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WO2025030757A1 - Procédé de culture du coton upland, récepteur de transformation génétique efficace, et son utilisation - Google Patents

Procédé de culture du coton upland, récepteur de transformation génétique efficace, et son utilisation Download PDF

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Publication number
WO2025030757A1
WO2025030757A1 PCT/CN2023/141832 CN2023141832W WO2025030757A1 WO 2025030757 A1 WO2025030757 A1 WO 2025030757A1 CN 2023141832 W CN2023141832 W CN 2023141832W WO 2025030757 A1 WO2025030757 A1 WO 2025030757A1
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Prior art keywords
callus
cotton
genetic transformation
regeneration
screening
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PCT/CN2023/141832
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English (en)
Chinese (zh)
Inventor
林忠旭
乐愉
张献龙
王涛
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Huazhong Agricultural University
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Huazhong Agricultural University
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Publication of WO2025030757A1 publication Critical patent/WO2025030757A1/fr
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • 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
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid

Definitions

  • the present invention belongs to the technical field of plant breeding, and in particular relates to a cultivation method and application of an upland cotton efficient genetic transformation receptor.
  • Cotton is an important fiber and oil crop in the world and plays a pivotal role in my country's economic development. Traditional hybrid breeding methods can no longer meet the needs of cotton genetic improvement, and molecular breeding is a hot spot for cotton germplasm innovation. Somatic embryogenesis uses the totipotency of plant cells to recapitulate zygotic embryo morphogenesis. The application of modern molecular biotechnology is mostly based on plant tissue culture technology to succeed, so tissue culture technology has become crucial in the breeding of new cotton varieties. Steward and Reinert induced carrot phloem cells by means of suspension culture and obtained somatic embryos. Further culture obtained the first regenerated plant, opening a new era of exploring how to regenerate plants. In 1912, Robins added different types of substances to the culture medium and used the culture medium to culture the root tips of cotton to obtain green stems and roots. This was the first report on cotton tissue culture.
  • YZ-1 chicken-foot-leaved upland cotton variety 'Yu Zao 1'
  • YZ-1 has super-chicken-foot leaves and poor agronomic traits, so it is difficult to apply transgenics with it as the receptor in production practice.
  • cotton somatic embryogenesis and plant regeneration are highly dependent on cotton genotype, based on the reported regeneration culture systems of different cotton varieties, a regeneration system related to the parents is selected to evaluate the regeneration ability of the constructed population, and attempts are made to locate sites related to somatic embryogenesis and regeneration ability, and these sites are targetedly introduced into varieties with excellent agronomic traits, which can broaden the selection of excellent varieties as transgenic receptors.
  • One of the purposes of the present invention is to provide a method for cultivating an efficient genetic transformation receptor of upland cotton, comprising the following steps: material creation ⁇ high regeneration material screening ⁇ high genetic transformation material screening, wherein the material creation comprises: hybridizing 'Emian 22' as the female parent and 'Yu Zao 1' as the male parent, and then selfing for multiple generations to construct a genetically stable F 9 generation recombinant inbred line.
  • the high regeneration material screening includes: performing callus induction, embryo differentiation and plant regeneration culture on the hypocotyls of sterile seedlings of the genetically stable F9 generation recombinant inbred line to screen out high regeneration materials.
  • steps include:
  • Callus induction using two sets of callus induction culture systems, IBA+KT and/or 2,4-D+KT, to culture the hypocotyls of the sterile seedlings;
  • the embryonic callus is inoculated into a differentiation medium and a rooting medium in sequence, and hydroponically cultured to obtain regenerated plants.
  • the cotton regeneration ability traits include: callus induction rate, callus subculture reproduction capacity, callus embryo emergence time, and callus embryo emergence rate.
  • Callus induction rate number of callus blocks/total number of induced callus blocks ⁇ 100%;
  • Subculture reproduction capacity of callus tissue callus weight after 25 days of subculture - callus weight at 0 days of subculture;
  • Callus embryogenesis time the earliest time when embryonic callus appears in a single family
  • Embryo emergence rate of callus tissue the number of blocks with embryonic callus/the total number of subculture blocks ⁇ 100%.
  • the screening of the highly genetically transformed material includes: using the Agrobacterium-mediated cotton genetic transformation system on the highly regenerative material, using fluorescent protein as a reporter gene, obtaining transgenic plants, and screening transgenic lines with a genetic transformation efficiency of not less than 80%.
  • screening steps of high genetic transformation materials are as follows:
  • culture media including sterile seedling germination medium, Agrobacterium activation medium, co-culture medium, callus induction medium, somatic embryo differentiation medium, and plant rooting medium;
  • the step also includes: investigating the agronomic traits of the high genetic transformation material, and finally screening out regenerated materials with excellent agronomic traits and high regeneration ability and genetic transformation efficiency.
  • the present invention also aims to provide a method for cultivating a high-efficiency genetic transformation receptor for upland cotton and its application in cultivating a high-efficiency genetic transformation receptor for upland cotton.
  • the present invention also aims to provide a method for cultivating a high-efficiency genetic transformation receptor for upland cotton, and obtain the high-efficiency genetic transformation receptor for upland cotton.
  • the purpose of the present invention is to provide a cultivation method and application of an upland cotton efficient genetic transformation receptor.
  • the invention uses Hubei province's high-yield broadleaf variety 'Emian 22' (E22) as a female parent and chicken-foot-leaf variety 'Yu Zao 1' (YZ-1) with high regeneration ability as a male parent for hybridization, and then self-pollinates for multiple generations to construct a genetically stable F9 recombinant inbred line, utilizes a culture system of two different hormone combinations of IK (IBA+KT) and DK (2,4-D+KT) to evaluate the regeneration ability of the population, and identifies the genetic transformation efficiency of the screened high-regeneration broadleaf cotton to obtain a cotton receptor with high genetic transformation efficiency, and further investigates agronomic traits to prepare a cotton receptor with excellent agronomic traits and high genetic transformation efficiency, thereby broadening the renewable upland cotton genotype, providing excellent receptor materials for cotton genetic transformation, and
  • FIG1 is a technical route of the present invention.
  • FIG2 is a flow chart of the regeneration capacity evaluation under the IK and DK systems of the present invention.
  • Figure 3 is a diagram of the culture process of the two culture systems of IK and DK in the present invention.
  • Figures (a) and (b) show the regeneration process of the IK system and the DK system, respectively, wherein A is a cut hypocotyl segment cultured on a callus induction medium, B is the induced non-embryonic callus, C is the embryonic callus, D is the differentiated embryoid, E is the differentiated rooted seedling, and F is the hydroponic regenerated plant.
  • Figure 4 is a frequency distribution histogram of CSC, CET and CRE in callus tissue.
  • (a) and (b) show the frequency distribution of CSC, CET and CRE in callus tissue under IK and DK systems, and the frequency distribution of CSC, CET and CRE in callus tissue of regenerative families under both IK and DK systems, respectively.
  • A is CSC
  • B is CET
  • C is CRE.
  • Figure 5 is an overview of regenerated plants from high regeneration families.
  • FIG. 6 is a diagram of the genetic transformation process of the high regeneration broadleaf family.
  • the high regeneration broadleaf cotton family was cultured in sterile seedlings, and RFP was used as a reporter gene and transformed using the Agrobacterium-mediated cotton genetic transformation system.
  • A is the cultured sterile seedlings
  • B is the co-culture of the cut hypocotyl segments and the Agrobacterium transformed with the RFP gene
  • C is the transformation to the screening medium for callus induction
  • D is the induced non-embryonic callus
  • E is the differentiated embryonic callus and embryoids
  • F is the differentiated regenerated plant
  • G is the hydroponic transgenic T0 plant.
  • Figure 7 is a diagram for identifying the efficiency of RFP transformation in high regeneration broadleaf family.
  • the obtained transgenic T0 generation plants were positively identified, and the RFP expression of transgenic leaves was observed using a fluorescent stereomicroscope.
  • A, B, C, and D are pictures under white light
  • E, F, G, and H are corresponding pictures under fluorescence.
  • a and E are embryonic callus during culture
  • B and F are embryoids during culture
  • C and G are leaves of transgenic plants
  • D and H are leaves of transgenic plants.
  • the left side shows positive plants
  • the right side shows transgenic negative plants.
  • hypocotyls of seeds of the YE family and the parents E22 and YZ-1 were subjected to callus induction, embryo differentiation and plant regeneration culture to screen out high regeneration materials.
  • the steps are as follows:
  • the hypocotyls of sterile seedlings were cut into 0.5 cm-0.8 cm segments and inoculated on two callus induction media (IK and DK).
  • the regeneration capacity evaluation flow chart under the IK and DK systems is shown in Figure 2, where the IK system Includes: MS medium + 1mg/L IBA + 0.2mg/L KT; DK system includes: MS medium + 1mg/L2,4-D + 0.2mg/L KT.
  • Subculture was performed every 25-30 days using their respective systems until embryonic callus appeared, and then transferred to differentiation medium (MS macroelements 50 ml, trace elements 10 ml, iron salts 10 ml, inositol 10 ml, potassium nitrate 50 ml, L-Gly 1 ml, B5 organic matter 1 ml, IBA 1 ml, KT 0.3 ml, glutamine 1 g, asparagine 0.5 g, glucose 30 g, Phytagel 2.6 g, pH 6.1-6.2, ddH 2 O to 1 liter) until somatic embryos and seedlings were cultured; the seedlings were transferred to rooting medium (MS macroelements 25 ml, trace elements 5 ml, iron salts 5 ml, inositol 10 ml, L-Gly 1 ml, B5 organic matter 1 ml, glucose 15 g, Phytagel 2.6 g, pH 5.90-5.9, ddH
  • each system was used for subculture every 25 days, and the callus induction rate (CIF) was calculated.
  • the proliferation weight of each family was weighed at the first subculture (25 days), and the average value was calculated to calculate the callus subculture fecundity (CSC).
  • CSC callus subculture fecundity
  • the number of callus blocks was randomly reduced during the subculture process.
  • the callus embryo emergence time (CET) and the number of callus embryo emergence blocks and bottles were counted to calculate the callus embryo emergence rate (CRE).
  • CCT callus embryo emergence time
  • CRE callus embryo emergence rate
  • Callus induction rate (CIF): number of callus blocks/total number of induced callus blocks ⁇ 100%;
  • CCT Callus embryogenesis time
  • CRE Callus embryo emergence rate
  • the DK system is: 2.1g-7.4g, with an average value of 4.8g, and the IK system is: 2.8g-5.7g, with an average value of 4.2g; for the CET trait, the DK system is: 75d-154d, and the IK system is: 67d-116d; for the CRE trait, the DK system is: 33.3%-100%, with an average value of 86.3%, and the IK system is: 25%-100%, with an average value of 82.4%. Based on the statistical results of the above three traits, the DK system has a faster callus proliferation, and the earliest embryonic time of the IK system is earlier than that of the DK system. It can be seen that excessive callus proliferation does not accelerate the appearance of embryonic callus. In terms of the selection of the culture system for the YE family, the IK system is more suitable.
  • the plants with callus induction rate higher than 98%, close to 100%, callus subculture reproduction capacity higher than 4.2g, callus embryo generation time no higher than 116d, and callus embryo generation rate higher than 82.4% were selected to obtain the high regeneration material.
  • the high regeneration plant overview is shown in FIG5 .
  • Example 2 Identification of genetic transformation efficiency of high regenerated broadleaf cotton.
  • the genetic transformation efficiency of the obtained high regeneration broadleaf cotton family was analyzed, and transgenic plants were obtained by using the red fluorescent protein (RFP) as a reporter gene and the Agrobacterium-mediated cotton genetic transformation system, and the process diagram is shown in Figure 6.
  • the specific steps are as follows:
  • IBA+KT (IK) culture system For lines that can regenerate under both systems, the IBA+KT (IK) culture system is used; for lines that can only regenerate under the IK system or the DK system, the IK or DK system is used.
  • Subculture is carried out every 25d-30d using their respective systems until the emergence of The embryonic callus tissue is then transferred to a differentiation medium until somatic embryos and seedlings are cultured. The seedlings are transferred to a rooting medium and cultured until transgenic plants are obtained. The transgenic plants obtained are hydroponically cultivated and hardened, and then transplanted into soil pots in a greenhouse and the transgenic plants finally obtained in each family are counted.
  • the expression of red fluorescent protein in the plants was observed using a stereo fluorescence microscope, and the transgenic positive plants were counted to analyze the genetic transformation efficiency.
  • the transgenic broadleaf family YE3 with a genetic transformation efficiency of no less than 80% was screened, and its genetic transformation efficiency was 82.9%.
  • the method of the present invention can eventually screen out cotton receptors with high genetic transformation efficiency, wherein the highest genetic transformation efficiency can reach 82.9%.
  • the main agronomic traits of the broad-leaved families with higher genetic transformation efficiency and the maternal parent E22 and the paternal parent YZ-1 were further investigated, including fiber length, fiber strength, fiber fineness, fiber elongation, fiber uniformity and lint percentage, as shown in Table 1.

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Abstract

La présente invention concerne un procédé de culture pour un récepteur de transformation génétique efficace du coton upland, comportant : l'utilisation de 'E cotton 22' (E22) comme parent femelle et de 'Yuzao No. 1' cotton (YZ-1) comme parent mâle pour construire une lignée de consanguinité recombinée F9 génétiquement stable, la réalisation de l'induction, de la différenciation et de l'embryogenèse de cals, et la culture régénérative de plantes sur l'hypocotyle de semis aseptique de la lignée de consanguinité recombinée F9 génétiquement stable, et le criblage d'un matériel hautement régénératif. Le procédé comporte les étapes suivantes : utilisation de deux combinaisons d'hormones différentes, à savoir , IK(IBA+KT) et DK(2,4-D+KT), comme système de culture d'induction de cals pour cultiver l'hypocotyle aseptique de la plantule, réaliser une culture pour obtenir un cal embryonnaire, réaliser un criblage statistique sur les caractères de capacité de régénération d'un coton pour obtenir le matériel hautement régénérable, inoculer le cal embryonnaire dans un milieu de culture de différenciation, inoculer une plantule générée par différenciation dans un milieu de culture d'enracinement, et enfin réaliser une culture à l'eau pour obtenir une plante régénérative.
PCT/CN2023/141832 2023-08-04 2023-12-26 Procédé de culture du coton upland, récepteur de transformation génétique efficace, et son utilisation Pending WO2025030757A1 (fr)

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CN202310974030.1A CN116751811B (zh) 2023-08-04 2023-08-04 一种陆地棉高效遗传转化受体的培育方法及应用

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Publication number Priority date Publication date Assignee Title
CN116751811B (zh) * 2023-08-04 2024-06-21 华中农业大学 一种陆地棉高效遗传转化受体的培育方法及应用

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US20040009601A1 (en) * 2002-07-15 2004-01-15 The Regents Of The University Of California Methods for the regeneration and transformation of cotton
CN106801065A (zh) * 2015-11-25 2017-06-06 华中农业大学 一种提高棉花再生及转化效率的方法及应用
CN116751811A (zh) * 2023-08-04 2023-09-15 华中农业大学 一种陆地棉高效遗传转化受体的培育方法及应用

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JIN, SHUANGXIA: "Optimization of Cotton Genetic Transformation System and Creation of Mutants", CHINA DOCTORAL DISSERTATIONS/MASTER'S THESES FULL-TEXT DATABASE (PHD) (AGRICULTURAL SCIENCE AND TECHNOLOGY), 1 June 2006 (2006-06-01), XP093276417 *
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