Tissue culture method for rapidly propagating Rucheng small yellow ginger and application of tissue culture method

Abstract

The invention relates to the field of plant tissue culture, and particularly discloses a tissue culture method for rapidly propagating Ru-city small yellow ginger and application of the tissue culture method in order to simplify a Ru-city small yellow ginger tissue culture system and achieve the purposes of reducing production cost, saving labor and time and reducing pollution in seedling transferring and culture processes. The tissue culture method for rapidly propagating the Ru-cheng small yellow ginger comprises the following steps: inoculating an integrated culture medium with a Ru-cheng small yellow ginger explant for culture to complete callus induction and seedling differentiation so as to obtain a Ru-cheng small yellow ginger tissue culture seedling; the integrated culture medium is not replaced in the culture process. The tissue culture method for rapidly propagating the Ru-cheng small yellow ginger has the characteristics of simplicity in operation, low cost, time saving and labor saving, and has important application and popularization values.

Description

Technical Field

[0001] The present invention relates to the field of plant tissue culture, and particularly to a tissue culture method for rapid propagation of Rucheng small yellow ginger and its application. Background Art

[0002] Ginger is an economic crop with extremely high economic value. In addition to containing physiological active substances such as gingerone and gingerol, it also contains proteins, polysaccharides, vitamins and various trace elements, integrating nutrition, flavoring and health care. Since ancient times, it has been regarded by medical experts as a health care product with both medicinal and edible properties, and has various health care functions such as dispelling cold, removing dampness, warming the stomach and accelerating blood circulation. There are many local varieties of ginger, which can be divided into small ginger, large ginger, mountain ginger (wild ginger) and other varieties. Habitually, small ginger is called "small yellow ginger". The cut surface of small yellow ginger is pure yellow, with a strong spicy taste, rich in ginger oil and ginger juice, tender meat quality and low fiber content, which is superior to large ginger in terms of taste, color and medicinal effect.

[0003] Rucheng small yellow ginger is a local excellent variety widely cultivated in Rucheng, Hunan in the past hundred years. At present, the rhizomes of small yellow ginger are generally used as ginger seeds in production, and ginger farmers are used to planting with self-retained rhizomes (the rhizomes harvested in the current year are used as sowing materials for the next year). These rhizomes carry pathogenic microorganisms and are parasitized by pests to varying degrees. The main diseases of Rucheng small yellow ginger include bacterial wilt, basal stem rot, anthracnose, leaf blight, sheath blight, etc., and the main pests include root-knot nematodes, ginger borers, ginger maggots, prodenia litura and underground pests, etc. With the continuation of planting time and the expansion of planting area, the generation accumulation of pests and diseases on the rhizomes has caused the germplasm degradation of Rucheng small yellow ginger, and the stress resistance has been greatly reduced. In the light case, the yield of small yellow ginger is reduced by 10%-20%, and in the severe case, the yield reduction reaches more than 50%, and even a complete failure of the harvest occurs, causing huge economic losses to ginger farmers. Therefore, cultivating high-quality ginger seeds (seedlings) without bacteria and pests has become one of the important measures to reduce pests and diseases and achieve increased production and income.

[0004] Tissue culture is an important method for rapidly breeding sterile and high-quality seedlings by using the totipotency of plant cells. So far, in vitro cultivation systems of Zingiberaceae plants such as Alpinia malaccensis, Kaempferia galanga, Alpinia zerumbet and Alpinia officinarum have been established based on tissue culture technology. However, in the established tissue culture system, it is necessary to go through three stages: callus induction, proliferation culture and rooting culture. The composition and proportion of the medium and its plant growth regulators need to be changed in each culture stage, resulting in high production costs, time-consuming and laborious, and increasing the risk of contamination during the process of transplanting seedlings and culturing. Summary of the Invention

[0005] In order to simplify the tissue culture system of Rucheng small yellow ginger, achieve the purpose of reducing production costs, saving labor and time, and reducing pollution during the process of transplanting seedlings and cultivation, the present invention uses a kind of culture medium to complete the process of dedifferentiation of explants to form callus and differentiation into seedlings, greatly simplifies the seedling raising process, and establishes a high-quality and efficient tissue culture and propagation method for Rucheng small yellow ginger.

[0006] The present invention provides a tissue culture method for rapid propagation of Rucheng small yellow ginger, which includes the following steps: inoculating the Rucheng small yellow ginger explants onto an integrated culture medium for cultivation to obtain Rucheng small yellow ginger tissue culture seedlings; the integrated culture medium is not replaced during the cultivation process;

[0007] The types of plant growth regulators in the integrated culture medium are cytokinin and auxin; the cytokinin is 6-benzyladenine, and the auxin is any one of 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid.

[0008] In the above method, the content of the cytokinin in the integrated culture medium is 1 mg / L - 6 mg / L, and the content of the auxin in the integrated culture medium is 0.1 mg / L - 0.5 mg / L.

[0009] In the above method, preferably, the cytokinin is 6-benzyladenine, and its content in the integrated culture medium is 2 mg / L; the auxin is naphthaleneacetic acid, and its content in the integrated culture medium is 0.5 mg / L.

[0010] In the above method, the integrated culture medium is a solid culture medium obtained by adding sucrose, solidifying agent and plant growth regulators to the MS basic culture medium.

[0011] In the above method, the MS basic culture medium is obtained by dissolving MS dry powder in water, and the amount of MS dry powder used is 4.74 g / L.

[0012] In the above method, the solidifying agent of the integrated culture medium is agar.

[0013] In the above method, the content of sucrose in the integrated culture medium is 30 g / L, the content of agar is 8 g / L, and the pH value is 5.8 - 6.0.

[0014] In the above method, the small yellow ginger explants are aseptic shoot tips, aseptic stem segments or aseptic seedlings.

[0015] The aseptic shoot tips, aseptic stem segments or aseptic seedlings come from A1, A2 or A3:

[0016] A1. Collect the shoot tips of the rhizomes of Rucheng small yellow ginger, and obtain aseptic shoot tips after disinfection;

[0017] A2. Collect stem segments from the above-mentioned Rucheng small yellow ginger tissue culture seedlings to obtain aseptic stem segments;

[0018] A3. Sub-divide the cluster shoots of the tissue culture seedlings of Rucheng small yellow ginger to obtain aseptic seedlings.

[0019] The shoot tip refers to the bud tip containing the growth point of the ginger rhizome. The shoot tip is a bud tip with a length of 0.4 cm - 0.6 cm, preferably 0.5 cm in length.

[0020] The stem segment refers to the stem segment obtained by cutting off the roots and leaves of the tissue culture seedlings of small yellow ginger and taking the stem base. The stem segment is the stem base with a length of 0.8 cm - 1.2 cm, preferably 1.0 cm in length.

[0021] In the method of the present invention, when the shoot tip is used as the explant, callus is formed on the culture medium, and then buds and roots are redifferentiated to become complete seedlings. The whole process takes 2 to 4 months, and almost all the surviving explants can develop into seedlings after 6 months. If the aseptic stem segment is selected as the explant, the culture time for completing callus induction and seedling morphogenesis can be shortened to 40d - 60d. If the aseptic seedlings obtained by sub-dividing the clustered aseptic tissue culture seedlings are selected as the explants for subculture and proliferation, the culture time can be shortened to 30d - 45d.

[0022] In the above method, the disinfection is preferably carried out by disinfecting with 75% alcohol for 15 s, then disinfecting with 0.1% HgCl 2 solution for 15 min, and finally disinfecting with 75% alcohol for 15 s.

[0023] In the above method, the culture is carried out under the conditions of a temperature of 24°C - 26°C, a light time of 10 h / d, and a light intensity of 90 μmol·m -2 ·s -1 of.

[0024] The tissue culture method for rapid propagation of Rucheng small yellow ginger of the present invention further includes the step of acclimatizing and transplanting the tissue culture seedlings of Rucheng small yellow ginger.

[0025] The present invention also protects the application of the above method in the production of Rucheng small yellow ginger.

[0026] A tissue culture method for rapid propagation of Rucheng small yellow ginger provided by the present invention can induce the explant to form callus on the integrated culture medium, and then redifferentiate to form complete seedlings. After acclimatizing, they are planted in the field and conventional water and fertilizer management is carried out, and then the germ-free original seed tubers of small yellow ginger can be harvested. This technology has the characteristics of simple operation, low cost, time-saving and labor-saving. Therefore, the technology of the present invention has important application and popularization value. Description of the Drawings

[0027] Figure 1This shows the effects of different types and concentrations of plant growth regulators in the culture media treated with P1, P2, P3, and P4 in Example 1 of the present invention on the formation of callus and shoot differentiation of explants.

[0028] Figure 2 This shows the growth conditions of tissue culture seedlings of integrated culture media of Zingiber officinale Rosc. var. wenchangense transplanted into plug trays and fields in Example 2 of the present invention. Specific Embodiments

[0029] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0030] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0031] The Zingiber officinale Rosc. var. wenchangense used in this embodiment is a locally excellent variety that has been widely cultivated in Rucheng, Hunan for nearly a hundred years and can be purchased in the market.

[0032] Example 1. Sterilization and Effects of Plant Growth Regulators on Callus Induction and Differentiation of Zingiber officinale Rosc. var. wenchangense

[0033] The tissue culture in this embodiment was carried out in the tissue culture room of the Nanling Modern Seed Industry Research Institute of Xiangnan University, and the field cultivation was carried out in Shushuyuan Village, Quanshui Town, Rucheng County, Chenzhou City, Hunan Province. The specific steps are as follows:

[0034] 1. Effects of Different Disinfection Methods on the Sterilization Efficiency and Growth of Explants

[0035] The rhizomes of Zingiber officinale Rosc. var. wenchangense were cut into appropriate sizes for easy cleaning. After the rhizomes were cleaned, stem tips with a length of 0.5 cm were cut as explants. The dirt and soil on the explants were brushed with a brush dipped in dishwashing liquid, and then the explants were soaked in a 0.5% chlorothalonil solution for 20 min, rinsed with running water for 2 h, and rinsed with sterile water. Then, they were combined and disinfected with 75% (volume percentage) alcohol and 0.1% (mass percentage) mercuric chloride solution (the solute is HgCl 2 , and the solvent is water).

[0036] The disinfection time of 75% alcohol was set to 3 durations of 15 s, 30 s, and 45 s, and 0.1% HgCl 2The solution disinfection time was set to 3 durations of 12 min, 15 min, and 18 min, and 9 disinfection combinations of S1, S2, S3, S4, S5, S6, S7, S8, and S9 were set, as shown in Table 1 for details. The S1 treatment (S, sterilization) was 75% ethanol for 15 s → 0.1% mercuric chloride for 12 min → 75% ethanol for 15 s, that is, first disinfect with 75% ethanol for 15 s, then disinfect with 0.1% mercuric chloride for 12 min, and finally disinfect with 75% ethanol for 15 s. The other treatments were carried out in the same way.

[0037] Table 1 Effects of different disinfection methods on the sterilization efficiency and growth status of explants

[0038] Treatment Disinfection method (treatment + treatment time) Number of explants Contamination rate (%) Survival rate (%) S1 75% ethanol for 15 s → 0.1% mercuric chloride for 12 min → 75% ethanol for 15 s 30 26.67％ 6.67％ S2 75% ethanol for 30 s → 0.1% mercuric chloride for 12 min → 75% ethanol for 30 s 31 3.23％ 6.45％ S3 75% ethanol for 45 s → 0.1% mercuric chloride for 12 min → 75% ethanol for 45 s 31 3.23％ 6.45％ S4 75% ethanol for 15 s → 0.1% mercuric chloride for 15 min → 75% ethanol for 15 s 28 0％ 17.86％ S5 75% ethanol for 30 s → 0.1% mercuric chloride for 15 min → 75% ethanol for 30 s 30 33.33％ 6.67％ S6 75% ethanol for 45 s → 0.1% mercuric chloride for 15 min → 75% ethanol for 45 s 30 0％ 3.33％ S7 75% ethanol for 15 s → 0.1% mercuric chloride for 18 min → 75% ethanol for 15 s 30 6.67％ 3.33％ S8 75% ethanol for 30 s → 0.1% mercuric chloride for 18 min → 75% ethanol for 30 s 31 0％ 6.45％ S9 75% ethanol for 45 s → 0.1% mercuric chloride for 18 min → 75% ethanol for 45 s 31 0％ 0％

[0039] Inoculate onto the MS solid medium without any plant growth regulator, observe the contamination situation 2 weeks after inoculation, and count the survival rate 3 months after inoculation.

[0040] The results showed that the treatment S4 of disinfecting with 75% alcohol for 15 s, then with 0.1% mercuric chloride solution for 15 min, and finally with 75% alcohol for 15 s had a decontamination success rate of up to 100% (contamination rate was 0%) for the explants and also had the highest culture survival rate for the explants.

[0041] Select the disinfection method of S4 for subsequent experiments.

[0042] 2. Effects of different plant growth regulators and ratios on the growth of explants

[0043] Using the MS solid medium (the pH value of the general plant medium can be 5.8 - 6.0, and the pH value of the medium used in the examples of the present invention is 6.0) as the basic medium, add plant growth regulators (phytohormone) to prepare the media of 4 treatments of P1, P2, P3, and P4 in Table 2. The specific types of plant growth regulators added are cytokinin and auxin. The cytokinin is 6 - BA (6 - benzyladenine), and the auxin is any one of 2,4 - D (2,4 - dichlorophenoxyacetic acid) and NAA (naphthaleneacetic acid).

[0044] The composition and concentration of the plant growth regulator in the P1 treatment medium were 6 - BA 1.0 mg / L + 2,4 - D 0.5 mg / L. That is, the P1 treatment medium was a medium obtained by adding 6 - BA and 2,4 - D to the MS solid medium (pH value 6) as the basic medium. The concentration of 6 - BA in this medium was 1.0 mg / L, and the concentration of 2,4 - D was 0.5 mg / L. The preparation methods of the media for other treatments were carried out in the same way.

[0045] Table 2 Effects of Different Plant Growth Regulators and Their Ratios on Dedifferentiation and Shoot Differentiation of Ginger Shoot Tips

[0046]

[0047]

[0048] After the explants were sterilized by the S4 method, they were respectively inoculated onto the media of 4 treatments, namely P1, P2, P3, and P4, with 10 replicates set, and 1 explant was inoculated on each medium per replicate. Under the conditions of 24℃ - 26℃, a light duration of 10 h / d, and a light intensity of 90 μmol·m -2 ·s -1 , they were cultured for 60 d.

[0049] The results are shown in Table 2 and Figure 1 , indicating that different combinations of cytokinins and auxins at different concentrations significantly affect the growth and development of explants. The explants in P1 swelled, and yellowish-white flaky structures appeared around the explants. These structures were thick and brittle, and were callus. The explants in P3 grew thick roots at the base, and there were yellowish-white flaky structures around them, which were callus. The explants could grow buds and roots in the P2 and P4 treatments, but the roots in P2 were thinner, shorter, and more numerous, similar to a villous structure, while the roots in P4 were thicker, longer, and fewer. These results indicate that a combination of appropriate concentrations of 6-BA and 2,4-D or NAA can induce the formation of callus and differentiate adventitious buds and adventitious roots.

[0050] 3. Effects of Different Plant Growth Regulators and Their Ratios on the Proliferation of Cluster Shoots

[0051] Using the MS solid medium as the basic medium, plant growth regulators were added to prepare the media of 16 treatments, namely T1 - T16 in Table 3. The specific plant growth regulators added were cytokinins and auxins. The cytokinin was 6-BA, and the auxin was any one of 2,4-D and NAA.

[0052] 4 replicates were set, and 3 explants were inoculated on each medium per replicate.

[0053] The medium of T1 treatment was prepared by adding 6-BA and NAA to the MS solid medium (pH value 6) as the basic medium. The concentration of 6-BA in this medium was 1 mg / L, and the concentration of NAA was 0.1 mg / L. The preparation methods of the media for other treatments are shown in Table 3.

[0054] Table 3 Effects of Different Combinations of Plant Growth Regulators on the Proliferation of Ginger Seedlings

[0055]

[0056] Note: Different lowercase letters after the proliferation coefficient values indicate significant differences (P<0.05).

[0057] The aseptic tissue culture seedlings obtained in the above step 2 were cultured and propagated (tillered), and then divided into individual plants and inoculated onto the culture media of 16 treatments from T1 to T16. Then, they were cultured at 24°C - 26°C, with a light duration of 10 h / d and a light intensity of 90 μmol·m -2 ·s -1 for 45 d, and the proliferation coefficient (the multiple of the increase in seedlings after inoculation and culture of clustered buds) was counted.

[0058] The results are shown in Table 3. Compared with the MS solid medium without plant growth regulators (proliferation coefficient 1.56±0.2e), the application of plant growth regulators significantly increased the proliferation coefficient. When using the combination of 6-BA and NAA, the proliferation result of treatment T6 was better; when using the combination of 6-BA and 2,4-D, the proliferation effect of T9 was the best; in this experiment, the proliferation coefficient of treatment T9 (6-BA 1 mg / L + 2,4-D 0.1 mg / L) was the largest, reaching up to 4.44, and well-developed roots grew at the base of the clustered seedlings.

[0059] Example 2: Rapid propagation of Rucheng small yellow ginger seedlings with an integrated culture medium

[0060] 1. Effects of the integrated culture medium on the dedifferentiation of explants to form callus and the differentiation into seedlings

[0061] The explants can be induced to form callus and differentiated into normal seedlings and proliferated in the MS medium containing different combinations of plant growth regulators and ratios (Table 2, Table 3, Figure 1 ). Then, can the integrated culture medium be used to make the explants dedifferentiate to form callus and differentiate into a larger number of seedlings with good growth status? That is, to complete the entire tissue culture process (callus induction, proliferation, and rooting) on one medium.

[0062] (1) Using the MS solid medium as the basic medium, plant growth regulators (phytohormone) were added to prepare the culture media of 22 treatments, ICM1 - ICM22 (ICM, integrated culture medium) in Table 4. The specific types of plant growth regulators added were the cytokinin 6-BA and the auxins NAA and 2,4-D. The specific preparation method is shown in Table 4. The MS solid medium without plant growth regulators (pH value 6) was used as the control (CK) medium.

[0063] Using the tissue-cultured seedlings obtained in Example 1 as materials, cut off the roots and leaves, and take 1-cm-long stem segments at the base of the stem as explants. Inoculate the explants onto the culture media of 22 treatments from ICM1 to ICM22, and use the MS solid medium (pH value 6) without plant growth regulators as the control medium, for a total of 23 media.

[0064] The explants inoculated onto each medium were respectively treated with the following two light modes:

[0065] Dark + light culture: After 10 days of dark culture, normal light culture for 30 days.

[0066] Light culture: Normal light culture for 40 days.

[0067] Set 3 replicates, and inoculate 4 explants of each medium and each light mode in each replicate.

[0068] The culture temperature for both light modes was 24°C - 26°C. For normal light culture, the light was 10 h / d, and the light intensity was 90 μmol·m -2 ·s -1 . After 40 days of culture, observe and calculate the callus induction and budding rate (the number of explants forming callus / the number of inoculated explants) and the multiplication coefficient of cluster buds (the multiple of the increase in seedlings after inoculation and culture of explants). The culture medium was not replaced during the whole culture process.

[0069] In the culture media containing different concentrations and ratios of 6-BA and NAA or 6-BA and 2,4-D, all combinations with 6-BA concentration of 1 mg / L - 6 mg / L and NAA concentration of 0.1 mg / L - 0.5 mg / L or 6-BA concentration of 1 mg / L - 6 mg / L and 2,4-D concentration of 0.1 mg / L - 0.5 mg / L could induce the stem segments to form callus. Taking whether adventitious buds could be induced on the callus as the standard, the callus induction and budding rate of the stem segments were statistically analyzed. Taking the number of ginger seedlings with a plant height of more than 2 cm and unfolded leaves formed from the callus induction of the stem segments as the standard, the emergence number of the stem segments was statistically analyzed. During the 40-day culture, it was found that there were differences in the efficiency of callus formation and bud induction of the stem segments on different culture media and the number of ginger seedlings formed after bud induction (Table 4).

[0070] For the induction of adventitious buds from callus, continuous normal light culture for 40 d was superior to dark culture for 10 d + light culture for 30 d in all 6-BA and NAA combination media except ICM5 and ICM7, and in all 6-BA and 2,4-D combination media except ICM17 and ICM18. When different auxins with the same concentration were selected under the same cytokinin 6-BA concentration, the adventitious bud induction rate of 2,4-D in all combinations of normal light culture was lower than that of NAA for the callus of stem segments. It should be noted that adventitious buds could also be induced from the callus of stem segments in the medium without adding any plant growth regulators, and the adventitious bud induction rate was significantly higher than that in the combination media supplemented with 6-BA and 2,4-D (Table 4).

[0071] Table 4 Effects of integrated media on adventitious bud induction and seedling formation from ginger stem segments

[0072]

[0073]

[0074] Note: Different lowercase letters after the values in the same column represent significant differences (P < 0.05).

[0075] For the number of normal seedlings formed from adventitious buds, continuous normal light culture for 40 d was superior to dark culture for 10 d + light culture for 30 d in all 6-BA and NAA combination media except ICM1. In the 6-BA and 2,4-D combination media, the results of continuous normal light culture for 40 d and dark culture for 10 d + light culture for 30 d were both poor. When different auxins with the same concentration were selected under the same cytokinin 6-BA concentration, the number of seedlings promoted by 2,4-D in all combinations of normal light culture was lower than that of NAA, and even no ginger seedlings could be formed in ICM18. Adventitious bud induction and normal seedling formation from the callus of stem segments could also occur in the medium without adding any plant growth regulators, but the number of ginger seedlings was significantly lower than that in most combinations supplemented with 6-BA and NAA, and significantly higher than that in most combinations supplemented with 6-BA and 2,4-D. It should be noted that a higher concentration of plant growth regulators would lead to a higher degree of vitrification of the seedlings formed from adventitious buds, weaker growth state, and affect subsequent culture and planting.

[0076] (2) Harvest fresh rhizomes at 6 months old from the field. After primary cleaning, cut stem tips with a length of 0.5 cm as explants. After sterilization of the explants by the method of S4 in Table 1, inoculate them into the integrated medium (containing MS + 6-BA 2 mg / L + NAA 0.5 mg / L) (ICM9 in Table 2), and then at 24℃ - 26℃, the light time is 10 h / d, and the light intensity is 90 μmol·m -2 ·s -1Cultivate for 120 d under the following conditions. The results show that for the explants in the integrated culture medium, the contamination rate is 0%, the mortality rate is 9.52%, the callus induction rate is 85.71%, and the cluster seedling differentiation rate is 71.43%.

[0077] After the explants are sterilized by the method of S4 in Table 1, they are inoculated into the integrated culture medium (containing MS + 6-BA 1 mg / L + 2,4-D 0.2 mg / L) (ICM16 in Table 2), and then cultured at 24°C - 26°C, with a light time of 10 h / d and a light intensity of 90 μmol·m -2 ·s -1 Cultivate for 120 d under the following conditions. The results show that for the explants in the integrated culture medium, the contamination rate is 0%, the mortality rate is 15.00%, the callus induction rate is 65.00%, and the cluster seedling differentiation rate is 35.00%.

[0078] These results indicate that whether it is the stem segment of the tissue-cultured seedling or the shoot tip of the rhizome of the field ginger, they can dedifferentiate into callus in the integrated culture medium and differentiate into complete seedlings (ginger seedlings with complete tissue structures such as roots, stems, and leaves and in good growth states), and the optimal integrated culture medium is ICM9.

[0079] 2. Cultivation of tissue-cultured seedlings in the integrated culture medium

[0080] Transfer the tissue-cultured seedlings (in-vitro seedlings) obtained in Step 1 of Example 2 to the greenhouse for hardening off (at 25°C, with a light time of 12 h / d) for 7 - 10 d. Wash the culture medium adhering to the root base of the ginger seedlings, and transplant them into a plug tray filled with disinfected substrate, maintaining an appropriate temperature (15°C - 25°C) and appropriate moisture; when the ginger seedlings grow 2 - 3 new leaves after 20 d of cultivation, transplant the seedlings to the field (Shushuyuan Village, Quanshui Town, Rucheng County, Hunan Province) on April 29. Photos of subsequent growth stages are shown in Figure 2 . Conduct conventional water and fertilizer management and manual weeding, and harvest on November 4. After measurement, the rhizome yield per 667 m 2 is about 1900 kg. Compared with the results of planting with the rhizome of Rucheng small yellow ginger as the material, the tissue-cultured seedlings produced by the present invention perform well during the whole growth process, with developed roots, thick stems, dark green leaves, and no diseases occurring.

[0081] In the method of the present invention, when the shoot tip is used as the explant, it forms callus on the culture medium, and then differentiates into buds and roots to become complete seedlings. The whole process takes 2 to 4 months, and almost all the survived explants can develop into seedlings after 6 months. If the sterile stem segment is selected as the explant, the culture time for completing callus induction and seedling morphogenesis can be shortened to 40 d - 60 d. If the sterile seedlings obtained by dividing the clustered sterile tissue-cultured seedlings are selected as the explants for subculture and proliferation, the culture time can be shortened to 30 d - 45 d.

[0082] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principles of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the appended claims below.

Claims

1. A tissue culture method for rapid propagation of Rucheng small yellow ginger, characterized in that: The method comprises the following steps: inoculating Rucheng small yellow ginger explants into an integrated culture medium for cultivation to obtain Rucheng small yellow ginger tissue culture seedlings; the integrated culture medium is not replaced during the cultivation process; The types of plant growth regulators in the integrated culture medium are cytokinins and auxins; the cytokinins are 6-benzyladenine, and the auxins are any one of 2,4-dichlorophenoxyacetic acid and naphthylacetic acid.

2. The method according to claim 1, characterized in that The content of the cytokinin in the integrated culture medium is 1-6 mg / L, and the content of the auxin in the integrated culture medium is 0.1-0.5 mg / L.

3. The method according to claim 2, characterized in that The cytokinin is 6-benzyladenine, and its content in the integrated culture medium is 2 mg / L; the auxin is naphthylacetic acid, and its content in the integrated culture medium is 0.5 mg / L.

4. The method according to claim 3, characterized in that The integrated culture medium is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to the MS basic culture medium.

5. The method according to claim 4, characterized in that The coagulant of the integrated culture medium is agar.

6. The method according to claim 5, characterized in that The integrated culture medium contains 30 g / L sucrose, 8 g / L agar, and a pH value of 5.8-6.

0.

7. The method according to any one of claims 1 to 6, characterized in that: The small yellow turmeric explant is a sterile stem tip, a sterile stem segment or a sterile seedling.

8. The method according to claim 7, characterized in that The sterile stem tips, sterile stem segments or sterile seedlings are from A1, A2 or A3: A1. Collect the stem tips of the rhizomes of Rucheng ginger and obtain sterile stem tips after disinfection; A2, collecting stem segments from the Rucheng Xiaohuangjiang tissue culture seedlings to obtain sterile stem segments; A3, dividing the clump seedlings of the Rucheng Xiaohuangzingiber officinale tissue culture seedlings to obtain sterile seedlings.

9. The method according to claim 8, characterized in that The disinfection is performed by using 75% alcohol for 15 seconds, then using 0.1% HgCl2 solution for 15 minutes, and finally using 75% alcohol for 15 seconds.

10. Application of the method according to any one of claims 1 to 9 in the production of Rucheng small yellow ginger.

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