CN110786238A - Ranunculus Sclerotii somatic embryo induction method and somatic embryo mediated genetic transformation method - Google Patents
Ranunculus Sclerotii somatic embryo induction method and somatic embryo mediated genetic transformation method Download PDFInfo
- Publication number
- CN110786238A CN110786238A CN201910884952.7A CN201910884952A CN110786238A CN 110786238 A CN110786238 A CN 110786238A CN 201910884952 A CN201910884952 A CN 201910884952A CN 110786238 A CN110786238 A CN 110786238A
- Authority
- CN
- China
- Prior art keywords
- ranunculus
- culture medium
- somatic
- somatic embryo
- sclerotii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for inducing somatic embryos of ranunculus lanceolatus and a method for mediating genetic transformation of the somatic embryos. The method for inducing the somatic embryos of ranunculus chinensis comprises the following steps: culturing the aseptic seedlings of the ranunculus Sclerotii; inducing the root, stem (without axillary bud) and leaf of Ranunculus Sclerotis to generate somatic embryos by using a somatic embryo induction culture medium SE-M under a dark culture condition; and (3) inducing the somatic embryos to root and grow seedlings on a rooting culture medium RO-M. The invention can provide a stable receptor system and a verification platform for the research of the molecular mechanism of the somatic embryogenesis of the ranunculus Sclerotii, the stress resistance of the ranunculus Sclerotii, the new generation of soil repair resources and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing somatic embryos of ranunculus lanceolatus and a method for mediating genetic transformation of the somatic embryos.
Background
Ranunculaceae is a Chinese herbal medicine, and is rich in secondary metabolites with high medicinal value, including alkaloid, tannin, flavonoids, isoflavone and protocatechuic aldehyde. Poisonous buttercup has long been used in traditional Chinese medicine to prevent replication of hepatitis B virus HBV and herpes simplex virus 1 HSV-1, and to treat jaundice, rheumatic pain, asthma and urinary incontinence. Meanwhile, ranunculus chinensis is also a very effective pesticide for controlling pests such as drosophila melanogaster and tribolium castaneum. In addition, the ranunculus Sclerotii has strong sewage treatment capacity, can absorb a large amount of nitrogen and phosphorus, and accumulates and monitors the content change of heavy metals such as copper, lead, iron, zinc and the like in soil.
The research on Ranunculaceae plants in the prior art mostly focuses on the aspects of activity detection, toxicity and active component analysis of secondary metabolites of the Ranunculaceae plants, removal and purification of heavy metals and organic pollutants in soil, development mechanisms of floral organs of ornamental Ranunculaceae plants such as diploid black grass, aquilegia japonica and the like. The conventional aseptic seedling culture process of various Ranunculaceae plants has some technical difficulties, such as seed germination rate, synchronous rate of germination vigor, subculture of aseptic seedlings and the like. No existing system can be referred to about the Ranunculaceae plant somatic embryogenesis and the mediated efficient regeneration, the conventional regeneration system and the genetic transformation system. The establishment of systems such as efficient regeneration and genetic transformation mediated by somatic embryos of ranunculus lanceolatus has a plurality of technical bottlenecks: such as culture medium screening, seed germination acceleration, plant growth regulator type and concentration screening, optimal explant screening, agrobacterium infection period selection and the like. Therefore, no research report is reported on the efficient regeneration and genetic transformation system mediated by the somatic embryos of ranunculus chinensis.
Disclosure of Invention
The invention aims to provide a method for inducing a somatic embryo of ranunculus chinensis
Still another object of the present invention is to provide a method for somatic embryo-mediated genetic transformation of ranunculus sieboldii.
The method for inducing the somatic embryos of ranunculus Sclerotii according to the embodiment of the invention comprises the following steps of:
(1) sterilizing the seed of the ranunculus Sclerotii, and sowing the seed in a sterile seedling (MS) culture medium for sterile seedling culture;
(2) selecting a vegetative organ of the aseptic seedling as an explant, placing the explant on a somatic embryo induction culture medium, and inducing a somatic embryo structure, wherein the somatic embryo induction culture medium comprises the following components: MS salt, B5 organic, 10.0mg/L NAA, 30g/L sucrose, 3.6g/L gellan gum, and the pH of the somatic embryo induction culture medium is 5.8;
(3) transferring the induced somatic embryos of ranunculus Sclerotii into a rooting culture medium, performing vertical plate culture, and inducing the embryos to root, wherein the rooting culture medium comprises the following components: 1/2MS salt, 1/2B5 organic, 0.1mg/L NAA, 15g/L sucrose, 7.8g/L agar powder, and the pH value of the rooting medium is 5.8;
(4) hardening and transplanting the seedlings.
According to the method for inducing the somatic embryos of the ranunculus Sclerotii, in the step (1), healthy and plump ranunculus Sclerotii seeds are selected, surface disinfection is carried out for 0.5-1min by using 75% ethanol in volume ratio, and the ranunculus Sclerotii seeds are washed for 3-5 times by using sterile water; sterilizing with 2.5% ammonium hypochlorite for 8-10min, washing with sterile water for 3-5 times, and sterilizing the seeds.
According to the method for inducing the Ranunculus Sclerotii cell embryo, the MS culture medium comprises the following components: MS salt, B5 organic matter, 30g/L cane sugar and 7.8g/L agar powder, and the pH value of an MS culture medium is 5.8.
According to the method for inducing the somatic embryos of ranunculus Sclerotii, disclosed by the specific embodiment of the invention, in the step (1), after seeds are sowed in an MS culture medium, pregermination is carried out for 2-3d at the temperature of 4 ℃, and then the seeds are incubated in the dark at the temperature of 25 +/-1 ℃ until the seed shells are exposed to be white; the plates were then incubated at 25 ℃ for 16h light/8 h dark at a light level of 120. mu. mol.m-2s-1Culturing under the conditions of (1).
According to the method for inducing the somatic embryos of ranunculus lanceolatus, in the step (2), the roots, stems or leaves of aseptic seedlings are selected as explants.
A method for somatic embryo-mediated genetic transformation of ranunculus lanceolatus according to an embodiment of the present invention comprises the steps of:
(1) and (2) sterilizing the seed of the ranunculus Sclerotii, sowing the seed in an MS culture medium, and performing aseptic seedling culture, wherein the MS culture medium comprises the following components: MS salt, B5 organic matter, 30g/L sucrose and 7.8g/L agar powder, wherein the pH value of an MS culture medium is 5.8;
(2) selecting a nutritive organ of the aseptic seedling as an explant, and infecting the explant by using an agrobacterium transformation medium in a transformation infection medium;
(3) spreading the infected explants in a co-culture medium for co-culture;
(4) transferring the explants after co-culture into a somatic embryo induction culture medium containing antibiotics to perform induction generation of somatic embryos and screening of resistant somatic embryos, wherein the somatic embryo induction culture medium comprises the following components: MS salt, B5 organic, 10.0mg/L NAA, 30g/L sucrose, 3.6g/L gellan gum, and the pH of the somatic embryo induction culture medium is 5.8;
(5) placing the resistant somatic embryos in a rooting culture medium, and inducing the embryo bodies to root under the illumination condition, wherein the rooting culture medium comprises the following components: 1/2MS salt, 1/2B5 organic, 0.1mg/L NAA, 15g/L sucrose, 7.8g/L agar powder, and the pH value of the rooting medium is 5.8;
(6) hardening and transplanting the seedlings.
According to the method for the somatic embryo-mediated genetic transformation of the ranunculus lanceolatus, in the step (2), a transformation and infection medium comprises the following components: MS salt, B5 organic, 15g/L glucose, pH of transformation and infection medium 5.8.
According to the method for the somatic embryo-mediated genetic transformation of ranunculus lanceolatus in the embodiment of the invention, in the step (3), the co-culture medium comprises the following components: MS salt, B5 organic matter, 40mg/L acetosyringone, 30g/L sucrose and 7.8g/L agar powder, and the pH value of the co-culture medium is 5.8.
According to the method for somatic embryo-mediated genetic transformation of Ranunculus Sclerotis in the embodiment of the invention, in the step (4), the somatic embryo induction medium contains 100mg/L kanamycin sulfate and 500mg/L carbenicillin sodium.
According to the method for the somatic embryo-mediated genetic transformation of the ranunculus Sclerotii in the embodiment of the invention, in the step (5), the resistant somatic embryo is placed at the temperature of 25 ℃, the illumination is 16 h/8 h in darkness and the illumination is 120 mu mol-2s-1Inducing rooting under the condition.
The invention has the beneficial effects that:
the method can quickly and efficiently induce the somatic embryo structure of the ranunculus Sclerotii, can successfully transform the exogenous gene by an explant infection-somatic embryo induction method, and the established somatic embryo-mediated genetic transformation system of the ranunculus Sclerotii has the characteristics of quickness, high efficiency, low chimeric rate, stable inheritance and the like.
Drawings
FIG. 1 shows the different developmental stages of the somatic embryos of Ranunculus sieboldii; wherein A is a plurality of somatic embryo structures induced by a ranunculus Scleratus leaf explant; b is a spherical embryo; c is a spherical-heart-shaped transition embryo; D. e, F and G are heart-shaped embryos; h is a torpedo embryo; I. j, K and L are cotyledonary embryos;
FIG. 2 shows somatic embryo-mediated regeneration of Ranunculus sieboldii; wherein A is a somatic embryo structure of different developmental stages induced by a ranunculus Scleratus leaf explant; b is a somatic embryo development process, showing cotyledon embryos with different cotyledon numbers; c is a mature embryo; d is the rooting induction of somatic embryos; e is a somatic embryo regenerated seedling; f is a regeneration plant of ranunculus Sclerotii; f1 and F2 are the regenerated plant flower organs of ranunculus Sclerotii;
FIG. 3 showsExpression of RcLEC1-B resulted in dwarfing of Ranunculus Sclerotii and floral organ malformation; wherein A, A1, A2 and B are the process of genetic transformation of Ranunculus Sclerotii, and kanamycin sulfate (Kan) at different stages is induced from root, stem and leaf+) A positive embryo body; a is a Ranunculus Sclerotii root explant; a1 is Ranunculus Scleroticus explant; a2 and B are Ranunculus Sclerotii implants; c1 is GUS staining of Ranunculus Sceleratus positive embryo; c2 is GUS staining of Ranunculus Sceleratus positive floral organ; d and D1 are wild plants of Ranunculus Sceleratus (15D); e and E1 are Ranunculus Sclerotii null plants (pCAMBIA2300, 15 d); d2 is Ranunculus Sceleratus wild plant (30D); e2 is Ranunculus Scleroticus Idaeculatus (pCAMBIA2300, 30 d); f and F1 are RcLEC1-B-OE Ranunculus Scleroticus plants with dwarfing phenotype (15 d); f2 is RcLEC1-B-OE Ranunculus Sceleratus (30 d); g is RcLEC1-B-OE Ranunculus Sclerotii plant (15d) with cuticle deficiency phenotype;
FIG. 4 shows the floral morphology and number of RhLEC 1-B-OE Sclerotium Toxicodendri and wild type plants, wherein A has 5 sepals, 5 petals and 16 stamens and 1 total pistil; B-B16 is RcLEC1-B-OE Ranunculus Scleroticus organ; B. b2, B3, B4, B5, B6, B7 and B10 are petal-shaped structures with deformity in the organ of Ranunculus Scleroticus of RcLEC 1-B-OE; b4 is a transition structure between petal and sepal in the organ of Ranunculus Sclerotii of RcLEC 1-B-OE; B-B16 is RcLEC1-B-OE Ranunculus Scleroticus organ without petal and stamen structure;
FIG. 5 shows the somatic embryo induction of Ranunculus Sclerotis in the early development stage, wherein A-C is the somatic embryo of Ranunculus Sclerotis in the early development stage; A1-C1 is the somatic embryo of the young ranunculus Sclerotii; a and A1 are root explants; b and B1 are stem explants; c and C1 are leaf explants.
Detailed Description
The culture media used in the present invention are shown in table 1:
TABLE 1 abbreviations, names and compositions of the media used in the present invention
Example 1
(1) And (3) sterile seedling culture: selecting healthy and plump Ranunculus Sclerotis seed, sterilizing the surface of the seed with 75% (v/v) ethanol for 30s-1min, and washing with sterile water for 3-5 times; sterilizing with 2.5% ammonium hypochlorite for 8-10min, and washing with sterile water for 3-5 times; and (3) sowing the disinfected seeds into a tissue culture bottle containing an improved MS culture medium, accelerating germination for 2-3d at a low temperature (4 ℃), and then incubating the seeds (25 +/-1 ℃) in a dark condition until the seed shells are exposed to be white.
The culture dish was exposed to 16h light/8 h dark at 25 ℃ with a light intensity of 120. mu. mol.m-2s-1Culturing under the tissue culture conditions of (1).
(2) When the tissue culture seedling of the ranunculus Sclerotii grows to 4-5 true leaves, selecting three explants of root, stem (without axillary bud) and leaf of the ranunculus Sclerotii, placing the three explants on a somatic embryo induction culture medium SE-M (somatic embryo induction medium), and inducing the somatic embryo structure.
As shown in fig. 1, the development of the somatic embryos of ranunculus spinosus belongs to the classical SE pathway, which proceeds through proembryo-globular embryo-heart embryo-torpedo embryo-cotyledon embryo.
(3) Transferring the induced somatic embryo of ranunculus Sclerotii into a rooting culture medium RO-M (rooting medium), and performing vertical plate culture to induce the embryo to root and grow seedlings.
As shown in fig. 2, a single embryo can induce a normal root system, the induction rate of the root system of the embryo is 100%, and a single seedling can be formed; can normally develop and bloom after being cultured.
Example 2
(1) And (3) sterile seedling culture: selecting healthy and plump Ranunculus Sclerotis seed, sterilizing the surface of the seed with 75% (v/v) ethanol for 30s-1min, and washing with sterile water for 3-5 times; sterilizing with 2.5% ammonium hypochlorite for 8-10min, and washing with sterile water for 3-5 times; sowing the disinfected seeds in a tissue culture bottle containing an improved MS culture medium, accelerating germination for 2-3d at low temperature (4 ℃), and then incubating the seeds (25 +/-1 ℃) in the dark until the seed shells are exposed to be white;
the culture dish was exposed to 16h light/8 h dark at 25 ℃ with a light intensity of 120. mu. mol.m-2s-1Culturing under the tissue culture conditions of (1);
(2) firstly, carrying out agrobacterium transformation medium SU-M (Suspensions medium) infection on three explants, namely a root, a stem (without axillary buds) and a leaf of the ranunculus carinatus, and continuously shaking for 8-10min during the infection to ensure that the explants are fully contacted with the transformation medium;
taking the infected explant out of the transformation medium, firstly sucking redundant transformation medium by using sterile filter paper, flatly paving the explant in a culture medium CC-M (Co-culture medium) for Co-culture, and then placing the explant in a constant-temperature incubator at 25 ℃ for Co-culture treatment for 72 h;
(3) transferring the cocultured Ranunculus Sclerotii explants into a somatic embryogenesis-screening medium SEIS-M to perform induction of somatic embryos and screening of resistant somatic embryos.
As shown in FIG. 3, three explants, root, stem and leaf, induced somatic embryo structure, with leaf explant as the best recipient explant. GUS staining results showed that the embryo bodies were stained blue in the presence of X-gluc substrate, as in C1 in FIG. 3, and the single flowers of the positive seedlings were also stained blue, as in C2 in FIG. 3.
(4) Placing the resistant somatic embryos in a rooting culture medium RO-M under the condition of illumination (25 ℃, 16h of illumination/8 h of darkness and illumination intensity of 120 mu mol.m)-2s-1) Inducing the embryo to take root and grow seedling;
(5) opening bottles for hardening 7d of resistant buds induced by somatic embryos, washing off agar blocks remained in root systems by using a small stream of clear water, transplanting the young ranunculus Sclerotii seedlings into a culture medium (large vermiculite: flower soil: 1), and performing preservative film moisture culture under weak light. And after 15d, removing the preservative film, and maintaining in a relatively humid environment.
Example 3
(1) Infecting the leaf explant of Ranunculus Sclerotii with Agrobacterium transformation medium SU-M (RcLEC1-B-OE), and shaking for 8-10 min; spreading the infected explants in a co-culture medium CC-M, and carrying out dark co-culture treatment at 25 ℃ for 72 h;
(2) transferring the leaf explants of the Ranunculus Sclerotii after co-culture into a somatic embryogenesis-screening culture medium SEIS-M for screening resistant somatic embryos;
(3) placing the Sclerotinia Sclerotidis resistant somatic embryo in a rooting culture medium RO-M to induce the embryo to take root and grow seedlings;
(4) overexpression of RcLEC1-B also resulted in dwarfing of Ranunculus Sclerotis (F, F2 in FIG. 3), leaf malformation (F1 and G in FIG. 3), and teratization of floral organs by the above transgenic method; as shown in FIG. 4, the abnormal transformation of flower organs is mainly manifested by the number change and morphological change of petals, sepals and stamens. The result of the research shows that RcLEC1-B can cause the dwarfing of transgenic Arabidopsis thaliana and the malformation of floral organs.
In the process of inducing the somatic embryo structure by placing the explants on a somatic embryo induction culture medium SE-M, the influence of NAA with different concentrations on inducing the somatic embryos by the explants of the root, the stem and the leaf of ranunculus lanceolatus is examined, and the results are shown in Table 2 and FIG. 5.
TABLE 2 Effect of NAA at different concentrations on the induction of somatic embryos by the root, stem and leaf explants of Ranunculus Sclerotii
Note: the Duncan test was performed using SPSS16.0 and the mean and standard error of the number of somatic embryos produced was calculated for 300 explants on 30 dishes. Capital and lowercase letters indicate significant differences at 1% and 5% probability levels, respectively.
As shown in FIG. 5, the influence of NAA with different concentrations on inducing somatic embryos of the root, stem and leaf explants of the ranunculus Sclerotii is longitudinally analyzed, and the induced embryo yield of NAA with the concentration of 10mg/L is the highest and is obviously higher than the concentrations of 0mg/L, 1.0mg/L, 2.5mg/L, 5.0mg/L, 20.0mg/L and the like. Based on the data in table 2, the induction effect of three different explants was analyzed transversely, the leaf was the best embryo-inducing explant, and a single explant could produce 213.63 embryos, which are far greater than 66.03 from root explants and 126.47 from stem explants.
Claims (10)
1. The method for inducing the somatic embryos of ranunculus chinensis is characterized by comprising the following steps of:
(1) sterilizing the seed of the ranunculus Sclerotii, sowing the seed in an aseptic seedling culture medium, and performing aseptic seedling culture;
(2) selecting a vegetative organ of a sterile seedling as an explant, placing the explant on a somatic embryo induction culture medium, and inducing a somatic embryo structure, wherein the somatic embryo induction culture medium comprises the following components: MS salt, B5 organic, 10.0mg/L NAA, 30g/L sucrose, 3.6g/L gellan gum, and the pH of the somatic embryo induction culture medium is 5.8;
(3) transferring the induced Ranunculus Sceleratus somatic embryo into a rooting culture medium for culturing, and inducing the embryo body to root, wherein the rooting culture medium comprises the following components: 1/2MS salt, 1/2B5 organic, 0.1mg/L NAA, 15g/L sucrose, 3.6g/L gellan gum, the pH of the rooting medium is 5.8;
(4) hardening and transplanting the seedlings.
2. The method of inducing somatic embryos of ranunculus Sclerotii according to claim 1, wherein the sterile shoot medium comprises the following components: MS salt, B5 organic matter, 30g/L cane sugar, 7.8g/L agar powder, and the pH value of the aseptic seedling culture medium is 5.8.
3. The method for inducing the somatic embryo of ranunculus Sclerotii as claimed in claim 1, wherein in step (1), healthy and plump seeds of ranunculus Sclerotii are first surface-sterilized with 75% by volume of ethanol for 0.5-1min and then rinsed with sterile water for 3-5 times; sterilizing with 2.5% ammonium hypochlorite for 8-10min, and washing with sterile water for 3-5 times.
4. The method for inducing the somatic embryo of ranunculus Sceleratus as claimed in claim 1, wherein in step (1), after the seeds are sown in the sterile seedling culture medium, the seeds are germinated for 2-3 days at 4 ℃, incubated in the dark at the temperature of 25 +/-1 ℃ until the seed shells are exposed to white, and then the seeds are exposed to light at 25 ℃, 16 h/8 h in the dark and the illumination is 120 μmol-2s-1Culturing under the conditions of (1).
5. The method for inducing somatic embryos of ranunculus Sclerotii as claimed in claim 1, wherein in step (2), the root, stem or leaf of the sterile plantlet is selected as the explant.
6. A method for somatic embryo-mediated genetic transformation of ranunculus lanceolatus, comprising the steps of:
(1) sterilizing the seed of the ranunculus Sclerotii, sowing the seed in an aseptic seedling culture medium, and performing aseptic seedling culture;
(2) selecting a nutritive organ of the aseptic seedling as an explant, and infecting the explant by using an agrobacterium transformation medium in a transformation infection medium;
(3) spreading the infected explants in a co-culture medium for co-culture;
(4) transferring the explants after co-culture into a somatic embryo induction culture medium containing antibiotics to perform induction generation of somatic embryos and screening of resistant somatic embryos, wherein the somatic embryo induction culture medium comprises the following components: MS salt, B5 organic, 10.0mg/L NAA, 30g/L sucrose, 7.8g/L agar powder, and the pH value of the somatic embryo induction culture medium is 5.8;
(5) placing the resistant somatic embryos in a rooting culture medium, and inducing the embryo bodies to root under the illumination condition, wherein the rooting culture medium comprises the following components: 1/2MS salt, 1/2B5 organic, 0.1mg/L NAA, 15g/L sucrose, 7.8g/L agar powder, and the pH value of the rooting medium is 5.8;
(6) hardening and transplanting the seedlings.
7. The method for somatic embryo-mediated genetic transformation of ranunculus sieboldii as claimed in claim 6, wherein in step (2), the transformation invasion medium comprises the following components: MS salt, B5 organic, 15g/L glucose, pH of transformation and infection medium 5.8.
8. The method for somatic embryo-mediated genetic transformation of ranunculus sieboldii as claimed in claim 6, wherein in step (3), the co-culture medium comprises the following components: MS salt, B5 organic matter, 40mg/L acetosyringone, 30g/L sucrose and 7.8g/L agar powder, and the pH value of the co-culture medium is 5.8.
9. The method of claim 6, wherein in step (4), the somatic embryo induction medium contains 100mg/L kanamycin sulfate and 500mg/L carbenicillin sodium.
10. The method of claim 6, wherein in step (5), the resistant somatic embryos are subjected to a light intensity of 120 μmol.m at 25 ℃ for 16h light/8 h dark-2s-1Inducing rooting under the condition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210193136.3A CN114561421B (en) | 2019-08-03 | 2019-09-19 | Genetic transformation method of ranunculus |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019107140227 | 2019-08-03 | ||
| CN201910714022 | 2019-08-03 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210193136.3A Division CN114561421B (en) | 2019-08-03 | 2019-09-19 | Genetic transformation method of ranunculus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110786238A true CN110786238A (en) | 2020-02-14 |
| CN110786238B CN110786238B (en) | 2022-04-08 |
Family
ID=69427344
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210193136.3A Active CN114561421B (en) | 2019-08-03 | 2019-09-19 | Genetic transformation method of ranunculus |
| CN201910884952.7A Active CN110786238B (en) | 2019-08-03 | 2019-09-19 | Ranunculus Sclerotii somatic embryo induction method and somatic embryo mediated genetic transformation method |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210193136.3A Active CN114561421B (en) | 2019-08-03 | 2019-09-19 | Genetic transformation method of ranunculus |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114561421B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103314850A (en) * | 2013-06-02 | 2013-09-25 | 周口师范学院 | Method of establishing regeneration system and genetic transformation system of wild tomato solanum sities |
| CN103314849A (en) * | 2013-06-02 | 2013-09-25 | 周口师范学院 | Wild tomato Solanum sitiens somatic embryo induction method and somatic embryo genetic transformation method |
| CN104542280A (en) * | 2014-12-24 | 2015-04-29 | 临沂大学 | Pulsatilla tongkangensis somatic embryogenesis and plant regeneration method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101427050B1 (en) * | 2013-09-23 | 2014-08-06 | 순천대학교 산학협력단 | Antibacterial compositions containing Ranunculus sceleratus extracts or fractions thereof under light intensity |
| CN105165621B (en) * | 2015-10-10 | 2017-04-26 | 内蒙古农业大学 | Buffalo currant stem tip regeneration and propagation method |
| CN107683767A (en) * | 2017-10-15 | 2018-02-13 | 陈金水 | A kind of tissue cultivation rapid breeding method of pennywort |
-
2019
- 2019-09-19 CN CN202210193136.3A patent/CN114561421B/en active Active
- 2019-09-19 CN CN201910884952.7A patent/CN110786238B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103314850A (en) * | 2013-06-02 | 2013-09-25 | 周口师范学院 | Method of establishing regeneration system and genetic transformation system of wild tomato solanum sities |
| CN103314849A (en) * | 2013-06-02 | 2013-09-25 | 周口师范学院 | Wild tomato Solanum sitiens somatic embryo induction method and somatic embryo genetic transformation method |
| CN104542280A (en) * | 2014-12-24 | 2015-04-29 | 临沂大学 | Pulsatilla tongkangensis somatic embryogenesis and plant regeneration method |
Non-Patent Citations (1)
| Title |
|---|
| 李洁等: "石龙芮毛茛愈伤组织诱导及再生体系建立研究", 《中国园艺文摘》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561421B (en) | 2023-11-17 |
| CN114561421A (en) | 2022-05-31 |
| CN110786238B (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fernández et al. | In vitro culture of ornamental ferns | |
| Isikalan et al. | In vitro micropropagation of almond (Amygdalus communis L. cv. Nonpareil) | |
| Ju et al. | High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.) | |
| Lone et al. | Plant propagation through tissue culture-a biotechnological intervention | |
| Govindaraju et al. | High frequency plant regeneration in Ashwagandha (Withania somnifera (L.) Dunal): an important medicinal plant | |
| Shekhawat et al. | Role of phytohormones and nitrogen in somatic embryogenesis induction in cell culture derived from leaflets of Azadirachta indica | |
| Dilaver et al. | Breaking seed dormancy and micropropagation of perennial vulneraria milkvetch (Astragalus vulnerariae DC.) | |
| Ozel et al. | Efficient micropropagation of English shrub rose “Heritage” under in vitro conditions | |
| CN110786238B (en) | Ranunculus Sclerotii somatic embryo induction method and somatic embryo mediated genetic transformation method | |
| Pogorzelec et al. | Pollen viability and tissue culture initiation of Salix lapponum, an endangered species in Poland | |
| CA2276003A1 (en) | Method for rapid maturation and cultivation of ginseng plants regenerated from somatic embryo cultures | |
| Masanga et al. | An optimized protocol for high frequency regeneration of selected groundnut (Arachis hypogaea L) varieties from East Africa using cotyledons | |
| Pierik et al. | Vegetative propagation | |
| CN103314849A (en) | Wild tomato Solanum sitiens somatic embryo induction method and somatic embryo genetic transformation method | |
| CN110024694B (en) | Method for rapidly inducing redifferentiation of peanut young leaves to form adventitious buds | |
| Sahijram et al. | Somatic embryogenesis | |
| KR100398749B1 (en) | Method for mass propagation of Wild Korean ginseng (Panax ginseng C.A.Meyer) by biotechnological technique | |
| Xu et al. | Efficient in vitro plant regeneration of Pinellia ternata (Thunb) Breit | |
| Nhut et al. | Latest applications of thin cell layer (TCL) culture systems in plant regeneration and morphogenesis | |
| Hassanein et al. | Essential factors for in vitro regeneration of rose and a protocol for plant regeneration from leaves | |
| CN110710451B (en) | Physalis alkekengi root tuber induction generation method and root tuber-like body-mediated genetic transformation method | |
| Sidhu et al. | Optimization of explants, media, plant growth regulators and carbohydrates on callus induction and plant regeneration in Citrus jambhiri Lush. | |
| Jamir et al. | Plantlet regeneration of naga king chilli (Capsicum Chinense JACQ.) from nodal segments through in vitro technique | |
| ABUBAKAR et al. | INVITRO-PLANT REGENERATION FROM MATURED SEED EXPLANT OF LOCAL AND IMPROVED RICE (ORYZA SATIVA L.) VARIETIES | |
| Kost et al. | Regeneration of fertile plants from excised immature zygotic embryos of Arabidopsis thaliana |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |