CN117363486A - Culture medium for asexual proliferation of gulfweed and application thereof - Google Patents
Culture medium for asexual proliferation of gulfweed and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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Abstract
The invention discloses a culture medium for asexual propagation of gulfweed and application thereof, mainly comprising a USAES culture medium, a GSES culture medium and an SES culture medium used in three stages of asexual propagation of gulfweed. The invention mainly selects edible seaweed sargassum fusiforme in gulfweed as a research object, and judges the application effect of the culture medium by using the relative growth rate of sargassum fusiforme pseudoroot filaments cultured by the culture medium and the induction rate of callus/adventitious bud and the regeneration number of branch segments as indexes. Experiments prove that the Sargassum fusiforme cultivated by the culture medium has higher relative growth rate and Fv/Fm, the optimal relative growth rate can reach (4.11+/-0.424)%/day, and the Fv/Fm can reach 0.679+/-0.0328.
Description
Technical Field
The invention relates to the field of seaweed cultivation, in particular to a culture medium for asexual propagation of gulfweed and application thereof.
Background
The gulfweed is a perennial large-scale economic seaweed of the genus gulfweed of the order Phaeophyta, the order Fucales and the family gulfweed, mainly grows in low-tide zones, is widely distributed in Zhejiang, shandong and Fujian in China, has huge economic value, and can be used for ecological restoration or used as a marine algae plant to keep the stability of marine ecological environment.
At present, the gulfweed cultivation industry mainly faces two serious challenges, the first is that the environment is changed, the global air temperature is continuously increased, the pH value and the nutrient salt level in the seawater are continuously changed, the existing living state of the gulfweed is changed, the difficulty of gulfweed cultivation is increased, and the yield is reduced. The second is artificial reason, as people increasingly find the wonderful use of gulfweed in different industries, the demands of people are increased, finally people excessively catch, the natural growth speed and trend of gulfweed cannot catch up with the demands of people, and the final result is that people need to explore a culture medium to culture artificial gulfweed, but the proper culture medium is lacking in the seaweed culture process, and finally the seaweed yield is low. For the above reasons, the present invention has been developed to provide a medium suitable for asexual propagation of gulfweed.
The uniconazole is called a triazole type cytochrome P450 enzyme inhibitor, is used for biosynthesis of t-zeatin and biosynthesis of brassinosteroids, inhibits abscisic acid (ABA), has strong plant growth inhibition activity, can induce callus formation, increases an antioxidant system, delays aging of leaves, reduces electrolyte leakage rate of leaf cells, increases superoxide dismutase activity, reduces malondialdehyde accumulation, improves photosynthetic efficiency and increases yield. Gibberellin can promote the overall plant length and height, and especially can elongate the stem of dwarf mutant or physiological dwarf plants; the present invention can shorten the cell division interval, promote DNA replication, promote the increase of IAA synthesis level (increase in synthesis, decrease in oxidation), and thus regulate plant growth by using these two plant growth regulators as the main components of the culture medium.
Disclosure of Invention
The invention aims to solve the defects of the prior art, provides a culture medium for asexual propagation of gulfweed, and provides the optimal physiological condition of gulfweed by adjusting the components of the culture medium in a staged manner, and simultaneously can effectively inhibit the propagation of mixed bacteria and mixed algae.
The culture medium for asexual propagation of gulfweed comprises the following steps:
(1) Preparation of USAES culture medium
The USAES medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, UIZ, agar and sucrose. During weighing, the medicine is required to be accurately weighed on an electronic balance (the precision reaches 0.001), the medicine is dissolved by ultrapure water, a glass rod is continuously stirred, the volume of a volumetric flask is fixed after the medicine is dissolved, and finally, the medicine is filtered and sterilized by a filter head with the size of 0.45 mu m. When preparing USAES culture medium, the seawater used must be filtered seawater, agar must be fully heated and dissolved in microwave oven, and stirred while heating so as to reduce the phenomenon of nonuniform agar density, then the ES concentrate, fe-solution and PII-solution are added in sequence, finally agar and sucrose are added, then after uniformly mixing, the pH value is regulated by using portable pH meter, after autoclaving, the culture medium is placed in ultra-clean workbench for ultraviolet disinfection for 20min, when the temperature of the culture medium is reduced to about 60 deg.C, uniconazole (gun head used, etc. are required for high-pressure steam sterilization), after the culture medium is naturally cooled and solidified, the uniconazole sucrose agar seawater enrichment culture medium obtained by adopting the above-mentioned preparation is mainly used for inducing the generation of adventitious buds in initial stage of culture.
(2) Preparation of GSES Medium
The GSES medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, GA, sucrose, and sodium bicarbonate. When preparing GSES culture medium, the seawater is filtered seawater, the components are mixed uniformly, pH is adjusted by a portable pH meter, and packaged into 1L triangular bottles, and autoclaved. After the culture medium is cooled to normal temperature, GA is added, sucrose and sodium bicarbonate are added in the state of the algae body when the algae body is cultivated, and the gibberellin sucrose seawater enrichment culture medium obtained by the steps is mainly used for the rapid proliferation process of the induced adventitious buds from the USAES culture medium to the initial stage of the liquid culture medium.
(3) Preparation of SES Medium
The SES culture medium mainly comprises 5 parts including ES concentrate, fe-solution, PII-solution, sucrose and germanium dioxide. When preparing SES culture medium, the seawater is filtered seawater, the components are mixed, pH is adjusted by portable pH meter, packaged into 1L triangular flask, and autoclaved. When the algae is cultivated, the state of the algae is observed, sucrose and germanium dioxide are added, and the sucrose seawater culture medium is obtained through the preparation steps and is mainly used for the subsequent adaptation process of the regenerated larvae in normal seawater cultivation.
Preferably, the culture medium is mainly used for inducing callus/adventitious buds of gulfweed and subsequent seedling of gulfweed.
Preferably, the USAES culture medium comprises 0.9mg/L UIZ;
preferably, the GSES medium comprises 1.5mg/L GA;
in the invention, agar is required to be heated and dissolved by a microwave oven, packaged and finally sterilized, and hormone is required to be added when a solid culture medium is not solidified after sterilization and is gently shaken and homogenized in an ultra-clean workbench.
The advantages of the invention include:
1. the explant cultivated by the culture medium is bright yellow in color, has no other impurities, can grow up, and a large number of black spots appear on the Sargassum fusiforme segments cultivated by the sea water normally, and the death and decay problems can occur when the sargassum fusiforme segments cannot grow up;
2. the invention adopts staged culture, can provide proper culture conditions according to the growth state of gulfweed, changes the conventional fixed culture mode, has simple operation and can create great economic value with lower cost;
3. finally, compared with other culture mediums, the USAES culture medium can be used for germplasm preservation, and the later germination and growth of algae are not affected; meanwhile, the plant growth regulator can greatly promote the germination and proliferation of algae, and remarkably improve the yield.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
Fig. 1: regeneration morphology type after sargassum fusiforme pseudoroot culture is performed by using USAES culture medium.
Fig. 2: relative growth rate after cultivation of sargassum fusiforme pseudoroot filaments with different uniconazole concentrations (0. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M).
Fig. 3: callus-like/adventitious bud induction rate and shoot regeneration bud number after culturing sargassum fusiforme pseudoroot filaments with different uniconazole concentrations (0. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M).
Fig. 4: the morphological change pattern of culture with the culture medium of the invention compared with normal seawater culture. (a: 3 parallel groups of sargassum fusiforme pseudoroots cultured with the medium of the present invention; b: a group of sargassum fusiforme pseudoroots cultured with normal seawater).
Fig. 5: RGR, fv/Fm and rETRm of sargassum fusiforme pseudoroot cultured with the medium of the present invention.
Detailed Description
The present invention will be further described with reference to the drawings and the specific embodiments, and it should be noted that the embodiments of the present application and the features of the embodiments may be combined with each other without collision.
The present invention will be described in further detail with reference to specific examples.
Example 1:
a culture medium for asexual propagation of sargassum fusiforme, which is characterized by comprising the following steps:
(1) Pretreatment of materials
2022.3 harvesting Sargassum fusiforme from Sargassum fusiforme culture base in the region of the cave in the state of Winter in Zhejiang province, taking Sargassum fusiforme back to laboratory with a low-temperature preservation box at 4deg.C, removing surface-attached miscellaneous algae and plankton from the collected Sargassum fusiforme with a writing brush and tweezers, rinsing with filtered and sterilized natural seawater, and selecting Sargassum fusiforme with relatively consistent growth state. The selected Cyrtymenia Sparsa algae were temporarily cultivated and domesticated in a plant incubator (Percivale-36 HO, america) for 7 days.
(2) Preparation of USAES culture medium
The solid medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, UIZ, agar and sucrose. During weighing, the medicine is required to be accurately weighed on an electronic balance (the precision reaches 0.001), the medicine is dissolved by ultrapure water, a glass rod is continuously stirred, the volume of a volumetric flask is fixed after the medicine is dissolved, and finally, the medicine is filtered and sterilized by a filter head with the size of 0.45 mu m. When preparing USAES culture medium, the seawater is filtered seawater, agar is heated and dissolved in microwave oven to reduce the phenomenon of uneven agar density, sequentially adding ES concentrate, fe-solution and PII-solution, adding agar and sucrose, mixing, regulating pH with portable pH meter, sterilizing under high pressure, standing on a vertical horse, ultraviolet sterilizing for 20min, and cooling to 60 deg.C, and adding uniconazole (0 μM, 3 μM, 5 μM, 7 μM) for natural cooling and solidifying.
(3) Preparation of GSES Medium
The GSES medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, GA, sucrose, and sodium bicarbonate. When preparing GSES culture medium, the seawater is filtered seawater, the components are mixed uniformly, pH is adjusted by a portable pH meter, and packaged into 1L triangular bottles, and autoclaved. After the culture medium is cooled to normal temperature, GA is added, the state of the algae is observed when the algae is cultivated, sucrose and sodium bicarbonate are added, and an air pump is used for air blowing in the cultivation process.
(4) Preparation of SES Medium
The SES culture medium mainly comprises 5 parts including ES concentrate, fe-solution, PII-solution, sucrose and germanium dioxide. When preparing GSES culture medium, the seawater is filtered seawater, the components are mixed uniformly, pH is adjusted by a portable pH meter, and packaged into 1L triangular bottles, and autoclaved. During the culture of algae, sucrose and germanium dioxide are added to observe the state of algae, and air is blown by an air pump during the culture process.
Sargassum fusiforme material is cultured in USAES culture medium at 19deg.C under 3000lx condition.
After culture in the medium, the sargassum fusiforme segments showed 3 different calli, including friable morula-like calli, hard globular structure and filiform regeneration structure, as shown in fig. 1.
When germinated shoots and explants obtained by solid culture were cultured in GSES medium-SES medium, the results of the experiment showed that RGR was highest (3.1.+ -. 0.312)% (FIG. 2) and the callus/adventitious bud induction rate and the number of regenerated shoots were also highest for Sargassum fusiforme cultured with 3. Mu.M UIZ (FIG. 3).
Example 2:
and (5) taking the sargassum fusiforme bodies back to a laboratory by using a 4 ℃ low-temperature preservation box after harvesting the sargassum fusiforme bodies for 2022.12 months, removing surface-attached miscellaneous algae and plankton from the collected sargassum fusiforme bodies, washing the sargassum fusiforme bodies clean by using filtered and sterilized natural seawater, and selecting the sargassum fusiforme bodies which are healthy and have relatively consistent growth states. The selected Sargassum fusiforme bodies were acclimatized temporarily in a plant incubator (Percivale-36 HO, america). Temporary culture conditions are as follows: the photoperiod is L, D=12h:12h at 19 ℃ and 5000lx, the salinity of the culture solution is 26 per mill, and the continuous aeration culture is carried out by using an air pump.
2) Preparation of USAES culture medium
The solid medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, UIZ, agar and sucrose. During weighing, the medicine is required to be accurately weighed on an electronic balance (the precision reaches 0.001), the medicine is dissolved by ultrapure water, a glass rod is continuously stirred, the volume of a volumetric flask is fixed after the medicine is dissolved, and finally, the medicine is filtered and sterilized by a filter head with the size of 0.45 mu m. When preparing USAES culture medium, the seawater used must be filtered seawater, agar must be fully heated and dissolved on microwave oven to reduce the phenomenon of uneven agar density, then the ES concentrate, fe-solution and PII-solution are added in sequence, finally agar and sucrose are added, then after mixing uniformly, the pH value is regulated by means of portable pH meter, after high-pressure sterilization the culture medium is placed into ultra-clean workbench to make UV sterilization for 20min, when the temperature of culture medium is reduced to about 60 deg.C, finally 3 mu M uniconazole is added into the ultra-clean workbench by means of pipette, and after the culture medium is naturally cooled and solidified.
(3) Preparation of GSES Medium
The GSES medium mainly comprises 6 parts including ES concentrate, fe-solution, PII-solution, GA, sucrose, and sodium bicarbonate. When preparing GSES culture medium, the seawater is filtered seawater, the components are mixed uniformly, pH is adjusted by a portable pH meter, and packaged into 1L triangular bottles, and autoclaved. After the culture medium is cooled to normal temperature, 1.5mg/L GA is added, sucrose and sodium bicarbonate are added when the algae are cultivated, and an air pump is used for air blowing in the cultivation process.
(4) Preparation of SES Medium
The SES culture medium mainly comprises 5 parts including ES concentrate, fe-solution, PII-solution, sucrose and germanium dioxide. When preparing SES culture medium, the seawater is filtered seawater, the components are mixed, pH is adjusted by portable pH meter, packaged into 1L triangular flask, and autoclaved. During the culture of algae, sucrose and germanium dioxide are added to observe the state of algae, and air is blown by an air pump during the culture process.
Finally, the morphological, length and weight changes of the algae are recorded during the cultivation.
According to the change of the form of the graph a and the graph b in the graph 4, the culture medium and the operation method can obviously detoxify and ensure that the sargassum fusiforme grows into seedlings normally after tissue culture. The relative growth rate of the sargassum fusiforme cultivated by the culture medium after seedling formation can reach 4.11% +/-0.004, and fv/Fm and rETRm are 0.679+/-0.0328 and 49.72+/-6.749 (figure 5).
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. A culture medium for asexual propagation of gulfweed, characterized in that: the culture medium comprises a USAES culture medium, a GSES culture medium and an SES culture medium, wherein the USAES culture medium comprises an ES concentrated solution, fe-solution, PII-solution, UIZ, agar and sucrose; the GSES culture medium comprises ES concentrated solution, fe-solution, PII-solution, GA, sucrose and sodium bicarbonate; the SES culture medium comprises ES concentrated solution, fe-solution, PII-solution, sucrose and germanium dioxide.
2. The medium of claim 1, wherein: the USAES culture medium is an uniconazole sucrose agar seawater enrichment culture medium and is used for inducing the generation of adventitious buds in the initial stage of the asexual propagation culture of gulfweed; the GSES culture medium is gibberellin sucrose seawater enrichment culture medium and is used for the rapid proliferation process of inducing adventitious buds from the USAES culture medium to the initial stage of the liquid culture medium stage; the SES culture medium is a sucrose seawater enrichment culture medium and is used for the adaptation process of placing the regenerated larvae in normal seawater culture in the GSES culture medium.
3. A method for preparing the culture medium of claim 1, characterized in that:
the preparation steps of the USAES culture medium comprise:
(1) The filtered seawater is adopted;
(2) Preparing mother solution of each component of the culture medium: including the preparation of ES concentrate, fe-solution, PII-solution and others; wherein the ES concentrate comprises 0.9g NaNO 3 0.2g of beta-sodium glycerophosphate and 20 mu g of vitamin B 12 Vitamin B1 mg 1 1.5g of Tris buffer solution, adding ultrapure water, stirring with a glass rod, dissolving to a constant volume of 100ml, and placing in a brown bottle for preservation; the Fe-solution comprises 0.4g Fe (NH) 4 ) 2 (SO 4 ) 2 .6H 2 O、0.3gNa 2 EDTA, then adding ultrapure water, stirring with a glass rod, dissolving to a volume of 100ml, and placing in a brown bottle for preservation; PII-solution comprises 0.5g Na 2 EDTA、30mgFeCL 3 .6H 2 O、90mgMnSO 4 .H 2 O、17mgZnSO 4 .7H 2 O、2.5mg CoSO4.7H2O、0.5gH 3 BO 3 Then adding ultrapure water, stirring with a glass rod, dissolving to a constant volume of 100ml, and placing in a brown bottle for preservation; UIZ dissolving with dimethyl sulfoxide, filtering with 0.45 μm filter head, sterilizing, and storing in 4deg.C refrigerator;
(3) Weighing 7-8g of agar and 7g of sucrose into a beaker, adding 10ml of ES concentrate, 1ml of Fe-solution and 1ml of PII-solution into the beaker, stirring the mixture to dissolve the mixture by using a glass rod, fixing the volume to 1L by using pumped and filtered seawater, fully heating the mixture in a microwave oven, and fixing the volume to 1L again by using the seawater after the agar is fully dissolved;
(4) Testing the pH value by adopting a portable pH meter, adjusting the pH value until the pH value is 7.8, sterilizing by utilizing an autoclave after the adjustment is finished, and then immediately taking the culture medium to an ultra-clean workbench for ultraviolet sterilization for 20min;
(5) When the temperature of the culture medium is reduced to about 60 ℃, adding Uniconazole (UIZ) into a gun head sterilized by high-pressure steam in an ultra-clean workbench before the culture medium is not solidified, slightly shaking uniformly, and then placing the culture medium to be naturally cooled and solidified;
the preparation steps of the GSES culture medium comprise:
(1) The filtered seawater is adopted;
(2) Preparing mother solution of each component of the culture medium: including the preparation of ES concentrate, fe-solution, PII-solution and others; wherein the ES concentrate comprises 0.9g NaNO 3 0.2g of beta-sodium glycerophosphate and 20 mu g of vitamin B 12 Vitamin B1 mg 1 1.5g of Tris buffer solution, adding ultrapure water, stirring with a glass rod, dissolving to a constant volume of 100ml, and placing in a brown bottle for preservation; the Fe-solution comprises 0.4g Fe (NH) 4 ) 2 (SO 4 ) 2 .6H 2 O、0.3gNa 2 EDTA, then adding ultrapure water, stirring with a glass rod, dissolving to a volume of 100ml, and placing in a brown bottle for preservation; PII-solution comprises 0.5g Na 2 EDTA、30mgFeCL 3 .6H 2 O、90mgMnSO 4 .H 2 O、17mgZnSO 4 .7H 2 O、2.5mg CoSO4.7H2O、0.5gH 3 BO 3 Then adding ultrapure water, stirring with a glass rod, dissolving to a constant volume of 100ml, and placing in a brown bottle for preservation; GA medicine is dissolved in 95% alcohol and 0.4% alcohol is adoptedFiltering with 5 μm filter head, sterilizing, preparing, and storing in 4deg.C refrigerator;
(3) Weighing 3g of sucrose into a beaker, adding 10ml of ES concentrate, stirring 1ml of Fe-solution and 1ml of PII-solution glass rod until the mixture is fully dissolved, and fixing the volume to 1L by using the seawater filtered by pumping;
(4) Testing the pH value by adopting a portable pH meter, regulating the pH value until the pH value is 7.8, performing high-pressure sterilization by utilizing an autoclave after the regulation is finished, and then taking the culture medium to an ultra-clean workbench for ultraviolet sterilization for 20min;
(5) Adding GA when the temperature of the culture medium is reduced to normal temperature in an ultra-clean workbench, and finally adding 400mg/L NaHCO into the culture medium 3 Sodium bicarbonate is added in the amount of (2);
preparation of the SES Medium
(1) The filtered seawater is adopted;
(2) Preparing mother solution of each component of the culture medium: including the preparation of ES concentrate, fe-solution, PII-solution and others; wherein the ES concentrate comprises 0.3g NaNO 3 0.05g of beta-sodium glycerophosphate and 5. Mu.g of vitamin B 12 Vitamin B0.25 mg 1 2.5ug of vitamin H and 0.3g of Tris buffer solution, adding ultrapure water, stirring with a glass rod, dissolving to a volume of 100ml, and placing in a brown bottle for preservation; fe-solution includes 0.
09gFe(NH 4 ) 2 (SO 4 ) 2 .6H 2 O、0.07gNa 2 EDTA, then adding ultrapure water, stirring with a glass rod, dissolving to a volume of 100ml, and placing in a brown bottle for preservation; PII-solution comprises 0.1g Na 2 EDTA、7.5mgFeCL 3 .6H 2 O、20mgMnSO 4 .H 2 O、4mgZnSO 4 .7H 2 O、0.7mg CoSO4.7H2O、0.13gH 3 BO 3 Then adding ultrapure water, stirring with a glass rod, dissolving to a constant volume of 100ml, and placing in a brown bottle for preservation;
(3) Weighing 1g of sucrose into a beaker, adding 10ml of ES concentrate, stirring 1ml of Fe-solution and 1ml of PII-solution glass rod until the sucrose is fully dissolved, fixing the volume to 1L by using the seawater filtered by pumping, and sterilizing by high-pressure steam after regulating the pH;
(4) When the algae are cultivated, the state of the algae is observed, and 1mg/L germanium dioxide is added.
4. The use of the medium according to claim 1 or 2 for the induction of callus/adventitious buds of gulfweed and subsequent stages of gulfweed seedling, characterized in that: the USAES culture medium is used for inducing generation of adventitious buds in the early stage of gulfweed; the GSES culture medium is used for the rapid proliferation process of adventitious buds; the SES culture medium is used for the adaptation process of the subsequent regenerated larvae to normal seawater culture.
5. A method of preparing a culture medium according to claim 3, wherein: UIZ of the USAES medium comprises 0.9mg/L, and GA of the GSES medium comprises 1.5mg/L.
6. A method of preparing a culture medium according to claim 3, wherein: the amount of dimethyl sulfoxide in UIZ should be greater than one ten thousandth of the volume of the medium after the UIZ is added to the medium.
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