CN113207691B - Method for establishing seashore paspalum tissue culture regeneration system - Google Patents
Method for establishing seashore paspalum tissue culture regeneration system Download PDFInfo
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Images
Classifications
-
- 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
- A01H4/008—Methods for regeneration to complete plants
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for establishing a seashore paspalum tissue culture regeneration system, which can overcome the season limitation of explant supply so as to greatly shorten the experimental period. According to the method, the creeping stems of the seashore paspalum are used as explants, the creeping stems of the seashore paspalum are cleaned, sterilized, cut into small sections with sections and inoculated into an induction culture medium SI for culturing for 20-30 d to obtain callus, then the callus is transferred into a subculture medium SS for culturing for 5-10 d, then the callus is transferred into a differentiation culture medium SR for culturing for 15-20 d, and when seedlings grow to 2-3 cm, the seedlings are transplanted to obtain the regeneration system. Is suitable for popularization and application in the field of biotechnology.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a seashore paspalum tissue culture regeneration system.
Background
Seashore Paspalum (Paspalum vaginatum Sw.) also known as Hawaiian is a perennial herb of the Panicum (Paniacea) Panicum (Paspalum) family of the Gramineae family. The perennial warm-season lawn grass growing on the seaside, which is warm in nature, has stolons and rhizomes, is propagated sexually, pollinated by cross flowers and has a certain degree of self-incompatibility, and takes diploid as the main part (2n ═ 20). The color of the fertilizer is dark green, the fertilizer requirement is less, the plant diseases and insect pests are less, the low pruning resistance is realized, and the drought resistance, the weak light resistance and the soil barreness resistance are realized. Are widely used in sports fields and leisure green systems ranging from warm to subtropical, tropical areas of the world.
The seashore paspalum is used as a halophyte, has strong salt tolerance, can strictly regulate the intake of sodium, can tolerate the soluble salt content of 34g/L in soil, and is the most salt-tolerant grass species in the prior lawn grass. Has great potential as pioneer plant, improves saline-alkali soil in China and repairs coastal mudflats in east. Meanwhile, the seashore paspalum has stronger drought resistance, can effectively take needed water and nutrient from a severe environment, can rapidly develop a root system, can fix sand dunes and repair land, and has important functions in the aspects of biological repair of the environment and regeneration of land resources; it has low requirement on water quality, can be irrigated by seawater, sewage and the like, and greatly saves fresh water resources. Therefore, the key gene for regulating and controlling the stress resistance of seashore paspalum is separated and identified, the molecular regulation mechanism of the key gene is analyzed, the molecular approach and the regulation network of the stress resistance are disclosed, functional genes with application values are mined from the molecular regulation mechanism, the theoretical basis is laid for the molecular design and breeding of other crops, excellent gene resources are provided, and the method has important significance for high and stable yield of the crops, improvement of the quality of the crops and the like.
With the development of molecular biology, many experiments involving gene function verification require genetic transformation by tissue culture. In the past, young flower spikes or mature seeds of seashore paspalum are mostly used as explants for callus induction, and then complete plants are obtained through differentiation. Moreover, only one commercial seed direct-seeding type seashore paspalum variety exists in the world at present. Therefore, the existing tissue culture mode has the defect that the selection of explants is limited by seasons and material varieties, so that enough explants cannot be provided for experiments all year round, and the experimental process is greatly hindered.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for establishing a seashore paspalum tissue culture regeneration system, which can overcome the season limitation of explant supply so as to greatly shorten the experimental period.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for establishing the seashore paspalum tissue culture regeneration system comprises the following steps:
A. the method comprises the steps of taking a seashore paspalum stolon as an explant, cleaning and sterilizing the seashore paspalum stolon, cutting the washed and sterilized seashore paspalum stolon into small sections with sections, inoculating the small sections into an induction culture medium SI, and culturing the small sections for 20-30 days to obtain callus, wherein the induction culture medium SI is prepared from MS culture medium salt, 30g/L maltose, 1000mg/L acid hydrolyzed casein, B5Vitamins, 20mg/L sorbitol, 3.0 mg/L2, 4-D and 2.5g/L plant gel, wherein the pH value of the induction medium SI is 5.8;
B. transferring the callus into a subculture medium SS for culturing for 5-10 d, wherein the subculture medium SS is prepared from MS medium salt, 30g/L maltose, 1000mg/L acid hydrolyzed casein and B5Vitamins, 20mg/L sorbitol, 2.0 mg/L2, 4-D and 2.5g/L plant gel, wherein the pH value of the subculture medium SS is 5.8;
C. transferring the callus processed in the step B into a differentiation medium SR for culturing for 15-20 days, wherein the differentiation medium SR consists of an MS medium, 10mg/L sorbitol, 30g/L sucrose, 1.0 mg/L6-BA and 2.5g/L plant gel, and the pH value of the differentiation medium SR is 5.8;
D. and transplanting the seedlings when the seedlings grow to 2-3 cm.
Further, in the step a, the washing and sterilizing treatment of the seashore paspalum stolons was performed as follows: firstly, cleaning the surface of the creeping stem of the seashore paspalum with tap water, then putting the creeping stem of the seashore paspalum into a bottle, washing the bottle with distilled water for 3 times, then putting the bottle into 1 percent NaClO solution for soaking for 30min, finally taking out the creeping stem of the seashore paspalum, washing the creeping stem of the seashore paspalum with double distilled water for 5-7 times, putting the bottle into sterilized filter paper, and airing the bottle for redundant water.
Further, in the step A, cleaning and sterilizing the creeping stems of the seashore paspalum, cutting the creeping stems into small sections with the length of 3cm saved, inoculating the small sections into the induction culture medium SI, culturing for 10-15 days, subtracting new buds on the creeping stem nodes of the seashore paspalum, replacing the new induction culture medium SI, and continuously culturing for 10-15 days to obtain the callus.
Further, the salts of MS medium contained in the induction medium SI are formed from KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, the content of each component is as follows: KNO3Is 1900mg/L, NH4NO3The content of the active carbon is 1650mg/L,MgSO4·7H2o is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H2O is 27.8 mg/L.
Further, the salts of MS medium contained in the subculture medium SS are replaced with KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H2O is 27.8 mg/L.
Further, the MS medium contained in the differentiation medium SR was KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2The composition comprises O, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, wherein the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H227.8mg/L of O, 2.0mg/L of glycine, 0.1mg/L of thiamine hydrochloride, 0.5mg/L of pyridoxine hydrochloride, 0.5mg/L of nicotinic acid and 100mg/L of inositol.
Further, B contained in the induction medium SI and the subculture medium SS5The vitamins consist of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, and the content of each component is as follows: thiamine hydrochloride 10mg/L, pyridoxine hydrochloride 1mg/L, nicotinic acid 1mg/L, inositol 100 mg/L.
Further, in step a, the ambient temperature of the induction culture is 25 ℃, and the culture mode is dark culture.
The invention has the beneficial effects that: the invention adopts the creeping stem section of the seashore paspalum with nodes as the explant, the creeping stem section of the seashore paspalum with nodes is inoculated into an induction culture medium SI after being cleaned and sterilized to be cultured to obtain the callus, and the callus is prepared by MS culture medium salt, 30g/L maltose, 1000mg/L acid casein hydrolysate, B5The induction culture medium SI consisting of vitamins, 20mg/L sorbitol, 3.0 mg/L2, 4-D and 2.5g/L plant gel can greatly improve the induction rate and shorten the induction culture time, the induction culture can be completed only 20-30 days, then the callus is transferred into a callus subculture medium SS for culture, and the callus is subjected to secondary culture by MS medium salt, 30g/L maltose, 1000mg/L casein hydrolysate, B5Vitamin, 20mg/L mountainThe subculture medium SS consisting of the pyrinol, 2.0 mg/L2, 4-D and 2.5g/L plant gel can enable callus to be proliferated quickly and to be good in quality, subculture can be completed only by 5-10D, the subculture medium SS is transferred to a differentiation culture medium SR for culture, the callus differentiation culture medium SR consisting of an MS culture medium, 10mg/L sorbitol, 30g/L sucrose, 1.0 mg/L6-BA and 2.5g/L plant gel can greatly improve the differentiation rate and shorten the differentiation culture time, the differentiation culture can be completed only by 15-20D, when the seedling grows to 2-3 cm, the seedling is transplanted, an efficient seashore paspalum tissue culture regeneration system is established by the method, a foundation is laid for establishing an efficient genetic transformation system, and a technical reference is provided for the excellent variety of seashore paspalum, meanwhile, the time limit of explant supply is overcome, and the whole period of the method only needs 40-60 days, so that the experimental period is greatly shortened.
Drawings
FIG. 1 is a diagram showing the callus induction of seashore paspalum according to the embodiment of the present invention;
FIG. 2 is a diagram showing the secondary culture of seashore paspalum calluses according to the embodiment of the present invention;
FIG. 3 is a diagram showing callus differentiation of seashore paspalum according to the embodiment of the present invention;
FIG. 4 is a diagram of seedlings of seashore paspalum according to the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
The invention adopts the creeping stem section of the seashore paspalum with nodes as the explant, the creeping stem section of the seashore paspalum with nodes is inoculated into an induction culture medium SI after being cleaned and sterilized to be cultured to obtain the callus, and the callus is prepared by MS culture medium salt, 30g/L maltose, 1000mg/L acid casein hydrolysate, B5The induction culture medium SI consisting of vitamins, 20mg/L sorbitol, 3.0 mg/L2, 4-D and 2.5g/L plant gel can greatly improve the induction rate and shorten the induction culture time, the induction culture can be completed only 20-30 days, then the callus is transferred into a callus subculture medium SS for culture, and the callus is subjected to secondary culture by MS medium salt, 30g/L maltose, 1000mg/L casein hydrolysate, B5Vitamins, vitamin D and vitamin D,20mg/L sorbitol, 2.0 mg/L2, 4-D and 2.5g/L plant gel form a subculture medium SS, which can make callus proliferate fast and have good quality, the subculture can be completed only by 5-10D, and then the subculture medium SS is transferred to a differentiation medium SR for culture, the differentiation medium SR consisting of an MS medium, 10mg/L sorbitol, 30g/L sucrose, 1.0 mg/L6-BA and 2.5g/L plant gel can greatly improve the differentiation rate and shorten the differentiation culture time, the differentiation culture can be completed only by 15-20D, when the seedling grows to 2-3 cm, the seedling is transplanted, the efficient paspalum maritime tissue culture regeneration system is established by the method, the foundation of the efficient genetic transformation system for establishing paspalum maritime, and the technical reference is provided for the excellent variety of paspalum maritime, meanwhile, the limitation of explant supply is overcome, and meanwhile, the whole period of the method only needs 40-60d, so that the experimental period is greatly shortened. Specifically, the method for establishing the seashore paspalum tissue culture regeneration system comprises the following steps:
A. the method comprises the steps of taking a seashore paspalum stolon as an explant, cleaning and sterilizing the seashore paspalum stolon, cutting the washed and sterilized seashore paspalum stolon into small sections with sections, inoculating the small sections into an induction culture medium SI, and culturing the small sections for 20-30 days to obtain callus, wherein the induction culture medium SI is prepared from MS culture medium salt, 30g/L maltose, 1000mg/L acid hydrolyzed casein, B5Vitamin, 20mg/L sorbitol, 3.0 mg/L2, 4-D, 2.5g/L plant gel composition, pH of the induction culture medium is 5.8, MS culture medium in the induction culture medium SI has high salt concentration, can accelerate growth of calluses of seashore paspalum and meet requirements of seashore paspalum for mineral nutrition, maltose and sorbitol are carbon sources and also play a role of osmolyte, induction of embryogenic calluses of seashore paspalum can be enhanced, acid hydrolyzed casein is rich in various amino acids, acid hydrolyzed casein with concentration of 1000mg/L and B5The vitamins are favorable for keeping the activity of the calluses of the seashore paspalum, 3.0mg/L of 2,4-D can induce the embryogenic calluses which are compact in structure, light yellow in color and easy to differentiate, the plant gel is good in transparency and small in dosage, and the vitrification phenomenon of the calluses of the seashore paspalum can be effectively prevented;
B. transferring the callus into a subculture medium SS for culturing for 5-10 d, wherein subculture is carried outThe culture medium SS is composed of MS culture medium salt, maltose of 30g/L, acid hydrolyzed casein of 1000mg/L, and B5Vitamins, 20mg/L sorbitol, 2.0 mg/L2, 4-D and 2.5g/L plant gel, wherein the pH value of the subculture medium SS is 5.8, the salt concentration of an MS culture medium in the subculture medium SS is high, the growth of the calli paleacei callus can be accelerated, the requirement of the calli paleacei on mineral nutrition can be met, maltose and sorbitol are carbon sources and play roles of osmolytes, the induction of the embryogenic calli paleacei can be enhanced, the acid hydrolyzed casein is rich in various amino acids, and the acid hydrolyzed casein and the B gel with the concentration of 1000mg/L are obtained5The vitamins are beneficial to keeping the activity of the calluses of the seashore paspalum, the reduction of the concentration of 2,4-D (2.0mg/L) can effectively maintain the embryogenic calluses which are compact in structure, light yellow in color and easy to differentiate, the plant gel has good transparency and small dosage, and the vitrification phenomenon of the calluses of the seashore paspalum can be effectively prevented;
C. transferring the callus processed in the step B into a differentiation medium SR for culturing for 15-20 days, wherein the differentiation medium SR consists of an MS medium, 10mg/L sorbitol, 30g/L sucrose, 1.0 mg/L6-BA and 2.5g/L plant gel, the pH of the differentiation medium SR is 5.8, the MS medium in the differentiation medium SR has high salt concentration and can meet the mineral nutrition requirement of seashore paspalum, the maltose and the sorbitol are carbon sources and play a role of osmolyte to prevent the vitrification phenomenon of the seashore paspalum callus, and the 1.0 mg/L6-BA can effectively promote the embryogenic callus of the seashore paspalum to be differentiated into stem buds;
D. and transplanting the seedlings when the seedlings grow to 2-3 cm.
In the process of the method for establishing the tissue culture regeneration system of the seashore paspalum, in the step A, the treatment mode of cleaning and sterilizing the stolon segments with the seashore paspalum knots can adopt various modes, and in order to ensure that the cleaning effect and the sterilization effect are optimal, the invention adopts the following modes: firstly, cleaning the surface of the creeping stem of the seashore paspalum with tap water, then putting the creeping stem of the seashore paspalum into a bottle, washing the bottle with distilled water for 3 times, then putting the bottle into 1 percent NaClO solution, dripping Tween-201 drops into the bottle, soaking the bottle for 30min, finally taking out the creeping stem of the seashore paspalum, washing the creeping stem of the seashore paspalum with double distilled water for 5 to 7 times, putting the bottle on sterilized filter paper, and airing the bottle for redundant water.
In the step A, the stoloniferous stem segments with the knots of the seashore paspalum are cleaned, sterilized, inoculated into an induction culture medium and cultured for 10-15 days, new buds emitted from the stoloniferous stem segments of the seashore paspalum are subtracted, and the new induction culture medium is replaced to continue culturing for 10-15 days to obtain the callus. Removing the new buds on the stolon segment of the seashore paspalum, which is beneficial to the induction of the callus. In general, the creeping stem segments of seashore paspalum are cleaned, sterilized, inoculated into an induction culture medium and cultured for 12 days, new buds on the nodes of the creeping stem segments of seashore paspalum are subtracted, the induction culture medium is replaced by a new one and continuously cultured for 12 days to obtain calluses, and the effect of the new buds obtained in the final differentiation stage is optimal.
The salts of MS culture medium contained in the induction and subculture are KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4·7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4 & 5H2O is 0.025mg/L, CoCl2 & 6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4 & 4H2O is 27.8 mg/L.
B contained in the induction and subculture5The vitamins comprise thiamine hydrochloride 10mg/L, pyridoxine hydrochloride 1mg/L, nicotinic acid 1mg/L, and inositol 100 mg/L.
Contained in the differentiation medium SRMS culture medium consisting of KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2The composition comprises O, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, wherein the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H227.8mg/L of O, 2.0mg/L of glycine, 0.1mg/L of thiamine hydrochloride, 0.5mg/L of pyridoxine hydrochloride, 0.5mg/L of nicotinic acid and 100mg/L of inositol. The formula is specially prepared for the seashore paspalum, and is more suitable for tissue culture of the seashore paspalum.
In order to achieve better effect of the induction culture, the environment temperature of the induction culture is 25 ℃, and the culture mode is dark culture.
Examples
Cleaning and sterilizing a plurality of creeping stem sections of seashore paspalum, inoculating the creeping stem sections into the induction culture medium SI, culturing in a dark environment at 25 ℃ for 12 days, subtracting new buds emitted from the creeping stem sections of the seashore paspalum band knots, replacing the new induction culture medium SI, and continuously culturing for 12 days to obtain a seashore paspalum callus induction condition graph shown in figure 1, observing the quality of callus, counting the number of the callus, calculating the callus induction rate, wherein the callus induction rate is as high as 90 percent, and the callus has high growth speed, best quality, light yellow, compact and large (diameter is larger than 1cm) particles.
The subculture mainly has the function of expanding the callus, and is more beneficial to culturing more regeneration plants. Transferring the callus obtained by the culture of the induction culture medium SI into the subculture medium SS, performing dark culture in a culture room for 10 days to obtain a seashore paspalum callus subculture condition diagram shown in figure 2, observing the quality of the callus, counting the final weight of the callus, and calculating the proliferation efficiency, wherein the proliferation efficiency is the percentage of the weight increase of one callus in 10 days, the proliferation efficiency is as high as 2000%, and the callus is light yellow compact particles.
Transferring the callus processed by the subculture medium SS into the differentiation medium SR, and culturing at 25 + -2 deg.C under 1000lx illumination intensity for 20d to obtain the callus differentiation condition diagram of seashore paspalum shown in FIG. 3, wherein the green seedling differentiation rate is up to 90%.
And (5) transplanting the seedlings when the seedlings grow to 2-3 cm as shown in figure 4.
In the present invention, min represents minutes, D represents days, NaClO represents sodium hypochlorite, 2,4-D represents 2, 4-dichlorophenoxyacetic acid, and 6-BA represents 6-benzylaminopurine.
Claims (7)
1. A method for establishing a tissue culture regeneration system of seashore paspalum is characterized by comprising the following steps:
A. the method comprises the steps of taking a seashore paspalum stolon as an explant, cleaning and sterilizing the seashore paspalum stolon, cutting the washed and sterilized seashore paspalum stolon into small sections with sections, inoculating the small sections into an induction culture medium SI, and culturing the small sections for 20-30 days to obtain callus, wherein the induction culture medium SI is prepared from MS culture medium salt, 30g/L maltose, 1000mg/L acid hydrolyzed casein, B5Vitamins, 20mg/L sorbitol, 3.0 mg/L2, 4-D and 2.5g/L plant gel, wherein the pH value of the induction medium SI is 5.8; b is5The vitamins consist of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, and the content of each component is as follows: thiamine hydrochloride is 10mg/L, pyridoxine hydrochloride is 1mg/L, nicotinic acid is 1mg/L, and inositol is 100 mg/L;
B. transferring the callus into subculture medium SSCulturing for 5-10 days, wherein the subculture medium SS is prepared from MS medium salt, 30g/L maltose, 1000mg/L acid hydrolyzed casein, and B5Vitamins, 20mg/L sorbitol, 2.0 mg/L2, 4-D and 2.5g/L plant gel, wherein the pH value of the subculture medium SS is 5.8; b is5The vitamins consist of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, and the content of each component is as follows: thiamine hydrochloride is 10mg/L, pyridoxine hydrochloride is 1mg/L, nicotinic acid is 1mg/L, and inositol is 100 mg/L;
C. transferring the callus processed in the step B into a differentiation medium SR for culturing for 15-20 days, wherein the differentiation medium SR consists of an MS medium, 10mg/L sorbitol, 30g/L sucrose, 1.0 mg/L6-BA and 2.5g/L plant gel, and the pH value of the differentiation medium SR is 5.8;
D. and transplanting the seedlings when the seedlings grow to 2-3 cm.
2. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: in step a, the procedure for washing and sterilizing the stolons of seashore paspalum was as follows: firstly, cleaning the surface of the creeping stem of the seashore paspalum with tap water, then putting the creeping stem of the seashore paspalum into a bottle, washing the bottle with distilled water for 3 times, then putting the bottle into 1 percent NaClO solution for soaking for 30min, finally taking out the creeping stem of the seashore paspalum, washing the creeping stem of the seashore paspalum with double distilled water for 5-7 times, putting the bottle into sterilized filter paper, and airing the bottle for redundant water.
3. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: in the step A, cleaning and sterilizing the creeping stems of the seashore paspalum, cutting the creeping stems into small sections with the length of 3cm saved, inoculating the small sections into an induction culture medium SI, culturing for 10-15 days, subtracting new buds on the creeping stem nodes of the seashore paspalum, replacing the new induction culture medium SI, and continuously culturing for 10-15 days to obtain the callus.
4. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: the induction culture medium SI containsThe MS culture medium has the salinity of KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H2O is 27.8 mg/L.
5. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: the salt content of MS culture medium contained in the subculture medium SS is KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H2O is 27.8 mg/L.
6. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: the MS culture medium contained in the differentiation culture medium SR is KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2The composition comprises O, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, wherein the content of each component is as follows: KNO3Is 1900mg/L, NH4NO31650mg/L, MgSO4 & 7H2O is 370mg/L, KH2PO4170mg/L of CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO36.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2EDTA 37.3mg/L, FeSO4·4H227.8mg/L of O, 2.0mg/L of glycine, 0.1mg/L of thiamine hydrochloride, 0.5mg/L of pyridoxine hydrochloride, 0.5mg/L of nicotinic acid and 100mg/L of inositol.
7. The method for establishing a tissue culture regeneration system for seashore paspalum according to claim 1, wherein: in step A, the environmental temperature of the induction culture is 25 ℃, and the culture mode is dark culture.
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