In vitro culture method and application of red fiber shrimp seaweed root system

Abstract

This invention provides an in vitro root culture method and application for red-fiber shrimp seaweed, a plant tissue culture technique. This method addresses the difficulty in accurately studying the physiological state of roots and leaves of intact red-fiber shrimp seaweed plants grown hydroponically in the laboratory. The in vitro root culture method includes the following steps: collecting healthy red-fiber shrimp seaweed plants as explants, culturing them in an incubator, selecting healthy plants for a first trimming, disinfecting them, and then trimming them a second time. The plants are then inoculated onto culture plates containing an induction medium for root induction culture until root primordia germinate, and then transferred to culture plates containing a subculture medium for in vitro root culture. This method can be applied to studies of red-fiber shrimp seaweed root physiology.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to an in vitro culture method and application of a red fiber shrimp seaweed root system. Background Art

[0002] Seagrasses are a type of submerged plant that lives in seawater and has important ecological services. However, current research on the physiology of seagrasses lags far behind that of terrestrial plants and seaweeds. Phyllospadix )Seagrass is a type of seaweed that grows on rocky coasts. It relies on the mucus secreted by its roots to firmly fix itself on the rocks to resist erosion by wind and waves.

[0003] Red Fiber Shrimp Seaweed ( Phyllospadix iwatensis ) is a dominant species in the rocky coastal seagrass beds of the Yellow Sea and Bohai Sea in my country. Its robust roots make it an excellent material for studying seagrass root physiology. Furthermore, because its roots absorb nutrients and anchor it to the rock to protect it from wind and wave erosion, its root system is of great research value. However, since both its leaves and roots absorb nutrients, accurate studies of the physiological state of its roots and leaves are difficult to conduct by hydroponically cultivating intact plants in the laboratory. Therefore, to ensure accurate physiological experiments on this seagrass, separate cultivation of its roots and leaves is necessary. Traditional indoor seawater cultures of seagrasses in the genus Scutellaria are insufficient for accurate root physiological studies. Therefore, the challenge of isolating the roots of this seagrass in vitro to obtain roots suitable for physiological research is a pressing issue. Summary of the Invention

[0004] The present invention addresses the technical problem that it is difficult to accurately conduct root physiology research on intact red fiber shrimp seaweed plants hydroponically cultivated in the laboratory. The present invention provides an in vitro culture method and application of red fiber shrimp seaweed roots. The present invention uses the adventitious root primordium of red fiber shrimp seaweed as an explant and induces its normal growth and development through tissue culture technology, thereby achieving in vitro culture of the red fiber shrimp seaweed roots, providing excellent material for root physiology research.

[0005] To achieve the above-mentioned object, the present invention adopts a technical solution: an in vitro culture method for red fiber shrimp seaweed roots, comprising the following steps: collecting healthy red fiber shrimp seaweed plants as explants, selecting healthy plants after culturing in an incubator, performing a first pruning, disinfecting, and a second pruning, then inoculating the plants in a culture plate containing an induction culture medium for root induction culture until root primordia germinate, and then transferring the plants to a culture plate containing a subculture culture medium for in vitro root culture.

[0006] In some embodiments, the incubator culture conditions are as follows: the explant, retaining at least 10 nodes closest to the stem apex and four leaves at the stem apex, is placed in a culture bottle filled with natural seawater and cultured in an incubator under a light intensity of 3000 lux, a light:dark cycle of 8 hours:16 hours, and a temperature of 10°C. After 3 days of incubation, the natural seawater is replaced with sterilized natural seawater and culture continues in the incubator. The sterilized seawater is replaced daily, and the incubator temperature is increased by 1°C every 2 days until it reaches 15°C, and culture is continued for 7 days. Preferably, the explant is a healthy red fiber shrimp seaweed plant collected in spring when the water temperature is approximately 10°C.

[0007] In some embodiments, the first pruning step includes the following steps: plants that are still healthy after being cultured in an incubator are selected, and the first pruning is performed on a clean bench, the leaves are peeled off to expose the stem tip, and only 4 nodes and 4 internodes near the stem tip are left, and there are two clusters of newly emerged adventitious root primordia on the first and second nodes near the stem tip.

[0008] In some embodiments, the disinfection step includes the following steps: placing the stem tip obtained after the first pruning into a new sterilized culture bottle, rinsing it with sterilized natural seawater for 3-5 times, drying the surface moisture of the stem tip with sterilized filter paper, placing it in a 10% sodium hypochlorite solution for disinfection for 15 minutes, then taking it out, and rinsing it with sterilized natural seawater for 5 times; wherein, the rinsing step with sterilized natural seawater and the disinfection step in the 10% sodium hypochlorite solution are both carried out under vacuum filtration conditions.

[0009] In some embodiments, the second pruning step includes the following steps: placing the explant in sterilized natural seawater with 100 μM VC for a second pruning, cutting off the stem tip, and leaving only 2 nodes and 3 internodes near the stem tip.

[0010] In some embodiments, the root induction culture step includes the following steps: inoculating the explants after the second trimming into a six-well culture plate filled with induction medium, incubating one explant per well at 15°C in the dark, and waiting for root primordia to germinate. The root primordia germinate after 3 weeks of culture.

[0011] In some embodiments, the induction medium is liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 1 mg / L 1 BA, and 2 mg / L NAA.

[0012] In some embodiments, the in vitro root culture step includes the following steps: after root primordia germinate, they are transferred to a six-well culture plate containing a subculture medium for culture. When the roots reach 5 mm in length, they are cut from the stem and cultured further. After 60 days of culture, the roots may grow to 20.5 mm.

[0013] In some embodiments, the secondary culture medium is liquid MS medium supplemented with 200 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA.

[0014] Compared with existing technologies, the advantages and positive effects of the present invention are as follows: The present invention uses the adventitious root primordium of red-fiber shrimp seaweed as an explant and induces its normal growth and development through tissue culture techniques, thereby achieving in vitro culture of the red-fiber shrimp seaweed root system, providing excellent material for research on its root physiology. The present invention significantly reduces the contamination rate during explant surface disinfection by temporarily cultivating the red-fiber shrimp seaweed indoors; the use of a vacuum filter for surface disinfection ensures thorough disinfection; pruning the explant in sterilized natural seawater supplemented with VC prevents browning of the explant during pruning; and the use of cell culture plates for culturing reduces the risk of cross-contamination of the explant, conserves culture medium, and facilitates observation of contamination and replacement of culture medium. DETAILED DESCRIPTION

[0015] In order to be able to understand the features and technical contents of the embodiments of the present invention in more detail, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the embodiments described below are only part of the embodiments of the present invention, rather than all the embodiments of the present invention. In the following technical description, for the convenience of explanation, a plurality of details are provided to provide a full understanding of the disclosed embodiments. However, one or more embodiments can still be implemented without these details. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection requested by the present invention.

[0016] The embodiment of the present invention provides an in vitro culture method and application of the red fiber shrimp seaweed root system. The present invention uses the adventitious root primordium of the red fiber shrimp seaweed as an explant and induces its normal growth and development through tissue culture technology, thereby realizing the in vitro culture of the red fiber shrimp seaweed root system, providing excellent material for its root physiology research.

[0017] The in vitro culture method of the red fiber shrimp seaweed root system of the present invention comprises the following steps:

[0018] Incubator culture: Healthy red fiber shrimp seaweed plants were collected in spring when the water temperature was around 10°C. They were washed with seawater and brought back to the laboratory. Healthy plants, retaining at least 10 nodes closest to the stem tip and 4 leaves at the stem tip, were placed in culture bottles filled with natural seawater and cultured in an incubator with a light intensity of 3000 lux, a light:dark cycle of 8 hours:16 hours, and a temperature of 10°C. After culturing for 3 days, the natural seawater was replaced with sterilized natural seawater and continued to be cultured in the incubator. The sterilized seawater was replaced daily, and the incubator temperature was increased by 1°C every 2 days until it reached 15°C. Temporarily culturing red fiber shrimp seaweed indoors can greatly reduce the contamination rate of the explant surface disinfection.

[0019] Initial selection of explants and first pruning: Healthy plants were selected after culturing in sterile seawater at 15°C for 7 days. The first pruning was performed on a clean bench. Leaves were removed to expose the stem tip, leaving only the four nodes and four internodes near the stem tip. Two clusters of newly emerged adventitious root primordia were present on both the first and second nodes near the stem tip.

[0020] Explant disinfection: Place the stem tip with four internodes obtained after the first pruning into a new sterilized culture bottle, rinse with sterilized natural seawater 3-5 times, dry it with sterilized filter paper, and then place it in a 10% sodium hypochlorite solution for disinfection for 15 minutes. Rinse it again with sterilized natural seawater 5 times. Use a vacuum filter to filter both the disinfection and rinsing process to ensure thorough disinfection.

[0021] Second explant pruning: Place the explant in sterilized natural seawater with 100 μM VC for the second pruning to prevent browning of the explant during pruning. Cut off the stem tip, leaving only two nodes and three internodes near the stem tip.

[0022] Root induction culture: The explants after the second pruning were inoculated into six-well culture plates containing induction medium and cultured at 15°C in the dark until root primordia germinated. The induction medium was liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 1 mg / L IBA, and 2 mg / L NAA. Using cell culture plates for culture can reduce the chance of explant cross-contamination, conserve culture medium, and facilitate observation of contamination and replacement of culture medium.

[0023] In vitro root culture: After root primordia germinate, they are transferred to subculture medium and cultured in six-well culture plates. When the roots grow to 5 mm, they are cut from the stem and cultured again. The subculture medium is liquid MS medium supplemented with 200 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA.

[0024] In order to more clearly and in detail introduce the in vitro culture method and application of the red fiber shrimp seaweed root system provided by the embodiments of the present invention, the following description will be made in conjunction with specific examples.

[0025] Example 1

[0026] The in vitro culture method of the red fiber shrimp seaweed root system of this embodiment includes the following steps:

[0027] Explant selection: Healthy red fiber shrimp seaweed plants were collected in spring when the water temperature was around 10°C, washed with seawater and brought back to the laboratory;

[0028] Incubator culture: Place healthy plants, retaining the 10 nodes closest to the stem tip and the four leaves at the stem tip, in a 1000 ml beaker filled with natural seawater. After culturing in a light incubator for 3 days, replace the natural seawater with sterilized natural seawater and continue culturing in the incubator. Raise the incubator temperature by 1°C every 2 days until it reaches 15°C, and replace the sterilized seawater daily. The culture conditions are: light intensity 3000 lux, light:dark = 8h:16h, temperature 10°C.

[0029] Initial selection of explants and first pruning: Healthy plants were selected after culturing in sterile seawater at 15°C for 7 days. The first pruning was performed on a clean bench. Leaves were removed to expose the stem tip, leaving only the four nodes and four internodes near the stem tip. Two clusters of newly emerged adventitious root primordia were present on both the first and second nodes.

[0030] Explant disinfection: Place the stem tip with four internodes after the first pruning into a new sterilized culture bottle, rinse with sterilized natural seawater five times, dry it with sterilized filter paper, and then sterilize it in a 10% sodium hypochlorite solution for 15 minutes. Rinse it again with sterilized natural seawater five times. Both the 10% sodium hypochlorite solution disinfection and the sterilized natural seawater rinse should be filtered with a vacuum filter.

[0031] Second pruning of explants: The explants were placed in sterilized natural seawater supplemented with 100 μM VC for the second pruning, and the stem tip was cut off, leaving only the two nodes and three internodes near the stem tip;

[0032] Root induction culture: Explants after the second pruning were inoculated into six-well culture plates containing induction medium and cultured at 15°C in the dark. Root primordia germinated after approximately 3 weeks, with a germination rate of 31.2%. The induction medium was liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 1 mg / L IBA, and 2 mg / L NAA.

[0033] In vitro root culture: After the root primordium germinates, it is transferred to the subculture medium and continued to be cultured in a six-well culture plate. When the root grows to 5 mm, the root is cut from the stem and cultured. After 60 days, the root can grow to 20.5 mm. The subculture medium is a liquid MS medium supplemented with 200 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA.

[0034] Comparative Example 1

[0035] The difference from Example 1 is that the frequency of replacing the sterilized seawater in the incubator cultivation step of this comparative example is to replace the sterilized seawater every 2 days. Specifically, the 10 nodes closest to the stem tip and the 4 leaves at the stem tip of the healthy plant are retained and placed in a 1000 ml beaker filled with natural seawater. After culturing in a light incubator for 3 days, the natural seawater is replaced with sterilized natural seawater and continued to be cultured in the incubator. The incubator temperature is increased by 1°C every 2 days to 15°C, and the sterilized seawater is replaced every 2 days. The culture conditions are: light intensity 3000 lux, light: dark = 8h:16h, and temperature 10°C.

[0036] Comparative Example 2

[0037] The difference from Example 1 is that the frequency of replacing the sterilized seawater in the incubator cultivation step of this comparative example is to replace the sterilized seawater every 3 days. Specifically, the 10 nodes closest to the stem tip and the 4 leaves at the stem tip of the healthy plant are retained and placed in a 1000 ml beaker filled with natural seawater. After culturing in a light incubator for 3 days, the natural seawater is replaced with sterilized natural seawater and continued to be cultured in the incubator. The incubator temperature is increased by 1°C every 2 days to 15°C, and the sterilized seawater is replaced every 3 days. The culture conditions are: light intensity 3000 lux, light: dark = 8h:16h, and temperature 10°C.

[0038] Comparative Example 3

[0039] The difference from Example 1 is that in this comparison, in the explant disinfection step, no vacuum filter was used for filtration during the disinfection in 10% sodium hypochlorite solution and the rinsing with sterile natural seawater. Specifically, the stem tip with four internodes after the first pruning was placed in a new sterilized culture bottle, rinsed with sterile natural seawater five times, dried with sterile filter paper, and then placed in a 10% sodium hypochlorite solution for disinfection for 15 minutes, and then rinsed with sterile natural seawater five times.

[0040] Comparative Example 4

[0041] The difference from Example 1 is that in the second pruning step of the explants in this comparison, the explants were placed in sterilized natural seawater with 0 VC added for the second pruning, and the stem tips were cut off, leaving only 2 nodes and 3 internodes near the stem tips.

[0042] Comparative Example 5

[0043] The difference from Example 1 is that in the second trimming step of the explants in this control, the explants were placed in 50 μM VC sterilized natural seawater for the second trimming, and the stem tip was cut off, leaving only two nodes and three internodes near the stem tip.

[0044] Comparative Example 6

[0045] The difference from Example 1 is that the induction medium in the root induction culture step of this comparative example is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 5 mg / L NAA. Specifically, the explants after the second pruning were inoculated into a six-well culture plate containing an induction medium and cultured at 15 ° C. in the dark. After about 3 weeks, the root primordia germinated, and the germination rate was 0. The induction medium was a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 5 mg / L NAA.

[0046] Comparative Example 7

[0047] The difference from Example 1 is that the induction medium in the root induction culture step of this comparative example is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA. Specifically, the explants after the second pruning were inoculated in a six-well culture plate containing an induction medium and cultured at 15 ° C. in the dark. After about 3 weeks, the root primordia germinated with a germination rate of 3.6%, wherein the induction medium was a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA.

[0048] Comparative Example 8

[0049] The difference from Example 1 is that the induction medium in the root induction culture step of this comparative example is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 0.5 mg / L NAA. Specifically, the explants after the second pruning were inoculated into a six-well culture plate containing an induction medium and cultured at 15 ° C. in the dark. After about 3 weeks, the root primordia germinated with a germination rate of 0.9%, wherein the induction medium was a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, and 0.5 mg / L NAA.

[0050] Comparative Example 9

[0051] The difference from Example 1 is that the induction medium in the root induction culture step of this comparative example is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 0.2 mg / L IBA, and 2 mg / L NAA. Specifically, the explants after the second pruning were inoculated in a six-well culture plate containing an induction medium and cultured at 15 ° C in the dark. After about 3 weeks, the root primordia germinated with a germination rate of 8.3%, wherein the induction medium is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 0.2 mg / L IBA, and 2 mg / L NAA.

[0052] Comparative Example 10

[0053] The difference from Example 1 is that the induction medium in the root induction culture step of this comparative example is a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 2 mg / L IBA, and 2 mg / L NAA. Specifically, the explants after the second pruning were inoculated in a six-well culture plate containing an induction medium and cultured at 15 ° C in the dark. After about 3 weeks, the root primordia germinated with a germination rate of 16.4%, wherein the induction medium was a liquid MS medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 2 mg / L IBA, and 2 mg / L NAA.

[0054] 30 healthy explants were inoculated in Example 1 and Comparative Examples 1-2, respectively, and cultured in an incubator. The contamination rates after one week of culture are shown in Table 1.

[0055] Table 1 Contamination rate of explants after 1 week of culture in the incubator of Example 1 and Comparative Examples 1-2

[0056]

[0057] 30 explants were respectively selected from Example 1 and Comparative Example 3 for disinfection treatment, and the contamination rates are shown in Table 2.

[0058] Table 2 Contamination rate of explants after disinfection in Example 1 and Comparative Example 3

[0059]

[0060] 25 explants were respectively selected from Example 1 and Comparative Examples 4-5 for the second pruning. The browning rates after pruning are shown in Table 3.

[0061] Table 3 Browning rate of explants after the second pruning of Example 1 and Comparative Examples 4-5

[0062]

[0063] After the explants of Example 1 and Comparative Examples 6-10 were subjected to root induction culture, the induction rates were shown in Table 3.

[0064] Table 4 Root induction culture induction rate of Example 1 and Comparative Examples 6-10

[0065]

[0066] As can be seen from the above, compared with Example 1, the frequency of replacing sterilized seawater in the incubator culturing step in Comparative Examples 1-2 is lower than that in Example 1, and the contamination rate of the explants is significantly higher than that in Example 1; compared with Example 1, no vacuum filter is used for filtration during disinfection and rinsing in Comparative Example 3, and the contamination rate of the explants is significantly higher than that in Example 1; compared with Example 1, in the second explant pruning step, the sterilized seawater conditions of the explants in Comparative Examples 4-5 are different, the amount of VC added in the sterilized seawater in Comparative Example 4 is 0, and the amount of VC added in the sterilized natural seawater in Comparative Example 5 is 50 μM, both of which are lower than that in Example 1, and the browning rate of the explants is significantly higher than that in Example 1; compared with Example 1, the IBA and NAA in the culture medium in Comparative Examples 6-10 are different, and the root induction rate is significantly lower than that in Example 1. In summary, Example 1 used the culture method of the present invention to culture the roots of red fiber shrimp seaweed in vitro, which greatly reduced the contamination rate and browning rate of the explants. The germination rate after root induction culture reached 31.2%, and the roots after in vitro root culture could grow to 20.5 mm, providing excellent material for the study of the root physiology of red fiber shrimp seaweed.

[0067] Finally, it should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only the better feasible embodiments of the present invention. The above-mentioned embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, evolutions and improvements made by ordinary technicians in this field to the technical solutions of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for in vitro culture of red fiber shrimp seaweed roots, characterized in that: The method comprises the following steps: collecting healthy red fiber shrimp seaweed plants as explants, selecting healthy plants after culturing in an incubator, performing a first pruning, disinfecting, and a second pruning, cutting off the stem tip of the explants to leave only two nodes and three internodes near the stem tip, inoculating the plants into a culture plate filled with an induction culture medium, performing root induction culture at 15°C in the dark until root primordia germinate, and then transferring the plants to a culture plate filled with a subculture culture medium for in vitro root culture, wherein the induction culture medium is a liquid MS culture medium supplemented with 1000 mg / L hydrolyzed casein, 30 g / L sucrose, 1 mg / L IBA, and 2 mg / L NAA.

2. The in vitro culture method of red fiber shrimp seaweed root system according to claim 1, characterized in that: The incubator culture conditions are as follows: after retaining at least 10 nodes closest to the stem tip and 4 leaves at the stem tip, the explant is placed in a culture bottle filled with natural seawater and cultured in an incubator with a light intensity of 3000 lux, light: dark = 8h:16h, and a temperature of 10°C. After culturing for 3 days, the natural seawater is replaced with sterilized natural seawater and continued to be cultured in the incubator. The sterilized seawater is replaced daily, and the incubator temperature is raised by 1°C every 2 days until the incubator temperature reaches 15°C, and the culture is continued for 7 days.

3. The in vitro culture method of red fiber shrimp seaweed root system according to claim 1, characterized in that: The first pruning step includes the following steps: plants that are still healthy after being cultured in an incubator are selected, and the first pruning is performed on a clean workbench, the leaves are peeled off to expose the stem tip, and only 4 nodes and 4 internodes near the stem tip are left, and there are two clusters of newly emerged adventitious root primordia on the first and second nodes near the stem tip.

4. The in vitro culture method of red fiber shrimp seaweed root system according to claim 1, characterized in that: The disinfection step comprises the following steps: placing the stem tip obtained after the first pruning into a new sterilized culture bottle, rinsing it with sterilized natural seawater for 3-5 times, drying the surface moisture of the stem tip with sterilized filter paper, placing it in a 10% sodium hypochlorite solution for disinfection for 15 minutes, taking it out, and then rinsing it with sterilized natural seawater for 5 times; wherein, the rinsing step with sterilized natural seawater and the disinfection step with the 10% sodium hypochlorite solution are both carried out under vacuum filtration conditions.

5. The in vitro culture method of red fiber shrimp seaweed roots according to claim 1, characterized in that: The second trimming step was performed by placing the explants in sterile natural seawater supplemented with 100 μM VC.

6. The in vitro culture method of red fiber shrimp seaweed root system according to claim 1, characterized in that: The in vitro root culture step includes the following steps: after the root primordium germinates, it is transferred to a six-well culture plate filled with subculture medium for culture; when the root grows to 5 mm, the root is cut off from the stem and cultured further.

7. The in vitro culture method of red fiber shrimp seaweed roots according to claim 1, characterized in that: The secondary culture medium was liquid MS medium supplemented with 200 mg / L hydrolyzed casein, 30 g / L sucrose, and 2 mg / L NAA.

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