Tissue culture and rapid propagation method for fructus amomi seedlings

Abstract

The invention discloses a villous amomum fruit seedling tissue culture rapid propagation method, which is characterized in that villous amomum fruit seeds are used as a material, MS (Murashige and Skoog) is used as a basic culture medium, growth hormones with different proportions are added, adventitious buds are induced to proliferate and propagate, a rapid propagation technical system of villous amomum fruit is established, and after kinetin is added into the induction and proliferation culture medium, the germination speed is high, the germination rate is high, the buds are robust, and the proliferation coefficient is high; after the indoleacetic acid is added into the rooting culture medium, the rooting is fast, the rooting rate is high, the roots are many, the stems are thick, the leaf emergence is fast, the leaves are fleshy and good in growth vigor, and the transplanting survival rate is greatly improved. According to the method, the problems that the fructus amomi seeds are not easy to store and the propagation of the fructus amomi is influenced are solved, the problems that the division propagation rate of the fructus amomi is low and large-scale planting is limited are solved, large-scale propagation of the fructus amomi can be carried out in a short time through the method, and the production and planting requirements are met.

Description

Technical Field

[0001] This invention belongs to the technical field of plant cultivation methods, specifically relating to a method for rapid propagation of Amomum villosum seedlings via tissue culture. Background Technology

[0002] Amomum villosum is one of the four famous southern Chinese medicinal herbs, possessing high medicinal value. In recent years, the price of Amomum villosum has continued to rise, leading to a surge in farmers' enthusiasm for its cultivation and a significant increase in planting area, making it a key medicinal herb for understory economic development in many planting regions. Conventional propagation methods for Amomum villosum can be divided into sexual and asexual reproduction. Sexual reproduction uses seeds; asexual reproduction uses division. However, Amomum villosum seeds are difficult to store, and the division propagation rate is very limited, with each plant producing only a maximum of 15-20 new plants per year, which restricts large-scale cultivation. As an important medicinal herb with high demand, in vitro rapid propagation is a large-scale, rapid, and efficient method with advantages unmatched by conventional propagation.

[0003] Existing methods for tissue culture of Amomum villosum mainly use bamboo shoots as material, which results in a high contamination rate and requires improvement in propagation rate. The availability of bamboo shoots is limited, with peak reproductive capacity in spring. Furthermore, the size and quality of the bamboo shoots directly affect the tissue culture results, making it impossible to meet the demands of large-scale Amomum villosum cultivation in a short period. Therefore, to address this issue, a rapid propagation technology for Amomum villosum tissue culture is urgently needed to achieve rapid propagation and meet production needs. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapid propagation of Amomum villosum seedlings through tissue culture. This method uses Amomum villosum seeds as material and solves the problem that Amomum villosum seeds are difficult to store, which affects the propagation of Amomum villosum. It also solves the problem that the current propagation rate of Amomum villosum is low, which limits large-scale planting.

[0005] The technical solution adopted in this invention is: a rapid propagation method for Amomum villosum seedlings by tissue culture, using Amomum villosum seeds as material and MS as the basic culture medium, and inducing the proliferation and propagation of adventitious buds by adding different proportions of growth hormones, thereby establishing a rapid propagation technology system for Amomum villosum.

[0006] The technical solution adopted in this invention is also characterized by:

[0007] Furthermore, the method for rapid propagation of Amomum villosum seedlings through tissue culture is implemented according to the following steps: Step 1: Preparation of sterile seedlings: Select mature cardamom fruits, remove the outer shell, take plump kernels, disinfect the surface and inoculate them in MS medium to obtain sterile seedlings; Step 2, bud induction: Inoculate the rootstock portion (approximately 1 cm) of the sterile seedlings obtained in Step 1 into a bud induction medium supplemented with hormones for induction culture; Step 3, adventitious bud proliferation: Cut short stem segments (approximately 0.5 cm) with buds from the regenerated buds obtained in Step 2, vertically cut them from the center of the buds, and inoculate them into a proliferation medium supplemented with hormones for proliferation culture; Step 4, Adventitious bud rooting: Inoculate the clustered buds obtained in Step 3 into a rooting medium for rooting culture; Step 5, Culture Conditions: The formulations and culture conditions of the culture media for each stage in steps 1 to 4 are set according to the different culture stages.

[0008] Furthermore, the specific method for surface disinfection in step 1 is as follows: disinfect with 75% alcohol for 5.0 min, then disinfect with 0.1% HgCl2 for 8.0 min, and finally rinse with sterile water 4 to 5 times.

[0009] Furthermore, in step 2, the bud induction medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: A1: 6-BA 0.5 mg / L +KT 0.1 mg / L +IAA 0.5 mg / L; A2: 6-BA 1.0 mg / L + KT0.1 mg / L +IAA 0.5 mg / L; A3: 6-BA 2.0 mg / L + KT 0.1 mg / L +IAA 0.5 mg / L.

[0010] Furthermore, in step 3, the proliferation medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: B1: 6-BA 2.0 mg / L + KT0.2 mg / L +IAA 1.0 mg / L; B2: 6-BA 3.0 mg / L + KT 0.2mg / L + IAA 1.0 mg / L; B3: 6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L; B4: 6-BA 5.0 mg / L + KT 0.2 mg / L +IAA 2.0 mg / L.

[0011] Furthermore, in step 4, the rooting medium is 1 / 2 MS medium supplemented with IAA and / or NAA, and its formulation is any one of the following: C1: IAA 0.2 mg / L; C2: IAA 0.5 mg / L + NAA 0.5 mg / L; C3: IAA 0.5 mg / L + NAA 0.2 mg / L; C4: IAA 0.2 mg / L + NAA 0.5 mg / L; C5: IAA 0.5 mg / L; C6: IAA 0.2 mg / L + NAA 0.2 mg / L.

[0012] Furthermore, in step 1, the MS medium was supplemented with 30 g / L sucrose and 8.5 g / L carrageenan, with a pH of 5.7–5.8; the culture conditions were as follows: initial dark culture until the seeds showed white color, then switched to light culture, with a light duration of 16 h / d, a light intensity of 40 μmol / (m²·s), and a temperature of 26℃–28℃.

[0013] Furthermore, both the bud induction medium and the proliferation medium need to be cultured in the dark for 7 days before being switched to light culture. The light duration is 16 h / d, the light intensity is 1000–1200 lx, and the temperature is 26℃–28℃. Furthermore, the rooting medium was supplemented with 15 g / L sucrose and 9.0 g / L carrageenan, with a pH of 5.7–5.8; the culture conditions were: light intensity 3000 lx, light duration 16 h / d, and temperature 26℃–28℃.

[0014] The beneficial effects of this invention are: This invention provides a rapid propagation method for Amomum villosum using tissue culture with Amomum villosum seeds as the material. The tissue culture material is easy to obtain, the tissue culture technology is easy to operate, and the propagation efficiency is high. It can meet the planting needs of Amomum villosum at any time, solve the problem that Amomum villosum seeds are not easy to store and affect the propagation of Amomum villosum, and solve the problem that the low propagation rate of Amomum villosum by division limits the large-scale planting of Amomum villosum.

[0015] The rapid propagation method of Amomum villosum by tissue culture of the present invention uses Amomum villosum seeds as material, MS as the basic culture medium with different proportions of growth hormones to induce the proliferation and expansion of adventitious buds, establishes a rapid propagation technology system for Amomum villosum, opens up a new way for the expansion of its superior seedlings, and provides technical support for industrialized seedling production. Attached Figure Description

[0016] Figure 1 These are photos of bud induction 15 days after the invention, in Example 2 of this invention; Figure 2 This is a photograph of the buds after 20 days of proliferation in Example 4 of this invention; Figure 3 This is a photo of the tissue culture seedlings 40 days after rooting in Example 3 of this invention. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] This invention provides a method for rapid propagation of Amomum villosum seedlings by tissue culture. Using Amomum villosum seeds as material and MS as the basic culture medium, adventitious buds are induced to proliferate and expand by adding different proportions of growth hormones, thus establishing a rapid propagation technology system for Amomum villosum.

[0019] The specific steps are as follows: Step 1: Preparation of sterile seedlings: Select mature cardamom fruits, remove the outer shell, take plump kernels, disinfect the surface and inoculate them in MS medium to obtain sterile seedlings; The specific method for surface disinfection in step 1 is as follows: disinfect with 75% alcohol for 5.0 min, then disinfect with 0.1% HgCl2 for 8.0 min, and finally rinse with sterile water 4 to 5 times; In step 1, MS medium was supplemented with 30 g / L sucrose and 8.5 g / L carrageenan, with a pH of 5.7 to -5.8. The culture conditions were as follows: initial dark culture until the seeds showed white color, then switched to light culture, with a light duration of 16 h / d, a light intensity of 40 μmol / (m²·s), and a temperature of 26℃ to 28℃. Step 2: Bud induction: The rhizome part of the sterile seedlings obtained in Step 1 (cut about 1cm from the root) is inoculated into a bud induction medium supplemented with hormones for induction culture. In step 2, the bud induction medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: A1: 6-BA 0.5 mg / L +KT 0.1 mg / L+ IAA 0.5 mg / L; A2: 6-BA 1.0 mg / L + KT 0.1 mg / L + IAA 0.5 mg / L; A3: 6-BA 2.0 mg / L + KT 0.1 mg / L + IAA 0.5 mg / L; Step 3: Adventitious bud proliferation. Cut the regenerated buds obtained in Step 2 from the base of the bud (about 0.5cm), cut vertically from the center of the bud, and inoculate them into a proliferation medium supplemented with hormones for proliferation culture. In step 3, the proliferation medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: B1: 6-BA 2.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L; B2: 6-BA 3.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L; B3: 6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L; B4: 6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 2.0 mg / L; Both the bud induction medium and the proliferation medium need to be cultured in the dark for 7 days before being switched to light culture. The light duration is 16 h / d, the light intensity is 1000-1200 lx, and the temperature is 26℃~28℃. Step 4, Adventitious bud rooting: The clustered bud seedlings obtained in Step 3 are inoculated into rooting medium for rooting culture; In step 4, the rooting medium is 1 / 2 MS medium supplemented with IAA and / or NAA, and its formulation is any one of the following: C1: IAA 0.2 mg / L; C2: IAA 0.5 mg / L + NAA 0.5 mg / L; C3: IAA 0.5 mg / L + NAA 0.2 mg / L; C4: IAA 0.2 mg / L + NAA 0.5 mg / L; C5: IAA 0.5 mg / L; C6: IAA 0.2 mg / L + NAA 0.2 mg / L; The rooting medium was supplemented with 15 g / L sucrose and 9.0 g / L carrageenan, with a pH of 5.7-5.8; the culture conditions were: light intensity 3000 lx, light duration 16 h / d, and temperature 26℃~28℃. Step 5, Culture Conditions: The formulations and culture conditions of the culture media for each stage in steps 1 to 4 are set according to the different culture stages.

[0020] The present invention also provides cardamom tissue culture seedlings prepared according to the method.

[0021] The technical solution of the present invention will be further described below through embodiments and examples.

[0022] Example 1 1. Preparation of sterile seedlings: Select mature cardamom fruits, peel off their outer shells, leaving plump kernels, rinse with sterile water 3-4 times, place them on a clean bench to air dry naturally, then disinfect with 75% alcohol for 5.0 min, then disinfect with 0.1% HgCl2 for 8.0 min, rinse with sterile water 4-5 times, place them on sterile filter paper to absorb the surface moisture, and then inoculate them into MS medium and incubate in the dark to obtain sterile seedlings for induction.

[0023] 2. Bud Induction Method: Aseptic seedlings obtained from disinfected Amomum villosum seeds were grown for one month (approximately 4cm). Their stems (approximately 1cm long, cut from the root) were used as induction material and inoculated into bud induction media supplemented with different hormones. Ten bottles of each medium were inoculated, with four explants per bottle. Induction rate and growth status were recorded after 15 days. To ensure sufficient aseptic seedlings for subsequent experiments, the bud proliferation experiment was subcultured every 15-20 days, for a total of 2-3 subcultures.

[0024] 4. Adventitious bud propagation method: The regenerated buds obtained from the rhizomes of sterile seedlings are cut into short stem segments (about 0.5cm) with buds. The segments are vertically cut from the center of the bud and inoculated into different proliferation media containing different hormones. Ten bottles of each medium are inoculated, and five regenerated buds are inoculated into each bottle. The proliferation rate and growth status are recorded after 20 days.

[0025] 5. Adventitious bud rooting method: When the proliferating buds of Amomum villosum are 1-2cm long, cut off the robust and uniformly growing clustered buds and inoculate them into different rooting media. Inoculate 10 bottles for each treatment, with 4 plants in each bottle. After 40 days, count the rooting status and plant growth.

[0026] 6. Culture medium formulation and conditions: MS medium, with MS as the basal medium, supplemented with 30 g / L sucrose and 8.5 g / L carrageenan. After sterilization, the Amomum villosum seeds were initially cultured in the dark. Once the seeds showed signs of sprouting, they were subjected to light treatment using fluorescent lamps for 16 h / d at an illuminance of 40 μmol / (m²). 2 •s), culture temperature 26℃~28℃.

[0027] The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A1: 6-BA 0.5mg / L+KT 0.1 mg / L+IAA 0.5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0028] The bud proliferation medium, supplemented with hormones 6-BA, KT, and IAA on MS medium, has the following formulation: B1: 6-BA 2.0 mg / L+ KT 0.2 mg / L+ IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0029] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C1: IAA 0.2 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0030] The pH value of all the above culture media is 5.7 to 5.8.

[0031] 7. Harden off the rooted seedlings of Amomum villosum and transplant them.

[0032] Example 2 The difference between this embodiment and Embodiment 1 is that: The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A2: 6-BA 1.0 mg / L+KT 0.1 mg / L+IAA 0. 5mg / L; The bud induction medium required dark incubation for 7 days, followed by transfer to light incubation. The light duration was 16 hours / day, the light intensity was 1000-1200 lx, and the incubation temperature was 26℃-28℃. Results are as follows... Figure 1 As shown, after 15 days of bud induction, the bud base swelled, the bud turned green, and the bud height was 0.5-1cm. The buds were growing very well and could be used for propagation culture.

[0033] The bud proliferation medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: B2: 6-BA 3.0 mg / L+KT 0.2 mg / L+IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0034] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C2: IAA 0.5 mg / L+NAA0.5 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0035] The pH value of all the above culture media is 5.7 to 5.8.

[0036] The remaining steps and parameters are the same as in Example 1.

[0037] Example 3 The difference between this embodiment and Embodiment 1 is that: The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A3: 6-BA2.0mg / L+KT 0.1 mg / L+IAA 0.5 mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0038] The bud proliferation medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: B2: 6-BA 3.0 mg / L+KT 0.2 mg / L+IAA 1.0 mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0039] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C3: IAA 0.5 mg / L+ NAA0.2 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0040] The results are as follows Figure 3 As shown, after 40 days of rooting culture, the plant grows very well, with many thick roots, a sturdy stem, green and thick leaves, and a plant height of 4-5cm, and can be transplanted.

[0041] The pH value of all the above culture media is 5.7 to 5.8.

[0042] The remaining steps and parameters are the same as in Example 1.

[0043] Example 4 The difference between this embodiment and Embodiment 1 is that: The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A2: 6-BA 1.0mg / L+KT 0.1 mg / L+IAA 0.5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0044] The bud proliferation medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: B3: 6-BA5.0mg / L+KT 0.2 mg / L+IAA 1.0 mg / L; The bud proliferation medium required dark incubation for 7 days, followed by transfer to light incubation. The light duration was 16 hours / day, the light intensity was 1000-1200 lx, and the incubation temperature was 26℃-28℃. Results were as follows... Figure 2 As shown, after 20 days of bud proliferation culture, the multiplication rate reached over 5.0, with numerous robust bud clusters exhibiting excellent growth, making them suitable for rooting culture. The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C4:IAA 0.2 mg / L+NAA0.5 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0045] The pH value of all the above culture media is 5.7 to 5.8.

[0046] The remaining steps and parameters are the same as in Example 1.

[0047] Example 5 The difference between this embodiment and Embodiment 1 is that: The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A3: 6-BA 2.0 mg / L+KT 0.1 mg / L +IAA 0.5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0048] The bud proliferation medium, supplemented with hormones 6-BA, KT, and IAA on MS medium, has the following formulation: B3: 6-BA5.0 mg / L+KT 0.2 mg / L+IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0049] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C5: IAA 0.5 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0050] The pH value of all the above culture media is 5.7 to 5.8.

[0051] The remaining steps and parameters are the same as in Example 1.

[0052] Example 6 The difference between this embodiment and Embodiment 1 is that: The bud induction medium, prepared by adding hormones 6-BA, KT, and IAA to MS medium, has the following formulation: A3: 6-BA2.0mg / L+KT 0.1 mg / L+IAA 0.5 mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0053] The bud proliferation medium, supplemented with hormones 6-BA, KT, and IAA on MS medium, has the following formulation: B4: 6-BA5.0mg / L+KT 0.2 mg / L+IAA 2.0 mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0054] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: C6: IAA 0.2 mg / L+ NAA0.2 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0055] The pH value of all the above culture media is 5.7 to 5.8.

[0056] The remaining steps and parameters are the same as in Example 1.

[0057] Comparative Example 1 1. Treatment and disinfection method for preparing aseptic seedlings of Amomum villosum seeds: Select mature Amomum villosum fruits, peel off their outer shells, leaving plump kernels, rinse with sterile water 3-4 times, place them on a clean bench to air dry naturally, then disinfect with 75% alcohol for 5.0 min, then disinfect with 0.1% HgCl2 for 8.0 min, rinse with sterile water 4-5 times, place them on sterile filter paper to absorb the surface moisture, and then inoculate them in MS medium and incubate in the dark to obtain aseptic seedlings for induction.

[0058] 2. Bud Induction Method: Aseptic seedlings obtained from disinfected Amomum villosum seeds were grown for one month (approximately 4cm). Their stems (approximately 1cm long, cut from the root) were used as induction material and inoculated into bud induction media supplemented with different hormones. Ten bottles of each medium were inoculated, with four explants in each bottle. Induction rate and growth status were recorded after 15 days. To ensure sufficient aseptic seedlings for subsequent bud proliferation experiments, subculture was performed every 15-20 days, for a total of 2-3 subcultures.

[0059] 3. Adventitious bud propagation method: The regenerated buds obtained from the rhizomes of aseptic seedlings of Amomum villosum were cut into stem segments (about 0.5cm) with buds. The segments were vertically cut from the center of the buds and inoculated into different proliferation media containing different hormones. Ten bottles of each medium were inoculated, and five regenerated buds were inoculated into each bottle. The proliferation rate and growth status were counted after 20 days.

[0060] 4. Adventitious bud rooting method: When the proliferating buds of Amomum villosum are 1-2cm long, cut off the robust and uniformly growing clustered buds and inoculate them into different rooting media. Inoculate 10 bottles for each treatment, with 4 plants in each bottle. After 40 days, count the rooting status and plant growth.

[0061] 5. Culture medium formulation and conditions: MS medium, with MS basal medium supplemented with 30 g / L sucrose and 8.5 g / L carrageenan. After sterilization, the Amomum villosum seeds were initially cultured in the dark. Once the seeds showed signs of sprouting, they were subjected to light treatment using fluorescent lamps for 16 h / d at an illuminance of 40 μmol / (m²). 2 •s), culture temperature 26℃~28℃.

[0062] The bud induction medium, prepared by adding hormones 6-BA and IAA to MS medium, has the following formulation: a1: 6-BA 0.5mg / L +IAA 0.5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0063] The bud proliferation medium, supplemented with hormones 6-BA and IAA on MS medium, has the following formulation: b1: 6-BA 2.0 mg / L + IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0064] The rooting medium, based on 1 / 2 MS, contained 15 g / L sucrose and 9.0 g / L carrageenan, with the following formulation: c1: NAA 0.2 mg / L. The culture temperature was 26℃–28℃, the light intensity was 3000 lx, and the light intensity was 16 h / day. The pH of all the media was 5.7–5.8.

[0065] 6. Harden off the rooted seedlings of Amomum villosum and transplant them.

[0066] Comparative Example 2 The difference between this comparative example and Comparative Example 1 is: The bud induction medium, prepared by adding hormones 6-BA and IAA to MS medium, has the following formulation: a2: 6-BA 1.0 mg / L +IAA 0. 5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0067] The bud proliferation medium, supplemented with hormones 6-BA and IAA on MS medium, has the following formulation: b2: 6-BA 3.0 mg / L+IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0068] The rooting medium, with 1 / 2 MS as the basic medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: c2: NAA 0.5 mg / L; The culture temperature was 26℃~28℃, the light intensity was 3000 lx, and the continuous light exposure was 16h per day. The pH value of the culture medium was 5.7~5.8. All other steps and parameters were the same as those in Comparative Example 1.

[0069] Comparative Example 3 The difference between this comparative example and Comparative Example 1 is: The bud induction medium, prepared by adding hormones 6-BA and IAA to MS medium, has the following formulation: a3: 6-BA 2.0 mg / L +IAA 0. 5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0070] The bud proliferation medium, supplemented with hormones 6-BA and IAA on MS medium, has the following formulation: b3: 6-BA 5.0 mg / L +IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0071] The rooting medium, with 1 / 2 MS as the basic medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: c1: NAA 0.2 mg / L; The culture temperature was 26℃~28℃, the light intensity was 3000 lx, and the continuous light exposure was 16h per day. The pH value of the culture medium was 5.7~5.8.

[0072] The remaining steps and parameters are the same as those in Comparative Example 1.

[0073] Comparative Example 4 The difference between this comparative example and Comparative Example 1 is: The bud induction medium, prepared by adding hormones 6-BA and IAA to MS medium, has the following formulation: a3: 6-BA 2.0 mg / L +IAA 0. 5mg / L; The bud induction medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0074] The bud proliferation medium, supplemented with hormones 6-BA and IAA on MS medium, has the following formulation: b3: 6-BA 5.0 mg / L +IAA 1.0mg / L; The bud proliferation medium needs to be cultured in the dark for 7 days, and then transferred to light culture. The light duration is 16 hours / day, the light intensity is 1000~1200 lx, and the culture temperature is 26℃~28℃.

[0075] The rooting medium, with 1 / 2 MS as the basal medium, contains 15 g / L sucrose and 9.0 g / L carrageenan, and the formula is as follows: c2: NAA 0.5 mg / L; The cultivation temperature is 26℃~28℃, the light intensity is 3000 lx, and the continuous light exposure is 16h per day.

[0076] The pH value of all the above culture media is 5.7 to 5.8.

[0077] The remaining steps and parameters are the same as those in Comparative Example 1.

[0078] Table 1. Results of tissue culture in Examples 1-6 Germination time Germination rate Bud condition Proliferation rate Proliferation status Number of roots per plant Rooting status Example 1 20 days 100% Sprouting is fast, the buds are green, and the growth is good. 4.6 times Sprouting is relatively fast, the cluster of buds is relatively long, and the buds are relatively robust. 3.0 The plant grows at a moderate pace, roots relatively quickly, has thin and long roots, and a moderately sized stem. Example 2 15 days 100% Sprouting quickly, green buds, and vigorous growth 4.8 times Sprouting is relatively fast, the cluster of buds is relatively long, and the buds are relatively robust. 4.0 The plant grows at a moderate pace, roots quickly, has many roots, and a relatively thick stem. Example 3 19 days 100% It sprouts quickly, the buds are dark green, and it grows very well. 5.1 times Sprouting is rapid, with long clusters of buds and robust shoots. 4.2 The plant grows quickly, roots rapidly, has many thick roots, and a sturdy stem. Example 4 16 days 100% Fast germination, green buds, and vigorous growth 5.6 times Sprouting is rapid, with long and robust buds. 3.6 The plant grows relatively quickly and roots quickly; the stem is of moderate length. Example 5 18 days 100% It sprouts quickly, the buds are dark green, and it grows very well. 5.2 times Sprouting is rapid, with long and robust buds. 3.2 The plant grows at a moderate pace, roots relatively quickly, and has a moderately sized stem. Example 6 20 days 100% It sprouts quickly, the buds are dark green, and it grows very well. 5.0 times Sprouting is rapid, with long clusters of buds and robust shoots. 3.9 The plant grows quickly, roots relatively quickly, and has a moderate stem length. Table 2 Results of tissue culture in comparative examples 1-4

[0079] The cultivation results of Examples 1-6 and Comparative Examples 1-4 are shown in Tables 1 and 2, respectively. The results show that the budding time, budding rate, bud condition, proliferation rate, proliferation condition, number of roots per plant, and rooting condition of the Amomum villosum plants obtained by tissue culture rapid propagation of the present invention are significantly better than those of the existing methods.

[0080] The following is a data analysis of the culture medium formulations in Examples 1-6.

[0081] 1. Data Analysis The budding time is the number of days after the induced explants have reached 50% budding rate.

[0082] Germination rate (%) = number of germinated explants / number of inoculated explants × 100.

[0083] Bud length is the average bud length of the explant.

[0084] The number of buds is the average number of buds produced per explant.

[0085] Proliferation rate (multiple times) = total number of induced shoots / total number of inoculated explants.

[0086] The number of roots is the average number of roots per explant.

[0087] The data was processed using the DPS v9.50 data processing system to perform statistical analysis on the samples.

[0088] 2. Results and Analysis 2.1 Effects of different hormone combinations on adventitious bud induction in Amomum villosum When the sterile seedlings obtained after the disinfection treatment of Amomum villosum seeds grow to about 4 cm, cut off the base of the stem about 0.5-1.0 cm and inoculate it onto the bud induction medium with different hormone combinations for cultivation. Investigate the budding status every 3 days, and start to count the budding rate, bud length and growth status after 15 days.

[0089] Germination time is defined as the number of days after 50% of the explants have germinated. Germination rate is the proportion of germination from the inoculated explants. Bud length is the average bud length of the explants. Number of buds is the average number of buds from the explants. Table 3 shows that all three hormone combinations for adventitious bud induction induced germination, with a germination rate of 100%. A2 medium showed the best results, with faster germination, a larger number of buds, faster bud growth, and better vigor. A1 medium resulted in fewer buds, slower germination, weaker bud growth, and slower bud development. A3 medium resulted in faster germination, but fewer buds and slower growth, although the buds were robust. Analysis suggests that low concentrations of 6-BA hormone affected bud induction efficiency and bud growth, while high concentrations inhibited bud elongation. Therefore, A2 medium (6-BA 1.0 mg / L + KT 0.1 mg / L + IAA 0.5 mg / L) is more suitable for inducing adventitious buds from Amomum villosum seeds.

[0090] Table 3 Effects of different hormone combinations on adventitious bud induction in Amomum villosum seeds deal with Germination time / d Bud length / cm Number of buds (each) Germination rate / % Growth status A1 7 0.5 4.1 100 Slow budding, small buds, and slow growth A2 5 1.2 6.5 100 It sprouts quickly, the buds are thick and strong, and it grows rapidly. A3 5 0.8 5.6 100 It sprouts quickly and produces thick buds, but grows relatively slowly. 2.2 Effects of different hormone ratios on the proliferation of Amomum villosum The cardamom buds induced by the initial culture were transferred to a proliferation medium for further culture. After 30 days, the proliferation rate and bud growth were statistically analyzed. Roots began to form after 20 days of proliferation culture. Table 4 shows that the B1 medium had the lowest proliferation rate, while the B4 medium had the highest proliferation coefficient. Buds in the B1 medium were short, weak, with yellowish-green bud fibers and few roots. The B2 medium had a higher proliferation rate than the B1 medium, but the buds were still weak and had few roots. Buds in the B3 and B4 media were longer, stronger, greener, and had more roots, with higher proliferation coefficients. Analysis shows that the proliferation rate of cardamom buds gradually increased with increasing 6-BA concentration, and the buds grew more vigorously. Further increasing the IAA concentration could further improve the proliferation rate. Therefore, the optimal medium for cardamom bud proliferation is B3 medium (6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L), which has a high proliferation coefficient and good bud growth.

[0091] Table 4. Effects of different hormone combinations on the proliferation of Amomum villosum. deal with Proliferation rate / times Growth status B1 1.8 The buds are short, weak, thin, yellowish-green, and have few roots. B2 2.4 The buds are short, thin, and yellowish-green, with few roots. B3 4.3 The buds are robust and greenish, with many roots. B4 3.7 The buds are relatively long, robust, and lopsided, with many roots. 2.3 Effects of different hormone ratios on the rooting of Amomum villosum White root primordia begin to appear approximately 10 days after the rooting culture of Amomum villosum. Different hormone combinations have different effects on rooting. As shown in Table 5, C1 medium results in faster rooting, a larger number of roots, and faster plant growth, but the roots are thinner and the stem does not swell. C2 medium results in faster rooting, a large number of thick roots, a robust stem, and the stem base swells to twice its original size, but plant growth is not fast. C3 medium results in moderate plant growth, faster rooting, a smaller number of roots, a relatively robust stem, and the stem base swells to 1.5 times its original size. C4 medium results in slower plant growth, slower rooting, a smaller number of roots, a thinner stem, and the stem base swells to 0.5 times its original size. C5 medium results in slow plant growth, slow leaf emergence, slow rooting, a moderate stem size, and the stem base swells to 1.0 times its original size. C6 medium results in slow plant growth, slow rooting, a large number of roots, a thin stem, and the stem base swells to 0.5 times its original size. Comprehensive analysis shows that culture medium C3 (IAA 0.5 mg / L + NAA 0.2 mg / L) is most suitable for Amomum villosum rooting, resulting in more and stronger roots and greater swelling at the base of the stem, which is conducive to proliferation and propagation.

[0092] In the rooting culture of Amomum villosum, the hormone IAA promotes root growth, while the hormone NAA promotes thicker roots. The combined use of these two hormones yields the best rooting effect. Experimental results show that in C1 medium (IAA concentration 0.2 mg / L), plant growth is good and rooting is rapid, but the roots are relatively thin. In C5 medium (IAA concentration 0.5 mg / L), plant growth, leaf emergence, and rooting are slow. Therefore, when using IAA alone, excessive concentrations can inhibit rooting.

[0093] Table 5. Effects of different hormone combinations on the rooting of Amomum villosum. deal with Number of roots / plant Number of roots / plant Number of blades unfolded (in units) Longest root length cm Maximum plant height (cm) Growth status The base of the stem swells to a great extent C1 2.8 2.5 11 2.23 2.5 The plant grows quickly, roots rapidly, has thin and long roots, and a relatively thin stem. No increase C2 3.0 1.0 3 1.3 1.8 The plant grows normally, roots relatively quickly, and has a relatively thick stem. Increase by approximately 1.5 times C3 2.9 2.0 10 3.1 2.5 The plant grows relatively quickly, roots rapidly, has numerous and thick roots, and a sturdy stem. Increase by approximately 2 times C4 2.7 0.8 4 2.5 2 The plant grows slowly, produces leaves slowly, roots slowly, and has thin stems. Increase by approximately 0.5 times C5 1.8 3.0 6 0.77 1.6 The plant grows slowly, produces leaves slowly, roots slowly, and has a moderately sized stem. Increase by approximately 100%. C6 3.0 1.8 3 2.23 2.1 The plant grows slowly, roots slowly, has many roots, and has thin stems. Increase by approximately 0.5 times 3. Conclusion and Discussion Plant growth regulators are crucial factors influencing rapid tissue culture propagation. Different types and concentrations of plant growth regulators have varying effects on explant growth and differentiation. Therefore, the appropriate ratio of different plant growth regulators is key to establishing a rapid explant propagation system. In studies on the induction and proliferation of adventitious shoots, the added growth regulators mainly include KT, 6-BA, and IAA. The physiological functions of cytokinins are primarily to induce cell division and differentiation, as well as to induce shoot formation and promote shoot growth. KT activates cell division and induces shoot growth in tissues; 6-BA is a cost-effective cytokinin with strong thermal stability; and IAA promotes shoot growth and root formation, facilitating the full absorption of nutrients in the culture medium.

[0094] Studies have found that plant growth regulators can promote growth at low concentrations, inhibit growth at high concentrations, and even damage plants at extremely high concentrations. The results of this invention are consistent with these findings. High concentrations of cytokinin 6-BA reduce the efficiency of induced proliferation, high concentrations of auxin IAA inhibit plant growth, and appropriate concentrations of cytokinin KT promote bud-induced proliferation. The results of this experiment show that the A2 medium (6-BA 2.0 mg / L + KT 0.1 mg / L + IAA 0.5 mg / L) is suitable for inducing adventitious buds from Amomum villosum seeds; the optimal medium for Amomum villosum bud proliferation is the B3 medium (6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L), which has a high proliferation coefficient and good bud growth; the C3 medium (IAA 0.5 mg / L + NAA 0.2 mg / L) is most suitable for Amomum villosum rooting, resulting in numerous and robust roots, significant swelling at the stem base, and rapid seedling growth.

[0095] The present invention has been illustrated with the above embodiments to explain the detailed process of the invention. However, the present invention is not limited to the above content, that is, it does not mean that the present invention must rely on the above detailed process to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for rapid propagation of Amomum villosum seedlings via tissue culture, characterized in that, Using Amomum villosum seeds as material and MS as the basal culture medium, a rapid propagation technology system for Amomum villosum was established by inducing the proliferation and expansion of adventitious buds by adding different proportions of growth hormones.

2. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 1, characterized in that, The specific steps are as follows: Step 1: Preparation of sterile seedlings: Select mature cardamom fruits, remove the outer shell, take plump kernels, disinfect the surface and inoculate them in MS medium to obtain sterile seedlings; Step 2, bud induction: The stem base of the sterile seedlings obtained in Step 1 is inoculated into a bud induction medium supplemented with hormones for induction culture; Step 3, adventitious bud proliferation: Cut short stem segments with buds from the regenerated buds obtained in Step 2 and inoculate them into a proliferation medium supplemented with hormones for proliferation culture; Step 4, Adventitious bud rooting: Inoculate the clustered buds obtained in Step 3 into a rooting medium for rooting culture; Step 5, Culture Conditions: The formulations and culture conditions of the culture media for each stage in steps 1 to 4 are set according to the different culture stages.

3. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, The specific method for surface disinfection in step 1 is as follows: disinfect with 75% alcohol for 5.0 min, then disinfect with 0.1% HgCl2 for 8.0 min, and finally rinse with sterile water 4 to 5 times.

4. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, In steps 3 and 4, the bud induction medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: A1: 6-BA 0.5 mg / L +KT 0.1 mg / L +IAA 0.5 mg / L; A2: 6-BA 1.0 mg / L + KT0.1 mg / L +IAA 0.5 mg / L; A3: 6-BA 2.0 mg / L + KT 0.1 mg / L +IAA 0.5 mg / L.

5. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, In step 3, the proliferation medium is MS medium supplemented with 6-BA, KT, and IAA, and its formulation is any one of the following: B1: 6-BA 2.0 mg / L + KT0.2 mg / L +IAA 1.0 mg / L; B2: 6-BA 3.0 mg / L + KT 0.2mg / L + IAA 1.0 mg / L; B3: 6-BA 5.0 mg / L + KT 0.2 mg / L + IAA 1.0 mg / L; B4: 6-BA 5.0 mg / L + KT 0.2 mg / L +IAA 2.0 mg / L.

6. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, In step 4, the rooting medium is 1 / 2 MS medium supplemented with IAA and / or NAA, and its formulation is any one of the following: C1: IAA 0.2 mg / L; C2: IAA 0.5 mg / L + NAA 0.5 mg / L; C3: IAA 0.5 mg / L + NAA 0.2 mg / L; C4: IAA 0.2 mg / L + NAA 0.5 mg / L; C5: IAA 0.5 mg / L; C6: IAA 0.2 mg / L + NAA 0.2 mg / L.

7. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, In step 1, the MS medium is supplemented with 30 g / L sucrose and 8.5 g / L carrageenan, with a pH of 5.7-5.

8. The culture conditions are as follows: initial dark culture until the seeds show white color, then switch to light culture, with a light duration of 16 h / d, a light intensity of 40 μmol / (m²·s), and a temperature of 26-28℃.

8. The method for rapid propagation of Amomum villosum seedlings by tissue culture according to claim 2, characterized in that, Both the bud induction medium and the proliferation medium need to be cultured in the dark for 7 days before being switched to light culture. The light duration is 16 h / d, the light intensity is 1000–1200 lx, and the temperature is 26–28℃. The rooting medium was supplemented with 15 g / L sucrose and 9.0 g / L carrageenan, with a pH of 5.7–5.8; the culture conditions were: light intensity 3000 lx, light duration 16 h / d, and temperature 26–28℃.

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