Method for tissue culture breeding of amomum villosum

Abstract

The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for tissue culture and breeding of Amomum subulatum. The present application realizes large-scale factory production of Amomum subulatum by improving the synergistic effect of various factors such as a basic culture medium, exogenous plant hormones and exogenous alpha-ketoglutarate, so that the Amomum subulatum tissue culture seedlings maintain a suitable proliferation rate for a long time, and brownization occurs only after 21 generations of continuous transfer in the proliferation culture medium. In a relatively long production cycle, the Amomum subulatum tissue culture seedlings have a high proliferation rate, a low brownization rate, a weak degree of brownization, a high seedling proliferation rate and a high rooting rate, and the problem of brownization of the Amomum subulatum tissue culture seedlings is avoided.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically, it relates to a method for tissue culture propagation of cardamom. Background Technology

[0002] Cardamom (Alpinia hainanensis K.Schum.), also known as nutmeg or black cardamom, is a perennial herb belonging to the genus Alpinia in the family Zingiberaceae. It is mainly produced in Hainan, Guangdong, Guangxi, and Yunnan provinces. It has the effects of drying dampness, promoting qi circulation, warming the stomach, and stopping vomiting. Cardamom is also a traditional Chinese medicine and food ingredient, and a very important spice and seasoning. In recent years, with the increasing market demand for cardamom, the supply of cardamom seedlings has become a prominent issue.

[0003] In recent years, with the development of tissue culture technology, rapid propagation technology has also begun to be applied to the propagation and seedling cultivation of cardamom. For example, Wang Jun (Establishment of in vitro rapid propagation system for three ginger family flowers [D]. Zhongkai University of Agriculture and Engineering, 2017.) disclosed the establishment of an in vitro rapid propagation system for cardamom, selecting mature seeds as explants and inducing seed germination on MS medium. Callus tissue and clustered shoots were induced. Adventitious shoots were cut and transferred to shoot proliferation medium, with a proliferation ratio of 5.74 times. The shoots proliferated in large quantities in about 20 days, with an average plant height of 3.45 cm. Patent application number CN202210292799.0, publication number CN114568307A, entitled "A method for rapid propagation of seedlings using cardamom shoot tips", discloses a method for rapid propagation of seedlings using cardamom shoot tips, including induction of adventitious shoot differentiation, subculture proliferation, rooting induction, transplanting and management processes. The process of this invention is simple. It directly induces the differentiation of adventitious buds by using bamboo shoots or the stem tips of unflowered tillers as explants. The adventitious buds undergo subculture to form cluster buds, rooting, and transplanting. This process can produce a large number of healthy and high-quality seedlings suitable for production and cultivation in a short period of time. The cycle is short, and the seedlings obtained are of uniform physiological age and grow uniformly, making them suitable for factory seedling production and large-scale cultivation. This solves the demand for high-quality seedlings in artificial planting and plays an important role in promoting the development and utilization of cardamom and protecting wild resources.

[0004] However, our research on the production of cardamom tissue culture seedlings revealed that as the number of subculture generations increased, initially normal cardamom tissue culture seedlings gradually turned brown. Furthermore, the browning rate increased with each subculture generation, and severely browned seedlings exhibited extremely low proliferation and rooting rates, even dying, significantly hindering the large-scale industrial production of cardamom. Therefore, there is an urgent need to develop more effective cardamom tissue culture propagation methods. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a method for tissue culture propagation of cardamom. The method provided by this invention can solve the problem of browning of cardamom tissue culture seedlings within a long production cycle, and realize the large-scale industrial production of cardamom.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a method for tissue culture propagation of cardamom, comprising the following steps:

[0008] (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;

[0009] (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.

[0010] (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.

[0011] (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;

[0012] (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;

[0013] (F) Rooting culture: The cluster of shoots obtained in step (E) is cut into single shoots and inoculated into rooting culture medium;

[0014] The pre-culture medium uses modified MS medium as the basic medium and also includes:

[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 medium uses modified MS medium as the basic medium and also includes:

[0017] 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;

[0018] The proliferation medium uses modified MS medium as the basic medium and also includes:

[0019] 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;

[0020] The rooting medium is a 1 / 2 modified MS basal medium, and also includes:

[0021] 0.1–0.5 mg / L NAA, 5.0–8.0 mg / L α-ketoglutarate, 1.0 g / L activated charcoal, 20 g / L sucrose, and 6.0 g / L agar;

[0022] The modified MS medium comprises:

[0023] NH4NO3 1100mg / L, MgSO4·7H2O 370mg / L, KH2PO4 170mg / L, KNO3 1900mg / L, Ca(NO3)2·4H2O 1112mg / L, FeSO4·7H2O 27.8mg / L, Na2·EDTA 37.3mg / L, inositol 100mg / L, nicotinic acid 10mg / L, VB6 10mg / L, VB1 100mg / L, glycine 2mg / L, CuSO4·5H2O 0.025mg / L, H3BO3 6.2mg / L, MnSO4·H2O 16.9mg / L, CoCl2·6H2O 0.025mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25 mg / L, KI 0.83 mg / L.

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

[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 uses modified MS medium as the basic medium and further includes:

[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 medium uses modified MS medium as the basic medium and further includes:

[0029] 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.

[0030] Preferably, the proliferation medium uses modified MS medium as the basic medium and further includes:

[0031] 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.

[0032] Preferably, the rooting medium is a 1 / 2 modified MS basal medium, and further includes:

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

[0034] Preferably, the plant pretreatment further includes selecting healthy cardamom plants and refrigerating them at 4°C for 40 days before treatment.

[0035] Preferably, the culture conditions for the explant pre-culture are: dark culture at 4°C for 7 days.

[0036] Preferably, the culture conditions for the primary induction culture, proliferation culture and rooting culture are: a temperature of 20~22℃, a light intensity of 1500~2000 lux, a light duration of 10h / d, and a culture period of 30 days.

[0037] More preferably, the culture conditions further include placing the culture at a temperature of 20~22℃ and in the dark for 2 days before light culture.

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

[0039] This invention provides a method for tissue culture propagation of cardamom. In the explant pre-culture, primary induction culture, and proliferation culture steps, the basic culture medium is adjusted to a modified MS medium. In the modified MS medium, the NH4NO3 content is reduced, and the calcium salt is replaced with Ca(NO3)2·4H2O with increased content. Simultaneously, the contents of nicotinic acid, VB6, and VB1 are increased. Increasing organic matter content, increasing nitrate ion content, decreasing ammonium ion content, and increasing calcium salt content can promote substance transport to a certain extent, leading to faster cell division, vigorous growth, and reduced browning. The addition of TDZ, NAA, and α-ketoglutarate, among other components, allows TDZ to induce the synthesis and accumulation of endogenous hormones such as ethylene and auxin in plants, regulating plant growth and development by modulating hormone balance and promoting shoot differentiation. NAA promotes dedifferentiation of explant cells, disrupting the cell differentiation state. Through the regulation of its ratio with cytokinin, NAA can influence the differentiation direction of shoots and roots. Low concentrations used alone often promote root development, and it can work with other hormones to regulate growth and proliferation. In the complex process of plant growth and development, α-ketoglutarate plays a crucial role. It is widely involved in various physiological and biochemical reactions within plants. α-ketoglutarate promotes nitrogen assimilation, thereby providing sufficient nitrogen compounds for plant growth and contributing to robust development. By influencing hormone synthesis, α-ketoglutarate indirectly regulates plant growth and development patterns, enabling plants to better adapt to environmental changes. The addition of α-ketoglutarate effectively alleviates oxidation reactions caused by factors such as cutting, thus reducing browning to some extent. It also provides a new organic nitrogen source, addressing changes in the ratio and content of ammonium and nitrate nitrogen in cardamom. The addition of organic nitrogen sources also has a positive effect on inhibiting browning. It is evident that this invention, through the synergistic effect of various factors such as improved basic culture medium, exogenous plant hormones, and exogenous α-ketoglutarate, enables cardamom tissue culture seedlings to maintain a suitable proliferation rate over a long period. Furthermore, browning only occurs after 21 consecutive generations of transfer in the proliferation medium. This allows for the maintenance of a high proliferation rate over a relatively long production cycle. Simultaneously, the browning rate of the seedlings is low and weak, while the seedling proliferation rate and rooting rate are high, thus avoiding the browning problem of cardamom tissue culture seedlings and enabling large-scale industrial production of cardamom. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0041] Figure 1 This is a diagram of the pre-culture of explants in Example 1;

[0042] Figure 2 This is a diagram of the initial induction culture in Example 1;

[0043] Figure 3 This is a diagram of the proliferation culture after 22 generations of transfer in Example 1;

[0044] Figure 4 This is a diagram of rooting culture after 22 generations of transfer in Example 1;

[0045] Figure 5 This is a diagram showing the proliferation culture after 8 generations of transfer (scale 1).

[0046] Figure 6 This is a diagram of rooting culture after 8 generations of grafting at scale 1.

[0047] Figure 7 This is a diagram showing the proliferation culture after 7 generations of transfer (scale 2).

[0048] Figure 8 This is a diagram showing the rooting culture after 7 generations of grafting at scale 2.

[0049] Figure 9 This is a diagram showing the proliferation culture after 8 generations of transfer at scale 3;

[0050] Figure 10 This is a diagram of rooting culture after 8 generations of grafting at a scale of 3.

[0051] Figure 11 This is a diagram showing the proliferation culture after 8 generations of transfer at scale 4;

[0052] Figure 12 This is a diagram showing the rooting culture after 4 generations of grafting. Detailed Implementation

[0053] Current techniques for tissue culture propagation of cardamom involve simple experiments, with only a few generations of proliferation followed by rooting. This approach demonstrates advantages such as high proliferation rate and high rooting rate of acclimatized seedlings. However, when tissue culture technology is applied to large-scale industrial propagation, it involves dozens of generations of proliferation and transfer. After a limited number of generations of proliferation and transfer, existing techniques result in browning, with the browning rate gradually increasing. Moreover, browned tissue culture seedlings have extremely low proliferation and rooting rates, and may even die. When browning affects proliferation and rooting, these tissue culture seedlings must be discarded, and explants must be collected again for initial disinfection and restarting the process, which severely impacts production efficiency. In tissue culture, a higher proliferation rate is not always better. Excessively high proliferation rates often lead to mutations, weakness, and deformities in tissue culture seedlings, which can negatively impact production. The method provided by this invention allows cardamom tissue culture seedlings to maintain a suitable proliferation rate over a long period. Browning only occurs after 21 consecutive generations of transfer in the proliferation medium. At this point, re-collecting explants for induction will not affect production. This method can maintain a high proliferation rate over a long production cycle, while also ensuring a high rooting rate for browned seedlings. This avoids the browning problem in cardamom tissue culture seedlings and enables large-scale industrial production of cardamom.

[0054] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0055] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0056] The modified MS medium was used in the following examples and comparative examples, and its specific composition is as follows:

[0057] The modified MS medium was based on MS basal medium, with the NH4NO3 content reduced to 1100 mg / L, calcium salt replaced by Ca(NO3)2·4H2O and increased to 1112 mg / L. Simultaneously, the contents of nicotinic acid, vitamin B6, and vitamin B1 were increased. The specific composition was: 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, vitamin B6 10 mg / L, vitamin B1 100 mg / L, glycine 2 mg / L, CuSO4·5H2O 0.025mg / L, H3BO36.2mg / L, MnSO4·H2O16.9mg / L, CoCl2·6H2O 0.025mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, KI0.83mg / L.

[0058] Example 1

[0059] Cardamom plants were collected from the Wuding County Germplasm Resource Nursery of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, in March, and were processed as follows:

[0060] (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;

[0061] (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.

[0062] (C) Explant pre-culture: After removing the portion of the explant that had been exposed to disinfectant from the wound, the treated explants from step (B) were inoculated into a pre-culture medium. The pre-culture medium used modified MS medium as the basal medium and also 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, were incubated in the dark at 4°C for 7 days.

[0064] (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction medium to induce the formation of shoot clusters. The primary induction medium uses modified MS medium as the basic medium and also includes:

[0065] 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, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0066] (E) Proliferation Culture: The shoot clusters obtained in step (D) are cut into single shoots and transferred to a proliferation medium. Repeated cutting and proliferation culture are performed to obtain shoot clusters. The proliferation medium uses modified MS medium as the basic medium and also includes:

[0067] 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, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 hours / day for 30 days.

[0068] (F) Rooting Culture: The shoot clusters obtained in step (E) are cut into single shoots and inoculated into rooting medium. The rooting medium is 1 / 2 modified MS basal medium, which also includes:

[0069] 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, pH 5.8; cultured at 20-22℃, first in the dark and then under light for 2 days; light intensity 1500-2000 lux, light duration 10 h / d, cultured for 30 days.

[0070] Example 2

[0071] Cardamom plants were collected from the Wuding County Germplasm Resource Nursery of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, in March, and were processed as follows:

[0072] (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;

[0073] (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.

[0074] (C) Explant pre-culture: After removing the portion of the explant that had been exposed to disinfectant from the wound, the treated explants from step (B) were inoculated into a pre-culture medium. The pre-culture medium used modified MS medium as the basal medium and also included:

[0075] 2.0 mg / L TDZ, 0.3 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar, pH 5.8, were incubated in the dark at 4°C for 7 days.

[0076] (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction medium to induce the formation of shoot clusters. The primary induction medium uses modified MS medium as the basic medium and also includes:

[0077] 1.0 mg / L TDZ, 0.2 mg / L NAA, 10.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0078] (E) Proliferation Culture: The shoot clusters obtained in step (D) are cut into single shoots and transferred to a proliferation medium. Repeated cutting and proliferation culture are performed to obtain shoot clusters. The proliferation medium uses modified MS medium as the basic medium and also includes:

[0079] 0.6 mg / L TDZ, 0.1 mg / L NAA, 10.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 hours / day for 30 days.

[0080] (F) Rooting Culture: The shoot clusters obtained in step (E) are cut into single shoots and inoculated into rooting medium. The rooting medium is 1 / 2 modified MS basal medium, which also includes:

[0081] 0.5 mg / L NAA, 5.0 mg / L α-ketoglutarate, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, first in the dark and then under light for 2 days; light intensity 1500-2000 lux, light duration 10 h / d, cultured for 30 days.

[0082] Example 3

[0083] Cardamom plants were collected from the Wuding County Germplasm Resource Nursery of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, in March, and were processed as follows:

[0084] (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;

[0085] (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.

[0086] (C) Explant pre-culture: After removing the portion of the explant that had been exposed to disinfectant from the wound, the treated explants from step (B) were inoculated into a pre-culture medium. The pre-culture medium used modified MS medium as the basal medium and also included:

[0087] 2.0 mg / L TDZ, 0.3 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar, pH 5.8, were incubated in the dark at 4°C for 7 days.

[0088] (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction medium to induce the formation of shoot clusters. The primary induction medium uses modified MS medium as the basic medium and also includes:

[0089] 1.0 mg / L TDZ, 0.2 mg / L NAA, 27.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0090] (E) Proliferation Culture: The shoot clusters obtained in step (D) are cut into single shoots and transferred to a proliferation medium. Repeated cutting and proliferation culture are performed to obtain shoot clusters. The proliferation medium uses modified MS medium as the basic medium and also includes:

[0091] 0.6 mg / L TDZ, 0.1 mg / L NAA, 27.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0092] (F) Rooting Culture: The shoot clusters obtained in step (E) are cut into single shoots and inoculated into rooting medium. The rooting medium is 1 / 2 modified MS basal medium, which also includes:

[0093] 0.5 mg / L NAA, 8.0 mg / L α-ketoglutarate, 1.0 g / L activated carbon, 20 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, first in the dark and then under light for 2 days; light intensity 1500-2000 lux, light duration 10 h / d, cultured for 30 days.

[0094] Example 4

[0095] Cardamom plants were collected from the Wuding County Germplasm Resource Nursery of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, in March, and were processed as follows:

[0096] (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;

[0097] (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.

[0098] (C) Explant pre-culture: After removing the portion of the explant that had been exposed to disinfectant from the wound, the treated explants from step (B) were inoculated into a pre-culture medium. The pre-culture medium used modified MS medium as the basal medium and also included:

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

[0100] (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction medium to induce the formation of shoot clusters. The primary induction medium uses modified MS medium as the basic medium and also includes:

[0101] 1.5 mg / L TDZ, 0.1 mg / L NAA, 17.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0102] (E) Proliferation Culture: The shoot clusters obtained in step (D) are cut into single shoots and transferred to a proliferation medium. Repeated cutting and proliferation culture are performed to obtain shoot clusters. The proliferation medium uses modified MS medium as the basic medium and also includes:

[0103] 0.5 mg / L TDZ, 0.2 mg / L NAA, 17.0 mg / L α-ketoglutarate, 30 g / L sucrose and 6.0 g / L agar, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 hours / day for 30 days.

[0104] (F) Rooting Culture: The shoot clusters obtained in step (E) are cut into single shoots and inoculated into rooting medium. The rooting medium is 1 / 2 modified MS basal medium, which also includes:

[0105] 0.1 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, pH 5.8; cultured at 20-22℃, first in the dark and then under light for 2 days; light intensity 1500-2000 lux, light duration 10 h / d, cultured for 30 days.

[0106] Example 5

[0107] The other steps are the same as in Example 1, except that before pretreatment, healthy cardamom plants are selected and refrigerated at 4°C for 40 days.

[0108] Example 6

[0109] The other steps are the same as in Example 1, the only difference being...

[0110] The conditions for pre-culture of explants were adjusted from: dark culture at 4℃ for 7 days to culture at 20~22℃ with light intensity of 1500~2000 lux and a light duration of 10h / d for 7 days.

[0111] Example 7

[0112] The other steps are the same as in Example 1, except that 1.0 mg / L VC is added to the pre-culture medium.

[0113] Example 8

[0114] The other steps are the same as in Example 1, except that 2.0 mg / L VC is added to the pre-culture medium.

[0115] Example 9

[0116] The other steps are the same as in Example 1, except that the initial induction culture, proliferation culture and rooting culture are no longer carried out in the dark.

[0117] Example 10

[0118] Cardamom plants were collected from the Wuding County Germplasm Resource Nursery of the Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, in March. Before pretreatment, healthy cardamom plants were selected and refrigerated at 4℃ for 40 days for later use.

[0119] The process is as follows:

[0120] (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;

[0121] (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.

[0122] (C) Explant pre-culture: After removing the portion of the explant that had been exposed to disinfectant from the wound, the treated explants from step (B) were inoculated into a pre-culture medium. The pre-culture medium used modified MS medium as the basal 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, were incubated in the dark at 4℃ for 7 days.

[0124] (D) Primary induction culture: The explants cultured in step (C) are transferred to the primary induction medium to induce the formation of shoot clusters. The primary induction medium uses modified MS medium as the basic medium and also includes:

[0125] 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, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 h / day for 30 days.

[0126] (E) Proliferation Culture: The shoot clusters obtained in step (D) are cut into single shoots and transferred to a proliferation medium. Repeated cutting and proliferation culture are performed to obtain shoot clusters. The proliferation medium uses modified MS medium as the basic medium and also includes:

[0127] 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, pH 5.8; cultured at 20-22℃, initially in the dark and then under light for 2 days; then under 1500-2000 lux light for 10 hours / day for 30 days.

[0128] (F) Rooting Culture: The shoot clusters obtained in step (E) are cut into single shoots and inoculated into rooting medium. The rooting medium is 1 / 2 modified MS basal medium, which also includes:

[0129] 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, pH 5.8; cultured at 20-22℃, first in the dark and then under light for 2 days; light intensity 1500-2000 lux, light duration 10 h / d, cultured for 30 days.

[0130] Comparative Example 1

[0131] The steps are the same as in Example 1, except that α-ketoglutarate is not added to the primary induction medium, proliferation medium and rooting medium.

[0132] Comparative Example 2

[0133] The steps are the same as in Example 1, except that the modified MS medium is replaced with MS basic medium in the pre-culture medium, primary induction medium, proliferation medium and rooting medium.

[0134] Comparative Example 3

[0135] The steps are the same as in Example 1, except that the modified MS medium is replaced with B5 basic medium in the pre-culture medium, primary induction medium, proliferation medium and rooting 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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