CN109287482B - Special culture medium for tissue culture and rapid propagation of gardenia jasminoides - Google Patents
Special culture medium for tissue culture and rapid propagation of gardenia jasminoides Download PDFInfo
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- CN109287482B CN109287482B CN201811279135.0A CN201811279135A CN109287482B CN 109287482 B CN109287482 B CN 109287482B CN 201811279135 A CN201811279135 A CN 201811279135A CN 109287482 B CN109287482 B CN 109287482B
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- gardenia jasminoides
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/25—Dry fruit hulls or husks, e.g. chaff or coir
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/30—Growth substrates; Culture media; Apparatus or methods therefor based on or containing synthetic organic compounds
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a special culture medium for tissue culture and rapid propagation of gardenia jasminoides, which can improve the rooting rate and the transplanting survival rate of gardenia jasminoides. The promoter obtained by compounding the modified pine nut shells and the 1-deoxynojirimycin in a specific ratio is added into the conventional rooting culture medium, so that the rooting rate of gardenia jasminoides is improved, and particularly, the rooting time is shortened. And the post-forming rate after transplanting can reach 100%. In addition, the promoter disclosed by the invention is added into an MS culture medium, the addition amount is 1.0mg/L, and the promoter is used for culturing gardenia jasminoides callus, so that the content of jasminoidin in the callus can be greatly increased, and a new application is opened up for the promoter disclosed by the invention.
Description
Technical Field
The invention relates to a culture medium special for tissue culture and rapid propagation of gardenia jasminoides, belonging to the field of rapid propagation of traditional Chinese medicines
Background
The gardenia jasminoides is also called as gardenia jasminoides and gardenia jasminoides ellis, and fruits and roots are used as medicines, so that the effects of purging fire, detoxifying, clearing heat, promoting diuresis, cooling blood and dissipating blood stasis are achieved. Is distributed in Zhejiang, Jiangxi, Fujian, Hubei, Sichuan and Guizhou provinces, and the Yichun area in Jiangxi is mainly distributed in camphor trees, Fengcheng, Wan-carried and Fengxin provinces.
The traditional breeding mode of the gardenia jasminoides is plant division or cuttage, and the method has the advantages of low breeding coefficient, low speed and long breeding period, and greatly hinders the cultivation and popularization of the gardenia jasminoides. The tissue culture method can improve the propagation coefficient and shorten the culture time. However, the existing culture medium has some defects in the aspects of improving the transplanting survival rate and the content of effective components, and needs to be further improved.
Disclosure of Invention
The invention aims to provide a special culture medium for tissue culture and rapid propagation of gardenia jasminoides, which can improve the rooting rate and the transplanting survival rate of gardenia jasminoides.
The technical scheme of the invention is as follows:
a special culture medium for tissue culture and rapid propagation of gardenia jasminoides ellis is prepared by taking 1/2MS as a basic culture medium, adding 0.5mg/L of accelerator, 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 5.8-6.0.
The promoter is prepared by mixing modified pine nut shells and 1-deoxynojirimycin in a mass ratio of 5: 1.
The preparation method of the modified pine nut shells comprises the following steps: pulverizing pine nut shell to 40 mesh, soaking in modified solution for 12 hr, filtering, repeatedly washing the residue with distilled water until the washing liquid is neutral, and oven drying at 100 deg.C until the water content is below 8%.
The modified solution is an aqueous solution of triethylene diamine and choline chloride, wherein the mass fraction of the triethylene diamine is 6.5%, and the mass fraction of the choline chloride is 2.5%.
The pine nut shells contain various bioactive substances such as lignin, stilbenes, volatile oil, vitamins, palmatine, minerals, polysaccharide, protein, fat, flavonoids and the like. The invention adopts the pine nut shells as raw materials, changes waste into valuable, saves resources and also achieves the aim of protecting the environment.
The promoter obtained by compounding the modified pine nut shells and the 1-deoxynojirimycin in a specific ratio is added into the conventional rooting culture medium, so that the rooting rate of gardenia jasminoides is improved, and particularly, the rooting time is shortened. And the post-forming rate after transplanting can reach 100%.
In addition, the promoter disclosed by the invention is added into an MS culture medium, the addition amount is 1.0mg/L, and the promoter is used for culturing gardenia jasminoides callus, so that the content of jasminoidin in the callus can be greatly increased, and a new application is opened up for the promoter disclosed by the invention.
The invention has the advantages that: the promoter is added into a conventional 1/2MS culture medium, so that the rooting condition of gardenia jasminoides is improved, the rooting time is shortened, and the survival rate after transplanting is improved. And the promoter is added into a conventional MS culture medium, and can improve the content of geniposide in the gardenia callus.
Detailed Description
Example 1: a special culture medium for tissue culture and rapid propagation of gardenia jasminoides ellis is prepared by taking 1/2MS as a basic culture medium, adding 0.5mg/L of accelerator, 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 5.8-6.0.
The promoter is prepared by mixing modified pine nut shells and 1-deoxynojirimycin in a mass ratio of 5: 1.
The preparation method of the modified pine nut shells comprises the following steps: pulverizing pine nut shell to 40 mesh, soaking in modified solution for 12 hr, filtering, repeatedly washing the residue with distilled water until the washing liquid is neutral, and oven drying at 100 deg.C until the water content is below 8%.
The modified solution is an aqueous solution of triethylene diamine and choline chloride, wherein the mass fraction of the triethylene diamine is 6.5%, and the mass fraction of the choline chloride is 2.5%.
Example 2: a special culture medium for tissue culture and rapid propagation of gardenia jasminoides ellis is prepared by taking 1/2MS as a basic culture medium, adding 0.5mg/L of accelerator, 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 5.8-6.0.
The promoter is 1-deoxynojirimycin.
Example 3: a special culture medium for tissue culture and rapid propagation of gardenia jasminoides ellis is prepared by taking 1/2MS as a basic culture medium, adding 0.5mg/L of accelerator, 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 5.8-6.0.
The accelerant is a modified pine nut shell, and the preparation method comprises the following steps: pulverizing pine nut shell to 40 mesh, soaking in modified solution for 12 hr, filtering, repeatedly washing the residue with distilled water until the washing liquid is neutral, and oven drying at 100 deg.C until the water content is below 8%.
The modified solution is an aqueous solution of triethylene diamine and choline chloride, wherein the mass fraction of the triethylene diamine is 6.5%, and the mass fraction of the choline chloride is 2.5%.
Example 4: a special culture medium for tissue culture and rapid propagation of gardenia jasminoides ellis is prepared by taking 1/2MS as a basic culture medium, adding 0.5mg/L of accelerator, 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 5.8-6.0.
The promoter is prepared by mixing pine nut shells and 1-deoxynojirimycin in a mass ratio of 5:1 after being crushed to 40 meshes.
Example 5: the difference from example 1 is that:
the modified solution is a triethylene diamine aqueous solution, wherein the weight percentage of triethylene diamine is 6.5%.
The rest is the same as example 1.
Example 6: the difference from example 1 is that:
the modified solution is an aqueous solution of choline chloride, wherein the mass fraction of the choline chloride is 2.5%.
The rest is the same as example 1.
Example 7: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The formulation and preparation of the accelerator are the same as in example 1.
Example 8: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The accelerator was the same as in example 2.
Example 9: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The formulation and preparation of the accelerator are the same as in example 3.
Example 10: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The formulation and preparation of the accelerator are the same as in example 4.
Example 11: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The formulation and preparation of the accelerator are the same as in example 5.
Example 12: a culture medium for callus of fructus Gardeniae is prepared by taking MS as basic culture medium, and adding 1.0mg/L promoter. The formulation and preparation of the accelerator are the same as in example 6.
Experiment 1: 3-4cm long gardenia jasminoides clustering shoots with 3-5 leaves were cut into individual plants, and inoculated on the medium of examples 1-6, respectively, for root induction. A blank control group and a hormone control group are additionally arranged, and the culture medium of the blank control group is as follows: 1/2MS minimal medium +30g/L sucrose +7g/L agar, and the hormone control group medium is: 1/2MS minimal medium +0.2mg/L NAA (the best concentration for promoting the rooting of gardenia jasminoides ellis) 30g/L sucrose +7g/L agar. The culture temperature is (25+1) ° C, the relative humidity is 70%, the illumination intensity is 2400lx, and the illumination is carried out for 12h every day. Counting the rooting conditions of each group after 20d, and transplanting each group of gardenia seedlings to a matrix of humus soil: and (3) field soil: sand-soil is 3: 2: 1 (the diameter of the bottom of the nutrition pot is 8cm, the height of the nutrition pot is 10cm), after transplanting, watering thoroughly, and placing in a greenhouse. The temperature of the greenhouse is 15-30 ℃, the relative humidity is 11-65%, the light is shielded by 50%, and the survival rate is investigated after 2 weeks of planting.
TABLE 1 Effect of different media on Gardenia jasminoides rooting
| Grouping | 20d rooting percentage (%) | Shortest rooting time (d) |
| Blank control group | 34.3 | 11 |
| Hormone control group | 100 | 7 |
| Example 1 | 100 | 5 |
| Example 2 | 36.9 | 12 |
| Example 3 | 34.5 | 13 |
| Example 4 | 41.2 | 11 |
| Example 5 | 40.8 | 10 |
| Example 6 | 37.7 | 12 |
TABLE 2 Effect of different media on the survival rate of transplantation of Gardenia jasminoides Ellis
| Grouping | Survival Rate of transplantation (%) |
| Blank control group | 62 |
| Hormone control group | 95 |
| Example 1 | 100 |
| Example 2 | 62 |
| Example 3 | 65 |
| Example 4 | 64 |
| Example 5 | 60 |
| Example 6 | 64 |
Experiment 2: rinsing fructus Gardeniae seed with water, sterilizing with 75% (volume fraction, the same below) ethanol for 1 min, transferring into 2% sodium hypochlorite solution, sterilizing for 10min, washing with sterile water for 5 times, respectively inoculating on MS culture medium (MS +2.0 mg/L6 BA +0.50mg/L NAA + 2% sucrose + 0.6% agar) to germinate at induction temperature of 25+ -1 deg.C for 16h/d with light intensity of L200 Lux, and standing for 2 weeks to obtain sterile seedling. The young leaves of Gardenia jasminoides Ellis were cut into small pieces of 1 cm. times.1 cm with scissors, and inoculated into the culture medium of examples 7 to 12, respectively, to culture callus. A blank control group and a hormone control group are additionally arranged, and the culture medium of the blank control group is as follows: MS basic culture medium, hormone control group culture medium: MS minimal medium +1.0 mg/L6-BA. Culturing at 25 deg.C under 3000 Lx illumination for 14 hr per day for 30d to obtain callus, and measuring jasminoidin content in the callus by the following method:
(1) chromatographic conditions and system applicability tests for determining the content of geniposide in gardenia jasminoides callus are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the hexanenitrile-water (volume ratio 15: 85) is a mobile phase; the detection wavelength is 238 nm; the number of theoretical plates should not be less than 3000 calculated by gardenoside peak.
(2) Preparation of control solutions: taking appropriate amount of geniposide reference substance, precisely weighing, and adding methanol to obtain solution containing 0.1mg per lmL.
(3) Preparing gardenia callus extracting solution: weighing 25mg of callus dry powder, precisely weighing, placing in a 25mL measuring flask, adding methanol for dissolving, diluting to scale, and shaking up; precisely sucking 2mL of the solution, placing the solution in a 10mL measuring flask, adding methanol to dilute the solution to a scale, and shaking up the solution to obtain the product.
(4) The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
TABLE 3 Effect of different media on the content of geniposide in gardenia jasminoides callus
| Grouping | Content of geniposide (mg/g) |
| Blank control group | 17.23 |
| Hormone control group | 21.18 |
| Example 1 | 95.66 |
| Example 2 | 18.41 |
| Example 3 | 17.35 |
| Example 4 | 18.29 |
| Example 5 | 17.80 |
| Example 6 | 17.58 |
Claims (2)
1. A special culture medium for tissue culture and rapid propagation of gardenia jasminoides is characterized in that: 1/2MS is used as a basic culture medium, 0.5mg/L of accelerant, 30g/L of cane sugar and 7g/L of agar are added, and the pH value is 5.8-6.0;
the promoter is prepared by mixing modified pine nut shells and 1-deoxynojirimycin in a mass ratio of 5: 1;
the preparation method of the modified pine nut shells comprises the following steps: pulverizing pine nut shell to 40 mesh, soaking in modified solution for 12h, filtering, repeatedly washing filter residue with distilled water until the washing liquid is neutral, and oven drying at 100 deg.C until the water content is below 8%;
the modified solution is an aqueous solution of triethylene diamine and choline chloride, wherein the mass fraction of the triethylene diamine is 6.5%, and the mass fraction of the choline chloride is 2.5%.
2. The use of the promoter of claim 1 for increasing the content of geniposide in gardenia jasminoides callus, characterized in that: the promoter of claim 1 is added into MS culture medium at an amount of 1.0mg/L for culturing gardenia jasminoides callus.
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