A rapid breeding method of black ginger seedlings

Abstract

This invention discloses a rapid propagation method for black ginger seedlings. The method uses single buds from the underground rhizomes of black ginger as explants. The primary culture medium is: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8; the proliferation culture medium is: MS medium + 10.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8; the rooting culture medium is: MS medium + 0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8. After one cycle each in the primary culture medium and the proliferation medium, one explant can form a shoot cluster containing 20 single buds, which significantly improves the propagation coefficient compared to existing technologies. The tissue culture seedlings have a 100% rooting rate, a 95% survival rate after transplanting, and strong tillering ability, with a 95% survival rate after transplanting to the field. This invention shortens the seedling cultivation cycle, achieves high survival rates, saves costs by using culture bags, and facilitates transportation, making it directly applicable to large-scale commercial production.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a rapid propagation method for black ginger seedlings. Background Technology

[0002] Black ginger ( Kaempferia parviflora *Kaempferia ex Baker* is a perennial herb belonging to the genus *Kaempferia* in the ginger family (Zingiberaceae). It typically grows in sunny locations at altitudes of 500-700 meters with good ventilation. Black ginger is an important medicinal plant, used to treat wounds, fractures, and inflammation; when boiled, it is used internally to treat colds and sore throats. In recent years, the various pharmacological effects of black ginger have gradually attracted attention, such as anti-tumor, anti-inflammatory, antioxidant, vasodilatory and cardiovascular protective effects, anti-osteoarthritis effects, and enhancement of male sexual function. In addition, its beautiful foliage makes it suitable for ornamental purposes, and it can also be used as an indoor foliage plant and as ground cover in forests.

[0003] Black ginger has no above-ground stems, only rhizomes, making it unsuitable for propagation by cuttings and resulting in an extremely low seed production rate. It primarily relies on asexual reproduction, typically through rhizome division. However, due to its small bulb size, the division propagation coefficient is low. During cultivation, it is susceptible to severe diseases, with mortality rates reaching up to 50%, sometimes resulting in complete crop failure. Continuous cropping can easily lead to germplasm degradation, making it unsuitable for large-scale applications. Furthermore, wild resources are extremely scarce, and superior varieties cannot be mass-produced. Therefore, it is necessary to utilize biotechnology such as tissue culture to achieve rapid and efficient propagation to meet the demands of the medicinal and ornamental markets and achieve large-scale production. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of conventional seed-saving and seedling cultivation of black ginger, which suffers from low propagation rate and severe disease, by providing a method for black ginger propagation that can obtain a large number of seedlings with stable traits in a short period of time while maintaining the excellent characteristics of the mother plant.

[0005] The present invention provides a rapid propagation method for black ginger seedlings, comprising the following steps:

[0006] S1. Take the underground rhizomes of black ginger that are growing in the current year, clean the surface and dry them. Wipe them with a 75% ethanol aqueous solution and cut off the single buds to a diameter of 0.6 cm. Remove the epidermis and leaf sheath to complete the preparation of the explant.

[0007] S2. After disinfecting the explants, they were inoculated into the primary culture medium in a tissue culture flask to obtain adventitious shoot clusters; the primary culture medium was: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8;

[0008] S3. After cutting the bud clusters into 1 cm pieces, transfer them to the proliferation medium in the culture bag and culture to obtain bud clusters containing multiple single buds; the proliferation medium is: MS medium + 10.0 mg / L 6-BA + 0-2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8;

[0009] S4. When the single bud grows to 3-5 cm in height, cut off the single bud and inoculate it into the rooting medium in the culture bag to cultivate roots and form rooted seedlings; the rooting medium is: MS medium + 0.1-0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8;

[0010] S5. Transfer the seedlings that have grown to 6-8 cm in height, along with their culture bags, directly to natural diffused light conditions to harden them off, and transplant them at the appropriate time.

[0011] Preferably, the cleaning of the surface in step S1 involves cutting off the roots on the rhizome and cleaning the surface with a brush.

[0012] Preferably, the removal of the epidermis and leaf sheath in step S1 is achieved by scraping off the epidermis, leaf sheath, and surrounding brown tissue with a scalpel.

[0013] Preferably, the disinfection process in step S2 is as follows: soaking in a 75% ethanol aqueous solution for 1 min, rinsing with sterile water, soaking in a 0.1% HgCl2 solution for 8 min, and rinsing the soaking solution with sterile water.

[0014] Preferably, the cultivation conditions for steps S2 and S3 are: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, and light duration 12 h / d.

[0015] Preferably, the proliferation medium is: MS medium + 10.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8.

[0016] Preferably, the rooting medium is: MS medium + 0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8.

[0017] Preferably, step S5 is as follows: The seedlings, which have grown to 6-8 cm in height and have developed roots, are directly transferred to natural diffused light conditions for 30 days to harden off. Then, the seedlings are removed, the remaining culture medium on the roots is washed off with water, and they are transplanted into seedling trays. A 0.1% carbendazim solution is thoroughly watered, and a plastic film greenhouse is covered to maintain a temperature of 20℃-30℃ and a relative humidity of 70%-85%, avoiding direct sunlight. One week after transplanting, the film is removed, and a 0.1% carbendazim solution is sprayed alternately every 7 days. Regular watering and fertilization are also carried out. One month after transplanting into the seedling trays, the seedlings are transplanted to the field.

[0018] Preferably, the culture bag is made of plastic and has dimensions of 13 cm × 12 × 2 cm (height × length × width), with each bag containing 30-35 mL of culture medium.

[0019] The present invention has the following advantages over the prior art:

[0020] (1) Before using conventional tissue culture disinfection, the black ginger rhizomes were dried and wiped with cotton cloth dipped in 75% ethanol aqueous solution, which effectively inhibited the endophytic bacteria of the explants. The operation is simple.

[0021] (2) The present invention uses the buds of black ginger instead of stem segments for cultivation, which requires fewer mother plants and can quickly propagate seedlings. The propagation coefficient of the method of the present invention is 6 to 9. After one cycle of cultivation in the primary culture medium and the proliferation culture medium, one explant can form a bud cluster containing 20 single buds. The propagation coefficient of the method of the present invention is significantly improved compared with the prior art.

[0022] (3) This invention directly uses culture bags for rapid propagation of black ginger tissue culture, which saves costs and can be directly applied to large-scale commercial production, making transportation more convenient.

[0023] (4) The survival rate of the black ginger aseptic rooted seedlings obtained by the method of the present invention after transplanting is 95%, and the tillering ability is strong; while the mortality rate of black ginger planted by conventional seed saving and seedling raising methods reaches 50% in the second year, and there may even be no harvest. Attached Figure Description

[0024] Figure 1 This is the propagation culture bag seedling of black ginger in Example 1.

[0025] Figure 2 This is the rooting culture bag seedling of black ginger from Example 1.

[0026] Figure 3 This is a photo of black ginger seedlings after 10 days of hardening off in a greenhouse, as described in Example 1.

[0027] Figure 4 This is the growth of tissue culture seedlings three months after transplanting them into the field. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] In the following examples, the formulas for calculating the survival rate and contamination rate of explants are: Survival rate = (Number of surviving buds / Total number of inoculated buds) × 100%; Contamination rate = (Number of contaminated buds / Total number of inoculated buds) × 100%.

[0030] The culture bags used in the following examples are made of plastic and have dimensions of 13 cm × 12 × 2 cm (height × length × width). Each bag contains 30-35 mL of culture medium. After inoculating the culture material, the culture bags are heat-sealed.

[0031] Example 1

[0032] The black ginger propagation method of this embodiment includes the following steps:

[0033] (1) Explant preparation and disinfection: Take the underground rhizomes of vigorous black ginger from the current year, cut off the roots on the rhizomes, gently clean the surface with a brush, let it dry, wipe it clean with a cotton cloth dipped in 75% ethanol aqueous solution, cut off the single bud to a diameter of 0.6 cm, gently scrape off the epidermis, leaf sheath and surrounding brown tissue with a scalpel to complete the explant preparation, and then put it in sterile water for later use.

[0034] (2) Obtaining sterile shoots: Explants were soaked in 75% ethanol aqueous solution for 1 min, rinsed 5 times with sterile water, soaked in 0.1% HgCl2 solution for 8 min, and rinsed more than 5 times with sterile water to thoroughly remove the soaking solution. The explants were then inoculated into primary culture medium in tissue culture bottles. After 20 days of inoculation, the survival rate of explants was 75%, and the contamination rate was 25%. Shoot clusters containing 2-3 adventitious shoots were obtained after culturing in the primary culture medium for 2 months. The primary culture medium was: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, photoperiod 12 h / d.

[0035] (3) Proliferation of buds by division: The bud clusters were cut into pieces about 1 cm in size and then transferred to the proliferation medium in culture bags for proliferation culture. The proliferation medium was: MS medium + 10.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, light duration 12 h / d. After one month of culture, bud clusters containing 10-20 single buds were formed. Figure 1 The average multiplication rate was 9.15.

[0036] (4) Rooting culture: When the single bud in the bud cluster grows to 3-5 cm in height, the single bud is cut off and inoculated into the rooting medium in the culture bag for rooting culture. After one month of culture, a complete plant is formed. Figure 2 Each seedling developed 3-5 roots, 6-8 cm in length, achieving a 100% rooting rate, thus obtaining rooted seedlings. The rooting medium consisted of MS medium + 0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8. The rooted seedlings, reaching 6-8 cm in height, were then transferred directly to a hardening-off greenhouse and cultured under natural diffused light for 30 days.

[0037] (5) Transplanting and Management of Rooted Seedlings: After cultivation under natural diffused light, remove the rooted seedlings, wash off the remaining culture medium on the roots with water, transplant them into seedling trays, thoroughly water with a 0.1% carbendazim solution, cover with a plastic film arched shed, maintain temperature (20℃-30℃) and humidity (relative humidity 70%-85%), avoid direct sunlight, and uncover the film one week after transplanting. Figure 3 Spray with a 0.1% carbendazim solution every 7 days, and water and fertilize regularly.

[0038] (6) Cultivation and management in the field after transplanting: One month after transplanting into the seedling trays, the seedlings were transplanted to the field. Regular watering and fertilization were carried out. One month after transplanting to the field, the survival rate of the transplanted seedlings reached 95%. Figure 4 ).

[0039] Example 2

[0040] The black ginger propagation method of this embodiment includes the following steps:

[0041] (1) Explant preparation: Take the underground rhizomes of vigorous black ginger from the current year, cut off the roots on the rhizomes, gently clean the surface with a brush, let it dry, wipe it clean with a cotton cloth dipped in 75% ethanol aqueous solution, cut off the single bud to a diameter of 0.6 cm, gently scrape off the epidermis, leaf sheath and surrounding brown tissue with a scalpel to complete the explant preparation, and then put it in sterile water for later use.

[0042] (2) Obtaining sterile shoots: Explants were soaked in 75% ethanol aqueous solution for 1 min, rinsed 5 times with sterile water, soaked in 0.1% HgCl2 solution for 8 min, and rinsed more than 5 times with sterile water to thoroughly remove the soaking solution. The explants were then inoculated into primary culture medium in tissue culture bottles. After 20 days of inoculation, the survival rate of explants was 75%, and the contamination rate was 25%. Shoot clusters containing 2-3 adventitious shoots were obtained after culturing in the primary culture medium for 2 months. The primary culture medium was: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, photoperiod 12 h / d.

[0043] (3) Proliferation of buds by division: Bud clusters were cut into pieces approximately 1 cm in size and transferred to a proliferation medium in culture bags for further propagation. The proliferation medium consisted of MS medium + 10.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, and photoperiod 12 h / d. After one month of culture, bud clusters containing 10-20 single buds were formed, with an average proliferation ratio of 9.15.

[0044] (4) Rooting Culture: When the single buds in the bud cluster grow to 3-5 cm in height, the single buds are cut off and inoculated into the rooting medium in the culture bag for rooting culture. After one month of culture, complete plants are formed, and each seedling grows 3-5 roots with a root length of 6-8 cm. The rooting rate reaches 100%, and rooted seedlings are obtained. The rooting medium is: MS medium + 0.1 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8. The rooted seedlings that have grown to 6-8 cm in height are directly transferred to the hardening-off greenhouse along with the culture bag and cultured under natural diffused light for 30 days.

[0045] (5) Transplanting and management of rooted seedlings: After cultivation under natural diffused light, take out the rooted seedlings, wash off the culture medium remaining on the roots with water, transplant them into seedling trays, water them thoroughly with 0.1% carbendazim solution, cover them with plastic film arched sheds, maintain temperature (20℃-30℃) and humidity (relative humidity 70%-85%), avoid direct sunlight, uncover the film one week after transplanting, spray with 0.1% carbendazim solution every 7 days, and water and fertilize regularly.

[0046] (6) Cultivation and management after transplanting to the field: Transplant the seedlings to the field one month after transplanting them into the seedling trays, and water and fertilize them regularly.

[0047] Example 3

[0048] The black ginger propagation method of this embodiment includes the following steps:

[0049] (1) Explant preparation: Take the underground rhizomes of vigorous black ginger from the current year, cut off the roots on the rhizomes, gently clean the surface with a brush, cut off the single buds to a diameter of 0.6 cm, gently scrape off the epidermis, leaf sheath and surrounding brown tissue with a scalpel, rinse with running tap water for 30 min to complete the explant preparation, and then put them in sterile water for later use.

[0050] (2) Obtaining sterile shoots: Explants were soaked in 75% ethanol aqueous solution for 1 min, rinsed 5 times with sterile water, soaked in 0.1% HgCl2 solution for 10 min, and rinsed more than 5 times with sterile water to thoroughly remove the soaking solution. The explants were then inoculated into the primary culture medium in tissue culture bottles. After 20 days of inoculation, the survival rate of the explants was 25%, and the contamination rate was 75%. Shoot clusters containing 2-3 adventitious shoots were obtained after culturing in the primary culture medium for 2 months. The primary culture medium was: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, photoperiod 12 h / d.

[0051] (3) Proliferation of buds by division: Bud clusters were cut into pieces approximately 1 cm in size and transferred to proliferation medium in tissue culture bottles for proliferation culture. The proliferation medium consisted of MS medium + 10.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8. Culture conditions: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, and photoperiod 12 h / d. After one month of culture, bud clusters containing 9-14 single buds were formed, with an average proliferation multiple of 6.

[0052] (4) Rooting culture: When the single buds in the bud cluster grow to 3-5 cm in height, the single buds are cut off and inoculated into the rooting medium in the tissue culture bottle for rooting culture. After one month of culture, complete plants are formed, and each seedling grows 3-5 roots with a root length of 5-8 cm. The rooting rate reaches 100%, and rooted seedlings are obtained. The rooting medium is: MS medium + 0.1 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8. The rooted seedlings that have grown to 6-8 cm in height are directly transferred to the hardening-off greenhouse along with the tissue culture bottle and cultured under natural diffused light for 30 days.

[0053] (5) Transplanting and management of rooted seedlings: transplant the tissue culture seedlings to room temperature and harden them off with the cover partially open for 2 days and then with the cover fully open for 3 days. After hardening, take out the tissue culture seedlings, wash the culture medium from the roots, transplant them into seedling trays, water them thoroughly with 0.1% carbendazim solution, cover them with plastic film arched sheds, maintain the temperature (20℃-30℃) and humidity (relative humidity 70%-85%), avoid direct sunlight, uncover the film one week after transplanting, spray with 0.1% carbendazim solution every 7 days, and water and fertilize regularly.

[0054] (6) Cultivation and management after transplanting to the field: Transplant the seedlings to the field one month after transplanting them into the seedling trays, and water and fertilize them regularly.

[0055] Comparative Example 1: The impact of using culture bags on the commercial production of black ginger tissue culture seedlings

[0056] Table 1 compares the effects of culture bag cultivation (see Examples 1 and 2, where both proliferation and rooting cultures are conducted in culture bags) and traditional glass tissue culture bottle cultivation (Example 3) on the commercial production of black ginger tissue culture seedlings. Table 1 shows that culture bag cultivation reduces sterilization costs, labor costs, and cultivation space compared to glass tissue culture bottle cultivation, and is also easier to transport.

[0057] Table 1. Impact of different culture methods on the commercial large-scale production of black ginger tissue culture seedlings

[0058]

Claims

1. A rapid propagation method for black ginger seedlings, characterized in that, Includes the following steps: S1. Take the underground rhizomes of black ginger that are growing in the current year, clean the surface and dry them. Wipe them with a 75% ethanol aqueous solution and cut off the single buds to a diameter of 0.6 cm. Remove the epidermis and leaf sheath to complete the preparation of the explant. S2. After disinfection, the explants were inoculated into primary culture medium in tissue culture flasks to obtain adventitious shoot clusters. The primary culture medium was: MS medium + 5.00 mg / L 6-BA + 0.10 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.

8. The disinfection treatment was as follows: soaking in 75% ethanol aqueous solution for 1 min, rinsing with sterile water, soaking in 0.1% HgCl2 solution for 8 min, and rinsing thoroughly with sterile water. S3. After cutting the bud clusters into 1 cm pieces, transfer them to the proliferation medium in the culture bag and culture to obtain bud clusters containing multiple single buds; the proliferation medium is: MS medium + 10.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.8; S4. When the single bud grows to 3-5 cm in height, cut off the single bud and inoculate it into the rooting medium in the culture bag to cultivate roots and form rooted seedlings; the rooting medium is: MS medium + 0.1-0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.8; S5. Transfer the rooted seedlings that have grown to 6-8 cm in height, along with their culture bags, directly to natural diffused light conditions to harden them off, and transplant them at the appropriate time.

2. The method according to claim 1, characterized in that, The cleaning of the surface in step S1 involves cutting off the roots on the rhizome and cleaning the surface with a brush.

3. The method according to claim 1, characterized in that, The step S1, removing the epidermis and leaf sheath, involves scraping off the epidermis, leaf sheath, and surrounding brown tissue with a scalpel.

4. The method according to claim 1, characterized in that, The cultivation conditions for steps S2 and S3 are as follows: temperature 25℃±2℃, relative humidity 80%, light intensity 2000 lx, and light duration 12 h / d.

5. The method according to claim 1, characterized in that, The rooting medium is: MS medium + 0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar, pH 5.

8.

6. The method according to claim 1, characterized in that, Step S5 is as follows: Transplant the 6-8 cm tall rooted seedlings, along with their culture bags, directly to natural diffused light conditions for 30 days to harden them off. Then, remove the rooted seedlings, wash off any remaining culture medium from the roots with water, transplant them into seedling trays, thoroughly water them with a 0.1% carbendazim solution, cover them with a plastic film greenhouse, maintain a temperature of 20℃-30℃ and a relative humidity of 70%-85%, and avoid direct sunlight. One week after transplanting, uncover the film and spray with a 0.1% carbendazim solution every 7 days, watering and fertilizing regularly. One month after transplanting into the seedling trays, transplant the seedlings to the field.

7. The method according to claim 1, characterized in that, The culture bags are made of plastic and have dimensions of 13 cm × 12 × 2 cm (height × length × width). Each bag contains 30-35 mL of culture medium.

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