A method for rapid domestication of amla fruit

Abstract

The application provides a kind of quick domestication method of Edible Bird Nest fruit, comprising the following steps, using modified MS liquid medium is added to liquid medium I by antibiotic liquid;Modified MS liquid medium is mixed to prepare liquid medium II;Select the healthy stem segment containing thorn seat and sterilize;The sterilized branch is placed in liquid medium I and cultured to obtain the first generation of aseptic new shoots, and the first generation of aseptic new shoots is cut in liquid medium II to obtain the second generation of aseptic new shoots;The second generation of aseptic new shoots is transplanted to water culture for 5-7 days to obtain Edible Bird Nest fruit seedlings.The application uses modified MS+antibiotic liquid medium, and uses the cultivation method of new shoot transplanting water culture, which reduces the domestication time of Edible Bird Nest fruit, increases the root diameter, total root length and root surface area, and simultaneously, the number of lateral branches, lateral branch length and aboveground biomass are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a rapid domestication method for bird's nest fruit. Background Technology

[0002] Dragon fruit, native to Central America, is a hybrid of *Hylocereus undatus* and *Selenicereus grandiflorus*. It resembles the common yellow-skinned, white-fleshed dragon fruit, but is not the same as a typical yellow dragon fruit. Known as the "Queen of Dragon Fruits," dragon fruit is rich in nutrients, with a fibrous, smooth flesh resembling bird's nest. It is sweet, delicious, and juicy, containing plant-based albumin and anthocyanins, which are rare in other plants, as well as abundant vitamins and water-soluble dietary fiber. Compared to regular dragon fruit, dragon fruit takes 3-6 months from flowering to fruit ripening, and each fruit is lighter. Currently, dragon fruit seedlings are propagated through seedlings, cuttings, and grafting, but the propagation coefficient is low and limited by the amount of propagation material, resulting in insufficient production to meet market demand. Plant tissue culture technology can rapidly propagate large quantities of healthy seedlings of consistent quality, while preserving superior germplasm resources. Plant culture requires less material, has a high propagation coefficient, and is fast, significantly improving the efficiency of commercial seedling production. Currently, the tissue culture cycle of bird's nest fruit is relatively long, with a subculture growth cycle of 50-60 days and a rooting cycle of 30-35 days. This invention provides a rapid domestication method for bird's nest fruit, which improves the efficiency of commercial seedling production and has promising application prospects. Summary of the Invention

[0003] Therefore, this invention proposes a rapid domestication method for bird's nest fruit.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for rapid domestication of bird's nest fruit includes the following steps:

[0006] (1) Liquid culture medium I and liquid culture medium II were prepared using modified MS liquid culture medium;

[0007] Preferably, liquid culture medium I consists of modified MS liquid culture medium + NAA 0.5-0.8 mg / L + 6-BA 1.5 mg / L.

[0008] Composed of -2.5 mg / L + polyvinylpyrrolidone 45-55 mg / L + activated charcoal 0.1-0.2 g / L + sucrose 15-25 g / L + antibiotic 3-5 mg / L, pH value 5.8-6.0;

[0009] Preferably, the antibiotic in liquid culture medium I is streptomycin sulfate, with a volume concentration of 40-45 μg / mL, and the liquid culture medium I-II is stored at -20 to -25°C.

[0010] Preferably, liquid culture medium II consists of modified MS liquid culture medium + 6-BA 3-3.5 mg / L + IAA 0.4 g / L.

[0011] Composed of 0.6 mg / L sucrose 20-23 g / L + tryptone 0.2-0.4 g / L + activated charcoal 0.1-0.2 g / L, pH 5.8-6.0.

[0012] The preferred components and corresponding concentrations of the modified MS liquid culture medium are as follows: potassium nitrate 1700-1800 mg / L, ammonium nitrate 1500-1550 mg / L, magnesium sulfate heptahydrate 650-700 mg / L, anhydrous potassium dihydrogen phosphate 160-170 mg / L, calcium chloride dihydrate 550-600 mg / L, ferrous sulfate heptahydrate 40-45 mg / L, manganese sulfate tetrahydrate 15-18 mg / L, and zinc sulfate 8-10 mg / L. / L, boric acid 2-4mg / L, potassium iodide 0.5-0.7mg / L, copper sulfate pentahydrate 0.02-0.03mg / L, cobalt chloride hexahydrate 0.1-0.2mg / L, thiamine hydrochloride 5-7mg / L, nicotinic acid 2.0-2.3mg / L, pyridoxine hydrochloride 2.0-2.3mg / L, inositol 80-90mg / L, 6-benzylpurine 3.0-3.5mg / L, naphthaleneacetic acid 3.0-3.5mg / L.

[0013] The preferred modified MS liquid culture medium components and their corresponding concentrations are as follows: potassium nitrate 1750 mg / L, ammonium nitrate 1500 mg / L, magnesium sulfate heptahydrate 650 mg / L, anhydrous potassium dihydrogen phosphate 160 mg / L, calcium chloride dihydrate 600 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 3 mg / L, potassium iodide 0.6 mg / L, copper sulfate pentahydrate 0.02 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 6 mg / L, nicotinic acid 2.2 mg / L, pyridoxine hydrochloride 2.2 mg / L, inositol 85 mg / L, 6-benzylpurine 3.0 mg / L, and naphthaleneacetic acid 3.0 mg / L.

[0014] (2) Select healthy stem segments containing areoles for sterilization;

[0015] Preferred sterilization method: Select healthy stem segments containing areoles (6-7 cm in length) for sterilization. Place the stem segments on a clean bench and wash them 2-3 times with sterile water. Wipe the stem segments 2-3 times with 75 wt% ethanol cotton pads. Wash them 2-3 times with sterile water and soak them in 0.1% wt% HgCl2 for 10-12 minutes. Wash them 4-5 times with sterile water and wipe them 2-3 times with 2-5 wt% sodium hypochlorite cotton pads. Wash them 4-5 times with sterile water.

[0016] (3) The sterilized branches prepared by S2 were placed horizontally in liquid culture medium I to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into segments and cultured in liquid culture medium II to obtain the second generation of sterile new shoots.

[0017] Preferably, the first-generation aseptic new shoot culture conditions are as follows: the sterilized branches prepared by S2 are placed horizontally in liquid culture medium I and cultured for 25-28 days in a light intensity of 1600-2200 Lux, a temperature of 25-27℃, and a 16-hour light and 8-hour dark cycle mode; the second-generation aseptic new shoot culture conditions are as follows: the first-generation aseptic new shoots are cut into segments, and the middle segments of 0.8-1 cm are placed in liquid culture medium II and cultured under light for 25-28 days.

[0018] (4) Transplant the second generation of sterile new shoots into hydroponics for 5-7 days to obtain bird's nest fruit seedlings.

[0019] The preferred nutrient solution composition for hydroponic transplantation of new shoots includes: potassium nitrate 240-360 g / L, calcium nitrate 400-450 g / L, potassium sulfate 80-90 g / L, potassium dihydrogen phosphate 90-100 g / L, ammonium dihydrogen phosphate 5-10 g / L, magnesium sulfate 180-220 g / L, copper sulfate 0.3-0.5 g / L, ammonium molybdate 0.1-0.3 g / L, DTPA iron 12-15 g / L, zinc sulfate amino acids 0.5-0.8 g / L, and manganese sulfate amino acids 4.0-4.5 g / L.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) The seedlings of bird's nest fruit cultivated by the modified MS + antibiotic liquid culture medium and the hydroponic cultivation method of new shoot transplanting have good root development. The modified MS culture medium has added inositol, 6-benzylpurine and naphthaleneacetic acid and antibiotic liquid. While inhibiting bacteria, it also increases the growth of the roots of bird's nest fruit seedlings, which significantly increases the average root diameter, average total root length and average root surface area.

[0022] (2) The results of the test on the lateral branch index of the bird's nest fruit seedlings cultivated by the modified MS+antibiotic liquid culture medium and the hydroponic cultivation method of transplanting new shoots showed that the compound culture medium combined with the hydroponic cultivation method effectively improved the number of lateral branches, the length of lateral branches and the aboveground biomass of bird's nest fruit seedlings.

[0023] (3) The hydroponic method of this invention can improve the survival rate of bird's nest fruit in a short time. It does not require the use of high-cost resources such as soil and pesticides, and is easy to manage, saving time and production costs. Detailed Implementation

[0024] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0026] The full name of DTPA iron is: sodium ferric diethylenetriaminepentaacetate.

[0027] Streptomycin sulfate is an agricultural grade streptomycin sulfate, in which the active ingredient of streptomycin sulfate is 72%.

[0028] Preparation Example 1-1

[0029] Preparation of raw materials for modified MS liquid culture medium in Example 1-1:

[0030] Potassium nitrate 1750 mg / L, ammonium nitrate 1500 mg / L, magnesium sulfate heptahydrate 650 mg / L, anhydrous potassium dihydrogen phosphate 160 mg / L, calcium chloride dihydrate 600 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 3 mg / L, potassium iodide 0.6 mg / L, copper sulfate pentahydrate 0.02 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 6 mg / L, nicotinic acid 2.2 mg / L, pyridoxine hydrochloride 2.2 mg / L, inositol 85 mg / L, 6-benzylpurine 3.0 mg / L, naphthaleneacetic acid 3.0 mg / L.

[0031] Preparation Examples 1-2

[0032] Preparation of the raw material contents for the modified MS liquid culture medium in Examples 1-2:

[0033] Potassium nitrate 1700 mg / L, ammonium nitrate 1550 mg / L, magnesium sulfate heptahydrate 700 mg / L, anhydrous potassium dihydrogen phosphate 170 mg / L, calcium chloride dihydrate 550 mg / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate tetrahydrate 17 mg / L, zinc sulfate 9 mg / L, boric acid 4 mg / L, potassium iodide 0.7 mg / L, copper sulfate pentahydrate 0.03 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 7 mg / L, nicotinic acid 2.1 mg / L, pyridoxine hydrochloride 2.1 mg / L, inositol 80 mg / L, 6-benzylpurine 3.0 mg / L, naphthaleneacetic acid 3.0 mg / L.

[0034] Preparation Examples 1-3

[0035] Preparation of raw material contents for modified MS liquid culture medium in Examples 1-3:

[0036] Potassium nitrate 1800 mg / L, ammonium nitrate 1520 mg / L, magnesium sulfate heptahydrate 680 mg / L, anhydrous potassium dihydrogen phosphate 165 mg / L, calcium chloride dihydrate 550 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 4 mg / L, potassium iodide 0.7 mg / L, copper sulfate pentahydrate 0.03 mg / L, cobalt chloride hexahydrate 0.15 mg / L, thiamine hydrochloride 5 mg / L, nicotinic acid 2.3 mg / L, pyridoxine hydrochloride 2.3 mg / L, inositol 90 mg / L, 6-benzylpurine 3.5 mg / L, naphthaleneacetic acid 3.5 mg / L.

[0037] Preparation Example 2-1

[0038] The nutrient solution for hydroponic transplantation of new shoots in Example 2-1 consists of: potassium nitrate 300 g / L, calcium nitrate 400 g / L, potassium sulfate 80 g / L, potassium dihydrogen phosphate 100 g / L, ammonium dihydrogen phosphate 10 g / L, magnesium sulfate 200 g / L, copper sulfate 0.5 g / L, ammonium molybdate 0.2 g / L, DTPA iron 15 g / L, zinc sulfate amino acids 0.5 g / L, and manganese sulfate amino acids 4.0 g / L.

[0039] Preparation Example 2-2

[0040] The nutrient solution for hydroponic transplantation of new shoots in Example 2-2 consists of: potassium nitrate 250 g / L, calcium nitrate 420 g / L, potassium sulfate 80 g / L, potassium dihydrogen phosphate 90 g / L, ammonium dihydrogen phosphate 10 g / L, magnesium sulfate 220 g / L, copper sulfate 0.5 g / L, ammonium molybdate 0.3 g / L, DTPA iron 15 g / L, zinc sulfate amino acids 0.8 g / L, and manganese sulfate amino acids 4.5 g / L.

[0041] Preparation Examples 2-3

[0042] The nutrient solution for hydroponic transplantation of new shoots in Examples 2-3 consisted of: potassium nitrate 260 g / L, calcium nitrate 440 g / L, potassium sulfate 85 g / L, potassium dihydrogen phosphate 95 g / L, ammonium dihydrogen phosphate 6 g / L, magnesium sulfate 180 g / L, copper sulfate 0.3 g / L, ammonium molybdate 0.2 g / L, DTPA iron 12 g / L, zinc sulfate amino acids 0.5 g / L, and manganese sulfate amino acids 4.3 g / L.

[0043] Example 1

[0044] The modified MS liquid culture medium from Preparation Example 1-1 was used to prepare the acclimatization of bird's nest fruit, and the nutrient solution from the hydroponic transplantation of new shoots from Preparation Example 2-3 was used. The acclimatization included the following steps:

[0045] (1) Select a healthy stem with areoles and sterilize it. Place the stem segment on a clean bench and wash it 3 times with sterile water. Wipe the stem segment 3 times with 75wt% ethanol cotton pads. Wash it 3 times with sterile water and soak it in 0.1wt% HgCl2 for 10 minutes. Wash it 5 times with sterile water and wipe it 3 times with 3wt% sodium hypochlorite cotton pads. Wash it 4 times with sterile water.

[0046] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.6.

[0047] The modified MS basal medium consisted of 2.0 mg / L 6-BA, 50 mg / L polyvinylpyrrolidone, 0.1 g / L activated carbon, 15 g / L sucrose, and 4 mg / L streptomycin sulfate, with a pH of 5.8. It was prepared using Example 1-1, and liquid medium I was stored at -25°C.

[0048] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3 mg / L + IAA 0.4 mg / L + sucrose 20 g / L + tryptone 0.4 g / L + activated carbon 0.2 g / L, with a pH of 5.8. The modified MS basic medium is prepared in Example 1-1. Liquid culture medium I is stored at -25℃.

[0049] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 28 days in a cycle of 16h light (light intensity 2200 Lux) and 8h darkness at a temperature of 27℃ to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 28 days to obtain the second generation of sterile new shoots.

[0050] (5) The second generation of sterile new shoots were transplanted into hydroponics for 7 days to obtain bird's nest fruit seedlings. The nutrient solution used in the hydroponics was prepared according to Example 2-2.

[0051] Example 2

[0052] The modified MS liquid culture medium from Preparation Examples 1-2 was prepared using the same proportions of each ingredient. The nutrient solution from the hydroponic transplantation of new shoots from Preparation Example 2-2 was used to acclimate the bird's nest fruit, including the following steps:

[0053] (1) Select a 6.5cm healthy stem containing areoles and sterilize it. Place the stem segment on a clean bench and wash it twice with sterile water. Wipe the stem segment three times with 75wt% ethanol cotton pads. Wash it twice with sterile water and soak it in 0.1% wt HgCl2 for 12min. Wash it five times with sterile water and wipe it three times with 4wt% sodium hypochlorite cotton pads. Wash it five times with sterile water.

[0054] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.7.

[0055] The modified MS basal medium consisted of 1.5 mg / L 6-BA, 48 mg / L polyvinylpyrrolidone, 0.15 g / L activated carbon, 20 g / L sucrose, and 3 mg / L streptomycin sulfate, with a pH of 5.8-6.0. The modified MS basal medium was prepared using Examples 1-2, and liquid medium I was stored at -23°C.

[0056] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3.3 mg / L + IAA 0.6 mg / L + sucrose 20 g / L + 0.5 g / L tryptone + activated carbon 0.3 g / L, pH 6.0. The modified MS basic medium is prepared in Example 1-2. Liquid culture medium I is stored at -25℃.

[0057] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 26 days in a cycle of 16h light and 8h dark at a light intensity of 1800 Lux and a temperature of 25℃ to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 26 days to obtain the second generation of sterile new shoots.

[0058] (5) Transplant the second generation of sterile new shoots into hydroponics for 6 days to obtain bird's nest fruit seedlings. The nutrient solution used in the hydroponics was prepared according to Example 2-2.

[0059] Example 3

[0060] The modified MS liquid culture medium prepared using the same raw material content as in Preparation Examples 1-3 was used to acclimate the bird's nest fruit using the same nutrient solution composition as that used in hydroponic transplantation of new shoots in Preparation Example 1-1. The acclimatization process included the following steps:

[0061] (1) Select a healthy stem with areoles and sterilize it. Place the stem segment on a clean bench and wash it 3 times with sterile water. Wipe the stem segment 3 times with 75wt% ethanol cotton pads. Wash it 2 times with sterile water and soak it in 0.1% wt HgCl2 for 10 minutes. Wash it 4 times with sterile water and wipe it 2 times with 2wt% sodium hypochlorite cotton pads. Wash it 4 times with sterile water.

[0062] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.5-0.8 g / L.

[0063] The modified MS basal medium consisted of 1.5-2.5 mg / L 6-BA, 55 mg / L polyvinylpyrrolidone, 0.2 g / L activated carbon, 23 g / L sucrose, and 3 mg / L streptomycin sulfate, with a pH of 5.9. The modified MS basal medium was prepared using Examples 1-3, and liquid medium I was stored at -25°C.

[0064] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3.4 mg / L + IAA 0.4 mg / L + sucrose 20 g / L + tryptone 0.5 g / L + activated carbon 0.3 g / L, pH 6.0. The modified MS basic medium is prepared in Examples 1-3. Liquid culture medium I is stored at -25℃.

[0065] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 28 days in a light intensity of 2000 Lux, a temperature of 25℃, and a cycle of 16h light and 8h dark to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 28 days to obtain the second generation of sterile new shoots.

[0066] (5) Transplant the second generation of sterile new shoots into hydroponics for 5 days to obtain bird's nest fruit seedlings. The nutrient solution used in the hydroponics is prepared according to Example 1-1.

[0067] Comparative Example 1

[0068] The main difference between Comparative Example 1 and Example 1 is that inositol, 6-benzylpurine, and naphthaleneacetic acid were removed from the modified MS liquid culture medium.

[0069] The raw materials for the modified MS liquid culture medium in Comparative Example 1 were prepared with the following concentrations: potassium nitrate 1750 mg / L, ammonium nitrate 1500 mg / L, magnesium sulfate heptahydrate 650 mg / L, anhydrous potassium dihydrogen phosphate 160 mg / L, calcium chloride dihydrate 600 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 3 mg / L, potassium iodide 0.6 mg / L, copper sulfate pentahydrate 0.02 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 6 mg / L, nicotinic acid 2.2 mg / L, and pyridoxine hydrochloride 2.2 mg / L.

[0070] The modified MS liquid culture medium of Comparative Example 1 was prepared with the same proportions of each raw material. The nutrient solution used in the hydroponic transplantation of new shoots from Examples 2-3 was used to acclimate the bird's nest fruit, including the following steps:

[0071] (1) Select a healthy stem with areoles and sterilize it. Place the stem segment on a clean bench and wash it 3 times with sterile water. Wipe the stem segment 3 times with 75wt% ethanol cotton pads. Wash it 3 times with sterile water and soak it in 0.1wt% HgCl2 for 10 minutes. Wash it 5 times with sterile water and wipe it 3 times with 3wt% sodium hypochlorite cotton pads. Wash it 4 times with sterile water.

[0072] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.6.

[0073] The modified MS basal medium consisted of 2.0 mg / L 6-BA, 50 mg / L polyvinylpyrrolidone, 0.1 g / L activated carbon, 15 g / L sucrose, and 4 mg / L streptomycin sulfate, with a pH of 5.8. The modified MS basal medium was prepared using the formulation of Comparative Example 1, and liquid medium I was stored at -25°C.

[0074] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3 mg / L + IAA 0.4 mg / L + sucrose 20 g / L + tryptone 0.4 g / L + activated carbon 0.2 g / L, with a pH of 5.8. The modified MS basic medium adopts the formula of Comparative Example 1. Liquid culture medium I is stored at -25℃.

[0075] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 28 days in a light intensity of 2200 Lux, a temperature of 27℃, and a cycle of 16h light and 8h dark to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 28 days to obtain the second generation of sterile new shoots.

[0076] (5) The second generation of sterile new shoots were transplanted into hydroponics for 7 days to obtain bird's nest fruit seedlings. The nutrient solution used in the hydroponics was prepared according to Example 2-2.

[0077] Comparative Example 2

[0078] The main difference between Comparative Example 2 and Example 1 is that the transplanting hydroponics was replaced by direct cultivation in soil.

[0079] The modified MS liquid culture medium from Preparation Example 1-1 was used to prepare the acclimatization of bird's nest fruit, and the nutrient solution from the hydroponic transplantation of new shoots from Preparation Example 2-3 was used. The acclimatization included the following steps:

[0080] (1) Select a healthy stem with areoles and sterilize it. Place the stem segment on a clean bench and wash it 3 times with sterile water. Wipe the stem segment 3 times with 75wt% ethanol cotton pads. Wash it 3 times with sterile water and soak it in 0.1wt% HgCl2 for 10 minutes. Wash it 5 times with sterile water and wipe it 3 times with 3wt% sodium hypochlorite cotton pads. Wash it 4 times with sterile water.

[0081] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.6.

[0082] The modified MS basal medium consisted of 2.0 mg / L 6-BA, 50 mg / L polyvinylpyrrolidone, 0.1 g / L activated carbon, 15 g / L sucrose, and 4 mg / L streptomycin sulfate, with a pH of 5.8. It was prepared using Example 1-1, and liquid medium I was stored at -25°C.

[0083] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3 mg / L + IAA 0.4 mg / L + sucrose 20 g / L + tryptone 0.4 g / L + activated carbon 0.2 g / L, with a pH of 5.8. The modified MS basic medium is prepared in Example 1-1. Liquid culture medium I is stored at -25℃.

[0084] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 28 days in a light intensity of 2200 Lux, a temperature of 27℃, and a cycle of 16h light and 8h dark to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 28 days to obtain the second generation of sterile new shoots.

[0085] (5) Transplant the second generation of sterile new shoots into soil for cultivation and apply NPK-Mg-S compound fertilizer, which contains 15wt% N, 15wt% P, 15wt% K, 6wt% Mg, and 10wt% S.

[0086] Comparative Example 3

[0087] The main difference between Comparative Example 3 and Example 1 is that the antibiotic streptomycin sulfate was removed from liquid culture medium I, and the modified MS liquid culture medium was replaced with inositol 40 mg / L, 6-benzylpurine 1.5 mg / L, and naphthaleneacetic acid 1.5 mg / L instead of inositol 85 mg / L, 6-benzylpurine 3.0 mg / L, and naphthaleneacetic acid 3.0 mg / L. The other raw materials and their contents remained unchanged.

[0088] Preparation of raw material contents for the modified MS liquid culture medium in Comparative Example 3:

[0089] Potassium nitrate 1750 mg / L, ammonium nitrate 1500 mg / L, magnesium sulfate heptahydrate 650 mg / L, anhydrous potassium dihydrogen phosphate 160 mg / L, calcium chloride dihydrate 600 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 3 mg / L, potassium iodide 0.6 mg / L, copper sulfate pentahydrate 0.02 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 6 mg / L, nicotinic acid 2.2 mg / L, pyridoxine hydrochloride 2.2 mg / L, inositol 40 mg / L, 6-benzylpurine 1.5 mg / L, naphthaleneacetic acid 1.5 mg / L.

[0090] Includes the following steps:

[0091] (1) Select a healthy stem with areoles and sterilize it. Place the stem segment on a clean bench and wash it 3 times with sterile water. Wipe the stem segment 3 times with 75wt% ethanol cotton pads. Wash it 3 times with sterile water and soak it in 0.1% wt HgCl2 for 10 minutes. Wash it 5 times with sterile water and wipe it 3 times with 3wt% sodium hypochlorite cotton pads. Wash it 4 times with sterile water.

[0092] (2) Prepare liquid culture medium I, which consists of modified MS basal medium + NAA 0.6.

[0093] The modified MS basal medium consisted of 2.0 mg / L 6-BA, 50 mg / L polyvinylpyrrolidone, 0.1 g / L activated carbon, and 15 g / L sucrose, with a pH of 5.8. The modified MS basal medium was prepared using Example 1-1, and liquid medium I was stored at -25°C.

[0094] (3) Prepare liquid culture medium II. Liquid culture medium II consists of modified MS basic medium + 6-BA 3 mg / L + IAA 0.4 mg / L + sucrose 20 g / L + tryptone 0.4 g / L + activated carbon 0.2 g / L, with a pH of 5.8. The modified MS basic medium is prepared in Example 1-1. Liquid culture medium I is stored at -25℃.

[0095] (4) The sterilized branches prepared in step (1) were placed horizontally in liquid culture medium I and cultured for 28 days in a light intensity of 2200 Lux, a temperature of 27℃, and a cycle of 16h light and 8h dark to obtain the first generation of sterile new shoots. The first generation of sterile new shoots were cut into sections, and the middle section of 1cm was placed in liquid culture medium II and cultured under light for 28 days to obtain the second generation of sterile new shoots.

[0096] (5) The second generation of sterile new shoots were transplanted into hydroponics for 7 days to obtain bird's nest fruit seedlings. The nutrient solution used in the hydroponics was prepared according to Example 2-2.

[0097] test:

[0098] Sixty seedlings of *Acer negundo* were cultivated using the methods of the examples and comparative examples, with 10 seedlings in each group. After 93 days of cultivation, the seedlings in Examples 1-3 all had a height exceeding 25 cm, more than two lateral branches, and healthy roots, indicating they were ready for transplanting. Various indicators were measured after 93 days of cultivation. Sampling and marking were performed on both the aboveground and underground parts. Roots were rinsed with purified water until free of soil, and scanned using a flatbed scanner at a resolution of 400 dpi. Parameters such as root length, root surface area, and average root diameter were obtained and analyzed using WinRhizo software. At harvest, the number of lateral branches on the mother branch was recorded, and the total length of the lateral branches was measured. The branches were then gently rinsed with tap water to remove surface impurities, followed by three rinses with deionized water. After slight drying, the samples were cut into small pieces and placed in pre-labeled kraft paper envelopes, then placed in an oven. The samples were blanched at 105℃ for 30 minutes, then the temperature was adjusted to 80℃ and dried to constant weight. The dry weight of the branches was then measured.

[0099] (a) Root test

[0100] The results of the root index test of *Acer negundo* seedlings showed that, after 93 days, the average root diameter, average total root length, and average root surface area of ​​Examples 1-3 were significantly higher than those of Comparative Examples 1-3. Compared with Comparative Example 1 (modified MS liquid medium with inositol, 6-benzylpurine, and naphthaleneacetic acid removed), Examples 1-3 showed an increase of 0.09 mm (18.2%) in average root diameter, 31.17 cm (13.1%) in average total root length, and 85.3 cm² in average root surface area. 2 The increase was 20.1%. Compared with Comparative Examples 1-3, the average root diameter of *Acer negundo* seedlings in Examples 1-3 increased by 0.1 mm (an increase of 18.8%), the average total root length increased by 25.97 cm (an increase of 10.9%), and the average root surface area increased by 77.7 cm². 2 The average root diameter, average total root length, and average root surface area of ​​*Agrostis foetida* were increased by 18.3%. Therefore, the tissue culture method of *Agrostis foetida* of the present invention significantly increased the average root diameter, average total root length, and average root surface area of ​​*Agrostis foetida*, and this method significantly promoted the root growth of *Agrostis foetida* seedlings.

[0101] Table 1. Root Indicators of Bird's Nest Fruit Seedlings

[0102]

[0103] Note: a, b, and c represent statistically significant differences between groups (P < 0.05).

[0104] (ii) Lateral branch experiment

[0105] The results of the lateral branch index test of *Agrostis spp.* seedlings showed that, after 93 days, the average number of lateral branches, average lateral branch length, and average aboveground biomass of Examples 1-3 were significantly higher than those of Comparative Examples 1-3. Compared with Comparative Example 1 (modified MS liquid medium with inositol, 6-benzylpurine, and naphthaleneacetic acid removed), Examples 1-3 showed an increase of 1.3 average lateral branches, an increase of 12.1 cm in average lateral branch length, and an increase of 3.3 g / plant in average aboveground biomass. Compared with Comparative Examples 1-3, Examples 1-3 showed an increase of 1.2 average lateral branches, an increase of 10.2 cm in average lateral branch length, and an increase of 3.8 g / plant in average aboveground biomass. Therefore, the *Agrostis spp.* tissue culture method of the present invention significantly increased the average root diameter, average total root length, and average root surface area of ​​*Agrostis spp.*, and this method significantly promoted root growth of *Agrostis spp.* seedlings.

[0106] Table 2. Lateral branch indicators of *Acer buergerianum* seedlings

[0107]

[0108] Note: a, b, and c represent statistically significant differences between groups (P < 0.05).

[0109] Conclusion: The results of this study indicate that the seedlings of *Acer negundo* cultivated using the method of this invention exhibit good root and lateral branch development. Root index tests on *Acer negundo* seedlings showed that, after 93 days, the average root diameter, average total root length, and average root surface area of ​​Examples 1-3 were significantly higher than those of Comparative Examples 1-3. Compared to Comparative Examples 1-3, Examples 1-3 showed an increase of 0.1 mm in average root diameter (18.8%), an increase of 259.7 cm in average total root length (10.9%), and an increase of 77.7 cm² in average root surface area. 2 The number of lateral branches increased by 18.3%. The results of the lateral branch index test on *Agrostis spp.* seedlings showed that, after 93 days, the average number of lateral branches, average lateral branch length, and average aboveground biomass of Examples 1-3 were significantly higher than those of Comparative Examples 1-3. Compared with Comparative Examples 1-3, Examples 1-3 showed an increase of 1.2 average lateral branches, an increase of 10.2 cm in average lateral branch length, and an increase of 3.8 g / plant in average aboveground biomass. Therefore, the *Agrostis spp.* tissue culture method of the present invention increased the growth of roots and lateral branches in *Agrostis spp.* seedlings, and Examples 1-3 showed an earlier development time than Comparative Examples 1-3.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rapid domestication of bird's nest fruit, characterized in that, Includes the following steps: S1. Prepare liquid culture medium I and liquid culture medium II using modified MS liquid culture medium; The liquid culture medium I consists of modified MS liquid culture medium + NAA 0.5-0.8 mg / L + 6-BA 1.5-2.5 mg / L + polyvinylpyrrolidone 45-55 mg / L + activated carbon 0.1-0.2 g / L + sucrose 15-25 g / L + antibiotic 3-5 mg / L, with a pH of 5.8-6.0; The antibiotic in the liquid culture medium I is streptomycin sulfate, and the volume concentration of streptomycin sulfate is 40-45 μg / mL. The liquid culture medium II consists of modified MS liquid culture medium + 6-BA 3-3.5 mg / L + IAA 0.4-0.6 mg / L + sucrose 20-23 g / L + tryptone 0.2-0.4 g / L + activated carbon 0.1-0.2 g / L, with a pH of 5.8-6.0; The modified MS liquid culture medium components and their corresponding concentrations are as follows: potassium nitrate 1700-1800 mg / L, ammonium nitrate 1500-1550 mg / L, magnesium sulfate heptahydrate 650-700 mg / L, anhydrous potassium dihydrogen phosphate 160-170 mg / L, calcium chloride dihydrate 550-600 mg / L, ferrous sulfate heptahydrate 40-45 mg / L, manganese sulfate tetrahydrate 15-18 mg / L, zinc sulfate 8-10 mg / L. L, boric acid 2-4 mg / L, potassium iodide 0.5-0.7 mg / L, copper sulfate pentahydrate 0.02-0.03 mg / L, cobalt chloride hexahydrate 0.1-0.2 mg / L, thiamine hydrochloride 5-7 mg / L, nicotinic acid 2.0-2.3 mg / L, pyridoxine hydrochloride 2.0-2.3 mg / L, inositol 80-90 mg / L, 6-benzylpurine 3.0-3.5 mg / L, naphthaleneacetic acid 3.0-3.5 mg / L; S2. Select healthy stem segments containing areoles and sterilize them; S3. Place the sterilized branches prepared in S2 horizontally in liquid culture medium I to culture and obtain the first generation of sterile new shoots. Cut the first generation of sterile new shoots into segments and culture them in liquid culture medium II to obtain the second generation of sterile new shoots. S4. Transplant the second-generation sterile new shoots into hydroponics for 5-7 days to obtain bird's nest fruit seedlings; The nutrient solution for hydroponic transplantation of new shoots includes: potassium nitrate 240-360 g / L, calcium nitrate 400-450 g / L, potassium sulfate 80-90 g / L, potassium dihydrogen phosphate 90-100 g / L, ammonium dihydrogen phosphate 5-10 g / L, magnesium sulfate 180-220 g / L, copper sulfate 0.3-0.5 g / L, ammonium molybdate 0.1-0.3 g / L, DTPA iron 12-15 g / L, zinc sulfate amino acids 0.5-0.8 g / L, and manganese sulfate amino acids 4.0-4.5 g / L.

2. The method for rapid domestication of bird's nest fruit as described in claim 1, characterized in that, The liquid culture media I-II are stored at -20 to -25°C.

3. The method for rapid domestication of bird's nest fruit as described in claim 1, characterized in that, S1, the modified MS liquid culture medium components and their corresponding concentrations are as follows: potassium nitrate 1750 mg / L, ammonium nitrate 1500 mg / L, magnesium sulfate heptahydrate 650 mg / L, anhydrous potassium dihydrogen phosphate 160 mg / L, calcium chloride dihydrate 600 mg / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate tetrahydrate 16 mg / L, zinc sulfate 10 mg / L, boric acid 3 mg / L, potassium iodide 0.6 mg / L, copper sulfate pentahydrate 0.02 mg / L, cobalt chloride hexahydrate 0.1 mg / L, thiamine hydrochloride 6 mg / L, nicotinic acid 2.2 mg / L, pyridoxine hydrochloride 2.2 mg / L, inositol 85 mg / L, 6-benzylpurine 3.0 mg / L, naphthaleneacetic acid 3.0 mg / L.

4. The rapid domestication method for bird's nest fruit as described in claim 1, characterized in that, S2, the sterilization method, involves selecting a healthy stem segment of 6-7 cm containing areoles for sterilization, placing the stem segment on a clean bench, rinsing it 2-3 times with sterile water, wiping the stem segment 2-3 times with 75wt% ethanol cotton pads, rinsing it 2-3 times with sterile water, soaking it in 0.1% wtHgCl2 for 10-12 minutes, rinsing it 4-5 times with sterile water, wiping it 2-3 times with 2-5wt% sodium hypochlorite cotton pads, and rinsing it 4-5 times with sterile water.

5. The rapid domestication method for bird's nest fruit as described in claim 1, characterized in that, S3, the first generation of sterile new shoot culture conditions are as follows: the sterilized branches prepared in S2 are placed horizontally in liquid culture medium I and cultured for 25-28 days in a light intensity of 1600-2200 Lux, a temperature of 25-27℃, and a 16-hour light and 8-hour dark cycle mode; the second generation of sterile new shoot culture conditions are as follows: the first generation of sterile new shoots are cut into segments, and 0.8-1cm middle segments are taken and cultured in liquid culture medium II under light for 25-28 days.

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