CN111471640A - Separation and culture method of honeysuckle protoplast and special culture medium - Google Patents
Separation and culture method of honeysuckle protoplast and special culture medium Download PDFInfo
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- CN111471640A CN111471640A CN202010373363.5A CN202010373363A CN111471640A CN 111471640 A CN111471640 A CN 111471640A CN 202010373363 A CN202010373363 A CN 202010373363A CN 111471640 A CN111471640 A CN 111471640A
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- protoplast
- honeysuckle
- culture
- protoplasts
- enzymolysis
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a method for separating and culturing honeysuckle protoplast and a special culture medium, belonging to the technical field of plant tissue and cell culture. The method comprises the following steps: (1) sterilizing explants; (2) pretreatment of explants: pretreating the leaves in the plasmolysis solution for 60 min; (3) separating protoplast: mixing the leaves with the enzymolysis liquid, standing for enzymolysis for 4h, and then oscillating at low speed for enzymolysis for 4 h; (4) and (3) purifying protoplasts: purifying the separated protoplast by a precipitation method; (5) protoplast culture: the protoplast culture medium is adopted for liquid shallow layer culture. The honeysuckle protoplast with high yield and high activity is obtained by separating and purifying the honeysuckle test-tube seedling leaves as the explants, and the culture system of the protoplast is initially established, thereby having important significance for developing related biotechnology researches such as honeysuckle crossbreeding and genetic engineering.
Description
Technical Field
The invention relates to a method for separating and culturing honeysuckle protoplast and a special culture medium, belonging to the technical field of plant tissue and cell culture.
Background
Honeysuckle flower (honeysuckle flowerLonicera japonicaThunb) is also known as honeysuckle, honeysuckle and honeysuckle, and is a perennial semi-evergreen woody vine plant of the genus lonicera of the family loniceraceae. Honeysuckle flower is named as honeysuckle flower because its single leaf is opposite, its leaf is oval or long oval, its two sides are soft, and in the axilla of every leaf a small umbrella inflorescence is formed from two flowers, and its two sides are symmetrical, and its corolla is lip-shaped, and is white at first bloom and then turns into golden yellow. The traditional medicine considers that stems, leaves, vines and buds of honeysuckle can be used as medicines, particularly the buds are the best, and the dried buds or the initially opened flowers are the traditional Chinese medicine honeysuckle. Honeysuckle is known as a good medicine for clearing heat and removing toxicity from old times. It is sweet and cold in nature and fragrant, sweet and cold in nature and clearing heat without hurting stomach, and the fragrance is thorough and can eliminate pathogens. Flos Lonicerae can disperse wind-heat and clear away blood toxin, and can be used for treating various heat diseases such as fever, eruption, speckle, sore and carbuncle due to heat toxin, and pharynxIt has obvious effect on swelling and pain of throat, etc. The modern pharmacological action research shows that the main chemical components of the honeysuckle flower contain chlorogenic acid, isochlorogenic acid, flavonoid, volatile oil and other compounds, and have the effects of broad-spectrum bacteriostasis, inflammation diminishing, virus resisting, tumor resisting, fever relieving, liver protecting, hemostasis, lipid lowering, fertility resisting, oxidation resisting, immunoregulation and the like. According to incomplete statistics, honeysuckle is used in one third of the Chinese medicinal formulas in China. In addition, the leaves and flowers of the honeysuckle can be used as health-care tea, have pure taste, are sweet and tasty, have the effects of resisting bacteria, diminishing inflammation, clearing away heat and toxic materials, reducing blood pressure, reducing blood fat, preventing coronary heart disease and the like, and are popular traditional health-care drinks. In a word, honeysuckle is a medicinal and edible medicinal material, and the field of deep development of the honeysuckle is very wide.
Medicinal plants are economic plants with special purposes, and through thousands of years of application and development, the traditional Chinese medicine with a long history is formed in China, but at present, the ecological environment is seriously damaged, the quality of medicinal plant cultivars is degraded, wild medicinal plant resources are gradually reduced and even exhausted, so that the quality and the yield of the traditional Chinese medicine are urgently solved, and the new variety cultivation by using the modern biotechnology is expected to become an effective means for solving the problems. The honeysuckle is usually propagated in a cutting and sowing mode, the propagation speed is low, the actual production capacity cannot meet the market demand, particularly, the honeysuckle has few varieties, mainly comes from wild seed sources, the accumulation of active ingredients is less, and the quality of plants and products is low. Although the honeysuckle breeding work in China has achieved a certain achievement at present, through the breeding process, the fact that the traditional breeding method is heavily used in the honeysuckle improved variety breeding process is found, the new variety is produced with a large amount of manpower, material resources and financial resources, the period is long, the effect is not obvious, and the current production needs cannot be met. Therefore, on the basis of conventional breeding, the development of honeysuckle biotechnology breeding is the most practical and effective method.
Protoplasts are those parts of living material which are separated from the cell wall by plasmolysis. Plant protoplasts still maintain cell totipotency and have the totipotency of regenerating individuals similar to their parents under proper culture conditions. The protoplast has no cell wall, so that incompatibility obstacle is eliminated to some extent and cell hybridization is facilitated, and protoplast culture and cell fusion are one way to solve genetic material exchange at cell level. In addition, protoplasts are usually accompanied by the generation of a large number of clonal variations during the cultivation of regenerated plants, and economically valuable variants can be selected by appropriate selection. Protoplasts are ideal receptors for plant genetic engineering, and can create a new method for capturing exogenous genes, DNA fragments, organelles and chromosomes.
The protoplast culture of plants and the somatic cell hybridization thereof are effective means for creating new species and overcoming incompatibility, and can be used for genetic transformation to realize effective transfer of disease resistance, stress resistance, cytoplasmic male sterility and excellent quality characteristics. All technical links comprise protoplast separation, protoplast culture, somatic cell hybridization, generation of callus and regeneration of plants by hybrid cells and the like. Wherein the effective separation and culture of protoplasts are the basis of cell fusion and somatic cell hybridization. After the plant protoplast is separated and purified, a regeneration plant is generated by culturing the protoplast. The cultivation of plant protoplasts is influenced by many factors, such as the species, donor material, medium, cultivation method, cultivation density, growth hormone, etc. From protoplast to plant regeneration, the protoplast is divided, the callus grows and the plant is regenerated. Different culture mediums are needed in different stages of protoplast culture, and the culture mediums, namely, a culture medium for promoting the protoplast to restore cell walls, start division and maintain cell division, a culture medium for promoting callus to grow and a culture medium for inducing each organ to differentiate, are generally required to be replaced for several times. Among them, a medium that promotes initiation of protoplasts and maintains cell division is most important. However, the research on the separation, purification and culture of honeysuckle protoplasts is still blank.
Disclosure of Invention
In order to solve the problems, the invention provides a method for separating and culturing honeysuckle protoplasts and a special culture medium for culturing the honeysuckle protoplasts, and lays a solid foundation for the subsequent research on the aspects of fusion, genetic transformation and the like of the honeysuckle protoplasts.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for separating and culturing honeysuckle protoplast is characterized by comprising the following steps:
(1) disinfection of explants
Selecting tender leaves of strongly growing honeysuckle test-tube plantlets as explants, washing the explants in running water for 10min after being cleaned by detergent, then disinfecting the explants for 30s by using 75% alcohol, and then washing the explants by using 0.1% HgCl2Sterilizing the solution for 10min, and washing with sterile water for 3 times;
(2) pretreatment of explants
Placing the disinfected leaves in a culture dish, and adding a plasmolysis solution for pretreatment for 60 min;
(3) isolation of protoplasts
Placing the pretreated leaves on clean filter paper, sucking the residual solution, cutting into strips with the width of 1.0-2.0 mm, placing the strips into a sterile triangular flask filled with enzymatic hydrolysate, sealing the sterile triangular flask, standing for enzymolysis for 4 hours in the dark, and then placing the strips on a shaking table for low-speed oscillation enzymolysis for 4 hours;
(4) purification of protoplasts
Filtering the enzymolysis mixture by using a 300-mesh sterile mesh screen, removing tissues and cell clusters which are not completely enzymolyzed, subpackaging the filtrate into centrifuge tubes, centrifuging at 800rpm for 8min, discarding the supernatant, adding a protoplast cleaning solution, centrifuging at 800rpm for 5min, discarding the supernatant, repeatedly washing for 2-3 times, and finally washing for 1 time by using a protoplast culture medium to obtain purified protoplasts;
(5) performing protoplast culture, namely suspending the purified protoplast in a protoplast culture medium, adjusting the protoplast culture medium to a certain density, sucking 2-3 m L protoplast suspension, adding the protoplast suspension into a culture dish with the diameter of 60mm to form a shallow layer, sealing the shallow layer by using a sealing film, and performing static culture at the temperature of 25 +/-1 ℃ in the dark;
the protoplast culture medium comprises the following components: KNO31600~1800mg/L，NH4NO3500~600mg/L，MgSO4·7H2O 330~360mg/L，CaCl2·2H2O 450~500mg/L，KH2PO4160~180mg/L，MnSO4·4H2O20~25mg/L，ZnSO4·7H2O 7~9mg/L，H3BO311~14mg/L，KI 0.6~0.8mg/L，CuSO4·5H2O 0.022~0.026mg/L，CoCl2·6H2O 0.022~0.026mg/L，Na2MoO4·2H2O 0.20~0.30mg/L，FeSO4·7H2O53~58mg/L，Na260-64 mg/L of EDTA, 45-55 mg/L of inositol, 160-200 mg/L of hydrolyzed casein, 40-60 mg/L of glutathione, 14-17 mg/L of putrescine, 10.2-0.5 mg/L of vitamin B, 60.4-0.6 mg/L of vitamin B, 6-8 mg/L of vitamin C, 0.45-0.65 mg/L of nicotinic acid, 0.1-0.3 mu mol/587 of melatonin, 7.0-10.0 mg/L of anthocyanin, 20-40 ml/L of malt extract solution, 0.4-0.6 mg/L of NAA, 0.8-1.2 mg/L.8-2.2 mg/L of 2, 1.5-1.9 mg/L of titanium citrate, 100-150 mg/150 of fructose, 0.6 mg/L-0.5 mg/L of mannan, and 0.27-0.6 mg/L to L of mannitol.
In the step (2), the components of the plasmolysis separation liquid are as follows: KNO3190mg/L，CaCl2·2H2O 75mg/L，MgSO4·7H2O 125mg/L，KH2PO417.5mg/L，MnSO4·4H2O 2.23mg/L，H3BO30.62mg/L，KI0.083mg/L，Na2MoO4·2H2O 0.025mg/L，CoCl2·6H2O 0.0025mg/L，ZnSO4·7H2O 0.86mg/L，FeSO4·7H2O 2.79mg/L，Na23.73 mg/L of EDTA, 10.05mg/L of vitamin B, 60.05mg/L of vitamin B, 0.5 mg/L of nicotinic acid, 0.005 mg/L of biotin, 2.0 mg/L of glycine, 0.05 mg/L of folic acid, 10 mg/L3 of inositol, 3 mmol/L of tetrahydropyridine, 40 mg/L of tetrahydropyridine and 20 percent of sucrose.
In the step (3), the components of the enzymolysis solution are as follows: 2% of fiberCellulase, 0.5% pectinase, 0.25% eductase, 0.1% MES, 10 mmol/L CaCl2·2H2O，0.7mmol/L KH2PO40.6 mol/L mannitol, pH 5.8.
In the step (3), the enzymolysis liquid is filtered and sterilized by a 0.22 mu m microporous filter membrane.
In the step (3), the adding ratio of the leaves to the enzymolysis liquid is 1g to 10m L.
In the step (3), the enzymolysis temperature is 28 +/-1 ℃, and the rotation speed of a shaking table is 30-50 rpm.
In the step (4), the protoplast cleaning solution comprises the following components: KNO3101.0mg/L，MgSO4·7H2O246.0mg/L，CaCl2·2H2O 1480mg/L，KI 0.16mg/L，CuSO4·5H2O0.025 mg/L, and mannitol 0.6 mol/L.
In the step (5), the density of the protoplast suspension is 2.0 × 105And each m L.
The invention has the beneficial effects that:
(1) the method takes honeysuckle test-tube plantlet leaves as materials to separate and purify the protoplast, adopts the plasmolysis separation liquid to carry out pretreatment before separation, can increase the strength of cell membranes, achieves the purposes of improving the cell wall enzymolysis effect and reducing the damage of the protoplast, determines the components and the concentration of enzymolysis liquid, adopts the enzymolysis method of firstly standing and then oscillating at a low speed, can greatly improve the yield and the activity of the protoplast, and the yield of the obtained protoplast can reach 5.73 × 10 to the maximum extent6The maximum protoplast activity can reach 85.4% per gram.
(2) The invention carries out systematic research on factors influencing the honeysuckle protoplast culture, determines the special culture medium suitable for the honeysuckle protoplast culture, and obviously improves the division frequency and the cell mass formation frequency of the honeysuckle protoplast compared with the conventional culture medium. The honeysuckle protoplast isolation culture technology system is initially established, and has important significance for developing related biological technologies of honeysuckle (such as protoplast regeneration, protoplast fusion, transgenic research, preservation of germplasm resources and innovation research).
Detailed Description
The present invention is further illustrated by the following specific examples, which should be construed as merely illustrative, and not limitative of the remainder of the disclosure.
Example 1
A method for separating and culturing honeysuckle protoplast comprises the following steps:
(1) disinfection of explants
Selecting tender leaves of strongly growing honeysuckle test-tube plantlets as explants, washing the explants in running water for 10min after being cleaned by detergent, then disinfecting the explants for 30s by using 75% alcohol, and then washing the explants by using 0.1% HgCl2The solution was sterilized for 10min and finally rinsed 3 times with sterile water.
(2) Pretreatment of explants
Placing the disinfected leaves in a culture dish, and adding a plasmolysis solution for pretreatment for 60 min; the components of the plasmolysis separation liquid are as follows: KNO3190mg/L，CaCl2·2H2O 75mg/L，MgSO4·7H2O 125mg/L，KH2PO417.5mg/L，MnSO4·4H2O 2.23mg/L，H3BO30.62mg/L，KI 0.083mg/L，Na2MoO4·2H2O 0.025mg/L，CoCl2·6H2O0.0025mg/L，ZnSO4·7H2O 0.86mg/L，FeSO4·7H2O 2.79mg/L，Na23.73 mg/L of EDTA, 10.05mg/L of vitamin B, 60.05mg/L of vitamin B, 0.5 mg/L of nicotinic acid, 0.005 mg/L of biotin, 2.0 mg/L of glycine, 0.05 mg/L of folic acid, 10 mg/L3 of inositol, 3 mmol/L of tetrahydropyridine, 40 mg/L of tetrahydropyridine and 20 percent of sucrose.
(3) Isolation of protoplasts
Placing the pretreated leaves on clean filter paper, sucking dry the residual solution, cutting into strips with the width of 1.0-2.0 mm, placing the strips into a sterile triangular flask filled with enzymolysis liquid, wherein the adding ratio of the leaves to the enzymolysis liquid is 1g to 10m L, sealing the sterile triangular flask, standing for enzymolysis for 4h at 28 ℃ in the dark, placing the mixture on a shaking table, and carrying out low-speed oscillation enzymolysis for 4h at the rotating speed of 40rpm, wherein the enzymolysis liquid comprises 2% of celluloseEnzyme, 0.5% pectinase, 0.25% eductase, 0.1% MES, 10 mmol/L CaCl2·2H2O，0.7mmol/L KH2PO40.6 mol/L mannitol, pH 5.8, sterile filtered through a 0.22 μm microfiltration membrane.
(4) Purification of protoplasts
Filtering the enzymolysis mixture with 300 mesh sterile mesh screen to remove tissue and cell mass, subpackaging the filtrate into centrifuge tubes, centrifuging at 800rpm for 8min, discarding supernatant, and adding protoplast cleaning solution (comprising KNO3101.0mg/L，MgSO4·7H2O 246.0mg/L，CaCl2·2H2O 1480mg/L，KI 0.16mg/L，CuSO4·5H2O0.025 mg/L and mannitol 0.6 mol/L), centrifuging at 800rpm for 5min, discarding the supernatant, repeatedly washing for 2-3 times, and finally washing with a protoplast culture medium for 1 time.
Determination of the yield of protoplasts (pieces/g) = density of protoplasts (pieces/m L) × total volume of protoplast suspension (m L)/total mass of material (g) were determined microscopically by the haemocytometer method.
The activity of the protoplast is determined by detecting the protoplast strength by adopting a Fluorescein Diacetate (FDA) staining method, taking about 0.5m L of the purified protoplast, adding FDA with the concentration of 12 mu L of 5mg/m L, standing for 10min, observing under a fluorescence microscope, and obtaining the protoplast which emits green fluorescence and is active, wherein the calculation formula is as follows, the activity of the protoplast (%) = the number of the protoplasts emitting fluorescence/the total number of the protoplasts × 100%.
(5) And (3) protoplast culture, namely suspending the purified protoplast in a protoplast culture medium, adjusting the protoplast to a certain density, sucking 2-3 m L protoplast suspension, adding the protoplast suspension into a culture dish with the diameter of 60mm to form a shallow layer, sealing the shallow layer by using a sealing film, performing static culture at the temperature of 25 ℃ in the dark, adding 1 time of fresh protoplast culture medium every week, and gradually reducing the concentration of mannitol.
The protoplast culture medium had the following composition: KNO31700mg/L，NH4NO3550mg/L，MgSO4·7H2O345mg/L，CaCl2·2H2O 475mg/L，KH2PO4170mg/L，MnSO4·4H2O 22.5mg/L，ZnSO4·7H2O 8mg/L，H3BO312.5mg/L，KI 0.7mg/L，CuSO4·5H2O 0.024mg/L，CoCl2·6H2O 0.024mg/L，Na2MoO4·2H2O 0.25mg/L，FeSO4·7H2O 56mg/L，Na2EDTA 63 mg/L, inositol 50 mg/L, hydrolyzed casein 180 mg/L0, glutathione 50 mg/L1, putrescine 15.5 mg/L2, vitamin B10.35mg/L3, vitamin B60.5mg/L4, vitamin C7 mg/L5, nicotinic acid 0.55 mg/L6, melatonin 0.2. mu. mol/L7, anthocyanin 8.5 mg/L8, malt extract solution 30 ml/L9, NAA 0.5mg/L, 2, 4-D1.0mg/L/L, titanium citrate 1.7 mg/L, fructose 125 mg/L, xylan 125 mg/L, sucrose 30 g/L, mannitol 0.5 mol/L, pH adjusted to 5.8.
And (3) observing the regeneration and division conditions of the cell wall of the protoplast under an inverted microscope, counting the division frequency of the protoplast after culturing for 7d, and counting the formation frequency of the cell mass after culturing for 21 d.
Example 2 Effect of pretreatment time on honeysuckle protoplast isolation
For explant pretreatment in example 1, this experiment set 5 gradient pretreatment times, respectively: 0. 30min, 60min, 90min and 120 min. The explants pretreated at different times were subjected to protoplast isolation and purification, respectively, and the yield and viability of the protoplasts were determined (see table 1).
The results in table 1 show that the yield and activity of the honeysuckle protoplast both increase and decrease with the increase of the pretreatment time, and when the pretreatment time is 60min, the yield and activity of the honeysuckle protoplast both reach the maximum value, respectively 5.73 × 106Seed/g and 85.4%; with the further extension of the pretreatment time, the yield and activity of the honeysuckle flower are reduced sharply, so that the pretreatment time most suitable for the honeysuckle flower leaves is 60 min.
Example 3 Effect of different enzymatic hydrolysis methods on the isolation of Lonicera japonica protoplasts
For the separation of protoplasts in example 1, 3 enzymatic methods were set up in this experiment, which were: standing for enzymolysis, namely mixing the leaves with enzymolysis liquid, and standing for enzymolysis for 8 hours at 28 ℃ under the dark condition; shaking table for vibration enzymolysis, i.e. mixing the leaves with the enzymolysis solution, and placing on a shaking table (28 ℃, 40 rpm) for vibration enzymolysis for 8 h; standing and then oscillating for enzymolysis, namely mixing the leaves with the enzymolysis liquid, standing and carrying out enzymolysis for 4h, and then placing on a shaking table (28 ℃, 40 rpm) for oscillating and carrying out enzymolysis for 4 h. The protoplasts obtained by different enzymatic methods were purified and the yield and viability of the protoplasts were determined (see table 2).
The results in Table 2 show that the yield of protoplasts obtained by the enzymatic hydrolysis method with a preliminary standing followed by low-speed shaking was 5.68 × 106The number/g of the honeysuckle leaves is one, the protoplast activity is 84.8%, which is obviously higher than that of the standing enzymolysis and the oscillating enzymolysis, so that the enzymolysis method of firstly standing and then oscillating has the best separation effect on the protoplasts of the honeysuckle leaves.
EXAMPLE 4 Effect of culture Density on honeysuckle protoplast culture
For the protoplast culture in example 1, 5 protoplast culture densities of 0.5 × 10 were set for this experiment5Pieces/m L, 1.0 × 105Pieces/m L, 2.0 × 105Pieces/m L, 5.0 × 105Pieces/m L, 10.0 × 105The number of the cells per m L is that the honeysuckle protoplasts are respectively cultured in a liquid shallow layer by adopting the culture densities, the division frequency of the protoplasts is counted after 7 days of culture, and the cell mass formation frequency is counted after 21 days of culture (see table 3).
Different culture densities have a great influence on the culture of protoplasts, and the development of the protoplasts can be inhibited if the culture density is too high or too low. The results in Table 3 show thatThe division frequency of the honeysuckle protoplast is continuously increased when the culture density is increased to 2.0 × 105At the speed of seed/m L, the protoplast division frequency and the cell mass formation frequency reach the highest, respectively 9.73 percent and 2.52 percent, when the culture density is higher than 5.0 × 105At the speed of each m L, the protoplast is broken and browned, so the culture density of the honeysuckle protoplast is 2.0 × 105And each m L.
EXAMPLE 5 Effect of different media on honeysuckle protoplast culture
The culture medium is one of the key factors for the protoplast to grow normally and divide continuously. For protoplast culture in example 1, 4 media were set up for this experiment, respectively: the protoplast culture medium, MS culture medium, KM of the invention8The P culture medium and the KPR culture medium are added with hormone combination of NAA0.5mg/L, 2, 4-D1.0mg/L/L, osmotic pressure regulator of mannitol 0.5 mol/L and pH value regulated to 5.8, the 4 culture media are respectively adopted to carry out liquid shallow layer culture on the honeysuckle protoplast, the division frequency of the protoplast is counted after 7 days of culture, and the appearance frequency of cell clusters is counted after 21 days of culture (see Table 4).
The results in Table 4 show that there are significant differences in the frequency of protoplast division and plating rate when cultured in different media. In MS culture medium, the protoplast division frequency is lowest, only 1.26%, and almost no division can be continuously performed, and the cell mass formation frequency is 0.02%; at KM8In the P and KPR culture media, the division frequency of the protoplast is respectively 6.77 percent and 5.35 percent, the appearance frequency of the cell mass is respectively 1.53 percent and 1.44 percent, and the cell mass is obviously improved compared with the MS culture medium; in the medium of the present invention, the protoplast division frequency and the cell mass formation frequency were the highest, 9.85% and 2.60%, respectively, as compared with KM8Compared with the conventional culture mediums of P and KPR, the culture medium of the invention is further improved, so that the continuous division and growth of the honeysuckle protoplast are more facilitated by adopting the culture medium of the invention under the same culture conditions. The reason is analyzed, and the reason is analyzed,the protoplast culture medium of the invention is adjusted for macroelements, in particular NH4NO3、CaCl2·2H2The honeysuckle protoplast regeneration cell has the concentration of O, and has certain promotion effect on the division and growth of the honeysuckle protoplast regeneration cell by adding rich nutrient components and special components such as putrescine, melatonin, anthocyanin and titanium citrate.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope thereof. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for separating and culturing honeysuckle protoplast is characterized by comprising the following steps:
(1) disinfection of explants
Selecting tender leaves of strongly growing honeysuckle test-tube plantlets as explants, washing the explants in running water for 10min after being cleaned by detergent, then disinfecting the explants for 30s by using 75% alcohol, and then washing the explants by using 0.1% HgCl2Sterilizing the solution for 10min, and washing with sterile water for 3 times;
(2) pretreatment of explants
Placing the disinfected leaves in a culture dish, and adding a plasmolysis solution for pretreatment for 60 min;
(3) isolation of protoplasts
Placing the pretreated leaves on clean filter paper, sucking the residual solution, cutting into strips with the width of 1.0-2.0 mm, placing the strips into a sterile triangular flask filled with enzymatic hydrolysate, sealing the sterile triangular flask, standing for enzymolysis for 4 hours in the dark, and then placing the strips on a shaking table for low-speed oscillation enzymolysis for 4 hours;
(4) purification of protoplasts
Filtering the enzymolysis mixture by using a 300-mesh sterile mesh screen, removing tissues and cell clusters which are not completely enzymolyzed, subpackaging the filtrate into centrifuge tubes, centrifuging at 800rpm for 8min, discarding the supernatant, adding a protoplast cleaning solution, centrifuging at 800rpm for 5min, discarding the supernatant, repeatedly washing for 2-3 times, and finally washing for 1 time by using a protoplast culture medium to obtain purified protoplasts;
(5) performing protoplast culture, namely suspending the purified protoplast in a protoplast culture medium, adjusting the protoplast culture medium to a certain density, sucking 2-3 m L protoplast suspension, adding the protoplast suspension into a culture dish with the diameter of 60mm to form a shallow layer, sealing the shallow layer by using a sealing film, and performing static culture at the temperature of 25 +/-1 ℃ in the dark;
the protoplast culture medium comprises the following components: KNO31600~1800mg/L，NH4NO3500~600mg/L，MgSO4·7H2O 330~360mg/L，CaCl2·2H2O 450~500mg/L，KH2PO4160~180mg/L，MnSO4·4H2O 20~25mg/L，ZnSO4·7H2O 7~9mg/L，H3BO311~14mg/L，KI 0.6~0.8mg/L，CuSO4·5H2O 0.022~0.026mg/L，CoCl2·6H2O 0.022~0.026mg/L，Na2MoO4·2H2O 0.20~0.30mg/L，FeSO4·7H2O53~58mg/L，Na260-64 mg/L of EDTA, 45-55 mg/L of inositol, 160-200 mg/L of hydrolyzed casein, 40-60 mg/L of glutathione, 14-17 mg/L of putrescine, 10.2-0.5 mg/L of vitamin B, 60.4-0.6 mg/L of vitamin B, 6-8 mg/L of vitamin C, 0.45-0.65 mg/L of nicotinic acid, 0.1-0.3 mu mol/587 of melatonin, 7.0-10.0 mg/L of anthocyanin, 20-40 ml/L of malt extract solution, 0.4-0.6 mg/L of NAA, 0.8-1.2 mg/L.8-2.2 mg/L of 2, 1.5-1.9 mg/L of titanium citrate, 100-150 mg/150 of fructose, 0.6 mg/L-0.5 mg/L of mannan, and 0.27-0.6 mg/L to L of mannitol.
2. The method for culturing honeysuckle protoplasts according to claim 1, wherein in the step (2), the plasmolysis solution comprises the following components: KNO3190mg/L，CaCl2·2H2O 75mg/L，MgSO4·7H2O 125mg/L，KH2PO417.5mg/L，MnSO4·4H2O 2.23mg/L，H3BO30.62mg/L，KI 0.083mg/L，Na2MoO4·2H2O0.025mg/L，CoCl2·6H2O 0.0025mg/L，ZnSO4·7H2O 0.86mg/L，FeSO4·7H2O 2.79mg/L，Na23.73 mg/L of EDTA, 10.05mg/L of vitamin B, 60.05mg/L of vitamin B, 0.5 mg/L of nicotinic acid, 0.005 mg/L of biotin, 2.0 mg/L of glycine, 0.05 mg/L of folic acid, 10 mg/L3 of inositol, 3 mmol/L of tetrahydropyridine, 40 mg/L of tetrahydropyridine and 20 percent of sucrose.
3. The method for culturing protoplasts of honeysuckle flowers as claimed in claim 1, wherein in step (3), the components of the enzymatic hydrolysate are 2% cellulase, 0.5% pectinase, 0.25% eductase, 0.1% MES, 10 mmol/L CaCl2·2H2O，0.7mmol/L KH2PO40.6 mol/L mannitol, pH 5.8.
4. The method for culturing protoplasts of honeysuckle flower according to claim 1, wherein in the step (3), the enzymatic hydrolysate is sterilized by filtration through a 0.22 μm microporous membrane.
5. The method for culturing the honeysuckle protoplast according to claim 1, wherein in the step (3), the adding ratio of the leaves to the enzymolysis solution is 1g to 10m L.
6. The method for culturing the honeysuckle protoplast according to claim 1, wherein in the step (3), the temperature of enzymolysis is 28 ± 1 ℃, and the rotation speed of a shaker is 30-50 rpm.
7. The method for culturing honeysuckle protoplasts according to claim 1, wherein in the step (4), the composition of the protoplast cleaning solution is as follows: KNO3101.0mg/L，MgSO4·7H2O 246.0mg/L，CaCl2·2H2O1480mg/L，KI 0.16mg/L，CuSO4·5H2O0.025 mg/L, and mannitol 0.6 mol/L.
8. The method for culturing honeysuckle protoplasts according to claim 2, wherein in the step (5), the density of the protoplast suspension is 2.0 × 105And each m L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358722A (en) * | 2019-08-16 | 2019-10-22 | 中南林业科技大学 | Preparation method of litsea cubeba mesophyll cell protoplast |
| CN112251395A (en) * | 2020-10-29 | 2021-01-22 | 西南大学 | Separation method of loquat protoplast |
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
| CN115968887A (en) * | 2022-12-28 | 2023-04-18 | 重庆大学 | Plant immunity inducer and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602691A (en) * | 2003-09-30 | 2005-04-06 | 吴彦 | Organic macromolecular activated complex for inducing plant wide spectrum disease resistance |
| CN102388803A (en) * | 2011-08-12 | 2012-03-28 | 江苏省农业科学院 | Chilli cytoplasm male sterile line protoplast separation purification and callus forming method |
| WO2015099274A1 (en) * | 2013-12-27 | 2015-07-02 | 코웨이 주식회사 | Structure in which active material is inserted into de-differentiated plant protoplast, method for preparing same, and cosmetic composition containing same |
| CN105340750A (en) * | 2015-11-27 | 2016-02-24 | 山东中医药大学 | Honeysuckle tissue-culture-seedling culture medium and honeysuckle tissue-culture rapid propagation method |
| CN109156354A (en) * | 2018-10-10 | 2019-01-08 | 东北农业大学 | A kind of growth regulator inducing indigo fruit Callus of Leaf |
| CN109628376A (en) * | 2019-01-20 | 2019-04-16 | 华中农业大学 | A kind of preparation method of Dianthus caryophyllus protoplast |
-
2020
- 2020-05-06 CN CN202010373363.5A patent/CN111471640A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602691A (en) * | 2003-09-30 | 2005-04-06 | 吴彦 | Organic macromolecular activated complex for inducing plant wide spectrum disease resistance |
| CN102388803A (en) * | 2011-08-12 | 2012-03-28 | 江苏省农业科学院 | Chilli cytoplasm male sterile line protoplast separation purification and callus forming method |
| WO2015099274A1 (en) * | 2013-12-27 | 2015-07-02 | 코웨이 주식회사 | Structure in which active material is inserted into de-differentiated plant protoplast, method for preparing same, and cosmetic composition containing same |
| CN105340750A (en) * | 2015-11-27 | 2016-02-24 | 山东中医药大学 | Honeysuckle tissue-culture-seedling culture medium and honeysuckle tissue-culture rapid propagation method |
| CN109156354A (en) * | 2018-10-10 | 2019-01-08 | 东北农业大学 | A kind of growth regulator inducing indigo fruit Callus of Leaf |
| CN109628376A (en) * | 2019-01-20 | 2019-04-16 | 华中农业大学 | A kind of preparation method of Dianthus caryophyllus protoplast |
Non-Patent Citations (2)
| Title |
|---|
| GEORGES, D. 等: "Biotechnological breeding of ornamental honeysuckles (Lonicera, Caprifoliaceae).", ACTA HORTICULTURAE, no. 320, pages 127 - 132 * |
| 刘颖等: "忍冬原生质体分离条件研究", 中国实验方剂学杂志, vol. 18, no. 19, pages 125 - 129 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358722A (en) * | 2019-08-16 | 2019-10-22 | 中南林业科技大学 | Preparation method of litsea cubeba mesophyll cell protoplast |
| CN110358722B (en) * | 2019-08-16 | 2021-10-01 | 中南林业科技大学 | Preparation method of litsea cubeba mesophyll cell protoplast |
| CN112251395A (en) * | 2020-10-29 | 2021-01-22 | 西南大学 | Separation method of loquat protoplast |
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
| CN114214305B (en) * | 2022-01-18 | 2023-05-09 | 中国科学院东北地理与农业生态研究所 | Enzymatic hydrolysate for preparing lonicera caerulea protoplast and preparation method and application thereof |
| CN115968887A (en) * | 2022-12-28 | 2023-04-18 | 重庆大学 | Plant immunity inducer and preparation method and application thereof |
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