A tissue culture rapid propagation method for Poa pratensis L. var. alpigena

Abstract

The present invention belongs to the technical field of plant cultivation, and particularly relates to a method for rapid tissue culture propagation of alpine wild Kentucky bluegrass. The tissue culture medium for alpine wild Kentucky bluegrass of the present invention comprises a primary medium, a multiple shoot induction medium and a rooting medium; 6-BA and 2,4-D are added to the primary medium and the multiple shoot induction medium; NAA is added to the rooting medium. Under the combined action of 6-BA, 2,4-D and NAA in each medium, the seeds of wild Kentucky bluegrass are induced to germinate and tissue differentiation occurs, obtaining a large number of multiple shoots and tissue culture seedlings, which can not only maintain the original genetic traits and bio-ecological characteristics, but also rapidly propagate to obtain seedlings, realizing the rapid domestication and breeding of native grass seeds, and providing effective technical support for the preservation of excellent traits of wild grass seeds and the breeding of new varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant cultivation, and particularly relates to a method for rapid tissue culture propagation of alpine wild Kentucky bluegrass (Poa pratensis L.). Background Art

[0002] Annual bluegrass (Poa annua L.) is a plant of the Poaceae family and a perennial winter grass of the genus Poa. The culms of annual bluegrass are erect, smooth and glabrous; the leaves are flat or folded, soft in texture, and slightly rough at the edges; the panicle is broadly ovate, and the spikelets are ovate; the caryopsis is spindle-shaped; the flowering period is from April to May; the fruiting period is from June to July. Annual bluegrass likes a warm and dry environment, and is relatively drought-tolerant, shade-tolerant and cold-tolerant; it prefers slightly acidic to neutral soil; it can overwinter smoothly at low temperatures, has poor heat resistance, has a developed root system, and has strong reproductive and regeneration abilities. Annual bluegrass is distributed in Inner Mongolia, Shanxi, Hebei, Liaoning, Jilin, Heilongjiang and other places in China, and also in Asia, Europe, North America, etc. Annual bluegrass has the effects of clearing heat and detoxifying, promoting diuresis and detumescence, relieving cough, and lowering blood sugar, and is used to treat eczema, traumatic injuries, coughs, etc.; at the same time, the stems and leaves of annual bluegrass are soft and have high nutritional value, and it is an excellent forage. In addition, annual bluegrass has good homogeneity density and smoothness, and is often used for building various lawns.

[0003] Kentucky bluegrass is a species under the genus Poa of the Poaceae family. It is widely distributed in the temperate zones of Eurasia and North America. It is often distributed in high-altitude areas such as Heilongjiang, Jilin, Liaoning, Inner Mongolia, Hebei, Shanxi, Henan, Shandong, Shaanxi, Gansu, Qinghai, Xinjiang, Tibet, Sichuan, Yunnan, Guizhou, Hubei, Anhui, Jiangsu, Jiangxi, etc. in China. It is drought-resistant, cold-resistant and trampling-resistant, with soft and fine grass quality, bright and fresh green color, and a long green period. It is widely used as an ornamental lawn in public green spaces such as families, parks, hospitals, schools, etc., and can also be used as a facility lawn for golf courses, sports fields, dam slopes, etc. At the same time, it is tender, fresh green, has high nutritional value, good palatability, and is liked by horses, cows, sheep, etc., and is also an excellent forage grass.

[0004] Poa pratensis L. is a rhizomatous grass of the lower type. It can reproduce vegetatively through underground rhizomes and sexually through seeds. However, the rhizomes of wild Poa pratensis are short and have limited expansion ability. The seeds have low seed setting and germination rates, making it difficult to quickly obtain a sufficient number of seedlings for breeding, which has become a bottleneck in the creation of wild native grass germplasm resources and the breeding of new varieties. At the same time, Poa pratensis under different habitat conditions has very different morphological characteristics and stress-resistant physiology, and the excellent germplasm resources it contains are also very different, which also creates an opportunity for the breeding of new varieties of water-saving, drought-resistant and cold-tolerant Poa pratensis. The alpine wild Poa pratensis in the present invention is collected from the sunny slope of the mountain top at an altitude of 1500 - 2000 m in Inner Mongolia. The annual rainfall in this area is less than 300 mm, the soil is poor and arid, and the extreme winter temperature is below -30°C. Its drought and cold resistance far exceed that of ordinary Poa pratensis recognized by the public, and it has great scientific research and breeding value. However, under natural environmental conditions, the seed setting rate and seed germination rate of this grass species are extremely low, and the transplanted native seedlings grow slowly and are not easy to survive. Therefore, in view of the actual situation of the difficult collection of alpine grass seed resources, the limited number of seeds and seedlings obtained, and the difficulty of rapid propagation of the original species, proposing an efficient and practical technology to improve the germination rate and propagation coefficient of alpine wild Poa pratensis seeds is the key to breaking through the bottleneck of the germplasm resource creation, new variety breeding and original species rapid propagation technology system. Summary of the Invention

[0005] The object of the present invention is to provide a tissue culture and rapid propagation method for alpine wild Poa pratensis. Using the tissue culture medium provided by the present invention for the tissue culture of alpine wild Poa pratensis can not only improve the germination rate of alpine wild Poa pratensis, but also quickly obtain a large number of clustered buds and healthy tissue culture seedlings of Poa pratensis in a short period, improve the propagation efficiency, and improve the transplanting survival rate of tissue culture seedlings through indoor acclimation and transplanting substrate formula, so as to realize the efficient proliferation and rapid propagation of alpine wild Poa pratensis.

[0006] In order to solve the above problems, the present invention provides the following technical solutions:

[0007] The present invention provides a tissue culture medium for alpine wild Poa pratensis, and the tissue culture medium includes a primary medium, a clustered bud induction medium and a rooting medium;

[0008] The primary medium is based on the MS medium and further includes: 0.1 - 0.5 mg / L 6-BA, 0.8 - 1.0 mg / L 2,4-D, 26 - 30 g / L sucrose and 6.5 - 7.0 g / L agar;

[0009] The clustered bud induction medium is based on the MS medium and further includes: 0.1 - 0.5 mg / L 6-BA, 1.0 - 1.5 mg / L 2,4-D, 28 - 32 g / L sucrose and 6.5 - 7.5 g / L agar;

[0010] The rooting medium uses 1 / 2 MS medium as the basic medium and further includes: 0.2 - 0.8 mg / L NAA, 29 - 32 g / L sucrose, and 6.5 - 7.5 g / L agar.

[0011] Preferably, the pH value of the primary medium is 6.6 - 7.0; the pH value of the multiple shoot induction medium is 6.6 - 7.0; the pH value of the rooting medium is 6.6 - 7.0.

[0012] The present invention provides a tissue culture and rapid propagation method for Poa pratensis L. var. alpigena, and the tissue culture and rapid propagation method uses the tissue culture medium described in the above technical solution, including the following steps:

[0013] Inoculate Poa pratensis L. var. alpigena seeds on the primary medium for primary culture to obtain Poa pratensis L. var. alpigena seed buds;

[0014] Inoculate the shoot tips of the Poa pratensis L. var. alpigena seed buds on the multiple shoot induction medium for subculture and proliferation to obtain Poa pratensis L. var. alpigena plantlets without roots;

[0015] Transfer the Poa pratensis L. var. alpigena plantlets without roots to the rooting medium for rooting culture, and after the rooting culture, obtain complete tissue culture seedlings for acclimatization culture and transplanting and hardening off.

[0016] Preferably, the length of the shoot tips of the Poa pratensis L. var. alpigena seed buds is 0.5 - 1.0 cm.

[0017] Preferably, the temperatures of the primary culture, subculture and proliferation culture, and rooting culture are 23 - 27 °C respectively.

[0018] Preferably, the primary culture, subculture and proliferation culture, and rooting culture are all carried out under light - dark alternating conditions. The light - culture time in each light - dark alternating cycle is 10 - 12 h / d respectively, and the dark - culture time in each light - dark alternating cycle is 12 - 14 h / d respectively; the light intensity is 2000 - 2500 lx respectively.

[0019] Preferably, the time of the primary culture is 35 - 70 d, the time of the subculture and proliferation culture is 40 - 60 d, and the time of the rooting culture is 15 - 22 d.

[0020] Preferably, before the Poa pratensis L. var. alpigena seeds are inoculated into the primary medium, it further includes: rubbing the seeds, cleaning, and disinfecting them in sequence.

[0021] Preferably, the cleaning includes detergent cleaning and running - water rinsing. The detergent cleaning time is 10 - 20 min, and the running - water rinsing time is 25 - 35 min;

[0022] The disinfection method includes: after cleaning, first soak in 70%-75% alcohol by mass concentration for 25-35 s, then soak in 0.1% mercuric chloride solution for 8-10 min; finally rinse with sterile water 3-4 times.

[0023] Preferably, the acclimation culture and transplanting and hardening are completed indoors and in a greenhouse respectively. The composition of the substrate for transplanting and hardening includes peat, perlite and vermiculite; the volume ratio of peat, perlite and vermiculite is (5-6):(1-2):(1-2).

[0024] The beneficial effects of the present invention: The present invention provides a tissue culture medium for alpine wild Kentucky bluegrass. The tissue culture medium includes a primary medium, a multiple shoot induction medium and a rooting medium; the primary medium is based on MS medium and further includes: 0.1-0.5 mg / L 6-BA, 0.8-1.0 mg / L 2,4-D, 26-31 g / L sucrose and 6.5-7.0 g / L agar; the multiple shoot induction medium is based on MS medium and further includes: 0.1-0.5 mg / L 6-BA, 1.0-1.5 mg / L 2,4-D, 28-32 g / L sucrose and 6.5-7.5 g / L agar; the rooting medium is based on 1 / 2MS medium and further includes: 0.2-0.8 mg / L NAA, 29-32 g / L sucrose and 6.5-7.5 g / L agar.

[0025] In the tissue culture medium provided by the present invention, 2,4-D (2,4-dichlorophenoxyacetic acid) promotes the germination of alpine wild Kentucky bluegrass seeds and the formation of callus, and 6-BA (6-benzylaminopurine) stimulates cell division to promote the growth and development of multiple shoots of alpine wild Kentucky bluegrass; NAA (naphthaleneacetic acid) promotes the rooting of tissue culture seedlings of alpine wild Kentucky bluegrass; 2,4-D and 6-BA act on seed germination and multiple shoot differentiation, and NAA acts on the rooting stage. Under the combined action of the above three components, the formation, differentiation and rooting of rootless seedlings of alpine wild Kentucky bluegrass are induced, the propagation coefficient is increased, and a large number of tissue culture seedlings of alpine wild Kentucky bluegrass are obtained. The results of the examples show that a large number of multiple shoots and tissue culture seedlings obtained by using the tissue culture medium of the present invention grow well, and the propagation coefficient after proliferation culture reaches 3.8, and finally tissue culture plants of alpine wild Kentucky bluegrass with good consistency and strong growth can be obtained. It can be seen that the primary medium, multiple shoot induction medium and rooting medium described in the present invention have good application effects, and the components cooperate with each other, and tissue culture plants of alpine wild Kentucky bluegrass with good consistency and robustness can be obtained under the control of appropriate component concentrations. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the embodiments.

[0027] Figure 1 It is the external view of seeds of Poa pratensis L. var. alpigena Wirtg.

[0028] Figure 2 It is the situation on the 1st day when the seeds of Poa pratensis L. var. alpigena Wirtg. in Example 1 are inoculated in the primary medium for culture.

[0029] Figure 3 It is the seed germination situation on the 17th day when the seeds of Poa pratensis L. var. alpigena Wirtg. in Example 1 are inoculated in the primary medium for culture.

[0030] Figure 4 It is the seed germination situation on the 35th day when the seeds of Poa pratensis L. var. alpigena Wirtg. in Example 1 are inoculated in the primary medium for culture.

[0031] Figure 5 It is the situation where the seeds of Poa pratensis L. var. alpigena Wirtg. in Example 1 germinate and show cluster bud differentiation on the 59th day when inoculated in the primary medium for culture.

[0032] Figure 6 The situation on the 48th day when the rootless seedlings of Poa pratensis L. var. alpigena Wirtg. in Example 1 are cultured in the induction medium for cluster buds.

[0033] Figure 7 The situation on the 18th day when the rooted seedlings of Poa pratensis L. var. alpigena Wirtg. in Example 1 are cultured in the rooting medium.

[0034] Figure 8 The situation on the 20th day when the transplanted seedlings of Poa pratensis L. var. alpigena Wirtg. in Example 1 are cultured.

[0035] Figure 9 It is the seed germination situation on the 17th day when the seeds of Poa pratensis L. var. alpigena Wirtg. in Comparative Example 1 are cultured.

[0036] Figure 10 It is the survival situation of the domesticated seedlings of Poa pratensis L. var. alpigena Wirtg. in Example 1 after 7 days of field transplantation. Specific Embodiments

[0037] The present invention provides a tissue culture medium for Poa pratensis L. var. alpigena Wirtg., and the tissue culture medium includes a primary medium, a cluster bud induction medium, and a rooting medium.

[0038] In the present invention, the primary culture medium is based on MS medium and further comprises: 0.1 - 0.5 mg / L 6 - BA, 0.8 - 1.0 mg / L 2,4 - D, 26 - 30 g / L sucrose and 6.5 - 7.0 g / L agar; more preferably, it is based on MS medium and only additionally contains: 0.1 - 0.5 mg / L 6 - BA, 0.8 - 1.0 mg / L 2,4 - D, 26 - 31 g / L sucrose and 6.5 - 7.0 g / L agar. In the present invention, the concentration of 6 - BA in the primary culture medium is 0.1 - 0.5 mg / L, preferably 0.15 - 0.3 mg / L, and more preferably 0.2 mg / L. The concentration of 2,4 - D in the primary culture medium is 0.8 - 1.0 mg / L, preferably 0.9 - 1.0 mg / L, and more preferably 1.0 mg / L. The concentration of sucrose in the primary culture medium is 26 - 31 g / L, more preferably 30 g / L. The concentration of agar in the primary culture medium is 6.5 - 7.0 g / L, more preferably 7 g / L.

[0039] In the present invention, the pH value of the primary culture medium is preferably 6.6 - 7.0, and more preferably 6.80.

[0040] Adding plant growth regulators 6 - BA and 2,4 - D to the primary culture medium of the present invention can promote the seed germination of Poa pratensis L. var. spontanea Regel, and the germination rate of Poa pratensis L. var. spontanea Regel seeds can reach over 80%.

[0041] In the present invention, the multiple - shoot induction medium is based on MS medium and further comprises: 0.1 - 0.5 mg / L 6 - BA, 1.0 - 1.5 mg / L 2,4 - D, 28 - 32 g / L sucrose and 6.5 - 7.5 g / L agar; more preferably, it is based on MS medium and only additionally contains: 0.1 - 0.5 mg / L 6 - BA, 1.0 - 1.5 mg / L 2,4 - D, 28 - 32 g / L sucrose and 6.5 - 7.5 g / L agar. In the present invention, the concentration of 6 - BA in the multiple - shoot induction medium is 0.1 - 0.5 mg / L, preferably 0.2 - 0.4 mg / L, and more preferably 0.3 mg / L. The concentration of 2,4 - D in the multiple - shoot induction medium is 1.0 - 1.5 mg / L, preferably 1.2 - 1.5 mg / L, and more preferably 1.5 mg / L. The concentration of sucrose in the multiple - shoot induction medium is 28 - 32 g / L, more preferably 30 g / L. The concentration of agar in the multiple - shoot induction medium is 6.5 - 7.5 g / L, preferably 6.8 - 7.3 g / L, and more preferably 7.0 g / L. In the present invention, 6 - BA and 2,4 - D can jointly promote the formation of rootless seedlings of Poa pratensis L. var. spontanea Regel at an appropriate concentration ratio, and improve the propagation coefficient.

[0042] In the present invention, the pH value of the cluster bud induction medium is preferably 6.6 - 7.0, more preferably 6.80.

[0043] In the present invention, the rooting medium uses 1 / 2 MS medium as the basic medium and further comprises: 0.2 - 0.8 mg / L NAA, 29 - 32 g / L sucrose, and 6.5 - 7.5 g / L agar; more preferably, it uses 1 / 2 MS medium as the basic medium and only additionally contains: 0.2 - 0.8 mg / L NAA, 29 - 32 g / L sucrose, and 6.5 - 7.5 g / L agar. In the present invention, the concentration of NAA in the rooting medium is 0.2 - 0.8 mg / L, preferably 0.4 - 0.6 mg / L, more preferably 0.5 mg / L. The concentration of sucrose in the rooting medium is 29 - 32 g / L, preferably 28 - 30 g / L, more preferably 30 g / L. The concentration of agar in the rooting medium is 6.5 - 7.5 g / L, more preferably 7 g / L. In the present invention, NAA can promote the rooting of the rootless seedlings of Poa pratensis var. alpigena, and a large number of rooted seedlings of Poa pratensis var. alpigena can be obtained.

[0044] In the present invention, the pH value of the rooting medium is preferably 6.6 - 7.0, more preferably 6.80.

[0045] The Poa pratensis var. alpigena described in the present invention is a local species under the genus Poa. The solutions described in the present invention are mainly applicable to Poa pratensis var. alpigena in high-altitude areas, but also have certain reference significance for the tissue culture of other drought-tolerant Poa pratensis. The present invention has no special limitation on the source of the Poa pratensis var. alpigena.

[0046] The tissue culture medium provided by the present invention has good application effects, can improve the germination rate and propagation coefficient of Poa pratensis var. alpigena seeds, obtain a large number of tissue-cultured seedlings of Poa pratensis var. alpigena, and improve the propagation efficiency of the original seedlings of Poa pratensis var. alpigena.

[0047] The present invention has no special limitation on the sources of the 6-BA, 2,4-D, NAA, sucrose, and agar, and conventional commercially available products can be used. Wild grass seeds are very sensitive to the hormone concentration. Normally, a concentration change of 0.1 mg / L can bring significant differences in the results. Therefore, the concentration ranges of the primary medium, cluster bud induction medium, and rooting medium in the present invention need to be strictly controlled, otherwise it will have a serious impact on the tissue culture effect.

[0048] The present invention also provides a tissue culture rapid propagation method for Poa pratensis var. alpigena. The tissue culture rapid propagation method uses the tissue culture medium described in the above technical solution and comprises the following steps:

[0049] Inoculate the seeds of Poa pratensis L. var. alpigena into the primary culture medium for primary culture to obtain the buds of Poa pratensis L. var. alpigena seeds;

[0050] Inoculate the shoot tips of the obtained buds of Poa pratensis L. var. alpigena seeds into the multiple shoot induction medium for subculture and proliferation to obtain the rootless seedlings of Poa pratensis L. var. alpigena;

[0051] Transfer the obtained rootless seedlings of Poa pratensis L. var. alpigena to the rooting medium for rooting culture, and after the rooting culture, obtain the complete tissue culture seedlings for acclimatization culture and transplanting and hardening off.

[0052] In the present invention, there is no special limitation on the source of Poa pratensis L. var. alpigena. The Poa pratensis L. var. alpigena described in the present invention is a wild grass species, growing on the mountain tops at an altitude of 1500 - 2000 m, with insufficient rainfall throughout the year, poor and arid soil, and the extreme winter temperature being lower than -30 °C, and the growth environment is harsh. The collection site of the Poa pratensis L. var. alpigena described in the present invention is the sunny slope area of the mountain tops in Inner Mongolia region.

[0053] In the present invention, the seeds of Poa pratensis L. var. alpigena are inoculated into the primary culture medium for primary culture to obtain the buds of Poa pratensis L. var. alpigena seeds. The present invention uses the seeds of Poa pratensis L. var. alpigena as explants for cultivation. The seeds of Poa pratensis L. var. alpigena described in the present invention are preferably the seeds of Poa pratensis L. var. alpigena with plump grains.

[0054] Before inoculating the seeds of Poa pratensis L. var. alpigena described in the present invention into the primary culture medium, it also includes successively carrying out husk rubbing, cleaning and disinfection. There is no special limitation on the operation of the husk rubbing in the present invention, and it is preferably a conventional technical means. The husk rubbing in the present invention is preferably carefully rubbing off the epidermis on the surface of the seeds. The function of the husk rubbing: The seeds of common Poa pratensis can germinate normally without husk rubbing, but the seeds of Poa pratensis L. var. alpigena are very small and are also wrapped with bran on the outside. If not rubbed off before germination, it is easy to cause incomplete disinfection between the bran and the seed gaps, which is likely to lead to the deterioration of the culture medium and the failure of germination. In addition, after being wrapped with gauze, the disinfectant can be retained for a longer time, and the seeds are not easy to leak out from the gaps, which is beneficial to improving the disinfection effect. One layer of gauze is enough for wrapping.

[0055] After rubbing the seeds to remove the husks, the present invention preferably wraps the seeds with gauze and then performs cleaning and disinfection. When wrapping the seeds with gauze in the present invention, the number of layers of the gauze is preferably one layer. The cleaning in the present invention preferably includes detergent cleaning and running water rinsing, and the present invention has no special limitation on the specific methods of detergent cleaning and running water cleaning. In the present invention, the detergent is preferably dishwashing liquid, and more preferably low-concentration dishwashing liquid. The concentration of the low-concentration dishwashing liquid in the present invention is preferably obtained by diluting the dishwashing liquid 300 to 400 times. The dishwashing liquid in the present invention is a commercially available conventional dishwashing liquid. The cleaning time of the low-concentration dishwashing liquid in the present invention is preferably 10 to 20 minutes, and more preferably 15 minutes. The running water rinsing time in the present invention is preferably 25 to 35 minutes, and more preferably 30 minutes. The present invention has no special limitation on the water used for the running water rinsing, and conventional tap water can be used. The detergent cleaning and running water rinsing in the present invention can effectively clean the dust on the surface of the Poa annua seeds.

[0056] After cleaning, the present invention disinfects the seeds. The present invention has no special limitation on the disinfection method. The disinfectant used for disinfection in the present invention is preferably ethanol with a volume concentration of 70% to 75% and mercuric chloride solution with a mass concentration of 0.1%, and more preferably ethanol with a volume concentration of 75% and mercuric chloride solution with a mass concentration of 0.1%. When disinfecting the seeds in the present invention, it is preferably first soaked in 75% alcohol by mass for 25 to 35 seconds, more preferably 30 seconds, and then preferably soaked in 0.1% mercuric chloride solution for 8 to 10 minutes, more preferably 9 minutes; finally, it is preferably rinsed with sterile water 3 to 4 times, more preferably 4 times. The present invention has no special limitation on the sterile water, and it is preferably obtained by sterilizing distilled water. The dosages of the 75% alcohol and mercuric chloride in the present invention are both preferably sufficient to submerge the Poa annua seeds. The 0.1% mercuric chloride in the present invention is preferably obtained by diluting 1 g of mercuric chloride to 1000 mL. The ethanol in the present invention belongs to a medium-level disinfectant, which can kill bacterial vegetative cells but cannot kill bacterial spores; the mercuric chloride can kill various microorganisms, including bacterial vegetative cells, bacterial spores, viruses, fungi, etc., and its advantage is strong bactericidal effect; the combined disinfection with ethanol and mercuric chloride ensures the disinfection effect.

[0057] After cleaning and disinfecting the Poa annua seeds, the present invention preferably dries the Poa annua seeds wrapped in gauze with filter paper. After opening the gauze, the seeds are taken out, placed in a petri dish with filter paper, and the surface moisture is dried again, and then inoculated onto the primary medium to obtain Poa annua seeds buds of alpine wild grassland.

[0058] In the present invention, the temperature of the primary culture is preferably 23 to 27 °C, further preferably 24 to 26 °C, and more preferably 25 °C.

[0059] In the present invention, the primary culture is preferably carried out under the condition of alternating light and darkness; in the present invention, the light culture time of each light-dark cycle is preferably 10-12 h / d, more preferably 11-12 h / d, and still more preferably 12 h / d; the dark culture time in each light-dark cycle of the present invention is preferably 12-14 h / d, more preferably 12-13 h / d, and still more preferably 12 h / d; the intensity of the light in the present invention is preferably 2000-2500 lx, more preferably 2100-2400 lx, and still more preferably 2200 lx. Each light-dark cycle of the present invention is preferably 24 h.

[0060] The primary culture time of the present invention is preferably 35-70 d, more preferably 55-60 d, and still more preferably 58 d. When the seeds of alpine wild Kentucky bluegrass are subjected to primary culture for 35 d in the present invention, the germination rate can reach 80%, while for the ordinary germination method without using the primary culture medium of the present invention, the germination rate of the seeds after 35 d of culture is less than 5% (see Figure 9 ). Thus, it can be seen that the germination rate of the germination method of the present invention is significantly higher than that of the ordinary germination method.

[0061] There is no special limitation on the number of seeds of alpine wild Kentucky bluegrass inoculated in each primary culture flask in the present invention, and it can be selected according to the routine. In the prior art, generally, the explant tissue is collected from parts such as the leaves, stem nodes, stem segments, shoot tips, root tips, and flower spikes of the original plants. However, the collection of flower spikes is difficult, and the natural environment pollution of other parts during tissue culture is serious. It is not easy to disinfect after collection, and it is easy to be infected, resulting in poor tissue culture effect. In this solution, after the wild seeds are disinfected and germinated, the actively growing part of the shoot tip is collected, which greatly reduces the probability of tissue infection and improves the vitality and survival rate of the cultured tissue. Moreover, for the tissue culture technology that usually uses seeds as explants, it is necessary to germinate in a culture dish first and then take the seedling tissue. In this solution, after the collected seeds are disinfected once, seed germination and sampling of the shoot tip of the seedling are carried out on the same culture medium, which not only simplifies the operation procedure but also avoids the risk of contamination, greatly improving the tissue culture efficiency.

[0062] The present invention uses the seeds of alpine wild Kentucky bluegrass as explants for tissue culture and bud differentiation cultivation, and solves the technical problems of low germination rate and insufficient callus bud differentiation in the prior art when using seeds for conventional germination.

[0063] After the primary culture of Poa pratensis L. seeds from alpine wild grasslands, Poa pratensis L. seeds buds from alpine wild grasslands are obtained. The seed buds described in the present invention include buds (single buds) formed by the germination of seed embryos and differentiated buds (multiple buds) formed by the differentiation of callus generated near the seed hypocotyls. In the prior art, only one bud can be formed from the germination of one Poa pratensis L. seed, but in the present invention, the callus generated at the hypocotyl part during seed germination can form a new majority of differentiated buds, that is, 2-5 new buds can appear from 1 seed. Therefore, the Poa pratensis L. seeds buds from alpine wild grasslands in the present invention preferably include single buds formed by the embryo and multiple differentiated buds formed by the callus. When the seed buds are multiple differentiated buds, the present invention preferably cuts the multiple buds into single buds, and then cuts the shoot tips and inoculates them on an induction medium for subculture. The present invention preferably inoculates the shoot tips of single buds and multiple buds on a multiple shoot induction medium, which can improve the survival rate and propagation coefficient of the multiple shoot culture of Poa pratensis L. seeds from alpine wild grasslands.

[0064] Under the traditional propagation method, the germination rate and propagation coefficient of cultivated Poa pratensis L. are at most no more than 0.95, while the propagation of wild Poa pratensis L. according to the traditional method is much lower than this, even less than 0.05. Through one primary culture, the propagation coefficient of this solution can reach 3.8. If multiple subcultures are carried out after the primary culture, the propagation coefficient can reach 3.8 n (n is the number of subcultures), and the result shows an exponential growth, with an impressive propagation efficiency.

[0065] After obtaining the seed buds, the present invention inoculates the shoot tips of the Poa pratensis L. seeds buds from alpine wild grasslands on a multiple shoot induction medium for subculture and proliferation, obtaining Poa pratensis L. plantlets without roots; more preferably, inoculating the shoot tips of the buds formed by the germination of the seed embryos of the Poa pratensis L. seeds from alpine wild grasslands and the shoot tips of the differentiated buds formed by the differentiation of the callus generated near the seed hypocotyls on a multiple shoot induction medium for subculture and proliferation, obtaining Poa pratensis L. plantlets without roots.

[0066] In the present invention, it is preferred to cut the shoot tips from the embryos and differentiated buds of the callus of Poa pratensis L. from alpine wild grasslands. The length of the cut shoot tips is preferably 0.5-1 cm, and more preferably 0.6-0.8 cm.

[0067] In the present invention, the temperature for subculture proliferation culture is preferably 23 - 27°C, more preferably 24 - 26°C, and even more preferably 25°C. The subculture proliferation culture of the present invention is preferably carried out under light-dark alternating conditions; the time for light culture in each light-dark alternating cycle of the present invention is preferably 10 - 12 h / d, more preferably 11 - 12 h / d, and even more preferably 12 h / d; the time for dark culture in each light-dark alternating cycle of the present invention is preferably 12 - 14 h / d, more preferably 12 - 13 h / d, and even more preferably 12 h / d; the intensity of the light of the present invention is preferably 2000 - 2500 lx, more preferably 2100 - 2400 lx, and even more preferably 2300 lx. Each light-dark alternating cycle of the present invention is preferably 24 h.

[0068] The time for the subculture proliferation culture of the present invention is preferably 40 - 60 d, more preferably 43 - 53 d, and even more preferably 47 d.

[0069] After obtaining the rootless seedlings of Poa pratensis L. var. alpigena, the rootless seedlings of Poa pratensis L. var. alpigena of the present invention are inoculated onto a rooting medium for rooting culture. The present invention preferably inoculates onto the rooting medium for rooting culture when the height of the rootless seedlings is preferably 3 - 5 cm, more preferably 4 - 5 cm, and even more preferably 4 cm.

[0070] In the present invention, the temperature for rooting culture is preferably 23 - 27°C, more preferably 24 - 26°C, and even more preferably 25°C. The rooting culture of the present invention is preferably carried out under light-dark alternating conditions; the time for light culture in each light-dark alternating cycle of the present invention is preferably 10 - 12 h / d, more preferably 11 - 12 h / d, and even more preferably 12 h / d; the time for dark culture in each light-dark alternating cycle of the present invention is preferably 12 - 14 h / d, more preferably 12 - 13 h / d, and even more preferably 12 h / d. The intensity of the light of the present invention is preferably 2000 - 2500 lx, more preferably 2200 - 2400 lx, and even more preferably 2400 lx. Each light-dark alternating cycle of the present invention is preferably 24 h. The time for the rooting culture of the present invention is preferably 15 - 22 d, more preferably 15 - 20 d, and even more preferably 17 d.

[0071] After the rooting culture of the present invention, rooted seedlings are obtained. After the rooted seedlings meet the two conditions of having 3 - 5 roots and a root length of 3 - 4 cm, acclimatization culture and transplanting and hardening-off culture are carried out to improve the survival rate of the rooted seedlings.

[0072] After all the primary medium, multiple shoot induction medium and rooting medium of the present invention are sterilized, they are poured into tissue culture bottles. The present invention has no special limitation on the sterilization method, and a conventional method can be used.

[0073] After the rooting culture, the present invention conducts acclimatization culture and transplanting hardening. The acclimatization culture of the present invention is preferably completed indoors. The acclimatization culture of the present invention is preferably completed in the acclimatization room of the tissue culture seedling room. Before transplanting hardening, the present invention preferably conducts acclimatization culture on the rooted seedlings for 3 - 5 days, more preferably for 4 days. The temperature for indoor acclimatization of the present invention is preferably 26 - 28°C, and the light for indoor acclimatization of the present invention is preferably natural light. The present invention preferably transfers the rooted seedlings from the culture room to the acclimatization room. The temperature of the acclimatization room of the present invention is preferably 2 - 3°C higher than that of the culture room, and the temperature of the acclimatization room is preferably 26 - 28°C. The present invention preferably opens the bottle caps of the culture bottles for 3 - 5 days during indoor acclimatization. To prevent the rooted seedlings from wilting, when the air humidity in the acclimatization room reaches 70 - 80%, the present invention preferably sprays a 800-fold solution of chlorothalonil on the surface of the rooted seedlings.

[0074] After the indoor acclimatization of the tissue culture seedlings is completed, the present invention preferably conducts transplanting hardening culture. The transplanting culture of the present invention is preferably completed in a substrate plug tray. The transplanting hardening of the present invention preferably uses the healthy tissue culture seedlings after acclimatization. After washing and disinfecting the roots, they are transplanted into the substrate in the plug tray for culture. The time for transplanting hardening of the present invention is preferably 15 - 30 days, more preferably 20 - 25 days. The composition of the substrate of the present invention preferably includes peat, perlite, and vermiculite; the volume ratio of peat, perlite, and vermiculite is preferably (5 - 6):(1 - 2):(1 - 2), more preferably 6:2:2. After the acclimatized seedlings are transplanted, they are prone to drought and water shortage. After a large amount of watering, it is easy to cause poor ventilation and serious fungal infection, resulting in root rot and a significant reduction in the survival rate. Therefore, the water retention of the substrate determines the survival rate of the acclimatized seedlings after transplantation. It is necessary to rationally mix the substrate to greatly improve the survival rate of the acclimatized seedlings after transplantation. The selection of the volume ratio of peat, perlite, and vermiculite in the present invention can greatly improve the survival rate of the acclimatized seedlings after transplantation. The temperature for transplanting culture in the greenhouse of the present invention is preferably that the night temperature is preferably 23°C - 27°C, further preferably 24 - 26°C, more preferably 25°C. The humidity for transplanting culture of the present invention is preferably 70% - 80%, more preferably 75% - 80%. Before transplanting culture in the greenhouse, the present invention preferably disinfects the substrate with an 800-fold solution of chlorothalonil. The present invention has no special limitation on the disinfection method.

[0075] The transplanting culture container of the present invention is preferably a moisture-retaining seedling-raising container.

[0076] The present invention has no special limitation on the source and specification of the plug trays used in the transplanting cultivation, and conventional products can be adopted. The specification of the plug trays preferably used in the present invention is 3.8 cm × 3.8 cm × 5.5 cm. Preferably, one rooted seedling is transplanted into each hole of the plug trays in the present invention. In the embodiments of the present invention, the survival rate of the domesticated seedlings during transplantation is 100%. After the acclimatization of the seedlings in the greenhouse is completed, the plug tray seedlings can be transplanted into the field. During the processes of indoor domestication, greenhouse acclimatization and field transplantation, the survival rates of the tissue-cultured seedlings after indoor domestication and the plug tray seedlings after acclimatization in the greenhouse are both 100%. This result is the key to the ultimate realization of the propagation coefficient and also plays a decisive role in the implementation effect of the tissue culture rapid propagation technology system.

[0077] The indoor domestication cultivation and greenhouse transplanting acclimatization of the present invention can be regarded as two acclimatization stages. If the rooted seedlings in this solution do not undergo reasonable indoor domestication and greenhouse transplanting acclimatization cultivation, the survival rate of the tissue-cultured seedlings will be severely reduced. Among them, the indoor domestication of the bottle seedlings for 3 - 5 days and the greenhouse transplanting acclimatization cultivation for 15 - 30 days are the key. At the same time, combined with appropriate temperature and humidity control, the ideal acclimatization effect can be achieved.

[0078] The time of the primary culture, subculture proliferation culture, rooting culture, indoor domestication and greenhouse transplanting cultivation described in the present invention needs to be accurately controlled. The minimum time is the minimum requirement for the success of tissue culture rapid propagation, and the technical solution presents a better choice, which is the best solution after multiple experiments.

[0079] In the prior art, when raising seedlings by sowing, at most only one plant can be formed from one Poa pratensis L. var. anceps Gaud. wild in the alpine region. However, in the present invention, through one primary culture, one subculture and one rooting culture, 3 - 4 plants can be formed from 1 seed, and the propagation coefficient reaches 3.8. If multiple separations and an increase in the number of subculture generations are carried out on the basis of the primary culture, the propagation coefficient can show an exponential growth of 3.8 n times, significantly improving the propagation efficiency of Poa pratensis L. var. anceps Gaud.

[0080] Taking the mature seeds of Poa pratensis L. var. anceps Gaud. as explants, the present invention germinates the seeds and induces the callus to differentiate into buds through different hormone ratios, then obtains rootless seedlings through subculture proliferation culture, and then through rooting culture, indoor domestication, greenhouse acclimatization and field transplantation and other processes, a rapid propagation technology system of Poa pratensis L. is established. It can not only maintain the original genetic traits but also obtain a large number of seedlings in a short time, realizing the rapid propagation and application of excellent wild grass seed resources, and providing beneficial technical support for the preservation of excellent genetic traits of Poa pratensis L. var. anceps Gaud. wild in the alpine region, the creation of new germplasm resources of native grasses and the variety breeding.

[0081] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0082] The sources of the seeds of Poa pratensis L. var. mongolica (Rendle) C. Ling from the alpine wild grasslands in Inner Mongolia in Example 1 and Comparative Examples 1-9 are the same and are collected in the same batch.

[0083] Example 1

[0084] Step (1): Obtaining, cleaning, and disinfecting the explants. Select plump seeds of Poa pratensis L. var. mongolica (Rendle) C. Ling from the alpine wild grasslands in Inner Mongolia. The Poa pratensis L. var. mongolica (Rendle) C. Ling is collected from the sunny slopes on the mountaintops at an altitude of 1500-2000 m in Inner Mongolia. The annual rainfall in this area is less than 300 mm, the soil is barren and arid, and the extreme winter temperature is below -30°C. The appearance of the seeds is as shown in Figure 1 , the seeds are tiny. Carefully rub off the epidermis on the surface of the seeds, and then wrap them with gauze. Soak the seeds wrapped in gauze in a low-concentration detergent solution diluted 300 times for 15 min, rinse them under tap water for half an hour, then on a sterile workbench, treat them with 75% (v / v) ethanol for 30 s, and then disinfect them with 0.1% (w / v) mercuric chloride solution for 8-10 min, and rinse them 3-4 times with sterile water. Then blot the seeds wrapped in gauze with filter paper, carefully open the gauze, pick out the seeds with forceps, put them into a petri dish with filter paper, blot the water on the surface of the seeds again, and transfer them to the primary medium.

[0085] Step (2): Primary culture. On March 8, 2023, directly inoculate the disinfected seeds of Poa pratensis L. var. mongolica (Rendle) C. Ling in step (1) into the primary medium, as shown in Figure 2 . The composition of the primary medium is: based on MS as the basic medium, the medium also contains 0.2 mg / L 6-BA, 1.0 mg / L 2,4-D, 30 g / L sucrose, and 7.0 g / L agar. The pH of the medium is 6.8. Set up 4 parallel experiments.

[0086] Culture conditions: temperature 25±2°C, the primary culture is carried out under light-dark alternating conditions, the daily light culture time is 10 h, the daily dark culture time is 14 h, and the light intensity is 2200 lx. The primary culture time is 58 d. Among them, the germination situation of the seeds on the 17th day of the primary culture is shown in Figure 3 , and the germination situation of the seeds on the 35th day of the primary culture is shown in Figure 4 .

[0087] After 58 d of the primary culture of the seeds, the seeds germinate and seed buds are obtained. The seed buds include the buds formed by the germination of the seed embryos and the differentiated buds formed by the callus generated near the seed hypocotyls. The seed buds include single buds and multiple buds. When the length of the seed buds reaches 0.5-1 cm, subculture and proliferation culture of the cluster buds are carried out. The germination rate of the seeds on the 35th day of the primary culture reaches 80%.

[0088] Step (3): Subculture and proliferation culture of cluster buds. When the length of the shoot tip of the seeds is 0.5 - 1.0 cm on May 6th, cut the shoot tip of the seeds obtained in step (2) and transfer it to the cluster bud induction medium for continuous culture to obtain rootless seedlings. The culture situation of the shoot tip of the seeds and the differentiation of cluster buds on May 6th can be seen in Figure 5 ; On June 23rd, the cluster buds grew well. According to the following calculation formula of the propagation coefficient, the average value of the seed propagation coefficient was 3.8, as shown in Figure 6 . The cluster bud induction medium was based on MS medium, and only added 0.3 mg / L 6 - BA, 1.5 mg / L 2,4 - D, 30 g / L sucrose and 7.0 g / L agar. The subculture and proliferation culture was carried out under the condition of alternating light and dark. The light culture time was 10 h / d, and the dark culture time was 14 h / d; the light intensity was 2200 lx. The subculture and proliferation culture time of the cluster buds was 47 d.

[0089] Step (4): Rooting culture. On June 23rd, transfer the single rootless seedlings (the height of the rootless seedlings is 3 - 5 cm) in step (3) to the rooting medium for rooting culture. The composition of the rooting medium was based on 1 / 2 MS medium, and also added 0.5 mg / L NAA, 30 g / L sucrose and 7.0 g / L agar; the pH of the medium was 6.8.

[0090] Culture conditions: The temperature was 25 ± 2 °C. The rooting culture was carried out under the condition of alternating light and dark. The daily light culture time was 10 h, and the dark culture time was 14 h, and the light intensity was 2200 lx. The rooting culture time was 17 d.

[0091] On July 11th, it was observed that the rooted seedlings had 3 - 5 small white roots and the root length was 3 - 4 cm, and rooted seedlings were obtained, as shown in Figure 7 , and at this time, indoor acclimation could be carried out.

[0092] Step (5): Indoor acclimation: The acclimation of the rooted seedlings was carried out in the acclimation room. The rooted seedlings of Poa pratensis L. var. alpigena from the culture room were transferred to the acclimation room with a room temperature of 26 - 28 °C for acclimation. The light was natural light. The bottle caps were half - opened for 4 d. When the indoor air humidity was lower than 70%, the surface of the leaves was sprayed with 800 - fold chlorothalonil solution. The acclimated seedlings obtained after 4 d of acclimation could be transplanted to the greenhouse.

[0093] Step (6): Transplanting and acclimatizing seedlings. Transplant the acclimatized seedlings obtained in step (5) into a substrate for cultivation. When the acclimatized seedlings of Poa pratensis L. are taken out of the culture bottle, wash the agar from the roots, quickly dip them in a 800-fold chlorothalonil solution, and then transplant them into a plug tray. It is preferred to use a plug tray as a moisturizing seedling-raising container. When transplanting, ensure that the roots of the acclimatized Poa pratensis L. seedlings are stretched. After transplanting, compact the substrate, spray water on the leaves, then cover with a moisturizing cover, and transfer them to a greenhouse for continuous cultivation for 20 days. The temperature in the greenhouse is 25 °C, and the humidity in the greenhouse is 75%. The transplanting medium is peat, perlite and vermiculite, and the volume ratio of peat, perlite and vermiculite is 6:2:2. The single-hole specification of the plug tray is 3.8 cm × 3.8 cm × 5.5 cm. The survival rate of the acclimatized seedlings after 20 days of acclimatization in the greenhouse is 100%, see Figure 8 . After acclimatization and cultivation in the greenhouse, transplant them into the field. The results show that the survival rate reaches 100% 7 days after transplanting into the field, see Figure 10 .

[0094] Seed germination rate (%) = Number of germinated seeds / Total number of seeds × 100.

[0095] Calculation formula for multiplication coefficient: Y = X n . Where Y represents the multiplication coefficient, X represents the number of individuals multiplied by each seed per cycle, and n represents the number of cycles that can be multiplied throughout the year. Taking Example 1 as an illustration, when the annual multiplication cycle is n = 1, the specific statistical data is as follows, see Table 1.

[0096] Table 1 Multiplication coefficient of seeds of wild alpine Poa pratensis L. in four parallel experiments of Example 1

[0097]

[0098] Comparative Example 1

[0099] The source of the seeds of wild alpine Poa pratensis L. in Inner Mongolia is the same as that in Example 1. The seeds of wild alpine Poa pratensis L. in Comparative Example 1 and Example 1 are collected in the same batch.

[0100] For the seeds of wild alpine Poa pratensis L. in Comparative Example 1, the germination test was carried out according to the method of GB / T 2930.4-2017 "Rules for Seed Testing - Germination Test" promulgated on November 1, 2017 and implemented on May 1, 2018. The seed germination rate was only 4%. Since the germination rate of wild grass seeds is extremely low, 200 seeds were selected for germination during the test. The specific seed germination situation is shown in Figure 9 .

[0101] It can be seen from Example 1 and Comparative Example 1 that the seed germination rate of wild alpine Poa pratensis L. in Comparative Example 1 is only 4%, less than 5%, and the seed germination rate of wild alpine Poa pratensis L. in the technical solution of the present invention can reach 80%.

[0102] Comparative Example 2

[0103] Step (1): Obtaining, cleaning, and disinfecting the explants. The same as in Example 1.

[0104] Step (2): Primary culture: On March 8, 2023, the disinfected Poa annua seeds in Step (1) were directly inoculated onto the primary culture medium. The composition of the primary culture medium was: based on MS as the basic medium, the medium was further supplemented with only 0.6 mg / L 6-BA, 1.0 mg / L 2,4-D, 30 g / L sucrose, and 7.0 g / L agar. The pH of the medium was 6.8. 4 parallel experiments were set up.

[0105] Cultivation conditions: Temperature 25 ± 2°C, the primary culture was carried out under alternating light and dark conditions, the daily light cultivation time was 10 h, the daily dark cultivation time was 14 h, and the light intensity was 2200 lx. The primary culture time was 58 d.

[0106] After 58 d of primary culture of the seeds, after the seeds germinated, more clustered buds were differentiated from the callus, and some reached more than 6, but the health of the differentiated clustered buds was weak and the growth was poor. It was difficult for the cut shoot tips to survive after being inoculated onto the proliferation medium or the plants had poor growth after survival, mainly because the excessive 6-BA led to over-proliferation of the tissue, the single meristematic buds were thin and weak, and the health decreased. Therefore, the concentration of 6-BA in Comparative Example 3 should not be greater than 0.5 mg / L.

[0107] Comparative Example 3

[0108] Step (1): Obtaining, cleaning, and disinfecting the explants. The same as in Example 1.

[0109] Step (2): Primary culture. The same as in Example 1.

[0110] Step (3): Subculture and proliferation culture of clustered buds. The same as in Example 1.

[0111] Step (4): Rooting culture. The same as in Example 1.

[0112] Step (5): Transplanting culture. The rooted seedlings obtained in Step (4) were directly transplanted into a plug tray for substrate culture without acclimatization by opening the culture bottle cap. The transplanting culture conditions were the same as in Example 1. The transplanting culture survival rate dropped to 60%.

[0113] Compared with Example 1, Comparative Example 4 did not carry out indoor acclimatization, and the survival rate dropped to 60%. It can be seen that after the formation of rooted seedlings, 3 days of acclimatization by opening the culture bottle cap is crucial. If this step is missing and directly transplanted into a plug tray, the survival rate will be significantly reduced. Therefore, disinfection at this stage and indoor acclimatization under suitable temperature, humidity, and conditions are essential.

[0114] Comparative Example 4

[0115] Step (1) Acquisition, cleaning and disinfection of explants. Same as Example 1.

[0116] Step (2) Primary culture. Same as Example 1.

[0117] Step (3) Subculture and proliferation culture of clustered shoots. Same as Example 1.

[0118] Step (4) Rooting culture. Same as Example 1.

[0119] Step (5) Indoor acclimatization: Same as Example 1.

[0120] Step (6) Transplanting and hardening off: Transplant the acclimatized seedlings obtained in step (5) into a plug tray substrate for hardening off culture. When the acclimatized seedlings of Poa pratensis are taken out from the culture bottle, wash the agar on the roots, quickly dip them in a 800-fold chlorothalonil solution and then transplant them into the plug tray. Ensure that the roots of the acclimatized Poa pratensis seedlings are stretched during transplantation, compact the substrate, spray water on the leaves, cover with a moisture-holding cover, and then transfer them to a greenhouse for continued cultivation for 20 days. The temperature in the greenhouse is 25 °C and the humidity is 75%. The transplanting uses a moisture-holding seedling-raising container plug tray, and the transplanting medium is peat, perlite and vermiculite, and the volume ratio of peat, perlite and vermiculite is 5:0.5:0.5. The single-hole specification of the plug tray is 3.8 cm × 3.8 cm × 5.5 cm, and the survival rate of the acclimatized seedlings after transplantation is only 43%.

[0121] Comparative Example 5

[0122] Step (1) Acquisition, cleaning and disinfection of explants. Same as Example 1.

[0123] Step (2) Primary culture: On March 8, 2023, directly inoculate the disinfected Poa pratensis seeds in step (1) onto the primary culture medium. The composition of the primary culture medium is based on MS as the basic medium, and the medium is supplemented with 0.2 mg / L 6-BA, 0.7 mg / L 2,4-D, 30 g / L sucrose and 7.0 g / L agar. The pH of the medium is 6.8. Set 4 parallel experiments.

[0124] Culturing conditions: Temperature 25 ± 2 °C, the primary culture is carried out under light-dark alternating conditions, the daily light culture time is 10 h, the daily dark culture time is 14 h, and the light intensity is 2200 lx. The primary culture time is 58 days.

[0125] After 58 days of primary culture of the seeds, the seed embryos can germinate to obtain seed buds, but when the concentration of 2,4-D is lower than 0.8 mg / L, the differentiated buds of the callus produced by the germinated seeds are weak and cannot be subcultured.

[0126] Comparative Example 6

[0127] Step (1) Acquisition, cleaning and disinfection of explants. Same as Example 1.

[0128] Step (2) Primary culture: On March 8, 2023, the disinfected Poa pratensis seeds from step (1) were directly inoculated onto the primary culture medium. The composition of the primary culture medium was based on MS as the basic medium, supplemented with 0.2 mg / L 6-BA, 1.0 mg / L 2,4-D, 31 g / L sucrose, and 7.0 g / L agar. The pH of the medium was 6.8. Four parallel experiments were set up.

[0129] Cultivation conditions: Temperature 25 ± 2 °C, the primary culture was carried out under alternating light and dark conditions, the daily light cultivation time was 10 h, the daily dark cultivation time was 14 h, and the light intensity was 2200 lx. The primary culture time was 58 d.

[0130] After 58 d of primary culture of the seeds, the seeds germinated and seed buds were obtained. The seed buds included buds formed by the germination of the seed embryo and differentiated buds formed by the differentiation of callus generated near the seed hypocotyl. However, the fungal infection of the germinated seeds increased significantly, and the loss rate exceeded 30%.

[0131] Comparative Example 7

[0132] Step (1) Obtaining, cleaning, and disinfecting the explants. Same as Example 1.

[0133] Step (2) Primary culture. Same as Example 1.

[0134] Step (3) Subculture and proliferation culture of clustered buds. Same as Example 1. The only difference was that the clustered bud induction medium was based on MS as the basic medium, and also supplemented with only 0.3 mg / L 6-BA, 1.5 mg / L 2,4-D, 27 g / L sucrose, and 7.0 g / L agar.

[0135] The subculture and proliferation culture time of the clustered buds was 47 d. After 47 d of subculture and proliferation culture of the clustered buds, the fungal infection of the subculture and proliferation culture of the clustered buds increased, and the infection of the meristematic buds exceeded 20%.

[0136] Comparative Example 8

[0137] Step (1) Obtaining, cleaning, and disinfecting the explants. Same as Example 1.

[0138] Step (2) Primary culture. Same as Example 1.

[0139] Step (3) Subculture and proliferation culture of clustered buds. Same as Example 1.

[0140] Step (4) Rooting culture. Same as Example 1. The only difference was that the composition of the rooting medium was based on 1 / 2MS as the basic medium, and also supplemented with 0.15 mg / L NAA, 30 g / L sucrose, and 7.0 g / L agar; the pH of the medium was 6.8.

[0141] After rooting culture, the number of roots of the tissue-cultured seedlings is scarce, or no roots emerge. Even if roots emerge, the root length is less than 1 cm.

[0142] Comparative Example 9

[0143] Step (1) Obtaining, cleaning and disinfecting the explant. The same as Example 1.

[0144] Step (2) Primary culture. The same as Example 1.

[0145] Step (3) Subculture and proliferation culture of cluster buds. The same as Example 1.

[0146] Step (4) Rooting culture. The same as Example 1. The only difference is that the composition of the rooting medium is based on 1 / 2 MS as the basic medium, and additionally contains 0.85 mg / L NAA, 30 g / L sucrose and 7.0 g / L agar; the pH of the medium is 6.8.

[0147] After rooting culture, the small white roots of the tissue-cultured seedlings are thin and weak, and the survival rate of the acclimatized seedlings during the stage of transplanting to the greenhouse is less than 70%.

[0148] In summary, the tissue-culture rapid propagation method of the present invention uses the seeds of wild Poa pratensis L. in alpine regions as explants, and through different hormone types and concentration ratios, different functional media are prepared to induce seed germination and produce callus, so that cluster buds are differentiated and rooted, and then through bottle-seedling acclimatization and substrate hardening, transplantable seedlings are produced, establishing a rapid propagation technology system for Poa pratensis L., providing a practical basic technical solution for the breeding of new varieties of wild drought-resistant Poa pratensis L. in high-altitude regions and the rapid propagation of original seedlings.

[0149] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A tissue culture medium for Poa pratensis L. var. alpigena, characterized in that, The tissue culture medium consists of a primary medium, a clustered bud induction medium, and a rooting medium; The primary medium uses the MS medium as the basic medium and also adds: 0.1 - 0.5 mg / L 6 - BA, 0.8 - 1.0 mg / L 2,4 - D, 26 - 30 g / L sucrose, and 6.5 - 7.0 g / L agar; The clustered bud induction medium uses the MS medium as the basic medium and also adds: 0.1 - 0.5 mg / L 6 - BA, 1.0 - 1.5 mg / L 2,4 - D, 28 - 32 g / L sucrose, and 6.5 - 7.5 g / L agar; The rooting medium uses the 1 / 2MS medium as the basic medium and also adds: 0.2 - 0.8 mg / L NAA, 29 - 32 g / L sucrose, and 6.5 - 7.5 g / L agar.

2. The tissue culture medium according to claim 1, wherein, The pH value of the primary medium is 6.6 - 7.0; the pH value of the clustered bud induction medium is 6.6 - 7.0; the pH value of the rooting medium is 6.6 - 7.

0.

3. A tissue culture and rapid propagation method for Poa pratensis L. var. alpigena, characterized in that, The tissue culture rapid propagation method uses the tissue culture medium described in claim 1 or 2 and includes the following steps: Inoculate the seeds of Poa pratensis L. var. alpigena in the primary medium for primary culture to obtain the buds of Poa pratensis L. var. alpigena seeds; Inoculate the shoot tips of the buds of Poa pratensis L. var. alpigena seeds on the clustered bud induction medium for subculture and proliferation to obtain the rootless seedlings of Poa pratensis L. var. alpigena; Transfer the rootless seedlings of Poa pratensis L. var. alpigena to the rooting medium for rooting culture, and after the rooting culture, obtain the complete tissue - cultured seedlings for acclimatization culture and transplanting and hardening - off.

4. The tissue culture and rapid propagation method according to claim 3, characterized in that, The length of the shoot tips of the buds of Poa pratensis L. var. alpigena seeds is 0.5 - 1.0 cm.

5. The tissue culture rapid propagation method according to claim 3, characterized in that, The temperatures of the primary culture, subculture and proliferation culture, and rooting culture are 23 - 27 °C respectively.

6. The tissue culture rapid propagation method according to claim 3, wherein The primary culture, subculture and proliferation culture, and rooting culture are all carried out under the condition of alternating light and darkness. The light - culture time in each light - dark cycle is 10 - 12 h / d respectively, and the dark - culture time in each light - dark cycle is 12 - 14 h / d respectively; the light intensity is 2000 - 2500 lx respectively.

7. The tissue culture and rapid propagation method according to claim 3, characterized in that, The time of the primary culture is 35 - 70 d, the time of the subculture and proliferation culture is 40 - 60 d, and the time of the rooting culture is 15 - 22 d.

8. The tissue culture rapid propagation method according to claim 3, characterized in that, Before the seeds of Poa pratensis L. var. alpigena are inoculated into the primary medium, it also includes: rubbing the seeds, cleaning, and disinfecting them in sequence.

9. The tissue culture and rapid propagation method according to claim 8, wherein The cleaning includes detergent cleaning and running - water rinsing. The detergent cleaning time is 10 - 20 min, and the running - water rinsing time is 25 - 35 min; The disinfection method includes: after cleaning, first soak in 70% - 75% (mass concentration) alcohol for 25 - 35 s, then soak in 0.1% mercuric chloride solution for 8 - 10 min; finally rinse with sterile water 3 - 4 times.

10. The tissue culture and rapid propagation method according to claim 3, wherein The acclimatization culture and transplanting and hardening - off are completed indoors and in a greenhouse respectively. The composition of the substrate for transplanting and hardening - off includes peat, perlite, and vermiculite; the volume ratio of peat, perlite, and vermiculite is (5 - 6):(1 - 2):(1 - 2).

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