Method for promoting maturation of porphyra haitanensis shell protonema

Abstract

The invention provides a method for promoting maturation of porphyra haitanensis shell protonema, and belongs to the technical field of porphyra haitanensis seedling culture. The method for promoting the porphyra haitanensis shell protonema to be mature comprises the following steps that shells with the shell faces completely covered by porphyra haitanensis filamentous algae fall are cultured under the irradiation condition of green light, the wavelength of the green light is 510-550 nm, and the illumination intensity of the green light is 28-30 [mu] mol / m < 2 > / s-30 [mu] mol / m < 2 > / s-30 [mu] mol / m < 2 > / s-30 [mu] mol / m < 2 > / s. According to the method for promoting maturation of the porphyra haitanensis shell protonema, the maturation time of the porphyra haitanensis shell protonema can be remarkably shortened, and the diffusion amount and germination rate of porphyra haitanensis conchospores can be remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of Porphyra haitanensis seedling cultivation, and in particular relates to a method for promoting the maturation of Porphyra haitanensis shell filaments. Background Art

[0002] Cultivation of shell filaments of Porphyra is the only way to obtain the "seed" conchospores needed for the aquaculture of Porphyra. The development process of shell filaments has four cell development stages: filamentous nutrient algae filaments, algae filaments composed of swollen cells, nearly mature algae filaments composed of amorphous cells, and conchospore sac branches. At present, the shell filaments seedling technology is mainly used in the production process of Porphyra seedlings. A large number of released conchospores are obtained by cultivating mature shell filaments. After the conchospores attach to the net curtain and germinate, they are cultivated on the sea. In the different development processes of shell filaments, the external environment will directly affect the growth and development of shell filaments of Porphyra. In the cultivation stage of shell filaments of Porphyra, promoting the nutritional proliferation of Porphyra filaments and promoting the development process of Porphyra filaments are two different purposes. The nutritional proliferation and development process of shell filaments of Porphyra have different requirements for external environmental conditions. At present, there are no relevant reports on whether light quality affects the development process of shell filaments of Porphyra and how to use light quality to promote the development process of shell filaments of Porphyra. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for promoting the maturation of Porphyra haitanensis shell conchoceles, which can significantly shorten the maturation time of Porphyra haitanensis shell conchoceles and significantly improve the release amount and germination rate of conchospores.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for promoting the maturation of Porphyra haitanensis conchocelis, comprising the following steps: culturing a shell whose shell surface is completely covered by Porphyra haitanensis filamentous algae under green light irradiation conditions, wherein the wavelength of the green light is 510-550nm, and the illumination intensity of the green light is 28-30μmol / m 2 / s.

[0006] Preferably, the culture temperature is 28.5-29.5°C.

[0007] Preferably, the photoperiod of the green light irradiation is 10L:14D or 12L:12D.

[0008] Preferably, the method for preparing the shell whose shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis comprises the following steps: chopping the free filaments of Porphyra haitanensis to obtain nutritious algae filaments, transplanting the nutritious algae filaments to the inner surface of the shell, culturing in the dark for 3 to 5 days, resuming light culturing, washing the shell on the 7th to 10th day after transplantation, and culturing until the shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis.

[0009] Preferably, the length of the nutritional algae filaments is 40 to 60 μm.

[0010] Preferably, the illumination intensity of the illumination culture is 40 to 50 μmol / m 2 / s.

[0011] Preferably, the photoperiod of the light culture is 10L:14D or 12L:12D.

[0012] Preferably, during the whole process of preparing the shells whose shell surfaces are completely covered with filamentous algae colonies of Porphyra haitanensis, the culture temperature is 23.5-24.5°C.

[0013] Beneficial effects of the present invention:

[0014] The method for promoting the maturation of Porphyra haitanensis shell conchocelis provided by the present invention can not only significantly shorten the maturation time of Porphyra haitanensis shell conchocelis, but also significantly increase the release amount and germination rate of Porphyra haitanensis conchospores. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the illumination culture device, where ① is the seedling pool of Porphyra haitanensis, ② is the seedling hanging rod, ③ is the seedling rope, ④ is the seedling attachment base, ⑤ is the walking wheel, ⑥ is the lamp stand, ⑦ is the adjustment rope, ⑧ is the pulley, ⑨ is the pulley inside the lamp stand, ⑩ is the lamp board, For the lamp, Adjust the lift bracket for the light panel height, Adjust the hoist rope reel for the light panel height, Adjust lift motor for light panel height;

[0016] Figure 2 The results of cell morphology observation of different developmental stages of Porphyra haitanensis conchocelis under different light quality conditions, among which b~d are microphotographs of different developmental stages cultured under white light conditions; f~h are microphotographs of different developmental stages cultured under blue light conditions; j~l are microphotographs of different developmental stages cultured under red light conditions; n~p are microphotographs of different developmental stages cultured under green light conditions; among which the first column from left to right, i.e., a, e, i, m, are microphotographs of the filamentous vegetative algae filament stage, the second column is microphotographs of the algae filament stage composed of swollen cells, the third column is microphotographs of the algae filament stage composed of amorphous cells close to maturity, and the fourth column is microphotographs of the conchosporangium branch stage;

[0017] Figure 3 The photographic observation results of the shell filaments of Porphyra haitanensis under different light quality conditions, among which a and b are microscopic photos of the culture under white light conditions; c and d are microscopic photos of the culture under blue light conditions; e and f are microscopic photos of the culture under red light conditions; g and h are microscopic photos of the culture under green light conditions;

[0018] Figure 4 The results of conchospores released from the shell filaments of Porphyra haitanensis ripened under different light qualities, among which a is the result of conchospores released under white light; b is the result of conchospores released under blue light; c is the result of conchospores released under red light; d is the result of conchospores released under green light;

[0019] Figure 5 The statistical results of the release of conchospores of Porphyra haitanensis shell filaments under different light qualities, where W represents white light, B represents blue light, R represents red light, and G represents green light; when any two groups are compared in the bar graph, if the superscript letters are different, it means that there is a significant difference in the release between the two groups (p < 0.05); if the superscript letters are the same, it means that there is no significant difference in the release between the two groups;

[0020] Figure 6 is the germination rate of conchospores of Porphyra haitanensis shell filaments promoted by different light qualities, where W represents white light, B represents blue light, R represents red light, and G represents green light. When comparing any two groups in the bar graph, if the superscript letters are different, it indicates that there is a significant difference in the emission between the two groups (p < 0.05); if the superscript letters are the same, it indicates that there is no significant difference in the emission between the two groups. DETAILED DESCRIPTION

[0021] The present invention provides a method for promoting the maturation of Porphyra haitanensis conchocelis, comprising the following steps: culturing a shell whose shell surface is completely covered by Porphyra haitanensis filamentous algae under green light irradiation conditions, wherein the wavelength of the green light is 510-550nm, and the illumination intensity of the green light is 28-30μmol / m 2 / s.

[0022] In the present invention, the shell surface of which is completely covered by the filamentous algae of Porphyra haitanensis refers to the shell that has completed the first developmental stage in the development process of the shell filaments: the filamentous nutritional algae stage. The shell preferably comprises a clam shell. In the present invention, the culture temperature is preferably 28.5-29.5°C, more preferably 29°C; the photoperiod of the green light irradiation is preferably 10L:14D or 12L:12D, the 10L:14D refers to 10h light: 14h dark, the 12L:12D refers to 12h light: 12h dark. In the present invention, the green light is preferably a green light emitting LED lamp. In a specific embodiment of the present invention, it is preferred to use Figure 1The light culture device shown in the figure is used for culture under green light irradiation conditions. The light culture device provided by the present invention can control the irradiation intensity by adjusting the height and the number of lamps, and the light component module can be freely slid to change the irradiation angle. Specifically: the lamps Installed on the light board ⑩, the light board is The lifts composed of ⑦ and ⑧ are used for height adjustment. The lifts are preferably divided into two left and right ones. When they are operated simultaneously, the light board can be lifted and lowered smoothly. When one side is operated, the light board can be tilted and adjusted. The walking wheel ⑤ enables the entire set of LED light source equipment to be moved as needed.

[0023] In the present invention, the method for preparing the shell whose shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis preferably comprises the following steps: chopping the free filaments of Porphyra haitanensis to obtain nutrient algae filaments, transplanting the nutrient algae filaments to the inner surface of the shell, culturing in the dark for 3 to 5 days, resuming light culturing, washing the shell on the 7th to 10th day after transplantation, and culturing until the shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis.

[0024] The present invention does not specifically limit the specific equipment for shredding. In a specific embodiment of the present invention, the equipment used for shredding is a grinder, and the shredding is preferably to shred the free filaments into 40-60 μm, and more preferably to shred them into 50 μm. In the present invention, the cleaning is preferably to use a soft brush to clean the excess free filaments on the surface of the shell to remove impurities that may affect growth. When preparing shells whose shell surfaces are completely covered with Porphyra alba colonies, the light intensity of the light culture is preferably 40-50 μmol / m 2 / s, more preferably 44 to 48 μmol / m 2 / s; the light cycle of the light culture is preferably 10L:14D or 12L:12D. In the whole process of preparing the shells whose shell surface is completely covered with the filamentous algae colonies of Porphyra haitanensis, the culture temperature is preferably 23.5-24.5°C.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] Example 1

[0029] A method for promoting the maturation of Porphyra haitanensis shell filaments, comprising the following steps:

[0030] (1) The free filaments of Porphyra haitanensis were chopped into 40 μm nutrient algae filaments by a crusher, and the nutrient algae filaments were evenly transplanted to the inner surface of the clam shells, each of which was 4 cm long. Every 15 shells were divided into a group and placed in a rectangular plastic box with a size of 6 cm × 20 cm × 15 cm. The culture was carried out for 3 days in a dark light environment. After the dark treatment, the light was restored and the light intensity was set to 40 μmol / m 2 / s, the photoperiod was 12L:12D; on the 7th day after transplantation, use a soft-bristle brush to clean the excess free filaments on the shell surface to remove impurities that may affect growth; when cultured to 35 days, the shell surface was completely covered with filamentous algae; during this step and the entire culture process, the temperature was 24°C to ensure the most suitable growth conditions.

[0031] (2) The clam shells with the shell surface completely covered by filamentous algae were placed under green light of 510-550 nm for ripening. The ripening temperature was 29 °C and the light intensity (density) was 30 μmol / m 2 / s, and the photoperiod was 10L:14D.

[0032] Example 2

[0033] A method for promoting the maturation of Porphyra haitanensis shell filaments, comprising the following steps:

[0034] (1) The free filaments of Porphyra haitanensis were chopped into 60 μm algae filaments by a crusher and evenly transplanted to the inner surface of clam shells. The length of each clam shell was 4 cm. Every 15 shells were divided into a group and placed in a rectangular plastic box with a size of 6 cm × 20 cm × 15 cm. The culture was carried out for 4 days in a dark light environment. After the dark treatment, the light was restored and the light intensity was set to 50 μmol / m 2 / s, the photoperiod was 12L:12D; on the 9th day after transplantation, a soft-bristled brush was used to clean the excess free filaments on the shell surface to remove impurities that may affect growth; when cultured to the 35th day, the shell surface was completely covered with filamentous algae; during this step and the entire culture process, the temperature was 24.5℃ to ensure the most suitable growth conditions.

[0035] (2) The clam shells whose shell surfaces are completely covered with filamentous algae are connected in series with a cultivation rope, and the clam string is hung on a Figure 1 The light culture device shown in the figure performs ripening culture under green light irradiation conditions, the wavelength of the green light is 510-550nm, the temperature of the ripening culture is 28.5℃, and the light intensity (density) is 28μmol / m 2 / s, and the photoperiod was 12L:12D.

[0036] Example 3

[0037] A method for promoting the maturation of Porphyra haitanensis shell filaments, comprising the following steps:

[0038] (1) The free filaments of Porphyra haitanensis were chopped into 50 μm algae filaments by a crusher and evenly transplanted to the inner surface of clam shells. The length of each clam shell was 4 cm. Every 15 shells were divided into a group and placed in a rectangular plastic box with a size of 6 cm × 20 cm × 15 cm. The culture was carried out for 5 days in a dark environment. After the dark treatment, the light was restored and the light intensity was set to 45 μmol / m 2 / s, the photoperiod was 12L:12D; on the 10th day after transplantation, use a soft-bristle brush to clean the excess free filaments on the shell surface to remove impurities that may affect growth; when cultured to 35 days, the shell surface was completely covered with filamentous algae; during this step and the entire culture process, the temperature was 23.5℃ to ensure the most suitable growth conditions.

[0039] (2) The clam shells with the shell surface completely covered by filamentous algae were placed under green light of 510-550 nm for ripening. The ripening temperature was 29.5 °C and the light intensity (density) was 29 μmol / m 2 / s, and the photoperiod was 10L:14D.

[0040] Comparative Example 1

[0041] The difference from Example 1 is that the light in step (2) is white light of 400-760 nm, and the rest is the same as Example 1.

[0042] Comparative Example 2

[0043] The difference from Example 1 is that the light in step (2) is blue light 455-475 nm, and the rest is the same as Example 1.

[0044] Comparative Example 3

[0045] The difference from Example 1 is that the light in step (2) is red light 580-630nm, and the rest is the same as Example 1.

[0046] Example 4

[0047] During the ripening process of Example 1 and Comparative Examples 1 to 3, the surface moisture of the shell filaments of different developmental stages of each light quality group (filamentous nutrient algae filaments, algae filaments composed of swollen cells, nearly mature algae filaments composed of amorphous cells, and shell spore sac branches) was absorbed dry, and the shell filaments were broken into pieces of 1 cm×1 cm in size, and the shells were dissolved using Berlanti solution (200 ml of 10% nitric acid, 150 ml of 95% alcohol, and 150 ml of 0.5% chromic acid). The shells were completely immersed in the shell melting reagent for 90 seconds, and immediately washed with sea water 3 times after soaking. After washing, the shell filaments were promptly scraped and collected with tweezers for morphological observation and microscopic photography.

[0048] (1) Record the maturity of algal filaments under different light quality conditions. Figures 2-3 And as shown in Table 1.

[0049] Table 1 Growth and development of Porphyra haitanensis shell filaments under different light quality conditions

[0050]

[0051]

[0052] Depend on Figures 2-3 As can be seen from Table 1, under different light quality conditions, the morphology of the algae filaments of the shell filaments is highly consistent, but the color changes of the algae are significantly different. The algae filaments are generally arranged in long strips, while the star-shaped chromatophores are mainly concentrated in the central part, with only a few chromatophores scattered around. Compared with the color of the algae under white light, the color of the algae under blue light is fuller and appears dark red or even purple ( Figure 2 fh in the figure). However, under red light, the color of the algae gradually turns to dark yellow-brown ( Figure 2 jl in the figure), and the same color change can be observed in the shell filaments by naked eye ( Figure 3 As the ripening time increases, the cytochrome bodies of the algae under green light show a darker yellow-brown color ( Figure 2 np), the number of immature cells approaching maturity is greater than that of other light quality groups ( Figure 2 o in the figure), and the cell contents are fuller. The mature conchosporon branches under red light appear partially hollow ( Figure 2 l in the figure). In addition, under different light quality conditions, the timing of the development of expanded algal filaments and conchosporangium branches was different. Compared with white light, under green light ripening conditions, the shell filaments matured relatively early, 5d, 2d and 9d earlier than white light, blue light and red light, respectively (Table 1).

[0053] (2) Statistics on the release and germination rate of conchospores from conchospores

[0054] When there are about 10 conchospores in each field of view (10×10) after sampling and microscopic observation, the release of conchospores begins to be counted. Select 3 mature shells of similar size from each group and put them into a culture dish (9 cm in diameter) containing 50 mL of sterilized seawater. Set up 3 parallels for each experimental group. After 12:00 noon, stir the conchospore liquid in the culture dish and take samples, taking 1 mL of conchospore liquid each time. Under a microscope, use a plankton counting frame to count the release of conchospores of shell filaments ripened under different light qualities. Each counting frame randomly selects 10 fields of view for counting, and calculates the average value. The release of conchospores is obtained by combining the field of view radius r. The released conchospores are cultured in darkness for 3 days, transferred to white light and continued to be cultured for 3 days, and the germination rate of conchospores is counted once a day. The specific formula is as follows:

[0055] Conchospore release (pieces / mL) = A × 1000 / (3.14 × r 2 ×2)

[0056] Where A is the average number of conchospores randomly counted in 10 fields of view for each sample.

[0057] Germination rate (%) = (germinated conchospores / total number of conchospores) × 100

[0058] The results are as follows Figures 4 to 6 As shown in the figure. The conchospores released by the shell filaments under different light quality conditions are relatively consistent in shape, and are spherical. However, the color of the spores just released is obviously different. The conchospores released by the shell filaments under white light are reddish brown ( Figure 4 a), the conchospores released by the blue light group appear purple ( Figure 4 b), while the conchospores released by the red light group showed a dull yellow-brown color ( Figure 4 c), while the conchospores released by the green light group conchospores appeared dark yellow-brown ( Figure 4 d) in.

[0059] In addition, the release of conchospores from the shell filaments of Porphyra haitanensis ripened under different light quality conditions showed significant differences (p<0.05) ( Figure 5 ), the number of conchospores released by the shell filaments ripened by green light was the largest, which increased by 25.05%, 13.95% and 34.5% compared with white light, blue light and red light, respectively. After 1 day of cultivation under normal lighting conditions, the germination rate of conchospores released by the shell filaments ripened by blue light and green light was higher than that of the white light group (p<0.05), while there was no significant difference in the germination rate of conchospores between the white light group and the red light group (p>0.05). As the culture time reached 2 days, there was no significant difference in the germination rate of conchospores released by the shell filaments in the white light and blue light groups, but the germination rate of conchospores in the red light group was significantly lower (p<0.05). After 3 days of cultivation, the conchospores ripened by green light still maintained a high germination rate ( Figure 6 ).

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for promoting the maturation of Porphyra haitanensis shell filaments, characterized in that: The method comprises the following steps: culturing a shell whose shell surface is completely covered by filamentous algae of Porphyra haitanensis under green light irradiation conditions, wherein the wavelength of the green light is 510 to 550 nm, and the illumination intensity of the green light is 28 to 30 μmol / m 2 / s.

2. The method according to claim 1, characterized in that The culture temperature is 28.5-29.5°C.

3. The method according to claim 1, characterized in that The photoperiod of the green light irradiation is 10L:14D or 12L:12D.

4. The method according to claim 1, characterized in that The method for preparing the shell whose shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis comprises the following steps: chopping the free filaments of Porphyra haitanensis to obtain nutrient algae filaments, transplanting the nutrient algae filaments to the inner surface of the shell, culturing in the dark for 3 to 5 days, resuming light culturing, washing the shell on the 7th to 10th day after transplantation, and culturing until the shell surface is completely covered with filamentous algae colonies of Porphyra haitanensis.

5. The method according to claim 4, characterized in that The length of the nutritional algae filaments is 40 to 60 μm.

6. The method according to claim 4, characterized in that The illumination intensity of the light culture is 40 to 50 μmol / m 2 / s.

7. The method according to claim 4, characterized in that The photoperiod of the light culture is 10L:14D or 12L:12D.

8. The method according to claim 4, characterized in that During the whole process of preparing the shells whose shell surfaces are completely covered with filamentous algae colonies of Porphyra haitanensis, the culture temperature is 23.5-24.5°C.

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