A method for inducing test tube microrhizomes of Curcuma zedoaria tissue culture seedlings

Abstract

The present invention belongs to the field of agricultural biotechnology and discloses a method for inducing test tube microrhizomes of Curcuma australis tissue culture seedlings. The method comprises taking a cluster of proliferating buds of the terminal buds of Curcuma australis obtained by conventional tissue culture and inoculating them into a rhizome induction medium for rooting culture. After 30 days of culture, a tissue culture seedling with rhizomes is obtained. The rhizome induction medium comprises: 1 / 2 MS + 0.1-1.0 mg / L NAA + 30 g / L sucrose + 1-3 mg / L choline chloride + 1-3 mg / L sodium nitrophenolate, with a pH of 5.8, wherein the weight ratio of choline chloride to sodium nitrophenolate is 1:(0.3-2). The method induces the formation of test tube microrhizomes of Curcuma australis in vitro, directly transplants the sterile microrhizomes, and produces artificial microrhizomes for sowing. This method omits the green seedling hardening and transplanting step and reduces seedling losses caused by the green seedlings' strict humidity requirements. The test tube microrhizome seedlings are also convenient for storage and long-distance transportation, thereby improving the efficiency of rhizome plant seedling propagation.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture and rapid propagation, and particularly relates to a method for inducing test tube microrhizomes of Curcuma aromatica tissue culture seedlings. Background Art

[0002] Curcuma wenyujin (Wenyujin) is a cultivated cultivar of the genus Curcuma in the Zingiberaceae family. It is a perennial medicinal plant primarily grown in Ruian, Wenzhou City, Zhejiang Province. The plant is rich in a variety of functional metabolic components, including volatile oils, curcuminoids, and polysaccharides. These components possess anti-tumor, anti-inflammatory, antibacterial, antiviral, antithrombotic, hepatoprotective, neuroprotective, and antioxidant properties. Its main medicinal parts are the rhizome and tubers, which can be processed into three different types of Chinese medicinal materials depending on the part and method of processing: the main rhizome, cooked and sun-dried, is called "Wenyuzhu"; the tubers, cooked and sun-dried, are called "Wenyujin"; and the fresh lateral rhizome, cut longitudinally into thick slices and then sun-dried, is called "Pianjianghuang." Wenyujin is one of the renowned "Eight Flavors of Zhejiang" medicinal herbs.

[0003] Curcumol, curclione, and β-elemene, among other active anticancer ingredients in zedoary turmeric volatile oil, are being developed. A growing number of new anti-tumor, anti-inflammatory, and antibacterial pharmaceuticals are being developed using zedoary volatile oil as a raw material, demonstrating promising efficacy and minimal side effects. For example, the gynecological topical medicine Baofukang Suppositories contain zedoary volatile oil as a primary ingredient. Curcuma zedoaria is highly sought after among commercial zedoary products for its high volatile oil content and excellent quality. Consequently, zedoary turmeric is cultivated not only in its native Ruian, Wenzhou, Zhejiang, but also in other suitable provinces across China, such as Hainan, Yunnan, and Guizhou.

[0004] The traditional planting material of Curcuma zedoaria is rhizomes. Long-term asexual reproduction causes the plants to carry and accumulate viruses and be infected with soil-borne pathogens to varying degrees, resulting in species degeneration and uneven rhizome quality. Using rhizomes as seed stems has a low reproduction coefficient, consumes a large amount of rhizomes that could have been used as commodities, and has high seedling costs. The seedlings propagated by rhizomes are of varying quality, resulting in uneven rhizome quality and reduced yield, making it difficult to obtain safe, effective, stable and controllable Chinese medicinal materials, posing serious challenges to the sustainable development of the Curcuma zedoaria industry.

[0005] At present, the use of tissue culture rapid propagation for the propagation of Curcuma aromatica tissue culture seedlings is not restricted by seasons and can be produced all year round, with significantly improved propagation efficiency. It is an effective supplement to conventional field rhizome propagation seedlings. However, when Curcuma aromatica is planted with tissue culture seedlings, the main method is to transplant rooted green seedlings. The roots are thin and long. During the seedling hardening and transplanting process, the roots are easily injured and easily affected by humidity, causing seedling death. If the seedlings are not hardened, their transplant survival rate is low or not stable enough, which is not conducive to large-scale promotion and application. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for inducing test tube microrhizomes of Curcuma aromatica tissue culture seedlings. By inducing the formation of test tube microrhizomes of Curcuma aromatica in vitro, sterile microrhizomes are directly transplanted and artificial microrhizomes are produced for sowing, thereby omitting the green seedling hardening and transplanting steps and reducing seedling losses caused by the green seedlings' strict requirements for humidity. In addition, the test tube microrhizome seedlings are conducive to storage and long-distance transportation, thereby improving the efficiency of rhizome plant seedling breeding.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for inducing test tube microrhizomes of Curcuma aromatica tissue culture seedlings comprises the following steps: obtaining a Curcuma aromatica proliferation cluster bud obtained by conventional tissue culture, inoculating a single bud into a rhizome induction medium for rooting culture, and obtaining tissue culture seedlings with rhizomes after 30 days of culture; the rhizome induction medium comprises: 1 / 2MS + 0.1-1.0 mg / L NAA + 30 g / L sucrose + 1-3 mg / L choline chloride + 1-3 mg / L sodium nitrophenolate, with a pH of 5.8, wherein the weight ratio of choline chloride to sodium nitrophenolate is 1:(0.3-2).

[0009] In an optional embodiment, the concentration of choline chloride is 3 mg / L, and the concentration of sodium nitrophenolate is 1 mg / L.

[0010] In an optional embodiment, the concentration of choline chloride is 1 mg / L, and the concentration of sodium nitrophenolate is 2 mg / L.

[0011] In an optional embodiment, the concentration of choline chloride is 2 mg / L, and the concentration of sodium nitrophenolate is 3 mg / L.

[0012] In an optional embodiment, the concentration of choline chloride is 3 mg / L, and the concentration of sodium nitrophenolate is 2 mg / L.

[0013] In an optional embodiment, the tissue culture process of the Curcuma aromatica adventitious buds is:

[0014] 1) Disinfection and sterilization of explants

[0015] Take the rhizomes of the best strains of Curcuma zedoariae, disinfect them and place them in a light incubator at 25-27°C for germination. When the buds grow to 2-3 cm, cut the buds and disinfect them.

[0016] 2) Induction culture

[0017] The upper half of the leaves of the disinfected sprouts are removed, leaving a bud length of 0.8 to 1.2 cm. The single bud is cut longitudinally into two pieces, and the pieces are inserted into an induction medium for early primary bud germination induction culture for 7 to 10 days. The culture conditions are: temperature of 24 to 26° C., light intensity of 1000 to 2000 lx, and light duration of 10 to 12 hours per day to obtain primary buds. The induction medium comprises MS, 1.5 to 2.5 g / L phytogel, and 30 g / L sucrose, with a pH of 5.8.

[0018] 3) Proliferation culture

[0019] The primary buds obtained through the early induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 24-26°C, light intensity of 1500-2000 lx, and light duration of 6-8 hours per day; a large number of clustered buds are obtained after 30 days of culture, and a new subculture proliferation medium is replaced to continue proliferation culture, and a large number of clustered buds are obtained after 3-4 subculture proliferation cultures; the subculture proliferation medium comprises: MS+4-5 mg / L 6-BA+0.1-0.5 mg / L NAA+2.1-2.5 g / L phytogel+30 g / L sucrose, and the pH value is 5.8.

[0020] In an optional embodiment, the rooting culture conditions are: temperature of 25-27°C, dark culture for 3-5 days, then light culture, light intensity of 1500-2500lx, light time of 8-12h / d, relative humidity of 75%.

[0021] In an optional embodiment, in step 1), the rhizome is disinfected with a 0.1% carbendazim solution for 10 to 15 minutes; the sprout is first disinfected with 70% alcohol for 45 to 60 seconds, rinsed twice with sterile water, and then disinfected with 2% sodium hypochlorite for 15 to 20 minutes, and rinsed five times with sterile water.

[0022] In an optional embodiment, in step 2), the upper half of the leaves of the sprout containing the growth point after disinfection are removed.

[0023] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0024] (1) The present invention adds choline chloride and sodium nitrophenolate in an appropriate ratio to the rooting medium, and the two can complement each other to effectively induce the occurrence and expansion of the test tube microrhizomes of Curcuma zedoariae. The tissue culture rooted seedlings with rhizomes are used for planting, which not only maintains the genetic stability of excellent germplasm and improves production efficiency, but also improves the quality of its medicinal materials. In the rhizomes harvested from the tissue culture seedlings, the main effective medicinal active ingredients in the volatile oil of Curcuma zedoariae, such as germacron, curdadione, and curcumin, are all higher than those of the corresponding ordinary Curcuma zedoariae.

[0025] (2) By using the sterile microrhizomes of the present invention for direct transplanting and manufacturing microrhizome artificial seeds for sowing, the steps of hardening and transplanting green seedlings can be omitted, and the seedling losses caused by the strict humidity requirements of green seedlings can be reduced. In addition, the test tube microrhizome seedlings are convenient for storage and long-distance transportation, thereby improving the efficiency of root plant seedling breeding.

[0026] (3) The tissue culture rooted seedlings with rhizomes of the present invention have higher survival rates after transplantation and higher yields of rhizomes and tubers than conventional tissue culture rooted seedlings, which can effectively simplify the acclimatization and transplantation of tissue culture seedlings and promote their growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention relates to the extraction and germination of rhizomes of Curcuma aromatica in the embodiments of the present invention. A. Fresh rhizomes of Curcuma aromatica; B. Germination of rhizomes; C. Rhizome sprouting explants; D. Sterile buds.

[0028] Figure 2 3. This is a diagram showing the effects of different rooting culture media on rhizome differentiation induction of Curcuma aromatica in the examples of the present invention.

[0029] Figure 3 The effect of the in vitro microrhizomes of the present invention on the growth potential of the tissue culture seedlings of Curcuma zedoariae is shown in Figure 1. A shows the plant morphology of the tissue culture seedlings of Curcuma zedoariae after 60 days of potting; and B shows the growth and morphology of the underground roots and rhizomes after 90 days of potting. DETAILED DESCRIPTION

[0030] The following examples further describe the specific embodiments of the present invention. The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0031] The procedures for obtaining tissue culture buds in the following examples and control examples are the same, except that the components and concentration ratios of the culture medium are different when the tissue culture buds are subjected to rhizome induction culture. Figure 1 As shown, Figure 1 Figure A shows a fresh rhizome of Curcuma zedoaria taken from Hainan; Figure B shows the germination of the rhizome; Figure C shows the sprouts cut from the rhizome; Figure D shows the sterile buds obtained after the sprouts were proliferated and cultured; the obtained adventitious buds were used as the rhizome induction test for the examples and control examples.

[0032] 1. Curcuma zedoaria seedling breeding

[0033] Example 1

[0034] This embodiment provides a method for inducing microrhizomes of Curcuma aromatica tissue culture seedlings in vitro, comprising the following steps:

[0035] 1. Disinfection and sterilization of explants

[0036] In December 2023, rhizome explants were selected and introduced from healthy and high-yielding plants that have adapted to the ecological environment of Hainan. When collecting rhizomes, try to avoid wounds and maintain the integrity of the rhizomes. After cleaning and drying, use a 0.1% solution of a broad-spectrum fungicide, carbendazim (effective content 50% wettable powder), for preliminary surface disinfection for 10 minutes. Then, place the rhizomes in a (25±2)℃ light incubator for germination. When the buds grow to 3 cm, cut the buds for surface disinfection. The surface disinfection method is to first disinfect with 70% alcohol for 45 seconds, rinse with sterile water twice, and then disinfect with 2% sodium hypochlorite for 15 minutes, and rinse with sterile water 5 times.

[0037] 2. Induction Culture

[0038] The upper half of the leaves of the disinfected sprout containing the growth point are removed, leaving a bud length of 1 cm, and the single bud is cut into two pieces longitudinally. The pieces are inserted into an induction culture medium for early primary bud germination induction culture for 7 days under the following culture conditions: temperature of 26°C, light intensity of 1500 lx, and light duration of 12 hours per day; primary buds are obtained; the induction culture medium comprises MS+2.3 g / L plant gel+30 g / L sucrose, and has a pH value of 5.8.

[0039] 3. Proliferation Culture

[0040] The primary buds obtained by the early induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 26°C, light intensity of 1500 lx, and light duration of 8 hours per day; after culturing for 30 days, a large number of clustered buds are obtained, and a new subculture proliferation medium is replaced, and the clustered buds are cut and proliferation culture is continued, and a large number of clustered buds are obtained after three subculture proliferation cultures; the subculture proliferation medium includes: MS+5 mg / L 6-BA+0.5 mg / L NAA+2.3 g / L phytogel+30 g / L sucrose, and has a pH value of 5.8.

[0041] 4. Rooting Culture

[0042] The proliferating buds of Curcuma zedoaria are cut into single buds, and the excess tissue, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds of about 3 cm with the growth point retained. The buds are transferred to a rhizome induction medium by single bud grafting and cultured. The culture conditions are as follows: temperature of 25°C, dark culture for 3 days, followed by light culture with a light intensity of 2500 lx, a light duration of 8 hours / day, and a relative humidity of 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium comprises: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 3 mg / L + sodium nitrophenolate 1 mg / L, and a pH of 5.8.

[0043] 5. Transplanting

[0044] Transplant the tissue culture seedlings with rhizomes directly into small pots filled with substrate (vermiculite: nutrient soil = 1:1), slow down the growth of the seedlings under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water according to weather conditions, and water once a month with a 1000-fold diluted soluble fertilizer solution.

[0045] Example 2

[0046] This embodiment provides a method for inducing microrhizomes of Curcuma aromatica tissue culture seedlings in vitro, comprising the following steps:

[0047] 1. Disinfection and sterilization of explants

[0048] In December 2023, rhizome explants were selected and introduced from healthy and high-yielding plants that have adapted to the ecological environment of Hainan. When collecting rhizomes, try to avoid wounds and maintain the integrity of the rhizomes. After cleaning and drying, use a 0.1% solution of a broad-spectrum fungicide, carbendazim (effective content 50% wettable powder), for preliminary surface disinfection for 10 minutes. Then, place the rhizomes in a (25±2)℃ light incubator for germination. When the buds grow to 3 cm, cut the buds for surface disinfection. The surface disinfection method is to first disinfect with 70% alcohol for 45 seconds, rinse with sterile water twice, and then disinfect with 2% sodium hypochlorite for 15 minutes, and rinse with sterile water 5 times.

[0049] 2. Induction Culture

[0050] The upper half of the leaves of the disinfected sprout containing the growth point are removed, leaving a bud length of 1 cm, and the single bud is cut into two pieces longitudinally, and inserted into an induction culture medium for early primary bud germination induction culture for 7 days; the culture conditions are: temperature of 26°C, light intensity of 1500lx, and light duration of 12 hours / day; primary buds are obtained; the induction culture medium comprises MS+2.3g / L plant gel+30g / L sucrose, and has a pH value of 5.8.

[0051] 3. Proliferation Culture

[0052] The sterile primary buds obtained through the preliminary induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 26°C, light intensity of 1500 lx, and light duration of 8 hours per day; after culturing for 30 days, a large number of clustered buds are obtained, a new subculture proliferation medium is replaced, the clustered buds are cut and proliferation culture is continued, and a large number of clustered buds are obtained after three subculture proliferation cultures; the subculture proliferation medium comprises: MS+5 mg / L 6-BA+0.5 mg / L NAA+2.3 g / L phytogel+30 g / L sucrose, and has a pH value of 5.8.

[0053] 4. Rooting Culture

[0054] The proliferating buds of Curcuma zedoaria are cut into single buds, and the excess tissue, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds of about 3 cm with the growth point retained. The buds are transferred to a rhizome induction medium by single bud grafting and cultured. The culture conditions are as follows: temperature of 25°C, dark culture for 3 days, followed by light culture with a light intensity of 2000 lx, a light duration of 10 h / d, and a relative humidity of 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium comprises: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 1 mg / L + sodium nitrophenolate 2 mg / L, and a pH of 5.8.

[0055] 5. Transplanting

[0056] Transplant the tissue culture seedlings with rhizomes directly into small pots filled with substrate (vermiculite: nutrient soil = 1:1), slow down the growth of the seedlings under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water according to weather conditions, and water once a month with a 1000-fold diluted soluble fertilizer solution.

[0057] Example 3

[0058] This embodiment provides a method for inducing microrhizomes of Curcuma aromatica tissue culture seedlings in vitro, comprising the following steps:

[0059] 1. Disinfection and sterilization of explants

[0060] In December 2023, rhizome explants were selected and introduced from healthy and high-yielding plants that have adapted to the ecological environment of Hainan. When collecting rhizomes, try to avoid wounds and maintain the integrity of the rhizomes. After cleaning and drying, use a 0.1% solution of a broad-spectrum fungicide, carbendazim (effective content 50% wettable powder), for preliminary surface disinfection for 10 minutes. Then, place the rhizomes in a (25±2)℃ light incubator for germination. When the buds grow to 3 cm, cut the buds for surface disinfection. The surface disinfection method is to first disinfect with 70% alcohol for 45 seconds, rinse with sterile water twice, and then disinfect with 2% sodium hypochlorite for 15 minutes, and rinse with sterile water 5 times.

[0061] 2. Induction Culture

[0062] The upper half of the leaves of the disinfected sprout containing the growth point are removed, leaving a bud length of 1 cm, and the single bud is cut into two pieces longitudinally, and inserted into an induction culture medium for early primary bud germination induction culture for 7 days; the culture conditions are: temperature of 26°C, light intensity of 1500lx, and light duration of 12 hours / day; primary buds are obtained; the induction culture medium comprises MS+2.3g / L plant gel+30g / L sucrose, and has a pH value of 5.8.

[0063] 3. Proliferation Culture

[0064] The sterile primary buds obtained through the preliminary induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 26°C, light intensity of 1500 lx, and light duration of 8 hours per day; after culturing for 30 days, a large number of clustered buds are obtained, a new subculture proliferation medium is replaced, the clustered buds are cut and proliferation culture is continued, and a large number of clustered buds are obtained after three subculture proliferation cultures; the subculture proliferation medium comprises: MS+5 mg / L 6-BA+0.5 mg / L NAA+2.3 g / L phytogel+30 g / L sucrose, and has a pH value of 5.8.

[0065] 4. Rooting Culture

[0066] The proliferating buds of Curcuma zedoaria are cut into single buds, and the excess tissue, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds of about 3 cm with the growth point retained. The buds are transferred to a rhizome induction medium by single bud grafting and cultured. The culture conditions are as follows: temperature of 25°C, dark culture for 3 days, followed by light culture with a light intensity of 1500 lx, a light duration of 12 h / d, and a relative humidity of 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium comprises: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 2 mg / L + sodium nitrophenolate 3 mg / L, with a pH of 5.8.

[0067] 5. Transplanting

[0068] Transplant tissue-cultured seedlings with rhizomes directly into small pots filled with substrate (vermiculite:nutrient soil = 1:1). Allow them to grow under diffuse light for 10 days, then place the potted seedlings under normal light. Water each pot with deionized water based on weather conditions, and water once a month with a 1000-fold diluted soluble fertilizer solution.

[0069] Example 4

[0070] This embodiment provides a method for inducing microrhizomes of Curcuma aromatica tissue culture seedlings in vitro, comprising the following steps:

[0071] 1. Disinfection and sterilization of explants

[0072] In December 2023, rhizome explants were selected and introduced from healthy and high-yielding plants that have adapted to the ecological environment of Hainan. When collecting rhizomes, try to avoid wounds and maintain the integrity of the rhizomes. After cleaning and drying, use a 0.1% solution of a broad-spectrum fungicide, carbendazim (effective content 50% wettable powder), for preliminary surface disinfection for 10 minutes. Then, place the rhizomes in a (25±2)℃ light incubator for germination. When the buds grow to 3 cm, cut the buds for surface disinfection. The surface disinfection method is to first disinfect with 70% alcohol for 45 seconds, rinse with sterile water twice, and then disinfect with 2% sodium hypochlorite for 15 minutes, and rinse with sterile water 5 times.

[0073] 2. Induction Culture

[0074] The upper half of the leaves of the disinfected sprout containing the growth point are removed, leaving a bud length of 1 cm, and the single bud is cut into two pieces longitudinally, and inserted into an induction culture medium for early primary bud germination induction culture for 7 days; the culture conditions are: temperature of 26°C, light intensity of 1500lx, and light duration of 12 hours / day; primary buds are obtained; the induction culture medium comprises MS+2.3g / L plant gel+30g / L sucrose, and has a pH value of 5.8.

[0075] 3. Proliferation Culture

[0076] The sterile primary buds obtained through the preliminary induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 26°C, light intensity of 1500 lx, and light duration of 8 hours per day; after culturing for 30 days, a large number of clustered buds are obtained, a new subculture proliferation medium is replaced, the clustered buds are cut and proliferation culture is continued, and the subculture proliferation culture is repeated three times to obtain a large number of clustered buds; the subculture proliferation medium includes: MS+5 mg / L 6-BA+0.5 mg / L NAA+2.3 g / L phytogel+30 g / L sucrose, and the pH value is 5.8.

[0077] 4. Rooting Culture

[0078] The proliferating buds of Curcuma zedoaria are cut into single buds, and the excess tissue, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds of about 3 cm with the growth point retained. The buds are transferred to a rhizome induction medium by single bud grafting and cultured. The culture conditions are as follows: temperature of 25°C, dark culture for 3 days, followed by light culture with a light intensity of 1500 lx, a light duration of 12 h / d, and a relative humidity of 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium comprises: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 3 mg / L + sodium nitrophenolate 2 mg / L, with a pH of 5.8.

[0079] 5. Transplanting

[0080] Transplant the tissue culture seedlings with rhizomes directly into small pots filled with substrate (vermiculite: nutrient soil = 1:1), slow down the growth of the seedlings under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water according to weather conditions, and water once a month with a 1000-fold diluted soluble fertilizer solution.

[0081] Comparative test

[0082] Comparative Example 1

[0083] The process of obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the two plant regulators, choline chloride and sodium nitrophenolate, are not added to the rhizome induction medium. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L.

[0084] Comparative Example 2

[0085] The process for obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the concentration ratio between choline chloride and sodium nitrophenolate in the rhizome induction medium is different. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 3 mg / L + sodium nitrophenolate 0.6 mg / L.

[0086] Comparative Example 3

[0087] The process of obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the concentration ratio between choline chloride and sodium nitrophenolate in the rhizome induction medium is different. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 1 mg / L + sodium nitrophenolate 3 mg / L.

[0088] Comparative Example 4

[0089] The process for obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the concentration ratio between choline chloride and sodium nitrophenolate in the rhizome induction medium is different. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 2 mg / L + sodium nitrophenolate 0.2 mg / L.

[0090] Comparative Example 5

[0091] The process for obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the concentration ratio between choline chloride and sodium nitrophenolate in the rhizome induction medium is different. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 2 mg / L + sodium nitrophenolate 0.3 mg / L.

[0092] Comparative Example 6

[0093] The process of obtaining tissue culture seedlings in this control example is the same as that in Example 1 above, except that in the rooting culture step of this comparative example, the concentration ratio between choline chloride and sodium nitrophenolate in the rhizome induction medium is different. The composition of the rhizome induction medium is: 1 / 2MS + NAA 0.5 mg / L + sucrose 30 g / L + choline chloride 1 mg / L + sodium nitrophenolate 4 mg / L.

[0094] 2. Identification of tissue culture effects

[0095] 1. Identification of the effect of inducing differentiation of bud microrhizomes from Curcuma zedoaria tissue culture

[0096] To evaluate the effect of rhizome induction medium on the differentiation of microrhizomes from tissue cultured buds of Curcuma aromatica, the induction rate, length, and diameter of microrhizomes from tissue cultured buds of Curcuma aromatica in the above examples and control examples were observed and calculated. Microrhizome induction rate (%) = (number of buds induced into microrhizomes / number of original inoculated buds) × 100%. Rhizome length (cm), root length (cm), and rhizome diameter (mm) were measured with a vernier caliper. The results are shown in Tables 1 and Figure 2 .

[0097] Table 1 Effects of different rooting media on the differentiation of bud microrhizomes in tissue culture of Curcuma zedoaria

[0098] project Rhizome induction rate (%) Rhizome length (cm) Root diameter (mm) Root length (cm) Root diameter (mm) Example 1 46±2.6b 1.7±0.2a 6.8±0.1a 2.8±0.2e 4.5±0.2a Example 2 62±1.0a 1.8±0.2a 4.5±0.3b 4.3±0.3a 2.1±0.0b Example 3 20±2.0d 0.9±0.6b 3.1±0.2c 3.5±0.1cd 1.8±0.2c Example 4 28±0.6c 0.5±0.3c 2.8±0.2c 3.6±0.0cd 1.6±0.0cd Comparative Example 1 / / / 3.5±0.1d 0.4±0.1g Comparative Example 2 / / / 3.2±0.1de 1.1±0.1ef Comparative Example 3 / / / 3.4±0.4d 1.3±0.1ef Comparative Example 4 / / / 4.3±0.1b 0.7±0.2g Comparative Example 5 / / / 4.1±0.2bc 1.1±0.2f Comparative Example 6 / / / 3.4±0.0de 1.4±0.1de

[0099] *Note: Different lowercase letters in the same column of numbers in the table indicate significant differences (P<0.05).

[0100] The above-mentioned Wen Yu Jin tissue culture bud micro-rhizome induction differentiation effect shows that the rhizome induction medium of the present invention (Example 1, Example 2, Example 3, Example 4, Control Example 2, Control Example 3, Control Example 4, Control Example 5, Control Example 6) and the conventional rooting medium (Control Example 1) can both induce rooting, without obvious main roots, but only Example 1, Example 2, Example 3 and Example 4 can induce differentiation to form rhizomes longer than 0.5 cm (rhizomes with a length of less than 0.5 cm are not counted), and the induction rates between them are significantly different, with Example 2 having the highest induction rate of 62%, followed by Example 1, which reaches 46%. Among these four examples, the order in which rhizomes can be induced is: Example 2> Example 1> Example 4> Example 3.

[0101] Among them, Figure 2 In the figure, A is the rhizome induction of Example 1; B is the rhizome induction of Example 2; C is the rhizome induction of Example 3; D is the rhizome induction of Example 4; E is the rhizome induction of Control Example 1; F is the rhizome induction of Control Example 2; G is the rhizome induction of Control Example 3; H is the rhizome induction of Control Example 4; I is the rhizome induction of Control Example 5; and J is the rhizome induction of Control Example 6.

[0102] The length and thickness of microrhizomes are key indicators for measuring microrhizome growth and yield. Figure 2 It can be seen that the rhizome induction medium of Example 2 still significantly promotes the elongation of microrhizomes (e.g. Figure 2 There is no significant difference from Example 1 (as shown in Figure B). Figure 2 ), but both are significantly stronger than Example 3 (as shown in Figure A). Figure 2 C in FIG) and Example 4 (as shown in FIG Figure 2 (as shown in Figure D in the figure), while Example 1 was the most effective in promoting rhizome thickening, with the maximum rhizome diameter reaching 6.8 mm, followed by Example 2, both of which were significantly higher than Examples 3 and 4. Taking into account the rhizome induction rate, rhizome length, and rhizome thickness, the induction effects of the four examples were, in descending order: Example 1 > Example 2 > Example 3 > Example 4.

[0103] Compared with Examples 2-6, Examples 1-4 of the present invention can maintain the growth of rhizomes while improving the induction rate, indicating that only when the concentration ratio of the two plant regulators, choline chloride and sodium nitrophenolate, is added within a reasonable range can it be beneficial to the induction and differentiation of microrhizomes of Curcuma aromatica and promote the growth of rhizomes. When the concentration ratio exceeds this reasonable range, the formation of rhizomes cannot be induced.

[0104] 2. Identification of the Growth Morphology of Curcuma zedoaria Tissue Culture Buds

[0105] To identify the effect of the rhizome induction medium on the growth morphology of Curcuma australis tissue culture buds, the growth status of the Curcuma australis tissue culture buds during rhizome induction culture in the above examples and control examples was observed. The results are shown in Table 2.

[0106] Table 2 Effects of different rooting media on the growth status of Curcuma zedoaria tissue culture buds

[0107]

[0108]

[0109] The above-mentioned Curcuma aromatica tissue culture bud growth status shows that when the Curcuma aromatica tissue culture buds are subjected to rooting induction using ordinary rooting medium, the buds are small, the growth potential is poor, and the leaves are light green; when the rhizome induction medium of the present invention (Example 1, Example 2, Example 3, Example 4) is used, the growth status of the buds is better, with Example 1 and Example 2 being the best, and the buds grow vigorously, are thick, and the leaves are dark green; however, when the choline chloride and sodium nitrophenolate in the rhizome induction medium exceed the appropriate range (Control Example 2, Control Example 3, Control Example 4, Control Example 5, and Control Example 6), only the bud growth status of Control Example 2 and Control Example 4 is normal, with good growth and green leaves, while the growth status of the remaining buds is poor.

[0110] 3. Identification of the Growth Potential of Curcuma zedoaria Tissue Culture Seedlings

[0111] In order to identify the growth effect of the Curcuma zedoaria tissue culture seedlings with rhizomes after transplantation, the Curcuma zedoaria tissue culture seedlings in the above Example 1 and Control Example 1 were transplanted into pots for cultivation. The survival rate (%) was calculated after the 30th day, the absolute growth rate AGR (mm / d) was measured between the 31st and 40th days, and the number of sprouted leaves and tillers (pieces or pieces / plant) of the tissue culture seedlings were observed and counted on the 60th day, as well as the weight of the underground part (g, including rhizomes and tubers). Among them, the transplant survival rate (%) = (number of surviving plants / total number of transplanted plants) × 100%, AGR is the average value of 10 days; the growth of underground rhizomes and tubers (g) was measured by weighing on a balance. The results are shown in Table 3 and Figure 3 shown.

[0112] Table 3 Transplantation growth of Curcuma zedoaria tissue culture seedlings

[0113]

[0114] The above-mentioned Wenyujin tissue culture seedling transplant growth situation shows that, compared with Wenyujin conventional rooting tissue culture seedlings (reference example 1), the tissue culture rooting seedlings with rhizomes (Example 1) can significantly improve its transplant efficiency and growth potential at different stages. Compared with reference example 1 (potting with conventional tissue culture rooting seedlings), Example 1 (potting with rhizomes) has a survival rate of 100%, a growth rate of (1.7 ± 0.3) mm / d, a tiller number of (9.7 ± 1.5), a number of new leaves of (21.0 ± 3.6), and a weight of underground roots and rhizomes of (33.7 ± 1.3) g / plant, which are significantly increased by 36.4%, 112.5%, 83.0%, 103.9% and 83.2%, respectively. In summary, tissue culture rooting seedlings with rhizomes can effectively simplify the acclimatization and transplanting of tissue culture seedlings and promote growth.

[0115] After 60 days of potting, the morphology of the plants was observed. Figure 3As shown in Figure A (the left figure is the plant growth morphology diagram in Control Example 1; the right figure is the plant growth morphology diagram in Example 1), it can be seen from Figure A that the growth rate of the plant in Control Example 1 is slower. 、 The number of tillers and newly added leaves is small, indicating that the tissue culture seedlings obtained by the present invention can increase the growth rate of the plant when planted; after 90 days of potting, the underground roots and rhizomes of the Curcuma zedoaria tissue culture seedlings were taken out to observe their growth morphology. Figure 3 As shown in Figure B (the left figure is the plant growth morphology diagram in Control Example 1; the right figure is the plant growth morphology diagram in Example 1), it can be seen from Figure B that the number and weight of rhizomes and tubers in Control Example 1 are less than those in Example 1, indicating that the number of rhizomes is larger and thicker when the tissue culture seedlings of the present invention are used for planting.

[0116] Although the present invention has been described using the above preferred embodiments, they are not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. A method for inducing microrhizomes of Curcuma aromatica tissue culture seedlings in vitro, characterized in that: The buds of Curcuma zedoaria proliferated by conventional tissue culture were inoculated into rhizome induction medium for rooting, and tissue culture seedlings with rhizomes were obtained after 30 days of culture. The rhizome induction medium: 1 / 2 MS + NAA 0.1-1.0 mg / L + sucrose 30 g / L + choline chloride 1-3 mg / L + sodium nitrophenolate 1-3 mg / L, pH 5.8; Wherein, the concentration of the choline chloride is 3 mg / L, and the concentration of the sodium nitrophenolate is 1 mg / L; or the concentration of the choline chloride is 1 mg / L, and the concentration of the sodium nitrophenolate is 2 mg / L; or the concentration of the choline chloride is 2 mg / L, and the concentration of the sodium nitrophenolate is 3 mg / L; or the concentration of the choline chloride is 3 mg / L, and the concentration of the sodium nitrophenolate is 2 mg / L; The rooting culture conditions are as follows: temperature of 25-27° C., dark culture for 3-5 days, and then light culture, with a light intensity of 1500-2500 lx, a light duration of 8-12 h / d, and a relative humidity of 75%.

2. The method for inducing the microrhizomes of warm turmeric tissue culture seedlings in test tube according to claim 1, wherein The tissue culture process of the Curcuma aromatica adventitious buds is as follows: 1) Disinfection and sterilization of explants Take the rhizomes of the best strains of Curcuma zedoariae, disinfect them and place them in a light incubator at 25-27°C for germination. When the buds grow to 2-3 cm, cut the buds and disinfect them. 2) Induction culture The upper half of the leaves of the disinfected sprouts were removed, leaving a bud length of 0.8 to 1.2 cm. The single sprouts were cut longitudinally into two pieces, and the pieces were inserted into an induction medium for early primary bud germination induction culture for 7 to 10 days. The culture conditions were: temperature of 24 to 26°C, light intensity of 1000 to 2000 lx, and light duration of 10 to 12 hours per day to obtain primary buds. The induction medium consisted of MS + 1.5 to 2.5 g / L phytogel + 30 g / L sucrose, with a pH of 5.

8. 3) Proliferation culture The primary buds obtained through the early induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: temperature of 24-26°C, light intensity of 1500-2000 lx, and light duration of 6-8 h / day; after 30 days of culture, a large number of clustered buds are obtained, and a new subculture proliferation medium is replaced for continued proliferation culture, and a large number of clustered buds are obtained after 3-4 subculture proliferation cultures; the subculture proliferation medium comprises: MS + 4-5 mg / L 6-BA + 0.1-0.5 mg / L NAA + 2.1-2.5 g / L phytogel + 30 g / L sucrose, with a pH value of 5.

8.

3. The method for inducing the microrhizomes of warm turmeric tissue culture seedlings in test tube according to claim 2, wherein In step 1), the rhizome is sterilized with a 0.1% carbendazim solution for 10 to 15 minutes; the sprout is first disinfected with 70% alcohol for 45 to 60 seconds, rinsed twice with sterile water, and then disinfected with 2% sodium hypochlorite for 15 to 20 minutes, and rinsed five times with sterile water.

4. The method for inducing the microrhizomes of turmeric tissue culture seedlings in test tube according to claim 2, wherein: In step 2), the upper half of the leaves of the bud containing the growth point are removed after disinfection.

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