Katsumadai katsumadai tissue culture breeding method

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a katsumadai katsumadai tissue culture breeding method. Through the synergistic effect of various factors such as the improved basic culture medium, the exogenous plant hormone and the exogenous alpha-ketoglutaric acid, the tissue culture seedlings of the alpinia katsumadai can maintain the appropriate proliferation rate for a long time, browning can be generated only after 21 generations of continuous transfer in the proliferation culture medium, the high proliferation rate can be maintained in a long production period, and the tissue culture seedlings of the alpinia katsumadai can be obtained. Meanwhile, the browning rate is low, the browning degree is low, the seedling proliferation rate is high, the rooting rate is high, the problem of browning of the katsumade galangal seed tissue culture seedlings is solved, and large-scale industrialized production of the katsumade galangal seeds is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a method for tissue culture and breeding of Alpinia hainanensis K. BACKGROUND

[0002] Alpinia hainanensis K. Schum., also known as Alpinia hainanensis K. Schum., is a perennial herb of Zingiberaceae and Alpinia, mainly produced in Hainan, Guangdong, Guangxi and Yunnan, and has the effects of drying dampness, promoting qi and relieving vomiting. Meanwhile, Alpinia hainanensis K. Schum. is a traditional medicine and food of China, and is a very important spice and food seasoning. In recent years, with the increasing market demand for Alpinia hainanensis K. Schum., the supply of Alpinia hainanensis K. Schum. seedlings has become prominent.

[0003] In recent years, with the development of tissue culture technology, tissue culture and rapid propagation technology has also been applied to the propagation and seedling of Alpinia hainanensis K. Schum. For example, Wang Jun (Establishment of Three In Vitro Rapid Propagation Systems of Zingiberaceae Flowers[D]. Zhongkai College of Agriculture Engineering, 2017.) discloses a method for establishing an in vitro rapid propagation system of Alpinia hainanensis K. Schum. Mature seeds are selected as explants, and seed germination is induced on MS medium. Callus and multiple shoots are induced. The adventitious buds are cut and transferred to bud proliferation medium, and the proliferation multiple is 5.74 times. The buds proliferate in large quantities in about 20 days, and the average plant height is 3.45 cm. A patent with the application number CN202210292799.0 and the publication number CN114568307A and the title of a method for rapidly breeding seedlings by using Alpinia hainanensis K. Schum. stem tips discloses a method for rapidly breeding seedlings by using Alpinia hainanensis K. Schum. stem tips, which includes the processes of inducing and differentiating adventitious buds, subculture and proliferation, rooting induction, transplanting and management. The process flow of the present application is simple. The sprout or stem tip of a tillering plant that has not yet flowered is directly used as an explant to induce and differentiate adventitious buds. The adventitious buds are subjected to subculture and proliferation to form multiple shoots, rooting of the adventitious buds, transplanting and other processes, and a large number of healthy and high-quality seedlings that can be used for production and cultivation are obtained in a short time. The cycle is short, the physiological ages of the obtained seedlings are consistent, the growth is uniform, the seedlings are suitable for factory seedling and large-scale cultivation, the demand for excellent seedlings for artificial planting is met, and the development and utilization of Alpinia hainanensis K. Schum. and the protection of wild resources are promoted.

[0004] However, we found that, during the production of Alpinia hainanensis K. Schum. tissue culture seedlings, with the increase of the number of subculture generations, the originally normal Alpinia hainanensis K. Schum. tissue culture seedlings gradually appeared browning, and the browning rate gradually increased with the increase of the number of subculture generations. The proliferation rate and rooting rate of Alpinia hainanensis K. Schum. tissue culture seedlings with severe browning were extremely low or even dead, which seriously restricted the large-scale factory production of Alpinia hainanensis K. Schum. Therefore, it is urgent to develop a more effective method for tissue culture and breeding of Alpinia hainanensis K. Schum. SUMMARY

[0005] In order to overcome the problems in the prior art, the purpose of the present application is to provide a method for tissue culture and breeding of Amomum villosum, which can solve the problem of browning of Amomum villosum tissue culture seedlings in a long production cycle and realize large-scale industrial production of Amomum villosum.

[0006] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0007] The present application provides a method for tissue culture and breeding of Amomum villosum, comprising the following steps:

[0008] (A) Plant pretreatment: Amomum villosum plants are removed from the root system and outer leaves, soaked in soap water for 35 min, and then washed with tap water for 6.5 h;

[0009] (B) Disinfection of explants: in the super-clean workbench, the Amomum villosum plants treated in step (A) are soaked in 75% alcohol for 1.5 min, washed with sterile water for 4 times, soaked in sodium hypochlorite solution with effective chlorine of 1% for 26 min, washed with sterile water for 7 times, and the surface water is absorbed by sterile filter paper to obtain sterile explants;

[0010] (C) Pre-culture of explants: after the explants treated in step (B) are cut off and the wound is contacted with disinfectant, they are connected to the pre-culture medium;

[0011] (D) Primary induction culture: the explants cultured in step (C) are transferred to the primary induction culture medium to induce the formation of clumps;

[0012] (E) Proliferation culture: the clumps obtained in step (D) are cut into single buds and transferred to the proliferation culture medium for repeated cutting and proliferation culture to obtain clumps;

[0013] (F) Rooting culture: the clumps obtained in step (E) are cut into single buds and inoculated into the rooting culture medium;

[0014] The pre-culture medium takes the modified MS medium as the basic medium, and further comprises:

[0015] 2.0-3.0 mg / L TDZ, 0.1-0.3 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar;

[0016] The primary induction culture medium takes the modified MS medium as the basic medium, and further comprises:

[0017] 1.0-1.5 mg / L TDZ, 0.1-0.2 mg / L NAA, 10.0-27.0 mg / L α-ketoglutaric acid, 30 g / L sucrose and 6.0 g / L agar;

[0018] The proliferation culture medium takes the modified MS culture medium as the basic culture medium, and further comprises:

[0019] 0.5-0.6 mg / L TDZ, 0.1-0.2 mg / L NAA, 10.0-27.0 mg / L α-ketoglutaric acid, 30 g / L sucrose and 6.0 g / L agar;

[0020] The rooting culture medium takes 1 / 2 modified MS basic culture medium, and further comprises:

[0021] 0.1-0.5 mg / L NAA, 5.0-8.0 mg / L α-ketoglutaric acid, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar;

[0022] The composition of the modified MS culture medium comprises:

[0023] NH4NO31100 mg / L, MgSO4· 7H2O 370 mg / L, KH2PO4170 mg / L, KNO31900 mg / L, Ca(NO3)2· 4 H2O 1112 mg / L, FeSO4· 7H2O 27.8 mg / L, Na2· EDTA 37.3 mg / L, myo-inositol 100 mg / L, nicotinic acid 10 mg / L, VB6 10 mg / L, VB1 100 mg / L, glycine 2 mg / L, CuSO4· 5H2O 0.025 mg / L, H3BO36.2 mg / L, MnSO4· H2O 16.9 mg / L, CoCl2· 6H2O 0.025 mg / L, ZnSO4· 7H2O 8.6 mg / L, Na2MoO4· 2H2O 0.25 mg / L, KI 0.83 mg / L.

[0024] Preferably, the pre-culture medium takes the modified MS culture medium as the basic culture medium, and further comprises:

[0025] 2.0 mg / L TDZ, 0.3 mg / L NAA, 1.0-2.0 mg / L VC, 30 g / L sucrose and 6.0 g / L agar.

[0026] More preferably, the pre-culture medium takes the modified MS culture medium as the basic culture medium, and further comprises:

[0027] 2.0 mg / L TDZ, 0.3 mg / L NAA, 1.0 mg / L VC, 30 g / L sucrose and 6.0 g / L agar.

[0028] Preferably, the primary induction culture medium takes the modified MS culture medium as the basic culture medium, and further comprises:

[0029] 1.0 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L.

[0030] Preferably, the proliferation medium takes the modified MS medium as the basic medium, and further comprises:

[0031] 0.6 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L.

[0032] Preferably, the rooting medium takes the 1 / 2 modified MS medium as the basic medium, and further comprises:

[0033] 0.5 mg / L NAA, 6.0 mg / L α-ketoglutaric acid, activated carbon 1.0 g / L, sucrose 20 g / L and agar 6.0 g / L.

[0034] Preferably, the plant pretreatment further comprises, before the treatment, selecting the robustly grown piper methysticum plants, and storing in a refrigerator at 4 ℃ for 40 d.

[0035] Preferably, the culture condition of the explant pre-culture is dark culture at 4 ℃ for 7 d.

[0036] Preferably, the culture condition of the primary induction culture, the proliferation culture and the rooting culture is culture at 20-22 ℃, illumination 1500-2000 lux, illumination time 10 h / d, and culture for 30 d.

[0037] More preferably, the culture condition further comprises, before the illumination culture, dark culture at 20-22 ℃ for 2 d.

[0038] By the above technical solution, the present application can at least achieve the following beneficial effects:

[0039] The application provides a method for tissue culture of Amomum villosum. In the steps of explant pre-culture, primary induction culture and proliferation culture, the basic culture medium is modified MS culture medium. In the modified MS culture medium, the content of NH4NO3 is reduced, Ca(NO3)2·4H2O is used as calcium salt and the content of calcium salt is increased, and the contents of nicotinic acid, VB6 and VB1 are increased. Increasing the contents of organic matter, nitrate ions, reducing the content of ammonium ions and increasing the content of calcium salt can promote material transport to a certain extent, accelerate cell division, make the growth vigorous, and reduce the degree of browning. TDZ, NAA and alpha-ketoglutaric acid and other components are added. TDZ can induce the synthesis and accumulation of endogenous hormones such as ethylene and auxin in plants, regulate the growth and development process of plants by adjusting the hormone balance, and promote the differentiation of cluster buds. NAA can promote the dedifferentiation of explant cells and break the differentiation state of cells. By adjusting the ratio of cytokinin, NAA can affect the differentiation direction of buds and roots. When used alone at a low concentration, NAA often promotes root development and other hormone substances can jointly regulate growth and proliferation. In the complex process of plant growth and development, alpha-ketoglutaric acid plays a crucial role. It widely participates in various physiological and biochemical reactions in plants. Alpha-ketoglutaric acid promotes the assimilation of nitrogen in plants, thereby providing sufficient nitrogen-containing compounds for plant growth, helping plants to grow vigorously. Alpha-ketoglutaric acid indirectly regulates the growth and development pattern of plants by affecting hormone synthesis, so that plants can better adapt to environmental changes. The addition of alpha-ketoglutaric acid effectively alleviates the oxidation reaction caused by factors such as cutting, thereby reducing browning to a certain extent. The addition of a new organic nitrogen source to Amomum villosum changes the ratio and content of ammonium nitrogen and nitrate nitrogen. The addition of organic nitrogen source also has a positive effect on inhibiting browning. It can be seen that through the synergistic effect of improved basic culture medium, exogenous plant hormones and exogenous alpha-ketoglutaric acid and other factors, the Amomum villosum tissue culture seedlings can maintain a suitable proliferation rate for a long time. After 21 generations of continuous transfer in the proliferation culture medium, browning occurs. In a relatively long production cycle, high proliferation rate, low browning rate, weak browning degree, high seedling proliferation rate and high rooting rate can be maintained. The problem of browning of Amomum villosum tissue culture seedlings is avoided, and large-scale industrial production of Amomum villosum is realized. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments

[0041] Figure 1 It is the pre-culture diagram of the explant in Example 1.

[0042] Figure 2 It is the primary induction culture diagram in Example 1.

[0043] Figure 3 It is the proliferation culture diagram after 22 generations of transfer in Example 1.

[0044] Figure 4 Proliferation culture chart after subculture 8 generations for ratio 1

[0045] Figure 5 Proliferation culture chart after subculture 8 generations for ratio 1

[0046] Figure 6 Proliferation culture chart after subculture 8 generations for ratio 1

[0047] Figure 7 Proliferation culture chart after subculture 7 generations for ratio 2

[0048] Figure 8 Proliferation culture chart after subculture 7 generations for ratio 2

[0049] Figure 9 Proliferation culture chart after subculture 8 generations for ratio 3

[0050] Figure 10 Proliferation culture chart after subculture 8 generations for ratio 3

[0051] Figure 11 Proliferation culture chart after subculture 8 generations for ratio 4

[0052] Figure 12 Proliferation culture chart after subculture 8 generations for ratio 4 DETAILED DESCRIPTION

[0053] The prior art of tissue culture of Amomum villosum is based on simple experiments, only a few generations of proliferation, and then rooting, which shows the advantages of high proliferation and high rooting rate of domesticated seedlings. In fact, when the tissue culture technology is applied to large-scale factory cultivation, it will undergo dozens or hundreds of generations of proliferation subculture. After a limited number of generations of proliferation subculture in the prior art, browning occurs and the browning rate gradually increases. The proliferation rate and rooting rate of the browning tissue culture seedlings are extremely low or even dead. The browning of the proliferation tissue culture seedlings affects the proliferation and rooting, and the part of the tissue culture seedlings can only be abandoned, and the external explants are collected again for primary disinfection and re-starting, which seriously affects the production efficiency. When the tissue culture is cultured, it is not that the higher the proliferation rate is, the better. Too high proliferation rate often leads to variation, weakness, and malformation of the tissue culture seedlings, which affects production. The method provided by the present application enables the Amomum villosum tissue culture seedlings to maintain a suitable proliferation rate for a long time, and browning occurs only after 21 generations of continuous subculture in the proliferation medium. At this time, the re-collection of external explants for induction does not affect production. The high proliferation rate can be maintained for a long production cycle, and the browning seedlings have a high rooting rate, which avoids the problem of browning of Amomum villosum tissue culture seedlings, and enables large-scale factory production of Amomum villosum.

[0054] For further illustrating the present application, the technical solutions provided by the present application are described in detail below with reference to the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0055] Unless otherwise specified, the materials and reagents used in the present application are commercially available.

[0056] The modified MS medium used in the following examples and comparative examples has the following specific composition:

[0057] The modified MS medium is based on the MS basic medium, with the NH4NO3 content reduced to 1100 mg / L, Ca(NO3)2·4H2O used for calcium salt and the content increased to 1112 mg / L, and the contents of nicotinic acid, VB6 and VB1 increased. The specific composition is: NH4NO31100 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4170 mg / L, KNO31900 mg / L, Ca(NO3)2·4H2O 1112 mg / L, FeSO4·7H2O 27.8 mg / L, Na2·EDTA 37.3 mg / L, myo-inositol 100 mg / L, nicotinic acid 10 mg / L, VB610 mg / L, VB1100 mg / L, glycine 2 mg / L, CuSO4·5H2O 0.025 mg / L, H3BO36.2 mg / L, MnSO4·H2O 16.9 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, KI 0.83 mg / L.

[0058] Example 1

[0059] The piper longum plants were collected from the Germplasm Garden of Medicinal Plant Institute of Yunnan Academy of Agricultural Sciences in Wuding County, and the sampling time was March. The following steps were performed:

[0060] (A) Plant pretreatment: The piper longum plants were removed from the roots and outer leaves, soaked in soap water for 35 min, and then washed with tap water for 6.5 h;

[0061] (B) Disinfection of explants: In the clean bench, the piper longum plants treated in step (A) were soaked in 75% alcohol for 1.5 min, washed with sterile water 4 times, soaked in 1% sodium hypochlorite solution for 26 min, washed with sterile water 7 times, and the surface water was absorbed with sterile filter paper to obtain sterile explants;

[0062] (C) Pre-culture of explants: the explants treated in step (B) were cut to remove the part contacting the disinfectant and then transferred to the pre-culture medium. The pre-culture medium was based on the modified MS medium and further included:

[0063] 2.0 mg / L TDZ, 0.3 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar, pH 5.8, and dark incubation at 4°C for 7 days;

[0064] (D) Primary induction culture: the explants cultured in step (C) were transferred to the primary induction medium to induce the formation of clumps. The primary induction medium was based on the modified MS medium and further included:

[0065] 1.0 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; incubation at 20-22°C, first in dark and then under light, dark time 2 days; light intensity 1500-2000 lux, light time 10 h / d, culture for 30 days;

[0066] (E) Proliferation culture: the clumps obtained in step (D) were cut into single buds and transferred to the proliferation medium. The proliferation culture was repeated by cutting to obtain clumps. The proliferation medium was based on the modified MS medium and further included:

[0067] 0.6 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; incubation at 20-22°C, first in dark and then under light, dark time 2 days; light intensity 1500-2000 lux, light time 10 h / d, culture for 30 days;

[0068] (F) Rooting culture: the clumps obtained in step (E) were cut into single buds and inoculated into the rooting medium. The rooting medium was based on the 1 / 2 modified MS medium and further included:

[0069] 0.5 mg / L NAA, 6.0 mg / L α-ketoglutaric acid, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar, pH 5.8; incubation at 20-22°C, first in dark and then under light, dark time 2 days; light intensity 1500-2000 lux, light time 10 h / d, culture for 30 days.

[0070] Example 2

[0071] The plant of Amomum villosum was collected from the Germplasm Garden of Medicinal Plant Institute of Yunnan Academy of Agricultural Sciences in Wuding County, and the sampling time was March, and the following steps were performed:

[0072] (A) Plant pretreatment: the roots and outer leaves of the plant are removed, and the plant is soaked in soapy water for 35 min and then washed with tap water for 6.5 h;

[0073] (B) Disinfection of explants: the plant treated in step (A) is soaked in 75% alcohol for 1.5 min, washed with sterile water for 4 times, soaked in sodium hypochlorite solution with an effective chlorine of 1% for 26 min, washed with sterile water for 7 times, and the surface water is absorbed by sterile filter paper to obtain sterile explants;

[0074] (C) Pre-culture of explants: the explants treated in step (B) are cut to remove the part in contact with the disinfectant, and then transferred to the pre-culture medium. The pre-culture medium is based on modified MS medium and further includes:

[0075] 2.0 mg / L TDZ, 0.3 mg / L NAA, 30 g / L sucrose, and 6.0 g / L agar, pH 5.8, and dark culture at 4°C for 7 d;

[0076] (D) Primary induction culture: the explants cultured in step (C) are transferred to the primary induction medium to induce the formation of clumps. The primary induction medium is based on modified MS medium and further includes:

[0077] 1.0 mg / L TDZ, 0.2 mg / L NAA, 10.0 mg / L α-ketoglutaric acid, 30 g / L sucrose, and 6.0 g / L agar, pH 5.8; dark culture at 20-22°C, followed by light culture, dark culture time is 2 d; light intensity is 1500-2000 lux, light time is 10 h / d, and culture time is 30 d;

[0078] (E) Proliferation culture: the clumps obtained in step (D) are cut into single buds and transferred to the proliferation medium. The proliferation culture is repeated to obtain clumps. The proliferation medium is based on modified MS medium and further includes:

[0079] 0.6 mg / L TDZ, 0.1 mg / L NAA, 10.0 mg / L α-ketoglutaric acid, 30 g / L sucrose, and 6.0 g / L agar, pH 5.8; dark culture at 20-22°C, followed by light culture, dark culture time is 2 d; light intensity is 1500-2000 lux, light time is 10 h / d, and culture time is 30 d;

[0080] (F) Rooting culture: the clumps obtained in step (E) are cut into single buds and inoculated into the rooting medium. The rooting medium is based on 1 / 2 modified MS medium and further includes:

[0081] 0.5mg / L NAA, 5.0mg / L α-ketoglutaric acid, activated carbon 1.0g / L, sucrose 20g / L and agar 6.0g / L, pH 5.8; placed in temperature at 20~22℃, first in the dark and then in the light culture, dark time for 2d; illumination 1500~2000lux, illumination time 10h / d, culture for 30d.

[0082] Example 3

[0083] Piper longum plants were collected from the Germplasm Resource Garden of Medicinal Plant Institute of Yunnan Academy of Agricultural Sciences in Wuding County, and the sampling time was March, which was carried out by the following steps:

[0084] (A) Plant pretreatment: Piper longum plants were removed from the root system and outer leaves, soaked in soap water for 35min, and then washed with tap water for 6.5h;

[0085] (B) Disinfection of explants: In the clean bench, the Piper longum plants treated in step (A) were soaked in 75% alcohol for 1.5min, washed with sterile water 4 times, soaked in 1% sodium hypochlorite solution for 26min, washed with sterile water 7 times, and the surface water was absorbed with sterile filter paper to obtain sterile explants;

[0086] (C) Pre-culture of explants: After the explants treated in step (B) were cut off and the wound contacted with disinfectant, they were transferred to the pre-culture medium. The pre-culture medium used modified MS medium as the basic medium, and also included:

[0087] 2.0mg / L TDZ, 0.3mg / L NAA, sucrose 30g / L and agar 6.0g / L, pH 5.8, placed in dark at 4℃ for 7d;

[0088] (D) Primary induction culture: The explants cultured in step (C) were transferred to the primary induction medium to induce the formation of clumps. The primary induction medium used modified MS medium as the basic medium, and also included:

[0089] 1.0mg / L TDZ, 0.2mg / L NAA, 27.0mg / L α-ketoglutaric acid, sucrose 30g / L and agar 6.0g / L, pH 5.8; placed in temperature at 20~22℃, first in the dark and then in the light culture, dark time for 2d; illumination 1500~2000lux, illumination time 10h / d, culture for 30d;

[0090] (E) Proliferation culture: The clumps obtained in step (D) were cut into single buds and transferred to the proliferation medium. The proliferation culture was repeated to obtain clumps. The proliferation medium used modified MS medium as the basic medium, and also included:

[0091] 0.6 mg / L TDZ, 0.1 mg / L NAA, 27.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in temperature at 20-22℃, dark first and then light culture, dark time for 2d; light 1500-2000 lux, light time 10h / d, culture 30d;

[0092] (F) rooting culture: the single buds obtained in step (E) are cut and inoculated into rooting medium, the rooting medium is 1 / 2 modified MS basic medium, further comprising:

[0093] 0.5 mg / L NAA, 8.0 mg / L α-ketoglutaric acid, activated carbon 1.0 g / L, sucrose 20 g / L and agar 6.0 g / L, pH 5.8; placed in temperature at 20-22℃, dark first and then light culture, dark time for 2d; light 1500-2000 lux, light time 10h / d, culture 30d.

[0094] Example 4

[0095] The piper longum plants are collected from the germplasm resource garden of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Wuding County, and the sampling time is March, which is carried out by the following steps:

[0096] (A) plant pretreatment: the piper longum plants are removed from the root system and outer leaves, soaked in soap water for 35 min, and then washed with tap water for 6.5h;

[0097] (B) disinfection of explants: in the clean bench, the piper longum plants treated in step (A) are soaked in 75% alcohol for 1.5 min, washed with sterile water 4 times, soaked in 1% sodium hypochlorite solution for 26 min, washed with sterile water 7 times, and the surface water is absorbed by sterile filter paper to obtain sterile explants;

[0098] (C) pre-culture of explants: after the explants treated in step (B) are cut off and the wound is contacted with disinfectant, they are inoculated into the pre-culture medium, and the explant pre-culture medium is a modified MS medium as the basic medium, further comprising:

[0099] 3.0 mg / L TDZ, 0.1 mg / L NAA, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in dark at 4℃ for 7d;

[0100] (D) primary induction culture: the explants cultured in step (C) are transferred to the primary induction medium to induce the formation of clumps, and the primary induction medium is a modified MS medium as the basic medium, further comprising:

[0101] 1.5 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22°C, first in the dark and then in light culture, dark time for 2d; light 1500-2000 lux, light time 10h / d, culture for 30d;

[0102] (E) proliferation culture: the clumps obtained in step (D) are cut into single buds and transferred to the proliferation medium, repeated cutting for proliferation culture to obtain clumps, the proliferation medium takes the modified MS medium as the basic medium, and further includes:

[0103] 0.5 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22°C, first in the dark and then in light culture, dark time for 2d; light 1500-2000 lux, light time 10h / d, culture for 30d;

[0104] (F) rooting culture: the clumps obtained in step (E) are cut into single buds and inoculated into the rooting medium, the rooting medium takes the 1 / 2 modified MS basic medium, and further includes:

[0105] 0.1 mg / L NAA, 6.0 mg / L α-ketoglutaric acid, activated carbon 1.0 g / L, sucrose 20 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22°C, first in the dark and then in light culture, dark time for 2d; light 1500-2000 lux, light time 10h / d, culture for 30d.

[0106] Example 5

[0107] Other steps are the same as those in Example 1, the only difference is that before pretreatment, the healthy and strong piper nigrum plants are selected and placed in a refrigerator at a temperature of 4°C for 40d.

[0108] Example 6

[0109] Other steps are the same as those in Example 1, the only difference is that,

[0110] The pre-culture condition of the explant is changed from: placed in a temperature of 4°C and dark culture for 7d to: placed in a temperature of 20-22°C; light 1500-2000 lux, light time 10h / d, culture for 7d.

[0111] Example 7

[0112] Other steps are the same as those in Example 1, the only difference is that 1.0 mg / L VC is added in the pre-culture medium.

[0113] Example 8

[0114] Other than the steps of Example 1, the only difference is that 2.0 mg / L VC is added in the pre-culture medium.

[0115] Example 9

[0116] Other than the steps of Example 1, the only difference is that the initial induction culture, proliferation culture and rooting culture are no longer in dark culture.

[0117] Example 10

[0118] The plant of Amomum villosum was collected from the Germplasm Garden of Medicinal Plant Institute of Yunnan Academy of Agricultural Sciences in Wuding County. The sampling time was March. Before pretreatment, the healthy Amomum villosum plants were stored in a refrigerator at 4°C for 40 days, and were ready for use.

[0119] The following steps were taken:

[0120] (A) Plant pretreatment: The roots and outer leaves of Amomum villosum plants were removed, and the plants were soaked in soap water for 35 min and then washed with tap water for 6.5 h;

[0121] (B) Disinfection of explants: The Amomum villosum plants treated in step (A) were soaked in 75% alcohol for 1.5 min, washed with sterile water 4 times, soaked in 1% sodium hypochlorite solution for 26 min, washed with sterile water 7 times, and the surface water was absorbed with sterile filter paper to obtain sterile explants;

[0122] (C) Pre-culture of explants: After the explants treated in step (B) were cut to remove the part in contact with the disinfectant, they were transferred to the pre-culture medium. The pre-culture medium was based on modified MS medium and also included:

[0123] 2.0 mg / L TDZ, 0.3 mg / L NAA, 1.0 mg / L VC, 30 g / L sucrose and 6.0 g / L agar, pH 5.8, and dark culture at 4°C for 7 days;

[0124] (D) Initial induction culture: The explants cultured in step (C) were transferred to the initial induction medium to induce the formation of clumps. The initial induction medium was based on modified MS medium and also included:

[0125] 1.0 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22℃, first in the dark and then in the light culture, dark time for 2d; illumination 1500-2000 lux, illumination time 10h / d, culture for 30d;

[0126] (E) proliferation culture: the cluster buds obtained in step (D) are cut into single buds and transferred to the proliferation culture medium, and repeated cutting is performed to obtain cluster buds, the proliferation culture medium uses modified MS medium as the basic medium, and further comprises:

[0127] 0.6 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutaric acid, sucrose 30 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22℃, first in the dark and then in the light culture, dark time for 2d; illumination 1500-2000 lux, illumination time 10h / d, culture for 30d;

[0128] (F) rooting culture: the cluster buds obtained in step (E) are cut into single buds and inoculated into the rooting culture medium, and the rooting culture medium uses 1 / 2 modified MS basic medium, and further comprises:

[0129] 0.5 mg / L NAA, 6.0 mg / L α-ketoglutaric acid, activated carbon 1.0 g / L, sucrose 20 g / L and agar 6.0 g / L, pH 5.8; placed in a temperature of 20-22℃, first in the dark and then in the light culture, dark time for 2d; illumination 1500-2000 lux, illumination time 10h / d, culture for 30d.

[0130] Comparative Example 1

[0131] Other steps are the same as those of Example 1, the only difference is that α-ketoglutaric acid is not added in the primary induction culture medium, the proliferation culture medium and the rooting culture medium.

[0132] Comparative Example 2

[0133] Other steps are the same as those of Example 1, the only difference is that the modified MS medium is replaced by MS basic medium in the pre-culture medium, the primary induction culture medium, the proliferation culture medium and the rooting culture medium.

[0134] Comparative Example 3

[0135] Other steps are the same as those of Example 1, the only difference is that the modified MS medium is replaced by B5 basic medium in the pre-culture medium, the primary induction culture medium, the proliferation culture medium and the rooting culture medium.

[0136] Comparative Example 4

[0137] The steps are the same as in Example 1, except that the modified MS medium is replaced with WPM basic medium in the pre-culture medium, primary induction medium, proliferation medium and rooting medium.

[0138] In Examples 1-10 and Comparative Examples 1-4, the proliferation culture was continuously transferred; after browning occurred, the browned seedlings were transferred to proliferation culture medium and rooting culture medium for continued culture; the transfer generation number when the first tissue culture seedlings showed browning, the browning rate of that generation, and the rooting rate and proliferation multiple of the browned seedlings of that generation were observed and statistically analyzed.

[0139] Browning rate = (Number of browned seedlings / Number of inoculated seedlings) * 100%;

[0140] Brown seedling rooting rate = (Number of brown seedlings with roots / Number of inoculated seedlings) * 100%;

[0141] Multiplication factor = (Number of differentiated buds / Number of inoculated seedlings) * 100%;

[0142] The statistical results of different treatments are shown in Table 1:

[0143] Table 1 Statistical results of different treatments

[0144]

[0145] The results show that browning occurred in Examples 1-10 as early as 21 generations of propagation culture, with a browning rate below 10.3% and a weak degree of browning. The rooting rate of browned seedlings was above 61.7%, and the fold increase of browned seedlings was only slightly reduced, having little impact on large-scale production. This also allowed sufficient time for recollecting explants to establish a tissue culture system, indicating that adding an appropriate concentration of α-ketoglutarate has a significant effect on alleviating the generation number of browning in cardamom. The pre-culture state of explants in Example 1 is as follows: Figure 1 As shown, the initial induction culture state is as follows: Figure 2 As shown, the culture method according to the examples did not produce browning and had a high induction rate; in Example 1, browning occurred after 22 generations of transfer, and the proliferation culture was as follows. Figure 3 As shown, in Example 1, the rooting culture after transfer 22 was as follows: Figure 4 As shown, the proliferation rate and rooting rate were high, and browning was weak. In contrast, in Comparative Examples 1-4, browning appeared as early as 7 generations after proliferation culture, and the degree of browning was strong, with a browning rate exceeding 22.3%. The rooting rate of browned seedlings was less than 7.3%, and the proliferation ratio of browned seedlings was significantly reduced. Some tissue culture seedlings showed browning and death. The browned seedlings in Comparative Example 1 after 8 generations of transfer showed... Figure 5 As shown, the rooting culture of brown seedlings after 8 generations of transplantation in ratio 1 is as follows: Figure 6 As shown; Comparative Example 2, after 7 generations of transfer, the browned seedlings were cultured for propagation as follows. Figure 7 As shown, the rooting culture of brown seedlings after 7 generations of transplantation in ratio 1 is as follows:Figure 8 As shown; Comparative Example 3, after 8 generations of transfer, the browned seedlings were cultured for proliferation as follows. Figure 9 As shown, the rooting culture of brown seedlings after 8 generations of transplantation at ratio 3 is as follows: Figure 10 As shown; Comparative Example 4, after 8 generations of transplantation, the browned seedlings were cultured for proliferation. Figure 11 As shown, the rooting culture of brown seedlings after 8 generations of transplantation at ratio 4 is as follows: Figure 12 As shown, the browned seedlings in Comparative Examples 1-4 exhibited very low proliferation rates and rooting rates, and also showed browning and death. Therefore, the technical solution provided by this invention can maintain a high proliferation rate over a relatively long production cycle, while maintaining a low browning rate and weak degree of browning in the seedlings, as well as a high seedling proliferation rate and high rooting rate, thus avoiding the browning problem of cardamom tissue culture seedlings and enabling large-scale industrial production of cardamom.

[0146] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for tissue culture propagation of cardamom, characterized in that, Includes the following steps: (A) Plant pretreatment: Remove the roots and outer leaves of the cardamom plant, soak in soapy water for 35 minutes and then rinse under running tap water for 6.5 hours; (B) Explant disinfection: On a clean bench, soak the cardamom plants treated in step (A) in 75% alcohol for 1.5 min, rinse with sterile water 4 times, soak in sodium hypochlorite solution with 1% available chlorine for 26 min, rinse with sterile water 7 times, and absorb the surface moisture with sterile filter paper to obtain sterile explants. (C) Explant pre-culture: After removing the part of the explant that was in contact with the disinfectant after the wound was removed, the explant treated in step (B) was inoculated into the pre-culture medium. (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction culture medium to induce the formation of shoot clusters; (E) Proliferation culture: The cluster of shoots obtained in step (D) is cut into single shoots and transferred to the proliferation culture medium. The cutting is repeated to carry out proliferation culture to obtain cluster of shoots; (F) Rooting culture: The cluster of shoots obtained in step (E) is cut into single shoots and inoculated into rooting culture medium; The pre-culture medium uses modified MS medium as the basic medium and also includes: 2.0-3.0 mg / L TDZ, 0.1-0.3 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar; The primary induction medium uses modified MS medium as the basic medium and also includes: 1.0–1.5 mg / L TDZ, 0.1–0.2 mg / L NAA, 10.0–27.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar; The proliferation medium uses modified MS medium as the basic medium and also includes: 0.5-0.6 mg / L TDZ, 0.1-0.2 mg / L NAA, 10.0-27.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar; The rooting medium is a 1 / 2 modified MS basal medium, which also includes: 0.1-0.5 mg / L NAA, 5.0-8.0 mg / L α-ketoglutarate, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar; The modified MS medium comprises: NH4NO3 1100 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, KNO3 1900 mg / L, Ca(NO3)2·4H2O 1112 mg / L, FeSO4·7H2O 27.8 mg / L, Na2·EDTA 37.3 mg / L, inositol 100 mg / L, nicotinic acid 10 mg / L, VB6 10 mg / L, VB1 100 mg / L, glycine 2 mg / L, CuSO4·5H2O 0.025 mg / L, H3BO3 6.2 mg / L, MnSO4·H2O 16.9 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4· 2H2O 0.25mg / L, KI 0.83mg / L.

2. The method according to claim 1, characterized in that, The pre-culture medium uses modified MS medium as the basic medium and also includes: 2.0 mg / L TDZ, 0.3 mg / L NAA, 1.0–2.0 mg / L VC, 30 g / L sucrose and 6.0 g / L agar.

3. The method according to claim 2, characterized in that, The pre-culture medium uses modified MS medium as the basic medium and also includes: 2.0 mg / L TDZ, 0.3 mg / L NAA, 1.0 mg / L VC, 30 g / L sucrose and 6.0 g / L agar.

4. The method according to claim 1, characterized in that, The primary induction medium uses modified MS medium as the basic medium and also includes: 1.0 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar.

5. The method according to claim 1, characterized in that, The proliferation medium uses modified MS medium as the basic medium and also includes: 0.6 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar.

6. The method according to claim 1, characterized in that, The rooting medium is a 1 / 2 modified MS basal medium, which also includes: 0.5 mg / L NAA, 6.0 mg / L α-ketoglutarate, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar.

7. The method according to any one of claims 1 to 6, characterized in that, The plant pretreatment also includes selecting healthy cardamom plants and refrigerating them at 4°C for 40 days before treatment.

8. The method according to any one of claims 1 to 6, characterized in that, The culture conditions for the explants were as follows: they were cultured in the dark at 4°C for 7 days.

9. The method according to any one of claims 1 to 6, characterized in that, The culture conditions for the initial induction culture, proliferation culture and rooting culture are as follows: temperature at 20~22℃, light intensity at 1500~2000 lux, light duration at 10h / d, cultured for 30 days.

10. The method according to claim 9, characterized in that, The cultivation conditions also include placing the culture at a temperature of 20~22℃ and incubating it in the dark for 2 days before light cultivation.

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