CN113502257B - Drying marking method for spruce somatic embryos before germination - Google Patents
Drying marking method for spruce somatic embryos before germination Download PDFInfo
- Publication number
- CN113502257B CN113502257B CN202110953164.6A CN202110953164A CN113502257B CN 113502257 B CN113502257 B CN 113502257B CN 202110953164 A CN202110953164 A CN 202110953164A CN 113502257 B CN113502257 B CN 113502257B
- Authority
- CN
- China
- Prior art keywords
- spruce
- filter paper
- somatic embryos
- drying
- somatic
- 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.)
- Active
Links
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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Abstract
The invention relates to a drying and marking method before germination of somatic embryos of spruce shavings, which comprises the following steps: (1) Collecting and processing explants, collecting mature seeds of the spruce on sand, and sterilizing the seeds. (2) Inoculating the explant, stripping off embryo from the sterilized seed, inoculating to a prepared culture medium, and culturing to obtain the callus. (3) Differentiation of callus of spruce, inoculating the callus obtained in the step 2 into a differentiation medium, and inducing formation of somatic embryo. (4) preparation work before drying the somatic embryo. The culture dish, sterile water and filter paper which need to be sterilized are prepared in advance. (5) Drying the somatic embryos of the spruces by adopting a filter paper container method. The invention has the advantages that: can improve the germination rate and rooting and seedling rate of somatic embryos of the spruce shavings, and provides technical support for the preparation and germination of artificial seeds of the spruce shavings.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a desiccation marking method before germination of somatic embryos of spruce shadian.
Background
An intact somatic embryogenesis process involves four stages of embryogenic callus induction, proliferation and differentiation and germination of mature somatic embryos. The germination stage of mature somatic embryo is used as the final step of the whole technological system, and the yield of the final somatic embryo and the success or failure of the construction of the whole somatic embryo technological system are determined directly by the germination rate and germination quality of the somatic embryo. At present, in the germination stage of conifer somatic embryos, in order to improve the germination rate and germination quality of the somatic embryos, a common method at home and abroad is to carry out drying pretreatment before the somatic embryos germinate. In general, the phenomenon of water loss is the most direct response of the somatic embryo to the transition from the maturation stage to the germination stage, which can promote the transition of somatic embryos from morphological maturation to physiological maturation. However, currently, there is no reference to the artificial preparation and germination means for this tree species of spruce over sandy land.
Disclosure of Invention
According to the defect of the prior art, the invention provides the desiccation marking method before germination of the somatic embryos of the spruce shavings, and the proper desiccation treatment is carried out on the somatic embryos, so that the early germination of the somatic embryos in the differentiation medium can be inhibited, and the germination quantity and quality in the germination stage can be improved.
The invention is realized by the following technical scheme:
a desiccation marking method before germination of spruce somatic embryos is characterized in that: the drying marking method comprises the following steps:
stripping the mature seeds of the sterilized and disinfected spruce to obtain mature embryos of the spruce; inoculating the stripped mature embryo of the spruce shavings into a culture medium for culture to obtain callus; inoculating the obtained callus of the spruce to a differentiation medium, and inducing to form somatic embryos; and (3) drying and marking the somatic embryos of the spruce shavings by adopting a filter paper container method.
The filter paper container method is to utilize the combination of a culture dish and filter paper to carry out drying treatment, and then compare the drying treatment results of at least two filter paper container methods to determine the optimal drying treatment method.
One of the at least two filter paper container methods, namely the filter paper container method 1, is to place the somatic embryos of the fully developed spruce on a plastic culture dish with two layers of filter paper on the bottom, pad a layer of wet filter paper on the lowest surface of the plastic culture dish, pad a layer of dry filter paper on the wet filter paper, place the somatic embryos of the spruce on the dry filter paper, and seal the plastic culture dish by using a sealing film.
One of the at least two filter paper container methods, namely the filter paper container method 2, is to place the somatic embryos of the fully developed spruce on a plastic culture dish with two layers of dry filter paper on the bottom, then place the plastic culture dish in another glass culture dish, place a proper amount of sterile water in the glass culture dish, make the somatic embryos of the spruce in a relatively dry environment, place the somatic embryos of the spruce on the dry filter paper of the plastic culture dish, and then seal the glass culture dish with a sealing film.
At least three times of repetition are set for each desiccation treatment method, 20-30 individual cell embryos are placed in a culture dish for each desiccation treatment method, one culture dish is repeated, and then at least two desiccation treated somatic embryos are respectively placed in dark culture for culture, and morphological changes of the somatic embryos in the culture dish are observed.
Drying 0d, 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d and 30d by adopting a drying treatment mode of a filter paper container method 1, and respectively carrying out germination comparison experiments; three replicates were set for each treatment, and 20-30 fully developed somatic embryos of spruce sandy were placed in each plastic petri dish as one replicate to determine the optimal desiccation time.
The germination culture medium of somatic embryos of the spruce shavings is 1/2 MS +30 g/L of sucrose+6 g/L of agar, the pH of the culture medium is 5.8, the culture temperature is 23+/-1 ℃, the culture is carried out in the dark for 1 week and then is carried out under the illumination culture condition, and the number of somatic embryos which normally germinate is counted after three weeks.
Taking somatic embryos differentiated from the callus of the spruce shavings as materials, adopting a filter paper container method 1 drying treatment mode, and dividing the somatic embryos into two types according to the color change after drying treatment for 12d, wherein the two types are respectively a cotyledon, a hypocotyl and a radicle type without color change; and respectively carrying out germination comparison on the two types of somatic embryos, setting three repetitions for each treatment, and placing 20-30 somatic embryos which are complete in development on each plastic culture dish as one repetition for drying and marking the somatic embryos.
The invention has the advantages that: the germination rate of the somatic embryos of the spruce is improved, the rooting and seedling rate of the tissue culture seedlings of the spruce is improved, the germination rate of the somatic embryos of the spruce after the drying marking treatment is 84.2%, the rooting and seedling rate is 78%, the germination rate of the somatic embryos of the spruce without the drying marking treatment is 57.6%, and the rooting and seedling rate is 30.5%, so that technical guarantees are provided for the preparation and germination of artificial seeds of the spruce; can be used for referencing the tissue culture seedling way of other plant somatic embryogenesis, and has important popularization value.
Detailed Description
The following examples are provided to illustrate the features of the present invention and other related features in further detail to facilitate understanding by those skilled in the art:
examples: the method for drying and marking the spruce somatic embryos before germination in the embodiment comprises the following steps:
(1) Disinfection method of spruce explants on sand
The collected mature seeds of the spruce are treated, and the disinfection and sterilization treatment is carried out according to the following steps: soaking mature spruce seeds in tap water for 12 hours; sterilizing the soaked mature seeds with 75% alcohol on an ultra-clean workbench for 30 seconds; washing the seeds with sterile water for 3-5 times after the sterilization with 75% alcohol; sterilizing with 2% sodium hypochlorite for 8 min; after the sodium hypochlorite is sterilized, the water is used for washing 3 to 5 times.
(2) Method for inducing callus by spruce with sand
The basic culture medium of the callus induced by the spruce is a 1/2 LM culture medium added with gel, inositol, acid hydrolyzed casein, glutamine and sucrose, and the culture medium comprises 1/2 LM+0.6-1.8 mg/L6-BA+1.0-2.7 mg/L2, 4-D. The culture conditions are dark culture, and the culture temperature is 23+/-1 ℃.
(3) Culture of somatic embryos of spruce
The callus differentiation culture medium of the spruce is a 1/2 LM culture medium added with gel, inositol, acid hydrolyzed casein, glutamine, PEG4000 and sucrose, and the culture medium comprises 1/2 LM+18-26mg/L ABA. The culture conditions are dark culture, and the culture temperature is 23+/-1 ℃.
(4) Drying method
The somatic embryo differentiated from the callus of the spruce is used as a material, and two filter paper container methods are adopted for comparison of the desiccation method.
1) "Filter paper Container method 1" the fully developed somatic embryos are placed in a plastic petri dish (80X 24 mm) lined with two layers of filter paper, one layer of wet filter paper is lined on the lowest side of the plastic petri dish, one layer of dry filter paper is lined on the top side of the plastic petri dish, the somatic embryos are placed on the dry filter paper, and then the petri dish is sealed with a sealing film.
2) "Filter paper Container method 2" A fully developed somatic embryo was placed in a plastic culture dish (60X 12 mm) with two layers of dry filter paper on its bottom, then the plastic culture dish was placed in a larger glass culture dish (87X 15 mm) in which 10ml of sterile water was placed so that the somatic embryo was in a relatively dry environment, the somatic embryo was placed on the dry filter paper of the plastic culture dish, and then the glass dish was sealed with a sealing film. Setting three times of repetition for each desiccation treatment method, placing 20-30 individual cell embryos in a culture dish for each desiccation method, wherein one culture dish is repeated, then respectively placing at least two desiccation treated somatic embryos in dark culture for culture, and observing morphological changes of the somatic embryos in the culture dish. And determining the optimal drying treatment method according to the observation result.
(5) Comparison of drying time
And (3) taking somatic embryos differentiated from the callus of the spruce on the sand as a material, drying the somatic embryos by adopting a 'filter paper container method 1' drying mode, and then respectively carrying out germination comparison experiments after drying treatment of 0d, 3d, 6d, 9d, 12d, 15d, 18d, 21d, 24d, 27d and 30 d. Three replicates were set for each treatment, with 20-30 fully developed somatic embryos placed in each plastic petri dish as one replicate. The germination culture medium is 1/2 MS +30 g/L of sucrose+6 g/L of agar, the pH of the culture medium is 5.8, the culture temperature is 23+/-1 ℃, the culture medium is changed to the light culture condition after being subjected to dark culture for 1 week, the number of normally germinated somatic embryos is counted after 3 weeks, and the optimal desiccation time is determined.
(6) Drying marking of somatic embryos
The somatic embryos differentiated from the callus of the spruce are used as materials, a filter paper container method 1 is adopted for drying, and after 12d of drying treatment, the somatic embryos are divided into two types according to the color change after the drying treatment, namely, the cotyledon, the hypocotyl and the radicle types which are respectively in a color-change-free type and a green type. The two types of somatic embryos were subjected to germination comparison, each treatment was set with three replicates, and 20-30 fully developed somatic embryos were placed per plastic culture dish as one replicate. The germination medium and the culture conditions are carried out according to the step (5).
According to the drying marking method before germination of the somatic embryos of the spruce shavings, the drying mode of the filter paper container method 1 is optimal, and the optimal drying time period is 15-21d. The germination rate of the somatic embryos of the sand spruce can be improved after the somatic embryos of the sand spruce are dried, the germination rate of the somatic embryos of the sand spruce after drying and marking treatment is 84.2%, the rooting and seedling rate is 78%, and the germination rate of the somatic embryos of the sand spruce after drying and marking treatment is 57.6%, and the rooting and seedling rate is 30.5%. Therefore, the embodiment can greatly improve the germination rate and rooting seedling rate of the somatic embryos of the spruce on the sand, meet the market demand of the tissue culture seedlings of the spruce on the sand, and has important popularization value.
Although the foregoing embodiments have been described in some detail for the purposes of this invention, it will be appreciated by those skilled in the art that various modifications and changes may be made without departing from the scope of the invention, and thus, the details are not set forth herein.
Claims (1)
1. A desiccation marking method before germination of spruce somatic embryos is characterized in that: the drying marking method comprises the following steps:
stripping the mature seeds of the sterilized and disinfected spruce to obtain mature embryos of the spruce; inoculating the stripped mature embryo of the spruce shavings into a culture medium for culture to obtain callus; inoculating the obtained callus of the spruce to a differentiation medium, and inducing to form somatic embryos; drying and marking somatic embryos of the spruce with a filter paper container method 1;
"filter paper container method 1" refers to drying treatment using a combination of a culture dish and filter paper; placing the somatic embryo of the fully developed spruce on a plastic culture dish with two layers of filter paper on the bottom, filling a layer of wet filter paper on the lowest surface of the plastic culture dish, filling a layer of dry filter paper on the wet filter paper, placing the somatic embryo of the spruce on the dry filter paper, and sealing the plastic culture dish by using a sealing film.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110953164.6A CN113502257B (en) | 2021-08-19 | 2021-08-19 | Drying marking method for spruce somatic embryos before germination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110953164.6A CN113502257B (en) | 2021-08-19 | 2021-08-19 | Drying marking method for spruce somatic embryos before germination |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113502257A CN113502257A (en) | 2021-10-15 |
| CN113502257B true CN113502257B (en) | 2023-08-01 |
Family
ID=78015931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110953164.6A Active CN113502257B (en) | 2021-08-19 | 2021-08-19 | Drying marking method for spruce somatic embryos before germination |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113502257B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304034A (en) * | 2014-11-11 | 2015-01-28 | 新疆林科院造林治沙研究所 | Induction culture method and special induction culture medium for somatic embryo calluses of picea schrenkiana |
| CN108956679A (en) * | 2018-03-02 | 2018-12-07 | 中国林业科学研究院林业研究所 | A method of mark of correlation metabolin is sprouted based on NMR technology screening PIECA ASPERATA body embryo |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2069964C (en) * | 1989-10-23 | 1997-12-30 | Gerald S. Pullman | Method for reproducing conifers by somatic embryogenesis |
| US5491090A (en) * | 1994-02-09 | 1996-02-13 | Westvaco Corporation | Embryogenic coniferous liquid suspension cultures |
| CN101228848B (en) * | 2008-02-27 | 2010-06-02 | 东北林业大学 | Method for inducing plant regeneratation using somatic embryo of picea koraiensis nakai |
| CN102792888B (en) * | 2012-07-31 | 2014-05-14 | 中国林业科学研究院林业研究所 | Method for somatic embryogenesis and plant regeneration of Lijiang spruce |
| CN103497928B (en) * | 2013-09-30 | 2016-02-03 | 中国林业科学研究院林业研究所 | Drying method for treating before the sprouting of PIECA ASPERATA somatic embryo |
| CN105165618B (en) * | 2015-10-10 | 2017-07-14 | 内蒙古农业大学 | The method that Picea Mongolica somatic embryo occurs |
| CN106561451B (en) * | 2016-10-20 | 2018-09-21 | 黑龙江省林业科学研究所 | A kind of Picea koraiensis Nakai somatic embryo induction plant regeneration method and application |
| CN109652424B (en) * | 2018-12-27 | 2019-11-08 | 山东农业大学 | A kind of methods and applications that somatic embryos directly occur |
| CN110999785B (en) * | 2019-11-28 | 2021-08-31 | 内蒙古蒙荣园林绿化工程有限责任公司 | Synthetic method of artificial seeds of picea asperata |
-
2021
- 2021-08-19 CN CN202110953164.6A patent/CN113502257B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304034A (en) * | 2014-11-11 | 2015-01-28 | 新疆林科院造林治沙研究所 | Induction culture method and special induction culture medium for somatic embryo calluses of picea schrenkiana |
| CN108956679A (en) * | 2018-03-02 | 2018-12-07 | 中国林业科学研究院林业研究所 | A method of mark of correlation metabolin is sprouted based on NMR technology screening PIECA ASPERATA body embryo |
Non-Patent Citations (1)
| Title |
|---|
| 川西云杉早期体细胞胚胎发生机理研究;李青粉;中国博士学位论文全文数据库;D049-14 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113502257A (en) | 2021-10-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110073981B (en) | Method for obtaining poplar hybrid seedlings by utilizing embryo rescue | |
| CN112616673B (en) | Culture medium for screening peanut salt-tolerant bodies and method for screening peanut salt-tolerant bodies | |
| Barnabás | Anther culture of maize (Zea mays L.) | |
| CN113502257B (en) | Drying marking method for spruce somatic embryos before germination | |
| CN105519448A (en) | Culture method of radix astragali tissue culture seedlings | |
| CN110476807B (en) | Method for establishing sterile culture system of mature seed embryo of peony 'paeonia ostii' for oil | |
| CN104686334A (en) | Tissue culture and rapid propagation method for androsace longifolia | |
| CN115024220B (en) | Method for obtaining clivia miniata somatic embryo by thin-layer culture | |
| Gürel et al. | Development of an efficient plant regeneration system in sunflower (Helianthus annuus L.) | |
| CN1768576A (en) | Peony standardized quick-breeding method | |
| CN108184462A (en) | A kind of open country eggplant sapling stock breeding method and its method for grafting fruits and vegetables | |
| CN111226791B (en) | Method for directly inducing cluster buds to proliferate through peony cotyledonary nodes | |
| CN113303227A (en) | Method for cultivating new camellia variety seedlings by using immature hybrid seeds | |
| CN106577219A (en) | Plant transgenic seed screening method | |
| US7521237B2 (en) | Methods for promoting maturation of conifer somatic embryos | |
| CN114568305B (en) | Treatment method for improving regeneration efficiency by tissue culture of quercus acutissima | |
| CN110150150B (en) | Peony molecular breeding method | |
| CN114586683B (en) | Screening of feed bermuda grass germplasm for efficient induction of callus and regeneration | |
| CN108077075B (en) | Method for rapid propagation culture of clone by using Bawangchia aseptic seedling | |
| SU1541252A1 (en) | Method of producing rice regenerating plants | |
| CN116941529A (en) | Tobacco haploid plant doubling and rapid propagation integrated method | |
| CN1227968C (en) | Prepn of ripe microspore plumule for direct germination into plant | |
| CN116806698A (en) | Poison-free citrus seedling breeding method | |
| CN115885846A (en) | Rapid tissue culture regeneration system for white carrots | |
| CN115735768A (en) | Tissue culture medium and tissue culture method for Conus carneus |
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 |