A culture medium and method for inducing callus and regenerating tiger nuts.

Abstract

This invention relates to the field of plant tissue culture technology, specifically to a culture medium and method for inducing and regenerating tiger nut callus. This invention creatively combines growth regulators (2,4-D, 6-BA, NAA, and ZT) in the culture medium for three different stages of tiger nut callus induction, regeneration, and differentiation, and optimizes the selection of the best explants. This allows each component to fully synergistically enhance its effect at each stage, resulting in high callus induction rate, good growth status, low browning degree, good proliferation effect, and high differentiation rate and differentiation coefficient. Furthermore, no growth regulators are required during rooting induction, resulting in lower costs. This invention lays the foundation for tiger nut gene function research, variety genetic improvement, and germplasm resource optimization.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a culture medium and culture method for inducing callus regeneration from tiger nuts. Background Technology

[0002] Tiger nuts (Cyperus esculentus L.), also known as oilseed rape or tiger nut, are a high-quality, high-yield, and multi-purpose economic crop with rich oil, starch, sugar, protein, and various vitamins. Their tubers are rich in oil, starch, sugar, protein, and various vitamins. Tiger nuts are also currently the only known oilseed crop that stores oil in its tubers. The oil is rich in 12 fatty acids, including oleic acid, linoleic acid, linolenic acid, and palmitic acid, and has high nutritional value, making it a high-quality alternative to plant oils. Furthermore, tiger nuts have a large biomass, strong resistance and adaptability, and are drought-tolerant, flood-tolerant, and salt-tolerant, making them suitable for planting on marginal land. Therefore, developing the tiger nut industry not only benefits the improvement of the ecological environment but also helps increase my country's self-sufficiency rate in edible vegetable oil.

[0003] my country's tiger nut germplasm resources suffer from low genetic diversity and a lack of superior varieties. Furthermore, most tiger nut materials cultivated on a large scale in my country are triploid, exhibiting sterility, rarely flowering, or flowering without producing seeds. Propagation is largely limited to tubers, making them highly susceptible to problems such as varietal degeneration, genetic instability, quality decline, and severe bacterial contamination. Compared to conventional breeding methods, cell engineering breeding and genetic engineering breeding are more suitable for improving tiger nut varieties and promoting germplasm innovation. Much of this breeding work relies on the establishment of tissue culture regeneration systems, and tissue culture technology is also a prerequisite and foundation for gene function research. Therefore, constructing a tiger nut callus culture regeneration system is of great significance for the development and advancement of tiger nut breeding work.

[0004] The selection of explants, callus induction, proliferation, and differentiation are the four major factors to consider in plant tissue culture. The selection of explants is crucial to the success of tissue culture regeneration, as the cell meristematic capacity of the explants directly affects callus induction and regeneration. Commonly used explants include plant embryos, anthers, young spikelets, seeds, and young leaves. Some researchers have successfully induced callus using the base of tiger nut stem segments and root tips as explants, but callus induced from root tips cannot induce adventitious buds, and the base of the stem segment is prone to browning. The induction, proliferation, and differentiation of callus are important processes for plant regeneration. Adding appropriately balanced concentrations of growth regulators and cytokinin-based agents to the culture medium can effectively improve the callus induction rate, proliferation rate, and adventitious bud differentiation rate. Currently, many scholars have reported on the rapid propagation system of tiger nuts, but research on tiger nut callus induction and regeneration systems is relatively scarce and incomplete. There is a lack of detailed systematic research on tiger nut callus induction, proliferation and differentiation. Furthermore, existing research has many problems such as low callus emergence rate, easy browning of tiger nut callus, lack of research on callus proliferation culture, and low efficiency of callus differentiation into adventitious buds.

[0005] Therefore, how to provide a perfect and efficient method for inducing callus and regenerating tiger nuts is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the problems existing in the prior art, one of the objectives of this invention is to provide a culture medium for inducing callus and regenerating plants in tiger nuts, systematically optimizing the formulation of the culture medium for callus induction, proliferation, and differentiation, thereby improving the callus induction rate, proliferation rate, and adventitious bud differentiation rate.

[0007] Meanwhile, this invention also provides a method for inducing and regenerating tiger nut callus. It systematically studies the selection of explants for tiger nut callus induction, callus induction, proliferation and differentiation regeneration, and can efficiently induce callus. It establishes a complete tiger nut callus induction and plant regeneration system, providing certain technical and material support for tiger nut gene function research and variety genetic improvement.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0009] A culture medium for inducing callus and regenerating plants from tiger nuts includes a callus induction medium, a proliferation medium, and a differentiation medium; the induction medium, proliferation medium, and differentiation medium are all based on MS medium, with added growth regulators, trace elements, amino acids, anti-browning agents, carbon sources, and coagulants; pH 5.8;

[0010] The composition and mass concentration ratio of the growth regulator in the induction medium were: 2,4-D:6-BA:NAA:ZT = 25:6:12:9

[0011] The composition and mass concentration ratio of the growth regulator in the proliferation medium were: 2,4-D: 6-BA: NAA: ZT = 10: 9: 12: 4;

[0012] The composition and mass concentration ratio of growth regulators in the differentiation medium are: 6-BA:NAA = 6:9.

[0013] Preferably, the composition of trace elements, amino acids, anti-browning agents, carbon sources and coagulants in the induction medium, proliferation medium and differentiation medium are: 4 g / L PVP, 7 mg / L AgNO3, 0.5 g / L CH, 30 g / L sucrose and 7 g / L agar.

[0014] Preferably, the growth regulator composition in the induction medium is: 2.5 mg / L 2,4-D, 0.6 mg / L 6-BA, 1.2 mg / L NAA, and 0.9 mg / L ZT.

[0015] Preferably, the growth regulator composition in the proliferation medium is: 1.0 mg / L 2,4-D, 0.9 mg / L 6-BA, 1.2 mg / L NAA, and 0.4 mg / L ZT.

[0016] Preferably, the growth regulator composition of the differentiation medium is: 0.6 mg / L 6-BA and 0.9 mg / L NAA.

[0017] A method for inducing callus and regenerating plants from tiger nuts includes the following steps:

[0018] 1) Obtain sterile seedlings and select sterile seedling tissues as explants;

[0019] 2) Inoculate the explants obtained in step 1) into callus induction medium and induce culture to obtain callus tissue;

[0020] 3) Inoculate the callus tissue from step 2) into the proliferation medium and culture to obtain proliferated callus tissue;

[0021] 4) Inoculate the proliferated callus obtained in step 3) into differentiation medium and culture to obtain differentiated adventitious shoots;

[0022] 5) Inoculate the differentiated adventitious buds obtained in step 4) into the rooting medium, and carry out rooting culture to complete plant regeneration;

[0023] The callus induction medium, proliferation medium, and differentiation medium are the aforementioned media.

[0024] Optionally, the specific method for obtaining sterile seedlings in step 1) is as follows: Take tiger pea tubers that are uniform in size, plump, free of insect holes, and have complete buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days. Change the carbendazim treatment solution every 8 hours. After soaking, peel off the complete buds from the tubers. Under sterile conditions, treat the peeled buds with 75% ethanol for 1 minute, wash them once with sterile water, treat them with 5.0% NaClO for 1 minute, and wash them 3-5 times with sterile water. Inoculate the disinfected buds onto MS solid medium and place them in a light incubator at 30℃, 10000 lx light, and 14 h / d for about 20 days. Take sterile seedlings that are free of pollution and in good growth condition to obtain explants.

[0025] As a preferred method, the specific method for obtaining explants in step 1) is as follows: when tiger nut sterile seedlings grow to 4-5cm, take the tillering nodes of the healthy tiger nut sterile seedlings as explants.

[0026] Optionally, the culture conditions for steps 2) and 3) are both 25°C in the dark.

[0027] Step 4) The differentiation culture was carried out under the following conditions: 25℃, light intensity of 2000lx, and light duration of 12h / d.

[0028] Optionally, the cultivation method in step 5) is as follows: When the adventitious buds in step 4) grow to 3-5cm, they are transferred to the rooting medium MS in the form of single buds. After rooting culture for 20 days and hardening off in half-open culture bottles for 3 days, they are transplanted into sandy soil and cultured under the conditions of 30℃, light intensity of 12000lx, and light duration of 12h / d. During this period, watering is carried out as needed, and nutrient solution is applied once every 7 days to complete plant regeneration.

[0029] The beneficial effects of this invention are as follows: This invention creatively combines growth regulators in the culture medium for three different stages of tiger nut callus induction, regeneration, and differentiation, and optimizes the selection of the best explants, so that each component can fully synergistically enhance the effect at each stage. This results in a high callus induction rate, good growth status, low browning degree, good proliferation effect, and high differentiation rate and differentiation coefficient. At the same time, no growth regulators need to be added during rooting induction, which reduces costs. This invention can lay the foundation for tiger nut gene function research and optimization of germplasm resources for variety genetic improvement. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the description. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0031] Figure 1The effect of different tiger nuts explants on callus induction in Example 1.

[0032] Figure 2 The callus induction effects of the optimal and worst callus induction media combinations in Example 2 are shown.

[0033] Figure 3 The callus proliferation effect of the optimal and worst growth regulator combinations in the growth medium in Example 3 is shown.

[0034] Figure 4 The callus differentiation effect of the optimal and worst growth regulator combinations in the proliferation culture medium in Example 4 is shown.

[0035] Figure 5 This describes the process of tiger nut callus induction, proliferation, differentiation, and plant regeneration in Example 5. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0037] Example 1: Effects of different explants on callus induction in tiger nuts

[0038] This embodiment provides a method for inducing and culturing tiger nut callus, the method comprising the following steps:

[0039] (1) Take tiger pea tubers that are uniform in size, plump, free of insect holes, and with intact buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200rpm for 3 days, changing the carbendazim treatment solution every 8 hours. After soaking, peel off the intact buds of the tubers, place them in a 2mL centrifuge tube containing sterile water, and in a clean bench, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds on MS solid medium and place them in a light incubator at 30℃ and 10000lx light (14h / d) for about 20 days. Take sterile seedlings with good growth and no pollution for subsequent experiments.

[0040] 2) When tiger nut aseptic seedlings reached 4-5 cm in height, four tissue samples were taken from healthy tiger nut aseptic seedlings: young roots, young leaves, young leaf sheaths, and tillering nodes. These samples were inoculated into MS medium (pH 5.8) containing 2.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.6 mg / L NAA, 0.9 mg / L ZT, 4 g / L PVP, 7 mg / L AgNO3, 0.5 g / L CH, 30 g / L sucrose, and 7 g / L agar. Thirty explants were inoculated for each treatment, with three replicates. The plants were cultured at 25°C in the dark for 30 days. The callus growth was observed, and the callus induction rate was calculated. The optimal induction tissue was selected.

[0041] Callus induction rate (%) = Number of explants that produce callus / Total number of inoculated explants × 100.

[0042] The results showed that the callus induction rates of four types of explants—young roots, young leaves, young leaf sheaths, and tillers—from aseptic tiger nut seedlings differed significantly, with the order from highest to lowest being tillerers > young leaf sheaths > young roots > young leaves (Table 1). Figure 1 Tillering nodes are the best explants for inducing callus in tiger nuts.

[0043] Table 1. Effects of different explant types on callus induction.

[0044]

[0045]

[0046] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate highly significant differences between treatments (P<0.05).

[0047] Example 2: Effects of different combinations of plant growth regulators on callus induction

[0048] This embodiment provides a method for inducing and culturing tiger nut callus, the method comprising the following steps:

[0049] 1) Take uniformly sized, plump tiger nut tubers without insect holes and with intact buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days, changing the carbendazim treatment solution every 8 hours. After thorough soaking, peel off the intact buds from the tubers and place them in a 2mL centrifuge tube containing sterile water. In a clean bench, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds onto MS solid medium and place them in a light incubator at 30℃ and 10000lx light (14h / d) for about 20 days. Use sterile seedlings with good growth and no contamination for subsequent experiments.

[0050] 2) When the aseptic tiger nut seedlings grew to 4-5 cm, the tillers of the healthy aseptic tiger nut seedlings were inoculated into MS medium (pH 5.8) supplemented with different concentrations of 2,4-D (1.5, 2.0, 2.5, 3.0 mg / L), 6-BA (0.3, 0.6, 0.9, 1.2 mg / L), NAA (0.3, 0.6, 0.9, 1.2 mg / L), ZT (0.3, 0.6, 0.9, 1.2 mg / L), 4 g / L PVP, 7 mg / L AgNO3, 0.5 g / L CH (acid-hydrolyzed casein), 30 g / L sucrose, and 7 g / L agar. Each treatment was inoculated with 30 explants, and the inoculation rate was calculated in 3 replicates. The plants were cultured in the dark at 25℃ for 30 days. The callus growth was observed, and the callus induction rate was calculated (Table 2). Figure 2 ).

[0051] The results showed that the callus induction medium containing 2.5 mg / L 2,4-D, 0.6 mg / L 6-BA, 1.2 mg / L NAA, and 0.9 mg / L ZT as growth regulators exhibited a synergistic effect among the growth regulators, increasing the callus induction rate to 61.11%.

[0052] Table 2. Effects of different growth regulator compositions in culture media on the induction rate of tiger pea callus.

[0053]

[0054]

[0055] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate highly significant differences between treatments (P<0.05).

[0056] Example 3: Effects of different combinations of plant growth regulators on callus proliferation

[0057] 1) Take uniformly sized, plump tiger nut tubers without insect holes and with intact buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days, changing the carbendazim treatment solution every 8 hours. After thorough soaking, peel off the intact buds from the tubers and place them in a 2mL centrifuge tube containing sterile water. In a clean bench, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds onto MS solid medium and place them in a light incubator at 30℃ and 10000lx light (14h / d) for about 20 days. Use sterile seedlings with good growth and no contamination for subsequent experiments.

[0058] 2) When the aseptic tiger nut seedlings grow to 4-5cm, take the tillering nodes of the healthy aseptic tiger nut seedlings and inoculate them into MS medium (pH 5.8) with 2.5mg / L 2,4-D, 0.6mg / L 6-BA, 1.2mg / L NAA, 0.9mg / L ZT, 4g / L PVP, 7mg / L AgNO3, and 0.5g / L CH medium. Incubate at 25℃ in the dark to induce callus tissue.

[0059] 3) After tiger nut tillers were cultured on induction medium for 15 days, explants that produced callus were selected and transferred to MS medium supplemented with different concentrations of 2,4-D (0.5, 1.0, 1.5, 2.0 mg / L), 6-BA (0.3, 0.6, 0.9, 1.2 mg / L), NAA (0.3, 0.6, 0.9, 1.2 mg / L), ZT (0.4, 0.8, 1.2, 1.6 mg / L), 4 g / L PVP, 7 mg / L AgNO3, 0.5 g / L CH (acid-hydrolyzed casein), 30 g / L sucrose, and 7 g / L agar. Thirty callus tissues were inoculated for each treatment, and the treatment was repeated three times. The tissues were cultured at 25℃ in the dark for 30 days, and callus proliferation was observed and the callus proliferation rate was recorded (Table 3). Figure 3 ).

[0060] Proliferation rate (%) = Number of proliferating callus / Number of inoculated callus × 100.

[0061] The results showed that when the growth regulator composition in the proliferation culture medium was 1.0 mg / L 2,4-D + 0.9 mg / L 6-BA + 1.2 mg / L NAA + 0.4 mg / L ZT, the components could exert a synergistic effect, increasing the proliferation rate to 64.44%.

[0062] Table 3. Effects of different growth regulator compositions in culture media on the proliferation rate of tiger pea callus.

[0063]

[0064]

[0065] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate highly significant differences between treatments (P<0.05).

[0066] Example 4: Effect of 6-BA and NAA combination on callus differentiation

[0067] 1) Take uniformly sized, plump tiger nut tubers without insect holes and with intact buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days, changing the carbendazim treatment solution every 8 hours. After thorough soaking, peel off the intact buds from the tubers and place them in a 2mL centrifuge tube containing sterile water. In a clean bench, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds onto MS solid medium and place them in a light incubator at 30℃ and 10000lx light (14h / d) for about 20 days. Use sterile seedlings with good growth and no contamination for subsequent experiments.

[0068] 2) When the aseptic tiger nut seedlings grow to 4-5cm, take the tillering nodes of the healthy aseptic tiger nut seedlings and inoculate them into MS medium (pH 5.8) with 2.5mg / L 2,4-D, 0.6mg / L 6-BA, 1.2mg / L NAA, 0.9mg / L ZT, 4g / L PVP, 7mg / L AgNO3, and 0.5g / L CH medium. Incubate at 25℃ in the dark to induce callus tissue.

[0069] 3) After tiger nut tillers were cultured on induction medium for 15 days, explants with callus production were selected and transferred to MS medium (pH 5.8) containing 1.0 mg / L 2,4-D, 0.9 mg / L 6-BA, 1.2 mg / L NAA, 0.4 mg / L ZT, 4 g / L PVP, 7 mg / L AgNO3, and 0.5 g / L CH medium. The medium was then cultured and proliferated in the dark at 25°C.

[0070] 4) Light yellow, well-grown callus tissue was excised and inoculated into a basal medium consisting of MS + 4 g / L PVP + 7 mg / L AgNO3 + 0.5 g / L CH (acid-hydrolyzed casein) + 30 g / L sucrose + 7 g / L agar, supplemented with two plant growth regulators: 6-BA (0.3, 0.6, 0.9, 1.2 mg / L) and NAA (0.3, 0.6, 0.9, 1.2 mg / L). A total of 16 treatment combinations were established, with 30 callus tissues inoculated for each treatment, and the mixture was replicated three times. Callus differentiation was observed after 30 days of culture at 25℃, 2000 lx light intensity, and 12 h / d light duration. The callus differentiation rate and differentiation coefficient were then calculated (Table 4). Figure 4 ).

[0071] Callus differentiation rate (%) = Number of calluses producing adventitious buds / Number of inoculated calluses × 100;

[0072] Differentiation coefficient = Total number of adventitious buds / Total number of differentiated calluses.

[0073] The results showed that with increasing concentrations of 6-BA and NAA, the differentiation rate and coefficient of tiger pea callus tissue first increased and then decreased. The optimal growth state of adventitious shoots was observed under the combination of 0.6 mg / L 6-BA and 0.9 mg / L NAA. Figure 4 The callus tissue had the highest differentiation rate and differentiation coefficient, at 66.67% and 4.27, respectively (Table 4).

[0074] Table 4. Effects of different concentrations of 6-BA and NAA combinations on callus differentiation.

[0075]

[0076] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate highly significant differences between treatments (P<0.05).

[0077] Example 5: Method for callus induction and plant regeneration of tiger nuts

[0078] 1) Take uniformly sized, plump tiger nut tubers without insect holes and with intact buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days, changing the carbendazim treatment solution every 8 hours. After thorough soaking, peel off the intact buds from the tubers and place them in a 2mL centrifuge tube containing sterile water. In a clean bench, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds onto MS solid medium and place them in a light incubator at 30℃ and 10000lx light (14h / d) for about 20 days. Use sterile seedlings with good growth and no contamination for subsequent experiments.

[0079] 2) When the aseptic tiger nut seedlings grow to 4-5cm, take the tillering nodes of the healthy aseptic tiger nut seedlings and inoculate them into MS medium (pH 5.8) with 2.5mg / L 2,4-D, 0.6mg / L 6-BA, 1.2mg / L NAA, 0.9mg / L ZT, 4g / L PVP, 7mg / L AgNO3, and 0.5g / L CH medium. Incubate at 25℃ in the dark to induce callus tissue.

[0080] 3) After tiger nut tillers were cultured on induction medium for 15 days, explants with callus production were selected and transferred to MS medium (pH 5.8) containing 1.0 mg / L 2,4-D, 0.9 mg / L 6-BA, 1.2 mg / L NAA, 0.4 mg / L ZT, 4 g / L PVP, 7 mg / L AgNO3, and 0.5 g / L CH medium. The medium was then cultured and propagated in the dark at 25°C.

[0081] 4) Cut pale yellow, well-grown callus tissue and inoculate it into MS medium containing 0.6 mg / L 6-BA, 0.9 mg / L NAA, 4 g / L PVP, 7 mg / L AgNO3, 0.5 g / L CH (acid-hydrolyzed casein), 30 g / L sucrose, and 7 g / L. Induce adventitious shoots under the following conditions: temperature 25℃, light intensity 2000 lx, and light duration 12 h / d.

[0082] 5) Once the adventitious buds have grown to 3-5 cm, they are transferred as single buds to MS rooting medium. Rooting culture is carried out for 20 days, followed by 3 days of hardening off in half-open culture bottles. Then, they are transplanted into sandy soil and cultured at 30℃, with a light intensity of 12000 lx and a light duration of 12 h / d. Watering is done as needed, with nutrient solution applied every 7 days, until plant regeneration is complete. Figure 5 ).

[0083] In summary, tiger nut tillering nodes are the optimal explants for callus induction; 2.5 mg / L 2,4-D + 0.6 mg / L 6-BA + 1.2 mg / L NAA + 0.9 mg / L ZT is the optimal plant growth regulator combination for callus induction; 1.0 mg / L 2,4-D + 0.9 mg / L 6-BA + 1.2 mg / L NAA + 0.4 mg / L ZT is the optimal plant growth regulator combination for callus proliferation; and 0.6 mg / L 6-BA + 0.9 mg / L NAA is the optimal plant growth regulator combination for callus differentiation. The method of this invention produces callus with good growth status and high callus induction efficiency, proliferation, and differentiation.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for inducing callus and regenerating plants from tiger nuts, characterized in that, Including callus induction medium, proliferation medium and differentiation medium; The callus induction medium consisted of MS medium + 2.5 mg / L 2,4-D + 0.6 mg / L 6-BA + 1.2 mg / L NAA + 0.9 mg / L ZT + 4 g / L PVP + 7 mg / L AgNO3 + 0.5 g / L CH + 30 g / L sucrose + 7 g / L agar, pH=5.8; The proliferation medium was MS medium + 1.0 mg / L 2,4-D + 0.9 mg / L 6-BA + 1.2 mg / L NAA + 0.4 mg / L ZT + 4 g / L PVP + 7 mg / L AgNO3 + 0.5 g / L CH, 30 g / L sucrose + 7 g / L agar, pH=5.8; The differentiation medium was MS medium + 0.6 mg / L 6-BA + 0.9 mg / L NAA + 4 g / L PVP + 7 mg / L AgNO3 + 0.5 g / L CH + 30 g / L sucrose + 7 g / L agar, pH=5.8; The culture method using tiger nut callus induction and plant regeneration medium includes obtaining explants and first inoculating them into the induction medium, wherein the explants are tiger nut tillers.

2. A method for inducing callus and regenerating plants from tiger nuts, characterized in that, Includes the following steps: 1) Obtain sterile seedlings and select tillering nodes from sterile seedlings as explants; 2) Inoculate the explants obtained in step 1) into callus induction medium and induce culture to obtain callus tissue; 3) Inoculate the callus tissue from step 2) into the proliferation medium and culture to obtain proliferated callus tissue; 4) Inoculate the proliferated callus obtained in step 3) into differentiation medium and culture to obtain differentiated adventitious shoots; 5) Inoculate the differentiated adventitious buds obtained in step 4) into the rooting medium, and carry out rooting culture to complete plant regeneration; The callus induction medium, proliferation medium, and differentiation medium are the media as described in claim 1.

3. The method for inducing callus and regenerating plants from tiger nuts as described in claim 2, characterized in that, The specific method for obtaining sterile seedlings in step 1) is as follows: Take tiger pea tubers that are uniform in size, plump, free of insect holes, and have complete buds, soak them in a 0.2% carbendazim solution, and shake them in a constant temperature shaker at 30℃ and 200 rpm for 3 days. Change the carbendazim treatment solution every 8 hours. After soaking, peel off the complete buds from the tubers. Under sterile conditions, treat the peeled buds with 75% ethanol for 1 min, wash them once with sterile water, treat them with 5.0% NaClO for 1 min, and wash them 3-5 times with sterile water. Inoculate the sterilized buds onto MS solid medium and place them in a light incubator at 30℃, 10000 lx light, and 14 h / d for about 20 days. Take sterile seedlings that are free of pollution and in good growth condition to obtain explants.

4. The method for inducing callus and regenerating plants from tiger nuts as described in claim 3, characterized in that, In step 1), the specific method for obtaining explants is as follows: when tiger nut sterile seedlings grow to 4-5cm, take the tillering nodes of the healthy tiger nut sterile seedlings as explants.

5. The method for inducing callus and regenerating plants from tiger nuts as described in claim 4, characterized in that, Both steps 2) and 3) are cultured under 25°C dark conditions. Step 4) The culture conditions for differentiation culture are 25 ℃, light intensity of 2000 lx, and light duration of 12 h / d.

6. The method for inducing callus and regenerating plants from tiger nuts as described in claim 5, characterized in that, Step 5) The specific cultivation method is as follows: When the adventitious buds in step 4) grow to 3-5 cm, they are transferred to the rooting medium MS in the form of single buds. After rooting culture for 20 days and hardening off in half-open culture bottles for 3 days, they are transplanted into sandy soil and cultured under the conditions of 30 ℃, light intensity of 12000 lx and light duration of 12 h / d. During this period, watering is carried out as needed, and nutrient solution is applied once every 7 days to complete plant regeneration.

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