CN111201999B - Method for promoting growth of free filaments and formation of sporangial branches of porphyra haitanensis - Google Patents
Method for promoting growth of free filaments and formation of sporangial branches of porphyra haitanensis Download PDFInfo
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Abstract
The invention discloses a method for promoting the growth of porphyra haitanensis free filament and the formation of sporangium branches, which comprises the following steps: (1) taking a proper amount of porphyra haitanensis free filament, crushing by a crusher, and culturing at a proper environment temperature under illumination; (2) preparing a required first jasmonic acid culture solution; (3) placing the porphyra haitanensis free filament obtained in the step (1) in a first jasmonic acid culture solution for culturing to promote the growth of the porphyra haitanensis free filament; (4) preparing a second jasmonic acid culture solution; (5) and (2) putting the free filaments of the porphyra haitanensis obtained in the step (1) into a proper amount of second jasmonic acid culture solution, and culturing in a proper culture environment to promote the formation of sporangium branches of the porphyra haitanensis.
Description
Technical Field
The invention relates to a method for promoting growth and development of porphyra haitanensis free filaments, in particular to a technical method for promoting growth rate of porphyra haitanensis free filaments and improving formation of sporangium branches indoors.
Background
The life history of porphyra haitanensis includes the filament and frond stages. At present, laver seedling raising is basically carried out in a filament form under indoor artificial control conditions, conchospores are obtained by culturing the filament, and seedlings are picked, surfed and hung on the sea for culturing. However, in the long-term culture production, a lot of problems are found. The filament has long culture period and great management difficulty, and the formation of a large amount of sporangium branches is the key of laver seedling culture. Therefore, the method is very important for culturing the good porphyra haitanensis variety filaments in a short time and realizing the good porphyra haitanensis variety production.
The prior art for cultivating filament has made a major breakthrough, for example, patent No. CN103931482B "A method for promoting maturation and collecting seedling of Porphyra haitanensis shell filament" discloses that the concentration ratio of P/N is increased by adding appropriate amount of dipotassium hydrogen phosphate, which is changed from 5-10:1 to 1: 5-10. The black curtain cloth is used for shading the surface of the seedling raising pool, so that the illumination time is shortened to 8-10 hours, and the illumination intensity is reduced to 300-500Lx to promote the ripening of the shell filaments.
The patent with the publication number of CN 102334444A, "improving the growth rate of porphyra haitanensis filamentous algae by adopting optimized regulation and control culture conditions" discloses that the optimal conditions for improving the growth rate of porphyra haitanensis filamentous algae are that the temperature, the light intensity, the pH of a water body and the real number of illumination per day take median values; NaHCO 23Taking a concentration low value; the salinity, the concentration of the compound Vitamin and the concentration of the f/2 nutrient solution are taken as height values.
Besides external environmental factors such as temperature, light and nutrient salts, phytohormones can also regulate the growth and development of algae. Researches report that indoleacetic acid can promote the growth of free filaments of porphyra haitanensis, and is beneficial to the development of kelp spores and the rapid growth of leaves and the like. Jasmonic acid is one of the plant hormones, however, the influence of jasmonic acid on the growth and development of algae, especially the free filament of porphyra haitanensis, has not been reported in the prior art.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for promoting the growth of porphyra haitanensis free filament and the formation of sporangium branches, which shortens the growth period of the porphyra haitanensis and improves the formation of the sporangium branches of the porphyra haitanensis.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for promoting the growth of free filaments and the formation of sporangium branches of porphyra haitanensis is characterized by comprising the following steps: (1) pulverizing appropriate amount of Porphyra haitanensis free filament, culturing in culture solution with N: P of 10:1 at 20-25 deg.C under illumination of 0-30 μmol/m for 12L:12D2·s；
(2) Dissolving jasmonic acid in an alcohol solution to prepare a first jasmonic acid solution, and diluting seawater containing an MES culture medium with the first jasmonic acid solution to prepare a first jasmonic acid culture solution;
(3) placing the cultured Porphyra haitanensis free filament obtained in step (1) in the first jasmonic acid culture solution prepared in step (2), and placing in a culture medium with illumination period of 12L:12DCulturing in incubator at 20-25 deg.C for 5-30 days with illumination intensity of 0-30 μmol/m2S, to promote the growth of free filaments of porphyra haitanensis;
(4) dissolving jasmonic acid in an alcohol solution to prepare a second jasmonic acid solution, and diluting seawater containing an MES culture medium with the second jasmonic acid solution to prepare a second jasmonic acid culture solution;
(5) culturing the cultured Porphyra haitanensis free filament in an incubator with a light cycle of 8L:16D for 10-30 days at 20-29 deg.C under 0-30 μmol/m2S to promote the formation of Porphyra haitanensis sporangial shoots.
The further preferable technical scheme of the invention is as follows: in step (1), P is represented by C3H4Na3O7P or K2HPO3One or both of them.
The further preferable technical scheme of the invention is as follows: the concentration of the second jasmonic acid solution prepared in the step (4) is 0-5 mol/L.
The further preferable technical scheme of the invention is as follows: and (4) selecting an ethanol solution as the alcohol solution in the step (2) and the step (4).
The further preferable technical scheme of the invention is as follows: the concentration of the second jasmonic acid culture solution in the step (4) is 0.5-2 mu mol/L.
The further preferable technical scheme of the invention is as follows: in the step (5), the second jasmonic acid culture solution is replaced every 5 days.
The further preferable technical scheme of the invention is as follows: the concentration of the first jasmonic acid solution in the step (2) is 0-5 mol/L.
The further preferable technical scheme of the invention is as follows: the concentration of the first jasmonic acid culture solution in the step (2) is 0-1 mu mol/L.
The further preferable technical scheme of the invention is as follows: in step (1), N is replaced by NaNO3Provided is a method.
Compared with the prior art, the invention has the advantages that jasmonic acid is mixed with alcohol solution to prepare the needed jasmonic acid solution; diluting jasmonic acid solution with seawater containing culture medium to obtain jasmonic acid culture solution; the porphyra haitanensis is cultured by using the jasmonic acid culture solution, so that the growth of the free filament of the porphyra haitanensis and the formation of sporangium branches can be effectively promoted, the growth period of the free filament of the porphyra haitanensis can be greatly shortened, and the time required for the formation of the sporangium branches can be prolonged.
Drawings
The present invention will be described in further detail below with reference to the drawings and preferred embodiments, but those skilled in the art will appreciate that the drawings are only drawn for the purpose of illustrating the preferred embodiments and therefore should not be taken as limiting the scope of the invention.
FIG. 1 is a graph showing the degree of development of sporangium branches of Porphyra haitanensis cultured in the control group of example three for 15 days;
FIG. 2 is a graph showing the degree of development of sporangium branches in the experimental group of the third example after 15 days of cultivation of the free filament of Porphyra haitanensis;
FIG. 3 is the development degree of sporangium branch of Porphyra haitanensis in the control group of the sixth example after 25 days of culture;
FIG. 4 shows the degree of development of sporangial shoots of Porphyra haitanensis cultured in the experimental group of the sixth example for 25 days.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Those skilled in the art will appreciate that the description is illustrative only, and is not to be construed as limiting the scope of the invention.
It should be noted that: like reference numerals refer to like items in the following figures, and thus, once an item is defined in one figure, it may not be further defined and explained in subsequent figures.
The first embodiment and the second embodiment of the patent embodiment of the invention mainly illustrate the influence of jasmonic acid on the growth of porphyra haitanensis protonema, and the specific experimental mode is as follows:
the first embodiment is as follows:
firstly, the porphyra haitanensis is treatedCarrying out synchronous culture by a filament, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: the seawater culture solution of P10: 1 is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
and (2) continuously culturing the porphyra haitanensis free filament subjected to the synchronous culture for a period of time in a jasmonic acid-free environment, specifically: culturing the pre-cultured Porphyra haitanensis free filament in culture solution with N: P of 10:1 at 23 + -0.5 deg.C for 5-20 days with illumination period of 12L:12D and illumination intensity of 20 μmol/m2S. Preferably, the N and P distribution is formed by NaNO3And PC3H4Na3O7P is provided, and preferably the incubation time is 15 days.
Every 5 days, the Relative Growth Rate (RGR) was measured, and the change in Fresh Weight (FW) of algal bodies during cultivation was measured using the formula RGR (% & d)-1)＝[(ln Wt-ln Wo)/t]X 100. Where Wo is initial FW and Wt is FW after t days, and surface water was blotted with filter paper before weighing the algal bodies. On day 15, the growth rate of the free filaments of Porphyra haitanensis in the blank group (jasmonic acid untreated) was 6.77. + -. 0.68.
Experimental groups:
firstly, a jasmonic acid culture solution is additionally prepared to continuously culture the porphyra haitanensis free filament subjected to synchronous culture, which comprises the following specific steps: (1) dissolving jasmonic acid in an ethanol solution to prepare a first jasmonic acid solution with the concentration of 5 mol/L; (2) diluting the first jasmonic acid solution with seawater containing MES culture medium to obtain 1 μmol/L first jasmonic acid culture solution; (3) adding the first jasmonic acid culture solution into a culture box of the continuously cultured porphyra haitanensis free filament, culturing for 5-20 days, and replacing the first jasmonic acid culture solution every 5 days.
Wherein the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2·s。
Further, the Relative Growth Rate (RGR) was measured every 5 days, the change in Fresh Weight (FW) of algal bodies during cultivation was measured, and the formula RGR (% & d) was used-1)＝[(ln Wt-ln Wo)/t]X 100. Where Wo is initial FW and Wt is FW after t days, and surface water was blotted with filter paper before weighing the algal bodies.
On day 15, the growth rate of the Porphyra haitanensis free filament was 9.86. + -. 0.52.
Example two:
this example mainly illustrates the comparison of the influence of indoleacetic acid and jasmonic acid on the growth of porphyra haitanensis filaments, and the specific experimental manner is as follows:
firstly, performing synchronous culture on porphyra haitanensis free filaments, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: the seawater culture solution of P10: 1 is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
and (3) continuously culturing the porphyra haitanensis free filament subjected to the synchronous culture for a period of time in an indoleacetic acid environment, specifically: (1) dissolving indoleacetic acid in an ethanol solution to prepare an indoleacetic acid solution of 5 mol/L; (2) diluting the indoleacetic acid solution with seawater containing MES culture medium to obtain 1 mu mol/L indoleacetic acid culture solution; (3) adding indoleacetic acid culture solution into the incubator of the continuously cultured Porphyra haitanensis free filament, culturing for 5-20 days, and replacing indoleacetic acid culture solution every 5 days.
Wherein the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2·s。
Every 5 days, the Relative Growth Rate (RGR) was measured, and the change in Fresh Weight (FW) of algal bodies during cultivation was measured using the formula RGR (% & d)-1)＝[(ln Wt-ln Wo)/t]X 100. Where Wo is initial FW and Wt is FW after t days, and surface water was blotted with filter paper before weighing the algal bodies. On day 15, the growth rate of Porphyra haitanensis free filaments in the control group cultured with indoleacetic acid was found to be 7.54. + -. 0.4.
Experimental groups:
firstly, a jasmonic acid culture solution is additionally prepared to continuously culture the porphyra haitanensis free filament subjected to synchronous culture, which comprises the following specific steps: (1) dissolving jasmonic acid in an ethanol solution to prepare a first jasmonic acid solution with the concentration of 5 mol/L; (2) diluting the first jasmonic acid solution with seawater containing MES culture medium to obtain 1 μmol/L jasmonic acid culture solution; (3) adding the first jasmonic acid culture solution into a culture box of the continuously cultured porphyra haitanensis free filament, culturing for 5-20 days, and replacing the first jasmonic acid culture solution every 5 days.
Wherein the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2·s。
Further, the Relative Growth Rate (RGR) was measured every 5 days, the change in Fresh Weight (FW) of algal bodies during cultivation was measured, and the formula RGR (% & d) was used-1)＝[(ln Wt-ln Wo)/t]X 100. Where Wo is initial FW and Wt is FW after t days, and surface water was blotted with filter paper before weighing the algal bodies.
On day 15, the growth rate of the Porphyra haitanensis free filament was 9.86. + -. 0.52.
The third embodiment to the sixth embodiment of the patent embodiment of the invention mainly illustrate the influence of jasmonic acid on the formation of Porphyra haitanensis sporangium branches, and the specific experimental mode is as follows:
example three:
firstly, performing synchronous culture on porphyra haitanensis free filaments, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: the seawater culture solution of P10: 1 is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
the method comprises the following steps of cultivating sporangium branches of porphyra haitanensis free filament bodies: 1) increasing the phosphorus concentration in the sterilized seawater to a value of N: p is 1: 10, thereby obtaining a seawater culture solution; 2) placing the porphyra haitanensis free filament subjected to synchronous culture in advance in a jasmonic acid-free seawater culture solution; 3) culturing the seawater culture solution containing Porphyra haitanensis free filaments in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C under illumination intensity of 20 μmol/m for 10-30 days2·s。
Wherein N: p is 1: the seawater culture solution of 10 is N (NaNO)3)：P(K2HPO3)＝1:10；
Preferably, the seawater culture solution is replaced every 5 days.
Every 5 days, the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid-free culture solution was observed under a microscope.
Preferably, the development of Porphyra haitanensis free filamentous sporangium branches in jasmonic acid-free medium is observed under a microscope on day 15, as shown in FIG. 1.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, on day 15, the formation rate of Porphyra haitanensis free filament sporangium branches was 8% + -1.4.
Experimental groups:
the method for promoting formation of the Porphyra haitanensis free filament sporangium branches comprises the following steps: (1) dissolving jasmonic acid in ethanol solution to prepare a second jasmonic acid solution of 5 mol/L; (2) diluting the second jasmonic acid solution with seawater containing MES culture medium to obtain 1 μmol/L second jasmonic acid culture solution; (3) placing the thallus Porphyrae filaments into the second jasmonic acid culture solution, culturing in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C and illumination intensity of 20 μmol/m for 10-30 days2·s。
Preferably, in step (3), the second jasmonic acid culture solution is replaced every 5 days.
Every 5 days, the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid culture solution was observed under a microscope.
Preferably, the development of Porphyra haitanensis free filamentous sporangium branches in jasmonic acid-free medium is observed under a microscope on day 15, as shown in FIG. 2.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, on day 15, the formation rate of Porphyra haitanensis free filament sporangium branches was 12.4% + -2.9.
Example four:
firstly, toThe porphyra haitanensis free filament is synchronously cultured, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: the seawater culture solution of P10: 1 is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
cultivating sporangium branches of the porphyra haitanensis free filament: 1) increasing the phosphorus concentration in the sterilized seawater to a value of N: p is 1: 10, thereby obtaining a seawater culture solution; 2) placing the porphyra haitanensis free filament subjected to synchronous culture in advance in a jasmonic acid-free seawater culture solution; 3) culturing the seawater culture solution containing Porphyra haitanensis free filaments in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C under illumination intensity of 20 μmol/m for 10-30 days2·s。
Wherein N: p is 1: the seawater culture solution of 10 is N (NaNO)3)：P(K2HPO3)＝1:10。
Preferably, the seawater culture solution is replaced every 5 days.
Specifically, 10 visual fields are randomly selected under a microscope every 5 days for observation and recording the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid-free culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, on day 15, the formation rate of Porphyra haitanensis free filament sporangium branches was 8% + -1.4.
Experimental groups:
the method for promoting formation of the Porphyra haitanensis free filament sporangium branches comprises the following steps: (1) mixing jasmonic acid and an ethanol solution to prepare a second jasmonic acid solution of 5 mol/L; (2) diluting the second jasmonic acid solution with seawater containing the culture medium to prepare a 0.5 mu mol/L second jasmonic acid culture solution; (3) placing the Porphyra haitanensis free filament in the second jasmonic acid culture solution, culturing in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C under illumination intensity of 20 μmol/m for 10-30 days2·s。
Preferably, in step (3), the second jasmonic acid culture solution is replaced every 5 days.
Every 5 days, the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid culture solution was observed under a microscope.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, on day 15, the formation rate of free filamentous sporangium branches of Porphyra haitanensis was 8.8% + -2.2.
Example five:
firstly, performing synchronous culture on porphyra haitanensis free filaments, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: 10:1 (P) marine cultureThe nutrient solution is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
cultivating sporangium branches of the porphyra haitanensis free filament: 1) increasing the phosphorus concentration in the sterilized seawater to a value of N: p is 1: 10, thereby obtaining a seawater culture solution; 2) placing the porphyra haitanensis free filament subjected to synchronous culture in advance in a jasmonic acid-free seawater culture solution; 3) culturing the seawater culture solution containing Porphyra haitanensis free filaments in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C under illumination intensity of 20 μmol/m for 10-30 days2·s。
Wherein N: p is 1: the seawater culture solution of 10 is N (NaNO)3)：P(K2HPO3)＝1:10。
Preferably, the seawater culture solution is replaced every 5 days.
On day 15, the number of Porphyra haitanensis free filamentous sporangium branches in the jasmonic acid-free culture solution was observed under a microscope.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, the formation rate of sporangium branches of the obtained porphyra haitanensis free filament was 8% ± 1.4.
Experimental groups:
the method for promoting formation of the Porphyra haitanensis free filament sporangium branches comprises the following steps: (1) mixing jasmonic acid and an ethanol solution to prepare a second jasmonic acid solution of 5 mol/L; (2) diluting the second jasmonic acid solution with seawater containing the culture medium to prepare a 2 mu mol/L second jasmonic acid culture solution; (3) placing the Porphyra haitanensis free filament in the second jasmonic acid culture solution, and placing in an incubator with illumination period of 8L:16DCulturing at 28 + -0.5 deg.C for 10-30 days under the condition of illumination intensity of 20 μmol/m2·s。
Preferably, in step (3), the second jasmonic acid culture solution is replaced every 5 days.
On day 15, the number of Porphyra haitanensis free filamentous sporangium branches in jasmonic acid culture solution was observed under a microscope.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, the formation rate of sporangium branches of the obtained porphyra haitanensis free filament was 11.2% ± 2.2.
Example six:
firstly, performing synchronous culture on porphyra haitanensis free filaments, wherein the synchronous culture comprises the following specific steps: 1) placing N: 10:1 of a seawater culture solution; 2) taking a certain amount of porphyra haitanensis free filaments, crushing the porphyra haitanensis free filaments by a crusher, and placing the porphyra haitanensis free filaments in a culture box for culture until the amount of the cultivated porphyra haitanensis free filaments reaches the requirement of an experiment; wherein preferably, the temperature during culture is 23 + -0.5 deg.C, the illumination period is 12L:12D, and the illumination intensity is 20 μmol/m2S; 3) change N weekly: p is 10:1 culture medium.
Wherein N: the seawater culture solution of P10: 1 is N (NaNO)3)：P(C3H4Na3O7P) concentration ratio of 10: 1.
Control group:
cultivating sporangium branches of the porphyra haitanensis free filament: 1) increasing the phosphorus concentration in the sterilized seawater to a value of N: p is 1: 10, thereby obtaining a seawater culture solution; 2) placing the porphyra haitanensis free filament subjected to synchronous culture in advance in a jasmonic acid-free seawater culture solution; 3) will be placed with Porphyra haitanensis freelyCulturing the filament in seawater culture solution in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C and illumination intensity of 20 μmol/m for 10-30 days2·s。
Wherein N: p is 1: the seawater culture solution of 10 is N (NaNO)3)：P(K2HPO3)＝1:10。
Preferably, the seawater culture solution is replaced every 5 days.
After culturing for a period of time, the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid culture solution is observed under a microscope.
Preferably, the development of Porphyra haitanensis free filamentous sporangium branches in jasmonic acid-free medium is observed under a microscope on day 25, as shown in FIG. 3.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, the formation rate of sporangium branches of the obtained Porphyra haitanensis free filament was 37% + -13.5.
Experimental groups:
the method for promoting formation of the Porphyra haitanensis free filament sporangium branches comprises the following steps: (1) mixing jasmonic acid and an ethanol solution to prepare a second jasmonic acid solution of 5 mol/L; (2) diluting jasmonic acid solution with seawater containing culture medium to obtain 1 μmol/L second jasmonic acid culture solution; (3) culturing the second jasmonic acid culture solution in an incubator with illumination period of 8L:16D at 28 + -0.5 deg.C and illumination intensity of 20 μmol/m for 10-30 days2·s。
Preferably, in step (3), the second jasmonic acid culture solution is replaced every 5 days.
After culturing for a period of time, the number of Porphyra haitanensis free filament sporangium branches in the jasmonic acid culture solution is observed under a microscope.
Preferably, the development of Porphyra haitanensis free filamentous sporangium branches in jasmonic acid culture solution is observed under a microscope on day 25, as shown in FIG. 4.
Specifically, 10 visual fields are randomly selected under a microscope to observe and record the number of Porphyra haitanensis free filament sporangium branches in jasmonic acid culture solution, and then the formation rate of the Porphyra haitanensis free filament sporangium branches is calculated.
Wherein, the formation rate (%) of the Porphyra haitanensis free filament sporangium branches is equal to the number of the free filament sporangium branches/the total number of the free filament branches multiplied by 100 in 10 visual fields of microscopic examination, and the average value is taken.
In this example, the formation rate of sporangium branches of the obtained porphyra haitanensis free filament was 52% ± 15.1.
The method for promoting the growth of the free filament and the formation of the sporangial branch of the porphyra haitanensis provided by the invention is described in detail above, and the principle and the implementation mode of the invention are explained by applying specific examples, and the description of the examples is only used for helping to understand the invention and the core idea thereof. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (5)
1. A method for promoting the growth of free filaments and the formation of sporangium branches of porphyra haitanensis is characterized by comprising the following steps:
(1) pulverizing appropriate amount of Porphyra haitanensis free filament, culturing in culture solution with N: P of 10:1 at 20-25 deg.C under illumination of 12L:12D at 20-30 μmol/m2·s；
(2) Dissolving jasmonic acid in an alcohol solution to prepare a first jasmonic acid solution, and diluting seawater containing an MES culture medium with the first jasmonic acid solution to prepare a first jasmonic acid culture solution;
(3) placing the cultured Porphyra haitanensis free filament obtained in step (1) in the first jasmonic acid culture solution prepared in step (2), culturing in an incubator with illumination period of 12L:12D at 20-25 deg.C and illumination intensity of 20-30 μmol/m for 5-30 days2S, to promote the growth of free filaments of porphyra haitanensis;
(4) dissolving jasmonic acid in an alcohol solution to prepare a second jasmonic acid solution, and diluting seawater containing an MES culture medium with the second jasmonic acid solution to prepare a second jasmonic acid culture solution;
(5) culturing the cultured Porphyra haitanensis free filament in an incubator with a light cycle of 8L:16D for 10-30 days at 20-29 deg.C under 0-30 μmol/m2S, to promote the formation of Porphyra haitanensis sporangial shoots; the concentration of the second jasmonic acid solution prepared in the step (4) is 0-5 mol/L; the concentration of the first jasmonic acid solution in the step (2) is 0-5 mol/L; the concentration of the first jasmonic acid culture solution in the step (2) is 0-1 mu mol/L; the concentration of the second jasmonic acid culture solution in the step (4) is 0.5-2 mu mol/L.
2. The method of claim 1, wherein P in step (1) is selected from the group consisting of C and P is selected from the group consisting of C, P3H4Na3O7P or K2HPO3One or both of them.
3. The method of claim 1, wherein said alcohol solution of step (2) and step (4) is ethanol.
4. The method of claim 1, wherein in step (5), the second jasmonic acid solution is replaced every 5 days.
5. The method of claim 1, wherein in step (1) N is NaNO3Provided is a method.
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