CN113875588B - Method for promoting rapid in-vitro propagation of curcuma longa - Google Patents
Method for promoting rapid in-vitro propagation of curcuma longa Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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Abstract
The invention discloses a method for promoting rapid in vitro propagation of curcuma aromatica. The germination of the rhizome of the curcuma aromatica is realized after the rhizome of the curcuma aromatica is soaked in yucca extract solution. The invention discovers that the yucca extract can improve the germination rate of stem buds of the curcuma aromatica, promote the proliferation of clustered shoots of the curcuma aromatica and enlarge the propagation coefficient, and the lanthanum nitrate can effectively promote the rooting and seedling strengthening of the clustered shoots of the curcuma aromatica.
Description
The technical field is as follows:
the invention belongs to the field of flower propagation, and particularly relates to a method for promoting rapid in-vitro propagation of curcuma longa.
The background art comprises the following steps:
the Curcuma longa (Curcuma sichuanensis X.X.Chen) belongs to the genus Curcuma of the family Zingiberaceae, is a perennial herbaceous plant, is mainly produced in various regions along the coast of Minjiang river basin, and has very wide market development and utilization values. The curcuma longa inflorescence grows from the top, has longer flowering period, fresh and elegant flowers and tall and beautiful leaves, is suitable for being used as fresh cut flowers, water culture or pot flower plants or under-forest configured plants, and has high ornamental value. Meanwhile, Chuan depression hardware has the effects of activating blood and relieving pain, promoting qi circulation and resolving depression, clearing heart and cooling blood, and benefiting gallbladder and removing jaundice, and is clinically used for treating depression, tourette syndrome in children, Alzheimer disease, liver injury, hiccup and other diseases. As one of the sources of traditional Chinese medicinal materials, the Sichuan radix curcumae has been accepted by herbalists in the Tang generation as early as the source of traditional Chinese medicinal materials, and has high medicinal value. By utilizing the tissue culture rapid propagation technology, the germplasm resources of the existing curcuma aromatica can be purified and rejuvenated, the propagation of large-scale medicinal seedlings and ornamental seedlings is facilitated, and the production cost is reduced.
Yucca (Yucca smaliliana) is a plant of the genus Yucca of the family Liliaceae, and the Yucca extract is an extract from the stem and leaf of the plant. The yucca extract is used as a natural plant additive, is widely applied to animal breeding production, is used for adsorbing harmful gas to improve feeding environment, and promotes animals such as pigs and broiler chickens to absorb nutrient substances; the yucca extract is used as nutritional supplement, and has the advantages of no toxicity, no pollution, and no residue.
Disclosure of Invention
The invention aims to provide a method for promoting rapid in-vitro propagation of curcuma aromatica, which can improve the germination rate and the proliferation coefficient of rhizomes of curcuma aromatica.
The method for promoting the in-vitro rapid propagation of the curcuma aromatica comprises the steps of sprouting of rhizomes of the curcuma aromatica, inducing adventitious buds, proliferating cluster buds, and rooting and seedling strengthening of the cluster buds, wherein the sprouting of the rhizomes of the curcuma aromatica is the sprouting of the rhizomes of the curcuma aromatica after being soaked in yucca extract solution.
The concentration of the yucca extract solution is 2-8 g/L.
Preferably, the sprouting of the rhizome of the curcuma aromatica is realized by digging after the overground part of the curcuma aromatica completely retreats from winter, selecting a root material of the curcuma aromatica which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skin on the surface, cutting off fibrous roots on the surface, soaking for 0.5h by using a yucca extract solution, covering in sterilized river sand after soaking treatment, culturing at 30 ℃ in full darkness, and accelerating sprouting at 70 +/-10% RH.
Preferably, the proliferation of the multiple shoots is carried out by adding yucca extract to a proliferation medium. More preferably, the concentration of the yucca extract in the culture medium is 0.1-1 mg/L.
Preferably, the proliferation of the cluster buds is to transfer the cluster buds of the curcuma aromatica obtained by induction into a proliferation culture medium for culture, wherein the proliferation culture medium is MS +6-BA 6mg/L + NAA 0.2mg/L + Yucca extract 0.1-1 g/L +30g/L sucrose +7.5g/L agar.
Preferably, the method comprises the following specific steps:
A. rhizome germination of curcuma longa: digging after the overground part of the radix curcumae completely recedes from winter, selecting a root and stem material of the radix curcumae which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skins on the surface, cutting off fibrous roots on the surface, soaking for 0.5h by using 2g/L or 8g/L yucca extract solution, covering in autoclaved river sand after soaking, culturing at 30 ℃ in full darkness, and accelerating germination by 70 +/-10% RH;
B. induction of adventitious buds: after sterilization and disinfection, the sprouts on the rhizome of the Sichuan radix curcumae are inoculated on an induction culture medium for culture, wherein the induction culture medium is MS +6-BA 5mg/L + NAA 0.2mg/L +30g/L sucrose +7.5g/L agar, and the pH value is 5.8-5.9;
C. and (3) multiplication of cluster buds: transferring the induced adventitious buds of the curcuma longa into a multiplication medium for culture, wherein the formula of the multiplication medium comprises MS +6-BA 6mg/L + NAA 0.2mg/L + Yucca extract 0.1-1 g/L + sucrose 30g/L + agar 7.5 g/L;
D. rooting and strengthening the seedlings of the cluster buds: transferring the proliferated cluster buds to a rooting and seedling strengthening culture medium for culture, wherein the rooting and seedling strengthening culture medium is 1/2MS +6-BA 2mg/L + NAA 0.5mg/L + lanthanum nitrate 0.1-1 mg/L + sucrose +7.5g/L agar.
Preferably, in the step B, the rhizome of the Sichuan radix curcumae with the bud is taken out of the incubator, the surface of the rhizome of the Sichuan radix curcumae is washed clean by tap water, healthy and strong buds with the diameter not less than 0.5cm are cut by a scalpel, a proper amount of detergent is added into a clean tissue culture bottle, the mouth of the tissue culture bottle is tightly tied by a rubber band and a gauze and is placed under a faucet to flow a fine water flow for washing for 30min, after water is drained, the tissue culture bottle is soaked for 30s by an ethanol solution with the volume fraction of 75% on an ultraclean workbench and is washed for 3 times by sterile water, then the tissue culture bottle is soaked for 8min by mercury liter with the mass fraction of 0.1% on the ultraclean workbench and is washed for 4 times by the sterile water, each time is 2min, and finally, the water is sucked by sterile filter paper.
The MS culture medium and 1/2MS culture medium of the invention are the culture medium commonly used in the tissue culture field.
The yucca extract can improve the germination rate of the stem buds of the curcuma aromatica and increase the propagation coefficient, so that the rapid in-vitro propagation of the curcuma aromatica can be promoted, and the lanthanum nitrate can effectively promote the seedling strengthening of the curcuma aromatica, so that the appropriate conditions of the rapid in-vitro propagation technology of the curcuma aromatica can be explored, and some references are provided for the optimization and the improvement of the tissue culture rapid propagation technology of the curcuma aromatica.
Drawings
FIG. 1 is a diagram showing the effect of rhizome germination acceleration, wherein 1 is Curcuma longa Linne in sand storage; 2(CK group) is soaked in clear water; 3, soaking 1g/L of yucca extract; 4, soaking 2g/L of yucca extract; 5, soaking yucca extract at 8 g/L;
FIG. 2 is a graph showing the effect of proliferating multiple shoots;
FIG. 3 is a diagram showing the effect of rooting and strengthening the seedlings of the cluster buds.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The yucca extract is named yucca treasure, and is produced by Mexico agricultural chemical company and imported by Shanghai Youjiu biological technology Limited company.
Example 1
1. Soaking and pre-culturing Curcuma rhizome with Yucca extract
Digging after the overground part of the radix curcumae completely recedes from winter, selecting a root and stem material of the radix curcumae which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skins on the surface, cutting off fibrous roots on the surface, and soaking for 0.5h by using 1g/L of yucca extract aqueous solution (shown in table 1, CK is clear water), wherein 15 roots and stems are treated at each concentration. After soaking treatment, the mixture is put into autoclaved river sand to cover the river sand, and high-temperature germination acceleration (30 ℃, full dark culture, (70 +/-10)% RH) is carried out in an artificial climate incubator. After the root of the curcuma longa is cultured in the incubator for 18 days, the original dormant bud on the rhizome of the curcuma longa begins to sprout.
The results are shown in table 1 and fig. 1.
TABLE 1 influence of yucca extract soaking in water solution on germination of rhizome of Curcuma longa
As can be seen from Table 1 and FIG. 1, 1g/L Yucca extract can significantly improve the germination rate of the rhizome buds of Curcuma xanthorrhiza.
2. Induction of adventitious buds
Taking the rhizome of Curcuma longa with sprout out of the incubator, washing the surface with tap water, cutting the healthy and strong sprout with diameter not less than 0.5cm with scalpel, putting into a clean tissue culture bottle, adding appropriate amount of detergent, tightening the bottle mouth with rubber band and gauze, and placing under a faucet to flow fine water flow for washing for 30 min. After draining, soaking the mixture in 75 vol% ethanol solution for 30s on a clean bench, and washing with sterile water for 3 times. And then soaking the glass tube on an ultra-clean workbench for 8min by using 0.1% by mass of mercuric chloride, washing the glass tube with sterile water for 4 times, and sucking the water by using sterile filter paper for 2min each time. Cutting healthy and strong buds on the tuber of the Sichuan radix curcumae with a scalpel to be used as an explant, cutting the sterilized explant into proper sizes, sucking surface water with a sterilizing filter paper, inoculating the cut healthy and strong buds to an MS culture medium added with different hormones (Table 2, namely, each liter of the preparation method comprises the steps of adding 6-BA 5mg, NAA 0.2mg, sucrose 30g and agar 7.5g into a 1L MS culture medium, adjusting the pH to 5.8-5.9, and sterilizing for later use, or adding 6-BA 7mg, NAA0.1mg, sucrose 30g and agar 7.5g into a 1L MS culture medium, adjusting the pH to 5.8-5.9, and sterilizing for later use), and performing adventitious bud induction, wherein the culture temperature is (25 +/-3) DEG C, the illumination intensity is 1500-2000 lx, the illumination time is 12h/d, and the influence of different culture medium formulas on the adventitious bud induction is counted when the culture is carried out for 30 d.
TABLE 2 adventitious bud Induction at different combinations of plant growth regulator concentrations
The culture medium not shown in the table also contained 30g/L sucrose, 7.5g/L agar
As a result, as shown in Table 2, it can be seen from Table 2 that the group 1 medium obtained a higher induction rate.
3. Multiplication of cluster buds
Transferring the adventitious buds of Curcuma xanthorrhiza obtained by the induction in the step 2 to MS culture medium (shown in table 3) added with Yucca extract with different concentrations, and inoculating 1 bud per bottle. The culture conditions are as follows: culturing in a constant-temperature incubator at the temperature of (25 +/-3) DEG C, the illumination intensity of 1500-2000 lx and the illumination time of 12 h/d. After 30 days, the proliferation coefficient and the rooting number of the adventitious bud are counted. The formula of the culture medium is MS +6-BA 6mg/L + NAA 0.2mg/L +30g/L sucrose +7.5g/L agar, the amount of the added yucca extract is 0g/L, 0.1g/L, 0.5g/L and 1g/L respectively, and the pH value is 5.8-5.9. The preparation method comprises adding 6-BA + NAA + Yucca extract into MS culture medium according to their content, adjusting pH, and sterilizing. The results are shown in table 3 and fig. 2:
TABLE 3 proliferation of clustered shoots of Curcuma longa with addition of Yucca at various concentrations
The culture medium not shown in the table also contained 30g/L sucrose, 7.5g/L agar
As can be seen from Table 3 and FIG. 2, the multiplication factor of the clumpy buds was 1.25 in the medium without yucca extract, and the multiplication factor of the clumpy buds was 1.875 in the medium with 0.1g/L yucca extract, which was slightly increased compared to the CK group; in the culture medium added with 0.5g/L of yucca extract, the multiplication coefficient of the cluster buds is obviously increased and is the highest 3.14, and the average root number is 5.14; in the medium containing 1g/L of Yucca extract, the growth coefficient of the clumpy buds was significantly increased to 2.67 and the average number of roots was 4.67, compared to the medium of CK group. Thus, it can be seen that the yucca extract can increase the multiplication factor of the clumpy buds.
4. Rooting and seedling strengthening of cluster buds
And (3) dividing the cluster buds obtained in the step (3) into single strains, respectively inoculating the single strains to culture media (shown in table 4, containing 30g/L of sucrose and 7.5g/L of agar, wherein the specific preparation method comprises the steps of respectively adding 6-BA + NAA + lanthanum nitrate + sucrose + agar into 1/2MS culture media according to the content, adjusting the pH to 5.8-5.9, sterilizing for later use), inoculating 20 bottles of each culture medium, inoculating 1 cluster bud in each bottle of culture medium, and carrying out statistics on the average length and the thickness of the buds after culturing for 30 days. The culture conditions are as follows: culturing in a constant-temperature incubator at the temperature of (25 +/-3) DEG C, the illumination intensity of 1500-2000 lx and the illumination time of 12 h/d.
The results are shown in table 4 and fig. 3:
TABLE 4 influence of different concentrations of lanthanum nitrate on the growth of Curcuma rhizome
The culture medium not shown in the table also contained 30g/L sucrose, 7.5g/L agar
As can be seen from Table 4 and FIG. 3, 0.1-1 mg/L lanthanum nitrate can effectively promote the strong seedlings of the Curcuma longa L, wherein the effect is best when 0.5mg/L lanthanum nitrate is added, and the average length of the buds after 30 days is 6.057cm and the average thickness of the buds is 0.414 cm.
Example 2:
1. soaking and pre-culturing rhizome of Curcuma rhizome with Yucca schidigera extract
Digging after the overground part of the radix curcumae completely falls off the winter, selecting a root and stem material of the radix curcumae which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skins on the surface, cutting off fibrous roots on the surface, soaking for 0.5h by using 2g/L of yucca extract aqueous solution (shown in table 4, CK is clear water), and treating 15 roots and stems at each concentration. After soaking treatment, the mixture is put into autoclaved river sand to cover the river sand, and high-temperature germination acceleration (30 ℃, full dark culture, (70 +/-10)% RH) is carried out in an artificial climate incubator. After the root of the curcuma longa is cultured in the incubator for 18 days, the original dormant bud on the rhizome of the curcuma longa begins to sprout.
The results are shown in table 5 and fig. 1.
TABLE 5 influence of Yucca extract in aqueous solution on sprouting of Curcuma rhizome
As can be seen from Table 5 and FIG. 1, 2g/L Yucca extract can significantly improve the germination rate of the rhizome buds of Curcuma xanthorrhiza.
Other steps such as induction of adventitious bud, propagation of multiple shoots, rooting and seedling strengthening of multiple shoots were the same as in example 1.
Example 3:
1. soaking and pre-culturing Curcuma rhizome with Yucca extract
Digging after the overground part of the radix curcumae completely falls off the winter, selecting a root and stem material of the radix curcumae which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skins on the surface, cutting off fibrous roots on the surface, and soaking for 0.5h by using 8g/L yucca extract aqueous solution (shown in table 5, CK is clear water), wherein 15 roots and stems are treated at each concentration. After soaking treatment, the mixture is put into autoclaved river sand to cover the river sand, and high-temperature germination acceleration (30 ℃, full dark culture, (70 +/-10)% RH) is carried out in an artificial climate incubator. After the root of the curcuma longa is cultured in the incubator for 18 days, the original dormant bud on the rhizome of the curcuma longa begins to sprout.
The results are shown in table 6 and fig. 1.
TABLE 6 influence of soaking in Yucca extract aqueous solution on rhizome germination of Curcuma rhizome
As can be seen from the table 6 and the figure 1, the yucca extract of 8g/L can obviously improve the germination rate of the tuber buds of the Sichuan radix curcumae.
Other steps such as induction of adventitious bud, propagation of multiple shoots, rooting and seedling strengthening of multiple shoots were the same as in example 1.
Claims (1)
1. A method for promoting rapid in-vitro propagation of Curcuma rhizome is characterized by comprising the following specific steps:
A. rhizome germination of curcuma longa: digging after the overground part of the curcuma aromatica completely falls back to winter, selecting a root and stem material of the curcuma aromatica which is not rotten and has consistent size, cleaning cultivation soil, cleaning old leaves and outer skins on the surface, cutting off fibrous roots on the surface, soaking for 0.5h by using 2 g/L-8 g/L yucca extract solution, covering in autoclaved river sand after soaking, culturing at 30 ℃, and accelerating germination by 70 +/-10% RH;
B. induction of adventitious buds: sterilizing and disinfecting the sprouts on the rhizome of the curcuma longa, and inoculating the sprouts on an induction culture medium for culture, wherein the induction culture medium is MS +6-BA 5mg/L + NAA 0.2mg/L +30g/L + sucrose +7.5g/L agar, and the pH value is 5.8-5.9;
C. and (3) multiplication of cluster buds: transferring the induced adventitious buds of the curcuma longa into a multiplication medium for culture, wherein the formula of the multiplication medium comprises MS +6-BA 6mg/L + NAA 0.2mg/L + Yucca extract 0.1-1 g/L + sucrose 30g/L + agar 7.5 g/L;
D. rooting and strengthening the seedlings of the cluster buds: transferring the proliferated cluster buds to a rooting and seedling strengthening culture medium for culture, wherein the rooting and seedling strengthening culture medium is 1/2MS +6-BA 2mg/L + NAA 0.5mg/L + lanthanum nitrate 0.1-1 mg/L + sucrose +7.5g/L agar;
and B, sterilizing and disinfecting the sprouts on the roots and stems of the Sichuan radix curcumae, namely taking the roots and stems of the Sichuan radix curcumae with the sprouts out of an incubator, washing the surface of the roots and stems of the Sichuan radix curcumae clean by tap water, cutting healthy and strong sprouts with the diameter not less than 0.5cm by using a scalpel, putting the roots and stems of the Sichuan radix curcumae into a clean tissue culture bottle, adding a proper amount of detergent into the tissue culture bottle, tightly binding a bottle mouth with a rubber band and a gauze, placing the bottle mouth under a faucet, flowing a fine water stream for washing for 30min, draining water, soaking the bottle on an ultraclean workbench for 30s by using an ethanol solution with the volume fraction of 75%, washing the bottle with sterile water for 3 times, soaking the bottle on the ultraclean workbench for 8min by using mercuric chloride with the mass fraction of 0.1%, washing the bottle with the sterile water for 4 times, washing the bottle for 2min each time, and finally drying the water by using sterile filter paper.
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