-A2, Ms-B2 and Ms-D2, respectively.
  • Table 1 List of Rf genes, distribution in chromosomes and origin.
  • the authors of the present invention have developed a restorative line of fertility in wheat, constructed by introgression in wheat of restorative factors from H. chilense. Although in hemicigosis the addition of the short arm of chromosome 6H ch S is not an effective restorative, it has been found that double monosomal 6H ch 6D plants are fertile, as well as those that show the 6H ch S / 6DL translocation.
  • a chromosome 1 H ch with a deletion of almost the entire long arm also restores fertility and is effective in hemicigosis.
  • This line allows the production of hybrid wheat seeds through the establishment of three lines: an euplasmic line, is a normal flour wheat, the alloplastic line of wheat in cytoplasm of H. chilense (obtained by crossing tritordeo or amphiploid Hordeum chilense - ⁇ . ⁇ go by wheat, until the elimination of all the barley chromosomes, and the restorative line.
  • the alloplastic line is propagated by pollination with the maintenance line, so that the whole progeny presents male sterility, since the cytoplasm is derived from the female parental.
  • a first aspect of the invention refers to a wheat seed, hereinafter referred to as wheat seed of the invention, characterized in that its genetic material comprises: a. a fragment of chromosome 1 H ch from Hordeum chilense, b. a double monosomy for 6H ch and 6D, c. The translocation 6H ch S / 6DL, or any of its combinations.
  • the genetic material of the seed of the invention comprises the introgression of the 1 H ch chromosome fragment responsible for the restoration in hemicigosis and the short arm of the 6H ch chromosome.
  • a wheat cell hereinafter referred to as a wheat cell of the invention, characterized in that its genetic material comprises: a. a fragment of chromosome 1 H ch from Hordeum chilense, b. a double monosomy for 6H ch and 6D, c. The translocation 6H ch S / 6DL, or any of its combinations.
  • the genetic material of the cell of the invention comprises the introgression of the 1 H ch chromosome fragment responsible for the restoration in hemicigosis and the short arm of the 6H ch chromosome.
  • “Wheat” is understood herein as the aestivum species of the Genus Triticum, Triticeae Tribe, Pooideae Subfamily, Poaceae Family, Poales Order, Liliopsida Class, Phylum Streptophyta, Viridiplantae Kingdom.
  • translocation is meant the exchange between two or more chromosomes.
  • a wheat plant from now on the wheat plant of the invention, comprising a plurality of wheat cells of the invention, and / or produced after the growth of the wheat seed of the invention. invention.
  • Another aspect refers to a wheat cell culture of the invention, capable of regenerating the wheat plant of the invention.
  • the cells of the culture of the invention come from embryos, meristematic cells, pollen, leaves, roots, inclined root, anther, pistil, flower, seed, pod or stem of the plant of the invention.
  • cell culture herein refers to a culture of isolated cells of the same or different type of tissue, or a collection of such cells organized in parts of a plant or in tissues (tissue cultures).
  • Types of crops of this type are, for example, cultures of protoplasts, calli (group of callus or undifferentiated plant cells capable of regenerating an entire plant) and plant cells that are isolated from plants or parts of plants, such as embryos, protoplasts , meristematic cells, pollen, leaves or anthers.
  • a pollen grain is provided whose genetic material is a haploid derivative of the genetic material of the wheat cell of the invention.
  • Another aspect of the invention constitutes a method for producing hybrid wheat seeds comprising: to. Obtain an euplasmic line formed by hybrid plants between Hordeum chilense and Triticum sp. b. Obtain an alloplastic line formed by wheat plants with gene-cytoplasmic androsterility. C. obtain an androesteril restorative line formed by wheat plants of the invention, d. cross the line obtained in b) with the line obtained in c).
  • the hybrids between Hordeum chilense and wheat can be obtained by emasculating spikes of H. chilense and pollinating them with wheat pollen, the day after pollination a solution of gibberellic acid is applied at 75 ppm and after three weeks the embryo is rescued and grown in vitro, obtaining the hybrid. Chromosomal duplication of the latter gives rise to the amphiploid, the octoploid tritorid.
  • the Chilean Hordeum entry to be used must be line H1 of the collection of the Institute of Sustainable Agriculture. Other entries in the collection may not produce androsterility of the alloplastic line.
  • Fig. 1 PCR amplification products using the ETS marker k01062 that specifically amplifies chromosome 6H ch S in H. chilense and does not produce an amplification product in wheat.
  • the 6HchS T21 ditelosomal addition (T21AH1-11) is used as a positive control.
  • the T593 restorative line is amplified by this pair of primers, confirming that the 6HchS chromosome arm is responsible for the restoration of fertility.
  • Fig. 2 In situ hybridization of the apex cells of the metaphase roots, of the T593 restorative line (42 + 6H ch S 6H ch S).
  • the 6H ch S chromosomes show an intense brightness and are also detected by the pTa71 probes (they are indicated by arrows).
  • Fig. 3 PCR amplification products using the consensus primer pair of SSR-4 chloroplast.
  • a 25 bp polymorphism is distinguished between the chloroplast genomes of flour wheat and H. chilense.
  • Amplification fragments of the T218 male alloplasmic sterility line and the T593 fertile restored line confirm that they carry the H1 cytoplasm and that there is no transmission of the parental cytoplasm.
  • Fig. 4 Development of pollen gametogenesis in fertile T21 lines T593 (A1-A5) and in the male sterile line T218 (B1-B5).
  • A1, B1 Normal tetrade state after Ia meiosis in plants with male sterility and fertility.
  • A2, B2 Normal early uninucleated microspores are observed in both sterility and male fertility genotypes.
  • A3) Normal late uninucleated microspores during vacuolation of microspores in fertile lines.
  • A4) Binucleated pollen grains with vegetative and generative nuclei perfectly formed in fertile genotypes.
  • Two spikes of H. chilense were emasculated and just before pollination the stems were introduced into a culture with a nutrient solution to which gibberellic acid has been added in a proportion of 75 ppm.
  • the spikes were pollinated with pollen of T. aestivum cv . Ch ⁇ nese Spring and subsequently maintained in a controlled environment at 2O 0 C and continuous light.
  • the culture solution was renewed at intervals of 2 days with the stock solution without gibberellic acid.
  • the barley ears were protected with vented polyethylene bags.
  • the spikes of the hybrid plant are essentially like those of wheat, but longer and laxer than those of the Ch ⁇ nese Spring variety and, as in H. chilense, with short edges in both glumes and slogans.
  • the hybrid was treated with 0.3% colchicine by the decapitation technique and some spikes gave grains by self-fertilization.
  • the amphiploid thus obtained, HT21 was repeatedly crossed with the wheat parental T21, obtaining an alloplastic line called T218 (AABBDD). This line carried the cytoplasm of H. chilense, being totally sterile.
  • T218 was pollinated and crossed with the set of addition chromosome lines in Ch ⁇ nense Spring.
  • T21 of dysphonic addition of 6H ch S was backcrossed as maternal parental for the disomic addition of 6H ch S.
  • the ditelosomal addition of 6H ch S in T218 was selected and named T593 (AABBDD + 6H ch S 6H ch S).
  • T218 was crossed with 25 restorative lines from three sources USDA, ARS, Aberdeen (Pl 473558, Pl 473559, Pl 591553, Pl 591554 , Pl 591555, Pl 591556, Pl 554584, Citr 17452, Pl 383343, Pl 383344, Pl 473548, Pl 473549, Pl 473551, Pl 473552, Pl 473553, Pl 473555 and Pl 473556), CIMMYT (337405, 332258, 332231 and 332197 ) and lines provided by Dr. Chen, School of Biological Technology and Engineering, Guizhou Normal University, China (QR720, QR715, QR650 and QR3). The hybrids were grown for two successive years and fertility restoration was never observed.
  • Microsporogenesis was studied in different stages of development.
  • the cut ears are introduced into a mixture of 3 parts of absolute alcohol: 1 part of glacial acetic acid with a few drops of iron chloride (1 ml of a saturated aqueous solution of iron chloride per 200 mi of the mixture 3: 1).
  • the material was transferred to alcohol of 90, after 1-2 hours and stored at 4 0 C.
  • the anthers were stained with 0.1% acetocarmin.
  • the PCR (polymerase chain reaction) was carried out in 25 ⁇ l of reaction mixture as described in Chung & Staub (2003).
  • the ETS (expressed sequence tag) marker k01062 (Hagras et al. 2005; Nasuda et al. 2005) was used to identify the chromosome in the restorative line. PCR was carried out as described in Nasuda et al. (2005). All amplification products were resolved by agarose gel electrophoresis and visualized with ethidium bromide.
  • FISH In situ hybridization
  • the ends of the roots were prepared as described in the cytological observation section. The preparations were made as described by Prieto et al. (2001).
  • the pTa7 probe which contains a unit of 18S-5.8S-26S rDNA (8.9 kb) from T. aestivum (Gerlach & Bedbrook 1979) was labeled by transcription with biotin-11-dUTP (Roche Corporate, Basel, Switzerland) and mixed in the hybridization solution until the final concentration of 5 ng / ⁇ l. After examination of the hybridized nuclei with their respective DNA probe, the preparations were re-probed using the total genome of H. chilense as a probe. The total DNA of H.
  • chilense was labeled by transcription with digoxigenin-11-dUTP.
  • He In situ hybridization protocol was that of Cabrera et al. (2002). Biotin and digoxigenin labeled probes were detected with antidigoxigenin-FITC (Roche Corporate) and streptavidin-Cy3 conjugates (Sigma, St. Louis, MO, USA), respectively.
  • the chromosomes were stained and counted on DAPI (4 ' , 6-diamidino-2-phenylindole) 339 and mounted on Vectashield (Vector Laboratories, Inc.). They were visualized using a Leica epifluorescence microscope. The images were captured with a SPOT CCD camera using SPOT 2.1 software (Diagnostics Instruments, Inc., Sterling Heights, Ml, USA) and processed with Photoshop 7.0 software (Adobe Systems Inc., San Jose, CA, USA).
  • EST k01062 was amplified for the purpose of the present work in lines T21, H1, T218, T593, and the disomic addition of 6H ch in T21 (called T21AH1-11) (Fig. 1).
  • T21AH1-11 the disomic addition of 6H ch in T21
  • No amplification product was obtained when T21 and T218 were used as the standard, while a product of about 370 bp was obtained when the 6H ch lines were amplified with H1, T593 and T21.
  • This result confirms that the addition of the arm of chromosome 6H ch S is responsible for the restoration, and could imply that the arm of chromosome 6H ch S carries at least one restorative gene of fertility.
  • the fertile T593 line was also characterized by fluorescent in situ hybridization (FISH), genomic in situ hybridization (GISH) techniques in T593, using the genomic DNA of H. chilense H1 as a probe, clearly detected the dithyleosomal addition of the H chromosomes Chilean. (Fig. 2).
  • the pTa71 probe which contains an 18S-5.8S-26S rDNA unit (located in the nuclear organizing region of the Chromosomes (NORs)) combined with the genomic DNA probe of H. chilense was used to confirm the presence of the 6H ch S ditelosomal addition in the T593 restorative line (Fig. 2).
  • the NOR in Hordeum is located in the 5HS and 6HS chromosomal segments, so that the presence of the signal of the pTa71 probe on the chromosomes of H. chilense confirms the results of the molecular markers in identifying the 6H ch S ditelosomal addition.
  • Morphological characterization morphology, fertility and environment
  • T593 had a delay of 3-4 days in the anthesis when they were grown in the greenhouse and in the growth chamber under long day conditions, although the difference was 7 days when it was grown in the open field.
  • the plants Apart from the lack of viable pollen in the sterility line male, the plants exhibited no other floral or developmental abnormalities. No wrinkle or germination defect was observed in the seeds of the alloplastic lines.
  • the data in Table 2 show the results of comparing lines T21, T218, the ditelosomal addition of 6H ch S to T21 and T593, in terms of morphology and fertility in greenhouse conditions.
  • the 6H ch S T21 was included in the table to compare T593 vs 6H ch S T21, for example, the same genotypes in the cytoplasms of H1 and T21, respectively. By this comparison a better analysis of the effect of cytoplasm H1 is possible. It has been seen that the T218 line was about 18 cm shorter (in height) than the T21.
  • the T593 restorative line was about 10 cm shorter than the T21, in this table no significant differences were found, either with the T21 or with the 6H ch S T21.
  • the number of edges per plant was not statistically significant among genotypes.
  • the number of spikelets per spike was lower in the line with male sterility T218 than in any other genotype, while the restorative line T593 showed an intermediate value between T21 and 6H ch S T21.
  • the number of stems per plant was not significantly different between genotypes.
  • the number of spikelets per spike was lower in the line with male sterility T218 than in any other genotype, while the restorative line T593 shows an intermediate value between T21 and 6H ch S T21.
  • the degree of sterility in the T218 male sterility line was 100%, and the T593 restorative line showed approximately 68% of viable grains compared to T21, and 77% compared to the 6H ch S T21.

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Abstract

L'invention concerne une semence, une cellule et un groupe de cellules à androstérilité géno-cytoplasmique, comprenant des gènes restaurateurs de la fertilité provenant de chromosomes de Hordeum chilense. Lesdites cellules engendrent des plantes formant la lignée restauratrice ou font partie de celles-ci, et renferment dans leur matériel génétique un fragment du chromosme 1Hch, une double monosomie pour 6Hch et 6D, et/ou la translocation 6HchS/6DL.
PCT/ES2009/070416 2008-11-18 2009-10-02 Système d'androstérilité géno-cytoplasmique dans le blé Ceased WO2010058048A1 (fr)

Applications Claiming Priority (2)

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ESP200803286 2008-11-18
ES200803286A ES2343934B1 (es) 2008-11-18 2008-11-18 Sistema de androesterilidad genico-citoplasmica en trigo.

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098209A2 (fr) * 2001-06-04 2002-12-12 Northwest Plant Breeding Co. Systeme a base de cms pour plants hybrides de ble et production de semences
WO2007134122A2 (fr) * 2006-05-09 2007-11-22 The Curators Of The University Of Missouri Plateformes végétales de chromosomes artificiels au moyen d'un troncage du télomère

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002098209A2 (fr) * 2001-06-04 2002-12-12 Northwest Plant Breeding Co. Systeme a base de cms pour plants hybrides de ble et production de semences
WO2007134122A2 (fr) * 2006-05-09 2007-11-22 The Curators Of The University Of Missouri Plateformes végétales de chromosomes artificiels au moyen d'un troncage du télomère

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MARTIN A ET AL.: "The development of tritordeum: a novel cereal for food processing.", JOURNAL OF CEREAL SCIENCE., vol. 30, 1999, pages 85 - 95 *
MARTIN A.C ET AL.: "Male fertility restoration of wheat in Hordeum chilense cytoplasm is associated with 6HchS chromosome addition.", AUSTRALIAN JOURNAL OF AGRICULTURAL RESEARCH., vol. 59, no. 3, 11 March 2008 (2008-03-11), pages 206 - 213 *

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ES2343934A1 (es) 2010-08-12

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