Tissue culture method for botrychium ternatum spore induced prothallium

Abstract

The invention discloses a tissue culture method for botrychium ternatum spore induced prothallium. The culture method comprises the following steps: collecting botrychium ternatum with sporangium, drying, sieving and collecting spores, sequentially carrying out multiple times of disinfection and cleaning pretreatment on the collected spores by using 75% ethanol, sterile water, 2% sodium hypochlorite and the like in a sterile environment, uniformly coating a culture medium with treated spore suspension, and carrying out observation culture, the time from spore inoculation to prothallium formation is 24-26 days, and the time from spore inoculation to juvenile sporophyte formation is 47-63 days. The culture medium consists of the following components: a 1 / 8 MS culture medium, 5g / L of sucrose and 8g / L of agar. The culture medium is simple to prepare, can effectively induce spore germination and prothallus proliferation of the botrychium ternatum, can be further cultured to obtain sporophytes, can also be used for acclimatization and transplantation, shortens the seedling raising period, further realizes large-scale cultivation of the botrychium ternatum, and has important significance for expanded application of pteridophyte.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for inducing prothallus tissue culture from spores of *Pteris vittata*. Background Technology

[0002] *Sceptridium ternatum* (Thunb.) Lyon, also known as Little Spring Flower, One Cloud, Back Snake, Blood-Scattering Leaf, Sky-Breaking Cloud, and Snake-Not-Seeing, is a perennial herb belonging to the genus *Sceptridium* in the family Sceptridaceae. The whole plant is used medicinally and is a commonly used wild herb among ethnic minorities in southwestern and southern my country. As a folk medicine, *Sceptridium ternatum* is used by several ethnic minorities, including the Bai and Yi. Similarly, it plays an important role in traditional Chinese medicine. It enters the liver and lung meridians, is slightly cold in nature, and has a sweet and bitter taste. It can clear heat and detoxify, calm the liver and extinguish wind, stop cough, stop bleeding, and improve eyesight. Clinically, it is often used for infantile febrile convulsions, lung heat cough, and red eyes with corneal opacity, possessing extremely high medicinal development value. Currently, the resources of *Fernonia shadiola* are relatively scarce. Therefore, actively carrying out tissue culture of *Fernonia shadiola* can not only address the shortage of resources, but also enhance the application value of traditional Chinese medicine and contribute to the promotion of traditional Chinese medicine. It can also accelerate the large-scale cultivation of *Fernonia shadiola* and provide a basic guarantee for the development of related medicines and foods.

[0003] Currently, *Adiantum capillus-veneris* mainly reproduces naturally, or by collecting spores from December to January of the following year, processing them, and then sowing them in a seedbed. Once the seedlings reach 3cm in height, they can be transplanted. However, traditional propagation has high environmental requirements, and the spores take approximately 250-300 days to germinate, followed by 20-30 days for them to grow into seedlings. Therefore, spore germination is the most time-consuming part of the entire cultivation process. Environmentally, it also requires specific temperature and humidity control, making management cumbersome, and the germination rate is relatively low. Therefore, it is necessary to provide a method for cultivating *Adiantum capillus-veneris* that can shorten the germination time and improve the germination rate. Summary of the Invention

[0004] The purpose of this invention is to provide a tissue culture method for inducing prothallus formation using spores of *Adiantum capillus-veneris*. Using tissue culture for spore germination not only shortens the germination time but also significantly increases the germination rate. Based on this, off-season cultivation and selective cultivation can be carried out, thereby achieving large-scale cultivation. Not only is there no time constraint, but the quality of the resulting seedlings is also greatly improved.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A tissue culture method for inducing prothallus from spores of *Dryopteris shadiola* includes the following steps: Step 1: Collect spores. Place the leaves of the shady fern with sporangia into a sulfuric acid paper bag, mark it with a code, and place it in a well-ventilated and dry place until the spores fall off freely. Step 2: Sterilize the DNA purification column (specifications: 1ml adsorption column, 2ml collection tube), 1ml pipette tip, and ultrapure water at 121℃ for 20-30 minutes for later use. Step 3: Place lens cleaning paper on the folded sulfuric acid paper, pour the spores that have fallen freely from the sulfuric acid paper bag onto the lens cleaning paper, and gently shake the lens cleaning paper for simple filtration. The spores can pass through the pores of the lens cleaning paper, while impurities such as broken leaves, rings, and scales will remain on the lens cleaning paper, thus achieving the purpose of filtration. Step 4: On a sterile laminar flow hood, add 400 μl of 75% ethanol to the filter column using a 1 ml pipette, sterilize for 30 seconds, invert the purification column twice, place the filter column in a centrifuge and centrifuge at 1500 rpm for 1 min, remove the filter column and discard the waste liquid in the collection tube. Step 5: Place the filter column on the centrifuge tube rack, pour the filtered spores along the folds of the sulfuric acid paper into the sterilized adsorption column, add 400 μl of 75% ethanol, repeatedly aspirate the solution to suspend the spores for preliminary sterilization, and then centrifuge at 1500 rpm for 1 min. Step 6: Remove the filter column, discard the waste liquid, place the filter column on a centrifuge rack, add 400 μl of sterile water using a pipette, repeatedly aspirate and shake the solution to thoroughly wash the spores, centrifuge at 1500 rpm for 1 min, remove the filter column again, discard the waste liquid, and place the filter column on a centrifuge rack. Repeat at least 2 times; Step 7: Add 400 μl of 2% sodium hypochlorite solution to the pipette for 8 min to sterilize. Repeatedly aspirate and aspirate the solution to ensure complete sterilization of the spores. Then centrifuge at 1500 rpm for 1 min. Step 8: Remove the filter column and discard the waste liquid. Place the filter column on the centrifuge rack, open the filter column, add 400 μl of sterile water with a pipette, and repeatedly aspirate and suspend the spores. Rinse the spores that have been disinfected with 2% sodium hypochlorite solution thoroughly, and then centrifuge at 1500 rpm for 1 min. Repeat at least 3 times. Step 9: Use a 1ml pipette to add 600μl of sterile water to the adsorption column, repeatedly pipette to make it into a spore suspension, and then evenly inoculate the spore suspension onto the surface of the culture medium. Step 10: After inoculation, the culture medium was transferred to a dark room at 25°C and cultured in the dark for 3 days. Then it was transferred to a light-dark alternating environment for culture, with a light culture time of 16 hours / day and a dark culture time of 8 hours / day. The light intensity was 2000-2500 Lx. The spore germination was observed.

[0006] The sulfuric acid paper bags marked with the code described in this invention can also be placed in a 30°C oven to allow the spores to fall off freely.

[0007] The 2% sodium hypochlorite solution described in this invention is 10% sodium hypochlorite: sterile water = 1:4, W / W.

[0008] The culture medium described in this invention is a solid culture medium.

[0009] The solid culture medium described in this invention includes a culture medium based on 1 / 8MS medium, and further includes: sucrose 0-20 g / L and agar 8 g / L.

[0010] The 1 / 8MS culture medium described in this invention can also be any one of MS, 1 / 2MS, or 1 / 4MS culture medium.

[0011] Preferably, the sucrose concentration is 5 g / L and the agar concentration is 8 g / L in this invention.

[0012] The time from spore inoculation to the formation of prothallus in this invention is 24-26 days, and the time from spore inoculation to the formation of young sporophyte is 47-63 days.

[0013] The beneficial effects of this invention are: 1. Compared with traditional propagation, the propagation of *Ferns shadiola* in this invention uses tissue culture for spore germination, which not only shortens the germination time but also greatly improves the germination rate.

[0014] 2. Compared with traditional culture media, the culture medium used in this invention has a simple composition. An investigation of the composition of the culture medium revealed that the optimal ratio is 1 / 8 MS medium, 5 g / L sucrose, and 8 g / L agar. The prepared culture medium is stable and exhibits the best results. Furthermore, the spore germination time is short, the germination efficiency is high, and the resulting prothallus can further develop into sporophyte seedlings. After hardening and transplanting, shady fern plants can be obtained.

[0015] 3. Under the optimal culture medium ratio of this invention (1 / 8 MS medium, sucrose concentration of 5 g / L, agar concentration of 8 g / L) and the optimal disinfection time (8 min) for spore germination using 2% sodium hypochlorite solution, the spore contamination rate of *Adiantum capillus-veneris* was the lowest at 2.46%, and the prothallus induction rate was the highest at 93.44%. The time from spore inoculation to prothallus formation was 24–26 days, and the time from spore inoculation to sporophyte formation was 47–63 days.

[0016] 4. The tissue culture method of the present invention can be used for off-season cultivation, selective cultivation, and large-scale cultivation. It is not only unrestricted by time, but also greatly improves the quality of the seedlings obtained. Attached Figure Description

[0017] Figure 1 Examples 1-5: Prothallophyte of *Pteridium shadiola* (from left to right: Example 1, Example 2, Example 3, Example 4, Example 5). Detailed Implementation

[0018] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0019] Example 1: A tissue culture method for inducing prothallus from *Dryopteris shadiola* spores, the steps of which are as follows: Step 1: Collect spores. Place the leaves of the shady fern with sporangia into a sulfuric acid paper bag, mark it with a code, and place it in a ventilated and dry place or in a 30℃ oven until the spores fall off freely. Step 2: Sterilize the DNA purification column (specifications: 1ml adsorption column, 2ml collection tube), 1ml pipette tip, and ultrapure water at 121℃ for 20-30 minutes for later use. Step 3: Place lens cleaning paper on the folded sulfuric acid paper, pour the spores that have fallen freely from the sulfuric acid paper bag onto the lens cleaning paper, and gently shake the lens cleaning paper for simple filtration. The spores can pass through the pores of the lens cleaning paper, while impurities such as broken leaves, rings, and scales will remain on the lens cleaning paper, thus achieving the purpose of filtration. Step 4: On a sterile laminar flow hood, add 400 μl of 75% ethanol to the filter column using a 1 ml pipette, sterilize for 30 seconds, invert the purification column twice, place the filter column in a centrifuge at 1500 rpm for 1 min, remove the filter column, and discard the waste liquid in the collection tube. Step 5: Place the filter column on the centrifuge tube rack, pour the filtered spores along the folds of the sulfuric acid paper into the sterilized adsorption column, add 400 μl of 75% ethanol, repeatedly aspirate the solution to suspend the spores for preliminary sterilization, and then centrifuge at 1500 rpm for 1 min. Step 6: Remove the filter column, discard the waste liquid, place the filter column on a centrifuge rack, add 400 μl of sterile water using a pipette, repeatedly aspirate and shake the solution to thoroughly wash the spores, centrifuge at 1500 rpm for 1 min, remove the filter column again, discard the waste liquid, and place the filter column on a centrifuge rack. Repeat at least 2 times; Step 7: Add 400 μl of 2% sodium hypochlorite solution to the pipette for 8 min to sterilize. Repeatedly aspirate and aspirate the solution to ensure complete sterilization of the spores. Then centrifuge at 1500 rpm for 1 min. Step 8: Remove the filter column and discard the waste liquid. Place the filter column on the centrifuge rack, open the filter column, add 400 μl of sterile water with a pipette, and repeatedly aspirate and suspend the spores. Rinse the spores that have been disinfected with 2% sodium hypochlorite solution thoroughly, and then centrifuge at 1500 rpm for 1 min. Repeat at least 3 times. Step 9: Use a 1ml pipette to add 600μl of sterile water to the adsorption column, repeatedly pipette to make it into a spore suspension, and then evenly inoculate the spore suspension onto the surface of the culture medium. Step 10: After inoculation, the culture medium was transferred to a dark room at 25°C and cultured in the dark for 3 days. Then it was transferred to a light-dark alternating environment for culture, with a light culture time of 16 hours / day and a dark culture time of 8 hours / day. The light intensity was 2000-2500 Lx. The spore germination was observed.

[0020] Culture medium preparation: 1 / 8MS medium, sucrose: 0 g / L, agar 8 g / L.

[0021] The results are shown in Table 1. Figure 1 In this embodiment, the spores remained unchanged.

[0022] Example 2: A tissue culture method for inducing prothallus from *Dryopteris shadiola* spores Culture medium preparation: 1 / 8MS medium, sucrose: 5g / L, agar: 8g / L.

[0023] The remaining steps are the same as in Example 1.

[0024] The results are shown in Table 1. Figure 1 In this embodiment, the spore contamination rate was 2.46%, and the protothallus induction rate was 93.44%. The time from spore inoculation to protothallus formation was 24–26 days, and the time from spore inoculation to sporophyte formation was 47–63 days.

[0025] Example 3: A tissue culture method for inducing prothallus from *Dryopteris shadiola* spores Culture medium preparation: 1 / 8MS medium, sucrose: 10g / L, agar: 8g / L.

[0026] The remaining steps are the same as in Example 1.

[0027] The results are shown in Table 1. Figure 1 In this embodiment, the spore contamination rate was 5.38%, and the protothallus induction rate was 89.24%. The time from spore inoculation to protothallus formation was 24–26 days, and the time from spore inoculation to sporophyte formation was 50–67 days.

[0028] Example 4: A tissue culture method for inducing prothallus from *Dryopteris shadiola* spores Culture medium preparation: 1 / 8MS medium, sucrose: 15g / L, agar: 8g / L.

[0029] The remaining steps are the same as in Example 1.

[0030] The results are shown in Table 1. Figure 1 In this embodiment, the spore contamination rate was 5.94%, and the prothallus induction rate was 85.15%. The time from spore inoculation to prothallus formation was 26–32 days, and the time from spore inoculation to sporophyte formation was 45–61 days.

[0031] Example 5: A tissue culture method for inducing prothallus from *Dryopteris shadiola* spores Culture medium preparation: 1 / 8MS medium, sucrose: 20g / L, agar: 8g / L.

[0032] The remaining steps are the same as in Example 1.

[0033] The results are shown in Table 1. Figure 1 In this embodiment, the spore contamination rate was 5.45%, and the prothallus induction rate was 84.55%. The time from spore inoculation to prothallus formation was 24–31 days, and the time from spore inoculation to sporophyte formation was 43–65 days.

[0034] Spore contamination rate = (Number of contaminated germinated spores / Total number of germinated spores) * 100%; Prothallus induction rate = number of prothallus / total number of germinating spores * 100%.

[0035] Experiment 1: Investigating the germination of spores of *Pteris vittata* under different treatments. Tissue culture method for inducing prothallus from spores of *Pteris vittata*: Step 1: Collect spores. Place the leaves of the shady fern with sporangia into a sulfuric acid paper bag, mark it with a code, and place it in a ventilated and dry place or in a 30℃ oven until the spores fall off freely. Step 2: Sterilize the DNA purification column (specifications: 1ml adsorption column, 2ml collection tube), 1ml pipette tip, and ultrapure water at 121℃ for 20-30 minutes for later use. Step 3: Place lens cleaning paper on the folded sulfuric acid paper, pour the spores that have fallen freely from the sulfuric acid paper bag onto the lens cleaning paper, and gently shake the lens cleaning paper for simple filtration. The spores can pass through the pores of the lens cleaning paper, while impurities such as broken leaves, rings, and scales will remain on the lens cleaning paper, thus achieving the purpose of filtration. Step 4: On a sterile laminar flow hood, add 400 μl of 75% ethanol to the filter column using a 1 ml pipette, sterilize for 30 seconds, invert the purification column twice, place the filter column in a centrifuge at 1500 rpm for 1 min, remove the filter column, and discard the waste liquid in the collection tube. Step 5: Place the filter column on the centrifuge tube rack, pour the filtered spores along the folds of the sulfuric acid paper into the sterilized adsorption column, add 400 μl of 75% ethanol, repeatedly aspirate the solution to suspend the spores for preliminary sterilization, and then centrifuge at 1500 rpm for 1 min. Step 6: Remove the filter column, discard the waste liquid, place the filter column on a centrifuge rack, add 400 μl of sterile water using a pipette, repeatedly aspirate and shake the solution to thoroughly wash the spores, centrifuge at 1500 rpm for 1 min, remove the filter column again, discard the waste liquid, and place the filter column on a centrifuge rack. Repeat at least 2 times; Step 7: Add 400 μl of 2% sodium hypochlorite solution to the pipette for 6 or 8 minutes to sterilize. Repeatedly aspirate and aspirate the solution to ensure complete sterilization of the spores. Then centrifuge at 1500 rpm for 1 minute. Step 8: Remove the filter column and discard the waste liquid. Place the filter column on the centrifuge rack, open the filter column, add 400 μl of sterile water with a pipette, and repeatedly aspirate and suspend the spores. Rinse the spores that have been disinfected with 2% sodium hypochlorite solution thoroughly, and then centrifuge at 1500 rpm for 1 min. Repeat at least 3 times. Step 9: Use a 1ml pipette to add 600μl of sterile water to the adsorption column, repeatedly pipette to make it into a spore suspension, and then evenly inoculate the spore suspension onto the surface of the culture medium. Step 10: After inoculation, the culture medium was transferred to a dark room at 25°C and cultured in the dark for 3 days. Then it was transferred to a light-dark alternating environment for culture, with a light culture time of 16 hours / day and a dark culture time of 8 hours / day. The light intensity was 2000-2500 Lx. The spore germination was observed.

[0036] This experiment investigated the effects of different disinfection times of 2% sodium hypochlorite solution and different culture medium treatments on the germination of *Adiantum capillus-veneris* spores, as detailed in Tables 2 and 3.

[0037] Note: \ indicates that it has not yet sprouted. Results: Comparison of various data showed that the optimal culture medium composition of this invention was 1 / 8 MS medium, sucrose concentration: 5 g / L, agar concentration: 8 g / L, and the optimal sterilization time for spore germination using 2% sodium hypochlorite solution was 8 min. The spore contamination rate was 2.46%, and the protothallus induction rate was 93.44%. The time from spore inoculation to protothallus formation was 24–26 days, and the time from spore inoculation to sporophyte formation was 47–63 days. Under these conditions, the tissue culture method for *Adiantum capillus-veneris* spores was most effective, with a short spore germination time and high germination efficiency.

[0038] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for inducing prothallus tissue culture from *Dryopteris shadiola* spores, characterized in that, Includes the following steps: Step 1: Collect spores. Place the leaves of the shady fern with sporangia into a sulfuric acid paper bag, mark it with a code, and place it in a well-ventilated and dry place until the spores fall off freely. Step 2: Sterilize the DNA purification column, 1ml pipette tip, and ultrapure water at 121℃ for 20-30 minutes for later use; Step 3: Place lens cleaning paper on the folded sulfuric acid paper, pour the spores that have fallen freely from the sulfuric acid paper bag onto the lens cleaning paper, and gently shake the lens cleaning paper for simple filtration; Step 4: On a sterile laminar flow hood, add 400 μl of 75% ethanol to the filter column using a 1 ml pipette, sterilize for 30 seconds, invert the purification column twice, place the filter column in a centrifuge and centrifuge at 1500 rpm for 1 min, remove the filter column and discard the waste liquid in the collection tube. Step 5: Place the filter column on the centrifuge tube rack, pour the filtered spores along the folds of the sulfuric acid paper into the sterilized adsorption column, add 400 μl of 75% ethanol, repeatedly aspirate the solution to suspend the spores for preliminary sterilization, and then centrifuge at 1500 rpm for 1 min. Step 6: Remove the filter column, discard the waste liquid, place the filter column on the centrifuge rack, add 400 μl of sterile water with a pipette, repeatedly aspirate and shake the solution to thoroughly rinse the spores, centrifuge at 1500 rpm for 1 min, remove the filter column again, discard the waste liquid, place the filter column on the centrifuge rack, repeat at least 2 times. Step 7: Add 400 μl of 2% sodium hypochlorite solution to the pipette for 8 min to sterilize. Repeatedly aspirate and aspirate the solution to ensure complete sterilization of the spores. Then centrifuge at 1500 rpm for 1 min. Step 8: Remove the filter column and discard the waste liquid. Place the filter column on the centrifuge rack, open the filter column, add 400 μl of sterile water with a pipette, and repeatedly aspirate and suspend the spores. Rinse the spores that have been disinfected with 2% sodium hypochlorite solution thoroughly, and then centrifuge at 1500 rpm for 1 min. Repeat at least 3 times. Step 9: Use a 1ml pipette to add 600μl of sterile water to the adsorption column, repeatedly pipette to make it into a spore suspension, and then evenly inoculate the spore suspension onto the surface of the culture medium. Step 10: Transfer the inoculated culture medium to a dark room at 25℃ for 3 days of dark culture, and then transfer it to a light-dark alternating environment for culture. The light culture time is 16h / day and the dark culture time is 8h / day. The light intensity is 2000~2500Lx. Observe the spore germination.

2. The tissue culture method according to claim 1, characterized in that, The sulfuric acid paper bags marked with the code can also be placed in a 30°C oven to allow the spores to fall off freely.

3. The tissue culture method according to claim 1, characterized in that, The 2% sodium hypochlorite solution is composed of 10% sodium hypochlorite: sterile water = 1:4, W / W.

4. The tissue culture method according to claim 1, characterized in that, The culture medium is a solid culture medium.

5. The tissue culture method according to claim 4, characterized in that, The solid culture medium includes a medium based on 1 / 8 MS medium, and also includes a sucrose concentration of 0-20 g / L and an agar concentration of 8 g / L.

6. The tissue culture method according to claim 5, characterized in that, The 1 / 8 MS medium can also be any one of MS, 1 / 2 MS, or 1 / 4 MS medium.

7. The tissue culture method according to claim 5, characterized in that, The sucrose concentration in the solid culture medium is 5 g / L.

8. The tissue culture method according to claim 1, characterized in that, The time from spore inoculation to the formation of prothallus is 24–26 days, and the time from spore inoculation to the formation of young sporophyte is 47–63 days.

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