Method for improving survival rate of small yellow ginger tissue culture seedlings and application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for increasing the survival rate of small yellow ginger tissue culture seedlings and application thereof.In the early rooting stage of small yellow ginger, a seedling strengthening step is added, CH and PP333 with a certain concentration are added into a seedling strengthening culture medium, meanwhile, the MS concentration is increased to 1.5 times, leaf thickening and stem thickening of small yellow ginger can be promoted, and the survival rate of small yellow ginger tissue culture seedlings is increased. The seedlings are thicker and stronger and the root system is shortened during rooting of the small yellow ginger, so that the resistance of the small yellow ginger is improved, and the root system breakage during domestication of the small yellow ginger is reduced. Kanamycin with a proper concentration is added into the strong seedlings of the small yellow ginger and the rooting culture medium, so that the tissue culture seedlings of the small yellow ginger carry kanamycin with a certain concentration when being taken out of a bottle, the infection of pathogenic bacteria on the tissue culture seedlings of the small yellow ginger in the domestication process can be effectively inhibited, and the kanamycin in the seedlings is hardly detected after the tissue culture seedlings are domesticated for 2-3 months.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for improving the survival rate of zingiber officinale tissue culture seedlings and an application thereof. Background Art

[0002] The tuber of small yellow ginger (Zingiber offiinale Rosceoe) is rich in active ingredients such as curcumin, essential oil, gingerol, and gingerol, and has multiple functions such as dispelling cold, removing dampness, warming the stomach, and accelerating blood circulation. Small yellow ginger combines nutrition, seasoning, and health care. It is an economic crop that can be used as medicine and food, and has high economic value. The cut surface of small yellow ginger is pure yellow, with a spicy and strong taste. The flesh is relatively tender, tastes good, and has fine fibers. Compared with large ginger, it has an excellent taste.

[0003] Traditional methods of propagating small yellow ginger include: (1) using traditional tuber propagation to produce seedlings, which is very likely to cause outbreaks of seed-borne diseases of small yellow ginger. At the same time, multiple generations of tuber division propagation also lead to variety degeneration, resulting in a sharp drop in the yield of small yellow ginger and greatly affecting the quality of small yellow ginger. It is impossible to promote large-scale planting in production, affecting economic income and also failing to meet production needs. (2) Ginger tissue culture propagation method, which is the most effective method to improve yield and quality. The publication number is CN106962202A, and the name is: an artificial seed of small yellow ginger and a preparation method thereof, which discloses: after detoxification treatment of small yellow ginger ginger buds, cutting the stem tip as an explant and inoculating it on a budding medium, and waiting for the clustered buds to grow to about 0.5-0.8 cm as the material for artificial embryos; using MS+0.1 mg / L penicillin+0.2% sodium benzoate+3.0% sucrose as artificial endosperm, and using 2.0% CaCl2+2.0% chitosan+4.0% sodium alginate as artificial seed coat, to embed the artificial seeds of small yellow ginger. The artificial seeds of small yellow ginger obtained by the method have a high reproduction coefficient and are not easily infected by diseases such as ginger wilt, ginger eye spot, ginger root rot, and ginger rot. The publication number is CN116420617A, and the name is: A cost-saving and efficiency-enhancing production method for virus-free test tube seedlings of small yellow ginger. The method adopts a variety of disinfection and sterilization measures, and also provides nutrients to plant tissues during the treatment, effectively promoting the germination process. The targeted induction differentiation culture medium can provide rich nutrients, promote the wrapping and entanglement of the root base to promote its development, and provide slow-release nutrients to the root system throughout the growth period, promote the growth of small yellow ginger, and improve the quality of small yellow ginger. The publication number is CN117617122A, and the name is: A combined culture medium and culture method for virus-free and thin-layer culture of small yellow ginger. The stem tip of small yellow ginger is inoculated in the primary culture medium for primary culture to obtain virus-free tissue culture seedlings, and the virus-free tissue culture seedlings are subjected to cell thin-layer culture to obtain virus-free plants. The number of buds in the bud induction culture of this method is large, and the multiplication coefficient of the virus-free plant is 21, which significantly improves the multiplication coefficient and multiplication efficiency. The publication number is CN116686717A, and the name is: a method for breeding original stocks of detoxified stem tips of small yellow ginger, which comprises selecting healthy tubers of small yellow ginger with bright skin, plump yellow flesh and no diseases or insect pests for germination in a light incubator, placing them in a soilless culture medium sterilized by high pressure after disinfection, placing them in a light incubator for constant germination, selecting strong buds about 2 cm long after germination and heat treatment in the light incubator, disinfecting the stem tips of the explants, peeling off the young leaves from the stem tips under a microscope, and cutting the 0.6-0.8 cm stem tip growth point with a scalpel; step four: primary culture; step five: subculture.

[0004] As far as the current small yellow ginger is concerned, both the virus-free technology and the tissue culture technology are relatively mature. However, when we use the existing technology to produce virus-free or tissue culture seedlings of small yellow ginger, we find that the rooting seedlings of the existing technology have a low survival rate during domestication and grow slowly during domestication. At the same time, after multiple generations of domestication on the same base, there will still be a certain degree of ginger blight infection, accompanied by infection from other diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application for improving the survival rate of Zingiber officinale tissue culture seedlings in view of the problems existing in the prior art.

[0006] To achieve the above object, the technical scheme adopted by the present invention is: a method for improving the survival rate of turmeric tissue culture seedlings, taking turmeric tuber buds, inoculating them in a primary induction medium after cleaning and disinfection to induce clustered buds, cutting the induced clustered buds into single buds, inoculating them in a secondary proliferation medium and culturing them for 30 days; characterized in that it also includes:

[0007] (1) Seedling cultivation: Cut the subcultured buds into single buds, inoculate them into seedling medium, and culture them in an environment with a light intensity of 2000-3000 lx, a single day of light for 12 hours, and a temperature of 25±2℃ for 25-35 days;

[0008] The formula of the seedling medium is: 1.5MS medium + CH 40-50 mg / L + PP333 0.1-0.2 mg / L + kanamycin 0.8-1.0 mg / L;

[0009] The 1.5MS medium is: the amount of all elements in the MS is 1.5 times that of the original MS, and the sugar concentration remains unchanged;

[0010] (2) Rooting culture: After the seedlings have grown strong, the clustered buds are cut into single buds, inoculated into rooting medium, and cultured in an environment with a light intensity of 2000-3000 lx, a single day of light for 12 hours, and a temperature of 25±2°C for 30-40 days;

[0011] The formula of the rooting medium is: 1 / 2MS+IBA 0.3-0.5 mg / L+SA 15-20 mg / L+Kanamycin 4.0-5.0 mg / L;

[0012] The 1 / 2MS medium is: the concentration of macroelements is reduced to half of MS, and the other components remain unchanged;

[0013] (3) Rooting seedlings acclimatization: Wash the rooted seedlings with clean water and plant them in the mixed substrate. After transplanting, keep the substrate humidity at 55-65%. During this period, spray nutrient solution A every 5-7 days for 3-4 times in succession.

[0014] The formula of the nutrient solution A is: IBA 1.5-2.5 mg / L+SA 50-70 mg / L.

[0015] Furthermore, the formula of the seedling-strengthening culture medium is: 1.5MS culture medium + CH 45 mg / L + PP333 0.15 mg / L + kanamycin 0.9 mg / L.

[0016] Furthermore, the formula of the rooting medium is: 1 / 2MS+IBA 0.4mg / L+SA 17mg / L+kanamycin 4.5mg / L.

[0017] Furthermore, the mixed matrix is ​​a mixture of humus and sand in a weight ratio of 1:10.

[0018] The beneficial technical effects of the present invention are:

[0019] 1. During the domestication of the small yellow ginger tissue culture seedlings, when the air humidity is lower than 90%, it is very easy to cause the small yellow ginger tissue culture seedlings to dehydrate, and most of the dehydration is even irreversible. In order to ensure the air humidity, it is necessary to spray water multiple times. When spraying water multiple times, the substrate humidity is too wet, which is very easy to cause the occurrence of root rot and stem base rot of the small yellow ginger, and finally leads to the low domestication survival rate of the small yellow ginger tissue culture seedlings. The present invention adds a suitable concentration of SA to the rooting medium and sprays a certain concentration of SA during domestication management, which can induce drought stress resistance of small yellow ginger. It is speculated that its mechanism is to promote the closure of leaf stomata, thereby reducing the demand for air humidity during the domestication of the small yellow ginger tissue culture seedlings, and finally alleviates the contradiction between air humidity and substrate humidity during the domestication process, and avoids the occurrence of root rot and stem base rot caused by excessive moisture in the substrate.

[0020] 2. In the early stage of rooting of small yellow ginger, the present invention adds a seedling strengthening step, adds a certain concentration of CH and PP333 to its seedling strengthening culture medium, and increases the MS concentration to 1.5 times, which can promote the thickening of the leaves and stems of small yellow ginger, make the seedlings of small yellow ginger stronger and the root system shorter when taking root, thereby improving its own resistance and reducing the root damage during the domestication of small yellow ginger.

[0021] 3. The present invention adds a suitable concentration of kanamycin to the seedling and rooting medium of small yellow ginger, so that the small yellow ginger tissue culture seedlings carry a certain concentration of kanamycin when they are out of the bottle, which can effectively inhibit the infection of pathogens on the small yellow ginger tissue culture seedlings during the domestication process, and after the tissue culture seedlings are domesticated for 2 to 3 months, the seedlings can hardly detect kanamycin. At the same time, during the planting process after the domesticated seedlings are transplanted into the field, the incidence of ginger blight is reduced by more than 15% compared with the case where kanamycin is not added during the seedling and rooting culture. It is speculated that after adding kanamycin, although it is effectively degraded during the domestication process, the seedlings hardly carry pathogens, which is the main reason for the reduction in the incidence of ginger blight during field planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 This is the acclimation result of Example 1 of the present invention.

[0024] Figure 2 It is the acclimation result of Comparative Example 1 of the present invention.

[0025] Figure 3 It is the acclimation result of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A method for improving the survival rate of ginger tissue culture seedlings, comprising the steps of:

[0029] (1) Primary induction: Take the tuber buds of Ginger zingiberensis, rinse them with tap water for 30 min, wipe or soak them with 75% alcohol on a clean bench for 30 s, sterilize them with 10% sodium hypochlorite for 15 min, rinse them with sterile water for 4 times, dry them with sterile filter paper, and inoculate them into the primary induction medium (formula: MS+6-BA 0.6 mg / L +IBA 0.2 mg / L) to induce clustered buds.

[0030] (2) Subculture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + 6-BA 2mg / L + IBA 0.1mg / L) and cultured for 30 days.

[0031] (3) Seedling cultivation: Cut the subcultured clustered buds into single buds, inoculate them into seedling culture medium (formula: 1.5MS medium + CH 40 mg / L + PP333 0.1 mg / L + kanamycin 0.8 mg / L), and culture them in an environment with a light intensity of 2000 lx, a single day of light for 12 hours, and a temperature of 25±2℃ for 25 days.

[0032] Among them, 1.5MS culture medium is: the amount of all elements in MS is 1.5 times that of the original MS, and the sugar concentration remains unchanged.

[0033] (4) Rooting culture: After the seedlings have grown strong, the clustered buds are cut into single buds and inoculated into rooting medium (formula: 1 / 2MS + IBA 0.3mg / L + SA 15mg / L + kanamycin 4.0mg / L), and cultured in an environment with a light intensity of 2000lx, a single day of light for 12h, and a temperature of 25±2℃ for 30 days.

[0034] Among them, 1 / 2MS culture medium is: the concentration of major elements in MS is reduced to half of that in MS, and the other components remain unchanged.

[0035] (5) Rooting seedlings acclimatization: Wash the rooted seedlings with clean water and plant them in the mixed medium. After transplanting, keep the medium humidity at 55%. During this period, spray nutrient solution A (formula: IBA 1.5mg / L + SA 50mg / L) every 5 days for 3 consecutive times. Acclimatize in the greenhouse for 30 days. The results are as follows: Figure 1 shown.

[0036] Example 2

[0037] A method for improving the survival rate of ginger tissue culture seedlings, comprising the steps of:

[0038] (1) Primary induction: Take the tuber buds of Ginger zingiberensis, rinse them with tap water for 30 minutes, wipe or soak them with 75% alcohol on a clean bench for 30 seconds, sterilize them with 10% sodium hypochlorite for 15 minutes, rinse them with sterile water for 5 times, dry them with sterile filter paper, and inoculate them into the primary induction medium (formula: MS+6-BA 0.6mg / L +IBA 0.2mg / L) to induce clustered buds.

[0039] (2) Subculture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + 6-BA 2mg / L + IBA 0.1mg / L) and cultured for 30 days.

[0040] (3) Seedling cultivation: Cut the subcultured buds into single buds, inoculate them into seedling culture medium (formula: 1.5MS medium + CH 50 mg / L + PP333 0.2 mg / L + kanamycin 1.0 mg / L), and culture them in an environment with a light intensity of 3000 lx, a single day of light for 12 h, and a temperature of 25±2℃ for 35 days.

[0041] Among them, 1.5MS medium is: the amount of all elements in MS is 1.5 times that of the original MS, and the sugar concentration remains unchanged;

[0042] (4) Rooting culture: After the seedlings have grown strong, the clustered buds are cut into single buds and inoculated into rooting medium (formula: 1 / 2MS + IBA 0.5mg / L + SA 20mg / L + kanamycin 5.0mg / L), and cultured in an environment with a light intensity of 3000lx, a single day of light for 12h, and a temperature of 25±2℃ for 40 days.

[0043] Among them, 1 / 2MS medium is: the concentration of macroelements in MS is reduced to half of MS, and the other components remain unchanged;

[0044] (5) Rooting seedlings acclimatization: Wash the rooted seedlings with clean water and plant them in the mixed substrate. After transplanting, keep the substrate humidity at 65%. During this period, spray nutrient solution A (formula: IBA 2.5mg / L + SA 70mg / L) once every 7 days for 4 consecutive times.

[0045] Example 3

[0046] A method for improving the survival rate of ginger tissue culture seedlings, comprising the steps of:

[0047] (1) Primary induction: Take the tuber buds of Ginger zingiberensis, rinse them with tap water for 30 minutes, wipe or soak them with 75% alcohol on a clean bench for 30 seconds, sterilize them with 10% sodium hypochlorite for 15 minutes, rinse them with sterile water for 5 times, dry them with sterile filter paper, and inoculate them into the primary induction medium (formula: MS+6-BA 0.6mg / L +IBA 0.2mg / L) to induce clustered buds.

[0048] (2) Subculture: The induced clustered buds were cut into single buds and inoculated into subculture proliferation medium (formula: MS + 6-BA 2mg / L + IBA 0.1mg / L) and cultured for 30 days.

[0049] (3) Seedling cultivation: Cut the subcultured buds into single buds, inoculate them into seedling medium (formula: 1.5MS medium + CH 45 mg / L + PP333 0.15 mg / L + kanamycin 0.9 mg / L), and culture them in an environment with a light intensity of 2500 lx, a single day of light for 12 hours, and a temperature of 25±2℃ for 30 days.

[0050] Among them, 1.5MS culture medium is: the amount of all elements in MS is 1.5 times that of the original MS, and the sugar concentration remains unchanged.

[0051] (4) Rooting culture: After the seedlings have grown strong, the clustered buds are cut into single buds and inoculated into rooting medium (formula: 1 / 2MS + IBA 0.4mg / L + SA 17mg / L + kanamycin 4.5mg / L), and cultured in an environment with a light intensity of 2500lx, a single day of light for 12h, and a temperature of 25±2℃ for 35 days.

[0052] Among them, 1 / 2MS culture medium is: the concentration of major elements in MS is reduced to half of that in MS, and the other components remain unchanged.

[0053] (5) Rooting seedlings acclimatization: Wash the rooted seedlings with clean water and plant them in the mixed substrate. After transplanting, keep the substrate humidity at 60%. During this period, spray nutrient solution A (formula: IBA 2.0mg / L + SA 60mg / L) once every 6 days for 4 consecutive times.

[0054] Comparative Example 1

[0055] The method disclosed in Example 1 was used for primary induction and secondary proliferation. No seedling culture was performed. Conventional rooting culture was used and the plants were domesticated in a greenhouse for 30 days. During domestication, nutrient solution A was not sprayed. Figure 2 As shown, most of the seedlings died.

[0056] Comparative Example 2

[0057] The method disclosed in Example 1 was used for operation, and the roots were acclimated in the greenhouse for 30 days. During acclimation, the nutrient solution A was not sprayed. The results were as follows: Figure 3 shown.

[0058] The survival rates of the above-mentioned Examples 1-3 and Comparative Examples 1-2 were statistically analyzed after acclimation for 30 days. The results are shown in Table 1 below.

[0059] Table 1 Statistical results of survival rate

[0060] Survival rate Example 1 96.3% Example 2 92.8% Example 3 94.7% Comparative Example 1 18.5% Comparative Example 2 56.2%

[0061] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for improving the survival rate of turmeric tissue culture seedlings, wherein the turmeric tuber buds are taken, cleaned and disinfected, and then inoculated into a primary induction medium to induce clustered buds, the induced clustered buds are cut into single buds, inoculated into a secondary proliferation medium and cultured for 30 days; characterized in that: Also includes: (1) Seedling cultivation: Cut the subcultured buds into single buds, inoculate them into seedling medium, and culture them in an environment with a light intensity of 2000-3000 lx, a single day of light for 12 hours, and a temperature of 25±2℃ for 25-35 days; The formula of the seedling medium is: 1.5MS medium + CH 40-50 mg / L + PP333 0.1-0.2 mg / L + kanamycin 0.8-1.0 mg / L; The 1.5MS medium is: the amount of all elements in the MS is 1.5 times that of the original MS, and the sugar concentration remains unchanged; (2) Rooting culture: After the seedlings have grown strong, the clustered buds are cut into single buds, inoculated into rooting medium, and cultured in an environment with a light intensity of 2000-3000 lx, a single day of light for 12 hours, and a temperature of 25±2°C for 30-40 days; The formula of the rooting medium is: 1 / 2MS+IBA 0.3-0.5 mg / L+SA 15-20 mg / L+Kanamycin 4.0-5.0 mg / L; The 1 / 2MS medium is: the concentration of macroelements is reduced to half of MS, and the other components remain unchanged; (3) Rooting seedlings acclimatization: Wash the rooted seedlings with clean water and plant them in the mixed substrate. After transplanting, keep the substrate humidity at 55-65%. During this period, spray nutrient solution A every 5-7 days for 3-4 times in succession. The formula of the nutrient solution A is: IBA 1.5-2.5 mg / L+SA 50-70 mg / L.

2. The method according to claim 1, characterized in that: The formula of the seedling-strengthening culture medium is: 1.5MS culture medium+CH45mg / L+PP333 0.15mg / L+kanamycin 0.9mg / L.

3. The method according to claim 1, characterized in that: The formula of the rooting medium is: 1 / 2MS+IBA0.4mg / L+SA 17mg / L+kanamycin 4.5mg / L.

4. The method according to claim 1, characterized in that: The mixed matrix is ​​a mixture of humus soil and sandy soil in a weight ratio of 1:

10.

5. Application of the method according to any one of claims 1 to 4 in improving the survival rate of Zingiber officinale tissue culture seedlings.

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