Sterile seedlings of cyperus chinensis and cultivation method thereof

Abstract

The present invention relates to the field of plant tissue culture technology, and in particular to a sterile cyperus chinensis seedling and a cultivation method thereof. The method of the present invention selects cyperus chinensis bud points as explants. Compared with the whole tuber, the volume is smaller, the pollution source is reduced, and there is no need to additionally disinfect and culture the tuber. Only the stripped bud points need to be disinfected for 0.5 to 5.0 minutes. Specifically, a low mass concentration of 2.5%-7.5% sodium hypochlorite solution can be used for disinfection, which reduces the concentration and disinfection time of the disinfectant and reduces the damage of the disinfection process to the bud points. The method is simple to operate, time-saving and efficient, and effectively solves the problems of high contamination rate and low germination rate in the preparation process of cyperus chinensis sterile seedlings, thereby greatly improving the efficiency of cyperus chinensis sterile seedling preparation. Furthermore, the method of the present invention optimizes and selects culture media of different cultivation processes, has a short bud induction time, a high proliferation coefficient, and robust growth, and does not require the addition of any growth regulator during root induction, resulting in low cost.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a sterile cyperus oleifera seedling and a cultivation method thereof. Background Art

[0002] Cyperus esculentus is a high-quality, high-yield, and highly valuable new cash crop used for oil, grain, livestock, and animal feed. Cyperus esculentus oil is rich in 12 fatty acids, including oleic, linoleic, and palmitic acids. Its nutritional value is comparable to olive oil, and it possesses strong antioxidant properties, strengthens the spleen and stomach, and can prevent cardiovascular disease and other cardiovascular diseases such as hyperlipidemia, making it a high-quality edible oil. Furthermore, Cyperus esculentus exhibits strong resistance and adaptability, being tolerant to drought, waterlogging, and salinity, and can grow in my country's sandy, desert, and salinized soils. Developing the Cyperus esculentus industry will help increase my country's edible vegetable oil self-sufficiency rate and ensure national grain and oil security.

[0003] In my country, the cyperus chinensis (Cyperus chinensis) currently cultivated on a large scale is triploid and exhibits sterility, making it a vegetatively propagated crop. Long-term asexual propagation of cyperus chinensis is prone to varietal degeneration, genetic instability, quality decline, and severe bacterial contamination. This results in low genetic diversity and a lack of superior cultivars. Research into techniques such as rapid tissue propagation, somatic hybridization, and genetic transformation is urgently needed as effective avenues for purifying, improving, and enhancing cyperus chinensis germplasm. Therefore, establishing an efficient system for producing sterile seedlings of cyperus chinensis is a crucial prerequisite for these technologies. Cyperus chinensis relies on tubers for asexual propagation, and traditional sterile seedling production often uses intact tubers as explants. Cyperus chinensis tubers have a rough surface, with fibrous roots and scales, and are prone to harboring numerous bacteria. The disinfectant concentration required for sterile seedling production is high and the disinfection time is long. However, these bacteria are difficult to completely eliminate, resulting in a high contamination rate. Furthermore, even after prolonged high-concentration disinfection, tuber germination is slow and the germination rate is reduced, making sterile seedling production inefficient.

[0004] However, limited research is currently underway on the preparation of sterile seedlings and tissue culture regeneration in Cyperus esculentus. This has hindered the development of technologies such as the establishment of genetic transformation systems, somatic cell hybridization, and rapid propagation of superior germplasm. Therefore, establishing a comprehensive system for the preparation of sterile seedlings in Cyperus esculentus and improving their efficiency is crucial. Summary of the Invention

[0005] In order to overcome the defects of the prior art, one of the objects of the present invention is to provide a method for cultivating sterile seedlings of cyperus esculentus, using the bud points of cyperus esculentus tubers as explants and improving the preparation efficiency of sterile cyperus esculentus seedlings by optimizing the processes of disinfection, bud proliferation and rooting culture.

[0006] The second object of the present invention is to provide a sterile seedling of cyperus esculentus, which is cultivated using the cultivation method provided by the present invention and is used as a high-quality seedling of cyperus esculentus for germplasm resource cultivation.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0008] A method for cultivating sterile seedlings of cyperus chinensis, comprising the following steps:

[0009] 1) Soak the cyperus tubers with intact buds in sterile water containing a fungicide. Shake the tubers to remove surface scales, fibrous roots, and soil. Rinse the tubers periodically and replace with fresh sterile water containing a fungicide.

[0010] 2) peeling off the complete buds of the fully imbibed Cyperus oleifera tubers from step 1);

[0011] 3) removing moisture from the surface of the buds obtained in step 2), then soaking them in ethanol and then rinsing them with sterile water;

[0012] 4) Soak the buds treated in step 3) in disinfectant for 0.5 to 5.0 minutes, and then rinse with sterile water;

[0013] 5) inoculating the buds treated in step 4) into a culture medium to obtain seedlings;

[0014] 6) transferring the seedlings obtained in step 5) to a bud cluster proliferation medium to obtain bud clusters;

[0015] 7) The clustered buds obtained in step 6) are inoculated into a rooting medium to obtain sterile seedlings of Cyperus esculentus.

[0016] Optionally, the sterile water containing a bactericide is sterile water containing 2.0 g / L carbendazim.

[0017] Optionally, the ethanol in step 3) is 75% ethanol by volume, and the soaking time is 1 min.

[0018] Optionally, the disinfectant in step 4) is a sodium hypochlorite solution with a mass concentration of 2.5%-7.5%;

[0019] More preferably, the disinfectant is a sodium hypochlorite solution with a mass concentration of 5%, and the disinfection time is 1 minute.

[0020] Optionally, the culture medium in step 5) is MS culture medium.

[0021] Optionally, the bud proliferation medium in step 6) is MS medium supplemented with 0.5-2.0 mg / L 6-BA, 0.0-1.0 mg / L KT, and 0.0-0.3 mg / L NAA;

[0022] More preferably, the clump sprout proliferation medium is MS medium supplemented with 2.0 mg / L 6-BA and 1.0 mg / L KT.

[0023] Optionally, the rooting medium in step 7) is MS medium. It should be noted that the rooting medium used in step 7) of the method of the present invention does not require the addition of any plant growth regulator.

[0024] Optionally, in step 1), the interval for replacing the sterile water containing the fungicide is to rinse the tubers with sterile water and then replace with new sterile water containing the fungicide every 12 hours; and the shaking is performed at 30° C. and 220 rpm on a shaker for 72 hours.

[0025] Optionally, the culture conditions for the seedlings in step 5) and the clustered buds in step 6) are: room temperature 30±1° C., 14 h of light and 10 h of darkness; seedlings with a height of 3.0 to 4.0 cm are cultivated; and the clustered buds are cultivated for 30 days.

[0026] The invention discloses a sterile seedling of cyperus chinensis, which is cultivated by the above method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention selects the bud point of Cyperus esculentus as the explant, which is smaller in size than the entire tuber, thereby reducing the source of pollution. The bud point is directly peeled off from the tuber after imbibition, without the need for additional disinfection and cultivation of the tuber. The peeled bud point only needs to be disinfected for 0.5 to 5.0 minutes. Specifically, a sodium hypochlorite solution with a mass concentration of 2.5% to 7.5% can be used for disinfection, which reduces the concentration of the disinfectant and the disinfection time, and reduces the damage to the bud point caused by the disinfection process. The operation is simple, time-saving and efficient, and effectively solves the problems of high contamination rate and low germination rate in the preparation process of Cyperus esculentus sterile seedlings, thereby greatly improving the efficiency of the preparation of Cyperus esculentus sterile seedlings.

[0029] (2) The present invention uses sterile water containing a fungicide to soak the tubers during the imbibition process of the cyperus juncea tubers, and simultaneously utilizes the high-speed continuous oscillation of the shaker to effectively remove most of the dirt, impurities and bacteria on the tuber surface in advance, thereby significantly reducing contamination in subsequent processes;

[0030] (3) Furthermore, the present invention systematically studies the optimal culture medium required for the proliferation and rooting stages of the cluster buds of Cyperus esculentus. The cluster bud induction time is short, the proliferation coefficient is high, and the growth is robust. At the same time, no growth regulators need to be added during rooting induction, and the cost is low. This can lay the foundation for the efficient breeding of high-quality Cyperus esculentus seedlings and the optimization of germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the description. Together with the following specific embodiments, they are used to explain the present invention, but do not constitute a limitation of the present invention. In the accompanying drawings:

[0032] Figure 1 This is a flow chart of the method for cultivating sterile seedlings of Cyperus oleifera in Example 1;

[0033] Figure 2 This is the proliferation of cyperus oleifera clumps under different growth regulator combinations in Example 3, where numbers 1-16 are treatment numbers;

[0034] Figure 3 This is the root growth of cyperus oleifera clumps under different minimal media, IBA, and NAA combinations in Example 4. In the figure, numbers 1 and 5 are 1 / 2MS, 2-4 are 1 / 2MS+0.1mg / L IBA, 1 / 2MS+0.2mg / L IBA, and 1 / 2MS+0.3mg / L IBA, respectively; 6-8 are 1 / 2MS+0.1mg / L NAA, 1 / 2MS+0.2mg / L NAA, and 1 / 2MS+0.3mg / L NAA, respectively; 9 and 13 are MS, 10-12 are MS+0.1mg / L IBA, MS+0.2mg / L IBA, and MS+0.3mg / L IBA, respectively; 14-16 are MS+0.1mg / L NAA, MS+0.2mg / L NAA, and MS+0.3mg / L NAA, respectively;

[0035] Figure 4 This is the preparation of sterile seedlings of Cyperus oleifera in comparative example 2, and the ones in the red circle are germinated;

[0036] Figure 5 This is the preparation of sterile seedlings of Cyperus esculentus in Comparative Example 3, and the ones in the red circle are germinated. DETAILED DESCRIPTION

[0037] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0038] In the following examples and comparative examples

[0039] Contamination rate (%) = total number of contaminations / total number of inoculations × 100;

[0040] Germination rate (%) = total number of germinations / total number of inoculations × 100;

[0041] The sterile seedling acquisition rate (%) = total number of germinated seedlings without contamination / total number of inoculations × 100;

[0042] Proliferation coefficient = (total number of clustered buds - total number of inoculated buds) / total number of inoculated buds;

[0043] Average root length (cm) = total root length / total number of roots; average rooting rate (%) = number of rooted buds / total number of inoculated buds × 100;

[0044] Average number of roots (per plant) = total number of roots / total number of inoculated buds.

[0045] Example 1

[0046] This embodiment provides a method for cultivating aseptic seedlings of Cyperus oleifera. The schematic diagram of the operation process is shown in FIG. Figure 1 As shown, the specific steps are:

[0047] 1) Select plump, worm-free, and intact bud-forming Cyperus oleifera tubers, rinse thoroughly with pure water, and soak in sterile water containing 2.0 g / L carbendazim. Shake on a shaker at 30°C and 220 rpm for 72 h to remove scales, fibrous roots, and soil from the tuber surface. Rinse the tubers with sterile water every 12 h, replacing the sterile water with 2.0 g / L carbendazim.

[0048] 2) Rinse the fully imbibed Cyperus oleifera tubers from 1) with sterile water, carefully remove the intact buds with a scalpel blade, and place them in a Petri dish filled with sterile water for later use;

[0049] 3) In a laminar flow hood, blot the surface moisture of the buds with sterilized filter paper, soak them in 75% ethanol for 1 minute, and rinse three times with sterile water.

[0050] 4) Transfer the buds obtained in 3) to a 5.0% sodium hypochlorite solution and soak for 1.0 min, then rinse with sterile water 5-6 times;

[0051] 5) Inoculate the sterilized buds into MS medium and culture in a light incubator at 30 ± 1°C, with 14 h of light and 10 h of darkness for 10 days. Inoculate 30 buds per treatment, repeat three times, and calculate the contamination rate, germination rate, and sterile seedling yield rate.

[0052] 6) In a laminar flow hood, transfer the young shoots obtained in 5) to a bud cluster proliferation medium (MS + 2.0 mg / L 6-BA + 1.0 mg / L KT) and incubate in a light incubator at 30 ± 1°C, 14 h light, 10 h dark for 30 days to obtain bud clusters. Calculate the proliferation coefficient.

[0053] 7) In a clean bench, carefully select uniformly growing clusters of shoots with tweezers and inoculate them into rooting medium MS. Incubate for 10 days to induce rooting to obtain sterile Cyperus esculentus seedlings. Statistically analyze the average root length, average rooting rate, and average number of roots.

[0054] In this embodiment, the contamination rate was 11.57%, the germination rate was 94.77%, the sterile seedling acquisition rate was 83.85%, the cluster bud proliferation coefficient was 78.20, the average root length was 8.49 cm, the average rooting rate was 96.10%, and the average number of roots was 4.56 per plant.

[0055] Example 2

[0056] This example designed an experiment to verify the effect of different disinfection methods on the efficiency of sterile seedling cultivation of Cyperus esculentus. Specifically, different sodium hypochlorite concentrations and treatment times were set according to the grouping shown in Table 1. The seeds were cultivated according to steps 1) to 5) of the same cultivation method provided in Example 1. The contamination rate, germination rate, and sterile seedling yield were calculated. The results are shown in Table 2:

[0057] Table 1 Combination of different disinfection methods for cyperus juncea sprouts

[0058]

[0059] Table 2 Effects of different disinfection methods on the preparation of sterile seedlings of Cyperus esculentus sprouts

[0060]

[0061] Note: Mean ± standard error. Different letters in the table represent significance at the 0.01 level.

[0062] The results shown in Table 2 indicate that the germination rate and contamination rate of cyperus oleifera gradually decreased with increasing sodium hypochlorite concentration and treatment time. Excessively high sodium hypochlorite concentrations significantly inhibited germination, while low concentrations resulted in poor disinfection effectiveness. Treatment time had a more pronounced effect on germination and contamination rates than sodium hypochlorite concentration. At the same sodium hypochlorite concentration, a disinfection time exceeding 1 minute resulted in a sharp decrease in both germination and contamination rates.

[0063] In terms of comprehensive germination and contamination-free rates, the best effect was achieved by disinfecting with 5.0% sodium hypochlorite for 1 minute, with a germination rate of 94.77%, a contamination rate of only 11.57%, and a sterile seedling acquisition rate of 83.85%.

[0064] Example 3

[0065] This example designed an experiment to verify the effect of the bud proliferation medium on the bud proliferation coefficient. Parallel experiments were designed according to the different bud proliferation mediums shown in Table 3. The buds were cultivated according to the cultivation method shown in steps 1) to 6) of Example 1, and the proliferation coefficients were calculated. The results were as follows: Figure 2 The specific statistical results are shown in Table 4:

[0066] Table 3 Combinations of different plant growth regulators in Cyperus oleifera bud proliferation culture medium

[0067]

[0068] Table 4 Analysis results of orthogonal test of Cyperus chinensis bud proliferation

[0069]

[0070]

[0071] Note: k1, k2, k3 and k4 are the average proliferation coefficients of the four levels of growth regulators; R is the range.

[0072] Depend on Figure 2 The results shown in Table 4 showed that the effects of the three plant growth regulators on bud proliferation were NAA>6-BA>KT, and NAA inhibited the proliferation of buds of Cyperus esculentus, while 6-BA and KT promoted it. The best combination of growth regulators was 2.0 mg / L 6-BA+1.0 mg / L KT, and the bud proliferation coefficient could reach 78.20.

[0073] Example 4

[0074] This example designs an experiment to verify the effect of different rooting culture media on the rooting of cyperus chinensis buds. The culture medium composition and test results are shown in the figure. Figure 3 As shown in Table 5, the following were cultivated according to the cultivation method provided in Example 1:

[0075] Table 5 Rooting of Cyperus oleifera buds in different basic culture media, IBA and NAA combinations

[0076]

[0077]

[0078] Note: Mean ± standard error. Different letters in the table represent significance at the 0.01 level.

[0079] Figure 3The results shown in Table 5 indicate that the average rooting rate, average root number, and average root length using MS as the basal medium were superior to those using 1 / 2 MS. The addition of IBA and NAA both inhibited root growth, with NAA exhibiting a greater degree of inhibition than IBA. MS without growth regulators was the optimal rooting medium, with an average rooting rate of 96.10%, an average root number of 4.56 roots per plant, and an average root length of 8.49 cm.

[0080] Comparative Example 1

[0081] This comparative example provides a method for cultivating sterile seedlings of Cyperus esculentus. The difference from Example 1 is that no buds are picked, and the tubers are directly inoculated into a culture medium for cultivation. The germination rate, contamination rate, and sterile seedling acquisition rate are statistically analyzed. The specific operating steps and parallel experiment design are as follows:

[0082] 1) Select plump, worm-free, and intact budded Cyperus oleifera tubers. Rinse thoroughly with pure water and soak in sterile water containing 2.0 g / L carbendazim. Shake at 30°C and 220 rpm for 72 hours to remove surface scales, fibrous roots, and soil. Rinse the tubers with sterile water every 12 hours, replacing the sterile water with fresh 2.0 g / L carbendazim.

[0083] 2) Wash the imbibed tubers with sterile water, soak them in 75% ethanol for 1 minute in a laminar flow hood, and then wash them three times with sterile water.

[0084] 3) Transfer the tubers obtained in 2) to sodium hypochlorite solutions of varying concentrations for the appropriate time periods, followed by 5-6 rinses with sterile water. Four sodium hypochlorite concentrations (2.5%, 5.0%, 7.5%, and 10%) and disinfection times (30, 60, 90, and 120 min) were used. Thirty tubers were inoculated into each treatment, repeated three times.

[0085] 4) The sterilized individual tubers were inoculated onto MS medium and cultured at 30 ± 1°C, 14 h of light, and 10 h of darkness for 10 days. The germination rate, contamination rate, and sterile seedling yield were calculated. The results are shown in Table 6.

[0086] Table 6 Effects of different disinfection methods on the preparation of sterile seedlings of Cyperus esculentus tubers

[0087]

[0088]

[0089] Note: Mean ± standard error. Different letters in the table represent significance at the 0.01 level.

[0090] The results shown in Table 6 above show that the optimal tuber disinfection method was 7.5% sodium hypochlorite disinfection for 60 minutes, with a germination rate of 79.73%, a contamination rate of 36.83%, and a sterile seedling acquisition rate of 51.57%. Compared with Example 1, the germination rate decreased by 15.04%, the contamination rate increased by 25.26%, and the sterile seedling acquisition rate decreased by 32.28%.

[0091] Comparative Example 2

[0092] This comparative example provides a method for cultivating sterile seedlings of Cyperus esculentus, and the specific steps are as follows:

[0093] 1) Select plump tubers with intact buds and no insect holes. Remove the fibrous roots and scales on the surface. Rinse with tap water and soak for 3 days, changing the water frequently.

[0094] 2) Soak in 75% ethanol for 1 minute and rinse 5-6 times with sterile water;

[0095] 3) Soak in 7.5% sodium hypochlorite for 50 minutes and rinse 1-2 times with sterile water;

[0096] 4) Wash 2-3 times with sterile water supplemented with 0.05% Tween-20 and 500 mg / L cephalexin, and finally wash 2-3 times with sterile water;

[0097] 5) Place the tubers on sterile paper to absorb the surface moisture of the tubers, and transfer them to terminal bud induction medium MS + 0.2 mg / L NAA + 0.5 mg / L 6-BA + 500 mg / L cephalosporin + 30 g / L sucrose + 8.0 g / L agar, pH 5.8, and culture for about 1 week;

[0098] 6) Cut the stem tip into single buds, soak them in 70% ethanol for 30 seconds, soak them in 0.1% mercuric chloride for 10 minutes, wash them three times with sterile water containing 0.05% Tween-20, rinse them five times with sterile water, place them on sterile paper to dry the surface moisture, and inoculate them on MS + 0.05 mg / L NAA + 0.5 mg / L 6-BA medium. Inoculate 6-8 single buds per bottle, and inoculate 5 bottles. On the 10th day, calculate the germination rate, contamination rate, and sterile seedling yield rate. Figure 4 The specific results are shown in Table 7.

[0099] Comparative Example 3

[0100] This comparative example provides a method for cultivating sterile seedlings of Cyperus esculentus, and the specific steps are as follows:

[0101] 1) Select plump cyperus seeds, rinse them 3-5 times with sterile water, and soak them in 2.0 g / L carbendazim for 24 hours.

[0102] 2) Rinse 3-5 times with sterile water, soak in 0.2% mercuric chloride in a clean bench for 30 minutes, rinse 3-5 times with sterile water, wrap with sterile wet gauze, place in a culture dish (humidity 100%), and culture in a germination box for 3-4 days (culture temperature 25-26°C);

[0103] 3) When the buds are 2-3 mm long, remove the buds, treat with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, treat with 0.1% mercuric chloride for 15 minutes, and rinse with sterile water 3-5 times;

[0104] 4) Place the buds on sterile filter paper to absorb surface moisture. In a clean bench, peel off the shoot apex growth point under a dissecting microscope at 40x magnification and inoculate it into MS + 0.6 mg / L 6-BA + 0.25 mg / L NAA medium. Place in an artificial climate chamber and culture for 10 days (culture temperature 26-28°C, humidity 70%, light intensity 2000 lx, light duration 12 h). Inoculate 6-8 shoot apex growth points per bottle. Inoculate 5 bottles and calculate the germination rate, contamination rate, and sterile seedling yield rate. Figure 5 The specific results are shown in Table 7.

[0105] The test results of Comparative Examples 2 and 3 are shown in Table 7:

[0106] Table 7 Effects of different methods on the preparation of sterile seedlings of Cyperus esculentus

[0107]

[0108] As can be learned from Table 7, when utilizing mercuric chloride solution to sterilize the terminal bud stripped, terminal bud is very susceptible to damage, causes germination rate to drop sharply, sterile seedling acquisition rate is lower, the sterile seedling acquisition rate of comparative example 2,3 is only 20.15%, 17.14% respectively, is far lower than the sterile seedling acquisition rate 83.85% of embodiment of the present invention 1;In addition, comparative example 2 and 3 are compared with embodiment of the present invention 1 operation cost time long, all need first to carry out disinfection and terminal bud induction culture to tuber, comparative example 2 needs to soak 50min with 7.5% sodium hypochlorite to tuber, comparative example 3 needs to soak 30min with 0.2% mercuric chloride, this step disinfection treatment also can cause damage to tuber, causes tuber germination rate to reduce, and the present invention directly strips bud point with the tuber after imbibition, tuber does not have disinfection treatment, ensures its germination ability, saves time and is efficient;Comparative example 3 is compared with the present invention operation complicated and difficulty height, needs to use dissecting mirror during operation, extremely difficult operation.Therefore, the cyperus juncea sterile seedling cultivation method of the present invention is better than existing other technical schemes.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for cultivating sterile seedlings of Cyperus oleifera, characterized in that: The following steps are included: 1) Soak the cyperus tubers with intact buds in sterile water containing a fungicide. Shake the water to remove surface scales, fibrous roots, and soil. Rinse the tubers periodically and replace with fresh sterile water containing a fungicide. 2) Peeling off the intact buds of the fully imbibed Cyperus oleifera tubers from step 1; 3) Remove the surface moisture of the buds obtained in step 2), then soak them in ethanol and rinse with sterile water; 4) Soak the buds treated in step 3) in disinfectant for 0.5-5.0 minutes, then rinse thoroughly with sterile water. 5) Inoculating the buds treated in step 4) into the culture medium to obtain seedlings; 6) transferring the seedlings obtained in step 5) to a bud cluster proliferation medium to obtain bud clusters; 7) inoculating the clustered buds obtained in step 6) into a rooting medium to obtain sterile seedlings of Cyperus esculentus; In step 4), the disinfectant is a 5% sodium hypochlorite solution, and the disinfection time is 1 minute. The bud proliferation medium in step 6) is MS medium + 2.0 mg / L 6-BA + 1.0 mg / L KT; The rooting medium in step 7) is MS medium.

2. The method for cultivating sterile seedlings of cyperus oleifera according to claim 1, wherein The sterile water containing the bactericide is sterile water containing 2.0 g / L carbendazim.

3. The method for cultivating sterile seedlings of cyperus oleifera according to claim 1, wherein: The ethanol in step 3) is 75% by volume, and the soaking time is 1 min.

4. The method for cultivating sterile seedlings of cyperus oleifera according to claim 1, wherein The culture medium in step 5) is MS medium.

5. The method for cultivating sterile seedlings of cyperus oleifera according to claim 1, wherein: In step 1), the interval for replacing the sterile water containing the fungicide is to rinse the tubers with sterile water every 12 hours and then replace with new sterile water containing the fungicide; The shaking was carried out at 30°C and 220 rpm for 72 h.

6. The method for cultivating sterile seedlings of Cyperus oleifera according to claim 1, wherein: The cultivation conditions for the seedlings in step 5) and the clustered buds in step 6) are: room temperature of 30±1°C, 14 hours of light and 10 hours of darkness; seedlings with a height of 3.0-4.0 cm are obtained; and the clustered buds are cultivated for 30 days.

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