Tissue culture and rapid propagation method for hemarthria procumbens

Abstract

The invention discloses a tissue culture and rapid propagation method for hemarthria procumbens. The tissue culture and rapid propagation method comprises the following steps: (1) carrying out sterile treatment on hemarthria procumbens seeds; (2) germination: putting the seeds on an inoculated germination culture medium; (3) induction: cutting a hypocotyl by 1-2cm and putting the hypocotyl into an induction culture medium; (4) proliferation and differentiation: putting into a proliferation and differentiation culture medium; and (5) rooting: finally putting into a rooting culture medium. According to the tissue culture and rapid propagation technology, a large number of hemarthria prostrata plant individuals can be obtained within a short time, dependence on wild hemarthria prostrata resources can be reduced through the tissue culture and rapid propagation technology, protection of the wild plant resources is facilitated, and therefore the requirements of the market for Chinese herbal medicine raw materials are met, and the tissue culture and rapid propagation technology has the technical prospect of large-scale production of pasture raw materials. Meanwhile, the tissue culture and rapid propagation technology of the hemarthria procumbens has wide application prospects in the aspects of promoting sustainable development of the pasture industry, protecting plant resources and promoting scientific research.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for rapid propagation of sand whip grass tissue culture. Background Art

[0002] Sand Whip Grass is a perennial herb of the genus Sand Whip Grass of the Poaceae family. Sand Whip Grass has well-developed vegetative branches and a long vegetative period. During the vegetative reproduction period, it has high crude protein and nitrogen-free extract content and a lot of crude fiber content. It is a medium-quality perennial forage grass on sandy grasslands in desert areas. It is also a good sand-fixing plant and has a high utilization value in desert areas. The traditional breeding method of Sand Whip Grass takes a long time to breed, reproduces slowly, and has a single breeding method. Therefore, it is very important to develop a tissue culture system suitable for the rapid and large-scale reproduction of Sand Whip Grass. Summary of the invention

[0003] In order to solve the problem of the reproduction and breeding of sand whip grass, the present invention provides a method for rapid propagation of sand whip grass through tissue culture. The sand whip grass tissue culture rapid propagation technology of the present invention can break through the traditional breeding means, solve the problems of long breeding years, single breeding methods, and slow reproduction, and can realize a rapid propagation system of multiple generations in one year through the tissue culture method, thereby solving the problem of breeding shortage. To this end, the present invention adopts the following technical solutions:

[0004] A method for rapid propagation of Herba Cyperi tissue culture of the present invention comprises the following steps:

[0005] (1) Sterile treatment of sand whip grass seeds;

[0006] (2) Germination culture: placing on an inoculated germination medium; the germination medium is MS medium supplemented with naphthaleneacetic acid NAA, gibberellic acid GA3 and activated carbon AC;

[0007] (3) Induction culture: 1-2 cm hypocotyl cuts are placed in an induction medium; the induction medium is MS medium supplemented with auxin 2AD, 6-benzylpurine 6-BA, naphthaleneacetic acid NAA and activated carbon AC;

[0008] (4) Proliferation and differentiation culture: placing in a proliferation and differentiation medium; the proliferation and differentiation medium is a modified MS medium supplemented with 6-benzylpurine 6-BA, naphthylacetic acid NAA, zeatin ZT and AC;

[0009] (5) Rooting culture: Finally, the rooting medium is placed in a 1 / 2 MS medium with indolebutyric acid (IBA) and activated carbon (AC). Indolebutyric acid can promote root growth and form a root system.

[0010] For the above method, it is preferred that in step (1) the seed sterilization treatment is to soak the sand whip grass seeds in 75% (v / v) alcohol and 0.1 wt% mercuric chloride successively.

[0011] For the method described above, it is preferred that the germination medium composition of step (2) is: MS medium + NAA (0.1-0.5 mg / L) + GA3 (0.08-0.2 mg / L) + AC (0.5-1.5 mg / L), and the more preferred composition is: MS medium + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L.

[0012] For the method described above, it is preferred that the composition of the induction medium in step (3) is: MS medium + 2AD (0.5-1.2 mg / L) + 6-BA (0.1-0.5 mg / L) + NAA (0.1-0.5 mg / L) + AC (0.5-1.5 mg / L), and the more preferred composition is: MS medium + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L.

[0013] For the method described above, it is preferred that the proliferation and differentiation medium in step (4) is composed of: modified MS medium (ammonium and nitrogen are reduced by 1 / 10, micro-molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) + 6-BA (0.2-0.8mg / L) + NAA (0.02-0.08mg / L) + ZT 0.01mg / L + AC (0.5-1.5mg / L). A more preferred composition is: modified MS medium (ammonium and nitrogen are reduced by 1 / 10, micro-molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) + 6-BA 0.5mg / L + NAA 0.05mg / L + ZT 0.01mg / L + AC 1mg / L. Zeatin accounts for 1 / 5 of the entire culture medium. Using the modified MS medium as the basis is more suitable for the growth of sand whip grass. In addition, zeatin ZT can better promote the division and differentiation of sand whip grass cells.

[0014] For the method described above, it is preferred that the rooting medium composition of step (5) is: 1 / 2 MS + IBA (0.2-0.8 mg / L) + AC (0.1-0.5 mg / L), and the more preferred composition is: 1 / 2 MS + IBA 0.5 mg / + AC 0.2 mg / L.

[0015] Preferably, in the above-mentioned method, the culture conditions of step (2) germination culture, step (3) induction culture, step (4) proliferation and differentiation culture, and step (5) rooting culture can all be: 23-26°C, 6-8 hours / day, and light intensity of 1000-4000 lux. Generally, for common plants, the light exposure time is preferably more than 12 hours, but the inventors have found that the light exposure time of the present invention is too long and is not conducive to the tissue culture growth of sand whip grass, and the light intensity can be preferably 3000 lux.

[0016] Preferably, in the above-mentioned method, the time for germination culture in step (2) and induction culture in step (3) is preferably 12-20 days, and optimally 15-16 days, while the time for proliferation and differentiation culture in step (4) is preferably 50-80 days, and optimally 60-65 days, and the time for rooting culture in step (5) is preferably 25-40 days, and optimally 30-35 days.

[0017] As a more preferred technical solution, the tissue culture rapid propagation method of the sand whip grass of the present invention comprises the following steps:

[0018] (1) Seed sterilization treatment: Soak the seeds of Sand Whip Grass in 75% (volume) alcohol for 10-30 seconds, then rinse with sterile water, then soak in 0.1wt% mercuric chloride for 0.5-2 minutes, and then rinse with sterile water;

[0019] (2) Germination culture: Place on the inoculated germination medium and expose to light at 23-26℃ for 6-8 hours with a light intensity of 1000-4000 lux; the germination medium composition is: MS medium + NAA 0.3mg / L + GA3 0.1mg / L + AC 1mg / L;

[0020] (3) Induction culture: Cut the hypocotyl into 1-2 cm pieces and place them in the induction culture medium. Expose them at 23-26℃ for 6-8 hours with a light intensity of 1000-4000 lux. The composition of the induction culture medium is: MS medium + 2AD 1mg / L + 6BA 0.2mg / L + NAA 0.2mg / L + AC1mg / L.

[0021] (4) Proliferation and differentiation culture: Place in proliferation and differentiation medium, illuminate at 23-26°C for 6-8 hours, light intensity 1000-4000 lux; the proliferation and differentiation medium composition is: modified MS medium + 6-BA 0.5mg / L + NAA 0.05mg / L + ZT0.01mg / L + AC 1mg / L;

[0022] (5) Rooting culture: Finally, place the seeds in rooting medium and expose them to light at 23-26℃ for 6-8 hours with a light intensity of 1000-4000 lux. The composition of the rooting medium is: 1 / 2 MS medium + IBA 0.5mg / +AC 0.2mg / L.

[0023] The beneficial effects of the present invention are:

[0024] 1. The tissue culture rapid propagation technology of the present invention is not limited by time and space: from a seed to germination and then callus induction, rooting can be achieved through continuous differentiation of callus, so that a large number of sand whip grass plants can be obtained in a relatively short period of time (about 3-4 months), and multiple generations of reproduction can be completed in one year, thus meeting breeding needs.

[0025] 2.Technological prospects: large-scale production of pasture materials.

[0026] 3. Protect wild resources: Through tissue culture rapid propagation technology, we can reduce dependence on wild sand whip grass resources and help protect wild plant resources.

[0027] 4. In general, the tissue culture and rapid propagation technology of Sand Whip Grass has broad application prospects in promoting the sustainable development of the forage industry, protecting plant resources and promoting scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are pictures of proliferation, differentiation and rooting conditions. A is a photo of proliferation and differentiation, and B is a photo of rooting. DETAILED DESCRIPTION

[0029] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0030] In the embodiment of the present invention, IBA is indolebutyric acid, NAA is naphthylacetic acid, 6-BA is 6-benzylpurine, 2AD is auxin, ZT is zeatin, GA3 is gibberellic acid, and AC is activated carbon. The improved MS medium is improved on the basis of the MS medium, wherein the ammonium nitrogen is reduced by 1 / 10, the micro-molybdenum is reduced by 1 / 10, and the calcium is reduced by 1 / 5. Specifically, the ammonium nitrogen is reduced by 1 / 10, which means that the amount of the culture medium component NH4NO3 (ammonium nitrate) is reduced by 1 / 10, the micro-molybdenum is reduced by 1 / 10, which means that the amount of the culture medium component Na2MoO4·2H2O (sodium molybdate) is reduced by 1 / 10, and the calcium is reduced by 1 / 5, which means that the amount of the culture medium component CaCl2·2H2O (calcium chloride) is reduced by 1 / 5.

[0031] Example 1

[0032] The tissue culture rapid propagation method of Sand Whip Herb comprises the following steps:

[0033] (1) Seed sterilization treatment: The seeds of the sand whip grass collected from Alxa League, Inner Mongolia Autonomous Region (Alxa Ecosystem Research Center, Inner Mongolia Autonomous Region) were soaked in 75% (v / v) alcohol for 20 seconds, rinsed once with sterile water, and then soaked in 0.1wt% mercuric chloride for 1 minute and rinsed three times with sterile water;

[0034] (2) Germination culture: Place the seeds treated in step (1) on a germination medium for inoculation and expose to light at 24°C for 6 hours / day with a light intensity of 3000 lux. The composition of the germination medium is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. Inoculation should be carried out in a fume hood to ensure sterility. Place the seeds in the germination medium and culture for about 7 days. Small white buds can be observed growing on the top of the seeds. After 15-16 days of culture, the seed buds grow to about 2-3 cm and can be induced. The selection of the germination medium allows the sand whip grass seeds to germinate well, and the germination of the sand whip grass seeds further proves that the seeds are active, so that subsequent induction can be carried out to form callus.

[0035] (3) Induction culture: When the buds are 2-3 cm long, cut the hypocotyls by 1-2 cm and place them in the induction culture medium. Expose them at 24°C for 6 hours a day with a light intensity of 3000 lux. The composition of the induction culture medium is: MS culture medium 10mg / L+2AD 1mg / L+6-BA0.2mg / L+NAA 0.2mg / L+AC 1mg / L. After the seeds germinate, cut the hypocotyls by 1-2 cm and place them in the induction culture medium. In the induction culture medium, the hypocotyls will grow callus tissue in small pieces. After growing for about half a month (15-16 days), they can be proliferated and differentiated.

[0036] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium, and light them for 6 hours / day at 24°C with a light intensity of 3000 lux. The composition of the proliferation and differentiation medium is: modified MS medium 10mg / L (ammonium nitrogen reduced by 1 / 10, micro molybdenum reduced by 1 / 10, calcium reduced by 1 / 5) + 6BA 0.5mg / L + NAA 0.05mg / L + ZT 0.01mg / L + AC 1mg / L. Callus tissue will proliferate and differentiate in large quantities. The calli need to be separated and placed in different culture media. After about one month of culture, stems and leaves can be observed growing on the calli. Figure 1 A. At this time, fresh culture medium needs to be replaced to provide nutrients for its growth in time. After about two months of culture, a large amount of callus tissue proliferates and differentiates.

[0037] (5) Rooting culture: Finally, place the roots in the rooting medium and expose them to light for 6 hours / day at 24℃ with a light intensity of 3000 lux. The rooting medium composition is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / + AC 0.2mg / L. After being placed in the rooting medium for about 20 days, roots will grow. Figure 1 B. After about 30 days when the roots are strong, the plant will be a complete plant and can be permanently preserved or hardened.

[0038] Example 2

[0039] The tissue culture rapid propagation method of Sand Whip Herb comprises the following steps:

[0040] (1) Seed sterilization treatment: the same as in Example 1;

[0041] (2) Germination culture: Place the seeds treated in step (1) on a germination medium for inoculation and expose to light at 23°C for 8 hours / day with a light intensity of 3000 lux. The composition of the germination medium is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. Inoculation must be carried out in a fume hood to ensure sterility. The selection of the germination medium allows the sand whip grass seeds to germinate well. The seeds germinate after 15 days of culture. The germination of the sand whip grass seeds further proves that the seeds are active, so that subsequent induction can be carried out to form callus.

[0042] (3) Induction culture: When the buds are 2-3 cm, cut the hypocotyl into 1-2 cm pieces and place them in the induction culture medium. Expose the cells to 8 hours of light per day at 23°C with a light intensity of 3000 lux. The composition of the induction culture medium is: MS medium 10 mg / L + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L. Callus tissue will grow after 15 days of induction culture.

[0043] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium and light them for 8 hours / day at 23℃ with a light intensity of 3000 lux. The composition of the proliferation and differentiation medium is: modified MS medium 10mg / L (ammonium and nitrogen are reduced by 1 / 10, micro molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) + 6BA 0.5mg / L + NAA 0.05mg / L + ZT 0.01mg / L + AC 1mg / L. The proliferation and differentiation culture is carried out for about 60 days, and a large amount of callus tissue undergoes proliferation and differentiation.

[0044] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 8 hours / day at 23℃ with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After about 30 days of rooting culture, the plants will take root in the medium and become complete plants.

[0045] Example 3

[0046] A method for rapid propagation of sand whip grass tissue culture, comprising the following steps:

[0047] (1) Seed sterilization treatment: the same as in Example 1;

[0048] (2) Germination culture: The seeds treated in step (1) were placed on a germination medium for inoculation and illuminated at 25°C for 7 hours / day with a light intensity of 3000 lux. The germination medium composition is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. After 18 days of germination culture, the seeds germinated, and subsequent induction to form callus was performed.

[0049] (3) Induction culture: When the buds are 2-3 cm, cut the hypocotyl into 1-2 cm pieces and place them in the induction culture medium. Expose them at 25°C for 7 hours per day with a light intensity of 3000 lux. The composition of the induction culture medium is: MS medium 10 mg / L + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L. Callus tissue will grow after 18 days of induction culture.

[0050] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium and light them for 7 hours / day at 25℃ with a light intensity of 3000 lux. The composition of the proliferation and differentiation medium is: modified MS medium 10mg / L (ammonium and nitrogen are reduced by 1 / 10, micro molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) + 6BA 0.5mg / L + NAA 0.05mg / L + ZT 0.01mg / L + AC 1mg / L. The proliferation and differentiation culture is carried out for about 65 days, and a large amount of callus tissue undergoes proliferation and differentiation.

[0051] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 7 hours / day at 25℃ with a light intensity of 3000 lux. The composition of the rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After about 30 days of rooting culture, the plants will take root in the medium and become complete plants.

[0052] Comparative Example 1

[0053] A method for tissue culture of sand whip grass comprises the following steps:

[0054] (1) Seed sterilization treatment: the same as in Example 1;

[0055] (2) Germination culture: the same as in Example 1;

[0056] (3) Induction culture: the same as in Example 1;

[0057] (4) Proliferation and differentiation culture: The proliferation and differentiation culture uses unmodified MS medium as the basic medium, and the specific composition is: MS medium 10mg / L + 6BA 0.5mg / L + NAA 0.05mg / L + ZT 0.01mg / L + AC 1mg / L. The callus tissue proliferates and differentiates in small amounts. After one month of culture, no stems and leaves are seen in the callus tissue. After about 50 days of culture, a small amount of callus tissue leaves appear, and rooting culture is carried out;

[0058] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 6 hours / day at 24°C with a light intensity of 3000 lux. The composition of the rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / + AC 0.2mg / L. After being placed in the rooting medium, a very small number of tiny roots will grow in about 35 days, and then the tiny roots will grow slowly after 10-15 days of cultivation.

[0059] By comparing Example 1 and Comparative Example 1, the improved MS medium is more suitable for the proliferation and differentiation growth of the callus of the sand whip grass of the present application than the unimproved MS medium, so that it can quickly grow callus, which is then conducive to its rooting and growth.

[0060] Comparative Example 2

[0061] A method for tissue culture of sand whip grass comprises the following steps:

[0062] (1) Seed sterilization treatment: the same as in Example 1;

[0063] (2) Germination culture: the same as in Example 1;

[0064] (3) Induction culture: the same as in Example 1;

[0065] (4) Proliferation and differentiation culture: Proliferation and differentiation culture uses low-concentration modified MS medium 6 mg / L (ammonium and nitrogen are reduced by 1 / 10, micro-molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) as the basic culture medium. The specific composition is: modified MS medium 6 mg / L + 6BA 0.5 mg / L + NAA 0.05 mg / L + ZT 0.01 mg / L + AC 1 mg / L. After about three months of culture, a small amount of callus tissue leaves will appear, and subsequent rooting culture will be carried out.

[0066] (5) Rooting culture: Finally, place the plants in rooting medium and expose them to light for 6 hours per day at 24°C with a light intensity of 3000 lux. The composition of the rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / + AC 0.2mg / L. A small amount of roots can be seen after about 60 days of culture.

[0067] Comparative Example 3

[0068] A method for tissue culture of sand whip grass comprises the following steps:

[0069] (1) Seed sterilization treatment: the same as in Example 1;

[0070] (2) Germination culture: the same as in Example 1;

[0071] (3) Induction culture: the same as in Example 1;

[0072] (4) Proliferation and differentiation culture: The proliferation and differentiation culture uses a high-concentration modified MS medium 15 mg / L (ammonium and nitrogen are reduced by 1 / 10, micro-molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5) as the basic medium. The specific composition is: modified MS medium 15 mg / L + 6BA 0.5 mg / L + NAA 0.05 mg / L + ZT 0.01 mg / L + AC 1 mg / L. After 20 days of culture, incomplete bean sprout-like (deformed) stems and leaves can be seen in the callus tissue. After about 50 days of culture, a large number of incomplete and deformed bean sprout-like leaves can be seen in the callus tissue. At the same time, the callus tissue becomes significantly thicker and is accompanied by a small number of nodules, and rooting culture is carried out.

[0073] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 6 hours per day at 24°C with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After about 20 days of culture, a large number of tuberous roots can be seen.

[0074] By comparing Example 1, Comparative Example 2 and Comparative Example 3, low-concentration modified MS culture medium may lead to slower cell division, reduced biomass accumulation, affect cell growth, delay or inhibit cell differentiation, and lead to incomplete tissue or organ formation; while high-concentration modified MS culture medium may accelerate cell division, increase biomass accumulation, induce overgrowth, form excessive callus or cell clumps (nodules), inhibit normal differentiation, lead to abnormal structure or incomplete development, interfere with normal morphogenesis, lead to deformed or overgrown tissues, and affect the healthy growth of plant cells.

[0075] Comparative Example 4

[0076] According to the method of Example 1, during the proliferation and differentiation culture in step (4), the dosage of ammonium nitrogen, micro-molybdenum and calcium in the MS culture medium was improved. The other aspects were the same as in Example 1. The results are shown in Tables 1-3 below:

[0077] Table 1 Optimization selection test of improved MS medium

[0078] Serial number Ammonium Nitrogen Micro Molybdenum calcium result 1 Increase 1 / 10 Increase 1 / 10 Increase 1 / 5 Callus cannot form and grows slowly. The reason is that the increase of ammonium nitrogen, trace molybdenum and calcium is toxic to the plant and inhibits its growth. 2 Increase 1 / 10 Increase 1 / 10 Minus 1 / 5 Callus cannot form and grows slowly. The reason is that the increase of ammonium nitrogen and trace molybdenum is toxic to plants and inhibits their growth. 3 Increase 1 / 10 Increase 1 / 10 constant Callus cannot form and grows slowly. The reason is that the increase of ammonium nitrogen and trace molybdenum is toxic to plants and inhibits their growth. 4 Increase 1 / 10 Minus 1 / 10 Increase 1 / 5 Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth. 5 Increase 1 / 10 Minus 1 / 10 Minus 1 / 5 Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth. 6 Increase 1 / 10 Minus 1 / 10 constant Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth. 7 Increase 1 / 10 constant Increase 1 / 5 Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth. 8 Increase 1 / 10 constant Minus 1 / 5 Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth. 9 Increase 1 / 10 constant constant Callus cannot form and grows slowly because the increase in ammonium nitrogen is toxic to the plant and inhibits its growth.

[0079] Table 2 Optimization selection test of improved MS medium

[0080] Serial number Ammonium Nitrogen Micro Molybdenum calcium result 1 Minus 1 / 10 Increase 1 / 10 Increase 1 / 5 Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 2 Minus 1 / 10 Increase 1 / 10 Minus 1 / 5 Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 3 Minus 1 / 10 Increase 1 / 10 constant Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 4 Minus 1 / 10 Minus 1 / 10 Increase 1 / 5 Increased calcium can cause competition with other ions, leading to nutritional imbalance. 5 Minus 1 / 10 Minus 1 / 10 Minus 1 / 5 The callus proliferated in large quantities and grew stems and leaves after one month of culture (equivalent to the modified MS medium used in Example 1). 6 Minus 1 / 10 Minus 1 / 10 constant Excessive calcium levels cause ion competition and can easily lead to nutritional imbalance. 7 Minus 1 / 10 constant Increase 1 / 5 Increased calcium can cause competition with other ions, leading to nutritional imbalance. 8 Minus 1 / 10 constant Minus 1 / 5 Excessive amounts of molybdenum can cause toxicity and inhibit growth. 9 Minus 1 / 10 constant constant Excessive amounts of molybdenum and calcium can cause toxic effects, lead to ion competition, inhibit growth and lead to nutritional imbalance.

[0081] Table 3 Optimization selection test of improved MS medium

[0082] Serial number Ammonium Nitrogen Micro Molybdenum calcium result 1 constant Increase 1 / 10 Increase 1 / 5 Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 2 constant Increase 1 / 10 Minus 1 / 5 Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 3 constant Increase 1 / 10 constant Callus cannot form because increasing the amount of molybdenum causes toxicity, inhibits growth and even leads to nutritional imbalance. 4 constant Minus 1 / 10 Increase 1 / 5 Excess calcium causes ion competition, leading to nutritional imbalance. 5 constant Minus 1 / 10 Minus 1 / 5 Excessive amounts of ammonium nitrogen can cause certain toxicity to plants and inhibit their growth. 6 constant Minus 1 / 10 constant Excessive amounts of ammonium nitrogen and calcium affect its growth and development. 7 constant constant Increase 1 / 5 Increased calcium leads to competition with other ions and nutritional imbalance. 8 constant constant Minus 1 / 5 Excessive amounts of ammonium nitrogen can cause certain toxicity to plants and inhibit their growth. 9 constant constant constant The callus proliferated and differentiated slightly, but no leaves were observed after one month of culture (equivalent to unmodified MS medium, such as comparative example 1)

[0083] As can be seen from Tables 1-3, when the MS medium is improved, in which ammonium nitrogen is reduced by 1 / 10, molybdenum is reduced by 1 / 10, and calcium is reduced by 1 / 5 as the basic medium for proliferation and differentiation, calluses can proliferate in large quantities and grow normal stems and leaves after about one month of culture.

[0084] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A method for rapid propagation of sand whip grass tissue culture, characterized in that: The steps include: (1) Sterilization treatment of sand whip grass seeds; (2) Germination culture: placing on an inoculated germination medium; the germination medium is MS medium supplemented with 0.1-0.5 mg / L NAA, 0.08-0.2 mg / L GA3 and 0.5-1.5 mg / L AC; (3) Induction culture: 1-2 cm hypocotyl cuts were placed in induction medium; the induction medium is MS medium supplemented with 0.5-1.2 mg / L 2AD, 0.1-0.5 mg / L 6-BA, 0.1-0.5 mg / L NAA and 0.5-1.5 mg / L AC; (4) Proliferation and differentiation culture: placing in a proliferation and differentiation medium; the proliferation and differentiation medium is a modified MS medium supplemented with 0.2-0.8 mg / L 6-BA, 0.02-0.08 mg / L NAA, 0.01 mg / L ZT and 0.5-1.5 mg / L AC; the modified MS medium is a MS medium in which the amount of ammonium nitrate is reduced by 1 / 10, the amount of sodium molybdate is reduced by 1 / 10, and the amount of calcium chloride is reduced by 1 / 5; (5) Rooting culture: Finally, place in rooting medium, which is 1 / 2 MS medium supplemented with 0.2-0.8 mg / L IBA and 0.1-0.5 mg / L AC; In the above steps, NAA is naphthylacetic acid, GA3 is gibberellic acid, AC is activated carbon, 2AD is auxin, 6-BA is 6-benzylpurine, ZT is zeatin, and IBA is indolebutyric acid.

2. The method according to claim 1, characterized in that In the step (1), the seed sterilization treatment is to soak the sand whip grass seeds in 75% alcohol and 0.1% mercuric chloride in sequence.

3. The method according to claim 1, characterized in that The germination medium composition of step (2) is: MS medium + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L.

4. The method according to claim 1, characterized in that The induction medium composition of step (3) is: MS medium + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L.

5. The method according to claim 1, characterized in that The proliferation and differentiation medium in step (4) is composed of: modified MS medium + 6-BA 0.5 mg / L + NAA 0.05 mg / L + ZT 0.01 mg / L + AC 1 mg / L.

6. The method according to claim 5, characterized in that The rooting medium composition of step (5) is: 1 / 2 MS medium + IBA 0.5 mg / L + AC 0.2 mg / L.

7. The method according to claim 1, characterized in that The culture conditions of step (2), step (3), step (4) and step (5) are: 23-26° C., 6-8 hours of light per day, and a light intensity of 1000-4000 lux.

8. The method according to claim 1, characterized in that The culture time of step (2) and step (3) is 12-20 days.

9. The method according to claim 1, characterized in that The culture time of step (4) is 50-80 days.

10. The method according to claim 1, characterized in that The culture time of step (5) is 25-40 days.

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