CN114747486A - Somatic embryogenesis and plant regeneration method for gardenia jasminoides ellis - Google Patents
Somatic embryogenesis and plant regeneration method for gardenia jasminoides ellis 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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Abstract
The invention is suitable for the technical field of plant cultivation, and provides a gardenia somatic embryogenesis and plant regeneration method, which comprises the following steps: step S1: selecting and pretreating explants; step S2: induction of embryogenic callus; step S3: differentiating and proliferating somatic cell embryos; step S4: and (3) germinating and culturing somatic cell embryos. The method has the advantages that the used plant materials are few, the method is not limited by seasons, the maximization of the propagation coefficient can be achieved in a short time, the seedling raising efficiency can be improved, the production cost is saved, the operation is simple and easy, the repeatability is high, and the method is suitable for annual tests or production; the transplanting survival rate of the nursery stock is ensured, and the quality of the nursery stock is ensured; realizes green production, optimizes the production flow of gardenia seedlings and is worthy of popularization and application.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a gardenia somatic embryogenesis and plant regeneration method.
Background
Gardenia belongs to evergreen shrubs of gardenia in Rubiaceae, and is the first medicinal and edible plant resource issued by Ministry of health. The gardenia leaf, the flower and the fruit can be viewed, the flower and the fruit can also be eaten, the active ingredients of the fruit have medicinal value, and the gardenia fruit is a multifunctional tree species which integrates greening, honey source, eating, medicinal and ecological restoration. At present, most of the gardenia is wild resources, the artificial damage is very serious, the good gardenia germplasm resources are seriously lost, and the healthy development of the gardenia industry is restricted, so that an efficient and high-quality resource breeding and conservation way is required to protect the good gardenia germplasm resources and improve the quantity and quality of gardenia seedlings.
At present, the rapid propagation of gardenia mostly adopts a cuttage rapid propagation mode and a tissue culture rapid propagation mode of 'propagating buds by buds', but the cuttage propagation coefficient is small, the period is long, the variation is easy, and the tissue culture mode of 'propagating buds by buds' has complex operation and high cost. Somatic embryogenesis is one of tissue culture and rapid propagation technologies, can realize rapid propagation of tens of times or even hundreds of times of excellent resources, can maintain excellent genetic characters of plants, fully makes up for the defects of the propagation modes, and is an ideal propagation mode.
Disclosure of Invention
The embodiment of the invention aims to provide a gardenia somatic embryogenesis and plant regeneration method, and aims to solve the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a gardenia somatic embryogenesis and plant regeneration method comprises the following steps:
step S1: explant selection and pretreatment: soaking immature fruits in warm water with detergent, cleaning, washing with running water, washing with sterile water, soaking in 75% ethanol solution for 30s, washing with sterile water, soaking in 0.1% mercuric chloride for 10min, washing with sterile water, and drying with sterile filter paper,
in order to achieve the purpose, the invention provides the following technical scheme:
a gardenia somatic embryogenesis and plant regeneration method comprises the following steps:
step S1: explant selection and pretreatment: soaking immature fruits in warm water and a detergent, then cleaning the fruits by brushing, then placing the fruits under running water for washing for later use, placing the cleaned fruits on a super-clean workbench, washing the fruits by using sterile water, then soaking the fruits in 75% alcohol solution for 30s, washing the fruits by using sterile water, then soaking the fruits by using 0.1% mercuric chloride for 10min, washing the fruits by using the sterile water, finally sucking water on the surfaces of the fruits by using sterile filter paper, and removing peels for later use;
step S2: induction of embryogenic callus: under the aseptic condition, cutting the disinfected immature fruits into slices, and putting the slices into an embryonic callus induction culture medium for culture; the embryogenic callus induction culture medium takes improved MS as a basic culture medium, 60g/L banana puree, 50-80 g/L coconut milk, 0.65% agar powder and 3% white granulated sugar are sequentially added, 0.1-0.25 mg/L6-BA and 0.05-0.15 mg/L2, 4-D are respectively added, the pH value of the culture medium is 5.8, the culture medium is placed in a sterilization pot, after being taken out, 1ml of tea saponin solution with the concentration of 20mg/ml is added by a filtration sterilization method under the aseptic condition, the mixture is uniformly mixed and then is subpackaged in an inoculation bottle, and the well-treated gardenia fruit slices are inoculated;
step S3: somatic embryo differentiation and proliferation culture: taking improved MS as a basic culture medium, sequentially adding 60g/L of banana puree, 50-80 g/L of coconut milk, 0.65% of agar powder and 3% of white granulated sugar, and respectively adding 0.25-0.5 mg/L of 6-BA, 0.1-0.25 mg/L of 2,4-D and 6mg/L of riboflavin VB2The pH of the medium was 5.8,placing in a sterilizing pot, inoculating the induced embryonic callus to a somatic embryo differentiation and proliferation culture medium under the aseptic condition;
step S4: and (3) germinating and culturing somatic embryos: taking improved MS as a basic culture medium, sequentially adding 60g/L of potato juice, 0.65% of agar powder and 1.5% of white granulated sugar, and adding 20ml/L of weeping willow leaching liquor and 3mg/L of VB2The culture medium has pH of 5.8, and is placed in a sterilizing pot, and somatic embryos induced and differentiated are transferred into the culture medium under aseptic conditions.
Further, the improved MS culture medium is as follows: basic MS culture medium, 0.16-0.18 g/L calcium nitrate and 0.01-0.03 g/L sodium sulfate.
Further, the sterilization temperature of the sterilization pot is 121 ℃, and the sterilization time is 15 min.
Further, in the step S2, the disinfected immature fruit is cut into slices and cultured in an embryonic callus induction culture medium under the weak blue light, the illumination intensity is 1500LX, and the culture temperature is 20-26 ℃.
Further, in step S2, the preparation method of the tea saponin solution includes: weighing 5g of tea saponin, dissolving with ethanol, and metering to a volumetric flask of 250ml with sterile water for later use.
Further, in the step S4, transferring the somatic embryos induced and differentiated into a culture medium under an aseptic condition, performing dark culture for 15 days, and then transferring into LED illumination culture, wherein the culture temperature is 20-26 ℃, the illumination is 12h/d, and the illumination intensity is 2000 LX.
Further, in step S4, the preparation method of the weeping willow leaching solution comprises: taking new fresh weeping willow branches of 1 year, crushing the fresh weeping willow branches by a crusher, soaking 500g of weeping willow branch powder in 500ml of distilled water for 1 day, performing ultrasonic extraction for 2 hours by using 100W of ultrasonic wave, and centrifuging to obtain supernatant for later use.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method has the advantages of less plant materials, no limitation of seasons, short production period, maximization of the propagation coefficient in a short time, improvement of the seedling raising efficiency, saving of the production cost, simple and easy operation, strong repeatability and suitability for annual tests or production;
(2) the rapid propagation system provided by the invention is constructed by a somatic embryogenesis way, the roots of aseptic seedlings do not pass through a callus culture stage, the seedling roots are strong, cannot fall off and are distributed in a radial shape, the transplanting survival rate of the seedlings is ensured, and the quality of the seedlings is ensured;
(3) the plant exogenous hormones and the bacteriostatic agent used in the invention mostly adopt plant extract, and the required plant resources are widely distributed and easily obtained, so that the green production is realized, the production cost is reduced, the production flow of gardenia seedlings is optimized, and the further popularization and application of the technology are facilitated.
Drawings
FIG. 1 is a schematic diagram of the germination and seedling rate of somatic embryos of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
The gardenia somatic embryogenesis and plant regeneration method provided by one embodiment of the invention comprises the following steps:
step S1: explant selection and pretreatment: soaking immature fruits in warm water, adding a detergent, soaking, cleaning, washing under running water for later use, placing the cleaned fruits on a super-clean workbench, washing with sterile water, soaking in 75% alcohol solution for 30s, washing with sterile water, soaking with 0.1% mercuric chloride for 10min, washing with sterile water, and finally sucking water on the surfaces of the fruits with sterile filter paper to remove peels for later use;
step S2: induction of embryogenic callus: under the aseptic condition, cutting the disinfected immature fruits into slices, and putting the slices into an embryonic callus induction culture medium for culture; the embryogenic callus induction culture medium takes improved MS as a basic culture medium, 60g/L banana puree, 50-80 g/L coconut milk, 0.65% agar powder and 3% white granulated sugar are sequentially added, 0.1-0.25 mg/L6-BA and 0.05-0.15 mg/L2, 4-D are respectively added, the pH value of the culture medium is 5.8, the culture medium is placed in a sterilization pot, after being taken out, 1ml of tea saponin solution with the concentration of 20mg/ml is added by a filtration sterilization method under the aseptic condition, the mixture is uniformly mixed and then is subpackaged in an inoculation bottle, and the well-treated gardenia fruit slices are inoculated;
step S3: somatic embryo differentiation and proliferation culture: taking improved MS as a basic culture medium, sequentially adding 60g/L banana puree, 50-80 g/L coconut milk, 0.65% agar powder and 3% white granulated sugar, and respectively adding 0.25-0.5 mg/L6-BA, 0.1-0.25 mg/L2, 4-D and 6mg/L riboflavin VB2Setting the culture medium pH value at 5.8, setting in a sterilizing pot, and inoculating the induced embryonic callus onto the somatic embryo differentiating and proliferating culture medium under aseptic condition;
step S4: and (3) germinating and culturing somatic embryos: taking improved MS as a basic culture medium, sequentially adding 60g/L of potato juice, 0.65% of agar powder and 1.5% of white granulated sugar, and adding 20ml/L of weeping willow leaching liquor and 3mg/L of VB2The culture medium has pH value of 5.8, and is placed in a sterilizing pot, and somatic embryos induced and differentiated are transferred into the culture medium under aseptic condition.
In the embodiment of the invention, in step S1, good gardenia germplasm which is robust in growth and free from plant diseases and insect pests is selected as a mother plant. In the last 9 months, the fruits are not ripe and picked and brought back to the laboratory. Soaking in 25 deg.C warm water and detergent for 30min, cleaning with soft brush, and washing under running water. Putting the cleaned fruits on a super-clean workbench, washing the fruits for 2-3 times by using sterile water, then soaking the fruits in 75% alcohol solution for 30s, washing the fruits for 2-3 times by using the sterile water, finally soaking the fruits for 10min by using 0.1% mercuric chloride, washing the fruits for 4-5 times by using the sterile water, sucking the water on the surfaces of the fruits by using sterile filter paper, and removing peels for later use; in step S2, cutting the disinfected immature fruits into slices with the thickness of 2-3 mm, and putting the slices into an embryonic callus induction culture medium for culture; in step S3, the induced embryogenic callus is inoculated to a somatic embryo differentiation and proliferation medium under aseptic conditions, the culture conditions being the same as the embryogenic callus induction process.
As a preferred embodiment of the present invention, the modified MS medium is: basic MS culture medium + 0.16-0.18 g/L calcium nitrate + 0.01-0.03 g/L sodium sulfate.
In the examples of the present invention, the modified MS medium was: adjusting the zinc sulfate in the MS minimal medium to be 8.75mg/L, and simultaneously adding 0.16-0.18 g/L of calcium nitrate and 0.01-0.03 g/L of sodium sulfate.
As a preferred embodiment of the invention, the sterilization temperature of the sterilization pot is 121 ℃, and the sterilization time is 15 min.
In a preferred embodiment of the present invention, in step S2, the disinfected immature fruit is sliced and cultured in the embryogenic callus induction medium under the weak blue light, the illumination intensity is 1500LX, and the culture temperature is 20-26 ℃.
As a preferred embodiment of the present invention, in step S2, the preparation method of the tea saponin solution comprises: weighing 5g of tea saponin, dissolving with ethanol, and metering to a volumetric flask of 250ml with sterile water for later use.
In the embodiment of the present invention, in step S2, the preparation method of the banana puree includes: peeling banana, and breaking cell wall to obtain banana paste; the preparation method of the coconut milk comprises the following steps: cleaning coconut, drilling, sucking coconut juice, placing in a container, opening coconut shell with a tool, taking out coconut pulp, and breaking coconut juice and coconut pulp in a wall breaking machine to obtain thick coconut milk.
In a preferred embodiment of the present invention, in step S4, transferring the somatic embryos induced and differentiated into a culture medium under sterile conditions, dark culturing for 15d, and then transferring to LED illumination culture, wherein the culture temperature is 20-26 ℃, the illumination time is 12h/d, and the illumination intensity is 2000 LX.
As a preferred embodiment of the present invention, in step S4, the preparation method of the weeping willow leaching solution comprises: taking new fresh weeping willow branches of 1 year, crushing the fresh weeping willow branches by a crusher, soaking 500g of weeping willow branch powder in 500ml of distilled water for 1 day, performing ultrasonic extraction for 2 hours by using 100W of ultrasonic wave, and centrifuging to obtain supernatant for later use.
In the embodiment of the present invention, in step S4, the preparation method of the potato juice comprises: and (3) preparing the peeled fresh potatoes into pulp by using a wall breaking machine, and filtering to obtain juice.
Comparative experiment
1.1 Experimental methods
1.1.1 method I: the method of the invention.
1.1.2 method II:
(1) the explant selection and pretreatment methods are the same as those of the present invention.
(2) Induction of embryogenic callus
Under the aseptic condition, cutting the disinfected immature fruits into slices with the thickness of 2-3 mm, and putting the slices into an embryonic callus induction culture medium for culture under the weak blue light (1500LX) at the culture temperature of 20-26 ℃.
The embryogenic callus induction culture medium takes modified MS as a basic culture medium, 0.65% agar powder and 3% white granulated sugar are added, 0.1-0.25 mg/L of 6-benzylaminopurine (6-BA) and 0.05-0.15 mg/L of 2, 4-dichlorophenoxyacetic acid (2,4-D) are respectively added, the pH value of the culture medium is 5.8, and the culture medium is placed in a sterilization pot and sterilized at the high temperature of 121 ℃ for 15 min. Inoculating the treated gardenia fruit slices under aseptic conditions.
The MS culture medium is improved as the invention.
(3) Somatic embryo differentiation and proliferation culture
Taking improved MS as a basic culture medium, adding 0.65% of agar powder and 3% of white granulated sugar, respectively adding 0.25-0.5 mg/L of 6-BA and 0.1-0.25 mg/L of 2,4-D, sterilizing the culture medium at a high temperature of 121 ℃ for 15min, inoculating the induced embryonic callus to a somatic embryo differentiation and proliferation culture medium under the aseptic condition, wherein the culture condition is the same as the induction process of the embryonic callus.
(4) Somatic embryo germination culture
Taking improved MS as a basic culture medium, adding 0.65% of agar powder and 1.5% of white granulated sugar, adding 0.5mg/L of ABA and 0.5mg/L of GA3, wherein the pH value of the culture medium is 5.8, sterilizing at the high temperature of 121 ℃ for 15min, and transferring the somatic embryos induced and differentiated into the culture medium under the aseptic condition, wherein the culture condition is the same as that of the invention.
1.1.3 method III:
(1) the explant selection and pretreatment methods are the same as those of the present invention.
(2) Induction of embryogenic callus
Under the aseptic condition, cutting the disinfected immature fruits into slices with the thickness of 2-3 mm, and putting the slices into an embryonic callus induction culture medium for culture under the weak LED light (1500LX) at the culture temperature of 20-26 ℃.
The embryogenic callus induction culture medium takes MS as a basic culture medium, and is similar to the method II.
(3) Somatic embryo differentiation and proliferation culture
The procedure was the same as procedure II except that the light conditions were low LED light (1500LX) and the minimal medium was MS medium.
(4) Somatic embryo germination culture
MS is used as a basic culture medium, 0.65% of agar powder and 1.5% of white granulated sugar are added, the pH value of the culture medium is 5.8, and the culture medium is sterilized at the high temperature of 121 ℃ for 15 min. Transferring the somatic embryo obtained by induction differentiation into the culture medium under the aseptic condition, wherein the culture condition is the same as that of the invention.
1.2 statistical methods
(1) Induction of embryogenic callus
In each of the methods I, II and III, 30 bottles of inoculation are repeated for 3 times, the total number of the inoculation is 270 bottles, and after 30 days of inoculation, the infection rate and the induction rate of different methods are respectively counted and averaged, and the average value is shown in Table 1.
(2) Somatic embryo differentiation and proliferation culture
After 30 days of culture, the differentiation and proliferation times of the somatic embryos of the methods I, II and III were counted respectively. For each method, 30 bottles were counted randomly, and the average values of differentiation rate and proliferation multiple were taken, as shown in table 2.
(3) Somatic embryo germination culture
And after 30 days of culture, respectively counting the germination and seedling rate of the somatic embryos of the method I, the method II and the method III. Each method randomly counts 30 bottles, and takes the average value of germination and seedling rate, which is shown in figure 1.
1.3 analysis and results
(1) Induction of embryogenic callus
TABLE 1 embryogenic callus induction and infection rates
| Experimental method | Infection Rate (%) | Inductivity (%) |
| Method I (invention) | 9.12±1.24 | 91.68±3.14 |
| Method II | 31.66±0.37 | 67.39±2.55 |
| Method III | 33.99±0.84 | 58.26±2.95 |
As can be seen from Table 1, in all of the 3 methods of embryogenic callus culture, there was infection, and the treatment with the lowest infection rate was 9.12% in the present invention, which was reduced by 22.54 and 24.87 percentage points, respectively, compared with method II and method III; the embryogenic callus induction rates of the 3 methods are greatly different, wherein the highest induction rate still belongs to the method, and can reach 91.68%, and secondly belongs to the method II (the induction rate is 67.39%), and the embryogenic callus induction rate of the method III is the minimum, and is only 58.26%.
(2) Somatic embryo differentiation and proliferation culture
TABLE 2 comparison of somatic embryo differentiation and proliferation
| Experimental methods | Differentiation Rate (%) | Fold of proliferation |
| Method I (invention) | 81.02±2.37 | 5.7±0.5 |
| Method II | 75.45±1.26 | 4.93±1.05 |
| Method III | 50.16±0.95 | 2.96±0.89 |
As can be seen from Table 2, the effects of the 3 methods on the differentiation and proliferation of somatic embryos are greatly different, the differentiation rates are from large to small, namely method I (the invention) > method II > method III, and the proliferation multiples also have the same change trend. The differentiation rate and proliferation rate of method I were 81.02% and 5.7 times, respectively, which are slightly superior to those of method II, but the differentiation rate was higher by about 20% and proliferation rate was increased by 2.74 times, compared with method III. Thus, method I (the invention) has a good effect on both somatic embryo differentiation and proliferation.
(3) Somatic embryo germination culture
As can be seen from FIG. 1, in the 3 somatic embryo germination culture methods, the germination and seedling rate of the somatic embryos of the method I and the method II is over 90%, and is about 10% higher than that of the somatic embryos of the method III. Although the difference between the somatic embryo germination and seedling rate is not obvious in the method I (the invention) and the method II, the germination hormone used in the invention is plant extract, is green and environment-friendly, is easy to obtain, and has low cost, so that the method has higher competitiveness in popularization and application.
It can be seen from the above that the invention shows the superiority in each culture stage of gardenia somatic embryogenesis and plant regeneration, and is an ideal method for gardenia somatic embryogenesis and plant regeneration.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several variations and modifications without departing from the concept of the present invention, and these should be considered as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicability of the patent.
Claims (7)
1. A gardenia somatic embryogenesis and plant regeneration method is characterized by comprising the following steps:
step S1: explant selection and pretreatment: soaking immature fruits in warm water and a detergent, then cleaning the fruits by brushing, then placing the fruits under running water for washing for later use, placing the cleaned fruits on a super-clean workbench, washing the fruits by using sterile water, then soaking the fruits in 75% alcohol solution for 30s, washing the fruits by using sterile water, then soaking the fruits by using 0.1% mercuric chloride for 10min, washing the fruits by using the sterile water, finally sucking water on the surfaces of the fruits by using sterile filter paper, and removing peels for later use;
step S2: induction of embryogenic callus: under the aseptic condition, cutting the disinfected immature fruits into slices, and putting the slices into an embryonic callus induction culture medium for culture; the embryogenic callus induction culture medium takes improved MS as a basic culture medium, 60g/L banana puree, 50-80 g/L coconut milk, 0.65% agar powder and 3% white granulated sugar are sequentially added, 0.1-0.25 mg/L6-BA and 0.05-0.15 mg/L2, 4-D are respectively added, the pH value of the culture medium is 5.8, the culture medium is placed in a sterilization pot, after being taken out, 1ml of tea saponin solution with the concentration of 20mg/ml is added by a filtering sterilization method under the aseptic condition, after being uniformly mixed, the mixture is subpackaged in an inoculation bottle, and the well-treated gardenia fruit slices are inoculated;
step S3: somatic embryo differentiation and proliferation culture: taking improved MS as a basic culture medium, sequentially adding 60g/L of banana puree, 50-80 g/L of coconut milk, 0.65% of agar powder and 3% of white granulated sugar, and respectively adding 0.25-0.5 mg/L of 6-BA, 0.1-0.25 mg/L of 2,4-D and 6mg/L of riboflavin VB2Setting the culture medium pH value at 5.8, setting in a sterilizing pot, and inoculating the induced embryonic callus onto the somatic embryo differentiating and proliferating culture medium under aseptic condition;
step S4: and (3) germinating and culturing somatic embryos: taking improved MS as a basic culture medium, sequentially adding 60g/L of potato juice, 0.65% of agar powder and 1.5% of white granulated sugar, and adding 20ml/L of weeping willow leaching liquor and 3mg/L of VB2The culture medium has pH value of 5.8, and is placed in a sterilizing pot, and somatic embryos induced and differentiated are transferred into the culture medium under aseptic condition.
2. The method for somatic embryogenesis and plant regeneration of gardenia according to claim 1, wherein the modified MS medium is: basic MS culture medium + 0.16-0.18 g/L calcium nitrate + 0.01-0.03 g/L sodium sulfate.
3. The gardenia somatic embryogenesis and plant regeneration method according to claim 1, wherein the sterilization temperature of the sterilization pot is 121 ℃ and the sterilization time is 15 min.
4. The gardenia somatic embryogenesis and plant regeneration method according to claim 1, wherein in step S2, the disinfected immature fruits are cut into slices and placed into an embryogenic callus induction medium for cultivation under weak blue light, the illumination intensity is 1500LX, and the cultivation temperature is 20-26 ℃.
5. The gardenia somatic embryogenesis and plant regeneration method of claim 4, wherein in the step S2, the preparation method of the tea saponin solution is: weighing 5g of tea saponin, dissolving with ethanol, and metering to a volumetric flask of 250ml with sterile water for later use.
6. The gardenia somatic embryogenesis and plant regeneration method according to claim 1, wherein in step S4, the somatic embryos induced and differentiated are transferred into a culture medium under sterile conditions, dark culture is carried out for 15d, and then the somatic embryos are transferred into an LED illumination culture, the culture temperature is 20-26 ℃, the illumination is 12h/d, and the illumination intensity is 2000 LX.
7. The gardenia somatic embryogenesis and plant regeneration method of claim 6, wherein in the step S4, the preparation method of weeping willow leaching solution comprises: taking new fresh weeping willow branches of 1 year, crushing the fresh weeping willow branches by a crusher, soaking 500g of weeping willow branch powder in 500ml of distilled water for 1 day, performing ultrasonic extraction for 2 hours by using ultrasonic waves with the power of 100W, and centrifuging to obtain supernate for later use.
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| CN116548309A (en) * | 2023-05-15 | 2023-08-08 | 中南林业科技大学 | Induction method of gardenia embryogenic callus |
| CN116548309B (en) * | 2023-05-15 | 2024-04-16 | 中南林业科技大学 | Induction method of gardenia embryogenic callus |
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