Method for asexual propagation of sargassum fusiforme explants

Abstract

The application discloses a kind of Hizikia fusiformis explant asexual propagation seedling raising method, mainly related to the selection and pretreatment of Hizikia fusiformis algal body, the preparation method of sterile explant, the induction of adventitious bud and the process of adventitious bud rapid propagation into regenerated seedling.The breeding method proves that by changing the medium form and condition of Hizikia fusiformis explant, the physiological state of algal body can be better controlled, and after being cultured in solid medium for at least 17 days, it is best to be transferred to liquid medium, and the subsequent algal body can survive and proliferate normally.In the present application, the upper end of Hizikia fusiformis stem can also be induced to grow into seedlings, and the lower end of Hizikia fusiformis stem base can induce adventitious buds.The present application can effectively solve the problems of few sources of Hizikia fusiformis germplasm resources, unclear operation conditions, vulnerability of Hizikia fusiformis to external diseases and pests, difficulty in control, etc., and the experimental conditions are easy to control, which can provide certain reference for algal propagation and selection of dominant species.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a method for asexual propagation and seedling culture of Sargassum fusiforme explants. BACKGROUND

[0002] Sargassum fusiforme is a perennial macroeconomic seaweed widely distributed in Guangdong, Fujian, Zhejiang and other coastal areas of China, which has high nutritional, medicinal and economic value. The seaweed is rich in polysaccharides, polyphenols, proteins and inorganic salts, and is widely used in biological medicine, chemical industry, food and cosmetics industries, and has a huge demand. Therefore, how to quickly and efficiently cultivate healthy and high-yield Sargassum fusiforme has become a problem to be solved.

[0003] During the growth of Sargassum fusiforme, it is not only subjected to the invasion of plankton and various pathogenic microorganisms, but also affected by the growth environment. In severe cases, under extreme weather conditions such as high temperature, the loss of excellent germplasm resources of algae and high-temperature rotting may occur. Therefore, it is crucial to control the culture conditions during the cultivation process for the plant tissue culture of algae. By adjusting the temperature, salinity, light, and medium composition of the culture, the growth and germination rate of Sargassum fusiforme can be changed. Different algae grow at different PGRs, and the final regeneration seedlings are different. IAA, NAA and BAP are commonly used to induce callus of Kappaphycus alvarezii.

[0004] Currently, there are two main propagation methods for Sargassum fusiforme. One is the sexual reproduction of regenerating rhizoids through zygotes, and the other is asexual reproduction, which can be divided into direct regeneration of rhizoids and regeneration through tissue culture. Tissue culture regeneration includes callus culture, segment tissue culture, and protoplast culture to preserve and cultivate economic seaweed. Sargassum fusiforme seedlings include wild seedlings and artificial seedlings. However, there are some problems in the asexual propagation of Sargassum fusiforme, which leads to ineffective propagation of seedlings or limited sources of seedlings. Therefore, the present application uses tissue culture to propagate excellent strains and ensure the stability and heredity of excellent strains. SUMMARY

[0005] The purpose of the present application is to provide a method for asexual propagation and seedling culture of Sargassum fusiforme explants, which can efficiently induce the propagation of Sargassum fusiforme and solve the problems of insufficient seedling sources and low yield of Sargassum fusiforme.

[0006] The method for asexual propagation and seedling culture of Sargassum fusiforme explants provided by the present application comprises the following technical solutions:

[0007] A method for asexual propagation and seedling culture of Sargassum fusiforme explants, comprising the following steps:

[0008] (1) The selection and pretreatment of Sargassum fusiforme algal body:

[0009] After the Sargassum fusiforme algal body is collected, the fresh Sargassum fusiforme is transported to the laboratory by using a low-temperature cold storage box, and then the attached algae, molluscs, sludge and other attached matters of the Sargassum fusiforme are removed, and the Sargassum fusiforme is washed 3-4 times with seawater after being filtered by a 0.45 μm filter paper and being sterilized by high-pressure steam. The Sargassum fusiforme with good growth conditions is temporarily cultured in a sterile culture room for 7 days.

[0010] (2) Preparation of sterile explants:

[0011] The Sargassum fusiforme with good conditions is selected, and the selected Sargassum fusiforme is soaked in a mixed solution of 0.38% (by volume) NaClO and 0.5% (by volume) KI for 3 min, and then immediately washed quickly 3-4 times with sterilized seawater.

[0012] (3) Induction of adventitious buds

[0013] The culture medium, sterilized forceps, scissors and other required equipment required by the experiment are placed in the super-clean workbench, and ultraviolet disinfection is performed for 30 min, and the experimental algal body is cut to an appropriate size beside the alcohol lamp, and the upper end of the Sargassum fusiforme with an appropriate length and the lower end of the stem base are inoculated into the solid culture medium in the super-clean workbench, and the culture is carried out in a sterile culture room.

[0014] (4) Rapid proliferation of adventitious buds into regenerated seedlings

[0015] The explants with translucent color and formed callus / adventitious buds in the solid culture medium are selected and transferred to the liquid culture medium in the super-clean workbench, and the culture of the regenerated seedlings is carried out in a sterile culture room.

[0016] Further, the culture method is to first culture on a solid PES culture medium, and then culture in a liquid PES culture medium, and the solid culture medium needs to be replaced once a month, and the liquid culture medium needs to be replaced once every 3 days.

[0017] Further, the culture method is to culture in the solid culture medium for at least 17 days before being transferred to the liquid culture medium.

[0018] Further, during the culture process, a gas pump is used to continuously supply air for 24 hours.

[0019] Further, during the culture process, all the equipment used should be sterilized by high pressure, and the operation should be carried out in the super-clean workbench.

[0020] Through the above scheme and description, the present application has at least the following advantages:

[0021] (1) The present application adopts tissue culture technology, and can realize the process of small amount of detoxification in the solid culture stage, obtain healthy explants, stable genetic lines, and improve the resistance of subsequent Sargassum fusiforme regenerative young plants. Meanwhile, the culture conditions are flexible, and the material source is not limited by seasons and the like.

[0022] (2) Other experiments mostly use young plants to induce callus-like tissue / adventitious buds, while in the present experiment, Sargassum fusiforme adults can be directly used, and Sargassum fusiforme regenerative seedlings are successfully induced, the material is easy to obtain, the operation method is simple, the cost is low, and a large amount of manpower and material resources is saved.

[0023] (3) The regenerative buds are successfully induced from the lower end of the Sargassum fusiforme stem base, and the regenerative seedlings are successfully induced from the morphological upper end of the Sargassum fusiforme stem.

[0024] (4) The present culture method has been verified for many times to culture Sargassum fusiforme algal body tissue culture seedlings, and the best condition can be from 2 cm to about 9 cm.

[0025] (5) The light intensity and temperature of the present experiment process are regulated according to different states, and the Sargassum fusiforme can be kept in a good ecological environment according to the actual state. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to the specific embodiments of the present application and in combination with the drawings.

[0027] Figure 1 : Regenerative morphological change diagram of Sargassum fusiforme explants in different algal body parts (morphological upper end of stem, lower end of stem base, and rhizoid filament) in each stage of culture.

[0028] Table 1: Callus-like tissue / adventitious bud induction rate and branch segment bud number of tissue culture of different sources of Sargassum fusiforme algal body.

[0029] Table 2: Relative growth rate of tissue culture of different sources of Sargassum fusiforme algal body.

[0030] Figure 2 : Callus-like tissue / adventitious bud induction rate and branch segment bud number of Sargassum fusiforme in different culture batches (1, 2, 3, and 4).

[0031] Figure 3 : Relative growth rate of Sargassum fusiforme in different culture batches (1, 2, 3, and 4).

[0032] Figure 4 : Regenerative change diagram of the morphological upper end of Sargassum fusiforme stem and relative growth rate change.

[0033] Figure 5 : Morphological change process of Sargassum fusiforme algal body after 237 days of culture into young seedlings. DETAILED DESCRIPTION

[0034] The application will be further described below in connection with the drawings and specific examples, it should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0035] The application will be further described below in connection with the drawings and specific examples, it should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0036] Example 1:

[0037] (1) Pretreatment of Sargassum foliaceum algal body:

[0038] In April 2022, after the Sargassum foliaceum algal body was harvested, the fresh Sargassum foliaceum was transported to the laboratory using a low-temperature cold storage box, and then the attached algae, mollusks, sludge and other attachments attached to the Sargassum foliaceum were removed, and the Sargassum foliaceum was washed 3-4 times with seawater filtered by 0.45 μm filter paper and sterilized by high-pressure steam. Sargassum foliaceum algal body with good growth condition was selected for temporary culture in a sterile culture room for 7 days.

[0039] (2) Preparation of sterile explants:

[0040] Select Sargassum foliaceum algal body in good condition, immerse the selected Sargassum foliaceum algal body in a mixed solution of 0.38% (volume ratio) NaClO and 0.5% (volume ratio) KI for 3 min, and then immediately rinse it quickly 3-4 times with sterilized seawater.

[0041] (3) Induction of adventitious buds

[0042] Put the culture medium, sterilized forceps, scissors and other equipment needed in the experiment into the ultraclean workbench, and sterilize it under ultraviolet light for 30 min. Cut the experimental algal body to the appropriate size beside the alcohol lamp, and inoculate the upper end of the Sargassum foliaceum stem and the lower end of the stem base in the ultraclean workbench into the solid culture medium, and culture it in a sterile culture room.

[0043] (4) Rapid propagation of adventitious buds into regenerated seedlings

[0044] Select the explants with transparent color and formed callus / adventitious buds in the solid culture medium, and transfer them to the liquid culture medium in the ultraclean workbench on the 14th, 17th, 20th and 55th days of culture, and culture the regenerated seedlings in a sterile culture room.

[0045] (5) Record the morphological changes of the algal body during the culture process and observe and record the induction rate of callus / adventitious buds and the number of branch regenerations.

[0046] The induction rate of callus / adventitious buds of Sargassum foliaceum algal body = (the number of germinated branches / the total number of branches) * 100.

[0047] The number of regenerated buds of the branch of the Sargassum fusiforme alga = (the total number of germinated buds / the total number of branches).

[0048] The experimental results are shown in Figure 1 According to the culture conditions, the Sargassum fusiforme false root, stem morphology upper end, and stem base lower end all sprouted and could be followed by growing into regenerated seedlings.

[0049] As can be clearly seen from Table 1, the callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme alga from different explants are different, among which the callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme false root reach 100% and (27.43±4.576), the callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme stem morphology upper end reach (66.67±11.55)% and (3.6±0.848), and the callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme stem base lower end reach (33.33±11.547)% and (2.2±0.846). The relative growth rate is the largest for the false root filament, followed by the stem morphology upper end, and the last is the stem base lower end (Table 2).

[0050] Finally Figure 2 The callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme from different culture batches (1, 2, 3, 4) are shown, and it can be seen that the callus-like tissue / indeterminate bud induction rate and the number of regenerated buds of the Sargassum fusiforme regenerated alga of the second batch and later are much higher than those of the first batch, so it can be determined that at least 17 days of solid culture medium culture and then transfer to liquid culture medium can significantly increase the number of regenerated buds.

[0051] Figure 3 The RGR of the first, second, and third batches of Sargassum fusiforme alga segments increases with the extension of the induction time in the solid culture medium, and the fourth batch is the smallest, mainly due to the too short proliferation culture time in the liquid culture.

[0052] Example 2:

[0053] In December 2022, after the Sargassum fusiforme alga was harvested, the fresh Sargassum fusiforme was transported to the laboratory using a low-temperature cold storage box, and then the attached algae, mollusks, and sludge attached to the Sargassum fusiforme were removed, and the Sargassum fusiforme was washed 3-4 times with seawater filtered by 0.45 μm filter paper and sterilized by high-pressure steam. Healthy Sargassum fusiforme was selected for temporary culture for 7 days.

[0054] The upper end of the Sargassum foliatum stem is immersed in a mixed solution of 0.38% NaClO and 0.5% KI by volume ratio for 3 minutes, and then immediately rinsed with sterile seawater for 3-4 times. The upper end of the Sargassum foliatum stem with appropriate length is inoculated into PES solid medium in an ultraclean bench. In February 2023, the explants of the upper end of the Sargassum foliatum stem which have formed callus-like tissues / adventitious buds and have bright color in the solid medium are transferred to liquid PES medium in an ultraclean bench, and the medium is replaced every 3 days. The amount of the medium is gradually reduced to simulate the ecological adaptability of the Sargassum foliatum algae in the natural environment. The experimental results are shown in the morphology and the relative growth rate, and the calculation formula is:

[0055] RGR (%·d -1 )=ln(W t / W0) / t×100.

[0056] The experimental results are shown in Figure 4 By using the method of the present application, the upper end of the Sargassum foliatum stem is successfully induced into many small rod-shaped structures which can develop into air sacs in the later stage, and a small amount of leaves and stems are also grown. Similar findings have not been found in other related articles in China, and the relative growth rate in the later culture can reach 3.444%.

[0057] Example 3:

[0058] A method for culturing Sargassum foliatum seedlings, characterized by comprising the following steps:

[0059] On March 5, 2022, Sargassum foliatum algae were harvested from a Sargassum foliatum cultivation base in Dongtou District, Wenzhou City, Zhejiang Province, and then taken back to the laboratory in a 4℃ low-temperature storage box. The Sargassum foliatum algae were cleaned by removing the epiphytic algae and plankton on the surface with a brush and tweezers, and then rinsed with filtered and sterilized natural seawater. Healthy Sargassum foliatum algae with consistent growth state were selected. The selected Sargassum foliatum algae were temporarily cultured in a plant incubator (Percival E-36HO, America) for 7 days. The temporary culture conditions were as follows: 19℃, 5000lx, light cycle L:D=12h:12h, and culture liquid salinity 26‰. Continuous aeration was performed by using an air pump. After 7 days of domestication culture, the healthy Sargassum foliatum algae were washed with sterile seawater for 3 times, and then placed in a mixed solution of 0.38% NaClO and 0.5% KI by volume ratio under sterile conditions for 2 minutes. The Sargassum foliatum algae were washed with sterile water for 3 times, and then cut into appropriate lengths with a scalpel under the alcohol lamp. The Sargassum foliatum algae segments were cultured in PES solid medium under sterile conditions, and finally cultured in a sterile culture room. The culture conditions were as follows: salinity 25psu, temperature 18℃, light intensity 3000lx, and light-dark ratio 12:12.

[0060] The morphology of the algae was recorded during the culture.

[0061] According to Figure 5 It can be seen that the Hizikia algae experienced a total of about 11 months of culture, with 7 months of solid preservation, followed by verification of whether it could normally survive and grow, and then another 3 months of culture in the form of liquid medium. Among them, the solid medium should be replaced every 1 month, while the liquid medium should be replaced every 3-4 days.

[0062] Table 1: Induction rate of callus-like tissue / adventitious bud and the number of branch regeneration of Hizikia algae tissue culture from different sources.

[0063]

[0064] Table 2: Relative growth rate of Hizikia algae tissue culture from different sources.

[0065]

[0066] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for asexual propagation of Sargassum fusiforme explants for seedling raising, characterized in that, The method comprises the following steps: (1) selecting and pretreating the Sargassum foliaceum thalli, preparing sterile explants, inducing adventitious buds, and rapidly proliferating the adventitious buds into regenerated seedlings; (2) selecting the Sargassum foliaceum thalli which are in good condition after being temporarily cultured in a sterile culture room for 7 days, and then soaking the selected Sargassum foliaceum thalli in a mixed solution of 0.38% NaClO and 0.5% KI by volume ratio for 3 min, and then immediately rinsing the Sargassum foliaceum thalli in sterile seawater for 3-4 times to obtain sterile explants; (3) sterilizing the required devices, including culture medium, tweezers and scissors, in the ultraclean workbench for 30 min under ultraviolet light, and then cutting the Sargassum foliaceum explants to a length of about 1.5-2 cm beside an alcohol lamp, and then inoculating the cut Sargassum foliaceum stems into solid culture medium in the ultraclean workbench, and then culturing the Sargassum foliaceum explants in a sterile culture room; (4) selecting the explants which have formed callus-like tissues / adventitious buds and are in good color in the solid culture medium, and then transferring the selected explants into liquid culture medium in the ultraclean workbench, and then culturing 8-10 explants in 800 ml of liquid culture medium in a sterile culture room; and (5) recording the morphological changes of the Sargassum foliaceum thalli by using a camera, and recording the induction rate of callus-like tissues / adventitious buds and the number of regenerated branches by observation, and weighing to determine the relative growth rate.

2. The method of claim 1, wherein, The steps (2), (3), (4) and (5) need to be performed in the ultraclean workbench, and the operation tools need to be sterilized at 121 DEG C for 30 min, and then sterilized under ultraviolet light for 30 min.

3. The method of claim 1, wherein, The induction rate of callus-like tissues / adventitious buds of the Sargassum foliaceum thalli in step (5) is (the number of germinated branches / the total number of branches) * 100.

4. The method of claim 1, wherein, The number of regenerated buds of the branch of the Sargassum fusiforme alga in step (5) is equal to (total number of germinated buds / total number of branches).

5. The method of claim 1, wherein, The explants in steps (3)-(4) need to be induced to form callus-like tissues / adventitious buds on solid culture medium in a tissue culture manner, and then proliferated in a liquid culture manner.

6. The method of claim 5, wherein, During the culture process, an air pump is needed to continuously supply air, and after the Sargassum fusiforme explants are sterilized, they need to be washed with 3-5 times of sterilized seawater to completely wash away the residual sterilizing solution.

7. The method of claim 1, wherein, The relative growth rate of the Sargassum foliatum in step (5) is calculated by the following formula: .

8. The method of claim 1, wherein, The basic culture medium used in the culture is PES culture medium.

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