Method for producing copperling white ginger seedling efficiently throughout the year

Abstract

The application discloses a kind of Tongling white ginger seedling year efficient production method, including with Tongling white ginger excellent strain current year young tender tuber as explant material, after surface disinfection, cut into tuber with bud point, inoculation in bud point germination medium carries out bud point germination culture;After the germination of bud point is stripped from tuber, is inoculated in adventitious bud induction medium and carries out adventitious bud induction culture;The adventitious bud cluster induced is directly transferred to proliferation culture medium and carries out the proliferation culture of adventitious bud;After the proliferation of adventitious bud is separated from bud cluster and is transferred to adventitious bud elongation, seedling and adventitious root induction synchronous medium and carries out adventitious bud elongation, seedling and adventitious root induction synchronous culture, then domestication, washing and surface disinfection are carried out and carry out seedling culture.The method of the application is efficient and can maintain the excellent characteristics of Tongling white ginger mother plant, provides certain technical support for the germplasm preservation, seedling factory production and germplasm creation of Tongling white ginger excellent strain.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a method for year-round high-efficiency production of Tongling white ginger seedlings. Background Technology

[0002] Tongling white ginger, as an asexually propagated crop, is currently mainly produced through tuber selection and propagation. However, long-term asexual reproduction and repeated cropping have led to serious disease accumulation, reduced yield and quality, and other genetic degradation problems in Tongling white ginger. Furthermore, the lack of effective breeding techniques results in a long breeding cycle, limited propagation methods, and a scarcity of high-quality, multi-resistant germplasm resources. These issues severely restrict the sustainable development of the Tongling white ginger industry.

[0003] Plant tissue culture, as a modern biotechnology, has advantages such as low material requirements, high propagation efficiency, high propagation coefficient, no seasonal or material limitations, and the ability to achieve large-scale seedling production in a short period. Currently, research on the virus-free propagation of *Gynostemma pentaphyllum* using stem tips as material has been reported. For example, Chinese patent application CN115769786A discloses a method for obtaining regenerated *Gynostemma pentaphyllum* seedlings using alternating tissue culture. This method uses *Gynostemma pentaphyllum* as material, germinating buds to obtain 0.5–1 cm shoots; in a clean bench, 0.5–1 mm stem tip explants are placed sequentially in four stages of induction culture medium to induce adventitious buds; once the adventitious buds have grown 3–5 leaves… After leaf formation, the ginger is transferred to a rooting medium for rooting culture. This significantly shortens the induction time of adventitious buds and increases the induction rate. Using a modified culture medium formula with alternating tissue culture, no special hormones are required; only 6-BA and NAA are needed. A large number of regenerated plants can be obtained within 30 days. This method is characterized by simple processing steps, short induction time, and high induction rate. However, during propagation, problems exist such as high initial culture contamination rate, low survival rate, low proliferation coefficient, low field transplant survival rate, and high production costs, making it difficult to promote and apply in production. These problems severely limit the large-scale propagation, promotion, and subsequent variety improvement of Tongling white ginger seedlings. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to use plant tissue culture technology to efficiently produce Tongling white ginger seedlings.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] A method for year-round high-efficiency production of Tongling white ginger seedlings includes the following steps:

[0007] S1. Using the young tubers of the current year from the superior strain of Tongling white ginger as explant material, the surface was disinfected, and then the tubers with buds were cut and inoculated into the bud germination medium for bud germination culture.

[0008] S2. After the sprouting buds in S1 are separated from the tuber, they are inoculated onto the adventitious bud induction medium for adventitious bud induction culture.

[0009] S3. The adventitious shoot clusters induced in S2 were directly transferred to the proliferation medium for adventitious shoot proliferation culture;

[0010] S4. The adventitious buds proliferated in S3 were separated from the bud cluster and transferred to the adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture medium for adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture to obtain complete regenerated plants.

[0011] S5. After the complete regenerated plants obtained in S4 are domesticated, cleaned and surface disinfected, they are cultured into seedlings.

[0012] Preferably, in S1, the culture time is 2 weeks; in S2, the culture time is 3 weeks; in S3, the culture time is 4 weeks; and in S4, the culture time is 3 weeks.

[0013] Preferably, in S1, the surface disinfection includes the following steps: rinsing the explant material with running water for 30 minutes, then wiping the surface with 75% ethanol; then rinsing with sterile water 5 to 8 times, then disinfecting with 0.1% (w / v) mercuric chloride solution for 9 to 18 minutes; then rinsing with sterile water 3 to 5 times, then rinsing with sterile water containing 300 to 500 mg / L cephalosporin 6 to 8 times, and finally absorbing the surface moisture with sterile filter paper.

[0014] Preferably, in S1, the bud germination medium is DKW + 0.1-1.0 mg / L Meta-Topolin + 0.2-0.5 mg / L GA3 + 3.0% (w / v) sucrose + 0.7% (w / v) agar, pH = 5.8.

[0015] Preferably, in S2, the adventitious bud induction medium is DKW + 1.0-5.0 mg / L LMeta-Topolin + 3.0% (w / v) sucrose + 0.7% (w / v) agar, pH = 5.8.

[0016] Preferably, in S3, the proliferation medium is DKW + 0.05-0.5 mg / L TDZ + 0.5-2.0 mg / L LMeta-Topolin + 3.0% (w / v) sucrose + 0.7% (w / v) agar, pH = 5.8.

[0017] Preferably, in S4, the synchronous medium for inducing adventitious bud elongation, seedling strengthening, and adventitious root induction is DKW + 0.5-1.0 mg / L Meta-Topolin + 0.1-0.5 mg / L IBA + 2.0% (w / v) sucrose + 0.7% (w / v) agar, pH = 5.8.

[0018] Preferably, S5 specifically includes the following steps: the complete regenerated plant obtained in S4 is acclimatized under natural conditions for 2 weeks, taken out and washed with running water, then placed in an aqueous solution containing 0.05% to 0.2% potassium permanganate for 10 to 15 minutes, and then transplanted into a seedling tray for seedling cultivation.

[0019] Preferably, the seedling tray is filled with a mixed nutrient substrate; the mixed nutrient substrate is a mixture of Pinsbury potting soil and vermiculite in a volume ratio of 2-4:1-3.

[0020] Preferably, in S1-S4, the cultivation conditions are: temperature 25±2℃, light intensity 2500~3000lx, and photoperiod 16 / 8h (light / dark); in S5, the seedling cultivation conditions are: temperature 20~28℃, humidity 75%~85%, and light intensity 1800~4000lx.

[0021] The advantages of this invention are:

[0022] (1) The method for year-round production of Tongling white ginger seedlings provided by the present invention induces adventitious buds through direct organogenesis, ultimately obtaining complete regenerated plants. The obtained regenerated plants can maintain the genetic stability of the excellent characteristics of the parent plant.

[0023] (2) The year-round production method of Tongling white ginger seedlings provided by the present invention has the advantages of high propagation efficiency, high propagation coefficient (each explant can produce 23.8 adventitious buds after one propagation) and high transplant survival rate, which provides important technical support for the industrialized production and germplasm creation of superior Tongling white ginger seedlings. Attached Figure Description

[0024] Figure 1 The young tubers of the current year from the superior and healthy strain of Tongling white ginger used in Example 2 of this invention;

[0025] Figure 2 This refers to the Tongling white ginger tuber that sprouted buds after being cultured on a sprouting medium for 2 weeks in Example 2 of this invention;

[0026] Figure 3 The buds of Tongling white ginger isolated in Example 2 of this invention germinated and formed adventitious buds after being cultured in an adventitious bud induction medium for 1 week;

[0027] Figure 4 The adventitious buds obtained by culturing the buds of Tongling white ginger isolated in Example 2 of this invention in an adventitious bud induction medium for 3 weeks;

[0028] Figure 5 The adventitious bud clusters of Tongling white ginger obtained in Example 2 of this invention were cultured on a proliferation medium for 3 weeks.

[0029] Figure 6 The complete regenerated plant obtained in Example 2 of this invention after culturing adventitious buds on a synchronized medium for elongation, seedling strengthening, and adventitious root induction for 3 weeks;

[0030] Figure 7 This refers to the tissue culture regenerated plants of Tongling white ginger in Example 2 of the present invention, which were acclimatized for 2 weeks and transplanted for 1 month.

[0031] Figure 8 The adventitious buds obtained by culturing the buds of Tongling white ginger isolated in Example 3 of this invention in an adventitious bud induction medium for 3 weeks;

[0032] Figure 9 The adventitious shoots obtained in Example 5 of this invention were obtained in a medium supplemented with 8.0 mg / L Meta-Topolin. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0035] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0036] Example 1

[0037] A method for year-round high-efficiency production of Tongling white ginger seedlings, the specific operation is as follows:

[0038] (1) At the end of August, young tubers of the current year from a superior and healthy strain of Tongling white ginger provided by Anhui Tongque Erqiao Biotechnology Co., Ltd. were used as explant materials. After rinsing with running water for 30 minutes, the surface was wiped with 75% ethanol. Then, the tubers were placed in 250ml culture bottles in a sterile operating table, rinsed 8 times with sterile water, and then sterilized with 0.1% (w / v) mercuric chloride solution for 9 minutes. After rinsing 5 times with sterile water, the tubers were rinsed 8 times with sterile water containing 500mg / L cephalosporin. After absorbing the surface moisture with sterile filter paper, the tubers were cut into tubers with buds and set aside.

[0039] (2) The tubers with buds, which had been disinfected in step (1), were inoculated into DKW germination medium supplemented with 0.1 mg / L Meta-Topolin, 0.2 mg / L GA3, 3.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. Germination was cultured in a constant-temperature incubator at 25 ± 2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8 h (light / dark). After 2 weeks of light culture, the contamination rate of the explants was 9.2%, and 87.4% of the buds germinated from the tubers of *Ginger indicum*.

[0040] (3) After the sprouting buds of *Glehnia littoralis* from step (2) were separated from the explants, they were inoculated into DKW adventitious bud induction medium supplemented with 1.0 mg / L Leta-Topolin, 3.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. Adventitious buds were induced in a constant-temperature culture room at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8h (light / dark). After one week of light culture, buds sprouted; after three weeks of light culture, adventitious bud clusters were induced, with an induction rate as high as 83.5%, and an average of 3.4 adventitious buds per explant.

[0041] (4) The adventitious shoot clusters obtained in step (3) were transferred to DKW adventitious shoot proliferation medium supplemented with 0.05 mg / L TDZ, 0.5 mg / L Leta-Topolin, 3.0% (w / v) sucrose and 0.7% (w / v) agar, pH=5.8 and cultured in a constant temperature culture room at 25±2℃, light intensity of 2500~3000lx and photoperiod of 16 / 8h (light / dark); after 3 weeks of light culture, an average of 8.6 adventitious shoots were produced per explant.

[0042] (5) After one week of continued light culture, the obtained adventitious shoots were separated from the shoot clusters and transferred to DKW synchronous medium supplemented with 0.5 mg / L Leta-Topolin, 0.1 mg / L IBA, 2.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. The adventitious shoots were simultaneously cultured in a constant-temperature incubator at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8h (light / dark). After three weeks of light culture, the adventitious shoots achieved synchronized rooting while simultaneously strengthening and elongating, resulting in robust, fully regenerated plants. The adventitious root induction rate was as high as 90.6%, with an average of 3.3 adventitious roots per explant, and the average height of the regenerated plants was 3.4 cm.

[0043] (6) Place the seedling bottles containing the complete regenerated plants obtained in step (5) under natural conditions for acclimatization for 2 weeks. Take out the regenerated plants, wash them with running water to remove the culture medium from the plant roots, and then soak them in an aqueous solution with a mass fraction of 0.05% potassium permanganate for 10 minutes. Then transplant them into seedling trays containing mixed nutrient substrate and acclimatize them in a culture room with a temperature of 20-28℃, a humidity of 75%-85%, and a light intensity of 1800-4000 lx. The mixed nutrient substrate is a mixture of Pinscher potting soil and vermiculite with a volume ratio of 2:1. After 4 weeks of culture, the transplant survival rate of the regenerated plants is 92.3%.

[0044] Example 2

[0045] A method for year-round high-efficiency production of Tongling white ginger seedlings, the specific operation is as follows:

[0046] (1) At the end of August, young tubers of the current year from the superior healthy strains of Tongling white ginger provided by Anhui Tongque Erqiao Biotechnology Co., Ltd. were used as explant materials. Figure 1 After rinsing with running water for 30 minutes, wipe the surface with 75% ethanol; then place in a 250ml culture flask on a sterile operating table, rinse 8 times with sterile water, then disinfect with 0.1% (w / v) mercuric chloride solution for 12 minutes; then rinse 5 times with sterile water, and finally rinse 8 times with sterile water containing 400mg / L cephalosporin. After blotting the surface moisture with sterile filter paper, cut into tubers with buds for later use.

[0047] (2) The tubers with buds, sterilized in step (1), were inoculated into DKW germination medium supplemented with 0.5 mg / L Meta-Topolin, 0.3 mg / L GA3, 3.0% (w / v) sucrose, and 0.7% (w / v) agar, pH=5.8, and cultured in a constant temperature incubator at 25±2℃, light intensity of 2500-3000 lx, and a photoperiod of 16 / 8h (light / dark). After 2 weeks of light culture, the contamination rate of the explants was 1.3%, and 100% of the buds germinated from the tubers of *Ginger indicum* var. *tonglingensis*. Figure 2 ).

[0048] (3) After detaching the sprouting Tongling white ginger buds from the explants in step (2), they were inoculated into DKW adventitious bud induction medium supplemented with 3.0 mg / L Leta-Topolin, 3.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. The induction of adventitious buds was carried out in a constant-temperature culture room at 25±2℃, a light intensity of 2500–3000 lx, and a photoperiod of 16 / 8 h (light / dark). After one week of light culture, buds sprouted and adventitious buds formed. Figure 3 After 3 weeks of light cultivation, the adventitious bud induction rate reached 96.4%, with an average of 6.9 adventitious buds produced per explant. Figure 4 ).

[0049] (4) The adventitious shoot clusters obtained in step (3) were transferred to DKW adventitious shoot proliferation medium supplemented with 0.2 mg / L TDZ, 1.0 mg / L Leta-Topolin, 3.0% (w / v) sucrose and 0.7% (w / v) agar, pH=5.8, and cultured in a constant temperature culture room at 25±2℃, light intensity of 2500~3000lx, and photoperiod of 16 / 8h (light / dark); after 3 weeks of light culture, an average of 23.8 adventitious shoots were produced per explant. Figure 5 ).

[0050] (5) After one week of continued light culture, the obtained adventitious shoots were separated from the shoot clusters and transferred to DKW synchronous medium supplemented with 0.75 mg / L Leta-Topolin, 0.3 mg / L IBA, 2.0% (w / v) sucrose, and 0.7% (w / v) agar, pH=5.8. The adventitious shoots were cultured simultaneously in a constant temperature incubator at 25±2℃, light intensity of 2500–3000 lx, and a photoperiod of 16 / 8h (light / dark) for simultaneous growth, strengthening, and root induction. After three weeks of light culture, the adventitious shoots achieved synchronized rooting while strengthening and elongating, resulting in robust, complete regenerated plants. The root induction rate was as high as 100%, with an average of 6.5 adventitious roots per explant, and the average height of the regenerated plants was 5.7 cm. Figure 6 ).

[0051] (6) Place the seedling bottles containing the complete regenerated plants obtained in step (5) under natural conditions for acclimatization for 2 weeks. Take out the regenerated plants, wash them with running water to remove the culture medium from the plant roots, and then soak them in an aqueous solution containing 0.1% potassium permanganate for 12 minutes. Then transplant them into seedling trays containing mixed nutrient substrate and acclimatize them in a culture room with a temperature of 20-28℃, a humidity of 75%-85%, and a light intensity of 1800-4000 lx. The mixed nutrient substrate is a mixture of Pinsbury potting soil and vermiculite in a volume ratio of 3:2. After 4 weeks of culture, the survival rate of the regenerated plants after transplanting is 100%. Figure 7 ).

[0052] Example 3

[0053] A method for year-round high-efficiency production of Tongling white ginger seedlings, the specific operation is as follows:

[0054] (1) At the end of August, young tubers of the current year from a superior and healthy strain of Tongling white ginger provided by Anhui Tongque Erqiao Biotechnology Co., Ltd. were used as explant materials. After rinsing with running water for 30 minutes, the surface was wiped with 75% ethanol; then placed in 250ml culture bottles in a sterile operating table, rinsed 5 times with sterile water, and then sterilized with 0.1% (w / v) mercuric chloride solution for 15 minutes; then rinsed 5 times with sterile water, and then rinsed 8 times with sterile water containing 400mg / L cephalosporin. After blotting the surface moisture with sterile filter paper, the tubers were cut into tubers with buds for later use.

[0055] (2) The tubers with buds, which had been disinfected in step (1), were inoculated into DKW germination medium supplemented with 1.0 mg / L Meta-Topolin, 0.5 mg / L GA3, 3.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. The buds were cultured in a constant-temperature incubator at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8h (light / dark). After 2 weeks of light culture, the contamination rate of the explants was 0.7%, and 85.7% of the buds germinated from the tubers of *Ginger indicum*.

[0056] (3) After detaching the sprouting buds of *Gynostemma pentaphyllum* from the explants in step (2), they were inoculated into DKW adventitious bud induction medium supplemented with 5.0 mg / L Leta-Topolin, 3.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. Adventitious buds were induced in a constant-temperature culture room at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8h (light / dark). After one week of light culture, buds sprouted and adventitious buds formed; after three weeks of light culture, the adventitious bud induction rate reached 88.6%, with an average of 4.7 adventitious buds per explant. Callus formation was observed at the base of the explants. Figure 8 As shown, it hinders the growth of adventitious buds in the later stages.

[0057] (4) The adventitious shoot clusters obtained in step (3) were transferred to DKW adventitious shoot proliferation medium supplemented with 0.5 mg / L TDZ, 2.0 mg / L Leta-Topolin, 3.0% (w / v) sucrose and 0.7% (w / v) agar, pH=5.8, and the adventitious shoots were cultured in a constant temperature culture room at 25±2℃, light intensity of 2500~3000lx, and photoperiod of 16 / 8h (light / dark); after 3 weeks of light culture, an average of 13.7 adventitious shoots were produced per explant.

[0058] (5) After one week of continued light culture, the obtained adventitious shoots were separated from the shoot clusters and transferred to DKW synchronous medium supplemented with 1.0 mg / L Leta-Topolin, 0.5 mg / L IBA, 2.0% (w / v) sucrose, and 0.7% (w / v) agar at pH 5.8. The adventitious shoots were cultured simultaneously in a constant-temperature incubator at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 16 / 8h (light / dark). After three weeks of light culture, the adventitious shoots achieved synchronized rooting while simultaneously strengthening and elongating, resulting in robust, complete regenerated plants. The adventitious root induction rate was as high as 100%, with an average of 7.4 adventitious roots per explant, and the average height of the regenerated plants was 4.5 cm.

[0059] (6) Place the seedling bottles containing the complete regenerated plants obtained in step (5) under natural conditions for acclimatization for 2 weeks. Take out the regenerated plants, wash them with running water to remove the culture medium from the roots, and then soak them in an aqueous solution with a mass fraction of 0.1% potassium permanganate for 12 minutes. Then transplant them into seedling trays containing mixed nutrient substrate and acclimatize them in a culture room with a temperature of 20-28℃, a humidity of 75%-85%, and a light intensity of 1800-4000 lx. The mixed nutrient substrate is a mixture of Pinsbury potting soil and vermiculite in a volume ratio of 2:3. After 4 weeks of culture, the survival rate of the regenerated plants after transplanting is 98.2%.

[0060] Example 4

[0061] This embodiment tested the effects of disinfectant type and disinfection time on the disinfection effect and regeneration ability of *Gynostemma pentaphyllum* tubers. At the end of August, young, healthy *Gynostemma pentaphyllum* tubers from superior strains provided by Anhui Tongque Erqiao Biotechnology Co., Ltd. were used as explants. After rinsing with running water for 30 minutes, the tubers were wiped with 75% ethanol. They were then placed in 250ml culture bottles on a sterile operating table and rinsed 8 times with sterile water. Different types and concentrations of disinfectants (0.1% (w / v) mercuric chloride solution, 20% (v / v) sodium hypochlorite solution, and 10% (v / v) hydrogen peroxide solution) were then used to treat the tubers for different times (9, 12, 15, and 18 minutes). After rinsing 5 times with sterile water, the tubers were rinsed 8 times with sterile water containing 400 mg / L cephalosporin. The surface moisture of the tubers was absorbed with sterile filter paper, and the tubers were cut into pieces with buds for later use. The disinfected tubers with buds were inoculated into the culture medium described in Example 2 for subsequent cultivation under the same conditions. The study found that the type of disinfectant and the disinfection time significantly affected the regeneration of buds and the contamination rate of ginger tubers. Among the three disinfectants tested, 0.1% mercuric chloride solution showed the best disinfection effect, followed by 20% sodium hypochlorite. With prolonged disinfection time, the contamination rate of the tubers gradually decreased, and the bud sprouting initially increased and then decreased with prolonged disinfection time. Specifically, when the tubers were disinfected with 0.1% mercuric chloride solution for 12 minutes, the contamination rate was only 1.3%, and 100% of the buds sprouted from the Tongling white ginger tubers (Table 1).

[0062] Table 1. Effects of disinfectant type and disinfection time on the regeneration of Tongling white ginger rhizomes.

[0063]

[0064] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0065] Example 5

[0066] This example tested the effects of the type and concentration of plant growth regulators on the induction of adventitious shoots in *Gynostemma pentaphyllum*. The buds of *Gynostemma pentaphyllum* that had been cultured on germination medium for 2 weeks in Example 2 were detached from the explants and inoculated into DKW adventitious shoot induction medium supplemented with different concentrations (1.0, 3.0, 5.0, and 8.0 mg / L) of cytokinins (KT, ZT, and Meta-Topolin), 3.0% (w / v) sucrose, and 0.7% (w / v) agar, at pH 5.8. The induction of adventitious shoots was carried out in a constant-temperature culture chamber at 25±2℃, a light intensity of 2500–3000 lx, and a photoperiod of 16 / 8 h (light / dark). The study found that among the three cytokinins tested, Meta-Topolin showed the best effect in inducing adventitious shoots in *Gynostemma pentaphyllum*. Furthermore, the induction efficiency gradually increased with increasing cytokinin concentration in the culture medium. The best induction effect was observed at a Meta-Topolin concentration of 3.0 mg / L, with an induction efficiency of up to 100%, producing an average of 6.9 adventitious shoots per explant. With further increases in Meta-Topolin concentration, the base of the *Gynostemma pentaphyllum* buds swelled, accompanied by callus formation, such as... Figure 9 As shown, this is not conducive to subsequent proliferation and elongation culture (Table 2).

[0067] Table 2. Effects of different types and concentrations of cytokinins on adventitious shoot induction in *Zingiber officinale* var. *tonglingensis*.

[0068]

[0069] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for annual production of Tongling white ginger seedlings, characterized by: The method comprises the following steps: S1, taking the current year's young and tender tubers of the excellent strain of Tongling white ginger as explant material, performing surface sterilization, then cutting the tubers with bud points, and inoculating the tubers into bud point germination culture medium to perform bud point germination culture; S2, after the germinated bud points in S1 are peeled off from the tubers, inoculating the bud points into adventitious bud induction culture medium to perform adventitious bud induction culture; the adventitious bud induction culture medium is DKW+1.0-5.0 mg / L Meta-Topolin+3.0% w / v sucrose+0.7% w / v agar, pH=5.8; S3, directly transferring the adventitious bud clusters induced in S2 into proliferation culture medium to perform adventitious bud proliferation culture; the proliferation culture medium is DKW+0.05-0.5 mg / L TDZ+0.5-2.0 mg / L Meta-Topolin+3.0% w / v sucrose+0.7% w / v agar, pH=5.8; S4, separating the adventitious buds proliferated in S3 from the bud clusters and transferring the adventitious buds into adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture medium to perform adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture, and obtaining complete regenerated plants; the adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture medium is DKW+0.5-1.0 mg / L Meta-Topolin+0.1-0.5 mg / L IBA+2.0% w / v sucrose+0.7% w / v agar, pH=5.8; S5, after the complete regenerated plants obtained in S4 are domesticated, washed and surface sterilized, performing seedling culture.

2. The method for annual production of Chongqing white ginger seedling according to claim 1, characterized in that: In S1, the culture time is 2 weeks; in S2, the culture time is 3 weeks; in S3, the culture time is 4 weeks; in S4, the culture time is 3 weeks.

3. The method for annual production of Chongqing white ginger seedling according to claim 1, characterized in that: In S1, the surface sterilization comprises the following steps: after the explant material is washed with flowing water for 30 min, the surface is wiped with 75% ethanol; then after the explant material is washed with sterile water for 5-8 times, the surface is sterilized with 0.1% w / v mercuric chloride solution for 9-18 min; then after the explant material is washed with sterile water for 3-5 times, the surface is washed with sterile water added with 300-500 mg / L cefotaxime for 6-8 times, and the surface water is absorbed with sterile filter paper.

4. The method for annual production of Chongqing white ginger seedlings according to claim 1, characterized in that: In S1, the bud point germination culture medium is DKW+0.1-1.0 mg / L Meta-Topolin+0.2-0.5 mg / L GA3+3.0% w / v sucrose+0.7% w / v agar, pH=5.

8.

5. The method for annual production of Chongqing Baijian seedling according to claim 1, characterized in that: S5 specifically comprises the following steps: The complete regenerated plants obtained in S4 are domesticated under natural conditions for 2 weeks, taken out, washed with flowing water, then soaked in an aqueous solution added with 0.05%-0.2% potassium permanganate for 10-15 min, and then transplanted into a seedling tray to perform seedling culture.

6. The method for annual production of Chongqing white ginger seedling according to claim 5, characterized in that: The seedling tray is provided with a mixed nutrient medium; the mixed nutrient medium is a mixture of Pinus nutrient soil and vermiculite in a volume ratio of 2-4:1-3.

7. The method for annual production of copperling white ginger seedling according to any one of claims 1-6, characterized in that: In S1-S4, the culture conditions are as follows: temperature 25±2℃, light intensity 2500-3000lx, light cycle 16h light / 8h dark; in S5, the conditions for seedling culture are as follows: temperature 20-28℃, humidity 75%-85%, light intensity 1800-4000lx.

Images