CN111286484A - Oncidium protoplast dissociation and culture method - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- 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 provides an oncidium protoplast dissociating and culturing method, which comprises the steps of taking oncidium protocorms as starting materials, and obtaining callus with vigorous growth and loose structure for dissociating protoplasts through callus induction culture and 4-5 times of subculture; cold treating the callus at 4 ℃ for 1d, then putting the callus into the mixed enzyme solution, oscillating and performing enzymolysis for 8-10 h by using a table concentrator, and purifying the protoplast by using an interface method, thereby obtaining a large amount of high-activity oncidium protoplasts; the culture medium is continuously used for solid-liquid double-layer culture, the obtained oncidium protoplast cells can normally grow and continuously divide to form small cell clusters, the division frequency of the oncidium protoplast cells reaches up to 10.16 percent, and the plating rate of the oncidium protoplast cells reaches up to 4.03 percent. The invention provides scientific basis for cell fusion, somatic cell hybridization, genetic transformation and germplasm innovation by using the oncidium protoplast.
Description
Technical Field
The invention belongs to the field of ornamental plant cell engineering, and particularly relates to an oncidium protoplast dissociation and culture method.
Background
Oncidium (A)Oncidium hybridum) The Luxiang orchid, dancing orchid, golden butterfly orchid, Oncales orchid, etc. are the general names of hybrid species in orchidaceae, oncidium and its kindred genera, and are mainly distributed in tropical and subtropical areas in central and south America, such as America, Mexico, Paraguay, Peru, Brazil, etc. Oncidium is a complex stem aerial orchid with large morphological change, and can be divided into two types with pseudobulb and two types without pseudobulb according to whether pseudobulb exists or not, and can be divided into three types of thin leaf, thick leaf and sword leaf according to the form of leaves. The flower of the oncidium has larger shape difference from a mini-type to a big-type, and has bright color and rich flower color, not only common yellow and brown colors, but also green, white, red, magenta, brown colors and the like, or a plurality of colors are mixed to form stripes or patches. The oncidium is mainly produced as cut flower and pot flower, has good flower branches, beautiful flower shape and bright color, can continuously bloom, and hasHigh ornamental value and strong market competitiveness, and particularly becomes one of popular potted flower types in the European and American markets. The main production area of the oncidium in China is in the Taiwan area, the industry is developed rapidly, but the germ plasm resources of the oncidium in the continental area of China are deficient, the cultivated varieties are the varieties bred by foreign breeding companies, and the varieties lack independent intellectual property rights.
Like other orchidaceae plants, oncidium is difficult to propagate in a natural state, and the seed germination rate is extremely low, so that the traditional cultivation depends on plant division propagation. But the division propagation coefficient is lower, and through long-term exploration of researchers on the rapid propagation method of high-quality seedlings of oncidium hybridum, tissue culture becomes the best method for propagation of seedlings of oncidium hybridum. Crossbreeding is the main breeding means of oncidium. Although some cultivars of oncidium with excellent traits have been successfully obtained in the past decades by cross breeding, they are still far less abundant in registration of new cultivars than other orchids such as butterfly orchid and dendrobii orchid, and the main reason for this is that oncidium has poor affinity for interspecific crosses within the genus and intergeneric crosses within the genus. Protoplast fusion can avoid specific gamete recognition reactions of species in fertilization, and can possibly break the sexual incompatibility limit in distant hybridization. Cell hybridization or cell reconstruction through the fusion between protoplasts is an important means of plant cell engineering, and has wide application prospect in the aspects of variety improvement and creation of new germplasm.
Plant protoplasts are naked cells which are obtained by removing cell walls from plant cells and are wrapped by plasma membranes, and are a morphological structural unit which forms cells, including cell membranes, cytoplasm in the membranes and other organelles with vital activities, and are the material basis of the vital activities of the cells. The plant protoplast provides a convenient genetic manipulation experimental system for basic research and genetic engineering of plant breeding. Culturing plant protoplast to study the formation of plant cell wall, further culturing to generate somatic clone, and even screening clone with excellent character from clone variant; the protoplast can be used for cell fusion and somatic cell hybridization to create a distant hybrid and obtain an homologous or heterologous triploid, a tetraploid and an amphidiploid, so that the method becomes a new way for breaking through species reproductive isolation; protoplasts are also ideal receptors for plant genetic engineering, and are now widely used in signal transduction, foreign gene introduction, and transient expression systems.
The culture of orchid protoplast and the fusion of somatic cells are the research field of orchid cell engineering technology, and this technological system opens up a new way for orchid breeding and has immeasurable value for the development of orchid industry. An effective protoplast separation and culture system is a prerequisite for protoplast fusion. Currently, the conventional method for isolating protoplasts is an enzymatic method. When protoplasts are separated by an enzymatic method, proper types, combinations and concentrations of hydrolytic enzymes are selected in addition to proper separation materials. In addition, the separation of plant protoplasts is also influenced by the time, temperature and value of enzymatic hydrolysis, the method of separation, and other factors. 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 donor material, medium composition, cultivation mode, cultivation density, etc. At present, researches on the separation and culture of orchid protoplasts mainly focus on several orchid, phalaenopsis and dendrobium, but researches on the orchid protoplasts are not reported yet. On the basis of the oncidium tissue culture, the research explores the influence of various factors on the yield and the activity of the separated oncidium protoplast, and carries out culture on the separated protoplast, thereby laying a foundation for researches on oncidium plant regeneration, protoplast fusion, genetic transformation and the like.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for dissociating and culturing an oncidium protoplast.
The invention solves the technical problems through the following technical scheme:
an oncidium protoplast dissociating and culturing method comprises the following steps:
1) induction of callus: cutting the oncidium protocorm into small pieces of 1-2 mm, inoculating the small pieces on a callus induction culture medium, and culturing for about 30 days at 24-26 ℃ in the dark;
2) subculturing the callus: transferring the induced callus onto a subculture medium for culturing, and carrying out selective subculture every 15-20 days, wherein the callus with vigorous growth and loose structure is obtained for dissociation of protoplasts after 4-5 times of subculture;
3) protoplast dissociation: performing cold treatment on the callus at 4 ℃ for 1d, then putting about 1g of callus into 10mL of mixed enzyme solution, and putting the mixed enzyme solution on a shaking table for 8-10 h in a shaking and enzymolysis way;
4) and (3) purifying protoplasts: after enzymolysis, filtering the enzymolysis liquid by using a sleeve funnel and a 200-mesh nylon net, adding a CPW-13% mannitol solution for washing, collecting filtrate in a centrifuge tube, centrifuging for 10min at 500r/min, discarding supernatant, suspending precipitates by using a CPW-25% sucrose solution, slowly adding the CPW-13% mannitol solution on a sucrose layer along the tube wall, centrifuging for 3min at 300r/min, forming a clear band between two liquid surfaces of sucrose and mannitol by protoplast, sucking out the protoplast band, placing the protoplast band in another centrifuge tube, adding a liquid protoplast culture medium, centrifuging for 10min at 500r/min, discarding supernatant, and obtaining purified protoplast;
5) and (3) culturing protoplasts: the purified protoplasts were resuspended in liquid protoplast medium at a density of 1X 105~3×105Per mL; adding 2mL of solid protoplast culture medium into a culture dish with the diameter of 6cm, adding 1mL of protoplast suspension after solidification, sealing by Parafilm, and culturing at 24-26 ℃ under a dark condition; after 3 weeks of culture, 4-6 drops of the medium for reducing blood pressure is added, and culture is continued until a multicellular mass visible to the naked eye is formed.
The formula of the callus induction culture medium in the step 1) is as follows: VW basic culture medium, Huabao No. 1 No. 3g/L, 6-BA1.0mg/L, NAA0.1mg/L, ferulic acid 10mg/L, cane sugar 40g/L and agar 6g/L, and the pH value of the culture medium is adjusted to 5.8.
The formula of the subculture medium in the step 2) is as follows: VW basic culture medium, Huabao No. 1 No. 3g/L, NAA3.0mg/L, IAA 2.0mg/L, 6-BA0.2mg/L, cane sugar 30g/L and agar 6 g/L; the pH of the medium was adjusted to 5.8.
The conditions of the subculture in the step 2) are as follows: the temperature is 24-26 ℃, the illumination intensity is 600-1000 lx, and the illumination time is 9-11 h/d.
The components of the mixed enzyme liquid in the step 3) are as follows: CPW solution, 1.5% cellulase, 0.2% pectinase, 10g/L polyvinylpyrrolidone, 2g/L betaine and 0.5mol/L mannitol, and adjusting the pH value to 5.6.
The temperature of the shaking table in the step 3) is 25-28 ℃, and the rotating speed is 30-50 r/min.
The formula of the liquid protoplast culture medium in the step 4) is as follows: KNO3800~1000mg/L，CaCl2·2H2O600~800mg/L，MgSO4·7H2O 630~720mg/L，KH2PO4150~160mg/L，H3BO33.2~3.8mg/L，KI0.37~0.45mg/L，Na2MoO4·2H2O 0.12~0.16mg/L，CoCl2·6H2O 0.015~0.020mg/L，MnSO4·4H2O 13~16mg/L，ZnSO4·7H2O 4.4~5.0mg/L，CuSO4·5H2O 0.02~0.03mg/L，FeSO4·7H2O13.7~14.5mg/L，Na218.8-19.6 mg/L of EDTA, 80-100 mg/L of inositol, 160-200 mg/L of hydrolyzed casein, 40-60 mg/L of asparagine, 40-60 mg/L of arginine, 10-20 mg/L of fumaric acid, 10-20 mg/L of choline, VB10.4~0.6mg/L，VB60.4-0.6 mg/L, 2.5-3.5 mg/L nicotinic acid, 0.05-0.07 mg/L biotin, 4.0-5.0 mg/L ascorbic acid, 0.2-0.5 mg/L5-aminolevulinic acid, 0.08-0.12 mg/L triacontanol, 0.03-0.04 mol/L glucose, 0.02-0.03 mol/L cellobiose, 0.4-0.6 mol/L mannitol, 50-80 ml/L coconut milk, 0.3-0.7 mg/L2, 4-D, 0.3-0.7 mg/L6-BA, 0.8-1.2 mg/L NAA, and the pH of the culture medium is adjusted to 5.8.
The solid protoplast culture medium in the step 5) comprises: 4.0-5.0 g/L agar is added to the liquid protoplast culture medium.
The medium for reducing the pressure in the step 5) comprises the following components: the concentration of mannitol in the liquid protoplast culture medium is reduced to 0.15-0.25 mol/L, and other components are unchanged.
The invention has the beneficial effects that:
1) oncidium isThe cotyledon plant is not easy to be degraded by enzyme liquid, so the callus induced by the oncidium protocorm is selected as the material of the free protoplast, and the free effect is better. The invention explores the enzyme liquid proportion and enzymolysis condition suitable for the dissociation of the oncidium protoplast, and purifies the protoplast by an interface method, and the yield of the obtained protoplast can reach 35.27 multiplied by 10 to the maximum5Per gram, and the activity of the protoplast is kept better. The culture medium and the culture method suitable for culturing the oncidium protoplast are also explored, after the culture, the oncidium protoplast cells can normally grow and continuously divide to form small cell clusters, the division frequency can reach 10.16 percent at most, and the plating rate can reach 4.03 percent at most.
2) The method successfully establishes the dissociation and culture system of the oncidium protoplast, and lays a foundation for the subsequent development of plant regeneration, somatic cell hybridization, genetic transformation and germplasm innovation of the oncidium.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1
1 Induction and subculture of callus
Induction of callus: cutting oncidium protocorm into 1-2 mm small pieces, inoculating on callus induction culture medium, and culturing at 25 deg.C in dark for about 30 d; the formula of the callus induction culture medium is as follows: VW basic culture medium, Huabao No. 1 No. 3g/L, 6-BA1.0mg/L, NAA0.1mg/L, ferulic acid 10mg/L, cane sugar 40g/L and agar 6g/L, and the pH value of the culture medium is adjusted to 5.8.
Subculturing the callus: transferring the induced callus onto a subculture medium for culturing, wherein the temperature is 25 ℃, the illumination intensity is 800lx, and subculture is carried out every 15-20 d, and the callus with vigorous growth and loose structure is obtained for dissociation of protoplasts after 4-5 times of subculture; the formula of the subculture medium is as follows: VW basic culture medium, Huabao No. 1 No. 3g/L, NAA3.0mg/L, IAA 2.0mg/L, 6-BA0.2mg/L, cane sugar 30g/L and agar 6 g/L; the pH of the medium was adjusted to 5.8.
2 isolation of protoplasts
Protoplast dissociation: performing cold treatment on the callus obtained by subculture at 4 ℃ for 1d, then putting about 1g of callus into 10mL of mixed enzyme solution, placing on a shaking table, performing shaking enzymolysis for 10h, wherein the temperature of the shaking table is 25 ℃, and the rotating speed is 40 r/min; the components of the mixed enzyme solution are as follows: CPW solution, 1.5% cellulase, 0.2% pectinase, 10g/L polyvinylpyrrolidone, 2g/L betaine and 0.5mol/L mannitol, and adjusting the pH value to 5.6.
And (3) purifying protoplasts: after enzymolysis, filtering the enzymolysis liquid by using a sleeve funnel and a 200-mesh nylon net, adding CPW-13% mannitol solution for washing, collecting filtrate in a centrifuge tube, centrifuging for 10min at 500r/min, discarding supernatant, suspending precipitate by using CPW-25% sucrose solution, slowly adding the CPW-13% mannitol solution on a sucrose layer along the tube wall, centrifuging for 3min at 300r/min, forming a clear band between two liquid surfaces of sucrose and mannitol by protoplast, sucking out the protoplast band, placing the protoplast band in another centrifuge tube, adding a liquid protoplast culture medium, centrifuging for 10min at 500r/min, discarding supernatant, and obtaining purified protoplast.
Determination of protoplast yield and viability
And observing and counting the yield of the protoplast under an inverted microscope by using a blood counting chamber, and repeating for 4-5 times. Protoplast viability was determined by the FDA (fluorescein diacetate) method, which was formulated with acetone to a concentration of 5mg/mL, as measured by 25. mu. LFDA: FDA was added to 1mL of protoplast, and the activity was measured under a fluorescent microscope after 5min at room temperature.
Protoplast viability (%) = number of fluorescent protoplasts in dark field/total number of protoplasts in bright field × 100%.
2.1 Effect of Cold treatment on the dissociation of oncidium protoplasts
Pretreatment before dissociation has a positive effect on maintaining the integrity of the protoplast and improving the yield of the protoplast. The callus of oncidium obtained by subculture was pretreated at 4 ℃ for 0.5d, 1d, 2d and 3d, respectively, and then dissociated and purified, and the influence of cold treatment time on the dissociation of the protoplasts was compared without cold treatment (0 d) as a control, as shown in table 1 below.
As shown in Table 1, the yield and viability of protoplasts increased gradually with the increase of the cold treatment time and reached the highest value at 1d of the cold treatment, at which the yield of protoplasts was 32.68X 105Per gram, the activity is 83.32%; with the continuous extension of the cold treatment time, the yield and the activity of the protoplast are obviously reduced, the yield and the activity are extremely low after the cold treatment for 3d, and the callus is possibly damaged by cold damage, so that the activity of the material is damaged, and the dissociation of the protoplast is influenced.
2.2 Effect of different enzymolysis time and temperature on the dissociation of oncidium protoplast
About 1g of callus is put into 10mL of mixed enzyme solution, and the mixed enzyme solution is placed on a shaking table for oscillating enzymolysis, the rotating speed is 40r/min, 5 enzymolysis times including 4h, 6h, 8h, 10h and 12h are set, 2 enzymolysis temperatures including 25 ℃ and 28 ℃ are set, 10 treatments are counted, and the influence of different enzymolysis times and enzymolysis temperatures on the dissociation of the oncidium protoplast is compared, as shown in the following table 2.
As can be seen from Table 2, the yield and viability of oncidium protoplasts showed a tendency to increase before decrease with increasing time of enzymolysis, whether at 25 ℃ or 28 ℃. Under the condition of 25 ℃, when the enzymolysis time is 10 hours, the yield and the activity of the oncidium protoplast reach the highest peak, which is 35.27 multiplied by 10 respectively5Seed/g and 86.25%; under the condition of 28 ℃, when the enzymolysis time is 8 hours, the yield and the activity of the oncidium protoplast reach the highest peak, respectively 33.05 multiplied by 105And each gram of protoplast is 84.65 percent, and the yield and the activity of the protoplast are reduced after the enzymolysis time is continuously prolonged. Therefore, when the enzymolysis temperature is higher, the enzymolysis time can be properly shortened. In conclusion, the enzyme digestion is carried out for 10h at 25 ℃ or for strips at 28 DEG CBetter effect can be obtained by enzymolysis for 8 hours under the condition, but the effect of the enzymolysis is better compared with that of the enzymolysis for 8 hours under the condition.
3 cultivation of protoplasts
And (3) culturing protoplasts: the purified protoplasts were resuspended in liquid protoplast medium at a density of 2X 105Per mL; adding 2mL of solid protoplast culture medium into a culture dish with the diameter of 6cm, adding 1mL of protoplast suspension after solidification, sealing the culture dish by Parafilm, and culturing at 25 ℃ in the dark; after 3 weeks of culture, adding 4-6 drops of a blood pressure reducing culture medium; the formula of the liquid protoplast culture medium is as follows: KNO3900mg/L，CaCl2·2H2O 700mg/L，MgSO4·7H2O 675mg/L，KH2PO4155mg/L，H3BO33.5mg/L，KI 0.41mg/L，Na2MoO4·2H2O 0.14mg/L，CoCl2·6H2O0.018mg/L，MnSO4·4H2O 14.5mg/L，ZnSO4·7H2O 4.7mg/L，CuSO4·5H2O 0.025mg/L，FeSO4·7H2O 14.1mg/L，Na219.2mg/L EDTA, 90mg/L inositol, 180mg/L hydrolyzed casein, 50mg/L asparagine, 50mg/L arginine, 15mg/L fumaric acid, 15mg/L choline, VB10.5mg/L，VB60.5mg/L, 3.0mg/L nicotinic acid, 0.06mg/L biotin, 4.5mg/L ascorbic acid, 0.35 mg/L5-aminolevulinic acid, 0.1mg/L triacontanol, 0.035mol/L glucose, 0.025mol/L cellobiose, 0.5mol/L mannitol, 65ml/L coconut milk, 0.5 mg/L2, 4-D, 6-BA0.5mg/L NAA1.0mg/L, adjusting the pH of the culture medium to 5.8; the solid protoplast culture medium is a liquid protoplast culture medium added with 4.5g/L agar; the blood pressure reducing culture medium is a liquid protoplast culture medium, wherein the concentration of mannitol is reduced to 0.2mol/L, and other components are unchanged.
Observation of protoplast cleavage status
Observing the growth and division conditions of the protoplast under an inverted microscope, and counting the division frequency of the protoplast when the protoplast is cultured for 10 days and counting the plate planting rate of the protoplast when the protoplast is cultured for 30 days.
Division frequency = (number of protoplasts that divided/number of protoplasts inoculated) × 100%
Plating rate = (number of cell clumps formed per plate/total number of protoplasts seeded per plate) × 100%
3.1 Effect of different media on the culture of oncidium protoplasts
The solid-liquid double-layer culture of the oncidium protoplast is carried out by respectively using 3 conventional culture media B5, KM8P and DPD and the protoplast culture medium of the invention, 2, 4-D0.5 mg/L, 6-BA0.5mg/L, NAA1.0mg/L and mannitol 0.5mol/L are added into 3 culture media B5, KM8P and DPD to keep consistent, and the density of the protoplast is 2 multiplied by 105Each/mL of the cells were cultured in the dark at 25 ℃ to compare the effect of different media on the culture of oncidium protoplasts, as shown in Table 3 below.
The type of the culture medium directly influences the division frequency of protoplasts, the plating rate, the appearance of small callus and the like. As can be seen from Table 3, when the culture medium of the present invention was used, the division frequency of the oncidium protoplast was 10.16%, and the plating rate was 4.03%, which were significantly higher than the division frequency and the plating rate when 3 media, B5, KM8P and DPD, were used, and thus it was found that the culture medium of the present invention was suitable as a culture medium for oncidium protoplasts, and the growth of the oncidium protoplast was the best, and it was able to divide continuously to form small cell masses.
3.2 Effect of different culture methods on the culture of oncidium protoplasts
The protoplast culture medium of the invention is respectively used for carrying out solid culture, liquid culture and solid-liquid double-layer culture on the oncidium protoplast, wherein the solid culture operation is as follows: mixing 1mL of protoplast suspension with 1mL of pre-melted (40 ℃) solid protoplast culture medium, adding a culture dish with the diameter of 6cm before cooling to prepare a flat plate, and culturing at 25 ℃ in the dark; the liquid culture operation is as follows: 2mL of the protoplast suspension was placed in a petri dish with a diameter of 6cm, sealed with Parafilm, and incubated at 25 ℃ in the dark. The effect of different culture regimes on the culture of oncidium protoplasts was compared, as shown in table 4 below.
As can be seen from Table 4, the culture effect of protoplasts is different in different culture modes, wherein the solid-liquid double-layer culture mode is adopted, the division frequency of the oncidium protoplast is 10.05%, the appearance frequency of cell clusters is 2.98%, and the cell clusters are significantly higher than that of the other 2 culture modes, so that the solid-liquid double-layer culture mode is a better culture mode when the culture mode of the oncidium protoplast is selected.
The method for liberating and culturing an oncidium protoplast of the invention has been described by specific examples, and those skilled in the art can use the contents of the invention to realize other corresponding purposes by appropriately changing the raw materials, process conditions and the like without departing from the contents of the invention, and all similar substitutions and modifications will be obvious to those skilled in the art and are considered to be included in the scope of the invention.
Claims (9)
1. An oncidium protoplast dissociating and culturing method comprises the following steps:
1) induction of callus: cutting the oncidium protocorm into small pieces of 1-2 mm, inoculating the small pieces on a callus induction culture medium, and culturing for about 30 days at 24-26 ℃ in the dark;
2) subculturing the callus: transferring the induced callus onto a subculture medium for culturing, and carrying out selective subculture every 15-20 days, wherein the callus with vigorous growth and loose structure is obtained for dissociation of protoplasts after 4-5 times of subculture;
3) protoplast dissociation: performing cold treatment on the callus at 4 ℃ for 1d, then putting about 1g of callus into 10mL of mixed enzyme solution, and putting the mixed enzyme solution on a shaking table for 8-10 h in a shaking and enzymolysis way;
4) and (3) purifying protoplasts: after enzymolysis, filtering the enzymolysis liquid by using a sleeve funnel and a 200-mesh nylon net, adding a CPW-13% mannitol solution for washing, collecting filtrate in a centrifuge tube, centrifuging for 10min at 500r/min, discarding supernatant, suspending precipitates by using a CPW-25% sucrose solution, slowly adding the CPW-13% mannitol solution on a sucrose layer along the tube wall, centrifuging for 3min at 300r/min, forming a clear band between two liquid surfaces of sucrose and mannitol by protoplast, sucking out the protoplast band, placing the protoplast band in another centrifuge tube, adding a liquid protoplast culture medium, centrifuging for 10min at 500r/min, discarding supernatant, and obtaining purified protoplast;
5) and (3) culturing protoplasts: the purified protoplasts were resuspended in liquid protoplast medium at a density of 1X 105~3×105Per mL; adding 2mL of solid protoplast culture medium into a culture dish with the diameter of 6cm, adding 1mL of protoplast suspension after solidification, sealing by Parafilm, and culturing at 24-26 ℃ under a dark condition; after 3 weeks of culture, 4-6 drops of the medium for reducing blood pressure is added, and culture is continued until a multicellular mass visible to the naked eye is formed.
2. The method for dissociating and culturing oncidium protoplasts according to claim 1, wherein the callus induction medium in the step 1) is prepared from the following components: VW basic culture medium, Huabao No. 1 No. 3g/L, 6-BA1.0mg/L, NAA0.1mg/L, ferulic acid 10mg/L, cane sugar 40g/L and agar 6g/L, and the pH value of the culture medium is adjusted to 5.8.
3. The method for dissociating and culturing oncidium protoplasts according to claim 1, wherein the formulation of the subculture medium in the step 2) is: VW basic culture medium, Huabao No. 1 No. 3g/L, NAA3.0mg/L, IAA 2.0mg/L, 6-BA0.2mg/L, cane sugar 30g/L and agar 6 g/L; the pH of the medium was adjusted to 5.8.
4. The method for dissociating and culturing an oncidium protoplast according to claim 1, wherein the conditions for subculture in the step 2) are as follows: the temperature is 24-26 ℃, the illumination intensity is 600-1000 lx, and the illumination time is 9-11 h/d.
5. The method for dissociating and culturing an oncidium protoplast according to claim 1, wherein the mixed enzyme solution in the step 3) comprises the following components: CPW solution, 1.5% cellulase, 0.2% pectinase, 10g/L polyvinylpyrrolidone, 2g/L betaine and 0.5mol/L mannitol, and adjusting the pH value to 5.6.
6. The method for dissociating and culturing an oncidium protoplast according to claim 1, wherein the temperature of the shaker in step 3) is 25-28 ℃ and the rotation speed is 30-50 r/min.
7. The method for dissociating and culturing oncidium protoplasts according to claim 1, wherein the liquid protoplast culture medium in the step 4) is prepared by: KNO3800~1000mg/L，CaCl2·2H2O 600~800mg/L，MgSO4·7H2O 630~720mg/L，KH2PO4150~160mg/L，H3BO33.2~3.8mg/L，KI 0.37~0.45mg/L，Na2MoO4·2H2O 0.12~0.16mg/L，CoCl2·6H2O 0.015~0.020mg/L，MnSO4·4H2O 13~16mg/L，ZnSO4·7H2O 4.4~5.0mg/L，CuSO4·5H2O 0.02~0.03mg/L，FeSO4·7H2O 13.7~14.5mg/L，Na218.8-19.6 mg/L of EDTA, 80-100 mg/L of inositol, 160-200 mg/L of hydrolyzed casein, 40-60 mg/L of asparagine, 40-60 mg/L of arginine, 10-20 mg/L of fumaric acid, 10-20 mg/L of choline, VB10.4~0.6mg/L，VB60.4-0.6 mg/L, 2.5-3.5 mg/L nicotinic acid, 0.05-0.07 mg/L biotin, 4.0-5.0 mg/L ascorbic acid, 0.2-0.5 mg/L5-aminolevulinic acid, 0.08-0.12 mg/L triacontanol, 0.03-0.04 mol/L glucose, 0.02-0.03 mol/L cellobiose, 0.4-0.6 mol/L mannitol, 50-80 ml/L coconut milk, 0.3-0.7 mg/L2, 4-D, 0.3-0.7 mg/L6-BA, 0.8-1.2 mg/L NAA, and the pH of the culture medium is adjusted to 5.8.
8. The method for dissociating and culturing oncidium protoplasts according to claim 1, wherein the solid protoplast culture medium in the step 5) is: 4.0-5.0 g/L agar is added to the liquid protoplast culture medium.
9. The method for dissociating and culturing oncidium protoplasts according to claim 1, wherein the reduced pressure culture medium in the step 5) is: the concentration of mannitol in the liquid protoplast culture medium is reduced to 0.15-0.25 mol/L, and other components are unchanged.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5573941A (en) * | 1988-03-17 | 1996-11-12 | University Of Delaware | Callus formation vanilla planifolia |
| CN101695283A (en) * | 2009-11-10 | 2010-04-21 | 北京师范大学 | Culture medium for tissue culture of pedicel buds of oncidium hybridum and tissue cultured seedling propagating method |
| CN105684896A (en) * | 2016-01-08 | 2016-06-22 | 玉林师范学院 | Dendrobium germplasm breeding method based on asymmetry protoplast hybridization technique |
-
2020
- 2020-05-06 CN CN202010372843.XA patent/CN111286484A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5573941A (en) * | 1988-03-17 | 1996-11-12 | University Of Delaware | Callus formation vanilla planifolia |
| CN101695283A (en) * | 2009-11-10 | 2010-04-21 | 北京师范大学 | Culture medium for tissue culture of pedicel buds of oncidium hybridum and tissue cultured seedling propagating method |
| CN105684896A (en) * | 2016-01-08 | 2016-06-22 | 玉林师范学院 | Dendrobium germplasm breeding method based on asymmetry protoplast hybridization technique |
Non-Patent Citations (2)
| Title |
|---|
| 孔庆彪;满若君;卜朝阳;: "文心兰组织培养初步研究" * |
| 崔广荣;: "文心兰组织培养及转基因研究进展" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111705033A (en) * | 2020-07-08 | 2020-09-25 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
| CN111705033B (en) * | 2020-07-08 | 2022-04-01 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
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