An efficient method for inducing callus and regeneration of Festuca glauca

Abstract

The present invention relates to the technical field of plant tissue culture, and specifically discloses a method for efficiently inducing callus and regeneration of Festuca glauca. In the present invention, the seeds of Festuca glauca "Frosty" are used as explants. After disinfection and sterilization, callus induction is carried out. By inducing callus maturation, embryogenic callus is selected as the receptor, and seed disinfection and pretreatment, callus induction, subculture, differentiation culture and seedling transplantation are carried out in sequence, and embryogenic callus with light yellow hard granular shape and high differentiation rate is successfully obtained. It has the advantages of simple operation, convenient material collection, significantly higher propagation coefficient than outdoors, and fast growth rate of seedlings. The present invention obtains Festuca glauca callus and regenerated seedlings with seeds as explants for the first time, and establishes a perfect and efficient regeneration system in Festuca glauca, laying a foundation for obtaining new varieties of Festuca glauca with excellent stress resistance traits and functional verification of key genes through transgenic technology.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for efficiently inducing callus and regeneration of Festuca glauca. Background Art

[0002] Festuca glauca is a perennial herb of the genus Festuca in the family Poaceae. It belongs to the cool-season blue series variety among ornamental grass species and has a cool color tone. Festuca glauca has the characteristics of tufted growth and dormancy in summer with high temperature and high humidity. It can be planted in patches in the garden or used as an edging plant, and can also play a unique role in flower bed or flower border configuration. It is an excellent potted plant.

[0003] Since Festuca glauca is not tolerant to high temperature and humidity, it is often adversely affected by abiotic stresses including drought, salt stress, high temperature, and low temperature. Therefore, it is of great significance to cultivate Festuca glauca varieties with strong stress resistance. Using genetic engineering to improve existing varieties is an important means, but the regeneration system of Festuca glauca has not been reported at home and abroad. Therefore, establishing a perfect regeneration system in Festuca glauca can lay a solid foundation for improving the stress resistance of Festuca glauca through transgenic technology in the field of genetic engineering in the future. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a method for inducing callus and differentiating and regenerating Festuca glauca using mature seeds of Festuca glauca, which has the advantages of simple operation, convenient material collection, significantly higher propagation coefficient than outdoors, and fast growth rate of seedlings. It can quickly propagate a large number of tissue culture seedlings with consistent traits, consistent growth, and robustness in a short time.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: a method for efficiently inducing callus and regeneration of Festuca glauca, comprising the following steps:

[0006] S1: Seed pretreatment: Remove and separate the glumes of the seeds, and after disinfection treatment and blotting to dry, obtain sterile seeds;

[0007] S2: Callus induction: Inoculate the sterile seeds obtained in S1 onto the callus induction medium to induce callus;

[0008] S3: Callus subculture: Transfer the callus obtained by culturing in S2 to the subculture medium for subculture;

[0009] S4: Callus differentiation: Transfer the callus obtained by culturing in S3 to the differentiation medium for differentiation culture;

[0010] S5: Rooting and transplanting: When the seedling height of the seedlings cultured in S4 reaches 4 - 5 cm, cut them and transfer them to the rooting medium, and transplant them when a large number of adventitious roots grow.

[0011] Further, in S1: The seed glumes were separated by oscillating and soaking in a 50% sulfuric acid solution for 25 minutes, washed twice with distilled water to remove the glumes; the disinfection treatment was: soaking in 50 mL of sodium hypochlorite solution for 10 min, gently shaken 2 - 3 times during this period, then washed 5 times with sterile water, placed on sterile filter paper to absorb moisture, and the seeds were longitudinally cut along the groove with a disinfected sterile scalpel and set aside on the ultra-clean workbench.

[0012] The 50% sulfuric acid solution was obtained by mixing concentrated sulfuric acid and water, v / v = 1:1; the sodium hypochlorite solution was obtained by mixing an aqueous sodium hypochlorite solution with an available chlorine concentration of 5.6% and water in a volume ratio of 1:98.

[0013] Further, the culture conditions in S2 were: temperature 25°C, in the dark, cultured for 20 d.

[0014] Further, the callus induction medium in S2 was based on MS medium, supplemented with 2,4-D, NAA, sucrose, and agar, and the addition amounts of 2,4-D, NAA, sucrose, and agar were 0 - 12.0 mg / L, 0.5 mg / L, 30.0 g / L, and 8.0 g / L respectively.

[0015] Further, the culture conditions in S3 were: temperature 25°C, in the dark, cultured for 28 d.

[0016] Further, the subculture medium in S3 was based on MS medium, supplemented with 2,4-D, NAA, sucrose, mannitol, and agar, and the addition amounts of 2,4-D, NAA, sucrose, mannitol, and agar were 1.0 - 12.0 mg / L, 0.5 mg / L, 30.0 g / L, 20 g / L, and 8.0 g / L respectively.

[0017] Further, the culture conditions in S4 were: temperature 25°C, light culture for 16.0 h per day, cultured for 25 d.

[0018] Further, the differentiation medium in S4 was based on MS medium, supplemented with 6-BA, NAA, sucrose, and agar, and the addition amounts of 6-BA, NAA, sucrose, and agar were 0 - 3.0 mg / L, 0.1 mg / L, 30.0 g / L, and 8.0 g / L respectively.

[0019] Further, the culture conditions in S5 were: temperature 25°C, light intensity 2500 - 3000 lx, light culture for 10 h per day, cultured for 25 - 30 days. When a large number of adventitious roots grew, the bottle cap was opened for acclimatization, and then transplanted into a flower pot filled with nutrient soil and continued to be cultured into seedlings in the greenhouse.

[0020] Furthermore, in step S5, the rooting medium is based on 1 / 2 MS medium and supplemented with NAA, sucrose, and agar, and the addition amounts of NAA, sucrose, and agar are 0 - 3.0 mg / L, 30.0 g / L, and 8.0 g / L respectively.

[0021] Furthermore, the pH values of the callus induction medium, subculture medium, differentiation medium, and rooting medium are all 5.8.

[0022] Furthermore, in step S2, the callus induction medium is based on MS medium and supplemented with 2,4 - D 8.0 mg / L, NAA 0.5 mg / L, sucrose 30.0 g / L, and agar 8.0 g / L.

[0023] Furthermore, in step S3, the subculture medium is based on MS medium and supplemented with 2,4 - D 4.0 mg / L, NAA 0.5 mg / L, sucrose 30.0 g / L, and agar 8.0 g / L.

[0024] Furthermore, in step S4, the differentiation medium is based on MS medium and supplemented with 6 - BA 1 mg / L, NAA 0.1 mg / L, sucrose 30.0 g / L, and agar 8.0 g / L.

[0025] Furthermore, in step S5, the rooting medium is based on 1 / 2 MS medium and supplemented with NAA 0.5 - 1.0 mg / L, sucrose 30.0 g / L, and agar 8.0 g / L.

[0026] The beneficial effects brought by the technical solution of the present invention are as follows: This method has the advantages of simple operation, convenient material collection, significantly higher propagation coefficient than outdoors, and fast growth rate of seedlings. It can quickly propagate a large number of tissue - cultured seedlings with consistent traits, consistent growth, and robustness in a relatively short time. Compared with previous studies, the survival rate of the tissue - cultured seedlings of the present invention is higher, and they are extremely easy to survive after acclimatization and transplantation outdoors, with a survival rate as high as 100%. The present invention uses sodium hypochlorite solution to disinfect the explants, which is more environmentally friendly than using mercuric chloride for disinfection in previous studies, and greatly reduces the poisoning risk to operators.

[0027] The present invention first obtained Festuca glauca callus and regenerated seedlings using seeds as explants, established a perfect and efficient regeneration system in Festuca glauca, laid a foundation for obtaining new Festuca glauca varieties with excellent stress - resistance traits and verifying the functions of key genes through transgenic technology, and has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effect of different 2,4 - D concentrations on the callus induction rate in callus induction culture

[0029] Figure 2Effects of different 2,4-D concentrations on callus formation rate in subcultured embryonic callus

[0030] Figure 3 Effects of different 6-BA concentrations on callus differentiation

[0031] Figure 4 Effects of different NAA concentrations on rooting rate

[0032] Figure 5 Seeds and callus tissue inoculated on callus induction medium (MS+2,4-D 8.0 mg / L)

[0033] Figure 6 Embryogenic callus obtained on subculture medium (MS+2,4-D 4.0 mg / L)

[0034] Figure 7 Green seedlings on differentiation medium (MS+6-BA 1.0 mg / L)

[0035] Figure 8 Blue fescue regeneration seedlings transplanted into pots DETAILED DESCRIPTION

[0036] Combine Figure 1-4 and attached Figure 5-8 The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Example

[0038] 1. Experimental plan of the embodiment of the present invention

[0039] 1 Materials and Methods

[0040] 1.1 Test materials

[0041] Mature seeds of blue fescue ‘Festina’ were used as explants.

[0042] 1.2 Culture medium

[0043] 1.2.1 Induction medium

[0044] MS medium + 2,4-D (0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 12 mg / L) + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8, with a total of 8 treatments, to verify the optimal 2,4-D concentration for callus induction of the Festuca glauca variety (Non-Stina).

[0045] 1.2.2 Subculture medium

[0046] MS medium + 2,4-D (1.0 mg / L, 2.0 mg / L, 4.0 mg / L, 8.0 mg / L, 12.0 mg / L) + NAA (0.5 mg / L) + sucrose (30.0 g / L) + mannitol (20.0 g / L) + agar (8.0 g / L), pH = 5.8, with a total of 5 treatments, to screen the optimal 2,4-D concentration in the subculture medium.

[0047] 1.2.3 Differentiation medium

[0048] MS medium + 6-BA (0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 3 mg / L) + NAA (0.1 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8, with a total of 5 treatments, to verify the optimal 6-BA concentration for callus differentiation of the Festuca glauca variety (Non-Stina).

[0049] 1.2.4 Rooting medium

[0050] 1 / 2 MS medium + NAA (0, 0.5, 1.0, 2.0, 3.0 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8.

[0051] 1.3 Experimental methods

[0052] 1.3.1 Material disinfection

[0053] For the mature and plump seeds of Festuca glauca, remove and separate the seed glumes, and after disinfection treatment and blotting to dry, obtain sterile seeds.

[0054] 1.3.2 Callus induction

[0055] For the air-dried mature seeds of Festuca glauca (after longitudinally cutting along the groove and making the cut surface contact the medium), inoculate them on the induction media of each treatment. The number of replicates (number of conical flasks) for each treatment in each experiment is 3 - 4 times. The number of explants inoculated into each conical flask is 20. After inoculation, place the conical flasks on a special tissue culture rack, and after dark culture at 25°C for 30 d, count the callus induction rate before transferring to the subculture medium.

[0056] 1.3.3 Callus Subculture

[0057] After about 30 days of induction, when the callus grows to a size of 2 - 3 mm in diameter, promptly dissect the callus and transfer it to a subculture medium containing different concentrations of 2,4 - D, and culture it in the dark at 25°C for 28 days. Then, before transferring to the differentiation medium, count the callus induction rate of embryogenic callus.

[0058] 1.3.4 Callus Differentiation

[0059] When the callus is dense in structure, granular, and light yellow, transfer it to the differentiation medium of each treatment. Green buds appear after 14 days, and rootless seedlings gradually form after 25 - 30 days of culture. Count the callus differentiation rate at 14 days, and count the average number of green buds before transferring to the rooting medium. The culture conditions during the differentiation culture are: temperature 25°C, light culture for 16.0 h per day, and culture for 25 days.

[0060] 1.3.5 Rooting and Transplanting

[0061] Transfer the rootless seedlings to the rooting medium. After 25 - 30 days of culture, a large number of adventitious roots are produced. Open the bottle cap to harden the seedlings. After 3 days of hardening, transplant them into flower pots filled with nutrient soil and continue to culture them into seedlings in the greenhouse.

[0062] 1.4 Data Statistics and Analysis

[0063] Callus induction rate = (Number of explants with callus growth) ÷ (Number of explants for callus induction) × 100%;

[0064] Embryogenic callus induction rate = (Number of callus pieces forming embryogenic callus) ÷ (Number of callus pieces for subculture) × 100%;

[0065] Average number of green buds = (Number of green buds formed) ÷ (Number of callus pieces for differentiation)

[0066] Differentiation rate = (Number of callus pieces with seedlings) ÷ (Number of callus pieces for differentiation) × 100%

[0067] Rooting rate = (Number of seedlings with roots) ÷ (Number of seedlings for rooting test) × 100%

[0068] II. Specific Steps of the Method for Efficient Induction of Callus and Regeneration of Festuca glauca in the Present Invention

[0069] 1. Seed Disinfection and Pretreatment

[0070] Remove the seed impurities. Place the seeds in a 50% sulfuric acid solution (obtained by mixing sulfuric acid and water in a volume ratio of 50:50) and soak them with shaking for 25 minutes. Wash them twice with distilled water to remove the bran. Then add them to 50 mL of sodium hypochlorite solution (obtained by mixing sodium hypochlorite aqueous solution with an available chlorine concentration of 5.6% and water in a volume ratio of 1:98) and soak for 10 min. Shake gently 2 - 3 times during this period, then wash with sterile water, place on sterile filter paper to absorb the moisture, and longitudinally cut the seeds with a sterilized sterile scalpel to obtain sterile seed materials.

[0071] 2. Callus induction of Festuca glauca

[0072] Place the dried mature seeds of Festuca glauca (after longitudinally cutting along the groove, the cut surface contacts the medium) on the induction medium of each treatment. The number of replicates (number of conical flasks) for each treatment in each experiment is 3 - 4 times. The number of explants inoculated into each conical flask is 20. After inoculation, place the conical flasks on a special tissue culture rack and culture them in the dark at 25°C for 20 d. Then, before transferring to the subculture medium, count the callus induction rate (see Figure 1 ). To verify the optimal 2,4-D concentration for inducing callus of Festuca glauca variety (Non-Stina). The formula of the induction medium is: MS medium + 2,4-D (0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 12 mg / L) + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8, with a total of 8 treatments.

[0073] It can be seen that the treatment method of MS + 2,4-D 8.0 mg / L ( Figure 5 ) belongs to the optimal selection scheme.

[0074] 3. Subculture of Festuca glauca callus and screening of embryogenic callus

[0075] After inducing for about 20 d, when the callus grows to a size of 1.0 - 2.0 cm in diameter, timely peel the callus and transfer it to the subculture medium containing different concentrations of 2,4-D, and culture it in the dark at 25°C for 28 d. Then, before transferring to the differentiation medium, count the callus induction rate of yellow granular embryogenic callus. Subculture medium: MS medium + 2,4-D (1.0 mg / L, 2.0 mg / L, 4.0 mg / L, 8.0 mg / L, 12.0 mg / L) + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8, with a total of 5 treatments, and screen the 2,4-D concentration in the optimal subculture medium.

[0076] It can be seen that as the concentration of 2,4-D increased from 1.0 mg / L to 12.0 mg / L, the induction rate of embryogenic callus remained at a relatively high level, and the induction rate of embryogenic callus was between 53% and 78%( Figure 2 ). The callus induction rate of the treatment with 4.0 mg / L was the highest, reaching 84%( Figure 6 ).

[0077] 4. Pre-culture of callus

[0078] Transfer the callus to a hypertonic medium and pre-culture it in the dark at 25 °C for 15 d. The hypertonic medium is a sterile medium obtained by adding 2,4-D, sucrose and agar to the MS medium. Among them, the addition amounts of 2,4-D, sucrose and agar are 4.0 mg / L, 60.0 g / L and 8.0 g / L respectively, and the pH is 5.8.

[0079] 5. Differentiation culture

[0080] Select embryogenic callus with a dense, granular and light yellow structure, transfer it to the differentiation medium of each treatment. Green buds appear after 14 d, and rootless seedlings gradually form after culturing for 25 - 30 d. The differentiation rate of callus is counted at 14 d, and the average number of green buds is counted before transferring the differentiated green buds to the rooting medium. The culture conditions during the differentiation culture are: temperature 25 °C, light culture for 16.0 h per day, and culture for 25 d. Differentiation medium: MS medium + 6-BA (0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 3 mg / L) + NAA (0.1 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8, a total of 5 treatments, and the optimal 6-BA concentration for the differentiation of Festuca glauca variety (Non-Stina) callus is screened.

[0081] When the concentration of 6-BA was 1.0 mg / L, the callus differentiation rate reached the highest and the number of green buds was the largest, which were 76.5% and 3.6( Figure 3 , Figure 7 ). Therefore, considering the differentiation rate and the average number of green buds, the optimal 6-BA concentration for the differentiation of Festuca glauca callus is 1.0 mg / L.

[0082] 6. Rooting culture and transplantation

[0083] Transfer the rootless seedlings to the rooting medium. A large number of adventitious roots are produced after culturing for 25 - 30 d. After rooting, open the bottle cap to harden the seedlings. After hardening for 3 d, transplant them into flower pots filled with nutrient soil and continue to culture them into seedlings in the greenhouse( Figure 8) The rooting medium is: 1 / 2 MS medium + NAA (0, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 3.0 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L), pH = 5.8.

[0084] III. Results and Analysis

[0085] 1. Appearance Morphological Differences in Callus Induction

[0086] After the seeds of Festuca glauca were inoculated on the induction media with different treatments, it was found that a small number of seeds began to germinate on the 2nd day. As the number of days increased, the buds grew continuously, and the formation of callus could be observed. After about 4 weeks of culture and observation, it was found that some seeds gradually produced a kind of white, transparent, water-soaked, soft, loose, and non-fixed morphological callus (non-embryogenic callus), which always maintained a loose water-soaked state during growth, did not differentiate, and gradually turned dark brown and died in the later stage of growth. Some seeds showed a kind of callus (embryogenic callus) with a relatively firm structure, opacity, rice grain shape, yellowish-white or light green color, which had good differentiation ability and was the callus that needed to be continuously subcultured in the experiment.

[0087] 2. Effects of Different 2,4-D Concentrations on the Callus Induction Rate in Callus Induction Culture

[0088] There were differences in the effects of different 2,4-D concentration treatments on the callus state ( Figure 1 ), and the multiple comparison results of the treatment means ( Figure 1 ) showed that 8 mg / L belonged to the optimal selection scheme, and the callus induction rate was 43.5%. It can be seen that when using the longitudinally cut seeds as explants, the treatment method of MS + 2,4-D 8.0 mg / L can be adopted ( Figure 5 ).

[0089] 3. Effects of Different 2,4-D Concentrations on the Callus Induction Rate of Embryogenic Callus in Subculture

[0090] The callus obtained from the longitudinally cut seeds on 8 kinds of induction media was transferred to the MS subculture media containing different concentrations of 2,4-D (one bottle for each of the five treatments) respectively, and the callus induction rate of embryogenic callus was counted after culturing for 28 days under light conditions. The comparison results of the means ( Figure 2 ) showed that as the 2,4-D concentration increased from 1.0 mg / L to 12.0 mg / L, the induction rate of embryogenic callus remained at a relatively high level, and the callus induction rate was between 53% and 84%. The treatment with 4.0 mg / L had the highest callus induction rate of embryogenic callus, reaching 84% ( Figure 6 ).

[0091] 4. Effects of 6-BA on the Differentiation of Callus

[0092] As can be seen Figure 3 from the figure, when the concentration of 6-BA is 0, the callus does not differentiate, indicating that 6-BA plays a great promoting role in the differentiation of callus. When the concentration of 6-BA is 1 mg / L, the differentiation rate of callus reaches the highest, which is 76%( Figure 7 ). A high green shoot regeneration rate is not necessarily the best differentiation medium. We also need to consider another factor, that is, the average number of green buds. The statistical results( Figure 3 ) show that the average number of green buds is the highest when the concentration of 6-BA is 1 mg / L and 2 mg / L. Therefore, considering the differentiation rate and the average number of green buds comprehensively, we believe that the optimal concentration of 6-BA for the differentiation of Festuca glauca callus is 1 mg / L.

[0093] 5. Rooting culture

[0094] As can be seen Figure 4 from the figure, when the concentration of NAA is in the range of 0.5 - 1.0 mg / L, the rooting medium is used to culture the differentiated green shoots without roots, and the rooting rate reaches 100%. Moreover, the roots generated in this concentration range grow robustly, and the regenerated seedlings grow well( Figure 4 ). Therefore, the suitable concentration range of NAA for rooting of Festuca glauca screened by us is 0.5 - 1.0 mg / L.

[0095] It should be noted that in this article, position terms such as up, down, left, and right are only for convenience of description. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0096] Of course, the above description is only the preferred embodiment of the present invention. The present invention is not limited to listing the above embodiments. It should be noted that all equivalent substitutions and obvious deformation forms made by any person skilled in the art under the guidance of this specification fall within the substantial scope of this specification and should be protected by the present invention.

Claims

1. A method for inducing, differentiating and regenerating callus of Festuca glauca, characterized in that, It includes the following steps: S1: Seed pretreatment: Remove the seed glumes, and after disinfection and moisture absorption, obtain sterile seeds; S2: Callus induction: Inoculate the sterile seeds obtained in S1 onto the callus induction medium to induce callus. Among them, the callus induction medium is MS medium + 8 mg / L 2,4-D + 0.5 mg / L NAA + 30.0 g / L sucrose + 8.0 g / L agar; S3: Callus subculture: Transfer the callus obtained by culturing in S2 to the subculture medium for subculture. Among them, the subculture medium is MS medium + 4 - 12 mg / L 2,4-D + 0.5 mg / L NAA + 30.0 g / L sucrose + 20 g / L mannitol + 8.0 g / L agar; S4: Callus differentiation: Transfer the callus to the hypertonic culture medium and pre-culture it in the dark at 25°C for 15 d. The hypertonic culture medium is MS medium + 4.0 mg / L 2,4-D + 60.0 g / L sucrose + 8.0 g / L agar. Select the embryogenic callus with a dense structure, granular shape, and light yellow color, and transfer it to the differentiation medium for differentiation culture. The differentiation medium is MS medium + 1 - 3.0 mg / L 6-BA + 0.1 mg / L NAA + 30.0 g / L sucrose + 8.0 g / L agar; S5: Rooting and transplantation: When the seedling height of the seedlings cultured in S4 reaches 4 - 5 cm, cut them and transfer them to the rooting medium. Transplant them when a large number of adventitious roots grow. The rooting medium is 1 / 2 MS medium + 0.5 - 1.0 mg / L NAA + 30.0 g / L sucrose + 8.0 g / L agar; In S1: Place the seeds in a 50% sulfuric acid solution and soak them with shaking for 25 minutes. After washing with distilled water, remove the glumes. The disinfection treatment is: soak them in 50 mL of sodium hypochlorite solution for 10 min, wash them with sterile water, place them on sterile filter paper to absorb moisture, and longitudinally cut the seeds along the groove with a sterilized sterile scalpel and set aside.

2. A method for inducing, differentiating and regenerating callus of Festuca glauca, according to claim 1, characterized in that The culture conditions in S2 are: temperature 25°C, dark, culture for 20 d.

3. A method for inducing, differentiating and regenerating the callus of Festuca glauca, as claimed in claim 1, wherein The culture conditions in S3 are: temperature 25°C, dark, culture for 28 d.

4. A method for inducing, differentiating and regenerating the callus of Festuca glauca, as claimed in claim 1, wherein The culture conditions during the differentiation culture in S4 are: temperature 25°C, light culture for 16.0 h per day, culture for 25 d.

5. A method for inducing, differentiating and regenerating the callus of Festuca glauca, as claimed in claim 1, wherein The culture conditions in S5 are: temperature 25°C, light intensity 2500 - 3000 lx, light culture for 10 h per day, culture for 25 - 30 days.

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