A disinfection method for seeds of single capsule of Panax japonicus

Abstract

The present application relates to a kind of sterilization methods of the seed of single capsule of Panax japonicus after cracking, belong to Panax japonicus tissue culture technical field, by harvesting completely mature Panax japonicus capsule, after capsule natural cracking, seed is all collected in the adsorption column of centrifugal tube, make adsorption column as the container of seed surface sterilization, in centrifugal tube, in turn, sterile water washing, alcohol soaking, sodium hypochlorite solution soaking and again sterile water washing, then directly from the seed of adsorption column bottom and sowing in the culture bottle of the germination medium placed, the method is integrated by the use of adsorption column, so that seed is always constrained in the same container, after sterilization, seed is directly taken from the bottom of adsorption column and sowing, completely eliminate the seed loss caused by transfer between container, seed utilization reaches 100%.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology of *Cymbidium goeringii*, specifically relating to a method for disinfecting seeds after a single capsule of *Cymbidium goeringii* has cracked open. Background Technology

[0002] Plants in the genus *Pleione* possess both ornamental and medicinal value. This genus features a typical pseudobulb structure, with pseudobulbs containing various active ingredients such as phenanthrene, bibenzyl, and benzyl succinate glycosides. It is listed in the Chinese Pharmacopoeia as the source plant for "Shan Ci Gu" (a type of medicinal mushroom), and its pseudobulbs are known as "ice balls." Due to severe resource depletion, all species in the genus *Pleione* were listed as Class II National Key Protected Wild Plants in 2021. *Pleione* has a typical pseudobulb structure; its seeds are as fine as dust, lack endosperm, and have incomplete embryo development, resulting in an extremely low germination rate under natural conditions. Artificial propagation is necessary through natural pseudobulb division or tissue culture. However, natural pseudobulb division has a low propagation coefficient, only multiplying 1-2 times per year, resulting in slow reproduction. Tissue culture technology offers rapid propagation and is a mature technique, currently the main method for artificial propagation of *Pleione*.

[0003] In aseptic procedures for tissue culture, the seeds of *Gynostemma pentaphyllum* are so fine as to be like dust that direct surface sterilization is impossible. Currently, three methods are typically used to obtain sterile seeds:

[0004] The first method involves harvesting immature capsules, disinfecting the surface of the unopened capsules, and then cutting them open to extract the seeds. The key to this method lies in controlling the maturity of the capsules. Seeds inside immature capsules are not fully developed, resulting in a low germination rate after disinfection.

[0005] The second method involves harvesting the capsules after they are fully mature. However, fully mature capsules are extremely prone to cracking. Once fully mature capsules crack, the seeds are directly exposed to the external environment and become contaminated by microorganisms, making them unsuitable for aseptic tissue culture. Therefore, the seeds need to be wrapped in gauze or nylon mesh bags and then soaked for sterilization. After sterilization, the operator needs to remove the seeds from the gauze or mesh bag into an intermediate container, and then transfer the seeds from the intermediate container to the surface of the culture medium for sowing using a pipette or tweezers. During this transfer process, a large number of seeds adhere to the fiber gaps of the gauze or mesh bag and cannot be removed, while some seeds are lost with the airflow or liquid flow during the transfer by tapping or rinsing. Therefore, this method is only suitable for operations with a large quantity of seeds. However, in the breeding and hybridization of *Cymbidium goeringii*, researchers often need to propagate by seed from individual capsules of superior single plants or hybrid offspring. The total number of seeds is extremely limited. Due to the high seed transfer loss rate after treatment with gauze or mesh bags, the remaining seeds are insufficient to complete the sowing of the germination medium, thus making it impossible to carry out subsequent aseptic tissue culture. This defect seriously hinders the breeding process of superior individuals and hybrid offspring of *Cymbidium goeringii*.

[0006] The third method also involves thoroughly sterilizing the seeds after the capsules have fully matured and split open. This involves placing the seeds in a centrifuge tube for sterilization; after sterilization, the tube opening needs to be plugged with gauze or cotton to filter out the liquid, and then the seeds are transferred from the centrifuge tube to the surface of the culture medium for sowing using a pipette. During this process, a large number of seeds adhere to the gauze or cotton and the centrifuge tube walls and cannot be detached, resulting in significant seed loss that affects sowing. Simultaneously, the sterilizing liquid is difficult to completely remove, impacting seed germination after sowing. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the present invention provides a method for disinfecting the seeds after the individual capsules of *Gnaphalium affine* crack open, so as to achieve the complete utilization of the seeds of the individual cracked capsules.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] The disinfection method includes the following steps:

[0010] 1) Seed collection: Select fully mature capsules of *Gnaphalium affine* after artificial self-pollination or hybridization of superior single plants. After the capsules naturally split open, fold a clean piece of paper into a funnel shape and collect all the seeds in the capsules into the adsorption column of the adsorption column set.

[0011] 2) Preparation of germination medium: The medium consists of MS + 1.0 mg / L NAA + 1.0 mg / L 6-BA + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.6–5.8, sterilized for later use;

[0012] 3) Seed surface disinfection: The adsorption column set is used as a container for seed surface disinfection. The adsorption column is washed with sterile water, soaked in alcohol, soaked in sodium hypochlorite solution, and washed with sterile water again in sequence. After each step, the adsorption column set is placed in a centrifuge for high-speed centrifugation to remove the liquid. After disinfection, the seeds are concentrated at the bottom of the adsorption column, and the liquid is collected in the centrifuge tube.

[0013] 4) Integrated sowing: With the seeds inside the adsorption column, remove and open the adsorption column, take the seeds from the bottom of the adsorption column and sow them in a culture bottle containing germination medium. Then place the culture bottle in a tissue culture room and culture until the seeds germinate and differentiate into leaves.

[0014] Furthermore, in step 1), the adsorption column assembly consists of a 1.5 mL centrifuge tube and an adsorption column containing a filter membrane.

[0015] Furthermore, in step 2), the sterilization temperature is 121°C and the sterilization time is 15 minutes.

[0016] Further, in step 3), the method of washing with sterile water is as follows: add 500 μL of sterile water to the adsorption column, place it in a shaker and shake for 1 minute, then place it in a centrifuge and centrifuge at 10000-12000 rpm at room temperature for 1 minute, remove the adsorption column, pour out the waste liquid in the centrifuge tube, and then put the adsorption column back into the centrifuge tube.

[0017] Further, in step 3), the alcohol soaking method is as follows: add 500 μL of 75% alcohol to the adsorption column, shake it in a shaker for 5 seconds, then place it in a centrifuge and centrifuge at 10,000 rpm for 30 seconds at room temperature. Remove the adsorption column, pour out the waste liquid in the centrifuge tube, and then put the adsorption column back into the centrifuge tube.

[0018] Further, in step 3), the method of soaking in sodium hypochlorite solution is as follows: add 500 μL of sodium hypochlorite solution with a volume fraction of 5% to the adsorption column, place it in a shaker and shake for 30 seconds, let it stand at room temperature for 6 minutes, inverting it several times every 30 seconds during this period, and then place it in a centrifuge and centrifuge at 10,000 rpm for 1 minute at room temperature. Remove the adsorption column, pour out the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube.

[0019] Furthermore, in step 4), the specific operation of the integrated seeding is as follows: remove the adsorption column from the centrifuge tube, keep the adsorption column cap closed, spray the outer surface of the adsorption column with 75% alcohol by volume, place it in a pre-sterilized clean bench, open the adsorption column cap, use tweezers to directly pick up the seeds from the bottom of the adsorption column and sow them in the culture bottle, sowing 100-120 seeds in each bottle, and gently shake the culture bottle after sowing to spread the seeds on the surface of the culture medium.

[0020] Furthermore, in step 4), the cultivation conditions are: temperature 25±1℃, relative humidity 40%–50%, light intensity 1800–2000 lx, light 12 h / dark 12 h, and cultivation for 60 days.

[0021] Furthermore, in step 4), after the seeds germinate and differentiate into leaves, they are transferred to a rooting medium for rooting culture.

[0022] This invention addresses the challenges of *Gnaphalium affine* seeds, which are as fine as dust and produce very few seeds per individual dehiscent capsule. It proposes an integrated sterilization and sowing container solution. Seeds undergo complete surface sterilization within an adsorption column. After sterilization, there's no need to transfer seeds from the column to other containers; instead, the column itself serves as the sowing container. Seeds are picked up from the bottom of the column and sown onto the germination medium surface. Simultaneously, high-speed centrifugation ensures thorough removal of the sterilizing solution, concentrating all seeds at the bottom of the column and preventing them from adhering to the column walls. Compared to existing methods using gauze or mesh bags and centrifuge tubes, where seed sterilization requires filtration to remove liquid, seeds may adhere to gauze fibers, be lost with the liquid flow, or adhere to the tube walls, resulting in a seed utilization rate of less than 10%. This method, through the integrated use of the adsorption column and high-speed centrifugation, allows for direct sowing from the bottom of the column after sterilization, completely eliminating seed loss caused by container transfer and filtration, achieving 100% seed utilization.

[0023] Furthermore, this invention incorporates both shaking and high-speed centrifugation at each disinfection and cleaning step within the adsorption column. Shaking ensures full contact between the disinfectant and the seeds, while high-speed centrifugation rapidly and thoroughly removes liquid, preventing any residual liquid from affecting seed viability. After disinfection, the seeds concentrate at the bottom of the adsorption column under centrifugal force, preventing seed loss due to adhering to the column walls. This facilitates precise sowing onto the germination medium surface using tweezers, further improving sowing efficiency and operational controllability. Attached Figure Description

[0024] Figure 1 A photograph showing the morphology of a single-flowered garlic orchid seed.

[0025] Figure 2 This is a photograph of the germination of *Hylocereus undatus* seeds using the disinfection method employed in this invention.

[0026] Figure 3 Photograph of the contaminated seeds of *Gnaphalium affine* (comparative example 1).

[0027] Figure 4 Photographs of the germination of *Gynostemma pentaphyllum* seeds (Comparative Example 2).

[0028] Figure 5 This is a photograph of the germination of *Gynostemma pentaphyllum* seeds, Comparative Example 3.

[0029] Figure 6 Photograph of the *Gnaphalium affine* seeds remaining on the gauze bag for comparison example 4. Example

[0030] 1) Seed collection: Select fully mature capsules of *Gnaphalium affine* after artificial self-pollination of superior single plants. After the capsules naturally split open, fold a clean piece of paper into a funnel shape and collect all the seeds in the capsules into the adsorption column of a 1.5 mL adsorption column kit to prevent the seeds from spilling.

[0031] 2) Preparation of germination medium: Prepare the medium according to the following composition: MS basal medium + 1.0 mg / L NAA + 1.0 mg / L 6-BA + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon. Adjust the pH to 5.6–5.8, sterilize at 121℃ for 15 minutes, and cool for later use after sterilization.

[0032] 3) Seed surface disinfection:

[0033] Sterile water washing: Add 500 μL of sterile water to the adsorption column, shake on a shaker for 1 minute, then place the adsorption column in a centrifuge and centrifuge at 10,000 rpm at room temperature for 1 minute. Remove the adsorption column, pour out the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube.

[0034] Alcohol soaking: Add 500 μL of 75% alcohol to the adsorption column, shake in a shaker for 5 seconds, centrifuge at 10,000 rpm for 30 seconds at room temperature, remove the adsorption column, pour out the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube.

[0035] Soaking in sodium hypochlorite solution: Add 500 μL of 5% sodium hypochlorite solution to the adsorption column, shake in a shaker for 30 seconds, and let stand at room temperature for 6 minutes. During this period, invert the adsorption column several times every 30 seconds to ensure that the seeds are in full contact with the disinfectant. After standing, place it in a centrifuge and centrifuge at 10,000 rpm for 1 minute at room temperature. Remove the adsorption column, pour out the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube.

[0036] Sterile water rinsing: Add 500 μL of sterile water to the adsorption column, shake for 30 seconds, place in a centrifuge, centrifuge at 12000 rpm for 1 minute at room temperature, remove the adsorption column, discard the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube. After sterilization, the seeds concentrate at the bottom of the adsorption column under the action of centrifugal force.

[0037] 4) Integrated sowing: With the seeds inside the adsorption column, remove the centrifuge tube from the centrifuge, keeping the adsorption column cap closed. Spray the outer surface of the adsorption column with 75% alcohol, place it in a pre-sterilized clean bench, open the adsorption column cap, and use tweezers to directly pick up a small amount of seeds from the bottom of the adsorption column and sow them on the surface of the culture bottle containing the germination medium described in step (2). During the operation, prevent the tweezers from touching the outer wall of the adsorption column. Sow 100–120 seeds per bottle. After sowing, gently shake the culture bottle to spread the seeds evenly on the surface of the germination medium. Place the culture bottle in a tissue culture room and culture it for 60 days under the conditions of temperature 25±1℃, relative humidity 40%–50%, light intensity 1800–2000 lx, and light 12 h / dark 12 h until the seeds germinate and differentiate into leaves.

[0038] Comparative Example 1, Soaking in sodium hypochlorite solution for 4 minutes: The operation was carried out according to steps 1) to 4) of Example 1, except that the room temperature standing time of the sodium hypochlorite solution soaking in step 3) was shortened from 6 minutes to 4 minutes, and the other operating parameters were exactly the same as those in Example 1.

[0039] Comparative Example 2, Soaking in sodium hypochlorite solution for 8 minutes: The operation was carried out according to steps 1) to 4) of Example 1, except that the room temperature standing time for soaking in sodium hypochlorite solution in step 3) was extended from 6 minutes to 8 minutes, and the other operating parameters were exactly the same as those in Example 1.

[0040] Comparative Example 3, Soaking in sodium hypochlorite solution for 10 minutes: The operation was carried out according to steps 1) to 4) of Example 1, except that the room temperature standing time for soaking in sodium hypochlorite solution in step 3) was extended from 6 minutes to 10 minutes, and the other operating parameters were exactly the same as those in Example 1.

[0041] Comparative Example 4, Gauze Mesh Bag Sterilization Method: The procedure was performed according to steps 1) and 2) of Example 1. Step 3) involved placing the seeds from the dehisced capsules in a gauze mesh bag, tying the bag tightly, gently rinsing it under running water for 1 minute, then transferring the mesh bag to a 75% (v / v) alcohol solution for 30 seconds, followed by a 5% (v / v) sodium hypochlorite solution for 6 minutes. Step 4) involved removing the sterilized mesh bag from the sterilization solution, opening the bag in a clean bench, and attempting to transfer the seeds adhering to the mesh bag fibers to the surface of the germination medium for sowing.

[0042] The seed treatment results for each embodiment and comparative example are shown in the table below (20 bottles were sown in each group of experiments):

[0043]

[0044] Seed utilization rate refers to the proportion of seeds that can actually be sown on germination medium after surface disinfection; germination rate refers to the proportion of seeds that germinate on germination medium; contamination rate refers to the proportion of seeds that become contaminated one week after sowing on germination medium.

[0045] The experimental results above show that, using the integrated disinfection and sowing method with adsorption column described in this invention, under the condition of soaking in sodium hypochlorite solution for 6 minutes, the seed utilization rate of a single dehiscent capsule of *Gynostemma pentaphyllum* reaches 100%, the germination rate is 73±9%, and the contamination rate is 10%. Since the seeds are always confined within the adsorption column, there is no need to transfer between containers throughout the entire process from disinfection to sowing, completely eliminating the risk of seed loss and achieving full utilization of the seeds from a single dehiscent capsule.

[0046] Comparative Example 1 shortened the sodium hypochlorite solution soaking time to 4 minutes. Although the seed utilization rate was still 100%, due to insufficient disinfection time and incomplete sterilization, the contamination rate reached 100% one week after sowing, and all seeds lost their germination ability. This indicates that a sodium hypochlorite solution soaking time of less than 6 minutes cannot meet the surface disinfection requirements of the cracked seeds of *Gynostemma pentaphyllum*.

[0047] Comparative Examples 2 and 3, which extended the sodium hypochlorite solution soaking time to 8 minutes and 10 minutes respectively, showed a significant decrease in contamination rate (5% and 0%, respectively), but a substantial drop in germination rate (32±11% and 7±6%, respectively). Furthermore, the leaf tips of the differentiated seedlings turned white, indicating that excessively long sodium hypochlorite treatment time caused irreversible damage to the cell viability of *Gynostemma pentaphyllum* seeds, leading to loss of germination ability. Therefore, the sodium hypochlorite solution soaking time should be strictly controlled to around 6 minutes.

[0048] Comparative Example 4 used the traditional gauze mesh bag disinfection method. During the disinfection and cleaning process, a large number of seeds were lost through the mesh bag openings with the water flow. After disinfection, a large number of seeds adhered to the mesh bag fibers and were difficult to detach during the transfer from the mesh bag to the culture medium, resulting in a seed utilization rate of less than 10%. Since the total number of seeds in a single capsule is extremely limited, the remaining seeds after this loss are insufficient to complete the sowing of the germination medium, making it impossible to carry out subsequent tissue culture operations in practice.

[0049] In summary, this invention provides an integrated operation process that completes all disinfection steps within centrifuge tubes and directly uses centrifuge tubes as sowing containers. This ensures thorough disinfection while completely eliminating seed loss during transfer between containers, achieving 100% utilization of individual dehiscent capsule seeds. This provides reliable technical support for the aseptic propagation of superior individuals and hybrid offspring of *Gynostemma pentaphyllum*.

Claims

1. A method for disinfecting seeds after the individual capsules of *Gnaphalium affine* have dehisced, characterized in that... Includes the following steps: 1) Seed collection: Select fully mature capsules of *Gnaphalium affine* after artificial self-pollination or hybridization of superior single plants. After the capsules naturally split open, fold a clean piece of paper into a funnel shape and collect all the seeds in the capsules into the adsorption column of the adsorption column set. 2) Preparation of germination medium: The medium consists of MS + 1.0 mg / L NAA + 1.0 mg / L 6-BA + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.6–5.8, sterilized for later use; 3) Seed surface disinfection: The adsorption column set is used as a container for seed surface disinfection. The adsorption column is washed with sterile water, soaked in alcohol, soaked in sodium hypochlorite solution, and washed with sterile water again in sequence. After each step, the adsorption column set is placed in a centrifuge for high-speed centrifugation to remove the liquid. After disinfection, the seeds are concentrated at the bottom of the adsorption column, and the liquid is collected in the centrifuge tube. 4) Integrated sowing: With the seeds inside the adsorption column, remove and open the adsorption column, take the seeds from the bottom of the adsorption column and sow them in a culture bottle containing germination medium. Then place the culture bottle in a tissue culture room and culture until the seeds germinate and differentiate into leaves.

2. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 2), the sterilization temperature is 121°C and the sterilization time is 15 minutes.

3. The method for disinfecting seeds after the individual capsules of *Gnaphalium affine* dehisce, as described in claim 1, is characterized in that... In step 3), the method of washing with sterile water is as follows: add 500 μL of sterile water to the adsorption column, place it in a shaker and shake for 1 minute, then place it in a centrifuge and centrifuge at 10000-12000 rpm at room temperature for 1 minute, remove the adsorption column, pour out the waste liquid in the centrifuge tube and put the adsorption column back into the centrifuge tube.

4. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 3), the alcohol soaking method is as follows: add 500 μL of 75% alcohol to the adsorption column, shake it in a shaker for 5 seconds, then place it in a centrifuge and centrifuge at 10,000 rpm for 30 seconds at room temperature. Remove the adsorption column, pour out the waste liquid in the centrifuge tube, and put the adsorption column back into the centrifuge tube.

5. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 3), the method for soaking in sodium hypochlorite solution is as follows: Add 500 μL of a 5% sodium hypochlorite solution to the adsorption column, shake in a shaker for 30 seconds, let stand at room temperature for 6 minutes, inverting the column several times every 30 seconds during this period, then place it in a centrifuge and centrifuge at 10,000 rpm for 1 minute at room temperature. Remove the adsorption column, discard the waste liquid in the centrifuge tube, and then put the adsorption column back into the centrifuge tube.

6. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 4), the specific operation of the integrated seeding is as follows: take the adsorption column out of the centrifuge tube, keep the adsorption column cap closed, spray the outer surface of the adsorption column with 75% alcohol by volume, place it in a pre-sterilized clean bench, open the adsorption column cap, use tweezers to directly pick up the seeds from the bottom of the adsorption column and sow them in the culture bottle, sowing 100-120 seeds in each bottle. After sowing, gently shake the culture bottle to spread the seeds on the surface of the culture medium.

7. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 4), the cultivation conditions are: temperature 25±1℃, relative humidity 40%–50%, light intensity 1800–2000 lx, light 12h / dark 12h, and cultivation for 60 days.

8. The method for disinfecting seeds after the individual capsule of *Gnaphalium affine* dehisces as described in claim 1, characterized in that... In step 4), after the seeds germinate and differentiate into leaves, they are transferred to a rooting medium for rooting culture.

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