Method for rapidly propagating free filaments of porphyra haitanensis

Abstract

The invention 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 the free filaments of the porphyra haitanensis comprises the following steps: placing the free filaments of the porphyra haitanensis in a container containing an MES culture solution, and carrying out irradiation aeration culture by adopting a blue light and blue-red combined light source or a white-blue combined light source, the illumination intensity of the irradiation is 18-22 [mu] mol / m < 2 > / s. According to the method, the purpose of rapid propagation of the free protonema of the porphyra haitanensis can be achieved within a short time by limiting the spectrum combination and culture conditions most suitable for rapid propagation of the porphyra haitanensis frond. The method provided by the invention can further improve the facility propagation seed production technology and accelerate the popularization and application of the new germplasm of the porphyra haitanensis.

Description

Technical Field

[0001] The invention belongs to the technical field of propagation of Porphyra haitanensis filaments, and in particular relates to a method for rapidly propagating free filaments of Porphyra haitanensis. Background Art

[0002] The marine economic algae Porphyra haitanensis has the characteristics of small size, fast proliferation, strong tolerance and easy artificial regulation in its conidia stage. After the free conidia of Porphyra haitanensis are crushed, the algal filaments are embedded in the shell matrix to form algal colonies, which is the free conidia transplantation technology. This technology is widely used in the research and development of new germplasm of Porphyra haitanensis because of its use of homozygous germplasm and simple seedling cultivation process. The new germplasm of Porphyra haitanensis currently bred through cell engineering mainly relies on this technology to cultivate shell conidia seedlings. Therefore, it is crucial to achieve a large-scale and rapid expansion of free conidia of Porphyra haitanensis and obtain healthy and high-quality conidia germplasm. At present, there is no relevant research in this field on how to achieve the purpose of rapid expansion of free conidia of Porphyra haitanensis. Summary of the invention

[0003] In view of this, an object of the present invention is to provide a method for rapidly propagating free filaments of Porphyra haitanensis.

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

[0005] The invention provides a method for rapidly propagating free filaments of Porphyra haitanensis, comprising the following steps: placing the free filaments of Porphyra haitanensis in a container containing MES culture solution, and irradiating and aerating the free filaments of Porphyra haitanensis with blue light, a blue-red combined light source, or a white-blue combined light source; the irradiation light intensity is 18-22 μmol / m 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 photoperiod of the irradiation is 12L:12D.

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

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

[0014] Beneficial effects of the present invention:

[0015] The method for rapidly propagating the free conchoceles of Porphyra haitanensis provided by the present invention can achieve the purpose of rapid propagation of the free conchoceles of Porphyra haitanensis in a short time by limiting the spectrum combination and culture conditions most suitable for the rapid proliferation of the algae of Porphyra haitanensis. The method of the present invention can further improve the facility-based propagation and seed production technology and accelerate the promotion and application of new germplasm of Porphyra haitanensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The growth morphology of free filaments of Porphyra haitanensis under monochromatic light conditions is observed, where a~f are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d cultured under white light, g~l are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d cultured under blue light, mr are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d cultured under red light, and sx are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d cultured under green light.

[0017] Figure 2 The results of observation on the growth morphology of free filaments of Porphyra haitanensis under the conditions of blue-red combined light quality are shown in Figure 1, where a~f are microscopic photos of 0d, 5d, 10d, 15d, 20d, and 25d cultured under 5B / 5R conditions; g~l are microscopic photos of 0d, 5d, 10d, 15d, 20d, and 25d cultured under 6B / 4R conditions; m~r are microscopic photos of 0d, 5d, 10d, 15d, 20d, and 25d cultured under 7B / 3R conditions; s~t are microscopic photos of 0d, 5d, 10d, 15d, 20d, and 25d cultured under 9B / 1R conditions;

[0018] Figure 3 The results of observation on the growth morphology of free filaments of Porphyra haitanensis under white-blue combined light quality conditions are shown in Figure 1, where a~f are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d of culture under 9W / 1B conditions; g~l are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d of culture under 8W / 2B conditions; m~r are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d of culture under 7W / 3B conditions; s~t are microphotographs of 0d, 5d, 10d, 15d, 20d, and 25d of culture under 5W / 5B conditions;

[0019] Figure 4is the average daily growth rate of free conchoceles of Porphyra haitanensis under different light quality conditions;

[0020] Figure 5 is the specific growth rate of free filaments of Porphyra haitanensis under different light quality conditions;

[0021] Figure 6 is the chlorophyll a content of free conchoceles of Porphyra haitanensis under different light quality culture conditions;

[0022] Figure 7 is the carotenoid content of free conchoceles of Porphyra haitanensis under different light quality culture conditions;

[0023] Figure 8 is the phycoerythrin content of free filaments of Porphyra haitanensis under different light quality culture conditions;

[0024] Fig. 9 is the phycocyanin content of free filaments of Porphyra haitanensis under different light quality culture conditions;

[0025] exist Figures 4 to 9 In the bar graph, the superscript letters represent whether the difference is significant. When any two groups are compared, if the superscript letters are different, it means that the two groups have a significant difference (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 DESCRIPTION

[0026] The invention provides a method for rapidly propagating free filaments of Porphyra haitanensis, comprising the following steps: placing the free filaments of Porphyra haitanensis in a container containing MES culture solution, and irradiating and aerating the free filaments of Porphyra haitanensis with blue light, a blue-red combined light source, or a white-blue combined light source; the irradiation light intensity is 18-22 μmol / m 2 / s.

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

[0028] In the present invention, the irradiation light cycle is preferably 12L:12D, and the 12L:12D refers to 12h light: 12h dark; the culture temperature is preferably 23.5-24.5°C, more preferably 24°C.

[0029] 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.

[0030] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

[0033] Example 1

[0034] A method for rapidly propagating free filaments of Porphyra haitanensis comprises the following steps: placing 3 g of free filaments of Porphyra haitanensis in a 500 mL conical culture bottle containing 300 mL of MES culture solution, and irradiating and aerating the bottle with 10 blue light emitting LED lamps; the wavelength of the blue light is 455 to 475 nm, and the illumination intensity of the irradiation is 20 μmol / m 2 / s, the irradiation light cycle is 12L:12D, and the culture temperature is 24°C.

[0035] Example 2

[0036] A method for rapidly propagating free filaments of Porphyra haitanensis comprises the following steps: placing 2.94 g of free filaments of Porphyra haitanensis in a 500 mL conical culture bottle containing 300 mL of MES culture solution, and irradiating and aerating the bottle with a blue-red combined light source; the blue-red combined light source is composed of 5 LED lamp tubes emitting blue light and 5 LED lamp tubes emitting red light, which are recorded 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 / m 2 / s, the irradiation light cycle is 12L:12D, and the culture temperature is 24.5°C.

[0037] Example 3

[0038] A method for rapidly propagating free filaments of Porphyra haitanensis comprises the following steps: placing 2.94 g of free filaments of Porphyra haitanensis in a 500 mL conical culture bottle containing 300 mL of MES culture solution, and irradiating and aerating the filaments with a white-blue combined light source; the white-blue combined light source is composed of 7 LED lamp tubes emitting white light and 3 LED lamp tubes emitting blue light, which is recorded 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 / m 2 / s, the irradiation light cycle is 12L:12D, and the culture temperature is 23.5°C.

[0039] Example 4

[0040] Comparison of the effects of different light sources on the vegetative proliferation (asexual proliferation) of free conchoceles of Porphyra haitanensis

[0041] 1.1 Synchronous culture of free conchoceles of Porphyra haitanensis

[0042] The free filaments of Porphyra haitanensis in the vegetative algal filament development stage were taken, crushed by a grinder, and placed in a 1L glass bottle filled with MES culture medium. After being protected from light for 3 days, the 12L:12D photoperiod was restored and continued to be cultured. The light intensity was set to 15μmol / m 2 / s, the temperature was kept stable at 24℃, and an air pump was used to aerate the culture. The culture medium was replaced regularly, with half of the culture medium replaced with fresh culture medium every 7 days. When the number of amplifications reached a certain level, the alginate was continued to be divided into bottles for propagation until the biomass of the algae met the biomass required for the experiment.

[0043] 1.2 Experimental light source setting and algae culture

[0044] The free filaments after pre-synchronized culture were weighed according to the same mass of 3g and placed in a 500mL conical culture bottle containing 300mL MES culture medium for aeration culture. These culture bottles were placed one by one in a culture rack, which was tightly sealed by black KT plates on all sides to ensure that the environment was completely opaque. The length, width and height of the culture rack were 1.0m, 0.5m and 0.5m respectively. The culture was carried out under the conditions of four light qualities (white light, blue light, red light and green light), different proportions of blue-red combined light sources (5B / 5R, 6B / 4R, 7B / 3R, 9B / 1R) and white-blue combined light sources (9W / 1B, 8W / 2B, 7W / 3B, 5W / 5B). The culture temperature was 24℃ and the photoperiod was 12L:12D. White light was used as the control group, with a total of 12 groups, and 3 parallels were set in each group. The wavelengths of white light, blue light, red light and green light are 510-550nm, 455-475nm, 580-630nm and 400-760nm respectively. By adjusting the number of lamps and the height of irradiation, the light intensity is maintained at 20μmol / m2 / s, and a PLA-20 plant light analyzer was used to measure light intensity and wavelength. The culture was continued for 25 days, and the fresh weight of the algae was weighed and the culture medium was replaced every 5 days.

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

[0046] Each time the algae fresh weight is measured, its specific growth rate (SGR) is calculated at the same time. The calculation formula is:

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

[0048] In the formula, S T is the fresh weight of algae on day T, S T-1 Represents the fresh weight of the algae in the previous time.

[0049] A Leica DMI8 optical microscope (Leica, Germany) was used to observe the morphology and take microscopic photos of free filaments cultured under different light quality combinations.

[0050] The microscopic photographic results are as follows Figure 1 to Figure 3 As shown. In the early stage of cultivation, the star-shaped chromatophores of the nutritional algae filaments are mainly concentrated in the central area, and a small amount of chromatophores are distributed in the periphery. The color of the algae under white light does not change significantly, and it maintains a normal growth morphology ( Figure 1 After 10 days of cultivation, the color of algae filaments under blue light is obviously darker than that under other monochromatic light ( Figure 1 However, the color of algal filaments under green and red light conditions is relatively dark, and the chromatophores of some cells appear light yellow ( Figure 1 v~x in the culture), and after 20 days of cultivation, some algae appeared hollow under red light ( Figure 1 In the combined light quality, the color of the cytochrome bodies of 7B / 3R and 8W / 2B algae bodies deteriorated significantly after 10 days of cultivation, and the contents of 7B / 3R algae filaments were hollow after 20 days of cultivation. After 25 days of cultivation, the color of algae filaments under the blue-white combined light source was brighter than that under the monochromatic light and blue-red combined light sources ( Figure 2 and Figure 3 ).

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

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

[0053]

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

[0055] In the early stage of cultivation, under various light quality treatment conditions, the free filaments of Porphyra haitanensis showed different growth trends, but under the combined light source 5W / 5B, its specific growth rate was significantly higher than that of other light quality groups ( Figure 5 ). After 10 days of cultivation, the algae growth rates of each light quality group began to differ, and the algae growth rates of the combined light sources 8W / 2B and 7W / 3B increased. After 15 days, the algae showed a rapid growth advantage under the monochromatic blue light and 5B / 5R combined light sources, and after 20 days, except for the combined light source 9W / 1B, the algae growth rates of other light quality groups slowed down ( Figure 5 ). During the 25-day culture period, the effects of different light quality conditions on algae weight gain were compared. It was found that under the 5W / 5B combination light source, the algae weight gain was the most obvious (Table 1), followed by monochromatic blue light and combination light source 5B / 5R. Both also showed a relatively stable and rapid growth rate. However, the algae under monochromatic red light showed negative growth ( Figure 5 By comparing the daily average weight gain rate of free filaments of Porphyra haitanensis under different light qualities, the results showed that after 25 consecutive days of cultivation, the daily average weight gain rate of algae under the combined light source 5W / 5B and blue light was significantly higher than that of other light quality groups ( Figure 4 ).

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

[0057] 1.4.1 Determination of chlorophyll content

[0058] First, accurately weigh 100 mg of fresh algae, place it in a methanol (100%) solution under dark light conditions, grind it thoroughly, and place it in a 4°C refrigerator overnight. The next day, high-speed centrifugation (10000r / min, 4°C, 10min) was performed using an Eppendorf5804 R4 centrifuge, and then the supernatant was taken. Finally, the absorbance of the sample was measured using a UV-visible 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 the figure, in the monochromatic light quality, the main photosynthetic pigment content of algae under blue light was significantly higher than that of other monochromatic light quality groups. In the combined light quality, the white-blue combined light quality 5W / 5B significantly promoted the accumulation of photosynthetic pigments. The carotenoid content of 9W / 1B and 7W / 3B was also high, while the chlorophyll a content of the combined light source 7B / 3R and the carotenoid content of the combined light source 6B / 4R were significantly lower than those of other light quality groups.

[0062] 1.4.2 Phycobiliprotein assay

[0063] Accurately weigh 100 mg of fresh algae and place it in a grinding container containing 8 mL of phosphate buffer (concentration 0.1 mol / L, pH 6.5). Grind thoroughly at 4°C in a dark environment and let stand overnight. The next day, centrifuge the homogenate at 19000 r / min for 15 minutes to separate the supernatant. Use a UV-visible spectrophotometer to measure the absorbance of the supernatant at wavelengths of 455, 564, 592, 618, and 645 nm. 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] Where V represents the extraction volume, and DW represents the dry weight of algae (g).

[0067] The results are as follows Figure 8 and Fig. 9 As shown in the figure, the contents of phycoerythrin (PE) and phycocyanin (PC) were significantly different under different light quality conditions. Among them, the PE content under white light, monochromatic blue light and 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 was not significantly different (P>0.05), and the PC content of algae under red and blue combined light quality was not significantly different (P>0.05), which was significantly lower than that of other light quality groups ( Fig. 9 ), the PC content of algae in the white-blue light quality 7W / 3B was significantly higher than that in other experimental groups (P<0.05) ( Fig. 9 ).

[0068] 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 rapidly propagating free filaments of Porphyra haitanensis, characterized in that: The method comprises the following steps: placing the free filaments of Porphyra haitanensis in a container containing MES culture solution, and irradiating and aerating the filaments with blue light, a blue-red combined light source, or a white-blue combined light source; the irradiation light intensity is 18 to 22 μmol / m 2 / s.

2. The method according to claim 1, characterized in that 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.

3. The method according to claim 1, characterized in that 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.

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

5. The method according to claim 2, characterized in that: The wavelength of the red light is 580-630 nm.

6. The method according to claim 3, characterized in that The wavelength of the white light is 510-550 nm.

7. The method according to claim 1, characterized in that The photoperiod of the irradiation was 12L:12D.

8. The method according to claim 1, characterized in that The culture temperature is 23.5-24.5°C.

9. The method according to claim 1, characterized in that: The mass volume ratio of the free filaments of Porphyra haitanensis to the MES culture solution is 2.94-3.06 g:300 mL.

Images