A method for promoting the maturation of porphyra haitanensis shell filament

Abstract

The application provides a method for promoting maturation of Porphyra haitanensis shell filament bodies, and belongs to the technical field of Porphyra haitanensis breeding. The method for promoting maturation of Porphyra haitanensis shell filament bodies comprises the following steps: culturing a shell with a shell surface completely covered by Porphyra haitanensis filament algae under green light irradiation, wherein the wavelength of the green light is 510-550 nm, and the light intensity of the green light is 28-30 μmol / m 2 / s. The method for promoting maturation of Porphyra haitanensis shell filament bodies can significantly shorten the maturation time of the Porphyra haitanensis shell filament bodies, and significantly improve the spore dispersal amount and germination rate of the Porphyra haitanensis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Porphyra haitanensis breeding, and particularly relates to a method for promoting maturation of Porphyra haitanensis shell filamentous body. BACKGROUND

[0002] Porphyra haitanensis shell filamentous body cultivation is the only way to obtain the seed crust spores required by Porphyra haitanensis cultivation industry. The development process of the shell filamentous body has four cell development stages: filamentous nutrient algal filament, algal filament composed of swollen cells, near-mature algal filament composed of unshaped cells, and crust spore cyst branch. At present, the shell filamentous body is mainly used in the breeding process of Porphyra haitanensis production, and a large number of dispersed crust spores are obtained by cultivating mature shell filamentous bodies. The crust spores are attached to the netting and germinated, and then the netting is hung on the sea for cultivation. In the different development processes of the shell filamentous body, the external environment directly affects the growth and development of the Porphyra haitanensis shell filamentous body. In the cultivation stage of the Porphyra haitanensis shell filamentous body, promoting the nutrient proliferation of the shell filamentous body and promoting the development process of the shell filamentous body are two different purposes. The nutrient proliferation of the shell filamentous body and the development process of the shell filamentous body have different requirements for external environmental conditions. At present, whether light quality affects the development process of the Porphyra haitanensis shell filamentous body and how to use light quality to promote the development process of the Porphyra haitanensis shell filamentous body have not been reported. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a method for promoting maturation of Porphyra haitanensis shell filamentous body, which can significantly shorten the maturation time of the shell filamentous body and significantly improve the dispersal amount and germination rate of the crust spores.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

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

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

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

[0008] Preferably, the method for preparing the shell with the shell surface completely covered by the filamentous algal mat of Porphyra haitanensis comprises the following steps: cutting the free filaments of Porphyra haitanensis to obtain the vegetative filaments, transplanting the vegetative filaments to the inner surface of the shell, dark culture for 3-5 days, light culture, cleaning the shell at 7-10 days after the transplantation, and culturing until the shell surface is completely covered by the filamentous algal mat of Porphyra haitanensis.

[0009] Preferably, the length of the vegetative filaments is 40-60 μm.

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

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

[0012] Preferably, the temperature of the culture during the whole process of preparing the shell with the shell surface completely covered by the filamentous algal mat of Porphyra haitanensis is 23.5-24.5℃.

[0013] The present application has the following advantages:

[0014] The method for promoting the maturation of the filamentous thalli of Porphyra haitanensis provided by the present application can not only significantly shorten the maturation time of the filamentous thalli of Porphyra haitanensis, but also significantly increase the spore dispersal amount and germination rate of the shell spores of Porphyra haitanensis. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of a light culture device, wherein ① is a Porphyra haitanensis breeding pond, ② is a seedling hanging rod, ③ is a seedling rope, ④ is an attached seedling base, ⑤ is a walking wheel, ⑥ is a lamp holder, ⑦ is an adjusting rope, ⑧ is a pulley, ⑨ is an inner pulley of the lamp holder, and ⑩ is a lamp plate, is a lamp tube, is a lamp plate height adjusting elevator support, is a lamp plate height adjusting elevator rope winder, is a lamp plate height adjusting elevator motor;

[0016] Figure 2 is the cell morphological observation result of the filamentous thalli of Porphyra haitanensis at different development stages under different light quality conditions, wherein b-d are micrographs of different development stages cultured under white light conditions; f-h are micrographs of different development stages cultured under blue light conditions; j-l are micrographs of different development stages cultured under red light conditions; and n-p are micrographs of different development stages cultured under green light conditions; wherein the first column from left to right, i.e., a, e, i, and m, are micrographs of the filamentous vegetative filament stage; the second column are micrographs of the algal filament stage composed of swollen cells; the third column are micrographs of the nearly mature algal filament stage composed of untypical cells; and the fourth column are micrographs of the conchospore branch stage;

[0017] Figure 3 The images show the photographic observations of the filamentous shells of *Porphyra yezoensis* under different light conditions. a and b are photomicrographs taken under white light; c and d are photomicrographs taken under blue light; e and f are photomicrographs taken under red light; and g and h are photomicrographs taken under green light.

[0018] Figure 4 The results of conchospore release from the shell filaments of *Porphyra yezoensis* under different light qualities are shown in the images. (a) shows conchospore release under white light; (b) shows conchospore release under blue light; (c) shows conchospore release under red light; and (d) shows conchospore release under green light.

[0019] Figure 5 The statistical results of conchospore release from the shell filaments of *Porphyra yezoensis* under different light qualities are shown. W represents white light, B represents blue light, R represents red light, and G represents green light. In the bar chart, if the superscript letters are different between any two groups, it indicates that there is a significant difference in the release amount between the two groups (p < 0.05). If the superscript letters are the same, it indicates that there is no significant difference in the release amount between the two groups.

[0020] Figure 6 The germination rate of conchospores from the shell filaments of *Porphyra yezoensis* under different light qualities is shown in the bar chart. W represents white light, B represents blue light, R represents red light, and G represents green light. In the bar chart, if the superscript letters are different between any two groups, it indicates that there is a significant difference in the amount of spores released between the two groups (p < 0.05). If the superscript letters are the same, it indicates that there is no significant difference in the amount of spores released between the two groups. Detailed Implementation

[0021] This invention provides a method for promoting the maturation of filamentous algae in *Porphyra yezoensis* shells, comprising the following steps: culturing shells whose shell surfaces are completely covered by *Porphyra yezoensis* filamentous algal colonies under green light irradiation conditions, wherein the wavelength of the green light is 510–550 nm and the light intensity of the green light is 28–30 μmol / m². 2 / s.

[0022] In this invention, a shell completely covered with filamentous algal colonies of *Porphyra yezoensis* refers to a shell that has completed the first developmental stage in the development of the shell's filamentous structure: the filamentous nutrient algal filament stage. The shell preferably includes a hard clam shell. In this invention, the cultivation 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, where 10L:14D refers to 10 hours of light: 14 hours of darkness, and 12L:12D refers to 12 hours of light: 12 hours of darkness. In this invention, the green light is preferably a green-emitting LED tube. In a specific embodiment of this invention, a preferred method is as follows: Figure 1The illustrated light-based culture device is used for cultivation under green light irradiation. The light-based culture device provided by this invention can control the irradiation intensity by adjusting the height and the number of lamps, and the light-based component modules can be freely slid to change the irradiation angle. Specifically: lamps... Placed on the light panel ⑩, the light panel is connected by... The lifting platform consisting of ⑦ and ⑧ is height-adjustable. Preferably, there are two lifting platforms, one on the left and one on the right. When they are running simultaneously, the light panel rises and falls smoothly. When running on one side, the light panel can be tilted. The traveling wheels ⑤ allow the entire LED light source equipment to be moved as needed.

[0023] In this invention, the preferred method for preparing a shell whose shell surface is completely covered by filamentous algal colonies of *Porphyra yezoensis* includes the following steps: chopping the free filamentous bodies of *Porphyra yezoensis* to obtain nutrient algal filaments, transplanting the nutrient algal filaments to the inner surface of the shell, culturing in the dark for 3-5 days, resuming light cultivation, cleaning the shell on the 7th-10th day after transplantation, and culturing until the shell surface is completely covered by *Porphyra yezoensis* filamentous algal colonies.

[0024] This invention does not specifically limit the equipment used for chopping. In one particular embodiment, a shredder is used, and the chopping is preferably performed to chop the free filaments to 40-60 μm, more preferably to 50 μm. In this invention, the cleaning is preferably performed by using a soft brush to clean the surface of the shell of excess free filaments to remove impurities that may affect growth. When preparing shells whose shell surfaces are completely covered by *Porphyra yezoensis* algae, the light intensity for the light cultivation is preferably 40-50 μmol / m². 2 / s, more preferably 44–48 μmol / m 2 / s; the photoperiod of the light cultivation is preferably 10L:14D or 12L:12D. Throughout the entire process of preparing shells whose shell surfaces are completely covered by *Porphyra yezoensis* filamentous algal colonies, the cultivation temperature is preferably 23.5–24.5℃.

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

[0026] Unless otherwise specified, the following embodiments are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] Example 1

[0029] A method for promoting the maturation of filamentous shells in *Porphyra yezoensis* includes the following steps:

[0030] (1) The free filaments of *Porphyra yezoensis* were shredded into 40 μm nutrient algal filaments using a shredder. These nutrient algal filaments were then evenly transferred to the inner surface of *Clam shells*, with each shell measuring 4 cm in length. Each group of 15 shells was placed in a rectangular plastic box measuring 6 cm × 20 cm × 15 cm. The shells were cultured in a dark environment for 3 days. After the dark treatment, the light was restored, and the light intensity was set to 40 μmol / m². 2 / s, photoperiod of 12L:12D; on the 7th day after transplantation, use a soft brush to clean the shell surface of excess free filaments to remove impurities that may affect growth; by the 35th day of cultivation, the shell surface is completely covered with filamentous algae; throughout this step of the entire cultivation process, the temperature is 24℃ to ensure the most suitable growth conditions.

[0031] (2) Clam shells whose shell surfaces were completely covered with filamentous algal colonies were placed under green light at 510–550 nm for ripening culture. The ripening culture temperature was 29℃ and the light intensity (density) was 30 μmol / m². 2 / s, with an optical period of 10L:14D.

[0032] Example 2

[0033] A method for promoting the maturation of filamentous shells in *Porphyra yezoensis* includes the following steps:

[0034] (1) The free filaments of *Porphyra yezoensis* were shredded into 60 μm algal filaments using a shredder. The algal filaments were then evenly transplanted onto the inner surface of *Clam shells*, with each shell measuring 4 cm in length. Each group of 15 shells was placed in a rectangular plastic box measuring 6 cm × 20 cm × 15 cm. The shells were cultured in a dark environment for 4 days. After the dark treatment, the light was restored, and the light intensity was set to 50 μmol / m². 2 / s, photoperiod of 12L:12D; on the 9th day after transplantation, use a soft brush to clean the shell surface of excess free filaments to remove impurities that may affect growth; by the 35th day of cultivation, the shell surface is completely covered with filamentous algae; throughout this step of the entire cultivation process, the temperature is 24.5℃ to ensure the most suitable growth conditions.

[0035] (2) String together clam shells whose shell surfaces are completely covered with filamentous algae using a culture rope, and hang the clam strings on a rack. Figure 1 The light-based culture apparatus shown was used for ripening culture under green light irradiation. The wavelength of the green light was 510–550 nm, the ripening culture temperature was 28.5 °C, and the light intensity (density) was 28 μmol / m³. 2 / s, with an optical period of 12L:12D.

[0036] Example 3

[0037] A method for promoting the maturation of filamentous shells in *Porphyra yezoensis* includes the following steps:

[0038] (1) The free filaments of *Porphyra yezoensis* were shredded into 50 μm algal filaments using a shredder. The algal filaments were then evenly transplanted onto the inner surface of *Clam shells*, with each shell measuring 4 cm in length. Each group of 15 shells was placed in a rectangular plastic box measuring 6 cm × 20 cm × 15 cm. The shells were cultured in a dark environment for 5 days. After the dark treatment, the light was restored, and the light intensity was set to 45 μmol / m². 2 / s, photoperiod of 12L:12D; on the 10th day after transplantation, use a soft brush to clean the shell surface of excess free filaments to remove impurities that may affect growth; by the 35th day of cultivation, the shell surface is completely covered with filamentous algae; throughout this step of the entire cultivation process, the temperature is 23.5℃ to ensure the most suitable growth conditions.

[0039] (2) Clam shells whose shell surfaces were completely covered with filamentous algal colonies were placed under green light at a wavelength of 510–550 nm for ripening culture. The ripening culture temperature was 29.5℃ and the light intensity (density) was 29 μmol / m². 2 / s, with an optical period of 10L:14D.

[0040] Comparative Example 1

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

[0042] Comparative Example 2

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

[0044] Comparative Example 3

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

[0046] Example 4

[0047] In the ripening process of Examples 1 and Comparative Examples 1-3, the surface moisture of the shell filaments of different developmental stages (filamentous nutrient algal filaments, algal filaments composed of swollen cells, nearly mature algal filaments composed of amorphous cells, and chitosporangia branches) of each photochemical group was dried, and fragments of 1cm×1cm size were picked out after being crushed. The shells were dissolved in Berlanni solution (200ml of 10% nitric acid, 150ml of 95% alcohol, and 150ml of 0.5% chromic acid) and the shells were completely immersed in the shell-dissolving agent for 90s. After immersion, the shells were immediately rinsed 3 times with seawater. After rinsing, the shell filaments were scraped and collected with tweezers and their morphology was observed and photographed under a microscope.

[0048] (1) Record the maturation of algal filaments under various light quality conditions. The results are as follows: Figures 2-3 As shown in Table 1.

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

[0050]

[0051]

[0052] Depend on Figures 2-3 As shown in Table 1, under different light quality conditions, the morphology of the shell-like filaments exhibits high uniformity; however, the color variations of the algae show significant differences. The filaments are generally arranged in long, thin strips, while the star-shaped chloroplasts are mainly concentrated in the central part, with only a few chloroplasts scattered around the periphery. Compared to the color of the algae under white light, the color of the algae under blue light is more saturated and appears deep red or even purplish-red. Figure 2 (fh in the middle). But under red light, the color of the algae gradually turns dark yellowish-brown ( Figure 2 (jl) The same color change was observed in the filamentous shell of the shell (jl) by the naked eye. Figure 3 As the ripening time increases, the cytochromes of the algae under green light exhibit a deeper yellowish-brown color. Figure 2 The number of near-mature amorphous cells in the np group was greater than that in other light quality groups. Figure 2 (o) and the cell contents are more full. Mature conchiocysts under red light show partial hollowing (o). Figure 2 (l) Furthermore, the timing of development into enlarged algal filaments and conchiocysts differed under different light quality conditions. Compared to white light, under green light maturation conditions, the shell filaments matured relatively earlier, maturing 5 days, 2 days, and 9 days earlier than under white light, blue light, and red light, respectively (Table 1).

[0053] (2) Statistics on the release and germination rate of shell filamentous conidia

[0054] When approximately 10 conidia were observed in each 10×10 field of view under a microscope, the release rate of conidia was recorded. Three mature shells of similar size were selected from each group and placed in a 50mL sterile seawater culture dish (9cm in diameter). Three replicates were set up for each experimental group. After 12:00 noon, the conidia solution in the culture dish was stirred thoroughly, and 1mL of conidia solution was sampled each time. The release rate of conidia from the filamentous shells maturing under different light qualities was recorded under a microscope using a plankton counting frame. Ten fields of view were randomly selected for each counting frame, and the average value was calculated. The release rate of conidia was determined by combining the field of view radius r. The released conidia were then incubated in the dark for 3 days, then transferred to white light for another 3 days. The germination rate of conidia was recorded daily using the following formula:

[0055] Conocyst release rate (cells / mL) = A × 1000 / (3.14 × r) 2 ×2)

[0056] In the formula, A is the average number of conchospores in 10 random fields of view for each sample.

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

[0058] The results are as follows Figures 4-6 As shown. The conchospores released from shell filaments ripened under different light conditions exhibited relatively consistent morphology, appearing spherical. However, the color of the newly released spores showed significant differences. Conchospores released from shell filaments ripened under white light appeared reddish-brown. Figure 4 In section a), the conchiospores released by the blue light group appear purplish-red. Figure 4 In section b), the conchispores released by the red light group exhibit a dull yellowish-brown color. Figure 4 c), while the conchospores released from the shell filaments of the Green Light Group are dark yellowish-brown ( Figure 4 (d)

[0059] In addition, the amount of conchospores released from the shell filaments of *Porphyra yezoensis* ripened under different light quality conditions showed significant differences (p<0.05). Figure 5 Green light-induced maturation of shell filaments resulted in the highest number of conchospores released, increasing by 25.05%, 13.95%, and 34.5% compared to white light, blue light, and red light, respectively. After 1 day of cultivation under normal light conditions, the germination rate of conchospores released from shell filaments matured under blue and green light was higher than that under white light (p<0.05), while there was no significant difference in germination rate between the white light and red light groups (p>0.05). After 2 days of cultivation, there was no significant difference in the germination rate of conchospores released from shell filaments between the white and blue light groups, but the germination rate of conchospores in the red light group was significantly lower (p<0.05). Even after 3 days of cultivation, the conchospores matured under green light maintained a high germination rate.Figure 6 ).

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for promoting the maturation of filamentous shells of *Porphyra yezoensis*, characterized in that, The process includes the following steps: culturing seashells whose shell surfaces are completely covered with filamentous algal colonies of *Porphyra yezoensis* under green light irradiation, wherein the wavelength of the green light is 510–550 nm and the light intensity is 28–30 μmol / m². 2 / s; The culture temperature is 28.5–29.5℃; The photoperiod of the green light irradiation is 10L:14D or 12L:12D.

2. The method according to claim 1, characterized in that, The method for preparing a shell whose shell surface is completely covered by filamentous algal colonies of Porphyra yezoensis includes the following steps: chopping the free filaments of Porphyra yezoensis to obtain nutrient algal filaments, transplanting the nutrient algal filaments to the inner surface of the shell, culturing in the dark for 3-5 days, resuming light cultivation, cleaning the shell on the 7th-10th day after transplantation, and culturing until the shell surface is completely covered by filamentous algal colonies of Porphyra yezoensis.

3. The method according to claim 2, characterized in that, The length of the nutrient algal filaments is 40–60 μm.

4. The method according to claim 2, characterized in that, The light intensity for the photoculture was 40–50 μmol / m². 2 / s.

5. The method according to claim 2, characterized in that, The photocycle for the light culture is 10L:14D or 12L:12D.

6. The method according to claim 2, characterized in that, Throughout the preparation of shells whose shell surfaces were completely covered by filamentous algal colonies of *Porphyra yezoensis*, the culture temperature was 23.5–24.5℃.

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