Culture medium and method for rapid propagation of tissue culture seedlings of Jingkexifengshou cut chrysanthemum

Abstract

The invention belongs to the field of plant tissue culture, and provides a culture medium for rapid propagation of Jingkexifengshou cut chrysanthemum tissue culture seedlings, the proliferation culture medium is based on MS basic culture medium, and is supplemented with: NAA, 6-BA, banana puree; and a method for rapid propagation of ornamental chrysanthemum tissue culture seedlings, including inoculating stem segments of ornamental chrysanthemum sterile seedlings into a proliferation culture medium to obtain adventitious buds, and then culturing the adventitious buds into ornamental chrysanthemum plants, the proliferation culture medium is based on MS basic culture medium, and is supplemented with: NAA, 6-BA, banana puree. Using the proliferation culture medium and method provided by the invention, adventitious bud proliferation culture can be performed using the stem segments of ornamental chrysanthemum sterile seedlings, and a large number of adventitious buds can be obtained in only about 50 days, and the propagation efficiency is very high, and the cost is low and the propagation efficiency is high.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and particularly relates to a proliferation culture medium for rapid propagation of "Jingke Xifengshou" cut flower ornamental chrysanthemum tissue culture seedlings, and a method for rapid propagation of "Jingke Xifengshou" cut flower ornamental chrysanthemum tissue culture seedlings using the culture medium. Background Art

[0002] The new chrysanthemum variety "Jingke Xifengshou" (Chrysanthemum×morifolium'Jingke Xifengshou') is based on the cut ornamental chrysanthemum CQ72 as the female parent, obtained through open pollination, and then continuously selected and bred. Its flowers are flat petals in a single round, about 3cm in diameter, and red stripes are distributed on the yellow ligulate flowers. It has good uprightness and good overall commercial quality, and is suitable for use as a multi-head cut ornamental chrysanthemum. The natural flowering period in Beijing is mid-to-late October. It is suitable for protected planting across the country.

[0003] As a new ornamental variety of cut ornamental chrysanthemum, "Jingke Xifengshou" has strong uprightness and good stress resistance, especially high resistance to white rust; the petals are single-wheeled and multi-colored, golden yellow (6B, Royal Horticultural Society color chart 6th edition) with dark red (N45B, Royal Horticultural Society color chart 6th edition) striped spots on the petals, bright color, good ornamental value, stable color, and little affected by temperature; the average flower diameter is 2.94cm, with 7-9 flower heads per branch, suitable for multi-head cut ornamental chrysanthemum; the plant grows moderately, with a plant height of about 80cm; the light response period is 7-7.5 weeks, and it is easy to regulate the flowering period. Since "Jingke Xifengshou" was launched, it has been favored by market customers, but due to the limited emergence rate of existing seedling propagation methods, the seedlings have been in short supply; therefore, in current scientific research and production applications, it is particularly important to develop a rapid propagation method for Jingke Xifengshou cut ornamental chrysanthemum with high proliferation rate, easy operation, and low cost. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a proliferation medium for rapid propagation of ornamental chrysanthemum tissue culture seedlings, wherein the proliferation medium is based on MS basic medium and is supplemented with: NAA, 6-BA, and banana puree.

[0005] The present invention also provides a method for rapid propagation of ornamental chrysanthemum tissue culture seedlings, the method comprising the steps of inoculating stem segments of sterile ornamental chrysanthemum seedlings into a proliferation medium to obtain adventitious buds, and then culturing the adventitious buds into ornamental chrysanthemum plants; wherein the proliferation medium is based on MS basic medium and is supplemented with: NAA, 6-BA, and banana puree.

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] 1. The present invention multiplies and cultivates adventitious buds on the aseptic stem segments of "Jingke Xifengshou" cut flower ornamental chrysanthemum, and a large number of adventitious buds can be obtained in only about 50 days, with a very high breeding efficiency.

[0008] 2. The adventitious bud proliferation medium of the present invention is added with banana puree containing a large amount of sugars and plant hormones, which synergizes with NAA and 6-BA to significantly increase the yield of adventitious buds.

[0009] 3. The NAA, 6-BA and banana puree added in the adventitious bud proliferation culture of the present invention have very low cost.

[0010] 4. The various steps and parameters of the present invention work synergistically to jointly improve the breeding efficiency of Jingke Xifengshou.

[0011] 5. The present invention is simple and easy to operate, low in cost, and suitable for wide use.

[0012] 6. The present invention is carried out in a tissue culture room and can be operated uninterruptedly throughout the year, thereby saving costs and achieving high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Schematic diagram of adventitious buds grown from a single stem segment after 50 days of adventitious bud proliferation culture in step (2) of Example 1.

[0014] Figure 2 :Will Figure 1 Schematic diagram of the adventitious buds after separation.

[0015] Figure 3 : Schematic diagram of adventitious buds and callus produced from a single stem segment after 50 days of adventitious bud proliferation culture in step (2) of comparative example 1.

[0016] Figure 4 : Figure 3 Schematic diagram of callus tissue.

[0017] Figure 5 : Schematic diagram of adventitious buds and callus produced from a single stem segment after 50 days of adventitious bud proliferation culture in step (2) of comparative example 2.

[0018] Figure 6 : Figure 5 Bottom view, i.e. schematic diagram of adventitious buds and callus tissue when looking up from the bottom of the bottle.

[0019] Figure 7 :Will Figure 5 Schematic diagram of the adventitious buds and callus tissues after being removed and displayed in the culture dish.

[0020] Figure 8 :Will Figure 5Schematic diagram showing the individual adventitious buds after they are separated. DETAILED DESCRIPTION

[0021] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0022] A first aspect of the present invention provides a proliferation culture medium for rapid propagation of ornamental chrysanthemum tissue culture seedlings; the culture medium is particularly suitable for "Jingke Xifengshou" cut ornamental chrysanthemum.

[0023] According to the first aspect of the present invention, the proliferation medium is based on MS basic medium and is supplemented with: NAA (naphthaleneacetic acid), 6-BA (6-benzylaminopurine), and banana puree.

[0024] According to the first aspect of the present invention, the final concentration of NAA in the above-mentioned proliferation medium is 1-4 mg / L, preferably 2 mg / L; the final concentration of 6-BA in the above-mentioned proliferation medium is 5-10 mg / L, preferably 7 mg / L; the final concentration of banana puree in the above-mentioned proliferation medium is 20-50 g / L, preferably 30 g / L.

[0025] Preferably, the banana puree is made from raw materials containing banana pulp and water; wherein the mass ratio of banana pulp to water is (1.2-2.0):1, preferably 1.5:1.

[0026] The use of the proliferation medium for rapid propagation of ornamental chrysanthemum tissue culture seedlings provided in the first aspect of the present invention in rapid propagation of ornamental chrysanthemum tissue culture seedlings also falls within the protection scope of the present invention. The ornamental chrysanthemum may preferably be "Jingke Xifengshou" cut ornamental chrysanthemum.

[0027] The second aspect of the present invention provides a method for rapid propagation of ornamental chrysanthemum tissue culture seedlings, which comprises inoculating stem segments of sterile ornamental chrysanthemum seedlings into the proliferation culture medium provided by the first aspect of the present invention to obtain adventitious buds, and then culturing the adventitious buds into ornamental chrysanthemum plants.

[0028] Wherein, the ornamental chrysanthemum may preferably be a "Jingke Xifengshou" cut ornamental chrysanthemum.

[0029] In a preferred embodiment, the method for rapid propagation of ornamental chrysanthemum tissue culture seedlings provided in the second aspect of the present invention comprises the following steps:

[0030] Step 1: Preparation of sterile seedlings:

[0031] The sterilized explants are inoculated into MS basic culture medium and cultured for 15 to 30 days (preferably 25 days) to obtain sterile ornamental chrysanthemum seedlings.

[0032] The conditions for the above-mentioned sterile seedling culture may include: a temperature of 22 to 26° C., a light intensity of 1000 to 2500 Lx (preferably 1500 Lx), and a lighting time of 12 to 18 h / d (preferably 16 h / d).

[0033] The sterilized explants can be prepared in the following manner:

[0034] (1) Select healthy plants of the current year that have not yet bloomed, remove the leaves, and cut the stems into stem segments with 2-3 axillary buds as explants; add the explants into a 3% by volume detergent solution and soak for 15 minutes; then shake on a shaker for 10 minutes and rinse with tap water for 30 minutes to obtain cleaned explants.

[0035] (2) In a clean bench, the cleaned explant is transferred to a sterile tissue culture bottle, rinsed with sterile water for 2 to 3 times, quickly rinsed and disinfected with a 75% alcohol solution, rinsed with sterile water for 2 to 3 times, and transferred to sterile absorbent paper or a sterile culture dish for use; then, the explant is disinfected in a 5% sodium hypochlorite solution for 12 to 15 minutes, rinsed with sterile water for 3 to 5 times, and finally placed on a sterile culture dish with sterile absorbent paper to obtain a sterilized explant.

[0036] Step 2: Adventitious bud proliferation culture:

[0037] The ornamental chrysanthemum sterile seedlings obtained in step 1 are placed in a proliferation culture medium and cultured for adventitious bud proliferation for 45 to 75 days (preferably 50 days) to obtain adventitious buds.

[0038] The proliferation culture medium is based on MS basic culture medium and is supplemented with NAA, 6-BA and banana puree.

[0039] In a preferred embodiment, the proliferation medium is: based on MS basic medium, NAA is added to a final concentration of 1-4 mg / L (preferably 2 mg / L); 6-BA is added to a final concentration of 5-10 mg / L (preferably 7 mg / L); banana puree is added to a final concentration of 20-50 g / L (preferably 30 g / L).

[0040] Preferably, the banana puree is made from raw materials containing banana pulp and water, wherein the mass ratio of banana pulp to water is (1.2-2.0):1, preferably 1.5:1.

[0041] In a preferred embodiment, the method for preparing the banana puree comprises any one of the following:

[0042] Method 1: Select bananas that are not fully ripe and have a certain hardness. Crush the banana pulp (cut into thin slices or small particles), add water and heat to boiling, boil the banana pulp to obtain boiled pulp, filter the boiled pulp, and retain the filtrate (paste), which is the banana puree.

[0043] Method 2: Choose fully ripe and softened bananas. Crush the banana pulp (you can use a blender to crush it) to obtain pulp paste, then add water to the pulp paste to obtain pulp suspension, then filter the pulp suspension and retain the filtrate (in a paste form), which is the banana puree.

[0044] Method 3: Select fully ripe and softened bananas, mix the banana pulp with water, and then crush them to obtain a pulp suspension, and then filter the pulp suspension to retain the filtrate (paste), which is the banana puree.

[0045] Method 4: Obtain cooked fruit pulp according to method 1, and the cooked fruit pulp is the above-mentioned banana puree.

[0046] Method 5: Obtain a pulp suspension according to method 2, which is the banana puree.

[0047] Method 6: Obtain a pulp suspension according to method 3, which is the above-mentioned banana puree.

[0048] The reason for adding banana puree to the above-mentioned proliferation medium is that banana puree contains a large amount of sugars, proteins, amino acids and other ingredients that can be fully utilized by plants and can provide energy for the division and differentiation of plant tissue cells; and bananas are rich in natural hormones, such as cytokinins, auxins, ethylene and gibberellins, which can promote plant growth, differentiation and callus induction. In addition, bananas can also improve the disease resistance of plants and avoid the invasion of bacteria and fungi. Therefore, the addition of banana puree is suitable for the culture medium for the rapid proliferation of sterile seedling stem segments of "Jingke Xifengshou" cut ornamental chrysanthemum. Banana puree synergistically acts with NAA and 6-BA, significantly enhancing the proliferation effect of the cut ornamental chrysanthemum variety in the proliferation culture medium for adventitious bud proliferation, thereby obtaining a large number of adventitious buds.

[0049] The culture conditions for the above-mentioned adventitious bud proliferation culture are: temperature of 22-28°C (preferably 26°C), light intensity of 1000-2500Lx (preferably 1500Lx), and light time of 12-18h / d (preferably 16h / d).

[0050] After 45 to 75 days of the above adventitious bud proliferation culture, the number of the obtained adventitious buds is no less than 46 times the number of the sterile seedling stem segments, and these adventitious buds can be used for the next step of rooting.

[0051] Step 3, rooting of adventitious buds: The adventitious buds obtained in the above step 2 are inoculated into a rooting medium for rooting culture. After 20 to 35 days (preferably 30 days), tissue culture seedlings (i.e., regenerated plants) are obtained.

[0052] The rooting medium is: based on 1 / 2MS basic medium, NAA is added to a final concentration of 0.05-0.3 mg / L (preferably 0.1 mg / L).

[0053] The culture conditions for the above-mentioned rooting culture are: temperature of 22-28°C (preferably 25°C), light intensity of 1500-2500Lx (preferably 2000Lx), and light duration of 12-18h / d (preferably 16h / d).

[0054] Through the operation of step three, at least 92% of the adventitious buds can take root and become tissue culture seedlings.

[0055] Step 4: Hardening and acclimatization: After hardening the tissue culture seedlings obtained in step 3 for 3 to 9 days (preferably 5 days), clean them and move them to a flat plate with a growth medium for acclimatization. After 8 to 20 days (preferably 14 days) of acclimatization, the "Jingke Xifengshou" cut flower ornamental chrysanthemum plants are obtained, which can be used for planting.

[0056] The growth matrix includes charcoal and vermiculite powder, and the mass ratio of charcoal to vermiculite powder is (1-3): (1-5), preferably 2: 3. The temperature of the acclimation treatment is 22-30°C, the humidity is 60-70%, and the ventilation volume is gradually increased during the acclimation treatment.

[0057] Through the seedling hardening and acclimatization operation in step 4, at least 92% of the tissue culture seedlings can survive after being transplanted.

[0058] The composition and content of the MS minimal medium used in the present invention are:

[0059] A large number of elements, including:

[0060] Potassium nitrate (KNO3): 1900mg / L, ammonium nitrate (NH4NO3): 1650mg / L, magnesium sulfate (MgSO4·7H2O): 370mg / L, potassium dihydrogen phosphate (KH2PO4): 170mg / L, calcium chloride (CaCl2·2H2O): 440mg / L.

[0061] Trace elements, including:

[0062] Manganese sulfate (MnSO4·H2O): 16.9 mg / L, zinc sulfate (ZnSO4·7H2O): 8.6 mg / L, boric acid (H3BO3): 6.2 mg / L, potassium iodide (KI): 0.83 mg / L, sodium molybdate (Na2MoO4·2H2O): 0.25 mg / L, copper sulfate (CuSO4·5H2O): 0.025 mg / L, cobalt chloride (CoCl2·6H2O): 0.025 mg / L.

[0063] Iron salts, including:

[0064] Disodium ethylenediaminetetraacetate dihydrate (Na2-EDTA): 37.3 mg / L, ferrous sulfate (FeSO4·4H2O): 27.8 mg / L.

[0065] Organic matter, including:

[0066] Glycine: 2.0mg / L, Pyridoxine hydrochloride: 0.5mg / L, Thiamine hydrochloride: 0.1mg / L, Niacin: 0.5mg / L, Creatine: 100mg / L.

[0067] Sucrose: 30 g / L, agar 3-10 g / L (preferably 5 g / L).

[0068] The 1 / 2MS basic culture medium used in the present invention is based on the above-mentioned MS basic culture medium, with the content of macroelements reduced to 1 / 2, while other components remain unchanged.

[0069] The pH values ​​of the MS basic culture medium and the 1 / 2MS basic culture medium used in the present invention are both 5.8-6.0.

[0070] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.

[0071] Example 1

[0072] This embodiment is used to illustrate the method for rapid propagation of "Jingke Xifengshou" cut ornamental chrysanthemum tissue culture seedlings provided by the present invention, comprising the following steps:

[0073] (1) Preparation of sterile seedlings: The sterilized explants were inoculated into MS basic medium and cultured for 25 days to obtain sterile seedlings. The conditions for the sterile seedling culture were as follows: the temperature was controlled at 22-26°C, the light intensity was 1500 Lx, and the light duration was 16 h / d.

[0074] Wherein, the sterilized explant is prepared by the following method:

[0075] Select healthy and unflowered plants of the current year, remove the leaves, and cut the stems into stem segments with 2 to 3 axillary buds as explants; add the explants to a 3% detergent solution by volume and soak for 15 minutes; then shake on a shaker for 10 minutes and rinse with tap water for 30 minutes. Transfer the washed explants to a sterile tissue culture bottle in a clean bench, rinse with sterile water 2 to 3 times, quickly rinse and disinfect with a 75% alcohol solution, rinse with sterile water 2 to 3 times, transfer to sterile absorbent paper or a sterile culture dish for standby use; then disinfect in a 5% sodium hypochlorite solution for 12 to 15 minutes, rinse with sterile water 3 to 5 times, and finally place on a sterile culture dish with sterile absorbent paper to obtain a sterilized explant.

[0076] (2) Adventitious bud proliferation culture: The sterile seedlings obtained in step (1) are placed in a proliferation culture medium and cultured for adventitious bud proliferation for 50 days to obtain adventitious buds.

[0077] The medium used for adventitious bud proliferation culture is: based on MS basic medium, supplemented with NAA 2mg / L, 6-BA 7mg / L, banana puree 30g / L, agar 5g / L. The conditions for adventitious bud proliferation culture are: temperature 26℃, light intensity 1500Lx, and light time 16h / d.

[0078] The preparation method of the banana puree is the above method 1, which is specifically as follows:

[0079] Cut 30.1g of banana pulp into thin slices or small particles, add 20g of water and boil until it is cooked. Filter out the solid matter with a filter, and the resulting paste-like filtrate is the banana puree.

[0080] After 50 days of adventitious bud proliferation culture, the number of adventitious buds obtained was 78 times the number of sterile seedling stem segments, and these adventitious buds can be used for the next step of rooting.

[0081] Figure 1 The adventitious buds obtained after 50 days of adventitious bud proliferation culture in step (2) are shown. It can be seen that after 50 days of adventitious bud proliferation culture, a large number of adventitious buds have grown on the same stem segment. Figure 2 Shows the Figure 1 After the adventitious buds in the stem are separated, it can be seen that after 50 days of adventitious bud proliferation culture, 78 adventitious buds have grown in the stem segment, and these adventitious buds can be used for rooting later. It can be seen that the above-mentioned proliferation culture medium used in the present invention can achieve the effect of rapid and efficient reproduction.

[0082] (3) Rooting of adventitious buds: The adventitious buds obtained in step (2) above were inoculated into a rooting medium and cultured for 30 days. Statistics showed that 95% of the adventitious buds could take root and obtain tissue culture seedlings (i.e., regenerated plants).

[0083] The rooting medium used for adventitious bud rooting is: based on 1 / 2MS basic medium, NAA 0.1mg / L is added. The culture conditions for adventitious bud rooting are: temperature 25℃, light intensity 2000Lx, and light time 16h / d.

[0084] (4) Acclimation step: After the tissue culture seedlings obtained in the above step (3) are acclimated for 5 days, they are cleaned and moved to a flat plate with a growth matrix (carbon soil and vermiculite powder in a mass ratio of 2:3) for acclimation. The acclimation temperature is 22-30°C and the humidity is 60-70%. The ventilation volume is gradually increased during the acclimation process. After 14 days of acclimation, it is found that 95% of the tissue culture seedlings can form plants for planting.

[0085] Example 2

[0086] Except for the following operations and parameters, the rest are the same as those in Example 1:

[0087] In step (1):

[0088] After 15 days of culture, sterile seedlings were obtained.

[0089] In step (2):

[0090] The culture medium used for adventitious bud proliferation culture is: based on MS basic medium, supplemented with NAA 1mg / L, 6-BA 5mg / L, banana puree 20g / L, and agar 5g / L.

[0091] The preparation method of the banana puree is the above method 2, which is specifically as follows:

[0092] 40 g of fully ripe and softened banana pulp was blended in a blender to obtain pulp paste, 33 g of water was added to the pulp paste to obtain pulp suspension, and the pulp suspension was filtered to retain the filtrate (paste), which was the banana puree.

[0093] The conditions for adventitious bud proliferation culture are: temperature 22℃, light intensity 2000Lx, and lighting time 12h / d.

[0094] After 45 days of adventitious bud proliferation culture, the number of adventitious buds obtained was 46 times the number of sterile seedling stem segments, and these adventitious buds can be used for the next step of rooting.

[0095] In step (3):

[0096] After 20 days of rooting culture, it was found that 92% of the adventitious buds could take root and obtain tissue culture seedlings (i.e., regenerated plants).

[0097] The rooting medium used for adventitious bud rooting is: based on 1 / 2MS basic medium, NAA0.05mg / L is added. The culture conditions for adventitious bud rooting are: temperature 22℃, light intensity 1500Lx, and light time 12h / d.

[0098] In step (4):

[0099] After 3 days of hardening and 12 days of acclimatization, it was found that 92% of the tissue culture seedlings could form plants for planting.

[0100] Example 3

[0101] Except for the following operations and parameters, the rest are the same as those in Example 1:

[0102] In step (1):

[0103] After 20 days of cultivation, sterile seedlings were obtained.

[0104] In step (2):

[0105] The culture medium used for adventitious bud proliferation culture is: based on MS basic medium, supplemented with NAA 1.5mg / L, 6-BA 6mg / L, banana puree 40g / L, and agar 5g / L.

[0106] The preparation method of the banana puree is the above method 3, which is specifically as follows:

[0107] 50 g of fully ripe and softened banana pulp and 28 g of water were mixed and crushed to obtain a pulp suspension, and the pulp suspension was filtered to retain the filtrate (paste), which was the banana puree.

[0108] The conditions for adventitious bud proliferation culture are: temperature 24℃, light intensity 2500Lx, and light time 14h / d.

[0109] After 55 days of adventitious bud proliferation culture, the number of adventitious buds obtained was 56 times the number of sterile seedling stem segments, and all of these adventitious buds could be used for rooting in the next step.

[0110] In step (3):

[0111] After 25 days of rooting culture, it was found that 93% of the adventitious buds could take root and obtain tissue culture seedlings (i.e., regenerated plants).

[0112] The rooting medium used for adventitious bud rooting is: based on 1 / 2MS basic medium, NAA0.2mg / L is added. The culture conditions for adventitious bud rooting are: temperature 28℃, light intensity 2500Lx, and light time 14h / d.

[0113] In step (4):

[0114] After 7 days of hardening and 10 days of acclimatization, it was found that 96% of the tissue culture seedlings could form plants for planting.

[0115] Example 4

[0116] Except for the following operations and parameters, the rest are the same as those in Example 1:

[0117] In step (1):

[0118] After 30 days of cultivation, sterile seedlings were obtained.

[0119] In step (2):

[0120] The culture medium used for adventitious bud proliferation culture is: based on MS basic medium, supplemented with NAA 2.5 mg / L, 6-BA 8 mg / L, banana puree 50 g / L, and agar 5 g / L.

[0121] The preparation method of the banana puree is the above method 4, which is specifically as follows:

[0122] 30 g of banana pulp that is not fully ripe and has a certain hardness is cut into thin slices or small particles, and then 20 g of water is added and heated to boiling, and the banana pulp is boiled to obtain boiled pulp, which is banana puree.

[0123] The conditions for adventitious bud proliferation culture are: temperature 28℃, light intensity 1500Lx, and light time 18h / d.

[0124] After 60 days of adventitious bud proliferation culture, the number of adventitious buds obtained was 62 times the number of sterile seedling stem segments, and all of these adventitious buds could be used for rooting in the next step.

[0125] In step (3):

[0126] After 35 days of rooting culture, statistics show that 99% of the adventitious buds can take root to obtain tissue culture seedlings (i.e., regenerated plants).

[0127] The rooting medium used for adventitious bud rooting is: based on 1 / 2MS basic medium, NAA 0.3mg / L is added. The culture conditions for adventitious bud rooting are: temperature 25℃, light intensity 2000Lx, and light time 18h / d.

[0128] In step (4):

[0129] After 9 days of hardening and 8 days of acclimatization, it was found that 98% of the tissue culture seedlings could form plants for planting.

[0130] Comparative Example 1

[0131] Except for the following operating parameters, the rest are the same as in Example 1:

[0132] In the proliferation medium of step (2) of Example 1, based on MS medium, NAA 2 mg / L, 6-BA 7 mg / L, 2,4-D 2 mg / L, agar 5 g / L were added, and banana puree was not added.

[0133] Figure 3 The figure shows the mass-like induced callus tissue obtained after 50 days of adventitious bud proliferation culture in step (2), which induced a very small number of abnormal adventitious buds, most of which showed curling of the leaves toward the back, irregularity, thickening of the leaves, and short adventitious buds. Very few adventitious buds became vitrified, yellowed and died. Figure 4 Shows Figure 3 The callus tissue in the culture can be seen as light green mass callus tissue with a whitish surface, a small amount of browning and vitrified edges.

[0134] It can be seen that although 2,4-D was added, if banana puree was not added, the number of adventitious buds that proliferated was very small and the quality was very poor, and it could not be used for subsequent adventitious bud rooting.

[0135] Comparative Example 2

[0136] Except for the following operating parameters, the rest are the same as in Example 1:

[0137] In the proliferation medium of step (2) of Example 1, MS medium was used as the base, NAA 0.5 mg / L, 6-BA 2 mg / L, agar 5 g / L were added, and banana puree was not added.

[0138] Figure 5 The adventitious buds and callus obtained after 50 days of adventitious bud proliferation culture in step (2) are shown. Figure 6 Shows Figure 5 Callus tissue in culture. Figure 7 Shown after transfer to the culture dish Figure 5 of callus tissue. Figure 8 Shows the Figure 5 After the adventitious buds in the stem were separated, it can be seen that after 50 days of adventitious bud proliferation culture, only 8 adventitious buds grew in the stem segment. The adventitious buds were clustered variant leaf phase adventitious buds. A large amount of light green and whitish mass callus tissue grew at the base of the adventitious buds, and the edges were vitrified; the leaves were accompanied by the variant characteristics of thickening, dwarfing, and hypertrophy.

[0139] It can be seen that if the concentrations of NAA and 6-BA are reduced and banana puree is not added, the number of adventitious buds that proliferate will be very small and the quality will be very poor, and they cannot be used for subsequent adventitious bud rooting.

[0140] Comparative Example 3

[0141] Except for the following operating parameters, the rest are the same as in Example 1:

[0142] In the proliferation medium of step (2) of Example 1, based on MS medium, 2 mg / L NAA, 7 mg / L 6-BA, 55 g / L banana puree and 5 g / L agar were added.

[0143] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus with only 35 adventitious buds growing out, and 12 of the adventitious buds showed at least one of the following conditions: being too short, growing abnormally, vitrified, and yellowing.

[0144] It can be seen that if the amount of banana puree is increased beyond the scope of the present invention, the proliferation number of adventitious buds will be greatly reduced, and the quality of the adventitious buds is very poor and cannot be used for subsequent adventitious bud rooting.

[0145] Comparative Example 4

[0146] Except for the following operating parameters, the rest are the same as in Example 1:

[0147] In the proliferation medium of step (2) of Example 1, based on MS medium, 2 mg / L NAA, 7 mg / L 6-BA, 15 g / L banana puree, and 5 g / L agar were added.

[0148] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus with only 28 adventitious buds growing out, and 18 of the adventitious buds showed at least one of the following conditions: being too short, growing abnormally, vitrified, and yellowing.

[0149] It can be seen that if the amount of banana puree is reduced beyond the scope of the present invention, the proliferation number of adventitious buds will be greatly reduced, and the quality of the adventitious buds is very poor and cannot be used for subsequent adventitious bud rooting.

[0150] Comparative Example 5

[0151] Except for the following operating parameters, the rest are the same as in Example 1:

[0152] In the proliferation medium of step (2) of Example 1, MS medium was used as the base, NAA 2 mg / L, 6-BA 7 mg / L, agar 5 g / L were added, and banana puree was not added.

[0153] After 50 days of adventitious bud proliferation culture, only 22 adventitious buds grew in the stem segment, and all the adventitious buds were clustered variant leaf phase adventitious buds, with a large number of light green and whitish mass callus tissues at the base, and vitrified edges; the leaves were accompanied by thickened, dwarfed, and hypertrophic variant characteristics.

[0154] It can be seen that if banana puree is not added to the proliferation medium, the number of adventitious bud proliferation will be greatly reduced, and the quality of the adventitious buds will be very poor and cannot be used for subsequent adventitious bud rooting.

[0155] Comparative Example 6

[0156] Except for the following operating parameters, the rest are the same as in Example 1:

[0157] In the proliferation medium of step (2) of Example 1, based on MS medium, 0.5 mg / L NAA, 7 mg / L 6-BA, 30 g / L banana puree, and 5 g / L agar were added.

[0158] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus without adventitious buds growing out.

[0159] It can be seen that if the concentration of NAA is reduced outside the range of the present invention, adventitious buds will not be obtained.

[0160] Comparative Example 7

[0161] Except for the following operating parameters, the rest are the same as in Example 1:

[0162] In the proliferation medium of step (2) of Example 1, based on MS medium, 5 mg / L NAA, 7 mg / L 6-BA, 30 g / L banana puree, and 5 g / L agar were added.

[0163] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus tissue and no adventitious buds were found to grow.

[0164] It can be seen that if the concentration of NAA is increased beyond the range of the present invention, adventitious buds will not be obtained.

[0165] Comparative Example 8

[0166] Except for the following operating parameters, the rest are the same as in Example 1:

[0167] In the proliferation medium of step (2) of Example 1, based on MS medium, 2 mg / L NAA, 4 mg / L 6-BA, 30 g / L banana puree, and 5 g / L agar were added.

[0168] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus tissue and no adventitious buds were found to grow.

[0169] It can be seen that if the concentration of 6-BA is reduced outside the range of the present invention, adventitious buds will not be obtained.

[0170] Comparative Example 9

[0171] Except for the following operating parameters, the rest are the same as in Example 1:

[0172] In the proliferation medium of step (2) of Example 1, based on MS medium, 2 mg / L NAA, 11 mg / L 6-BA, 30 g / L banana puree, and 5 g / L agar were added.

[0173] After 50 days of adventitious bud proliferation culture, the stem segment became a mass callus tissue and no adventitious buds were found to grow.

[0174] It can be seen that if the concentration of 6-BA is increased beyond the range of the present invention, adventitious buds will not be obtained.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for rapid propagation of Jingkexifengshou cut chrysanthemum tissue culture seedlings, characterized in that: The method comprises the steps of inoculating the stem segments of the sterile seedlings of Jingke Xifengshou cut chrysanthemum into a proliferation medium to obtain adventitious buds, and then culturing the adventitious buds into ornamental chrysanthemum plants; Wherein, the proliferation medium is based on MS basic medium and is supplemented with: NAA, 6-BA, banana puree; The final concentration of NAA in the proliferation medium is 1 to 2.5 mg / L; the final concentration of 6-BA in the proliferation medium is 5 to 8 mg / L; the final concentration of banana puree in the proliferation medium is 20 to 50 g / L; The banana puree is prepared from raw materials containing banana pulp and water; the mass ratio of banana pulp to water is (1.2-2.0):

1.

2. The method according to claim 1, characterized in that: The final concentration of NAA in the proliferation medium is 2 mg / L; the final concentration of 6-BA in the proliferation medium is 7 mg / L; and the final concentration of banana puree in the proliferation medium is 30 g / L.

3. The method according to claim 2, characterized in that: The mass ratio of the banana pulp to water is 1.5:

1.

4. The method according to claim 1 or 3, characterized in that: The operation time of inoculating the stem segments of the sterile seedlings of Jingke Xifengshou cut chrysanthemum into the proliferation culture medium to obtain adventitious buds is 45 to 75 days.

5. The method according to claim 4, characterized in that: The operation time of inoculating the stem segments of the sterile seedlings of Jingke Xifengshou cut chrysanthemum into the proliferation culture medium to obtain adventitious buds is 50 days.

6. The method according to claim 5, characterized in that: In the operation of inoculating the stem segments of the sterile seedlings of Jingke Xifengshou cut chrysanthemum into the proliferation culture medium to obtain adventitious buds, the culture conditions include: The temperature is 22-28℃; the light intensity is 1000-2500Lx; the lighting time is 12-18 h / d.

7. The method according to claim 6, characterized in that: The culture conditions include: temperature of 26°C; light intensity of 1500Lx; and light duration of 16 h / d.

8. The method according to claim 1, characterized in that: The method further comprises the step of rooting culture of the adventitious buds: inoculating the adventitious buds into a rooting culture medium, performing rooting culture, and obtaining tissue culture seedlings.

9. The method according to claim 8, characterized in that: The rooting medium used in the adventitious bud rooting culture contains: based on 1 / 2 MS basic medium, NAA is added to a final concentration of 0.05-0.3 mg / L.

10. The method according to claim 9, characterized in that: The rooting medium used in the adventitious bud rooting culture contains: based on 1 / 2 MS basic medium, NAA is added to a final concentration of 0.1 mg / L.

11. The method according to claim 10, characterized in that: In the adventitious bud rooting culture, the culture conditions include: The temperature is 22-28℃; the light intensity is 1500-2500Lx; the lighting time is 12-18 h / d.

12. The method according to claim 11, characterized in that: In the adventitious bud rooting culture, the culture conditions include: temperature of 25° C.; light intensity of 2000 Lx; and light duration of 16 h / d.

13. The method according to claim 12, characterized in that: The time for the adventitious bud rooting culture is 25 to 35 days.

14. The method according to claim 13, characterized in that: The time for the adventitious bud rooting culture is 30 days.

15. The method according to claim 14, characterized in that: The method further comprises the step of hardening and acclimating the tissue culture seedlings for 3 to 9 days, and then transferring the tissue culture seedlings to a growth medium for acclimation for 8 to 20 days to obtain Jingke Xifengshou cut chrysanthemum plants.

16. The method according to claim 15, characterized in that: The growth matrix includes charcoal and vermiculite powder, and the mass ratio of the charcoal to the vermiculite powder is (1-3): (1-5).

17. The method according to claim 16, characterized in that: The mass ratio of the charcoal clay to the vermiculite powder is 2:

3.

18. The method according to claim 17, characterized in that: The temperature of the acclimation treatment is 22-30° C., and the humidity is 60-70%.

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