CN110604056B - Tissue culture method for curcuma wenyujin - Google Patents
Tissue culture method for curcuma wenyujin Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a method for culturing curcuma wenyujin tissue, which mainly comprises the steps of tuber sand bed pregermination, adventitious bud induction, subculture multiplication, rooting culture, temporary planting and the like. By adopting the method, the pollution rate is less than 5 percent in the culture process, the adventitious bud induction rate reaches more than 129 percent, the bud multiplication factor reaches more than 7.5, the survival rate reaches more than 95 percent, and the volatile oil content reaches more than 5.4 percent. The method has simple operation and remarkable effect, can provide a large amount of high-quality Curcuma wenyujin seedlings in a short time, and is suitable for industrialized popularization and use.
Description
Technical Field
The invention relates to a method for tissue culture of curcuma wenyujin, belonging to the technical field of plant tissue culture.
Background
Curcuma wenyujin Y.H.Chen et C.Ling, also called Curcuma wenyujin, belongs to Curcuma of Zingiberaceae, perennial bulbous herbaceous flowers, plant height 100-120 cm, plant cluster, long and elliptical leaf, bright green, can be used as foliage plant for appreciation, and is suitable for garden cultivation or large pot culture. In the flowering period of 3-5 months, the cylindrical spike-shaped inflorescences are extracted from the rhizomes, the claret bracts are tightly arranged on the inflorescences, the duration of the flowering period is long, and the cylindrical spike-shaped inflorescences can be arranged as a flower mirror and a flower bed. The curcuma wenyujin is a similar round or irregular sheet with the diameter of 1.5-4 cm, the surface is gray yellow, the section is yellow brown to tan, and the curcuma wenyujin is often accompanied by yellow white or yellow brown powder, so the vascular bundle is more and obvious. Strong smell, slightly bitter and pungent taste. The turmeric root tuber is a famous medicinal material, the medicinal parts of the turmeric root tuber mainly comprise rhizome and root tuber, and the turmeric root tuber mainly comprises volatile oil, curcumin components, resins, saccharides and the like, and is a variety accepted by Chinese pharmacopoeia in multiple editions. The existing research shows that the curcuma wenyujin volatile oil has the activity of resisting plant pathogenic bacteria, and shows that the curcuma wenyujin volatile oil has certain application potential in the aspect of plant source bactericides.
With the research, the demand of the curcuma wenyujin resource is more and more. However, in cultivation and production, the curcuma wenyujin is mainly subjected to nutrition propagation through roots and stems, so that the seed consumption is large, the propagation coefficient is low, the virification is serious, the speed is slow, the quality is reduced, and the supply of resources is short of demand. In order to solve the problems in the production of curcuma wenyujin, a rapid propagation technology suitable for curcuma wenyujin is urgently needed to be developed. The plant tissue culture technology is a method for realizing rapid propagation by utilizing explants such as roots, stems, leaves, flowers and fruits of plants, and can effectively make up for the defects of the traditional culture method, but the tissue culture method of the curcuma wenyujin disclosed at present is not mature enough, and has a plurality of defects such as poor induction and proliferation effects, so that a method more suitable for tissue culture of the curcuma wenyujin is urgently needed to be found.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a method more suitable for tissue culture of curcuma wenyujin, which can obtain a large number of seedlings in a short period, rejuvenate a new excellent variety of curcuma wenyujin and provide a certain technical reference for large-scale mass propagation of curcuma wenyujin.
The technical scheme adopted by the invention is as follows:
a method for culturing Curcuma wenyujin tissue comprises the following steps:
s1: tuber sand bed germination acceleration
Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;
s2: explant acquisition
Selecting healthy plantlets, cutting off rhizome parts, disinfecting the plantlets with 75% alcohol for 30s, then disinfecting the plantlets with 0.1% mercuric chloride for 20min, washing the plantlets with sterile water for 3-5 times, and inoculating the plantlets in an MS culture medium containing 4-5 mg/L6-BA to obtain sterile explants;
s3: induction of adventitious buds
When the buds grow to 3-5 cm, inoculating the buds into an MS culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for induction culture;
s4: subculture multiplication
Cutting the new buds obtained by induction into small blocks with less than 5 lateral buds, and inoculating the small blocks into a culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for enrichment culture;
s5: rooting culture of test-tube plantlets
After subculture, inoculating the test-tube plantlet into an MS culture medium containing 0.5-1 g/L of active carbon and 0.5-1.0 mg/L of NAA for culture;
s6: temporary planting of test-tube plantlet
After rooting culture for 1 month, opening a bottle cap to ensure that the rooted seedlings are fully contacted with air, standing for at least 1 week, taking out the test-tube seedlings, and transplanting the test-tube seedlings into river sand matrix for culture;
the illumination conditions of the steps are as follows: the illumination intensity is 1600-2400 lx, the illumination time is 10-12 h/d, the ambient temperature is 25-28 ℃, and the ambient humidity is 40-90%.
Preferably, the MS culture medium obtained in the steps S2-S5 further contains 30-35 g/L of sucrose and 5-6 g/L of carrageenan.
Preferably, in step S3, the 6-BA content is 4-5 mg/L and the NAA content is 0.5 mg/L.
Preferably, the MS culture medium in the step S3 further contains 3-5 g/L alginate-derived oligosaccharide, 6.5-10.3 mg/L salicylic acid and 11-18 mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 640-680 mg/L.
Preferably, in step S4, the 6-BA content is 5mg/L and the NAA content is 0.5 mg/L.
Preferably, in step S5, the NAA content is 0.5 mg/L.
Preferably, the MS culture medium in the step S5 further contains 5-10 mg/L methyl jasmonate, and the concentration of calcium chloride is changed to 520-550 mg/L.
Preferably, in the steps S1 to S4, the LED is illuminated for 10-12 hours per day under the condition of red and blue light, the proportion of red and blue light is (4-5): 1, the illumination intensity is 1600-2000 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S5, illuminating for 10-12 hours per day under the white light condition, wherein the illumination intensity is 1800-2400 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; and step S6, the method is carried out under outdoor natural illumination conditions, wherein the ambient humidity is 80-90%, and the ambient temperature is 25-28 ℃.
Compared with the prior art, the invention has the beneficial effects that:
by adopting the method for warm zedoary tissue culture, the pollution rate is less than 5%, the induction rate reaches more than 129%, the bud multiplication multiple reaches more than 7.5, the survival rate reaches more than 95%, and the volatile oil content reaches more than 5.4%.
By improving the culture conditions and the culture medium, the pollution rate can be reduced to 2.1%, the induction rate can be increased to 158%, the multiplication factor can be increased to 11.37, more importantly, the volatile oil content can be increased to 8.22%, the actual requirements of people on the curcuma wenyujin can be met, and a large amount of high-quality raw materials can be provided for the curcuma wenyujin.
Drawings
FIG. 1: the effect chart of the tissue culture process of the curcuma aromatica is shown in the embodiment of the invention; A. inoculating an explant; B. differentiating the bud by the explant; C. differentiating the clustered shoots by the explant; D. multiplication of cluster buds; E. rooting seedlings; F. and (5) performing temporary planting.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1
A method for culturing Curcuma wenyujin tissue comprises the following steps:
s1: tuber sand bed germination acceleration
Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;
s2: explant acquisition
Selecting healthy plantlets, cutting off rhizome parts, sterilizing the plantlets with 75% alcohol for 30s, then sterilizing the plantlets with 0.1% mercuric chloride for 20min, cleaning the plantlets with sterilized water for 3-5 times, and inoculating the plantlets in an MS culture medium containing 30g/L of sucrose, 5g/L of carrageenan and 5mg/L of 6-BA to obtain sterile explants;
s3: induction of adventitious buds
When the buds grow to 3-5 cm, carrying out induction culture on the buds in an MS culture medium containing 30g/L of sucrose, 5g/L of carrageenan, 5mg/L of 6-BA and 0.5mg/L of NAA, and inoculating 1 bud in each bottle; the pH value of the culture medium is 5.5-5.8. And 4-5 d later, observing the growth condition of the buds, picking up the polluted bacterial seedlings, counting the pollution rate, continuously observing the induced differentiation condition of the adventitious buds of the curcuma wenyujin, and counting the induction rate of the buds after 30 d. The contamination rate (%) was the number of contaminated bottles/total bottles × 100%, and the adventitious bud induction rate (%) was the number of induced lateral buds/number of inoculated buds × 100%.
S4: subculture multiplication
Cutting the new buds obtained by induction into small blocks with the lateral buds not more than 5, inoculating the small blocks into a culture medium containing 30g/L of sucrose, 5g/L of carrageenan, 5mg/L of 6-BA and 0.5mg/L of NAA for enrichment culture, and inoculating 1-4 small blocks in each bottle; subculture 1 time every 20 d. Observing the proliferation and growth conditions of the buds, and counting the number of the proliferation buds and the proliferation multiple of the common turmeric. The number of proliferating shoots is the total number of shoots induced by the inoculated explants. The multiplication factor is the number of multiplied shoots/number of inoculated shoots.
S5: rooting culture of test-tube plantlets
After the subculture, the test-tube plantlets were inoculated into MS medium containing 30g/L sucrose, 5g/L carrageenan, 1g/L activated carbon and 0.5mg/L NAA for culture.
S6: temporary planting of test-tube plantlet
After rooting culture for 1 month, opening the bottle cap to ensure that the rooted seedlings are fully contacted with the air, standing for at least 1 week, taking out the test-tube seedlings, washing a root culture medium with clear water, and transplanting the test-tube seedlings into a river sand matrix for culture. And after 30 days, counting the transplanting survival rate of the curcuma wenyujin.
The illumination conditions of the steps are as follows: the white light source has illumination intensity of 2000-2200 lx, L/D of 10:14, ambient temperature of 25-28 ℃ and ambient humidity of 80-90%.
Example 2
A method for culturing Curcuma wenyujin tissue comprises the following steps:
s1: tuber sand bed germination acceleration
Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;
s2: explant acquisition
Selecting healthy plantlets, cutting off rhizome parts, sterilizing the plantlets with 75% alcohol for 30s, then sterilizing the plantlets with 0.1% mercuric chloride for 20min, cleaning the plantlets with sterilized water for 3-5 times, and inoculating the plantlets in an MS culture medium containing 35g/L of sucrose, 6g/L of carrageenan and 4mg/L of 6-BA to obtain sterile explants;
s3: induction of adventitious buds
When the buds grow to 3-5 cm, inoculating the buds into an MS culture medium containing 35g/L of sucrose, 6g/L of carrageenan, 2mg/L of 6-BA and 1.0mg/L of NAA for induction culture, and inoculating 1 bud per bottle; the pH value of the culture medium is 5.5-5.8. And 4-5 d later, observing the growth condition of the buds, picking up the polluted bacterial seedlings, counting the pollution rate, continuously observing the induced differentiation condition of the adventitious buds of the curcuma wenyujin, and counting the induction rate of the buds after 30 d. The contamination rate (%) was the number of contaminated bottles/total bottles × 100%, and the adventitious bud induction rate (%) was the number of induced lateral buds/number of inoculated buds × 100%.
S4: subculture multiplication
Cutting the new buds obtained by induction into small blocks with the lateral buds not more than 5, inoculating the small blocks into a culture medium containing 35g/L of sucrose, 6g/L of carrageenan, 2mg/L of 6-BA and 1.0mg/L of NAA for enrichment culture, and inoculating 1-4 small blocks in each bottle; subculture 1 time every 20 d. Observing the proliferation and growth conditions of the buds, and counting the number of the proliferation buds and the proliferation multiple of the common turmeric. The number of proliferating shoots is the total number of shoots induced by the inoculated explants. The multiplication factor is the number of multiplied shoots/number of inoculated shoots.
S5: rooting culture of test-tube plantlets
After the subculture, the test-tube plantlets were inoculated into MS medium containing 35g/L sucrose, 6g/L carrageenan, 0.5g/L activated carbon and 1.0mg/L NAA for culture.
S6: temporary planting of test-tube plantlet
After rooting culture for 1 month, opening the bottle cap to ensure that the rooted seedlings are fully contacted with the air, standing for at least 1 week, taking out the test-tube seedlings, washing a root culture medium with clear water, and transplanting the test-tube seedlings into a river sand matrix for culture. And after 30 days, counting the transplanting survival rate of the curcuma wenyujin.
The illumination conditions of the steps are as follows: the white light source has illumination intensity of 2000-2200 lx, L/D of 10:14, ambient temperature of 25-28 ℃ and ambient humidity of 40-50%.
Example 3
The difference between example 3 and example 1 is:
the MS culture medium of the step S3 also contains 3g/L alginate-derived oligosaccharide, 6.5mg/L salicylic acid and 11mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 640 mg/L.
The MS culture medium of the step S5 also contains 5mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 520 mg/L.
Step S1 to step S4, the LED is illuminated for 10 hours every day under the condition of red and blue light of the LED, the ratio of the red light to the blue light is 4:1, the illumination intensity is 1600-1800 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S5, illuminating for 10 hours every day under the white light condition, wherein the illumination intensity is 1800-2200 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S6, illuminating for 10 hours every day under the white light condition, wherein the illumination intensity is 1800-2200 lx, the ambient humidity is 80-90%, and the ambient temperature is 25-28 ℃.
Example 4
The difference between example 4 and example 1 is:
the MS culture medium of the step S3 also contains 5g/L alginate-derived oligosaccharide, 10.3mg/L salicylic acid and 18mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 680 mg/L.
The MS culture medium of the step S5 also contains 10mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 550 mg/L.
Step S1 to step S4, under the condition of LED red and blue light, the light is irradiated for 12 hours every day, the ratio of the red light to the blue light is 5:1, the light intensity is 2000-2200 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S5, illuminating for 12 hours every day under the white light condition, wherein the illumination intensity is 2200-2400 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S6, illuminating for 12 hours every day under the white light condition, wherein the illumination intensity is 2200-2400 lx, the ambient humidity is 80-90%, and the ambient temperature is 25-28 ℃.
Comparative example 1
A method for culturing Curcuma wenyujin tissue comprises the following steps:
s1: tuber sand bed germination acceleration
Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;
s2: explant acquisition
Selecting healthy plantlets, cutting off rhizome parts, sterilizing with 75% alcohol for 20s, then sterilizing with 0.2% mercuric chloride for 20min, cleaning with sterile water for 3-5 times, and inoculating in an MS culture medium containing 5mg/L6-BA to obtain sterile explants;
s3: induction of adventitious buds
When the buds grow to 3-5 cm, inoculating the buds into an MS culture medium containing 2.0 mg/L6-BA and 0.5mg/L NAA for induction culture, and inoculating 1 bud in each bottle; the pH value of the culture medium is 5.5-5.8. And 4-5 d later, observing the growth condition of the buds, picking up the polluted bacterial seedlings, counting the pollution rate, continuously observing the induced differentiation condition of the adventitious buds of the curcuma wenyujin, and counting the induction rate of the buds after 30 d. The contamination rate (%) was the number of contaminated bottles/total bottles × 100%, and the adventitious bud induction rate (%) was the number of induced lateral buds/number of inoculated buds × 100%.
S4: subculture multiplication
Cutting the new buds obtained by induction into small blocks with the lateral buds not more than 5, inoculating the small blocks into a culture medium containing 30g/L of sucrose, 0.4mg/L of TDZ and 0.5mg/L of NAA for enrichment culture, and inoculating 1-4 small blocks in each bottle; subculture 1 time every 20 d. Observing the proliferation and growth conditions of the buds, and counting the number of the proliferation buds and the proliferation multiple of the common turmeric. The number of proliferating shoots is the total number of shoots induced by the inoculated explants. The multiplication factor is the number of multiplied shoots/number of inoculated shoots.
S5: rooting culture of test-tube plantlets
After the subculture, the test-tube plantlets were inoculated in MS medium containing 0.5g/L of activated charcoal and 0.5mg/L of NAA and cultured.
S6: temporary planting of test-tube plantlet
After rooting culture for 1 month, opening the bottle cap to ensure that the rooted seedlings are fully contacted with the air, standing for 1 week, taking out the test-tube seedlings, washing a root culture medium with clear water, and transplanting the test-tube seedlings into river sand matrix for culture. And after 30 days, counting the transplanting survival rate of the curcuma wenyujin.
The illumination conditions of the steps are as follows: a white light source, wherein the illumination intensity is 1300-1500 lx, and the illumination time is 8 h/d;
test example:
(1) the method of the embodiment and the method of the comparative example are respectively adopted to carry out the tissue culture of the curcuma wenyujin, and the pollution rate, the adventitious bud induction rate, the adventitious bud multiplication multiple and the transplanting survival rate are counted. The results are shown in Table 1.
(2) Transplanting the provisionally planted plantlets into a field for planting, taking roots and stems of the plantlets after 6 months, and measuring the volatile oil content according to an XD method in an appendix of the first part of the pharmacopoeia of the people's republic of China 2015. The results are shown in Table 1.
TABLE 1
| Rate of contamination | Rate of induction of shoots | Multiple of bud multiplication | Survival rate | Content of volatile oil (mL/g) | |
| Practice ofExample 1 | 4.3％ | 137％ | 9.97 | 95％ | 5.57±0.03％ |
| Example 2 | 4.5％ | 129％ | 7.50 | 97％ | 5.40±0.07％ |
| Example 3 | 2.1％ | 158％ | 12.20 | 97％ | 7.89±0.05％ |
| Example 4 | 3.4％ | 155％ | 11.37 | 96％ | 8.22±0.04％ |
| Comparative example 1 | 6.6％ | 107％ | 4.12 | 97％ | 5.49±0.03％ |
The results show that the method for warm zedoary tissue culture has the pollution rate of less than 5 percent, the inductivity of more than 129 percent, the bud multiplication factor of more than 7.5, the survival rate of more than 95 percent and the volatile oil content of more than 5.4 percent.
In addition, the invention discovers that the pollution rate can be reduced to 2.1%, the induction rate can be improved to 158%, the multiplication factor can be improved to 11.37, and more importantly, the volatile oil content can be simultaneously improved to 8.22% by improving the culture conditions and the culture medium.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Claims (5)
1. A method for culturing Curcuma wenyujin tissue is characterized by comprising the following steps:
s1: tuber sand bed germination acceleration
Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;
s2: explant acquisition
Selecting healthy plantlets, cutting off rhizome parts, disinfecting the plantlets with 75% alcohol for 30s, then disinfecting the plantlets with 0.1% mercuric chloride for 20min, washing the plantlets with sterile water for 3-5 times, and inoculating the plantlets in an MS culture medium containing 4-5 mg/L6-BA to obtain sterile explants;
s3: induction of adventitious buds
When the buds grow to 3-5 cm, inoculating the buds into an MS culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for induction culture; the MS culture medium also contains 3-5 g/L alginate oligosaccharide, 6.5-10.3 mg/L salicylic acid and 11-18 mg/L methyl jasmonate, and the concentration of calcium chloride in the culture medium is changed to 640-680 mg/L;
s4: subculture multiplication
Cutting the new buds obtained by induction into small blocks with less than 5 lateral buds, and inoculating the small blocks into an MS culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for enrichment culture;
s5: rooting culture of test-tube plantlets
After subculture, inoculating the test-tube plantlet into an MS culture medium containing 0.5-1 g/L of active carbon and 0.5-1.0 mg/L of NAA for culture; the MS culture medium also contains 5-10 mg/L methyl jasmonate, and the concentration of calcium chloride is changed to 520-550 mg/L;
s6: temporary planting of test-tube plantlet
After rooting culture for 1 month, opening a bottle cap to ensure that the rooted seedlings are fully contacted with air, standing for at least 1 week, taking out the test-tube seedlings, and transplanting the test-tube seedlings into river sand matrix for culture;
step S1 to step S4, the LED is illuminated for 10-12 hours per day under the condition of red and blue light of the LED, the proportion of the red light to the blue light is (4-5): 1, the illumination intensity is 1600-2000 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S5, illuminating for 10-12 hours per day under the white light condition, wherein the illumination intensity is 1800-2400 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; and step S6, the method is carried out under outdoor natural illumination conditions, wherein the ambient humidity is 80-90%, and the ambient temperature is 25-28 ℃.
2. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein the MS culture medium in steps S2-S5 further contains sucrose 30-35 g/L and carrageenan 5-6 g/L.
3. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein in step S3, the content of 6-BA is 4-5 mg/L and the content of NAA is 0.5 mg/L.
4. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein the amount of 6-BA in step S4 is 5mg/L and the amount of NAA in step S is 0.5 mg/L.
5. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein in step S5, NAA content is 0.5 mg/L.
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