CN115125267B - Method for improving genetic transformation efficiency of oriental lily' Siberia - Google Patents
Method for improving genetic transformation efficiency of oriental lily' Siberia Download PDFInfo
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- 241000234435 Lilium Species 0.000 title claims abstract description 42
- 230000009466 transformation Effects 0.000 title claims abstract description 30
- 230000002068 genetic effect Effects 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 23
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 31
- 241000589158 Agrobacterium Species 0.000 claims abstract description 16
- 208000015181 infectious disease Diseases 0.000 claims abstract description 13
- 230000000408 embryogenic effect Effects 0.000 claims abstract description 8
- 238000003501 co-culture Methods 0.000 claims abstract description 6
- 230000004069 differentiation Effects 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 11
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 11
- 239000008272 agar Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 239000005720 sucrose Substances 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 10
- 230000000392 somatic effect Effects 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 230000009545 invasion Effects 0.000 claims description 6
- 239000012881 co-culture medium Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 4
- 230000030118 somatic embryogenesis Effects 0.000 claims description 4
- 208000027418 Wounds and injury Diseases 0.000 claims description 3
- 239000012869 germination medium Substances 0.000 claims description 3
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- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 230000017074 necrotic cell death Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 230000008929 regeneration Effects 0.000 claims description 3
- 238000011069 regeneration method Methods 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 2
- 230000006378 damage Effects 0.000 claims description 2
- 230000003203 everyday effect Effects 0.000 claims description 2
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- 208000014674 injury Diseases 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
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- 238000011161 development Methods 0.000 abstract description 2
- 230000018109 developmental process Effects 0.000 abstract description 2
- 230000013020 embryo development Effects 0.000 abstract description 2
- 230000006872 improvement Effects 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 239000012190 activator Substances 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 8
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 230000009261 transgenic effect Effects 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 241001634096 Lilium martagon Species 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 102100028908 Cullin-3 Human genes 0.000 description 1
- 101710094482 Cullin-3 Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
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Classifications
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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
Abstract
A method for improving genetic transformation efficiency of oriental lily Siberia belongs to the technical field of flower biology. The method optimizes the genetic transformation system of the 'Siberia' lily. Aiming at the phenomena of serious browning, extremely easy differentiation of adventitious buds and loss of embryogenic state in the culture process of the callus of the lily in Siberia, the temperature change treatment is utilized, the subculture period of the callus is prolonged to more than 33 days on the basis of ensuring embryogenesis, a stable and durable receptor material is provided for genetic transformation, and the transformation workload is greatly reduced. In the steps of agrobacterium infection and co-culture, the 'hydroxydiuron' is used for replacing the traditional acetosyringone as an activator to induce the gene expression of the Vir region of the agrobacterium, so that the conversion efficiency is improved by more than two times. The optimized genetic transformation system can be used for the genetic function verification in the fields of molecular genetic improvement, germplasm resource creation, genetic engineering breeding, lily development, propagation and the like of the oriental lily.
Description
Technical Field
The invention belongs to the technical field of flower biology, and particularly relates to a method for improving the genetic transformation efficiency of oriental lily 'Siberia'.
Background
Lily is an important ornamental, edible and medicinal plant, and the research of molecular biology thereof is rapidly developed. However, lily has large genome, high heterozygosity, different varieties and huge differences among different hybrid lines, and brings great difficulty to lily molecular breeding work. The establishment of a high-efficiency, stable and safe standardized lily molecular breeding technical system is a problem to be solved urgently in lily genetic engineering breeding.
In the genetic transformation study of lily, exogenous genes are usually inserted into the genome of lily by using methods such as an agrobacterium-mediated method, a gene gun method, a pollen tube channel method, an electric shock method and the like, wherein the agrobacterium-mediated genetic transformation is the most widely applied and most efficient transgenic method in the current lily transgenic study (Yan et al, 2019). To date, only a few lilies have achieved efficient and stable gene transformation, with the vast majority of stock and cultivars lacking genetic transformation systems.
Agrobacterium-mediated genetic transformation of monocots depends on a number of factors, including plasmids, agrobacterium strains, agrobacteriumvirActivation conditions for (virus) genes, plant genotypes, inoculation and co-culture conditions, screening and regeneration media, etc. (Cheng et al, 2003; lacroix B and Citovsky v., 2013). Genetic transformation of lily was studied by many researchers using agrobacterium-mediated methods, and genetic transformation systems were established with lily scales (Cohen A and Dalla-Favera r., 1992), leaves (Liu Juhua et al, 2003), calli (Wated et al, 1998), embryogenic calli (Tang Dongqin et al, 2003), somatic embryos (Yan et al, 2019), and the like, as receptor materials, respectively.
The oriental lily hybrid line has the characteristics of thick stems, upright flower stems, big flowers, positive color and fragrant smell, and is one of the most popular cut flowers worldwide. 'Siberia' is a lily main cultivated cut flower variety in the flower market in China, and has great sales. Generally, the cultivation is carried out in facilities, the cultivation is warm, the adaptability to light intensity is wider, weak light is resisted, and the buds are not easy to fall. At present, a genetic transformation system is established aiming at 'Oriental lily', but the problems of low transformation efficiency, long period, poor genetic stability and the like exist.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for improving the genetic transformation efficiency of lilium oriental hybrid.
The invention adopts the technical scheme that: the method for improving the genetic transformation efficiency of the oriental lily 'Siberia' is technically characterized by comprising the following process steps:
step 1, induction and preservation of embryogenic callus
Cutting small scales of lily tissue culture seedling in vigorous growth stage into 0.5. 0.5 cm 2 Inoculating in culture medium MS+2.0 mg/L PIC+30 g/L sucrose+7 g/L agar, and culturing at 25+ -1deg.C with relative humidity of 80-90% until yellow-green granular obvious callus is formed; transferring once in 33 days, separating the callus from the explant and cutting off brown part, and culturing at 20deg.C under complete darkness;
step 2, preparation of Agrobacterium invasion solution
Picking agrobacterium EHA105 single colony carrying plant expression vector, inoculating to strain containing 50 mg/L Kan and 50 mg.L -1 Shake culturing in YEB liquid medium of Rif to OD 600 Centrifuging at 5000 rpm for 10min at 0.6, and collecting thallus; taking 50 mL MS liquid culture medium containing 3 mu M CX to resuspend thalli, and oscillating the resuspension at 200 rpm for 2 h to obtain an infection liquid;
step 3, agrobacterium infection and Co-cultivation
Cutting pale yellow somatic embryo, culturing at 25deg.C for 10-15 days, and placing into Agrobacterium infection solution after hole pricking injury; slightly shaking the culture flask to enable the callus to be fully contacted with the infection liquid for 10min, taking out, rapidly sucking residual bacterial liquid on the surface of the tissue block, transferring the residual bacterial liquid to a co-culture medium MS+2.0 mg/L PIC+3 mu M CX+30 g/L sucrose+7 g/L agar for dark culture, wherein the culture temperature is 20+/-1 ℃;
step 4, screening of resistant callus
After 48 hours of co-culture, transferring the callus to MS+2. mg/L PIC+400 mg/L Cef+150 mg/L Kan+30 g/L sucrose+7 g/L agar medium for 2 weeks, transferring to the same medium, and transferring to the same medium for 15 days of continuous screening and degerming culture; removing the brown part of the callus block after each transfer;
step 5, regeneration of resistant callus
Resistance screening is carried out for more than 8 weeks, and the phenomena of browning and necrosis of the callus are gradually reduced; transferring healthy yellow resistant callus into germination medium MS+0.5 mg/L6-BA+0.88 g/L CaCl 2 Culturing in +30 g/L sucrose + g/L agar medium under light for 16 hr/8 hr darkness every day, and light intensity of 36 μmol.m -2 ·s -1 The culture temperature is 25+/-1 ℃; after 6 weeks, the calli regenerate into whole transformed plants via somatic embryogenesis.
In the scheme, 3 mu M hydroxydicarbonitrile is added to the invasion solution and the co-culture medium in the step 2 and the step 3 for improving the differentiation of the Lily somatic embryos of the 'Siberia'.
The beneficial effects of the invention are as follows: the invention provides a stable genetic transformation receptor for the lilium siberia, and reduces the transformation workload. Embryogenic callus cells of lilium oriental (Oriental lily) are vigorous in division, and the preservation time is only about 15 days under the conventional culture condition (25+/-1 ℃). The callus is cultured for more than 15 days, so that the callus is severely browned, meanwhile, the callus is extremely easy to redifferentiate adventitious buds, and the embryogenic state is lost. The genetic transformation system inhibits the germination of the lily siberian somatic embryos by temperature change treatment on the basis of ensuring the embryogenesis and the redifferentiation efficiency of the somatic embryos, so that the embryogenic state of the lily siberian somatic embryos is stably maintained for a long time, and a durable receptor material is provided for genetic transformation.
The invention obviously improves the genetic transformation rate of the oriental lily. In the infection and co-culture process, the 'hydroxydiuron' is used for replacing the traditional 'acetosyringone', so that the T-DNA is easier to insert into a genome and integrate with the genome, and the genetic transformation efficiency is improved by more than two times. The optimized genetic transformation system can be used for gene function verification in the fields of molecular genetic improvement of lily, germplasm resource creation, genetic engineering breeding, lily development and propagation and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the transformation process of lilium martagon 'siberia' and identification of resistant plants in an embodiment of the present invention;
FIG. 2 is a schematic representation of PCR identification of a lilium martagon 'Siberia' transgenic plant in an embodiment of the invention.
Detailed Description
The foregoing objects, features, and advantages of the invention will be more readily apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings, figures 1 and 2, and detailed description of the invention.
The transformation system established by the invention, which is adopted in the embodiment, carries the target geneBPMThe plant binary expression vector pRI101-ON-BPM is transformed to successfully obtain a transgenic plant.BPMThe gene-encoded proteins are modulators of the Cullin 3-based E3 ubiquitin ligase in animals and plants (Weber H and Hellmann h., 2009; chen et al, 2013; chico et al, 2020). The CUL3BPM E3 ubiquitin ligase is involved in regulating a variety of physiological processes. In order to describe the process steps of the invention in more detail, examples are illustrated below.
BPMGene transformed Oriental lily
Step 1, preparing agrobacterium tumefaciens invasion solution.
Will be carried alongBPMTransferring the plant binary expression vector of the gene into agrobacterium EHA105, selecting positive transformant clone, inoculating into 1 mL fresh YEB liquid culture medium containing 50 mg/L Rif and 50 mg/L Kan, shaking culture at 200 rpm for 12 h or more, sucking bacterial liquid according to the ratio of 1:50 was transferred to 50 ml liquid YEB medium containing 50 mg/L Rif and 50 mg/L Kan, and continued to culture until OD 600 The cells were collected by centrifugation at 5000 rpm for 10min at 0.6. The cells were resuspended in 50. 50 mL MS liquid medium containing 3. Mu.M CX, and the resuspension was further cultured with shaking at 200 rpm for 2 h as AgrobacteriumThe infection liquid is reserved.
And 2. Infection and co-culture.
In an ultra-clean workbench, the oriental lily embryogenic callus blocks with good growth state are immersed in agrobacterium tumefaciens invasion solution for 10min after being subjected to hole punching and wound, and the tissue blocks are continuously and lightly shaken to be fully contacted with the invasion solution. After the tissue block is taken out from the infection liquid, residual bacterial liquid on the surface of the tissue block is fully absorbed by sterile filter paper, and then transferred to a co-culture medium MS+2.0 mg/L PIC+3 mu M CX+30 g/L sucrose+7 g/L agar, wherein the culture temperature is 20+/-1 ℃ and the dark culture is 48 h.
And 3, screening and subculturing the resistant callus.
The co-cultured calli were transferred to MS+2.0 mg/L PIC+400 mg/L Cef+150 mg/L Kan+30 g/L sucrose+7 g/L agar medium for screening and degerming. After 2 weeks, transfer to the same medium followed by selection degerming, after which transfer is carried out once for 15 days, each transfer cutting off the brown part of the callus pieces (figure).
And 4, germination and seedling formation of somatic embryos.
Resistance screening is carried out for more than 8 weeks, and the phenomena of browning and necrosis of the callus are gradually reduced. Transferring healthy yellow resistant callus into germination medium MS+0.5 mg/L6-BA+0.88 g/L CaCl 2 Culturing in +30 g/L sucrose + g/L agar medium under light (16 h light/8 h dark per day, 36. Mu. Mol.m light intensity) -2 ·s -1 ) The culture temperature was 25.+ -. 1 ℃. After 6 weeks, the calli regenerated into whole transformed plants via somatic embryogenesis (figure).
And 5, PCR detection of the transformed plants.
Extraction of genomic DNA from resistant plantsBPMThe gene sequence is designed into a primer, PCR amplification is carried out by taking the genomic DNA of the lilium oriental 'siberia' as a template, and a wild plant is taken as a contrast, if the PCR result of the transformed plant is positive, the exogenous gene is integrated into the genome of the plant (attached drawing). The primer sequences for PCR detection of the transformed plants are as follows:
Primer F：5’-AGTTCTTTCGGCTTGTTG-3’
Primer R：5’-CTGAGCCTGAGCCTGAGATT-3’
the PCR reaction procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 1min, annealing at 58℃for 1min, extension at 72℃for 1min, 35 cycles; extending at 72 ℃ for 5 min.
Through a large number of experiments, the genetic transformation efficiency of the Siberia lily based on somatic embryogenesis is stabilized at 30.43%, and the method can be used for various works for obtaining transgenic lily in a large quantity.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. A method for improving the genetic transformation efficiency of lilium oriental 'siberia', which is characterized by comprising the following process steps:
step 1, induction and preservation of embryogenic callus
Cutting small scales of lily tissue culture seedling in vigorous growth stage into 0.5. 0.5 cm 2 Inoculating in culture medium MS+2.0 mg/L PIC+30 g/L sucrose+7 g/L agar, and culturing at 25+ -1deg.C with relative humidity of 80-90% until yellow-green granular obvious callus is formed; transferring once in 33 days, separating the callus from the explant and cutting off brown part, and culturing at 20deg.C under complete darkness;
step 2, preparation of Agrobacterium invasion solution
Picking agrobacterium EHA105 single colony carrying plant expression vector, inoculating to strain containing 50 mg/L Kan and 50 mg.L - 1 Shake culturing in YEB liquid medium of Rif to OD 600 Centrifuging at 5000 rpm for 10min at 0.6, and collecting thallus; taking 50 mL MS liquid culture medium containing 3 mu M CX to resuspend thalli, and oscillating the resuspension at 200 rpm for 2 h to obtain an infection liquid;
step 3, agrobacterium infection and Co-cultivation
Cutting pale yellow somatic embryo, culturing at 25deg.C for 10-15 days, and placing into Agrobacterium infection solution after hole pricking injury; slightly shaking the culture flask to enable the callus to be fully contacted with the infection liquid for 10min, taking out, rapidly sucking residual bacterial liquid on the surface of the tissue block, transferring the residual bacterial liquid to a co-culture medium MS+2.0 mg/L PIC+3 mu M CX+30 g/L sucrose+7 g/L agar for dark culture, wherein the culture temperature is 20+/-1 ℃;
step 4, screening of resistant callus
After 48 hours of co-culture, transferring the callus to MS+2. mg/L PIC+400 mg/L Cef+150 mg/L Kan+30 g/L sucrose+7 g/L agar medium for 2 weeks, transferring to the same medium, and transferring to the same medium for 15 days of continuous screening and degerming culture; removing the brown part of the callus block after each transfer;
step 5, regeneration of resistant callus
Resistance screening is carried out for more than 8 weeks, and the phenomena of browning and necrosis of the callus are gradually reduced; transferring healthy yellow resistant callus into germination medium MS+0.5 mg/L6-BA+0.88 g/L CaCl 2 Culturing in +30 g/L sucrose + g/L agar medium under light for 16 hr/8 hr darkness every day, and light intensity of 36 μmol.m -2 ·s -1 The culture temperature is 25+/-1 ℃; after 6 weeks, the calli regenerate into complete transformed plants via somatic embryogenesis;
and 3, adding 3 mu M hydroxydicarbonitrile into the counterstain solution and the co-culture medium for improving the differentiation of the Lily somatic embryo of Siberia.
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| CN103798140A (en) * | 2014-01-26 | 2014-05-21 | 浙江大学 | Culture method for significantly improving subculture proliferation rate of wild lily embryonic callus |
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| CN108300735A (en) * | 2018-03-13 | 2018-07-20 | 沈阳农业大学 | A kind of Lilium tenuifolium efficient genetic trasformation system based on somatic embryo occur |
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| CN103798140A (en) * | 2014-01-26 | 2014-05-21 | 浙江大学 | Culture method for significantly improving subculture proliferation rate of wild lily embryonic callus |
| CN107173236A (en) * | 2017-07-19 | 2017-09-19 | 合肥申沃园艺有限公司 | A kind of Lilium Germplasm method for tissue culture |
| CN108300735A (en) * | 2018-03-13 | 2018-07-20 | 沈阳农业大学 | A kind of Lilium tenuifolium efficient genetic trasformation system based on somatic embryo occur |
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| Title |
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| Hojatollah Abbasi等.Effect of ‘Chloroxynil’ on Agrobacterium-mediated transformation efficiency of Lilium cv ‘Manissa’.Scientia Horticulturae.2020,第271卷文章编号109404. * |
| 于世勇等.温度对植物体细胞胚胎发生影响的研究进展.防护林科技.2010,(第4期), * |
| 刘娜.两种百合体细胞胚高效再生与遗传转化体系优化.中国优秀硕士学位论文全文数据库 农业科技辑.2023,(第2023 年 第02期期), * |
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