A festuca mature seed callus induction medium, induction method and a method for establishing a regeneration system

Abstract

The application provides a kind of festuca ovina mature seed callus induction medium, induction method and the method for establishing regeneration system, belong to plant tissue culture technical field.The application provides a kind of callus induction medium, with MS medium as base medium, further include the following components with the following content: 2, 4-dichlorophenoxyacetic acid 4~6 mg / L, hydrolyzed casein 40~60 mg / L, abscisic acid 1~3 mg / L and kinetin 0.04~0.07 mg / L.The callus induction medium described in the application can efficiently induce mature seeds to form callus, and the induction rate of callus is more than 81%.The application selects festuca ovina mature seeds as explants for callus induction culture, which is convenient and low in cost, and lays a foundation for the research of festuca ovina gene editing.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a medium for inducing callus from mature seeds of Festuca aurantium, an induction method, and a method for establishing a regeneration system. Background Technology

[0002] Purple fescue ( Festuca rubra (L.) also known as red foxgrass, belongs to the genus Festuca of the subfamily Pooideae in the family Gramineae. Festuca (L.) Festuca amarum is an excellent cool-season turfgrass and a superior dual-purpose (forage and ecological) grass species, belonging to the perennial herbaceous plant family. It has a well-developed root system, abundant foliage, high nutritional value, good palatability, strong grazing tolerance, and excellent drought and cold resistance, making it a promising candidate for use in pastures in high-altitude and cold regions. Festuca amarum can also accelerate ecological restoration, revitalize degraded grassland vegetation, and fully realize the productive potential of grasslands, playing a significant role in strengthening grassland ecological environments and preventing desertification. In conclusion, Festuca amarum is a grass species with immense development potential.

[0003] Plant tissue culture, also known as aseptic plant culture or in vitro culture, is a technique based on the theory of totipotency of plant cells. It utilizes isolated plant organs, tissues, cells, and protoplasts under sterile conditions and suitable artificial culture media and temperatures to induce callus formation, adventitious buds, and adventitious roots, ultimately leading to the formation of complete plants. Tissue culture technology offers advantages such as rapid propagation, high production efficiency, preservation of the original variety's inherent shape and characteristics, and conservation of propagation materials. Currently, there are few reports on callus induction culture of mature seeds of *Festuca amurensis*. Summary of the Invention

[0004] In view of this, the present invention provides a callus induction culture medium, which uses mature seeds of red fescue as explant material and obtains high-quality callus by regulating exogenous hormones and culture medium formulation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a callus induction culture medium, wherein the callus induction culture medium is based on MS medium and further comprises the following components in the following amounts: 2,4-dichlorophenoxyacetic acid 4~6 mg / L, hydrolyzed casein 40~60 mg / L, abscisic acid 1~3 mg / L and kinetin 0.04~0.07 mg / L.

[0006] This invention provides a method for inducing callus formation in mature seeds of Festuca purpurea, wherein sterile mature seeds of Festuca purpurea are induced and cultured using the callus induction culture medium to obtain callus.

[0007] Preferably, the induction culture time is 30-60 days; The induction culture temperature is 23~27℃; the induction culture is a dark culture.

[0008] This invention provides a culture medium composition comprising the callus induction medium and the differentiation medium; The differentiation medium is based on 1 / 2 MS medium and also includes the following components in the following amounts: kinetin 0.01~0.03 mg / L and naphthaleneacetic acid 0.3~0.5 mg / L.

[0009] Preferably, it also includes a rooting medium; The rooting medium is MS medium containing 0.3~0.5 g / L naphthaleneacetic acid.

[0010] This invention provides a method for establishing a regeneration system of red fescue, which induces mature red fescue seeds to form callus tissue. The callus was differentiated and cultured using the differentiation medium in the culture medium composition to obtain regenerated seedlings; The regenerated seedlings were subjected to rooting culture to obtain regenerated plants.

[0011] Preferably, the rooting culture is performed using the rooting medium in the culture medium composition to culture the regenerated seedling.

[0012] Preferably, the differentiation culture time is 24-32 days; The differentiation culture temperature is 23~27℃; the differentiation culture is carried out under light; the photoperiod light-dark ratio of the light is (14~18):(6~10), and the light intensity is 1500~3000 lux.

[0013] Preferably, the rooting culture temperature is 23~27℃; the rooting culture is carried out under light; the photoperiod light-dark ratio of the light is (14~18):(6~10), and the light intensity is 2000~2500 lux.

[0014] Preferably, after the rooting culture, the process further includes hardening-off treatment; The substrate for the hardening treatment includes peat moss and vermiculite. Compared with the prior art, the present invention has the following advantages: This invention provides a callus induction medium based on MS medium, further comprising the following components in the following concentrations: 2,4-dichlorophenoxyacetic acid (2,4-D) 4-6 mg / L, hydrolyzed casein (CH) 40-60 mg / L, abscisic acid (ABA) 1-3 mg / L, and kinetin (KT) 0.04-0.07 mg / L. The callus induction medium of this invention can efficiently induce callus formation from mature seeds. In embodiments of this invention, the callus induction medium was also used to induce mature seeds, and results showed that the callus induction rate reached over 81%.

[0015] This invention provides a culture medium composition comprising a callus induction medium and a differentiation medium; the differentiation medium is based on 1 / 2 MS medium and further comprises the following components in concentrations: KT 0.01~0.03 mg / L and naphthaleneacetic acid (NAA) 0.3~0.5 mg / L. The callus induction medium in this invention's culture medium composition can efficiently induce mature seeds to form callus, which is then further differentiated into adventitious shoots using the differentiation medium. Results from embodiments of this invention show that, during differentiation culture, 35% of the callus induced from mature seeds can differentiate into adventitious shoots and produce regenerated plants; while the callus induction rate induced using young stem leaf sheaths as explants is only 28%, and the callus induced from leaf sheaths begins to differentiate into shoots after 10 days of subculture on the differentiation medium, with a differentiation rate of 17%.

[0016] This invention provides a method for establishing a regeneration system for *Festuca aurantium*. The method induces callus formation from mature *Festuca aurantium* seeds; the callus is differentiated and cultured using the differentiation medium in the culture medium composition to obtain regenerated seedlings; the regenerated seedlings are then subjected to rooting culture to obtain regenerated plants. This invention utilizes mature *Festuca aurantium* seeds as explants to induce callus and regulates the ratio of exogenous hormones in the callus induction medium to improve the callus induction rate. The method for establishing a mature *Festuca aurantium* seed callus induction and regeneration system constructed in this invention has significant advantages such as high proliferation efficiency, short culture cycle, and strong genetic stability, effectively maintaining the genetic traits and phenotypic characteristics of the original variety. This technical system provides an efficient technical support platform for cutting-edge fields such as plant gene editing, genetic transformation research, agronomic trait improvement, and germplasm resource innovation. Attached Figure Description

[0017] Figure 1 Morphological diagram of callus tissue induced from mature seeds of Festuca aurantiacus; Figure 2 A diagram showing the morphology of green seedlings differentiated from callus tissue of Festuca auriculata; Figure 3 A diagram showing the morphology of a rooted fescue seedling; Figure 4 This is a diagram showing the morphology of regenerated plants after transplanting of red fescue. Detailed Implementation

[0018] The present invention provides a callus induction culture medium, wherein the callus induction culture medium is based on MS medium and further comprises the following components in the following amounts: 2,4-D 4~6 mg / L, CH 40~60 mg / L, ABA 1~3 mg / L and KT 0.04~0.07 mg / L.

[0019] In this invention, the callus induction medium is based on MS medium, and preferably further comprises the following components in the following amounts: 2,4-D 4.5~5.5 mg / L, CH 45~55 mg / L, ABA 1.5~2.5 mg / L, and KT 0.05~0.06 mg / L; more preferably, it comprises the following components in the following amounts: 2,4-D 5 mg / L, CH 50 mg / L, ABA 2 mg / L, and KT 0.055 mg / L. The pH of the callus induction medium is preferably 5.7~6.0, more preferably 5.8~5.9, and most preferably 5.85. The 2,4-D component promotes cell elongation and division in plants; the CH component induces cell division, particularly in root and shoot tip meristems and callus tissue, synergistically regulating cell division with auxin and promoting cell differentiation, thus participating in plant organ formation; the ABA component inhibits excessive cell growth, resulting in compact and dense callus tissue; and the KT component promotes cell division and bud differentiation. This callus induction medium can effectively induce callus formation from mature seeds, achieving an induction rate of up to 81%.

[0020] This invention provides a method for inducing callus formation in mature seeds of Festuca purpurea, wherein sterile mature seeds of Festuca purpurea are induced and cultured using the callus induction culture medium to obtain callus.

[0021] In this invention, the mature fescue seeds preferably include plump fescue seeds. The sterilization reagent for the sterilization treatment of the mature fescue seeds preferably includes a sodium hypochlorite solution. The mass percentage of sodium hypochlorite in the sodium hypochlorite solution is preferably 35% to 45%, more preferably 40%. After sterilization, the seeds are preferably rinsed with sterile water and then dried with sterilized filter paper.

[0022] In this invention, the preferred temperature for the induction culture is 23-27°C, more preferably 24-26°C, and most preferably 25°C; the preferred light condition for the induction culture is dark culture. The preferred induction culture time is 30-60 days, more preferably 35-55 days, more preferably 40-50 days, and most preferably 45 days. These induction culture conditions are beneficial for promoting callus formation from mature fescue seeds.

[0023] This invention uses mature seeds of *Festuca aurantium* as explants for induction culture, resulting in a high callus induction rate. In one embodiment of this invention, to improve the callus acquisition rate, different materials (mature seeds, young stems and leaf sheaths) were subjected to induction culture. The results showed that the callus induction rate using mature seeds as explants was as high as 81%, while that of leaf sheaths was only 14-28%.

[0024] The present invention provides a culture medium composition comprising the callus induction medium and the differentiation medium; the differentiation medium is based on 1 / 2 MS medium and further comprises the following components in the following amounts: KT 0.01~0.03 mg / L and NAA 0.3~0.5 mg / L.

[0025] In this invention, the differentiation medium is based on 1 / 2 MS medium, preferably further comprising the following components in amounts: KT 0.015~0.025 mg / L and NAA 0.35~0.45 mg / L, more preferably comprising the following components in amounts: KT 0.02 mg / L and NAA 0.4 mg / L. The role of KT is to induce callus differentiation into shoots and promote cell proliferation; the role of NAA is to assist cytokinin in inducing callus differentiation into adventitious shoots, preventing excessive cell elongation and maintaining the normal morphology of the shoots. The differentiation medium can induce callus differentiation into adventitious shoots, with a differentiation rate reaching 35%.

[0026] In this invention, the culture medium composition preferably further includes a rooting medium; the rooting medium is preferably MS medium containing 0.3~0.5 g / L NAA, more preferably containing 0.35~0.45 g / L NAA, and most preferably containing 0.4 g / L NAA. When the NAA concentration is relatively high and the cytokinin concentration is low, it can strongly induce callus tissue or adventitious buds to differentiate into adventitious roots, realizing the "bud-to-root" process, and ultimately forming complete test-tube seedlings. The rooting medium of this component has a very good rooting effect.

[0027] In this invention, in order to effectively inhibit the growth of miscellaneous bacteria, TMT can be added to the culture medium, and the working concentration of TMT is preferably 200 mg / L.

[0028] This invention provides a method for establishing a regeneration system of red fescue, which induces mature red fescue seeds to form callus tissue. The callus tissue was differentiated and cultured using the differentiation medium in the culture medium composition to obtain regenerated seedlings; the regenerated seedlings were then subjected to rooting culture to obtain regenerated plants.

[0029] In this invention, the temperature for differentiation culture is preferably 23-27℃, more preferably 24-26℃, and most preferably 25℃; the differentiation culture is conducted under light; the photoperiod light-dark ratio of the light is (14-18):(6-10), more preferably (15-17):(7-9), and even more preferably 16:8; the light intensity for differentiation culture is preferably 1500-3000 lux, more preferably 1800-2800 lux, more preferably 1900-2700 lux, even more preferably 2000-2500 lux, and most preferably 2200 lux. The differentiation culture time is preferably 24-32 days, more preferably 26-30 days, more preferably 27-29 days, and most preferably 28 days. After differentiation culture, the regenerated seedlings grow to about 3 cm. The conditions of the differentiation culture are conducive to promoting the differentiation of adventitious buds from callus tissue.

[0030] In this invention, the rooting culture is performed using the rooting medium in the culture medium composition to cultivate the regenerated seedlings. Preferably, the regenerated seedlings are isolated into individual plants and inoculated into the rooting medium during the rooting culture. The temperature for the rooting culture is preferably 23-27°C, more preferably 24-26°C, and most preferably 25°C; the rooting culture is conducted under light; the photoperiod of the light is preferably the same as that of the differentiation culture. The light conditions are preferably fluorescent lamps with a light intensity of 2000-2500 lux, and most preferably 2200 lux. The rooting culture is preferably performed until the root length is 5-7 cm.

[0031] In this invention, the rooting culture preferably includes a hardening-off treatment. The hardening-off treatment preferably involves opening the rooted seedlings and hardening them off for 3 days before transplanting them into a hardening-off substrate. The hardening-off substrate includes peat moss and vermiculite. The volume ratio of peat moss to vermiculite is preferably (1~2):(1~2), more preferably 1:1.

[0032] In the tissue culture process of *Festuca purpurea*, factors affecting the success of tissue culture include explant source, culture medium composition, culture light, and culture temperature. This invention utilizes mature seeds and young stem and leaf sheaths of *Festuca purpurea* as explants to study the effects of different explants on callus induction and seedling differentiation, ultimately obtaining a method for establishing a *Festuca purpurea* regeneration system.

[0033] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Establishment of a callus induction and regeneration system for Festuca purpurea The seeds of *Festuca purpurea* were provided by the State Key Laboratory of Ecology and Plateau Agriculture and Animal Husbandry of the Three Rivers Source Region, jointly established by Qinghai University and Qinghai Province. Plump *Festuca purpurea* seeds were selected for cultivation, or appropriately sized young stem and leaf sheaths from *Festuca purpurea* seedlings were used as explants. The methods for establishing the *Festuca purpurea* callus induction and regeneration system are as follows.

[0035] 1. Explant disinfection treatment Select plump seeds of red fescue as explants. Disinfect the explants with 40% sodium hypochlorite (10% available chlorine concentration) for 5 min, wash with water 3 times, then disinfect with sodium hypochlorite for 10 min. Under sterile conditions in a laminar flow hood, rinse with sterile water 5 times, and then wipe the surface of the explants dry with sterilized filter paper.

[0036] 2. Inducing callus formation Mature seeds of *Festuca aurantium* were inoculated onto a callus-inducing medium and cultured in the dark at 25±2℃ for 1 to 2 months. This induced whitish, transparent callus suitable for subsequent experiments. The callus-inducing medium consisted of MS basal medium + 2,4-D (4 mg / L) + CH (50 mg / L) + ABA (2 mg / L) + KT (0.04 mg / L). The number of callus formed within 60 days was counted, and the callus induction rate was calculated.

[0037] 3. Subculture in differentiation medium The induced callus tissue was inoculated into differentiation medium to obtain regenerated seedlings. The differentiation medium consisted of 1 / 2 MS basal medium + KT (0.02 mg / L) + NAA (0.4 mg / L), pH: 5.8–5.9. The culture was conducted under 2500 lux light for 16 h / d and 8 h / d darkness at 23–27°C for 4 weeks until the seedlings reached approximately 3 cm in length. The differentiation and proliferation medium was cultured under 2200 lux light for 16 h / d and 8 h / d darkness at 25 ± 2°C. The number of differentiated tissues within 60 days of differentiation culture was counted, and the callus differentiation rate was calculated.

[0038] 4. Rooting Culture The differentiated regenerated seedlings were inoculated into a rooting medium for rooting induction. The rooting medium was MS basal medium + 0.5 g / L NAA, pH: 5.8~5.9, and cultured under ordinary fluorescent light (light intensity 2200 lux) at a temperature of 25±2℃ until roots were robust and 5-7 cm in length. The culture conditions for the rooting medium were: ordinary fluorescent light (light intensity 2200 lux) at a temperature of 23~27℃.

[0039] 5. Seedling hardening and transplanting After the test-tube seedlings were opened and hardened off for 3 days, the seedlings were removed, the roots were washed to remove any residual culture medium, and then transplanted to obtain complete regenerated plants.

[0040] Transplant the plant into a pot containing a 1:1 mixture of peat moss and vermiculite to obtain a fully regenerated plant.

[0041] Figure 1 Morphological diagram of callus tissue induced from mature seeds of Festuca aurantiacus; Figure 2 A diagram showing the morphology of green seedlings differentiated from callus tissue of Festuca auriculata; Figure 3 A diagram showing the morphology of a rooted fescue seedling; Figure 4 This is a diagram showing the morphology of regenerated plants after transplanting of red fescue.

[0042] Comparative Example 1 Methods for establishing a callus induction and regeneration system of Fescue 1. Explant disinfection treatment Select plump red fescue seeds, disinfect them with 40% sodium hypochlorite (10% available chlorine concentration) for 5 min, wash them 3 times with water, then disinfect them with sodium hypochlorite for 10 min. Under sterile conditions in a laminar flow hood, rinse them 5 times with autoclaved ddH2O, and then wipe the surface of the red fescue seeds dry with sterilized filter paper. Inoculate mature red fescue seeds onto seedling culture medium (MS medium) and culture for 20 days. Cut appropriately sized leaf sheaths from sterile red fescue seedlings as explants and inoculate them onto callus induction medium for callus culture.

[0043] 2. The methods for inducing callus formation, subculture in differentiation medium, rooting culture, and hardening and transplanting are the same as in Example 1.

[0044] Example 2 Statistics on callus induction rate and differentiation rate The callus induction rate and differentiation rate of Example 1 and Comparative Example 1 were statistically analyzed. The results showed significant differences in callus induction rates among different explants. The callus induction rate of mature seeds was 67%–81%, while that of leaf sheaths was only 14%–28% (Table 1). After the formation of obvious callus tissue, it was necessary to continue culturing for another month before it could be used for subsequent experiments. The number of callus tissues formed within 60 days was counted, and the callus induction rate was calculated using Formula I. The number of differentiated tissues within 60 days of differentiation culture was counted, and the callus differentiation rate was calculated using Formula II.

[0045] Callus induction rate (%) = (Number of callus generated / Number of explants) × 100% Formula I Adventitious shoot differentiation rate (%) = (Number of differentiated callus / Total number of callus) × 100% Formula II Table 1. Time and induction rate of callus induction using different explants.

[0046] Although both seed-induced and leaf-sheath-induced calluses can differentiate into shoots, their differentiation rates differ significantly. Seed-induced calluses show a differentiation rate of 27%–35% on differentiation medium, while leaf-sheath-induced calluses show a lower differentiation rate of approximately 9%–17% on differentiation medium.

[0047] Comparative Example 2 To screen for culture media that promote adventitious shoot differentiation, the effects of different differentiation media on the composition of different media and their influence on adventitious shoot differentiation were compared. The callus induced in Example 1 was inoculated into different differentiation media and cultured under the same conditions as the subculture conditions in Example 1. The results are shown in Table 2.

[0048] Table 2. Composition of different culture media and their effects on adventitious bud differentiation

[0049] It is evident that differentiation medium 4 is beneficial for promoting adventitious bud differentiation.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A callus induction culture medium, characterized in that, The callus induction medium is based on MS medium and also includes the following components in the following amounts: 2,4-dichlorophenoxyacetic acid 4~6 mg / L, hydrolyzed casein 40~60 mg / L, abscisic acid 1~3 mg / L and kinetin 0.04~0.07 mg / L.

2. A method for inducing callus formation in mature seeds of *Festuca purpurea*, characterized in that, Callus tissue was obtained by inducing the culture of sterile mature seeds of red fescue using the callus induction medium described in claim 1.

3. The method according to claim 2, characterized in that, The induction culture time is 30-60 days; The induction culture temperature is 23~27℃; the induction culture is a dark culture.

4. A culture medium composition, characterized in that, Includes the callus induction culture medium and differentiation culture medium as described in claim 1; The differentiation medium is based on 1 / 2 MS medium and also includes the following components in the following amounts: kinetin 0.01~0.03 mg / L and naphthaleneacetic acid 0.3~0.5 mg / L.

5. The culture medium composition according to claim 4, characterized in that, It also includes rooting culture medium; The rooting medium is MS medium containing 0.3~0.5 g / L naphthaleneacetic acid.

6. A method for establishing a regeneration system for red fescue, characterized in that, Inducing callus formation in mature seeds of Festuca purpurea using the method described in claim 2; The callus was differentiated and cultured using the differentiation medium in the culture medium composition of claim 4 to obtain regenerated seedlings; The regenerated seedlings were subjected to rooting culture to obtain regenerated plants.

7. The method for establishing according to claim 6, characterized in that, The rooting culture is performed by culturing the regenerated seedlings in the rooting medium of the culture medium composition of claim 5.

8. The method for establishing according to claim 6, characterized in that, The differentiation culture time is 24-32 days; The differentiation culture temperature is 23~27℃; the differentiation culture is carried out under light; the photoperiod light-dark ratio of the light is (14~18):(6~10), and the light intensity is 1500~3000 lux.

9. The method for establishing according to claim 6, characterized in that, The rooting culture temperature is 23~27℃; the rooting culture is carried out under light; the photoperiod light-dark ratio of the light is (14~18):(6~10), and the light intensity is 2000~2500 lux.

10. The method for establishing according to any one of claims 6 to 9, characterized in that, The rooting culture process also includes hardening-off treatment; The substrate for the hardening treatment includes peat moss and vermiculite.

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