CN119655170A - A method for inducing test tube micro-rhizomes of Curcuma australis tissue culture seedlings - Google Patents
A method for inducing test tube micro-rhizomes of Curcuma australis tissue culture seedlings Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural biology, and discloses a method for inducing micro-rootstock of a tissue culture seedling of curcuma wenyujin, which is obtained by conventional tissue culture, and is characterized in that a common curcuma wenyujin terminal bud cluster multiplication bud is inoculated into a rootstock induction culture medium for rooting culture, and the tissue culture seedling with rootstock is obtained after 30 days of culture, wherein the rootstock induction culture medium comprises 1/2MS+NAA 0.1~1.0mg/L+30 g/L of sucrose+1-3 mg/L of choline chloride+1-3 mg/L of sodium nitrophenolate, and the pH is 5.8, and choline chloride is 1 (0.3-2) of sodium nitrophenolate according to the weight ratio. The invention uses the aseptic micro-rootstock to directly transplant and manufacture the micro-rootstock artificial seeds for sowing by in vitro induction of the micro-rootstock of the test tube of the curcuma wenyujin, omits the link of hardening off the seedlings and temporary planting the seedlings and reduces the seedling loss caused by the severe humidity requirement of the green seedlings, and the micro-rootstock seedlings of the test tube are favorable for storage and long-distance transportation, thereby improving the seedling breeding efficiency of the rootstock plants.
Description
Technical Field
The invention belongs to the technical field of tissue culture and rapid propagation, and particularly relates to a method for inducing micro-rootstock of a tissue culture seedling of curcuma wenyujin.
Background
Curcuma wenyujin (Curcuma wenyujin) is a cultivar of Curcuma aromatica of Zingiberaceae, and is mainly produced in Ruian region of Wittice of Zhejiang for many years by using plant, and the plant is rich in Curcumae rhizoma volatile oil, curcumin and polysaccharide, and has effects of resisting tumor, inflammation, bacteria, virus, thrombosis, protecting liver, protecting nerve and oxidation. The medicinal parts are mainly rhizome and root tuber, and according to different parts and methods, 3 different traditional Chinese medicinal materials can be processed, namely, a main root stem is boiled and dried to be called as warm turmeric, a root tuber is boiled and dried to be called as warm turmeric, and a fresh side root stem is dried to be called as turmeric after being longitudinally cut into thick slices, wherein the warm turmeric is one of the famous genuine medicinal materials of Zhejiang eight.
The curcumenol (curcumol), curdione (curclione), beta-elemene (beta-elemene) and the like in the curcuma zedoary volatile oil are used as anticancer active ingredients, and the curcuma zedoary volatile oil is used as raw materials to develop a novel antitumor, anti-inflammatory and antibacterial pharmaceutical preparation with good curative effect and small side effect, and the like, for example, the main ingredient of the external-use medicine' Baofukang suppository for gynaecology is the curcuma zedoary volatile oil. The Curcuma zedoary is favored by the Curcuma zedoary commodity with high volatile oil content and good quality, so the Curcuma zedoary is cultivated in the Ruian original region of the original region of Zhejiang, and other suitable cultivated provinces in China such as Hainan, yunnan, guizhou and the like are introduced and enlarged.
The traditional planting materials of the curcuma aromatica are rhizomes, the plants are carried in different degrees, viruses are accumulated, soil-borne pathogens are infected, and the like due to long-term asexual propagation, so that the seed degeneration is caused, the quality of the rhizomes is uneven, the rhizomes are used as seed stems, the propagation coefficient is low, a large number of rhizomes which can be originally used as commodities are consumed, the cost of seedlings is high, the quality of the rhizomes is uneven, the yield is reduced due to the fact that the seedlings of the rhizomes are good, safe, effective, stable and controllable traditional Chinese medicinal materials are difficult to obtain, and the like, so that a serious challenge is brought to sustainable development of the curcuma aromatica industry.
At present, tissue culture seedling breeding of radix curcumae is carried out by utilizing tissue culture rapid propagation, the method is not limited by seasons, annual production can be carried out, the breeding efficiency is obviously improved, and the method is an effective supplementary means for breeding seedlings by using conventional field rhizomes, but when the radix curcumae is planted by using the tissue culture seedlings, the root is mainly transplanted by rooting green seedlings, the root is thin and long, the root is easy to be injured in the seedling hardening and transplanting process, the seedling is easy to be influenced by humidity to cause dead seedlings, and if the seedling hardening and the seedling hardening are not carried out through temporary planting, the transplanting survival rate is low or is not stable enough, and the method is not beneficial to large-area popularization and application.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a method for inducing the micro-rootstock of a test tube of a tissue culture seedling of curcuma wenyujin, which is characterized in that the micro-rootstock of the test tube is formed by in-vitro induction, the micro-rootstock of the test tube is directly transplanted by using aseptic micro-rootstock, artificial seeds are produced for sowing, the link of hardening off the green seedling and temporary planting is omitted, the seedling loss caused by the severe requirement of the green seedling on humidity is reduced, and the micro-rootstock seedling of the test tube is favorable for storage and long-distance transportation, so that the seedling breeding efficiency of the rootstock plant is improved.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A method for inducing micro-rootstock of tissue culture seedling of Curcuma wenyujin Y.H.Chen et C.Ling comprises taking proliferation cluster buds of Curcuma wenyujin Y.H.Chen et C.Ling obtained by conventional tissue culture, inoculating single buds into rootstock induction culture medium, rooting culture, and culturing for 30 days to obtain tissue culture seedling with rootstock; the rhizome induction culture medium comprises 1/2MS+NAA 0.1~1.0mg/L+30 g/L of sucrose+1-3 mg/L of choline chloride+1-3 mg/L of compound sodium nitrophenolate and pH 5.8, wherein the weight ratio of choline chloride to compound sodium nitrophenolate=1 (0.3-2).
In an alternative embodiment, the choline chloride is at a concentration of 3mg/L and the sodium nitrophenolate is at a concentration of 1mg/L.
In an alternative embodiment, the choline chloride is at a concentration of 1mg/L and the sodium nitrophenolate is at a concentration of 2mg/L.
In an alternative embodiment, the choline chloride is at a concentration of 2mg/L and the sodium nitrophenolate is at a concentration of 3mg/L.
In an alternative embodiment, the choline chloride is at a concentration of 3mg/L and the sodium nitrophenolate is at a concentration of 2mg/L.
In an alternative embodiment, the tissue culture process of the adventitious bud of Curcuma wenyujin is:
1) Sterilization of explants
Taking the rhizome of the Curcuma wenyujin Y.H.Chen et C.Ling, sterilizing, placing in a 25-27 ℃ illumination incubator to accelerate germination, and cutting the bud to sterilize when the bud grows to 2-3 cm;
2) Induction culture
Cutting off the upper half leaves of the sterilized sprouts, reserving buds with the length of 0.8-1.2 cm, longitudinally cutting the single buds into two pieces, inserting the pieces into an induction culture medium for carrying out primary fixed bud germination induction culture for 7-10 days, wherein the culture condition is that the temperature is 24-26 ℃, the illumination intensity is 1000-2000 lx, the illumination time is 10-12 h/day, and the primary fixed buds are obtained, wherein the induction culture medium is MS+1.5-2.5 g/L plant gel+30 g/L sucrose, and the pH value is 5.8;
3) Proliferation culture
Inoculating the primary fixed buds obtained through the early induction into a secondary proliferation culture medium for proliferation culture under the culture conditions that the temperature is 24-26 ℃, the illumination intensity is 1500-2000 lx, the illumination time is 6-8 h/day, a large number of cluster buds are obtained after 30 days of culture, the new secondary proliferation culture medium is replaced for continuous proliferation culture, a large number of cluster buds are obtained after 3-4 times of secondary proliferation culture, wherein the secondary proliferation culture medium comprises MS+4-5 mg/L6-BA+0.1-0.5 mg/L NAA+2.1-2.5 g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
In an alternative embodiment, the rooting culture is carried out under the conditions that the temperature is 25-27 ℃, the rooting culture is carried out for 3-5 days in a dark state, then the rooting culture is carried out under illumination, the illumination intensity is 1500-2500 lx, the illumination time is 8-12h/d, and the relative humidity is 75%.
In an optional implementation manner, in the step 1), the sterilizing treatment mode of the rootstalk is to sterilize with 0.1% carbendazim solution for 10-15 min, and the sterilizing treatment mode of the sprout is to sterilize with 70% alcohol for 45-60 s, to rinse with sterile water for 2 times, to sterilize with 2% sodium hypochlorite for 15-20 min, and to rinse with sterile water for 5 times.
In an alternative embodiment, in step 2), the top half of the leaf of the sprout, which after sterilization contains the growth point, is excised.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) According to the invention, the choline chloride and the compound sodium nitrophenolate which are properly proportioned are added into the rooting culture medium, so that the generation and expansion of micro-rhizomes of the curcuma wenyujin test tube can be effectively induced by supplementing each other, the tissue culture rooting seedlings with rhizomes are used for planting, the genetic stability of excellent germplasm can be maintained, the production efficiency is improved, the quality of medicinal materials is also improved, and in the rhizomes obtained by planting the tissue culture seedlings, the main effective medicinal active ingredients of the curcuma zedoary volatile oil, namely the germacrone, the curcuma zedoary diketone, the curcumin and the like are higher than those of the corresponding common curcuma wenyujin.
(2) The invention can omit the links of hardening off and temporary planting of green seedlings and reduce seedling loss caused by the severe humidity requirement of the green seedlings, and the test tube micro-rootstock seedlings are favorable for storage and long-distance transportation, thereby improving the seedling breeding efficiency of rootstock plants.
(3) The survival rate of the tissue culture root seedlings with the rootstock after transplanting and the yield of the rootstock and the root tuber are higher than those of the conventional tissue culture root seedlings, and the domestication and the transplanting of the tissue culture seedlings and the growth promotion can be effectively simplified.
Drawings
FIG. 1 shows the steps of obtaining the rhizome materials and accelerating germination of Curcuma wenyujin Y.H.Chen et C.Ling in the embodiment of the invention. A. Fresh rhizome of Curcuma wenyujin, germination accelerating of rhizome B, germination explant of rhizome C, and aseptic bud D.
FIG. 2 is a graph showing the effect of different rooting media on the root and stem differentiation induction of Curcuma wenyujin Y.H.Chen et C.Ling in the example of the invention.
FIG. 3 shows the effect of the isolated micro-rootstock of the present invention on the growth vigor of tissue culture seedlings of Curcuma wenyujin. A is plant form of the tissue culture seedling of Curcuma wenyujin 60 days after potting, and B is growth and form of the root and rhizome of the underground part of the tissue culture seedling Miao Penzai days after tissue culture.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
The following examples and comparative examples all have the same process of obtaining tissue culture buds, except that the composition and concentration ratio of the culture medium are different when the tissue culture buds are subjected to rhizome induction culture, the process of obtaining the tissue culture buds is shown in fig. 1, the graph A in fig. 1 shows fresh rhizomes of Curcuma wenyujin in Hainan regions, the graph B shows the germination accelerating condition of the rhizomes, the graph C shows sprouts cut from the rhizomes, the graph D shows sterile buds obtained after the germination is subjected to proliferation culture, and the obtained adventitious buds are used as rhizome induction tests of examples and comparative examples.
1. Seedling breeding of Curcuma wenyujin
Example 1
The embodiment provides a method for inducing micro-rootstock of a tissue culture seedling test tube of curcuma wenyujin, which comprises the following steps:
1. sterilization of explants
The method comprises the steps of selecting a root and stem explant which is introduced and is suitable for ecological environment, health and high yield in Hainan area in 12 months 2023 as an explant mother plant, avoiding wounds as much as possible and keeping the integrity of the root and stem during root and stem collection, cleaning, airing, carrying out primary surface sterilization for 10min by using a solution of broad-spectrum bactericide carbendazim (effective content 50% wettable powder) 0.1%, then placing the root and stem in a (25+/-2) DEG C illumination incubator for germination, cutting the bud for surface sterilization when the bud grows to 3cm, and carrying out surface sterilization by using 70% alcohol for 45s, using 2% sodium hypochlorite for sterilization for 15min and using 5 times of sterile water for sterilization.
2. Induction culture
Cutting off leaves at the upper half part of the disinfected sprouts containing growing points, reserving buds for 1cm, longitudinally cutting the single buds into two pieces, inserting the pieces into an induction culture medium, carrying out primary fixed bud germination induction culture for 7 days, wherein the culture condition is that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 12 h/day, and the primary fixed buds are obtained, wherein the induction culture medium is MS+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
3. Proliferation culture
Inoculating primary fixed buds obtained through early induction into a secondary proliferation culture medium for proliferation culture under the culture conditions that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 8 h/day, a large number of cluster buds are obtained after 30 days of culture, a new secondary proliferation culture medium is replaced, the cluster buds are cut for continuous proliferation culture, and a large number of cluster buds are obtained after 3 times of secondary proliferation culture, wherein the secondary proliferation culture medium comprises MS+5 mg/L6-BA+0.5 mg/L NAA+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
4. Rooting culture
Cutting the curcuma wenyujin into single buds after proliferation Cong Yati, cutting off redundant tissues at the base part of each bud, adventitious roots and upper sheath leaves, trimming to form single buds with a reserved growth point and about 3cm, inoculating the single buds to a rhizome induction culture medium for culture in a single bud grafting mode, wherein the culture condition is that the temperature is 25 ℃, the culture is carried out in a dark mode for 3 days, then the illumination is carried out, the illumination intensity is 2500lx, the illumination time is 8h/d, the relative humidity is 75%, and the tissue culture seedlings with rhizomes are obtained after 30 days of culture, wherein the rhizome induction culture medium is 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 3 mg/L+sodium nitrophenolate 1mg/L, and the pH is 5.8.
5. Transplanting
Transplanting the tissue culture seedlings with the roots into a small basin containing a matrix (vermiculite: nutrient soil=1:1), recovering the seedlings for 10d under scattered light, culturing the potted seedlings under normal light, quantitatively irrigating deionized water per basin according to weather conditions, and quantitatively irrigating 1 time per month with a soluble fertilizer solution diluted 1000 times.
Example 2
The embodiment provides a method for inducing micro-rootstock of a tissue culture seedling test tube of curcuma wenyujin, which comprises the following steps:
1. sterilization of explants
The method comprises the steps of selecting a root and stem explant which is introduced and is suitable for ecological environment, health and high yield in Hainan area in 12 months 2023 as an explant mother plant, avoiding wounds as much as possible and keeping the integrity of the root and stem during root and stem collection, cleaning, airing, carrying out primary surface sterilization for 10min by using a solution of broad-spectrum bactericide carbendazim (effective content 50% wettable powder) 0.1%, then placing the root and stem in a (25+/-2) DEG C illumination incubator for germination, cutting the bud for surface sterilization when the bud grows to 3cm, and carrying out surface sterilization by using 70% alcohol for 45s, using 2% sodium hypochlorite for sterilization for 15min and using 5 times of sterile water for sterilization.
2. Induction culture
Cutting off leaves at the upper half part of the disinfected sprouts containing growing points, reserving buds for 1cm, longitudinally cutting the single buds into two pieces, inserting the pieces into an induction culture medium, carrying out primary fixed bud germination induction culture for 7 days, wherein the culture condition is that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 12 h/day, and the primary fixed buds are obtained, wherein the induction culture medium is MS+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
3. Proliferation culture
Inoculating the sterile primary fixed buds obtained through the early induction into a secondary proliferation culture medium for proliferation culture under the culture conditions that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 8 h/day, a large number of cluster buds are obtained after 30 days of culture, a new secondary proliferation culture medium is replaced, the cluster buds are cut for continuous proliferation culture, and a large number of cluster buds are obtained after 3 times of secondary proliferation culture, wherein the secondary proliferation culture medium comprises MS+5 mg/L6-BA+0.5 mg/L NAA+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
4. Rooting culture
Cutting the curcuma wenyujin into single buds after proliferation Cong Yati, cutting off redundant tissues at the base part of each bud, adventitious roots and upper sheath leaves, trimming to form single buds with a reserved growth point and about 3cm, inoculating the single buds to a rhizome induction culture medium for culture in a single bud grafting mode, wherein the culture condition is that the temperature is 25 ℃, the culture is carried out in a dark mode for 3 days, then the illumination is carried out, the illumination intensity is 2000lx, the illumination time is 10h/d, the relative humidity is 75%, and the tissue culture seedlings with rhizomes are obtained after 30 days of culture, wherein the rhizome induction culture medium comprises 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 1 mg/L+sodium nitrophenolate 2mg/L and the pH is 5.8.
5. Transplanting
Transplanting the tissue culture seedlings with the roots into a small basin containing a matrix (vermiculite: nutrient soil=1:1), recovering the seedlings for 10d under scattered light, culturing the potted seedlings under normal light, quantitatively irrigating deionized water per basin according to weather conditions, and quantitatively irrigating 1 time per month with a soluble fertilizer solution diluted 1000 times.
Example 3
The embodiment provides a method for inducing micro-rootstock of a tissue culture seedling test tube of curcuma wenyujin, which comprises the following steps:
1. sterilization of explants
The method comprises the steps of selecting a root and stem explant which is introduced and is suitable for ecological environment, health and high yield in Hainan area in 12 months 2023 as an explant mother plant, avoiding wounds as much as possible and keeping the integrity of the root and stem during root and stem collection, cleaning, airing, carrying out primary surface sterilization for 10min by using a solution of broad-spectrum bactericide carbendazim (effective content 50% wettable powder) 0.1%, then placing the root and stem in a (25+/-2) DEG C illumination incubator for germination, cutting the bud for surface sterilization when the bud grows to 3cm, and carrying out surface sterilization by using 70% alcohol for 45s, using 2% sodium hypochlorite for sterilization for 15min and using 5 times of sterile water for sterilization.
2. Induction culture
Cutting off leaves at the upper half part of the disinfected sprouts containing growing points, reserving buds for 1cm, longitudinally cutting the single buds into two pieces, inserting the pieces into an induction culture medium, carrying out primary fixed bud germination induction culture for 7 days, wherein the culture condition is that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 12 h/day, and the primary fixed buds are obtained, wherein the induction culture medium is MS+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
3. Proliferation culture
Inoculating the sterile primary fixed buds obtained through the early induction into a secondary proliferation culture medium for proliferation culture under the culture conditions that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 8 h/day, a large number of cluster buds are obtained after 30 days of culture, a new secondary proliferation culture medium is replaced, the cluster buds are cut for continuous proliferation culture, and a large number of cluster buds are obtained after 3 times of secondary proliferation culture, wherein the secondary proliferation culture medium comprises MS+5 mg/L6-BA+0.5 mg/L NAA+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
4. Rooting culture
Cutting the curcuma wenyujin into single buds after proliferation Cong Yati, cutting off redundant tissues at the base part of each bud, adventitious roots and upper sheath leaves, trimming to form single buds with a reserved growth point and about 3cm, transferring the single buds to a rhizome induction culture medium for culture in a single bud grafting mode, wherein the culture condition is that the temperature is 25 ℃, the culture is carried out in a dark mode for 3 days, then the illumination is carried out, the illumination intensity is 1500lx, the illumination time is 12h/d, the relative humidity is 75%, and the tissue culture seedlings with rhizomes are obtained after 30 days of culture, wherein the rhizome induction culture medium comprises 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 2 mg/L+sodium nitrophenolate 3mg/L and the pH is 5.8.
5. Transplanting
Transplanting the tissue culture seedling with rhizome directly into a small basin with a matrix (vermiculite: nutrient soil=1:1), and then, growing the seedling for 10d under scattered light, and then, culturing the potted seedling under normal illumination. Deionized water was quantitatively poured per basin according to weather conditions, and 1 time was quantitatively poured with a 1000-fold diluted soluble fertilizer solution per month.
Example 4
The embodiment provides a method for inducing micro-rootstock of a tissue culture seedling test tube of curcuma wenyujin, which comprises the following steps:
1. sterilization of explants
The method comprises the steps of selecting a root and stem explant which is introduced and is suitable for ecological environment, health and high yield in Hainan area in 12 months 2023 as an explant mother plant, avoiding wounds as much as possible and keeping the integrity of the root and stem during root and stem collection, cleaning, airing, carrying out primary surface sterilization for 10min by using a solution of broad-spectrum bactericide carbendazim (effective content 50% wettable powder) 0.1%, then placing the root and stem in a (25+/-2) DEG C illumination incubator for germination, cutting the bud for surface sterilization when the bud grows to 3cm, and carrying out surface sterilization by using 70% alcohol for 45s, using 2% sodium hypochlorite for sterilization for 15min and using 5 times of sterile water for sterilization.
2. Induction culture
Cutting off leaves at the upper half part of the disinfected sprouts containing growing points, reserving buds for 1cm, longitudinally cutting the single buds into two pieces, inserting the pieces into an induction culture medium, carrying out primary fixed bud germination induction culture for 7 days, wherein the culture condition is that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 12 h/day, and the primary fixed buds are obtained, wherein the induction culture medium is MS+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
3. Proliferation culture
Inoculating the sterile primary fixed buds obtained through the early induction into a secondary proliferation culture medium for proliferation culture under the culture conditions that the temperature is 26 ℃, the illumination intensity is 1500lx, the illumination time is 8 h/day, a large number of cluster buds are obtained after 30 days of culture, a new secondary proliferation culture medium is replaced, the cluster buds are cut for continuous proliferation culture, and the secondary proliferation culture is carried out for 3 times to obtain a large number of cluster buds, wherein the secondary proliferation culture medium comprises MS+5 mg/L6-BA+0.5 mg/L NAA+2.3g/L plant gel+30 g/L sucrose, and the pH value is 5.8.
4. Rooting culture
Cutting the curcuma wenyujin into single buds after proliferation Cong Yati, cutting off redundant tissues at the base part of each bud, adventitious roots and upper sheath leaves, trimming to form single buds with a reserved growth point and about 3cm, inoculating the single buds to a rhizome induction culture medium for culture in a single bud grafting mode, wherein the culture condition is that the temperature is 25 ℃, the culture is carried out in a dark mode for 3 days, then the illumination is carried out, the illumination intensity is 1500lx, the illumination time is 12h/d, the relative humidity is 75%, and the tissue culture seedlings with rhizomes are obtained after 30 days of culture, wherein the rhizome induction culture medium is 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 3 mg/L+sodium nitrophenolate 2mg/L, and the pH is 5.8.
5. Transplanting
Transplanting the tissue culture seedlings with the roots into a small basin containing a matrix (vermiculite: nutrient soil=1:1), recovering the seedlings for 10d under scattered light, culturing the potted seedlings under normal light, quantitatively irrigating deionized water per basin according to weather conditions, and quantitatively irrigating 1 time per month with a soluble fertilizer solution diluted 1000 times.
Comparative test
Comparative example 1
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of the example 1, except that in the step of rooting culture, two plant regulators of choline chloride and sodium nitrophenolate are not added into the rhizome induction culture medium, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5mg/L and sucrose 30g/L.
Comparative example 2
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of example 1, except that in the step of rooting culture, the concentration ratio of choline chloride and sodium nitrophenolate in the rhizome induction culture medium is different, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 3 mg/L+sodium nitrophenolate 0.6mg/L.
Comparative example 3
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of example 1, except that in the step of rooting culture, the concentration ratio of choline chloride and sodium nitrophenolate in the rhizome induction culture medium is different, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 1 mg/L+sodium nitrophenolate 3mg/L.
Comparative example 4
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of example 1, except that in the step of rooting culture, the concentration ratio of choline chloride and sodium nitrophenolate in the rhizome induction culture medium is different, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 2 mg/L+sodium nitrophenolate 0.2mg/L.
Comparative example 5
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of example 1, except that in the step of rooting culture, the concentration ratio of choline chloride and sodium nitrophenolate in the rhizome induction culture medium is different, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 2 mg/L+sodium nitrophenolate 0.3mg/L.
Comparative example 6
The process of obtaining the tissue culture seedlings of the comparative example is the same as that of example 1, except that in the step of rooting culture, the concentration ratio of choline chloride and sodium nitrophenolate in the rhizome induction culture medium is different, and the ingredients of the rhizome induction culture medium are 1/2MS+NAA 0.5 mg/L+sucrose 30 g/L+choline chloride 1 mg/L+sodium nitrophenolate 4mg/L.
2. Identification of tissue culture Effect
1. Identification of tissue culture bud micro-rhizome induced differentiation effect of Curcuma wenyujin
In order to identify the effect of the rhizome induction culture medium on the induction differentiation of the micro-rhizomes of the tissue culture buds of the curcuma wenyujin, the induction rate, the length and the roughness of the micro-rhizomes of the tissue culture buds of the curcuma wenyujin in the above examples and comparative examples are observed and calculated. Wherein the induction rate (%) = (number of buds of induced micro rootstock/number of original inoculated buds) ×100%, the rootstock length (cm), root length (cm), rootstock thickness (mm) were measured with vernier calipers, and the results are shown in table 1 and fig. 2.
TABLE 1 Effect of different rooting media on tissue culture bud micro-rhizome differentiation of Curcuma wenyujin
| Project | Rhizome induction rate (%) | Rhizome length (cm) | Rhizome is thick (mm) | Root length (cm) | Root thickness (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 |
* Note that the same column of numbers in the table with different lower case letters indicates that the difference reaches a significant level (P < 0.05).
The root induction differentiation effect of the tissue culture buds of curcuma wenyujin is that the root induction culture medium (example 1, example 2, example 3, example 4, comparative example 2, comparative example 3, comparative example 4, comparative example 5 and comparative example 6) and the conventional rooting culture medium (comparative example 1) can induce rooting, but the root induction differentiation can be induced to form roots longer than 0.5cm (the length of the root is less than 0.5 cm), and the root induction rate of the root induction culture medium is up to 62% when the root induction culture medium is used for inducing the root induction culture buds of the curcuma wenyujin, and the root induction culture medium can induce rooting, but the root induction differentiation can induce the root induction culture medium to form roots longer than 0.5cm (the root length is less than 0.5 cm).
In FIG. 2, 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 comparative example 1, F is the rhizome induction of comparative example 2, G is the rhizome induction of comparative example 3, H is the rhizome induction of comparative example 4, I is the rhizome induction of comparative example 5, and J is the rhizome induction of comparative example 6.
The length and coarseness of the micro-rootstock are key indicators for measuring the growth and yield of the micro-rootstock, and as can be seen from the combination of table 1 and fig. 2, the rootstock induction medium of example 2 is still significant for promoting elongation of the micro-rootstock (as shown in fig. 2, panel B), and has no significant difference from example 1 (as shown in fig. 2, panel a), but is significantly stronger than example 3 (as shown in fig. 2, panel C) and example 4 (as shown in fig. 2), while the rootstock thickening promotion is most significant in example 1, with a diameter of 6.8mm at the maximum of the rootstock, and secondly, example 2, both of which are significantly higher than example 3 and example 4. The four examples were conducted in order of example 1> example 2> example 3> example 4, with the overall root induction rate, root length and root thickness being large.
Compared with examples 2-6, examples 1-4 of the invention can improve the induction rate and simultaneously maintain the growth of the rhizome, which shows that the concentration ratio of the choline chloride and the sodium nitrophenolate as two plant regulators can be within a reasonable range, and the induction differentiation of the micro rhizome of the curcuma wenyujin is facilitated and the growth of the rhizome is promoted, and the formation of the rhizome can not be induced when the concentration ratio exceeds the reasonable range.
2. Identification of growth morphological effect of tissue culture buds of Curcuma wenyujin
In order to identify the influence of the rhizome induction culture medium on the growth form of the tissue culture buds of Curcuma wenyujin Y.H.Chen et C.Ling, the growth state of the tissue culture buds of Curcuma wenyujin Y.H.Chen et C.Ling was observed in the above examples and comparative examples. The results are shown in Table 2.
TABLE 2 Effect of different rooting media on the growth status of tissue culture buds of Curcuma wenyujin
The above-mentioned growth states of tissue culture buds of Curcuma wenyujin are shown that when the conventional rooting medium is used for rooting induction of tissue culture buds of Curcuma wenyujin, the buds are small and have a poor growth vigor She Danlu, while when the rhizome induction medium of the present invention is used (example 1, example 2, example 3 and example 4), the growth states of buds are good, and the buds of example 1 and example 2 grow vigorously and robustly and She Nonglu, but when both choline chloride and sodium nitrophenolate in the rhizome induction medium are out of the proper range (comparative example 2, comparative example 3, comparative example 4, comparative example 5 and comparative example 6), only the growth states of buds of comparative example 2 and comparative example 4 are normal, good, and green, and the rest of buds are poor.
3. Identification of growth potential effect of tissue culture seedlings of curcuma wenyujin
To identify the growth effect of the tissue culture seedlings of Curcuma wenyujin Y.H.Chen et C.Ling having rootstock after transplanting, the tissue culture seedlings of Curcuma wenyujin Y.Ling of example 1 and comparative example 1 were transplanted into pots for cultivation, the survival rate (%) was counted after 30 days, the absolute growth rate AGR (mm/d) was measured between 31 to 40 days, the number of sprouting leaves and tillering (pieces or pieces/plant) of the tissue culture seedlings were observed and the growth conditions such as the weight of the underground part (g, rootstock and root tuber) were weighed. Wherein, the transplanting survival rate (%) = (the number of the survival plants/the total number of the transplanting plants) ×100%, AGR is the average value of 10 days, and the growth (g) of underground rootstocks and tubers is measured by weighing with a balance. The results are shown in Table 3 and FIG. 3.
TABLE 3 tissue culture seedling transplanting growth conditions of Curcuma wenyujin
The transplanting growth condition of the tissue culture seedlings of the curcuma wenyujin is shown that compared with the conventional rooting tissue culture seedlings of the curcuma wenyujin (comparative example 1), the transplanting efficiency and the growth vigor of the tissue culture rooting seedlings with roots can be obviously improved, compared with the conventional tissue culture rooting seedlings in a pot culture mode, the survival rate of the tissue culture seedlings with roots of the example 1 is 100%, the growth rate is (1.7+/-0.3) mm/d, the tillering number is (9.7+/-1.5), the number of newly added leaves is (21.0+/-3.6), the weight of the underground roots and the roots is (33.7+/-1.3) g/plant, and 36.4%, 112.5%, 83.0%, 103.9% and 83.2% are obviously improved. In conclusion, the tissue culture rooting seedling with the rhizome can effectively simplify domestication and transplanting of the tissue culture seedling and promote growth.
The results of observing the morphology of the plants after 60 days of potting of the tissue-cultured seedlings of Curcuma wenyujin are shown in the A graph in FIG. 3 (the left graph is the plant growth morphology graph in comparative example 1; the right graph is the plant growth morphology graph in example 1), and from the A graph, the plants in comparative example 1 have a slower growth rate of 、 tillers and fewer newly added leaves, which indicates that the growth rate of the plants can be improved when the tissue-cultured seedlings obtained by the invention are planted, and the root and rhizome of the underground part of the plants are taken out after 90 days of potting of the tissue-cultured seedlings of Curcuma wenyujin to observe the growth morphology thereof, and the results of the root and rhizome of the plants are shown in the B graph in FIG. 3 (the left graph is the plant growth morphology graph in comparative example 1; the right graph is the plant growth morphology graph in example 1), and from the B graph, the number and weight of the root and the rhizome are smaller than those in example 1, which indicates that the root and rhizome are more and thicker when the tissue-cultured seedlings of the invention are planted.
Although the present invention has been described with reference to the above preferred embodiments, it should be understood that the present invention is not limited to the above preferred embodiments, and various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the present invention.
Claims (9)
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