CN109554327B - Method for separating and purifying amomum villosum mesophyll protoplast - Google Patents
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Abstract
The invention discloses a method for separating and purifying amomum villosum mesophyll protoplasts, which comprises the steps of preparing and pretreating materials, separating the protoplasts by adopting an enzymolysis method, purifying the protoplasts by adopting a sedimentation method and a drifting method. According to the invention, through researching the factors influencing the yield and activity of the protoplast, such as whether the material is pretreated, the types and concentration of enzymes, the enzymolysis time, the concentration of an osmotic pressure regulator and the influence of different purification methods on the purification effect of the mesophyll cell protoplast, the experimental conditions for separating and purifying the actinolite mesophyll protoplast are optimized, so that the high-yield and high-activity actinolite protoplast is obtained, and a foundation is laid for cultivating new varieties of actinolite through somatic cell fusion.
Description
Technical Field
The invention relates to the technical field of protoplast separation and purification, in particular to a method for separating and purifying amomum villosum mesophyll protoplasts.
Background
Fructus amomi is one of famous four-south Chinese medicines, and the genuine product is zingiberaceae plant amomum villosumAmomum villosumLour, green shell sandAmomum Villosum Lour. var. xanthioidesT.L. Wu et Senjen, Hainan sandAmomumlongiligulareDry ripe fruit of t.l.wu. The amomum villosum is a mainstream variety, has pungent and warm nature and taste, enters spleen, stomach and kidney channels, has the effects of resolving dampness, stimulating appetite, warming spleen, relieving diarrhea, regulating qi, preventing miscarriage and the like, is commonly used for treating symptoms such as damp turbidity obstruction in middle energizer, abdominal fullness, hunger resistance, deficiency-cold in spleen and stomach, vomiting, diarrhea, pregnancy pernicious vomiting, threatened abortion and the like, and has a long medicinal and clinical use history in China.
In recent years, due to the development and expansion of fructus amomi processing industry, the supply of fructus amomi in the market is insufficient, and the price rises year by year. The development and utilization values of fructus amomi are more and more prominent, and the fructus amomi in spring is a valuable medicinal material with homology of medicine and food and has wide market prospect, long history of medication and eating, wide application and more prominent development and utilization values. The traditional Chinese medicine composition is directly used for various Chinese patent medicines, such as the pill of six monarch drugs of fructus amomi, the pill of stomach nourishing of fructus amomi, the pill of stomach stimulating and spleen invigorating, the powder of ginseng, tuckahoe and rhizoma atractylodis macrocephalae, and the like, and is also a main raw material of various health care products. However, special entomophilous agents such as leucorrhea bees or artificial pollination are needed in the planting process of the amomum villosum, so that the natural pollination rate is extremely low, the fruit drop rate is as high as 60% -70%, the yield is very low, the planting cost is improved, the amomum villosum is not in demand in the market, the low unit yield is a bottleneck in the development of the existing amomum villosum planting industry, and the screening and cultivation of high-yield and high-quality varieties are an effective way for solving the problem.
At present, the breeding technology of the amomum villosum adopts cross breeding, but because of the gene resource limitation, poor sexual hybridization affinity and the like, the traditional cross breeding technology has certain difficulty in cultivating high-yield and high-quality varieties. The plant somatic cell fusion technology can overcome the difficulty of poor distant hybridization affinity, and can open up a new way for screening new varieties of the Yangchun sand with high yield, high quality and strong stress resistance.
The successful separation and culture of protoplasts are the premise of somatic cell fusion, and no research reports exist at present about the relevant contents of the separation and purification of the amomum villosum protoplasts, and the factors influencing the yield and the activity of the protoplasts, such as the types and the concentrations of enzymes, the enzymolysis time, the osmotic pressure and the like involved in the separation and purification process are not clear. Therefore, the method needs to separate and purify the protoplast of the amomum villosum, screen and optimize experimental conditions so as to obtain the high-yield and high-activity amomum villosum protoplast and lay a foundation for cultivating new species of the amomum villosum through somatic cell fusion.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a method for separating and purifying the amomum villosum mesophyll protoplast, which is used for obtaining the high-yield and high-activity amomum villosum protoplast by screening and optimizing experimental conditions influencing the separation and purification of the amomum villosum mesophyll protoplast.
In order to achieve the purpose, the invention is realized by the following scheme:
a method for separating and purifying amomum villosum mesophyll protoplasts comprises the following steps:
s1, preparation and pretreatment of a material: preparing aseptic seedlings of amomum villosum, selecting healthy leaves with the leaf length of 30-40 mm, removing leaf veins, cutting the leaves into thin strips, and preparing the aseptic seedlings of amomum villosum according to the mass-to-volume ratio of 1 g: adding 15-30 mL of CPW solution containing 0.2-0.6M mannitol for pretreatment, and carrying out suspension culture for 1-3 h under the conditions of darkness, 26-30 ℃ and 80-150 r/min;
s2, separating protoplasts: adding the pretreated leaves into the pretreated leaves according to the mass volume ratio of 1 g: adding 10-25 mL of enzymolysis liquid, and carrying out oscillating enzymolysis for 8-13 h under the conditions of darkness, 25-27 ℃ and 30-90 r/min;
s3, purifying protoplasts: blowing and uniformly mixing the solution after enzymolysis, filtering the solution by using a 200-400-mesh cell sieve, and collecting filtrate; centrifuging the filtrate at 4 deg.C and 800r/min for 5min, and removing supernatant; suspending the protoplast by using 0.2-0.4 mL of CPW solution containing 0.5M mannitol, slowly adding 0.5-0.8 mL of 20% sucrose solution to the bottom of the solution, centrifuging at 4 ℃ and 800r/min for 5min, and collecting the protoplast zone at the interface of two phases; suspending the collected protoplast in 0.5-1.0 mL of 0.2M CaCl2•H2Centrifuging at 4 deg.C and 800r/min for 5min, and removing supernatant; adding protoplast culture solution, washing and precipitating, centrifuging at 4 deg.C and 800r/min for 5min, discarding supernatant, and collecting precipitate to obtain purified protoplast;
wherein, the enzymolysis solution in the step S2 contains the following components with final concentration: CPW solution containing 0.5M mannitol, 0.5-2.0% (w/v) cellulase and 0.5-1.0% (w/v) macerozyme; the pH value of the enzymolysis liquid is 5.8.
According to the invention, the experimental conditions for separating and purifying the leaf pulp protoplast of the amomum villosum are optimized by researching the factors influencing the yield and the activity of the protoplast of the amomum villosum, such as the selection of enzymolysis materials, whether the materials are pretreated, the types and the concentration of enzymes, the enzymolysis time, the concentration of an osmotic pressure regulator and the influence of different purification methods on the purification effect of the protoplast of the leaf pulp cells, so that the high-yield and high-activity amomum villosum protoplast is finally obtained.
Specifically, the invention selects tender leaves obtained from aseptic seedlings as enzymolysis materials, on one hand, the poison of sterilization reagents to leaves in sterilization can be avoided, and on the other hand, high-quality and high-yield protoplasts can be obtained more easily. The method adopts leaves with the leaf length of about 30-40 mm as a key link for dissociation and culture of the amomum villosum protoplast.
The invention finds that the yields of two groups of protoplasts obtained by direct enzymolysis without pre-wall separation and enzymolysis after pre-treatment have no significant difference, but the protoplasts of the group without pre-treatment have inferior dissociating effect and morphological integrity to the protoplasts of the group with pre-treatment. Therefore, the material is pretreated under the conditions of proper temperature and hypertonicity, and the method has a certain positive effect on the separation effect of the protoplast.
The invention researches the optimum enzyme type, concentration and enzymolysis condition of the amomum villosum protoplast, and finds that the enzymes required for dissociating and decomposing cell walls are different due to different plant cell walls and different structural compositions. The purpose of enzymatic digestion of protoplasts is to obtain the best results of protoplasts with the lowest enzyme concentration and the shortest digestion time. When the preparation method is used for carrying out enzymolysis on the sterile seedling leaves of the amomum villosum, the isolation enzyme R-10 and the cellulase R-10 are selected, when the mixture ratio of the enzyme mixed solution is 0.5-2.0% (w/v) of the cellulase and 0.5-1.0% (w/v) of the isolation enzyme, after oscillation enzymolysis is carried out for 8-13 hours under the conditions of 25-27 ℃ and 30-90R/min in the dark, the yield of the obtained protoplast is as high as 6.12 multiplied by 106 The activity of the protoplast is up to 81.21 percent per gram Fw. Further examining the effect of the ratio of the enzymes with different concentrations on the yield and viability of the protoplasts, it was found that the yield and viability of the protoplasts were the highest for each treatment when the ratio of the enzyme mixture solution was 1.5% (w/v) cellulase and 0.5% (w/v) macerase. It is speculated that high concentrations of cellulase may have a hydrolytic effect on free protoplasts, leading to reduced yield and viability. The invention inspects the enzymolysis time to find that the enzymolysis time is too short, the yield of the protoplast is low, the enzymolysis time is too long, and the yield and the activity of the protoplast are reduced on the contrary, so the optimal enzymolysis time is 10 h.
When the osmotic pressure concentration difference between the inside of the cell and the outside is too large, the protoplast expands or contracts, and cell membranes are ruptured, so that the cell death is caused; the protoplast can keep a spherical state by proper osmotic pressure. Therefore, the invention also preferably adjusts the concentration of the osmotic pressure regulator mannitol, and the result of the investigation test of the concentration of the mannitol shows that the 0.5M concentration of the mannitol can obtain protoplasts with higher yield and vitality.
The invention also compares the influence of three different purification methods, namely a sedimentation centrifugation method, a floating method and a method combining the sedimentation and the floating method, on the purification of the protoplast of the leaf flesh cells of the amomum villosum at the same time, and the result shows that the three methods can obviously reduce the content of impurity fragments in the protoplast suspension. Among them, the sedimentation method and the floating method are combined to achieve the best purification effect, the floating method is inferior in purification effect, and the simple sedimentation centrifugation method is the worst purification effect. Therefore, the invention adopts a method of combining sedimentation and drifting to purify the protoplast.
Preferably, the enzymolysis solution in step S2 contains the following components in final concentration: CPW solution containing 0.5M mannitol, 1.5% (w/v) cellulase, 0.5% (w/v) macerase.
Preferably, the preparation process of the enzymolysis solution in step S2 is as follows: adding cellulase and isolation enzyme into CPW solution containing 0.5M mannitol to constant volume, adjusting pH to 5.8 with 1M NaOH solution, filtering with 0.22 μ M pore size filter membrane, and sterilizing.
Preferably, the temperature of the enzymolysis in the step S2 is 25 ℃, and the time is 10 h.
Preferably, the cellulase is cellulase R-10 and the macerase is macerase R-10.
Preferably, the CPW solution in step S1 contains the following components in final concentrations: 101mg/L KNO3、27.2mg/L KH2PO4、0.025mg/L CuSO4·5H2O、0.16mg/L KI、246mg/L MgSO4·7H2O、1480mg/L CaCl2·2H2O; the pH of the CPW solution was 6.0.
Preferably, the protoplast culture solution in step S3 is MS minimal medium containing 0.2-0.6M mannitol.
As an alternative specific embodiment, the method for separating and purifying the amomum villosum mesophyll protoplast comprises the following specific steps:
s1, preparation and pretreatment of a material: inoculating the stem tip of a rhizome of the Yangchun sand long fruit cultivation type on an MS solid culture medium, inducing to obtain aseptic seedlings, performing light subculture for 2-3 times to obtain aseptic seedlings with strong green color and luxuriant leaves, and selecting healthy leaves with the leaf length of 30-40 mm for preparing protoplasts; removing veins of the sterile seedling leaves by using a sterilization blade, and cutting into thin strips with the thickness of about 0.5mm along the direction vertical to the veins, wherein the mass volume ratio of the thin strips is 1 g: adding a CPW solution containing 0.5M mannitol into 20mL of the suspension, performing pretreatment, and performing suspension culture for 1h at 28 ℃ and 120r/min in the dark to complete the separation of the pre-plasmoid wall;
s2, separating protoplasts: adding the pretreated leaves into the pretreated leaves according to the mass-to-volume ratio of 1 g: adding 15mL of enzymolysis solution, and carrying out shaking enzymolysis for 10h under the conditions of darkness, 25 ℃ and 60 r/min;
s3, purifying protoplasts: carefully blowing and uniformly mixing the solution after enzymolysis by using a shearing pipette tip to enable protoplasts to be dissociated in the enzymolysis solution, filtering by using a 200-400-mesh cell sieve, and collecting filtrate; centrifuging the filtrate at 4 deg.C and 800r/min for 5min, and removing supernatant; suspending the protoplast with 0.2mL of CPW solution containing 0.5M mannitol, slowly adding 0.6mL of 20% sucrose solution to the bottom of the solution in a centrifuge tube by using a long-needle syringe, centrifuging at 4 ℃ and 800r/min for 5min, and collecting the protoplast zone at the interface of two phases; the collected protoplasts were suspended in 0.8mL of 0.2M CaCl2•H2Centrifuging at 4 deg.C and 800r/min for 5min, and removing supernatant; adding protoplast culture solution, washing and precipitating, centrifuging at 4 deg.C and 800r/min for 5min, discarding supernatant, and collecting precipitate to obtain purified protoplast;
wherein, the enzymolysis solution in the step S2 contains the following components with final concentration: CPW solution containing 0.5M mannitol, 1.5% (w/v) cellulase, 0.5% (w/v) macerase; the pH of the enzymolysis liquid is 5.8.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the experimental conditions for separating and purifying the leaf-flesh protoplast of the amomum villosum are optimized by researching the factors influencing the yield and activity of the protoplast, such as whether the material is pretreated, the types and concentration of enzymes, the enzymolysis time, the concentration of an osmotic pressure regulator and the influence of different purification methods on the purification effect of the leaf-flesh cell protoplast, so that the amomum villosum protoplast with high yield and high activity is obtained, and a foundation is laid for cultivating new varieties of the amomum villosum through somatic cell fusion.
The method provided by the invention is simple and convenient to operate, the obtained amomum villosum protoplast has high yield and activity, the production cost is greatly saved, and the method has a wide application prospect.
Drawings
FIG. 1 shows the aphronychia foliata mesophyll cells before and after the pre-plasmolysis treatment; wherein, A is the control group of the Unpretreated Shaoyun leaf pulp cells, and B is the pretreated Shaoyun leaf pulp cells.
FIG. 2 shows the Yangchun sand mesophyll cells after enzymolysis; wherein, A is the control group of the Unpretreated Yangchun sand mesophyll cells, and B is the pretreated Yangchun sand mesophyll cells.
FIG. 3 is a photograph of the amomum villosum protoplasts (10X 10) before and after being stained with Evans Blue under an optical microscope; wherein, A is an undyed amomum villosum protoplast picture, and B is the actinolite protoplast picture dyed by Evans Blue.
FIG. 4 is the effect of enzymolysis time on the protoplast separation effect; wherein the lines in the graph represent the viability of the protoplasts and the bar graph represents the yield of the protoplasts.
FIG. 5 is the effect of mannitol concentration in the enzymatic hydrolysate on the protoplast separation effect; wherein the lines in the graph represent the viability of the protoplasts and the bar graph represents the yield of the protoplasts.
FIG. 6 shows the comparison of the purification of the Ascomfrey mesophyll protoplasts by different methods; wherein, A is unpurified mesophyll protoplast, B is mesophyll protoplast purified by sedimentation and centrifugation, C is mesophyll protoplast purified by floating method, and D is mesophyll protoplast purified by combining sedimentation and drifting method.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
And (3) determining the yield and activity of the protoplast:
a large number of light green globules with complete morphology, plump shape and clear internal tissue are observed under a microscope, counted by a blood counting chamber, each sample is counted for 3 times, and an average value is taken. Adjusting the density of the suspension to 5X 10 according to the measurement result5one/mL. And (4) detecting by adopting an Evans blue staining method. When the activity is measured, 20 mu L of purified protoplast is taken, 5 mu L of 1% Evans blue is added, and the protoplast which is not dyed into blue is observed and detected under an optical microscope, namely the protoplast with the activity. Counting by adopting a blood counting plate, counting the number of the undyed protoplasts and the total number of the protoplasts in each visual field, repeating each sample for 3 times, counting 5 visual fields in each repetition, and taking an average value.
Protoplast yield (pieces/g) = [ number of protoplasts isolated (pieces/mL) × volume after dilution (mL) ]/fresh weight of isolated leaf material (g)
Protoplast viability (%) = number of unstained protoplasts/total number of observed protoplasts × 100%
All data was analyzed using Excel 2003 and SPSS 21.0 data processing systems.
Example 1 method for separating and purifying amomum villosum mesophyll protoplast
The embodiment provides a method for separating and purifying amomum villosum mesophyll protoplasts, which comprises the following specific steps:
s1, preparation and pretreatment of a material: inoculating the stem tip of a rhizome of the Yangchun sand long fruit cultivation type on an MS solid culture medium, inducing to obtain aseptic seedlings, performing light subculture for 2-3 times to obtain aseptic seedlings with strong green color and luxuriant leaves, and selecting healthy leaves with the leaf length of 30-40 mm for preparing protoplasts; removing veins of the sterile seedling leaves by using a sterilization blade, and cutting into thin strips with the thickness of about 0.5mm along the direction vertical to the veins, wherein the mass volume ratio of the thin strips is 1 g: adding a CPW solution containing 0.5M mannitol into 20mL of the suspension, performing pretreatment, and performing suspension culture for 1h at 28 ℃ and 120r/min in the dark to complete the separation of the pre-plasmoid wall;
s2, separating protoplasts: adding the pretreated leaves into the pretreated leaves according to the mass-to-volume ratio of 1 g: adding 15mL of enzymolysis solution, and carrying out shaking enzymolysis for 10h under the conditions of darkness, 25 ℃ and 60 r/min;
s3, purifying protoplasts: carefully blowing and uniformly mixing the solution after enzymolysis by using a shearing pipette tip to enable protoplasts to be dissociated in the enzymolysis solution, filtering by using a 200-400-mesh cell sieve, and collecting filtrate; centrifuging the filtrate at 4 deg.C and 800r/min for 5min, and removing supernatant; suspending the protoplast with 0.2mL of CPW solution containing 0.5M mannitol, slowly adding 0.6mL of 20% sucrose solution to the bottom of the solution in a centrifuge tube by using a long-needle syringe, centrifuging at 4 ℃ and 800r/min for 5min, and collecting the protoplast zone at the interface of two phases; the collected protoplasts were suspended in 0.8mL of 0.2M CaCl2•H2Centrifuging at 4 deg.C and 800r/min for 5min, and removing supernatant; adding protoplast culture solution, washing and precipitating, centrifuging at 4 deg.C and 800r/min for 5min, discarding supernatant, and collecting precipitate to obtain purified protoplast;
wherein, the enzymolysis solution in the step S2 contains the following components with final concentration: CPW solution containing 0.5M mannitol, 1.5% (w/v) cellulase, 0.5% (w/v) macerase; the pH value of the enzymolysis liquid is 5.8. The preparation process of the enzymolysis liquid is as follows: adding cellulase and isolation enzyme into CPW solution containing 0.5M mannitol to constant volume, adjusting pH to 5.8 with 1M NaOH solution, filtering with 0.22 μ M pore size filter membrane, and sterilizing.
The CPW solution in step S1 contained the following final concentrations of the components: 101mg/L KNO3、27.2mg/L KH2PO4、0.025mg/L CuSO4·5H2O、0.16mg/L KI、246mg/L MgSO4·7H2O、1480mg/L CaCl2·2H2O; the pH of the CPW solution was 6.0.
And in the step S3, the protoplast culture solution is an MS minimal medium containing 0.2-0.6M mannitol.
The invention researches factors influencing the yield and activity of the amomum villosum protoplast, such as the selection of enzymolysis materials, whether the materials are pretreated, the types and concentration of enzymes, the enzymolysis time, the concentration of an osmotic pressure regulator and the influence of different purification methods on the purification effect of the mesophyll cell protoplast, optimizes the experimental conditions of the separation and purification of the amomum villosum mesophyll protoplast, and the specific research process is shown in the embodiment 2-6.
Example 2 Effect of pretreatment on protoplast isolation Effect
1. Experimental groups: taking sterile leaf of amomum villosum, tearing off the epidermis or cutting the leaf into 0.5mm slices by using a pair of tweezers, weighing 1g of the leaf into a triangular flask containing 20mL of CPW solution containing 13 percent mannitol, and performing suspension culture for 1h at 28 ℃ and 120r/min in a dark place to complete the separation of the pre-plasmoid wall. Control group: without pretreatment. The aphronychia foliata mesophyll cells before and after the pre-plasmolysis treatment are shown in FIG. 1.
2. And (3) respectively placing the experimental group and the control group in the step 1 into 15mL of enzymolysis liquid for enzymolysis. And after enzymolysis for 10h, the protoplast is respectively placed under a microscope to observe the yield and the activity of the protoplast. The results are shown in FIG. 2 and show that: the yields of two groups of protoplasts obtained by direct enzymolysis without pre-wall separation and enzymolysis after pre-treatment are not obviously different, but the protoplasts in the group without pre-treatment have the dissociating effect and the morphological integrity inferior to those in the group with pre-treatment. Therefore, the pretreatment of the material under the conditions of proper temperature and hypertonicity has a certain positive effect on the separation effect of the protoplast.
Example 3 Effect of different enzymatic hydrolysates on protoplast yield and viability
Cutting the sterile leaf of amomum villosum into 0.5mm, respectively putting the cut leaves into each enzymolysis liquid combination (table 1), carrying out low-speed oscillation enzymolysis for 10h in the dark, and observing the separation condition of protoplast under a microscope to determine the optimal enzyme concentration combination. The results are shown in table 1 and fig. 3. The results show that protoplasts can be obtained by separating combinations of cellulase and macerozyme at different concentrations. When the concentration of the isolation enzyme is 0.5%, the yield and the vitality of the protoplast have concentration effect on the cellulase, and when the concentration of the cellulase is 1.5%, the yield and the vitality of the protoplast are highest and are 6.12 multiplied by 106individual/g.Fw and 81.21%. When the concentration of the eductase is increased to 1.0 percent, the yield and the activity of the protoplast are the same as those of the protoplastThe concentration effect on cellulase appeared, and the peak still appeared at a cellulase concentration of 1.5%, but the activity of protoplasts was significantly reduced compared to the enzyme solution with 0.5% of the concentration of the isolated enzyme. Therefore, the composition of the enzymatic hydrolysate from which protoplasts were efficiently obtained was determined to be 1.5% cellulase +0.5% macerase. High concentrations of cellulase may have a hydrolytic effect on free protoplasts, resulting in reduced yield and viability.
Note 1: the above 8 kinds of enzymatic hydrolysate each contain 0.5M mannitol CPW solution, pH5.8, and are sterilized by filtration through a microporous filter with pore size of 0.22 μ M. Performing enzymolysis for 10h, and centrifuging for 5min at 1000 r.
Note 2: the data in the same column and different English letters are represented by Duncan,The difference is obvious by the s new complex pole difference method, and the following method is the same.
Example 4 Effect of enzymolysis time on protoplast yield and viability
In order to examine the influence of enzymolysis time on the yield and activity of protoplasts, enzymolysis solution with 1.0% cellulase +0.5% eductase was selected, and the leaf of amomum villosum was subjected to dark enzymolysis for 8, 9, 10, 11, 12 and 13 hours, respectively. The results are shown in FIG. 4. When enzymolysis is carried out for 8 hours, a large amount of protoplasts are found to be separated from the amomum villosum leaf blocks, and the number of the protoplasts is gradually increased along with the increase of time. The yield of protoplast is 6.12 multiplied by 10 hours when the enzymolysis is carried out for 10 hours6The cells/g-Fw, the activity was 81.13%. The protoplast yield then decreases as the enzymatic time increases. The protoplast activity and the enzymolysis time are also in a parabolic relation, and the highest peak of the activity is 11h and is 84.21 percent. The enzymolysis time is very important for separating the yield and the activity of the protoplast, if the enzymolysis time is too short, not only are materials and enzymolysis liquid wasted, but also the yield of the protoplast is low; however, the enzymolysis time is too long, and the yield and the activity are reduced.
With longer enzymolysis time, cell debris in the solution increases. From 11h of enzymolysis, the fragments are obviously increased. The optimal enzymolysis time is determined to be 10h by comprehensively considering three factors of the protoplast yield, the protoplast activity and the cell fragment number.
Example 5 Effect of mannitol concentration in enzymatic hydrolysate on protoplast yield and viability
After determining the kind and concentration of the enzymes in the enzymatic hydrolysate, the concentration of mannitol needs to be screened to maintain the optimal osmotic pressure. Adding 7%, 9% and ll% osmotic pressure regulator mannitol into mixed enzyme solution composed of CPW, 1.0% cellulase R-10 and 0.5% macerozyme R-l0, and performing dark enzymolysis for 10h to determine the yield and activity of protoplast. The results are shown in FIG. 5. Under different mannitol concentrations, the yield and the activity of the protoplast are obviously different. Within the range of mannitol concentration of 7% -9%, the separating effect of the mannitol concentration and the protoplast presents a parabolic variation trend, under the condition of lower mannitol concentration, the yield and the activity of the protoplast are both lower, and the yield and the activity of the protoplast are both improved along with the gradual increase of the mannitol concentration. When the concentration of mannitol is 9%, the yield and the activity reach the maximum value, and are respectively 6.08X 106g, Fw and 80.4 percent, and the obtained protoplast has basically consistent shape and size, clear edges, more inclusions and less fragments. The mannitol concentration is continuously improved, and the yield and the activity of the protoplast are obviously reduced. Therefore, the mannitol concentration in the enzymatic hydrolysate is preferably 9% (0.5M).
Example 6 Effect of different purification methods on the purification Effect of mesophyllic cell protoplasts
In the embodiment, the influence of the sedimentation centrifugation method, the floating method and the combination of the sedimentation method and the floating method on the purification of the leaf pulp cell protoplast of the amomum villosum are compared, and the purification effects of the sedimentation centrifugation method, the floating method and the combination of the sedimentation method and the floating method are compared. The method comprises the following specific steps:
and after the fine leaf strips are subjected to enzymolysis for 8-13 h, carefully sucking and releasing the enzymolysis solution for several times by using a shearing pipette tip to enable the protoplast to be dissociated in the enzymolysis solution, filtering by using a stainless steel screen mesh of 200-400 meshes, and collecting filtrate for later use.
(1) Purification of protoplasts by sedimentation
The filtrate was centrifuged at 800r/min at 4 ℃ for 5 min. The supernatant was carefully pipetted off and discarded, and the protoplasts were resuspended in 0.2mL of a CPW solution containing 0.5M mannitol, centrifuged again, the supernatant removed, and the process repeated 3 times. And finally, washing the protoplast by using a protoplast culture solution for 1 time, adjusting the protoplast to a certain density by using the protoplast culture solution, and then culturing.
(2) Floating method for purifying protoplast
Centrifuging the filtrate at 4 deg.C and 1000r/min for 5min, removing supernatant, and collecting precipitate (containing protoplast). Sucking about 3mL of 20% sucrose solution by using a narrow-mouth pipette, carefully inserting the pipette into the bottom of a centrifuge tube containing a protoplast suspension, floating the protoplast with strong vitality between 20% sucrose and 13% CPW, and sinking the broken cell residues into the bottom of the tube. The well-conditioned protoplasts were gently aspirated by a 200 μ L pipette (care was taken not to aspirate the lower layer of banana sugar solution as much as possible), placed in another clean centrifuge tube, 4mL of 13% CPW wash was added, centrifuged at 1000r/min for 5min, and the supernatant was discarded.
(3) Method for purifying protoplast by combining sedimentation method and drifting method
The filtrate was centrifuged at 800r/min at 4 ℃ for 5 min. Carefully sucking the supernatant with a pipette, discarding, suspending the protoplast with 0.2mL of CPW solution containing 0.5M mannitol, slowly adding 0.6mL of 20% sucrose solution to the bottom of the solution in the centrifuge tube with a long needle syringe, centrifuging at 4 ℃ and 800r/min for 5min, and collecting the protoplast zone at the interface of the two phases; the collected protoplasts were suspended in 0.8mL of 0.2M CaCl2•H2Centrifuging at 4 deg.C and 800r/min for 5min, and removing supernatant; adding protoplast culture solution, washing and precipitating, centrifuging at 4 deg.C and 800r/min for 5min, discarding supernatant, and collecting precipitate to obtain purified protoplast.
The results are shown in fig. 6 and show that: the three methods can obviously reduce the content of impurity fragments in the protoplast suspension. Among them, the purification effect by the combination of the sedimentation and the drift method is the best, the purification effect by the floating method is the second best, and the purification effect by the simple sedimentation centrifugation method is the worst. Thus, it was determined that protoplasts were purified by a combination of sedimentation and drift.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (5)
1. A method for separating and purifying amomum villosum mesophyll protoplasts is characterized by comprising the following steps:
s1, preparation and pretreatment of the material: preparing aseptic seedlings of amomum villosum, selecting healthy leaves with the leaf length of 30-40 mm, removing leaf veins, cutting the leaves into thin strips, and preparing the aseptic seedlings of amomum villosum according to the mass-to-volume ratio of 1 g: adding 15-30 mL of CPW solution containing 0.2-0.6M mannitol for pretreatment, and carrying out suspension culture for 1-3 h under the conditions of darkness, 26-30 ℃ and 80-150 r/min;
s2, separating protoplast: adding the pretreated leaves into the pretreated leaves according to the mass-to-volume ratio of 1 g: adding 10-25 mL of enzymolysis liquid, and carrying out oscillating enzymolysis for 8-13 h under the conditions of darkness, 25-27 ℃ and 30-90 r/min;
s3 purification of protoplasts: blowing and uniformly mixing the solution after enzymolysis, filtering the solution by using a 200-400-mesh cell sieve, and collecting filtrate; centrifuging the filtrate at 4 deg.C and 800r/min for 5min, and removing supernatant; suspending the protoplast by using 0.2-0.4 mL of CPW solution containing 0.5M mannitol, slowly adding 0.5-0.8 mL of 20% sucrose solution to the bottom of the solution, centrifuging at 4 ℃ and 800r/min for 5min, and collecting a protoplast zone at the interface of two phases; suspending the collected protoplast in 0.5-1.0 mL of 0.2M CaCl2•H2Centrifuging at 4 deg.C and 800r/min for 5min, and removing supernatant; adding protoplast culture solution, washing and precipitating, centrifuging at 4 deg.C and 800r/min for 5min, discarding supernatant, and collecting precipitate to obtain purified protoplast;
wherein, the enzymolysis liquid in the step S2 consists of the following components with final concentration: CPW solution containing 0.5M mannitol, 0.5-2.0% (w/v) cellulase R-10, 0.5-1.0% (w/v) macerozyme R-10; the pH value of the enzymolysis liquid is 5.8;
and in the step S3, the protoplast culture solution is an MS minimal medium containing 0.2-0.6M mannitol.
2. The method as claimed in claim 1, wherein the enzymolysis solution in step S2 contains the following components in final concentrations: CPW solution containing 0.5M mannitol, 1.5% (w/v) cellulase, 0.5% (w/v) macerase.
3. The method as claimed in claim 1, wherein the preparation process of the enzymolysis solution in step S2 is as follows: adding cellulase and isolation enzyme into CPW solution containing 0.5M mannitol to constant volume, adjusting pH to 5.8 with 1M NaOH solution, filtering with 0.22 μ M pore size filter membrane, and sterilizing.
4. The method of claim 1, wherein the temperature of the enzymolysis in step S2 is 25 ℃ and the time is 10 h.
5. The method of claim 1, wherein the CPW solution in step S1 comprises the following final concentrations of each component: 101mg/L KNO3、27.2mg/L KH2PO4、0.025mg/L CuSO4·5H2O、0.16mg/L KI、246mg/L MgSO4·7H2O、1480mg/L CaCl2·2H2O; the pH of the CPW solution was 6.0.
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| CN112251395A (en) * | 2020-10-29 | 2021-01-22 | 西南大学 | Separation method of loquat protoplast |
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