CN111727881A - Culture medium and culture method for promoting sterile germination of dendrobium officinale seeds - Google Patents
Culture medium and culture method for promoting sterile germination of dendrobium officinale seeds Download PDFInfo
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- CN111727881A CN111727881A CN202010552546.3A CN202010552546A CN111727881A CN 111727881 A CN111727881 A CN 111727881A CN 202010552546 A CN202010552546 A CN 202010552546A CN 111727881 A CN111727881 A CN 111727881A
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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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a culture medium and a culture method for promoting sterile germination of dendrobium officinale seeds, wherein the culture medium comprises the following components: 250g of potato juice, 0.8-1.2mg of 6-BA, 0.4-0.6mg of NAA, 0.1-0.2mg of lanthanum nitrate, 50-100mg of panax notoginseng saponins, 0.1-0.4g of oligopeptide, 0.2-0.5g of trehalose, 25-30g of cane sugar and 7-10g of agar, and the total volume of 1/2MS culture medium is up to 1000 mL. According to the invention, through optimization of the formula of the culture medium, the germination rate and the germination speed of the dendrobium officinale seeds in aseptic breeding culture are effectively improved, and the large-scale cultivation of the dendrobium officinale is facilitated.
Description
Technical Field
The invention relates to the technical field of aseptic culture of dendrobium officinale, in particular to a culture medium and a culture method for promoting aseptic germination of dendrobium officinale seeds.
Background
The dendrobium officinale is a herbal plant in the genus of dendrobium of the family of orchidaceae, has a long medicinal history, is rich in various active ingredients such as polysaccharide, alkaloid, amino acid and the like, has the effects of promoting the production of body fluid, moistening lung, tonifying kidney and improving eyesight, has good effects of resisting tumors, reducing blood sugar, resisting aging, enhancing immunity and the like, and is highly evaluated by the market. As the market demand gradually rises, the wild dendrobium officinale resources are gradually exhausted, and the price is gradually high. Therefore, artificial cultivation of dendrobium officinale becomes a main source, and the demand of seedlings for cultivating the dendrobium officinale is increasing. At present, the dendrobium officinale mainly adopts a tissue culture method, seeds are used as explants for aseptic breeding, the germination rate is low, the seed germination speed is slow, and a large amount of seedlings are difficult to obtain in a short time. Therefore, the germination rate and the germination speed of the dendrobium officinale seeds are improved, and the method has very important significance for large-scale cultivation of the dendrobium officinale.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a culture medium and a culture method for promoting sterile germination of dendrobium officinale seeds.
The invention provides a culture medium for promoting sterile germination of dendrobium officinale seeds, which comprises the following components: 250g of potato juice, 0.8-1.2mg of 6-BA, 0.4-0.6mg of NAA, 0.1-0.2mg of lanthanum nitrate, 5-20mg of panax notoginseng saponins, 0.1-0.4g of oligopeptide, 0.2-0.5g of trehalose, 25-30g of cane sugar and 7-10g of agar, and the total volume of 1/2MS culture medium is up to 1000 mL.
Preferably, the culture medium for promoting the sterile germination of the dendrobium officinale seeds comprises the following components: 230g of potato juice, 1mg of 6-BA, 0.5mg of NAA, 0.14mg of lanthanum nitrate, 15mg of panax notoginseng saponins, 0.25g of oligopeptide, 0.45g of trehalose, 28g of sucrose, 8g of agar and the balance of 1/2MS culture medium to 1000 mL.
Preferably, the molecular weight of the oligopeptide is 200-1000 Da.
Preferably, the oligopeptide is at least one of corn oligopeptide, soybean oligopeptide, rice oligopeptide and wheat oligopeptide.
An aseptic germination culture method for dendrobium officinale seeds adopts the culture medium for culture.
Preferably, the sterile germination culture method for the dendrobium officinale seeds comprises the following steps:
s1, taking the mature fruits of dendrobium officinale, and cleaning and disinfecting the mature fruits;
s2, inoculating the seeds in the fruits processed in the step S1 into the culture medium for culture.
Preferably, in step S1, the method of the sterilization process includes: soaking in 70-75% ethanol for 40-50s, and treating with 0.05-0.15% mercuric chloride solution for 15-30 min.
Preferably, in step S2, the cultivation method is: culturing at 24-26 deg.C under 1400-1800lux for 10-14h per day.
Wherein, 1/2MS culture medium is prepared by reducing macroelements of MS culture medium by half and keeping other macroelements unchanged, and comprises the following specific components:
KNO3950mg/L、NH4NO3825mg/L、KH2PO485mg/L、MgSO4.7H2O 185mg/L、CaCl2.2H2O220mg/L、KI 0.415mg/L、H3BO33.1mg/L、MnSO4.7H2O 11.15mg/L、ZnSO4.7H2O4.3mg/L、Na2MoO4.2H2O 0.125mg/L、CuSO4.5H2O 0.0125mg/L、CoCl2.6H2O0.0125mg/L, disodium edetate 37.3mg/L, FeSO4.7H227.8mg/L of O, 100mg/L of inositol, 10.1mg/L of vitamin B, 60.5mg/L of vitamin B, 0.5mg/L of nicotinic acid and 2.0mg/L of glycine.
The invention has the following beneficial effects:
in the invention, additives of lanthanum nitrate, panax notoginseng saponins, oligopeptide and trehalose with proper concentration are added into a conventional sterile germination culture medium for dendrobium officinale seeds. Lanthanum nitrate and panax notoginseng saponins can increase the permeability of a seed film, enhance the respiration of the seeds and accelerate the water absorption rate of the seeds, so that the germination speed and the germination rate of the seeds are effectively improved; the oligopeptide is a fragment which is composed of 2-6 amino acids and has the molecular weight of below 1000Da, is a high-efficiency transfer carrier, can be used as a nutrient component carrier, and can quickly meet the nutrient requirement in the seed germination process, thereby playing an auxiliary role and improving the germination speed and the germination rate of seeds. However, because the tolerance of the dendrobium officinale seeds to the salt solution is low, the high-concentration rare earth salt solution can cause osmotic stress and inhibit the germination of the seeds, the metabolism is enhanced due to the accelerated germination rate, the rapid oxygen consumption is caused, a large number of free radicals are generated, and the activity of the seeds is damaged. Therefore, the formula is reasonably optimized based on the selection of additives such as lanthanum nitrate, panax notoginseng saponins, oligopeptide and trehalose and the adjustment of the content of each additive, so that the efficient sterile germination culture medium for the dendrobium officinale seeds is obtained, the germination rate and the germination speed of the dendrobium officinale seeds in sterile breeding culture can be greatly improved, and the large-scale cultivation of the dendrobium officinale is facilitated.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
A culture medium for promoting sterile germination of dendrobium officinale seeds comprises the following components: 230g of potato juice, 6-BA1mg, 0.5mg of NAA, 0.14mg of lanthanum nitrate, 15mg of panax notoginseng saponins, 0.25g of oligopeptide, 0.45g of trehalose, 28g of sucrose, 8g of agar and the balance of 1/2MS culture medium to 1000 mL.
Wherein the oligopeptide is wheat oligopeptide with the molecular weight of 500-1000 Da.
An aseptic germination culture method for dendrobium officinale seeds comprises the following steps:
s1, taking mature fruits of dendrobium officinale, cleaning the epidermis with clear water, soaking the epidermis in 70% alcohol for 45S, washing with sterile water for 3 times, treating with 0.1% mercuric chloride solution for 20min, washing with sterile water for 10 times, and sucking the water of the epidermis with sterile filter paper;
s2, cutting the fruit processed in the step S1, uniformly sowing seeds in the fruit in the culture medium, and culturing under the conditions that the temperature is 25 ℃ and the illumination intensity is 1500lux, wherein the illumination time is 12 hours per day.
Example 2
A culture medium for promoting sterile germination of dendrobium officinale seeds comprises the following components: 220g of potato juice, 6-BA0.8mg, 0.4mg of NAA, 0.1mg of lanthanum nitrate, 5mg of panax notoginseng saponins, 0.1g of oligopeptide, 0.2g of trehalose, 25g of sucrose, 7g of agar and the balance of 1/2MS culture medium to 1000 mL.
Wherein the oligopeptide is wheat oligopeptide with the molecular weight of 500-1000 Da.
An aseptic germination culture method for dendrobium officinale seeds comprises the following steps:
s1, taking mature fruits of dendrobium officinale, cleaning the epidermis with clear water, soaking the epidermis with 70% alcohol for 50S, washing with sterile water for 3 times, treating with 0.05% mercuric chloride solution for 30min, washing with sterile water for 10 times, and sucking water of the epidermis with sterile filter paper;
s2, cutting the fruit processed in the step S1, uniformly sowing seeds in the fruit in the culture medium, and culturing under the conditions that the temperature is 24 ℃ and the illumination intensity is 1400lux, wherein the illumination time is 10 hours per day.
Example 3
A culture medium for promoting sterile germination of dendrobium officinale seeds comprises the following components: 250g of potato juice, 6-BA1.2mg, 0.6mg of NAA, 0.2mg of lanthanum nitrate, 20mg of panax notoginseng saponins, 0.4g of oligopeptide, 0.5g of trehalose, 30g of sucrose, 10g of agar and the balance of 1/2MS culture medium to 1000 mL.
Wherein the oligopeptide is wheat oligopeptide with the molecular weight of 500-1000 Da.
An aseptic germination culture method for dendrobium officinale seeds comprises the following steps:
s1, taking mature fruits of dendrobium officinale, cleaning the epidermis with clear water, soaking the epidermis with 75% alcohol for 40S, washing with sterile water for 3 times, treating with 0.15% mercuric chloride solution for 15min, washing with sterile water for 10 times, and sucking water of the epidermis with sterile filter paper;
s2, cutting the fruit processed in the step S1, uniformly sowing seeds in the fruit in the culture medium, and culturing under the conditions that the temperature is 26 ℃ and the illumination intensity is 1800lux, wherein the illumination time is 14 hours per day.
Comparative example 1
Comparative example 1 compared to example 1, the only difference is: adopting a conventional dendrobium officinale seed sterile germination culture medium, wherein the culture medium comprises the following components: 230g of potato juice, 1mg of 6-BA, 0.5mg of NAA, 28g of sucrose, 8g of agar and 1/2MS medium to make up to 1000 mL.
The culture method comprises the following steps:
s1, taking mature fruits of dendrobium officinale, cleaning the epidermis with clear water, soaking the epidermis in 70% alcohol for 45S, washing with sterile water for 3 times, treating with 0.1% mercuric chloride solution for 20min, washing with sterile water for 10 times, and sucking the water of the epidermis with sterile filter paper;
s2, cutting the fruit processed in the step S1, uniformly sowing seeds in the fruit into the conventional dendrobium officinale seed sterile germination culture medium, and culturing under the conditions that the temperature is 25 ℃ and the illumination intensity is 1500lux, wherein the illumination time is 12 hours per day.
And observing and recording germination conditions of the dendrobium seeds of the examples 1-3 and the comparative example 1, and recording germination rate and germination time, wherein the time when the seed embryo is enlarged and turned green and the seed coat is broken is counted as the germination time. The observation results are shown in table 1:
TABLE 1 germination of Dendrobium nobile seeds
| Experimental group | Percentage of germination (%) | Time of germination (d) |
| Example 1 | 98.2 | 5 |
| Example 2 | 97.6 | 7 |
| Example 3 | 96.3 | 7 |
| Comparative example 1 | 87.5 | 12 |
Therefore, the culture medium disclosed by the invention can effectively improve the germination rate and the germination speed of the dendrobium officinale seeds, and is suitable for tissue culture of the dendrobium officinale seeds.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A culture medium for promoting sterile germination of dendrobium officinale seeds is characterized by comprising the following components: 250g of potato juice, 0.8-1.2mg of 6-BA, 0.4-0.6mg of NAA, 0.1-0.2mg of lanthanum nitrate, 5-20mg of panax notoginseng saponins, 0.1-0.4g of oligopeptide, 0.2-0.5g of trehalose, 25-30g of cane sugar and 7-10g of agar, and the total volume of 1/2MS culture medium is up to 1000 mL.
2. The culture medium for promoting the sterile germination of the dendrobium officinale seeds as claimed in claim 1, wherein the culture medium comprises the following components: 230g of potato juice, 1mg of 6-BA, 0.5mg of NAA, 0.14mg of lanthanum nitrate, 15mg of panax notoginseng saponins, 0.25g of oligopeptide, 0.45g of trehalose, 28g of sucrose, 8g of agar and the balance of 1/2MS culture medium to 1000 mL.
3. The culture medium for promoting the sterile germination of the dendrobium officinale seeds as claimed in claim 1 or 2, wherein the molecular weight of the oligopeptide is 200-1000 Da.
4. The culture medium for promoting sterile germination of the dendrobium officinale seeds as claimed in any one of claims 1-3, wherein the oligopeptide is at least one of corn oligopeptide, soybean oligopeptide, rice oligopeptide and wheat oligopeptide.
5. An aseptic germination culture method of Dendrobium officinale seeds, which is characterized in that the culture medium of any one of claims 1-4 is adopted for culture.
6. The method for culturing the dendrobium officinale seeds in the sterile germination mode according to claim 5, wherein the method comprises the following steps:
s1, taking the mature fruits of dendrobium officinale, and cleaning and disinfecting the mature fruits;
s2, inoculating the seeds in the fruits processed in the step S1 into the culture medium for culture.
7. The method for culturing the dendrobium officinale seeds in the sterile germination mode according to claim 6, wherein in the step S1, the disinfection treatment method comprises the following steps: soaking in 70-75% ethanol for 40-50s, and treating with 0.05-0.15% mercuric chloride solution for 15-30 min.
8. The method for culturing the dendrobium officinale seeds as claimed in claim 6 or 7, wherein in the step S2, the culturing method comprises the following steps: culturing at 24-26 deg.C under 1400-1800lux for 10-14h per day.
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Application publication date: 20201002 |