Method for rapid propagation of free-living filaments of porphyra haitanensis

Abstract

The application provides a method for rapidly propagating free filaments of Porphyra haitanensis, and belongs to the technical field of Porphyra haitanensis filament propagation. The method for rapidly propagating free filaments of Porphyra haitanensis comprises the following steps: placing the free filaments of Porphyra haitanensis in a container containing MES culture solution, and performing irradiation and aeration culture by using a blue light, a blue-red combined light source or a white-blue combined light source; the irradiation intensity is 18-22 μmol / m 2 / s. The application can achieve the purpose of rapidly propagating the free filaments of Porphyra haitanensis in a short time by limiting the light spectrum combination and culture conditions most suitable for the rapid propagation of Porphyra haitanensis algae. The method can further improve the facility propagation and seed production technology, and accelerate the popularization and application of new Porphyra haitanensis germplasm.

Description

Technical Field

[0001] This invention belongs to the field of filamentous propagation technology of Porphyra yezoensis, and particularly relates to a method for rapidly propagating free filamentous tissues of Porphyra yezoensis. Background Technology

[0002] The marine economic algae *Porphyra yezoensis* is characterized by its small size, rapid proliferation, strong tolerance, and ease of artificial control during its filamentous stage. After the free filaments of *Porphyra yezoensis* are crushed, the algal filaments embed into the shell matrix, forming algal colonies—a technique known as free filament transplantation. This technique is widely used in the development of new *Porphyra yezoensis* germplasm due to its use of homozygous germplasm and simple seedling cultivation process. Currently, new *Porphyra yezoensis* germplasm bred through cell engineering mainly relies on this technique to cultivate shell filamentous seedlings. Therefore, achieving the rapid and large-scale expansion of *Porphyra yezoensis* free filaments and obtaining healthy, high-quality filamentous germplasm is crucial. Currently, there is no research in this field on how to achieve the rapid propagation of *Porphyra yezoensis* free filaments. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for rapidly propagating free filamentous structures of Porphyra yezoensis.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for rapidly propagating free filaments of *Porphyra yezoensis*, comprising the following steps: placing the free filaments of *Porphyra yezoensis* in a container containing MES culture medium, and irradiating and culturing them using blue light, a combination of blue and red light sources, or a combination of white and blue light sources; the light intensity of the irradiation is 18–22 μmol / m². 2 / s.

[0006] Preferably, the light quality ratio of blue light to red light in the blue-red combined light source is 5:5, 6:4, or 9:1.

[0007] Preferably, the light quality ratio of white light to blue light in the white-blue combined light source is 9:1, 7:3, or 5:5.

[0008] Preferably, the wavelength of the blue light is 455–475 nm.

[0009] Preferably, the wavelength of the red light is 580–630 nm.

[0010] Preferably, the wavelength of the white light is 510–550 nm.

[0011] Preferably, the irradiation period is 12L:12D.

[0012] Preferably, the culture temperature is 23.5–24.5°C.

[0013] Preferably, the mass-to-volume ratio of the free filaments of *Porphyra yezoensis* to the MES culture medium is 2.94–3.06 g: 300 mL.

[0014] The beneficial effects of this invention are:

[0015] The method for rapidly propagating free filaments of *Porphyra yezoensis* provided by this invention achieves rapid propagation of free filaments of *Porphyra yezoensis* in a short time by defining the most suitable spectral combination and culture conditions for the rapid proliferation of *Porphyra yezoensis* thallus. This method can further improve facility-based propagation and seed production technology and accelerate the promotion and application of new *Porphyra yezoensis* germplasm. Attached Figure Description

[0016] Figure 1 The images show the growth morphology of free filaments of *Porphyra yezoensis* under monochromatic light conditions. Images a through f are micrographs taken under white light for 0, 5, 10, 15, 20, and 25 days; images g through l are micrographs taken under blue light for 0, 5, 10, 15, 20, and 25 days; images mr are micrographs taken under red light for 0, 5, 10, 15, 20, and 25 days; and images sx are micrographs taken under green light for 0, 5, 10, 15, 20, and 25 days.

[0017] Figure 2 The images show the growth morphology of free filaments of *Porphyra yezoensis* under blue-red combined light conditions. Images a–f are micrographs taken at 0, 5, 10, 15, 20, and 25 days after cultivation under 5B / 5R conditions; images g–l are micrographs taken at 0, 5, 10, 15, 20, and 25 days after cultivation under 6B / 4R conditions; images m–r are micrographs taken at 0, 5, 10, 15, 20, and 25 days after cultivation under 7B / 3R conditions; and images s–t are micrographs taken at 0, 5, 10, 15, 20, and 25 days after cultivation under 9B / 1R conditions.

[0018] Figure 3 The images show the growth morphology of free filaments of *Porphyra yezoensis* under a white-blue combined light quality condition. Images a–f show the micrographs after 0, 5, 10, 15, 20, and 25 days of cultivation under 9W / 1B conditions; images g–l show the micrographs after 0, 5, 10, 15, 20, and 25 days of cultivation under 8W / 2B conditions; images m–r show the micrographs after 0, 5, 10, 15, 20, and 25 days of cultivation under 7W / 3B conditions; and images s–t show the micrographs after 0, 5, 10, 15, 20, and 25 days of cultivation under 5W / 5B conditions.

[0019] Figure 4The average daily growth rate of free filaments of *Porphyra yezoensis* under different light quality conditions;

[0020] Figure 5 The specific growth rate of free filaments of *Porphyra yezoensis* under different light quality conditions;

[0021] Figure 6 The chlorophyll a content of free filaments of Porphyra yezoensis under different light quality culture conditions;

[0022] Figure 7 The carotenoid content of free filaments of Porphyra yezoensis under different light quality culture conditions;

[0023] Figure 8 The phycoerythrin content of free filaments of Porphyra yezoensis under different light quality culture conditions;

[0024] Figure 9 The phycocyanin content of free filaments of Porphyra yezoensis under different light quality culture conditions;

[0025] exist Figures 4-9 In the bar chart, the superscript letters indicate whether the differences are significant. If the superscript letters are different between any two groups, it means that there is a significant difference between the two groups (P < 0.05). If the superscript letters are the same, it means that there is no significant difference between the two groups (P > 0.05). Detailed Implementation

[0026] This invention provides a method for rapidly propagating free filaments of *Porphyra yezoensis*, comprising the following steps: placing the free filaments of *Porphyra yezoensis* in a container containing MES culture medium, and irradiating and culturing them using blue light, a combination of blue and red light sources, or a combination of white and blue light sources; the light intensity of the irradiation is 18–22 μmol / m². 2 / s.

[0027] In this invention, the preferred mass-to-volume ratio of the *Porphyra yezoensis* free filaments to the MES culture medium is 2.94–3.06 g:300 mL, and the preferred container is a culture flask, more preferably a conical culture flask. In this invention, the blue light source is preferably a blue-emitting LED lamp; the blue-red combined light source is preferably composed of blue-emitting and red-emitting LED lamps; and the white-blue combined light source is preferably composed of white-emitting and blue-emitting LED lamps. In this invention, the preferred light quality ratio of blue to red light in the blue-red combined light source is 5:5, 6:4, or 9:1; and the preferred light quality ratio of white light to blue light in the white-blue combined light source is 9:1, 7:3, or 5:5. In this invention, the wavelength of the blue light is preferably 455-475nm, more preferably 465-470nm; the wavelength of the red light is preferably 580-630nm, more preferably 590-620nm; and the wavelength of the white light is preferably 510-550nm, more preferably 520-540nm.

[0028] In this invention, the irradiation light cycle is preferably 12L:12D, where 12L:12D refers to 12h light: 12h darkness; the culture temperature is preferably 23.5-24.5℃, more preferably 24℃.

[0029] 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.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] In the following embodiments, 5B / 5R refers to 5 blue-emitting LED tubes and 5 red-emitting LED tubes, and so on for 6B / 4R, 7B / 3R, and 9B / 1R. 9W / 1B refers to 9 white-emitting LED tubes and 1 blue-emitting LED tube, and so on for 8W / 2B, 7W / 3B, and 5W / 5B.

[0033] Example 1

[0034] A method for rapid propagation of free filaments of *Porphyra yezoensis* includes the following steps: placing 3g of free filaments of *Porphyra yezoensis* in a 500mL Erlenmeyer flask containing 300mL LMES culture medium, and irradiating and culturing the flask with aeration using 10 blue-emitting LED tubes; the wavelength of the blue light is 455-475nm, and the irradiation intensity is 20μmol / m². 2 / s, the irradiation light period is 12L:12D, and the culture temperature is 24℃.

[0035] Example 2

[0036] A method for rapid propagation of free filaments of *Porphyra yezoensis* includes the following steps: 2.94 g of free filaments of *Porphyra yezoensis* are placed in a 500 mL Erlenmeyer flask containing 300 mL of LMES culture medium, and cultured under aeration using a blue-red combined light source; the blue-red combined light source consists of 5 blue-emitting LED tubes and 5 red-emitting LED tubes, denoted as 5B / 5R; the wavelength of the blue light is 455–475 nm, the wavelength of the red light is 580–630 nm, and the total light intensity of the irradiation is 20 μmol / m³. 2 / s, the irradiation light period is 12L:12D, and the culture temperature is 24.5℃.

[0037] Example 3

[0038] A method for rapidly propagating free filaments of *Porphyra yezoensis* includes the following steps: 2.94 g of *Porphyra yezoensis* free filaments are placed in a 500 mL Erlenmeyer flask containing 300 mL of LMES culture medium, and cultured under aeration using a white-blue combined light source; the white-blue combined light source consists of 7 white LED tubes and 3 blue LED tubes, denoted as 7W / 3B; the wavelength of the white light is 510–550 nm, the wavelength of the blue light is 455–475 nm, and the total light intensity of the irradiation is 20 μmol / m³. 2 / s, the irradiation light period is 12L:12D, and the culture temperature is 23.5℃.

[0039] Example 4

[0040] Comparison of the effects of different light source irradiation on the vegetative proliferation (asexual proliferation) of free filaments of Porphyra yezoensis

[0041] 1.1 Synchronized culture of free filaments of Porphyra yezoensis

[0042] Free filaments of *Porphyra yezoensis* during its vegetative filament development stage were collected, pulverized, and placed in a 1L glass bottle containing MES culture medium. After 3 days in the dark, a 12L:12D photoperiod was resumed for further cultivation. The light intensity was set to 15 μmol / m². 2 The temperature was maintained at 24℃, and the algae were aerated using an aeration pump. The culture medium was changed regularly, with half of it replaced with fresh medium every 7 days. Once a certain number of algae were propagated, the algae were further propagated in separate bottles until the biomass of the algae met the experimental requirements.

[0043] 1.2 Experimental Light Source Setup and Algal Culture

[0044] Pre-synchronized free filaments were weighed at 3g increments and placed in 500mL Erlenmeyer flasks containing 300mL LMES medium under aeration. These flasks were then placed one by one into a culture rack, which was sealed tightly with black KT board to ensure a completely opaque environment. The rack's dimensions were 1.0m x 0.5m x 0.5m. Cultures were conducted under four light qualities (white, blue, red, and green), different ratios of blue-red light combinations (5B / 5R, 6B / 4R, 7B / 3R, 9B / 1R), and white-blue light combinations (9W / 1B, 8W / 2B, 7W / 3B, 5W / 5B). The culture temperature was 24℃, and the photoperiod was 12L:12D. White light served as the control group. A total of 12 groups were prepared, with three replicates per group. The wavelength ranges of white, blue, red, and green light are 510–550 nm, 455–475 nm, 580–630 nm, and 400–760 nm, respectively. The light intensity is maintained at 20 μmol / m² by adjusting the number of lamps and the illumination height.2 The light intensity and wavelength were measured using a PLA-20 plant light analyzer. The algae were cultured for 25 days, with the fresh weight of the algae measured and the culture medium replaced every 5 days.

[0045] 1.3 Growth determination and microscopic observation of free filaments

[0046] Each time the fresh weight of the algae is measured, its specific growth rate (SGR) is calculated simultaneously using the following formula:

[0047] SGR(%) = 100 × (lnS) T -lnS T-1 ) / 5

[0048] In the formula, S T For the fresh weight of the algae on day T, S T-1 This represents the previous fresh weight of the algae.

[0049] Morphological observation and microscopic photography of free filaments cultured under different combinations of light quality were performed using a Leica DMI8 optical microscope (Leica GmbH, Germany).

[0050] The results of the photomicrographs are as follows: Figures 1-3 As shown. In the early stages of cultivation, the star-shaped chloroplasts of the nutrient algal filaments are mainly concentrated in the central area, with a small number of chloroplasts distributed around the periphery. Under white light, the algae maintain little color change and retain their normal growth morphology. Figure 1 (a to f in the text). After 10 days of cultivation, the algal filaments under blue light showed a significantly deeper color than those under other monochromatic lights. Figure 1 (i~l). However, under green and red light conditions, the color of the algal filaments is relatively dull, and the chloroplasts of some cells appear pale yellow (i~l). Figure 1 (v~x) When cultured for 20 days, hollow structures appeared in some algae under red light. Figure 1 (q~r in the text). In the combined light quality, the color of the cytochromes of 7B / 3R and 8W / 2B algae significantly deteriorated after 10 days of cultivation, and by 20 days, hollowing of the contents of the algal filaments appeared in 7B / 3R. After 25 days of cultivation, the color of the algal filaments was more vibrant under the blue-white combined light source than under monochromatic light and the blue-red combined light source. Figure 2 and Figure 3 ).

[0051] The results of thallus fresh weight, daily average fresh weight growth rate and specific growth rate are shown in the figure. Figures 4-5 And Table 1.

[0052] Table 1 Fresh weight of *Porphyra yezoensis* free filaments under different light quality conditions

[0053]

[0054] Note: Different superscripts after the mean indicate significant differences (P<0.05).

[0055] In the early stages of cultivation, under various light quality treatments, the free filaments of *Porphyra yezoensis* showed varying degrees of growth. However, under the combined light source of 5W / 5B, its specific growth rate was significantly higher than that of other light quality groups. Figure 5 After 10 days of cultivation, differences in algal growth rates began to appear among the different light quality groups, with increased growth rates observed under the combined light sources 8W / 2B and 7W / 3B. After 15 days, monochromatic blue light and the 5B / 5R combined light source showed a rapid growth advantage, while after 20 days, except for the 9W / 1B combined light source which exhibited faster growth, the growth rates of algae in other light quality groups slowed down. Figure 5 During a 25-day cultivation period, the effects of different light quality conditions on algal weight gain were compared. It was found that the most significant weight gain occurred under a 5W / 5B combined light source (Table 1), followed by monochromatic blue light and the 5B / 5R combined light source, both of which exhibited relatively stable and rapid growth rates. However, algae under monochromatic red light showed negative growth. Figure 5 By comparing the daily average weight gain rate of free filaments of *Porphyra yezoensis* under different light qualities, the results showed that after 25 days of continuous cultivation, the daily average weight gain rate of algae under the combined light source 5W / 5B and blue light was significantly higher than that under other light quality groups. Figure 4 ).

[0056] 1.4 Determination of the content of major photosynthetic pigments and phycobiliproteins

[0057] 1.4.1 Chlorophyll content determination

[0058] First, accurately weigh 100 mg of fresh algae and place it in a 100% methanol solution under dark conditions. Grind thoroughly and incubate overnight at 4°C. The next day, centrifuge at high speed (10000 r / min, 4°C, 10 min) using an Eppendorf 5804 R4 centrifuge. Collect the supernatant and finally measure the absorbance of the sample using a UV-Vis spectrophotometer (UV-3200). The specific formula is as follows:

[0059] Chlorophyll a (μg / g) = 16.29 × (OD665 - OD750) - 8.54 × (OD652 - OD750)

[0060] Carotenoids (μg / g) = 7.6 × [(OD480 - OD750) - 8.54 × (OD510 - OD750)]

[0061] The results are as follows Figure 6 and Figure 7As shown, in monochromatic light quality, the content of major photosynthetic pigments in algae under blue light was significantly higher than in other monochromatic light quality groups. In combined light quality, the white-blue combined light quality 5W / 5B significantly promoted the accumulation of photosynthetic pigments. Furthermore, the carotenoid content was also high in 9W / 1B and 7W / 3B, while the chlorophyll a content in the combined light source 7B / 3R and the carotenoid content in the combined light source 6B / 4R were significantly lower than in other light quality groups.

[0062] 1.4.2 Phycobiliprotein Determination

[0063] Accurately weigh 100 mg of fresh algae and place it in a grinding container containing 8 mL of phosphate buffer (0.1 mol / L, pH 6.5). Grind thoroughly at 4°C in the dark and let stand overnight. The next day, centrifuge the homogenate at 19000 rpm for 15 min to separate the supernatant. Measure the absorbance of the supernatant at wavelengths of 455, 564, 592, 618, and 645 nm using a UV-Vis spectrophotometer. The specific formula is as follows:

[0064] Phycoerythrin (PE) (mg / g) = [(A564-A592) - 0.20 × (A455-A592)] / DW × V

[0065] Phycocyanin (PC) (mg / g) = ([(A618-A645)-0.51×(A592-A645)]) / DW×V

[0066] In the formula, V represents the extraction volume, and DW is the dry weight of the algae (g).

[0067] The results are as follows Figure 8 and Figure 9 As shown, the contents of phycoerythrin (PE) and phycocyanin (PC) differed significantly under different light quality conditions. Specifically, the PE content under white light, monochromatic blue light, and a blue-white combined light quality (9W / 1B) was significantly higher than that under other light quality groups (P<0.05). Figure 8 Except for monochromatic blue light, the PC content of algae under other monochromatic light qualities did not differ significantly (P>0.05), and the PC content of algae under red-blue combined light quality culture did not differ significantly (P>0.05), but was significantly lower than that under other light quality groups. Figure 9 The PC content of algae in the white-blue combined light quality 7W / 3B group was significantly higher than that in other experimental groups (P<0.05). Figure 9 ).

[0068] 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 rapidly propagating free filamentous structures of *Porphyra yezoensis*, characterized in that... The process includes the following steps: placing the free filaments of *Porphyra yezoensis* in a container containing MES culture medium, and irradiating and culturing them using a blue-red combined light source; the light intensity of the irradiation is 18~22 μmol / m². 2 / s; The light quality ratio of blue light to red light in the blue-red combined light source is 5:5 or 6:

4. The irradiation period is 12L:12D; The culture temperature is 23.5~24.5℃; The mass-to-volume ratio of the free filaments of *Porphyra yezoensis* to the MES culture medium was 2.94–3.06 g: 300 mL.

2. The method according to claim 1, characterized in that, The wavelength of the blue light is 455~475nm.

3. The method according to claim 1, characterized in that, The wavelength of the red light is 580~630nm.

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