Method for inducing curcuma wenyujin tissue culture seedling test tube micro rhizome

Abstract

The invention belongs to the technical field of agricultural biology, and discloses a method for inducing curcuma wenyujin tissue culture seedling test-tube micro rootstocks, which comprises the following steps: inoculating terminal bud cluster proliferation buds of curcuma wenyujin obtained by conventional tissue culture into a rootstock induction culture medium for rooting culture for 30 days to obtain tissue culture seedlings with rootstocks, the rhizome induction culture medium is prepared from 1 / 2MS, 0.1-1.0 mg / L of NAA, 30 g / L of cane sugar, 1-3 mg / L of choline chloride and 1-3 mg / L of compound sodium nitrophenolate, the pH is 5.8, and the weight ratio of choline chloride to compound sodium nitrophenolate is 1: (0.3-2). According to the method, the formation of the curcuma wenyujin test-tube micro rhizomes is induced in vitro, the sterile micro rhizomes are directly transplanted, and the micro rhizome artificial seeds are manufactured for sowing, so that a green seedling hardening and temporary planting link is omitted, and the seedling loss caused by strict requirements of green seedlings on humidity is reduced, and the test-tube micro rhizome seedlings are beneficial to storage and long-distance transportation; therefore, the seedling breeding efficiency of the rhizome plants is improved.

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 micro-rhizomes of Curcuma aromatica tissue culture seedlings. Background Art

[0002] Curcuma wenyujin is a cultivar of Curcuma genus of Zingiberaceae. It is a perennial medicinal plant, mainly produced in Ruian, Wenzhou City, Zhejiang Province. The plant is rich in various functional metabolic components such as volatile oil of Curcuma zedoariae, curcumin and polysaccharide, and has anti-tumor, anti-inflammatory and antibacterial, anti-viral, anti-thrombotic, liver-protective, nerve-protective and anti-oxidative effects. Its medicinal parts are mainly rhizomes and tubers. According to different parts and methods, it can be processed into three different Chinese medicinal materials: the main rhizome is boiled and dried, which is called "Wen Curcuma", the tubers are boiled and dried, which is called "Wen Curcuma", and the fresh lateral rhizomes are cut into thick slices and dried, which is called "Pian Jianghuang". Among them, "Wen Curcuma" is one of the famous local medicinal materials "Zhe Eight Flavors".

[0003] Curcumol, curclione, β-elemene and other active ingredients in the volatile oil of Curcuma are anti-cancer ingredients. More and more new anti-tumor, anti-inflammatory and antibacterial drugs with good efficacy and few side effects are developed using volatile oil of Curcuma as raw materials. For example, the main ingredient of Baofukang suppository, a good gynecological external medicine, is volatile oil of Curcuma. Among the commercial products of Curcuma, Wen Curcuma is highly favored for its high volatile oil content and good quality. Therefore, Wen Curcuma is not only cultivated in its original place of origin, Ruian, Wenzhou, Zhejiang, but also introduced and expanded in other suitable provinces in China such as Hainan, Yunnan, Guizhou and other places.

[0004] The traditional planting material of Curcuma aromatica 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 quality of seedlings propagated from rhizomes varies, resulting in uneven rhizome quality and reduced yield, making it difficult to obtain safe, effective, stable and controllable Chinese medicinal materials, all of which pose serious challenges to the sustainable development of the Curcuma aromatica industry.

[0005] At present, the use of tissue culture rapid propagation for the breeding of Curcuma aromatica tissue culture seedlings is not restricted by seasons and can be produced all year round. The breeding efficiency is significantly improved. It is an effective supplement to the 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. The roots are easily injured during the seedling hardening and transplanting process and are easily affected by humidity, causing seedling death. If the seedlings are not hardened, the transplanting 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 australis tissue culture seedlings, by inducing the formation of test tube microrhizomes of Curcuma australis in vitro, directly transplanting and manufacturing microrhizomes artificial seeds for sowing using sterile microrhizomes, omitting the steps of hardening and transplanting green seedlings and reducing the seedling losses caused by the strict requirements of green seedlings on humidity, and the test tube microrhizomes 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 micro-rhizomes of Curcuma australis tissue culture seedlings comprises the following steps: taking Curcuma australis proliferation cluster buds 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 culturing for 30 days; the rhizome induction medium comprises: 1 / 2MS+NAA 0.1-1.0 mg / L+sucrose 30 g / L+choline chloride 1-3 mg / L+sodium nitrophenolate 1-3 mg / L, with a pH value 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 australis 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℃ for germination. When the buds grow to 2-3cm, 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, and the single bud is cut into two pieces longitudinally, and 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 time of 10 to 12 hours per day to obtain primary buds; the induction medium includes: MS + 1.5 to 2.5 g / L plant gel + 30 g / L sucrose, and the pH value is 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: the temperature is 24-26 DEG C, the light intensity is 1500-2000lx, and the light time is 6-8h / day; after culturing for 30 days, a large number of clustered buds are obtained, and a new subculture proliferation medium is replaced to continue the proliferation culture, and a large number of clustered buds are obtained after 3-4 subculture proliferation cultures; the subculture proliferation medium: MS+4-5mg / L 6-BA+0.1-0.5mg / L NAA+2.1-2.5g / L plant gel+30g / 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, and then light culture, the light intensity is 1500-2500lx, the light time is 8-12h / d, and the relative humidity is 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 with sterile water twice, then disinfected with 2% sodium hypochlorite for 15 to 20 minutes, and rinsed with sterile water 5 times.

[0022] In an optional embodiment, in step 2), the upper half of the leaves of the sprouts containing the growth points 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 test tube microrhizomes of Curcuma australis. 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 zedoaria, such as germacron, curcuma dione, and curcumin, are all higher than those of the corresponding ordinary Curcuma australis.

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

[0026] (3) The survival rate of the tissue culture rooted seedlings with rhizomes after transplantation and the yield of rhizomes and tubers are higher than those of 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. explants of rhizomes sprouting; D. sterile buds.

[0028] Figure 2 Graph showing the effects of different rooting culture media on rhizome differentiation induction in 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 australis. A is the plant morphology of the tissue culture seedlings of Curcuma australis after 60 days of potting; B is the growth and morphology of the underground roots and rhizomes after 90 days of potting. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described in detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0031] The process of obtaining tissue culture buds in the following examples and control examples is 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 in the figure shows fresh rhizomes of Curcuma aromatica taken from Hainan; Figure B shows the germination of rhizomes; Figure C shows sprouts cut from rhizomes; Figure D shows sterile buds obtained after proliferation and cultivation of sprouts; the obtained adventitious buds are used as rhizome induction tests for embodiments and control examples.

[0032] 1. Breeding of Curcuma australis seedlings

[0033] Example 1

[0034] This embodiment provides a method for inducing test tube micro-rhizomes of Curcuma australis tissue culture seedlings, 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 superior 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, 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 sprout containing the growth point after disinfection are removed, leaving the sprout with a length of 1 cm, and the single sprout is cut into two pieces longitudinally, and 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 1500lx, and light duration of 12h / day; primary buds are obtained; the induction culture medium includes: MS+2.3g / L plant gel+30g / L sucrose, and the pH value is 5.8.

[0039] 3. Proliferation Culture

[0040] The primary buds obtained by the previous induction are inoculated into a subculture proliferation medium for proliferation culture, and the culture conditions are as follows: the temperature is 26°C, the light intensity is 1500lx, and the lighting time is 8h / 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 continued to be proliferated and cultured, and a large number of clustered buds are obtained after subculture proliferation culture for 3 times; the subculture proliferation medium includes: MS+5mg / L 6-BA+0.5mg / L NAA+2.3g / L plant gel+30g / L sucrose, and the pH value is 5.8.

[0041] 4. Rooting Culture

[0042] The proliferation cluster buds of Curcuma australis are cut into single buds, and the excess tissues, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds with a growth point of about 3 cm, and the buds are transferred to a rhizome induction medium for culture in a single bud grafting manner. The culture conditions are: the temperature is 25°C, dark culture is first carried out for 3 days, and then light culture is carried out, the light intensity is 2500lx, the light time is 8h / d, and the relative humidity is 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium is: 1 / 2MS+NAA 0.5mg / L+sucrose 30g / L+choline chloride 3mg / L+sodium nitrophenolate 1mg / L, and the pH is 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), acclimate under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water depending on 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 test tube micro-rhizomes of Curcuma australis tissue culture seedlings, 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 superior 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, 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 sprout containing the growth point after disinfection are removed, leaving the sprout with a length of 1 cm, and the single sprout 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 time of 12h / day; primary buds are obtained; the induction culture medium includes: MS+2.3g / L plant gel+30g / L sucrose, and the pH value is 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: the temperature is 26°C, the light intensity is 1500lx, and the lighting time is 8h / 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 to continue proliferation culture, and a large number of clustered buds are obtained after subculture proliferation culture for 3 times; the subculture proliferation medium includes: MS+5mg / L 6-BA+0.5mg / L NAA+2.3g / L plant gel+30g / L sucrose, and the pH value is 5.8.

[0053] 4. Rooting Culture

[0054] The proliferation cluster buds of Curcuma australis are cut into single buds, and the excess tissues, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds with a growth point of about 3 cm, and the buds are transferred to a rhizome induction medium in a single bud grafting manner for culture. The culture conditions are: the temperature is 25°C, dark culture is first carried out for 3 days, and then light culture is carried out, the light intensity is 2000lx, the light time is 10h / d, and the relative humidity is 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium is: 1 / 2MS+NAA 0.5mg / L+sucrose 30g / L+choline chloride 1mg / L+sodium nitrophenolate 2mg / L, and the pH is 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), acclimate under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water depending on 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 test tube micro-rhizomes of Curcuma australis tissue culture seedlings, 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 superior 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, 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 sprout containing the growth point after disinfection are removed, leaving the sprout with a length of 1 cm, and the single sprout 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 time of 12h / day; primary buds are obtained; the induction culture medium includes: MS+2.3g / L plant gel+30g / L sucrose, and the pH value is 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: the temperature is 26°C, the light intensity is 1500lx, and the lighting time is 8h / 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 to continue proliferation culture, and a large number of clustered buds are obtained after subculture proliferation culture for 3 times; the subculture proliferation medium includes: MS+5mg / L 6-BA+0.5mg / L NAA+2.3g / L plant gel+30g / L sucrose, and the pH value is 5.8.

[0065] 4. Rooting Culture

[0066] The proliferation cluster buds of Curcuma australis are cut into single buds, and the excess tissues, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds with a growth point of about 3 cm, and the buds are transferred to a rhizome induction medium in a single bud grafting manner for culture. The culture conditions are: the temperature is 25°C, dark culture is first carried out for 3 days, and then light culture is carried out, the light intensity is 1500lx, the light time is 12h / d, and the relative humidity is 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium is: 1 / 2MS+NAA 0.5mg / L+sucrose 30g / L+choline chloride 2mg / L+sodium nitrophenolate 3mg / L, and the pH is 5.8.

[0067] 5. Transplanting

[0068] Transplant the tissue culture seedlings with rhizomes directly into small pots filled with substrate (vermiculite: nutrient soil = 1:1), slow down 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 soluble fertilizer solution diluted 1000 times.

[0069] Example 4

[0070] This embodiment provides a method for inducing test tube micro-rhizomes of Curcuma australis tissue culture seedlings, 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 superior 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, 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 sprout containing the growth point after disinfection are removed, leaving the sprout with a length of 1 cm, and the single sprout 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 time of 12h / day; primary buds are obtained; the induction culture medium includes: MS+2.3g / L plant gel+30g / L sucrose, and the pH value is 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: the temperature is 26°C, the light intensity is 1500lx, and the lighting time is 8h / 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 to continue proliferation culture, and the subculture proliferation culture is repeated three times to obtain a large number of clustered buds; the subculture proliferation medium includes: MS+5mg / L 6-BA+0.5mg / L NAA+2.3g / L plant gel+30g / L sucrose, and the pH value is 5.8.

[0077] 4. Rooting Culture

[0078] The proliferation cluster buds of Curcuma australis are cut into single buds, and the excess tissues, adventitious roots and upper sheath leaves at the base of each bud are removed, and the buds are trimmed into single buds with a growth point of about 3 cm, and the buds are transferred to a rhizome induction medium for culture in a single bud grafting manner. The culture conditions are: the temperature is 25°C, dark culture is first carried out for 3 days, and then light culture is carried out, the light intensity is 1500lx, the light time is 12h / d, and the relative humidity is 75%. After 30 days of culture, tissue culture seedlings with rhizomes are obtained. The rhizome induction medium is: 1 / 2MS+NAA 0.5mg / L+sucrose 30g / L+choline chloride 3mg / L+sodium nitrophenolate 2mg / L, and the pH is 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), acclimate under scattered light for 10 days, and then culture the potted seedlings under normal light. Water each pot with deionized water depending on 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 the above-mentioned Example 1, except that in the step of rooting culture in this control example, the two plant regulators, choline chloride and sodium nitrophenolate, are not added to the rhizome induction medium, and the composition of the rhizome induction medium is: 1 / 2MS+NAA 0.5mg / L+sucrose 30g / L.

[0084] Comparative Example 2

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

[0086] Comparative Example 3

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

[0088] Comparative Example 4

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

[0090] Comparative Example 5

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

[0092] Comparative Example 6

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

[0094] 2. Identification of tissue culture effects

[0095] 1. Identification of the effect of inducing differentiation of bud micro-rhizomes in tissue culture of Curcuma australis

[0096] In order to identify the effect of rhizome induction medium on the differentiation of microrhizomes of tissue cultured buds of Curcuma australis, the induction rate, length and thickness of microrhizomes of tissue cultured buds of Curcuma australis in the above-mentioned examples and control examples were observed and calculated. Among them, microrhizome induction rate (%) = (number of buds induced into microrhizomes / number of original inoculated buds) × 100%; rhizome length (cm), root length (cm), and rhizome thickness (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 australis

[0098] project Rhizome induction rate (%) Root 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 Wenyujin tissue culture bud microrhizome 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 taproot, 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 are significantly different from each other, 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 microrhizomes growth and yield. Figure 2 It can be seen that the rhizome induction medium of Example 2 still significantly promotes the elongation of microrhizomes (such as Figure 2 There is no significant difference from Example 1 (as shown in Figure B in Figure 2 ), but both are significantly stronger than Example 3 (as shown in Figure A Figure 2 C in Figure ) and Example 4 (as shown in Figure Figure 2 The effect of promoting rhizome thickening in Example 1 is the most obvious, with the maximum diameter of the rhizome being 6.8 mm, followed by Example 2, both of which are significantly higher than Example 3 and Example 4. In terms of rhizome induction rate, rhizome length and rhizome thickness, the effects of the four examples are as follows: 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 micro-rhizomes of Curcuma aromatica and promote the growth of rhizomes. When it exceeds this reasonable range, the formation of rhizomes cannot be induced.

[0104] 2. Identification of the growth morphology of Curcuma australis tissue culture buds

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

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

[0107]

[0108]

[0109] The above-mentioned Wenyujin tissue culture bud growth status shows that when the Wenyujin tissue culture bud is subjected to rooting induction using ordinary rooting medium, the bud is 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 bud is better, with Example 1 and Example 2 being the best, and the bud growth is vigorous, sturdy, and the leaves are dark green; but 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, the growth is good, and the leaves are green, and the growth status of the remaining buds is poor.

[0110] 3. Identification of the growth potential of Curcuma wenyujin tissue culture seedlings

[0111] In order to identify the growth effect of the Curcuma australis tissue culture seedlings with rhizomes after transplanting, the Curcuma australis 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 was 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 (%) = (the number of surviving plants / the total number of transplanted plants) × 100%, and the 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 Tables 3 and Figure 3 shown.

[0112] Table 3 Growth of Curcuma zedoaria tissue culture seedlings transplanted

[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 periods. Compared with reference example 1 (potting with conventional tissue culture rooting seedlings), Example 1 (potting with rhizomes tissue culture seedlings) has a survival rate of 100%, a growth rate of (1.7±0.3) mm / d, a tillering number of (9.7±1.5), a number of newly added 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 improve the growth rate of the plants when planted; after 90 days of potting, the underground roots and rhizomes of the Wenyujin tissue culture seedlings were taken out to observe their growth morphology. The results are as follows 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, it is 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 test tube micro-rhizomes of Curcuma aromatica tissue culture seedlings, characterized in that: The buds of Curcuma australis proliferated by conventional tissue culture were inoculated into rhizome induction medium with single buds for rooting culture, and tissue culture seedlings with rhizomes were obtained after 30 days of culture. The rhizome induction medium comprises: 1 / 2MS+NAA 0.1-1.0 mg / L+sucrose 30 g / L+choline chloride 1-3 mg / L+sodium nitrophenolate 1-3 mg / L, with a pH value of 5.8, wherein the weight ratio of choline chloride to sodium nitrophenolate is 1:(0.3-2).

2. The method for inducing test tube micro-rhizomes of warm turmeric tissue culture seedlings according to claim 1, characterized in that: The concentration of the choline chloride is 3 mg / L, and the concentration of the sodium nitrophenolate is 1 mg / L.

3. The method for inducing test tube micro-rhizomes of warm turmeric tissue culture seedlings according to claim 1, characterized in that: The concentration of the choline chloride is 1 mg / L, and the concentration of the sodium nitrophenolate is 2 mg / L.

4. The method for inducing test tube micro-rhizomes of warm turmeric tissue culture seedlings according to claim 1, characterized in that: The concentration of the choline chloride is 2 mg / L, and the concentration of the sodium nitrophenolate is 3 mg / L.

5. The method for inducing test tube micro-rhizomes of Curcuma aromatica tissue culture seedlings according to claim 1, characterized in that: The concentration of the choline chloride is 3 mg / L, and the concentration of the sodium nitrophenolate is 2 mg / L.

6. The method for inducing test tube micro-rhizomes of warm turmeric tissue culture seedlings according to claim 1, characterized in that: The tissue culture process of the adventitious buds of Curcuma aromatica 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 25-27℃ light incubator for germination. When the buds grow to 2-3cm, cut the buds and disinfect them. 2) Induction culture The upper half of the leaves of the disinfected sprouts are removed, leaving a bud length of 0.8 to 1.2 cm, and the single bud is cut into two pieces longitudinally, and 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 time of 10 to 12 hours per day to obtain primary buds; the induction medium includes: MS + 1.5 to 2.5 g / L plant gel + 30 g / L sucrose, and the pH value is 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: the temperature is 24-26 DEG C, the light intensity is 1500-2000lx, and the light time is 6-8h / day; after culturing for 30 days, a large number of clustered buds are obtained, and a new subculture proliferation medium is replaced to continue the proliferation culture, and a large number of clustered buds are obtained after 3-4 subculture proliferation cultures; the subculture proliferation medium: MS+4-5mg / L 6-BA+0.1-0.5mg / L NAA+2.1-2.5g / L plant gel+30g / L sucrose, and the pH value is 5.

8.

7. The method for inducing test tube micro-rhizomes of warm turmeric tissue culture seedlings according to claim 1, characterized in that: The rooting culture conditions are as follows: the temperature is 25-27°C, dark culture is performed for 3-5 days, and then light culture is performed, the light intensity is 1500-2500lx, the light time is 8-12h / d, and the relative humidity is 75%.

8. The method for inducing test tube micro-rhizomes of Curcuma aromatica tissue culture seedlings according to claim 6, characterized in that: In step 1), the rhizome is sterilized with a 0.1% carbendazim solution for 10 to 15 minutes; the sprout is first sterilized with 70% alcohol for 45 to 60 seconds, rinsed with sterile water twice, then sterilized with 2% sodium hypochlorite for 15 to 20 minutes, and rinsed with sterile water 5 times.

9. The method for inducing test tube micro-rhizomes of Curcuma aromatica tissue culture seedlings according to claim 6, characterized in that: In step 2), the upper half of the leaves of the budding plants containing the growth points are removed after disinfection.

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