CN102505004B - Extraction and fusion method for strawberry protoplast - Google Patents
Extraction and fusion method for strawberry protoplast Download PDFInfo
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- CN102505004B CN102505004B CN2011103156107A CN201110315610A CN102505004B CN 102505004 B CN102505004 B CN 102505004B CN 2011103156107 A CN2011103156107 A CN 2011103156107A CN 201110315610 A CN201110315610 A CN 201110315610A CN 102505004 B CN102505004 B CN 102505004B
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Abstract
The invention provides an optimal isolation and fusion method for strawberry protoplasts, which utilizes strawberry leaves as research objects. According to the method, the protoplasts with high activity are obtained with both the pre-treatment method and the enzymolysis method, and 30-45% polyethylene glycol (PEG) 6000 is combined with high Ca<2+> method and high pH method so as to induce one-to-one protoplast fusion with improved probability. The method disclosed by the invention has the advantages of mild action, more than 85% of the activity of the isolated protoplast, one-to-one fusion rate up to 11.7%, simple operation, and possibility of forming hybrid cell. The method is suitable for inducing and culturing the new breed by overcoming distant hybridization barrier, and has certain significance in the biological breeding aspect.
Description
Technical field
The present invention relates to the plant species improvement technical field, i.e. a kind of extraction of strawberry protoplast and fusion method.
Background technology
The Fragaria Rosaceae (Rosaceae) Fragaria (Fragaria) plant, the perennial herb fruit tree, its economic worth and nutritive value are very high, and the plantation scope is wide.Since the eighties in 20th century, strawberry production has just had development rapidly, most kinds on China's strawberry production are drawn from external, variet complexity, and the ubiquity resistance is poor and quality under the degradation problem, the kind of real suitable China Different climate condition cultivation is few, the improved seeds that this just cultivates in the urgent need to the suitable China of seed selection.
At present, both at home and abroad the Strwberry Breeding protoplastis that mainly concentrates on cell suspension culture, the strawberry of stem tip culture, anther culture, somatic mutants screening, research on strawberry genetic engineering, the strawberry of strawberry is cultivated and cytogamy etc., and the application modern biotechnology is the important means of strawberry cultivars improvement.China aspect some of strawberry modern biotechnology breeding research (as the acquisition of haplobiont, the regeneration of transformed plant etc.) be in international most advanced level, and existence larger gap compared with going together abroad in many aspects, and the screening of resistance somatic mutants, protoplastis are cultivated and the gene transformation research of strawberry still belongs to blank.Along with further improving and the development of biotechnology of strawberry tissue culture technique, the problem that solves weather, freeze injury, insect pest will more be hopeful.
The protoplastis operation is the important component part in the modern biotechnology field, for Strwberry Breeding work opens up a new way.Hybrid between can producing kind, belong to by protoplast fusion, thus overcome the distant hybirdization obstacle, and in addition, protoplastis can also be used to separate and the purified mutant body.Height heterozygosity in strawberry heredity, karyomit(e) polyploidy and abundant fraises des bois resource, make the protoplastis operation become a kind of important means of Strwberry Breeding.
Summary of the invention
The object of the invention is to set up efficient strawberry protoplast separation, integration technology system, to improve the protoplastis vigor, fusion efficiencies, extraction and the fusion method of for New Strawberry Variety, cultivating the strawberry protoplast that technical support is provided.
For achieving the above object, the technical solution used in the present invention is: a kind of extraction of strawberry protoplast and fusion method is characterized in that step is as follows:
(1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, or secretly cultivate a week; Described MS substratum contains 6-benzyladenine (6-BA) 1.0-1.5mg/L, sucrose 1-1.5% and agar 0.7%;
(2) getting the pretreated blade of step (1) is cut into slice and removes vein; Described slice is 2.0mm * 2.0mm.
(3) material is mixed according to 1: 10 volume ratio with enzyme liquid, with the Parafilm sealing, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature, speed 60r/min condition, time 11-14h, and timing Microscopic observation enzymolysis situation; Described enzyme liquid consists of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 1.0-2.0% cellulase Cellulase Onozuka R-10+0.5-1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination.
(4) the mesophyll protoplast suspension of greenery east strawberry and rupee strawberry is respectively after 200 order nylon gauzes filter, centrifugal, remove supernatant liquor completely by enzymolysis respectively, the protoplastis of precipitation is suspended in to the CaCl of 2ml0.2mol/L
2In, with pipettor to centrifuge tube bottom slowly implantation quality concentration be centrifugal after 20% sucrose solution, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 6ml 0.2mol/L
2(removing impurity, purifying) suspends;
Described timing Microscopic observation enzymolysis situation is that enzyme liquid digested after 7 hours, every one hour, under inverted microscope, observes.
The protoplastis of purifying between the described interface that is taken at two phase liquid is with the precipitation at the bottom of the pipettor draft tube and the CaCl on sucrose solution and top
2Solution.
(5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean; Protoplastis concentration is 0.5 * 10
5Individual/mL, when six orifice plates dripped, every was 0.1mL.
(6) protoplastis of two kinds of strawberries is mixed with 1: 1 volume ratio, with the protoplastis that pipettor will mix, drop in 6 orifice plates, standing 10min, make protoplastis be attached to 6 orifice plate bottoms, observes its form;
(7) draw PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min observes the clustering phenomena of protoplastis, and adds up the shared percentage of aggregate, and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Described PEG solution composition is 30-45%PEG 6000+0.3mM glucose+8mM Cacl
2+ 0.7mMKH
2PO
4, it is 5.8 that KOH adjusts pH.
Described high Ca
2+, high pH washing lotion is 50mM CaCl
22H
2The O+0.2M glycine, it is 9-10 that NaOH adjusts pH.
The bright advantage of this is: the present invention is take the blade of strawberry as research material, the best approach that provides a kind of strawberry protoplast to separate and merge, and the protoplastis that the mode that combines by pre-treatment and enzymolysis obtains high vigor, utilize PEG in conjunction with high Ca
2+, high pH method induces both to merge, and has improved the probability that protoplastis merges one to one mutually.Present method action temperature and, the protoplastis vigor that separates is more than 85%, fusion rate can reach 11.7% one to one, easy and simple to handle, possessed the possibility that forms hybrid cell, be suitable for overcoming the distant hybridization obstacle and carry out inducing and cultivating of new variety, aspect Biology Breeding, have certain meaning.
Below in conjunction with embodiment, embodiments of the present invention are described in further detail.
The accompanying drawing explanation
Fig. 1 tentatively obtains the more protoplastis figure of impurity.
Fig. 2 is the strawberry protoplast figure that obtains after purifying.
Fig. 3 adds the protoplastis figure that assembles after PEG.
Fig. 4 is the cellular form figure after merging.
Embodiment
Embodiment 1
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, the MS substratum contains 6-benzyladenine (6-BA) 1.0mg/L, sucrose 1.5% and agar 0.7%;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 volume ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table at 26 ℃ of constant temperature, under speed 60r/min condition, carry out dark enzymolysis, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 1.0% cellulase Cellulase Onozuka R-10+0.5% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 14 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with 1: 1 volume ratio, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 30%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min observes the clustering phenomena of protoplastis, and adds up the shared percentage of aggregate; Interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 90%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10.4%.Referring to Fig. 1,2,3,4.
Embodiment 2
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, the MS substratum contains 6-benzyladenine (6-BA) 1.5mg/L, sucrose 1.5% and agar 0.7%;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 2.0% cellulase Cellulase Onozuka R-10+0.5% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 12 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 40%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 85%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10.7%.
Embodiment 3
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet dark cultivation a week under 26 ± 1 ℃ of conditions;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 1.0% cellulase Cellulase Onozuka R-10+1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 13 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 45%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 87%, and under inverted microscope, observing and calculate man-to-man fusion rate is 11.7%.
Embodiment 4
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet dark cultivation a week under 26 ± 1 ℃ of conditions;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 2.0% cellulase Cellulase Onozuka R-10+1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 11 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 45%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 89%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10.8%.
Embodiment 5
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, the MS substratum contains 6-benzyladenine (6-BA) 1.0mg/L, sucrose 1.5% and agar 0.7%;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 1.0% cellulase Cellulase Onozuka R-10+1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 13 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 30%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 88%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10.1%.
Embodiment 6
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, the MS substratum contains 6-benzyladenine (6-BA) 1.0mg/L, sucrose 1.5% and agar 0.7%;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 2.0% cellulase Cellulase Onozuka R-10+1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 11 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 40%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 85%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10.9%.
Embodiment 7
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet dark cultivation a week under 26 ± 1 ℃ of conditions;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 1.0% cellulase Cellulase Onozuka R-10+0.5% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 14 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 45%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 91%, and under inverted microscope, observing and calculate man-to-man fusion rate is 10%.
Embodiment 8
The extraction of strawberry protoplast and fusion method, step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet dark cultivation a week under 26 ± 1 ℃ of conditions;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to 1: 10 ratio with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table and carry out dark enzymolysis under 26 ℃ of constant temperature low speed (60r/min) condition, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the CaCl of N.F,USP MANNITOL+0.1-0.2%2-(N-morpholine) ethyl sulfonic acid MES+0.05mol/L of 2.0% cellulase Cellulase Onozuka R-10+1.0% polygalacturonase Pectolyase Y-23+0.6mol/L
2Combination, the enzymic digestion time is 11 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the ratio of 1: 1, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min left and right makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 40%PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min left and right, observe the clustering phenomena of protoplastis, and add up the shared percentage of aggregate. and interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling;
Trypan blue staining identifies that the activity of protoplastis is 87%, and under inverted microscope, observing and calculate man-to-man fusion rate is 11.2%.
Claims (1)
1. the extraction of a strawberry protoplast and fusion method is characterized in that step is as follows:
1) get respectively greenery east strawberry and rupee strawberry test-tube plantlet and move into the MS culture medium culturing 10 days that has reduced sucrose concentration, the MS substratum contains 6-benzyladenine (6-BA) 1.0mg/L, sucrose 1.5% and agar 0.7%;
2) get step 1) pretreated blade is cut into 2.0mm * 2.0mm size in small, broken bits little and removes vein with scissors;
3) material is mixed according to volume ratio 1:10 with enzyme liquid, with Parafilm, seal, the masking foil sealing, be placed on shaking table at 26 ℃ of constant temperature, under speed 60r/min condition, carry out dark enzymolysis, after 7 hours, every one hour Microscopic observation enzymolysis situation, enzyme liquid consisted of the N.F,USP MANNITOL of 1.0 % cellulase Cellulase Onozuka R-10+0.5% polygalacturonase Pectolyase Y-23+0.6mol/L+0.1-0.2% 2-(N-morpholine) CaCl of ethyl sulfonic acid MES+0.05mol/L
2Combination, the enzymic digestion time is 14 hours;
4) two kinds of strawberry mesophyll protoplast suspension are after 200 order nylon gauzes filter completely by enzymolysis, and the centrifugal 5min of the rotating speed of 600R/min, make the protoplastis precipitation, remove supernatant liquor, the protoplastis of precipitation is suspended in to the CaCl of 2ml 0.2mol/L
2In. with pipettor, to centrifuge tube bottom, slowly inject after 20% sucrose solution centrifugally, be taken at the protoplastis band of purifying between the interface of two phase liquid, with the CaCl of 3ml 0.2mol/L
2Suspend;
5) adopt trypan blue staining to identify the activity of protoplastis, triplicate is taken the mean, and adjusting protoplastis density is 0.5 * 10
5Individual/mL;
6) protoplastis of two kinds of strawberries is mixed with the 1:1 volume ratio, with the protoplastis that pipettor will mix, drop in 6 orifice plates, every is 0.1mL, and standing 10min makes protoplastis be attached to 6 orifice plate bottoms, observes its form;
7) draw 30% PEG solution, equal-volume drips on every protoplastis, rotates gently 6 orifice plates, and it is mixed, and static 10min observes the clustering phenomena of protoplastis, and adds up the shared percentage of aggregate; Interval 5min adds high Ca successively wherein
2+, high pH washing lotion washing, the fusion process of observing the cell mass of assembling.
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| CN103039356A (en) * | 2013-01-18 | 2013-04-17 | 通化师范学院 | Year-round strawberry hybridizing and breeding method |
| CN104762295A (en) * | 2015-03-18 | 2015-07-08 | 岭南师范学院 | Preparation method of acid-resistant salt-resistant facultative anaerobic bacillus |
| CN105647905B (en) * | 2015-12-08 | 2019-01-15 | 浙江万里学院 | The method for obtaining grape hybrid plant using protoplast asymmetric fusion technology |
| CN107964531B (en) * | 2016-10-19 | 2019-08-27 | 天津师范大学 | Duckweed living body method for preparing protoplast |
| CN107964530B (en) * | 2016-10-19 | 2019-08-27 | 天津师范大学 | Duckweed protoplast preparation method |
| CN107964529B (en) * | 2016-10-19 | 2021-03-02 | 天津师范大学 | Method for preparing Xiajugao grassland protoplast |
| CN114214264B (en) * | 2021-11-24 | 2024-02-02 | 浙江大学 | Strawberry protoplast separation and purification and gene transient expression method |
| CN115521897A (en) * | 2022-09-22 | 2022-12-27 | 鲁东大学 | Method for extracting protoplast from forest strawberry callus |
| CN115873782A (en) * | 2022-12-23 | 2023-03-31 | 中国农业科学院蔬菜花卉研究所 | Enzymolysis liquid, kit and method for preparing cabbage plant protoplast |
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