CN103461124A - Tissue culture and rapid propagation process for seedless golden silk jujube - Google Patents
Tissue culture and rapid propagation process for seedless golden silk jujube Download PDFInfo
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- CN103461124A CN103461124A CN2013104176889A CN201310417688A CN103461124A CN 103461124 A CN103461124 A CN 103461124A CN 2013104176889 A CN2013104176889 A CN 2013104176889A CN 201310417688 A CN201310417688 A CN 201310417688A CN 103461124 A CN103461124 A CN 103461124A
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Abstract
The invention discloses a tissue culture and rapid propagation process for seedless golden silk jujube, belongs to the technical field of agricultural biology, and relates to a plant tissue culture and rapid propagation technology. The process comprises formula matching for culture media at various stages, a preparation method for the culture media at various stages, and the tissue culture and rapid propagation process. By the establishment of the tissue culture and rapid propagation technology system, the propagation speed of the seedless golden silk jujube in Leling is greatly increased, the occurrence proportion of weak seedlings and disease seedlings is reduced, the popularization, the development and the utilization of new varieties are accelerated, domestic blank is filled, and the annual propagation coefficient can reach 2,000 to 15,000.
Description
Technical field
The invention belongs to agricultural biological technical field, relate to a kind of plant tissue culture fast breeding technique.
Background technology
Dezhou City is planted the little jujube history of existing more than 3000 year, and good cultivation tradition and higher culture technique are arranged, and has become one of center of the little jujube production of China.Leling jijithus in jujube producing region, Dezhou, be described as one of " Dezhou Triratna ", is Chinese gardening tradition famous product, well-known with high yield of fine quality.Because its sugar content is high, nutritious, convenient processing and edible, and extremely people's favor, supply falls short of demand in market, is well sold and in short supply kind international, domestic market always.Golden silk jujube is treasure in jujube especially, is one of the three large pillar ofs the economy in Dezhou, and people are increasing to the demand of Golden silk jujube, but its reproduction speed and output can not need by satisfying the market far away.
At present, Golden silk jujube still adopts the modes such as traditional root turion bar plant division, cuttage, grafting to breed, and reproduction rate is very low, and growth rate is slow, and weak sick seedling ratio is high, has directly restricted spread and the fast Development utilization of seedless golden jujube improved seeds.Therefore, the high power of Golden silk jujube is bred, Rapid Popularization becomes problem demanding prompt solution.
Summary of the invention
The purpose of this invention is to provide a kind of seedless golden jujube tissue-culturing rapid propagation technique, to solve, under prior art, the numerous coefficient of Golden silk jujube root is low, reproduction speed slow, can not meet grower's problems such as demand.
The technical solution adopted for the present invention to solve the technical problems is to provide the fast numerous technique of a kind of seedless golden jujube training, this technique comprises Plant Tissue Breeding stages medium compound method, condition of culture, a whole set of group culturation rapid propagating technology system of hardening domesticating method, the tissue-culturing rapid propagation of little jujube is selected the MS medium, and adding hormonal substance has BA(6-benzyl ammonia purine), the IAA(heteroauxin), the IBA(indolebutyric acid), the NAA(naa).
Its stages culture medium prescription (quality proportioning) is as follows:
First culture formula: MS+BA0.1～1.0mgL
-1+ IBA0.1～1.0mgL
-1+ sucrose 25～35gL
-1+ agar 6～8gL
-1.
Subculture is cultivated formula: MS+BA1.0～3.0mgL
-1+ NAA0.1～1.0mgL
-1+ sucrose 25～35gL
-1+ agar 6～8gL
-1.
Culture of rootage formula: 1/2MS+IAA0.1～1.0 mgL
-1+ IBA0.1～1.0 mgL
-1+ sucrose 15～25gL
-1+ agar 6～8gL
-1.
Stages medium compound method is as follows:
1. the just preparation of culture base
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its MS mother liquor that melts → get 10 times of 1.0L fully and 6-benzyl aminopurine, indolebutyric acid dissolving in pot → constant volume to 10L → pH value 5.7～6.1 → packing → carry out 115～121 ℃ of high temperature, high pressure 0.12～0.15Mpa, sterilizing 15～20 minutes → be cooled to solid → sterilized in the inoculation of transfer room superclean bench explant → proceeding to culturing room carries out aseptic culture.
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx, and the regular sterilization of attention culturing room.
2, the preparation of subculture medium
By above-mentioned raw materials proportioning weighing agar powder, add in the pot of dress 10L pure water boil → weighing sucrose to add in pot to add after it melts → get 1.0 L10 MS mother liquor doubly and 6-benzyl aminopurine, naa dissolving fully in pot → constant volume to 5.9～6.3 → packing of 10L → pH value → carry out 115～121 ℃ of high temperature, high pressure 0.12～0.15Mpa, solid → in the inoculation of transfer room superclean bench, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 15～20 minutes → be cooled to.
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx, note the regular sterilization of culturing room.Every 30～40 days subcultures once.
3, the preparation of root media
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its 1/2MS mother liquor that melts → get 10 times of 1.0L fully and heteroauxin, indolebutyric acid dissolving in pot → constant volume and carry out 115～121 ℃ of high temperature to 10L → pH value 5.9～6.3 → packing, high pressure 0.12～0.15Mpa, solid → at transfer room superclean bench inoculation individual plant, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 15～20 minutes → be cooled to.
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 2000～3500lx, note the regular sterilization of culturing room.
Its tissue-culturing rapid propagation processing step is as follows:
A, first culture
In 3～April, clip is given birth to high-quality, sturdy root turion bar then, and in culturing room, water planting is cultivated; 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx.Within 8～10 days, grow the water planting bud of 1.5～2cm stalwartness.Clip water planting bud, as explant, under aseptic condition, is first used 70～75% alcohol surface sterilization 30～60s, aseptic water washing 1～2 time; Put into again 0.1% mercuric chloride solution (adding several Tween 80s) and soak 8～10min, during constantly rock, then use aseptic water washing 5～6 times, cut the contacted wound of liquid.The water planting bud of 1.5～2.5cm is proceeded to 0.3～0.7cm in the first culture base of high-temperature sterilization, carry out aseptic culture in culturing room.
B, subculture are cultivated
The plants stems section of first culture is transferred into the subculture medium through high-temperature sterilization, 26～30 ℃ of cultivation temperature, intensity of illumination 2000～3500lx, light application time 14～18hd
-1, subculture cycle 35～40 days.
C, culture of rootage
The plants stems section that subculture is cultivated is transferred into the root media through high-temperature sterilization, 26～30 ℃ of cultivation temperature, intensity of illumination 2000～3500lx, light application time 14～18hd
-1.
The transplanting of D, rooting tube plantlet
Transplant in first 5～10 days and open gradually bottleneck, the air humidity in blake bottle is reduced gradually, test-tube plantlet progressively adapts to external environment; Take out gently test-tube plantlet with tweezers during transplanting, clean the root medium, be transplanted in the seedling-cultivating tray (4cm * 4cm) or pot for growing seedlings (d>=4cm) that matrix is housed, matrix is the peat composed of rotten mosses+rural area soil+vermiculite (1～2:1～2:2～4), and the front matrix of transplanting is 800～1000 times of carbendazim or 0.1%KMnO for palpus
4spray disinfectant.Water immediately permeablely after transplanting, and use the plastic foil moisturizing, relative moisture 90～98%, 26～28 ℃ of temperature.Ventilate gradually after one week, finally remove plastic foil, within every 7～10 days, spray one time of nutrition liquid, after planting, within 30～35 days, carry out outdoor transplanting.
Adopt good effect of the present invention to be: the foundation of this group culturation rapid propagating technology system has greatly improved the reproduction speed of Golden silk jujube, reduced the proportion of weak seedling, sick seedling, Rapid Popularization and the exploitation of new varieties have been accelerated, filled up domestic blank, made year reproduction coefficient can reach 2000～15000.
Embodiment
Embodiment 1
With the first culture base of preparation 10L, the 10L subculture medium, the 10L root media is example, its culture medium raw material formula and quality proportioning are:
First culture base: the MS that minimal medium is 10 times, 10 milligrams, 6-benzyl ammonia purine, 5 milligrams of indolebutyric acids, sucrose 270 grams, agar 75 grams;
Subculture medium: the MS that minimal medium is 10 times, 25 milligrams, 6-benzyl ammonia purine, 3 milligrams of naas, sucrose 280 grams, agar 65 grams;
Root media: the 1/2MS that minimal medium is 10 times, 8 milligrams of heteroauxins, 8 milligrams of indolebutyric acids, sucrose 170 grams, agar 68 grams.
The preparation method is as follows for its medium:
1. the just preparation of culture base
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its MS mother liquor that melts → get 10 times of 1.0L fully and 6-benzyl aminopurine, indolebutyric acid dissolving in pot → constant volume to 10L → 5.9 → packing → carry out 121 ℃ of high temperature, high pressure 0.125Mpa, sterilizing 20 minutes → be cooled to solid → sterilized in the inoculation of transfer room superclean bench explant → proceeding to culturing room carries out aseptic culture.
Culturing room's condition: 27 ℃ of cultivation temperature, light application time 15hd
-1, intensity of illumination 2500lx, and the regular sterilization of attention culturing room.
2, the preparation of subculture medium
By above-mentioned raw materials proportioning weighing agar powder, add in the pot of dress 10L pure water boil → weighing sucrose to add in pot to add after it melts → get 1.0 L10 MS mother liquor doubly and 6-benzyl aminopurine, naa dissolving fully in pot → constant volume to the 6.2 → packing of 10L → pH value → carry out high temperature of 120 DEG C, high pressure 0.125Mpa, solid → in the inoculation of transfer room superclean bench, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 20 minutes → be cooled to.
Culturing room's condition: 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, note the regular sterilization of culturing room.Every 35 days subcultures once.
3, the preparation of root media
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its 1/2MS mother liquor that melts → get 10 times of 0.5L fully and heteroauxin, indolebutyric acid dissolving in pot → constant volume and carry out 118 ℃ of high temperature to 10L → pH value 6.0 → packing packing, high pressure 0.125Mpa, solid → at transfer room superclean bench inoculation individual plant, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 20 minutes → be cooled to.
Culturing room's condition: 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, note the regular sterilization of culturing room.
Its tissue-culturing rapid propagation processing step is as follows:
A, first culture
In March, clip is given birth to high-quality, sturdy root turion bar then, and in culturing room, water planting is cultivated; 27 ℃ of cultivation temperature, light application time 15hd
-1, intensity of illumination 2500lx.Within 8 days, grow the water planting bud of 1.6cm stalwartness.Clip water planting bud, as explant, under aseptic condition, is first used 70% alcohol surface sterilization 45s, aseptic water washing 2 times; Put into again 0.1%HgCl
2soak 9min in solution (adding several Tween 80s), during constantly rock, then use aseptic water washing 6 times, cut the contacted wound of liquid.The water planting bud of 1.6cm is proceeded to the 0.4cm in the first culture base of high-temperature sterilization, carry out aseptic culture in culturing room.
B, subculture are cultivated
The plants stems section of first culture is transferred into the subculture medium through high-temperature sterilization, 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, subculture cycle 35 days.
C, culture of rootage
The plants stems section that subculture is cultivated is transferred into the root media through high-temperature sterilization, 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1.
The transplanting of D rooting tube plantlet
Transplant in first 6 days and open gradually bottleneck, the air humidity in blake bottle is reduced gradually, test-tube plantlet progressively adapts to external environment; Take out gently test-tube plantlet with tweezers during transplanting, clean the root medium, be transplanted in the seedling-cultivating tray (4cm * 4cm) that matrix is housed, matrix is the peat composed of rotten mosses+rural area soil+vermiculite (1.5:1.5:3.5), the front matrix of transplanting 800 times of carbendazim spray disinfectants for palpus.Water immediately permeablely after transplanting, and use the plastic foil moisturizing, relative moisture 95%, 27 ℃ of temperature.Ventilate gradually after one week, finally remove plastic foil, within every 8 days, spray one time of nutrition liquid, plant and within latter 32 days, carry out outdoor transplanting.
Embodiment 2
With the first culture base of preparation 10L, the 10L subculture medium, the 10L root media is example, its culture medium raw material formula and quality proportioning are:
First culture base: the MS that minimal medium is 10 times, 5 milligrams, 6-benzyl ammonia purine, 6 milligrams of indolebutyric acids, sucrose 300 grams, agar 72 grams;
Subculture medium: the MS that minimal medium is 10 times, 20 milligrams, 6-benzyl ammonia purine, 2 milligrams of naas, sucrose 300 grams, agar 72 grams;
Root media: the 1/2MS that minimal medium is 10 times, 6 milligrams of heteroauxins, 5 milligrams of indolebutyric acids, sucrose 230 grams, agar 70 grams.
The preparation method is as follows for its medium:
1. the just preparation of culture base
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its MS mother liquor that melts → get 10 times of 1.0L fully and 6-benzyl aminopurine, indolebutyric acid dissolving in pot → constant volume to 10L → adjust 118 ℃ of high temperature of 5.7 values → packing → carry out, high pressure 0.14Mpa, sterilizing 17 minutes → be cooled to solid → sterilized in the inoculation of transfer room superclean bench explant → proceeding to culturing room carries out aseptic culture.
Culturing room's condition: 28 ℃ of cultivation temperature, light application time 16hd
-1, intensity of illumination 1700lx, and the regular sterilization of attention culturing room.
2, the preparation of subculture medium
By above-mentioned raw materials proportioning weighing agar powder, add in the pot of dress 10L pure water boil → weighing sucrose to add in pot to add after it melts → get 1.0 L10 MS mother liquor doubly and 6-benzyl aminopurine, naa dissolving fully in pot → constant volume to the 6.0 → packing of 10L → pH value → carry out high temperature of 120 DEG C, high pressure 0.14Mpa, solid → in the inoculation of transfer room superclean bench, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 19 minutes → be cooled to.
Culturing room's condition: 30 ℃ of cultivation temperature, intensity of illumination 3200lx, light application time 14hd
-1, note the regular sterilization of culturing room.Every 35 days subcultures once.
3, the preparation of root media
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its 1/2MS mother liquor that melts → get 10 times of 0.5L fully and heteroauxin, indolebutyric acid dissolving in pot → constant volume and carry out 121 ℃ of high temperature to 10L → adjustment pH value → packing packing, high pressure 0.14Mpa, solid → at transfer room superclean bench inoculation individual plant, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 20 minutes → be cooled to.
Culturing room's condition: 29 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, note the regular sterilization of culturing room.
Its tissue-culturing rapid propagation processing step is as follows:
A, first culture
In April, clip is given birth to high-quality, sturdy root turion bar then, and in culturing room, water planting is cultivated; 28 ℃ of cultivation temperature, light application time 16hd
-1, intensity of illumination 2600lx.Within 9 days, grow the water planting bud of 1.8cm stalwartness.Clip water planting bud, as explant, under aseptic condition, is first used 70% alcohol surface sterilization 60s, aseptic water washing 1 time; Put into again 0.1%HgCl
2soak 10min in solution (adding several Tween 80s), during constantly rock, then use aseptic water washing 5 times, cut the contacted wound of liquid.The water planting bud of 1.8cm is proceeded to the 0.3cm in the first culture base of high-temperature sterilization, carry out aseptic culture in culturing room.
B, subculture are cultivated
The plants stems section of first culture is transferred into the subculture medium through high-temperature sterilization, 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, subculture cycle 37 days.
C, culture of rootage
The plants stems section that subculture is cultivated is transferred into the root media through high-temperature sterilization, 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1.
The transplanting of D rooting tube plantlet
Transplant in first 6 days and open gradually bottleneck, the air humidity in blake bottle is reduced gradually, test-tube plantlet progressively adapts to external environment; Take out gently test-tube plantlet with tweezers during transplanting, clean the root medium, be transplanted in the pot for growing seedlings (d>=4cm) that matrix is housed, matrix is the peat composed of rotten mosses+rural area soil+vermiculite (1.8:1.8:3.3), and before transplanting, matrix must be used 0.1%KMnO
4spray disinfectant.Water immediately permeablely after transplanting, and use the plastic foil moisturizing, relative moisture 95%, 27 ℃ of temperature.Ventilate gradually after one week, finally remove plastic foil, within every 7 days, spray one time of nutrition liquid, plant and within latter 33 days, carry out outdoor transplanting.
Embodiment 3
With the first culture base of preparation 10L, the 10L subculture medium, the 10L root media is example, its culture medium raw material formula and quality proportioning are:
First culture base: the MS that minimal medium is 10 times, 2 milligrams, 6-benzyl ammonia purine, 1 milligram of indolebutyric acid, sucrose 320 grams, agar 78 grams;
Subculture medium: the MS that minimal medium is 10 times, 11 milligrams, 6-benzyl ammonia purine, 1.3 milligrams of naas, sucrose 320 grams, agar 65 grams;
Root media: the 1/2MS that minimal medium is 10 times, 2 milligrams of heteroauxins, 2 milligrams of indolebutyric acids, sucrose 230 grams, agar 77 grams.
The preparation method is as follows for its medium:
1. the just preparation of culture base
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its MS mother liquor that melts → get 10 times of 1.0L fully and 6-benzyl aminopurine, indolebutyric acid dissolving in pot → constant volume to the 5.9 → packing of 10L → pH value → carry out 116 ℃ of high temperature, high pressure 0.12Mpa, sterilizing 20 minutes → be cooled to solid → sterilized in the inoculation of transfer room superclean bench explant → proceeding to culturing room carries out aseptic culture.
Culturing room's condition: 27 ℃ of cultivation temperature, light application time 15hd
-1, intensity of illumination 2500lx, and the regular sterilization of attention culturing room.
2, the preparation of subculture medium
By above-mentioned raw materials proportioning weighing agar powder, add in the pot of dress 10L pure water boil → weighing sucrose to add in pot to add after it melts → get 1.0 L10 MS mother liquor doubly and 6-benzyl aminopurine, naa dissolving fully in pot → constant volume to the 6.3 → packing of 10L → pH value → carry out 119 ℃ of high temperature, high pressure 0.125Mpa, solid → in the inoculation of transfer room superclean bench, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 20 minutes → be cooled to.
Culturing room's condition: 28 ℃ of cultivation temperature, intensity of illumination 3300lx, light application time 18hd
-1, note the regular sterilization of culturing room.Every 32 days subcultures once.
3, the preparation of root media
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its 1/2MS mother liquor that melts → get 10 times of 0.5L fully and heteroauxin, indolebutyric acid dissolving in pot → constant volume and carry out 121 ℃ of high temperature to 10L → adjustment pH value 6.3 → packing packing, high pressure 0.125Mpa, solid → at transfer room superclean bench inoculation individual plant, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 20 minutes → be cooled to.
Culturing room's condition: 26 ℃ of cultivation temperature, intensity of illumination 2200lx, light application time 15hd
-1, note the regular sterilization of culturing room.
Its tissue-culturing rapid propagation processing step is as follows:
A, first culture
In March, clip is given birth to high-quality, sturdy root turion bar then, and in culturing room, water planting is cultivated; 29 ℃ of cultivation temperature, light application time 15hd
-1, intensity of illumination 2500lx.Within 10 days, grow the water planting bud of 2cm stalwartness.Clip water planting bud, as explant, under aseptic condition, is first used 70% alcohol surface sterilization 55s, aseptic water washing 2 times; Put into again 0.1%HgCl
2soak 8min in solution (adding several Tween 80s), during constantly rock, then use aseptic water washing 5 times, cut the contacted wound of liquid.The water planting bud of 2cm is proceeded to the 0.6cm in the first culture base of high-temperature sterilization, carry out aseptic culture in culturing room.
B, subculture are cultivated
The plants stems section of first culture is transferred into the subculture medium through high-temperature sterilization, 27 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1, subculture cycle 38 days.
C, culture of rootage
The plants stems section that subculture is cultivated is transferred into the root media through high-temperature sterilization, 28 ℃ of cultivation temperature, intensity of illumination 2500lx, light application time 15hd
-1.
The transplanting of D rooting tube plantlet
Transplant in first 9 days and open gradually bottleneck, the air humidity in blake bottle is reduced gradually, test-tube plantlet progressively adapts to external environment; Take out gently test-tube plantlet with tweezers during transplanting, clean the root medium, be transplanted in the seedling-cultivating tray (4cm * 4cm) or pot for growing seedlings (d >=4cm) that matrix is housed, matrix is the peat composed of rotten mosses+rural area soil+vermiculite (2:2:2.5), the front matrix of transplanting 800 times of carbendazim spray disinfectants for palpus.Water immediately permeablely after transplanting, and use the plastic foil moisturizing, relative moisture 98%, 27 ℃ of temperature.Ventilate gradually after one week, finally remove plastic foil, within every 8 days, spray one time of nutrition liquid, plant and within latter 34 days, carry out outdoor transplanting.
Annotate: h-hour, Lx-lux, min-minute, s – second, d-days, cm-centimetre, L-liter, g-gram, mg-milligram.
Attached: tissue-culturing rapid propagation technique and traditional handicraft comparison sheet
Annotate: the year reproduction coefficient of tissue-culturing rapid propagation (it is example that the subculture of take is cultivated every bottle of 6 strains)=annual subculture coefficient (6 of explant survival rate (22%) *
6(subculture cycle number)) * cultivation robust plant rate (80%) * domestication survival rate (80%)=6671.
Claims (3)
1. a seedless golden jujube tissue-culturing rapid propagation technique, it is characterized in that this technique comprises Plant Tissue Breeding stages medium compound method, condition of culture, a whole set of group culturation rapid propagating technology system of hardening domesticating method, the tissue-culturing rapid propagation of little jujube is selected the MS medium, and adding hormonal substance has BA(6-benzyl ammonia purine), the IAA(heteroauxin), the IBA(indolebutyric acid), the NAA(naa); Its stages culture medium prescription (quality proportioning) is as follows:
First culture formula: MS+BA0.1～1.0mgL
-1+ IBA0.1～1.0mgL
-1+ sucrose 25～35gL
-1+ agar 6～8gL
-1;
Subculture is cultivated formula: MS+BA1.0～3.0mgL
-1+ NAA0.1～1.0mgL
-1+ sucrose 25～35gL
-1+ agar 6～8gL
-1;
Culture of rootage formula: 1/2MS+IAA0.1～1.0 mgL
-1+ IBA0.1～1.0 mgL
-1+ sucrose 15～25gL
-1+ agar 6～8gL
-1.
2. the described stages medium of a seedless golden jujube tissue-culturing rapid propagation technique compound method is characterized in that:
A is the preparation of culture base just
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its MS mother liquor that melts → get 10 times of 1.0L fully and 6-benzyl aminopurine, indolebutyric acid dissolving in pot → constant volume to 10L → pH value 5.7～6.1 → packing → carry out 115～121 ℃ of high temperature, high pressure 0.12～0.15Mpa, sterilizing 15～20 minutes → be cooled to solid → sterilized in the inoculation of transfer room superclean bench explant → proceeding to culturing room carries out aseptic culture;
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx, and the regular sterilization of attention culturing room;
The preparation of B subculture medium
By above-mentioned raw materials proportioning weighing agar powder, add in the pot of dress 10L pure water boil → weighing sucrose to add in pot to add after it melts → get 1.0 L10 MS mother liquor doubly and 6-benzyl aminopurine, naa dissolving fully in pot → constant volume to 5.9～6.3 → packing of 10L → pH value → carry out 115～121 ℃ of high temperature, high pressure 0.12～0.15Mpa, solid → in the inoculation of transfer room superclean bench, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 15～20 minutes → be cooled to;
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx, note the regular sterilization of culturing room, every 30～40 days subcultures are once;
The preparation of C root media
By above-mentioned raw materials proportioning weighing agar powder, add boil → weighing sucrose in the pot of dress 10L pure water to add in pot to add after its 1/2MS mother liquor that melts → get 10 times of 1.0L fully and heteroauxin, indolebutyric acid dissolving in pot → constant volume and carry out 115～121 ℃ of high temperature to 10L → pH value 5.9～6.3 → packing, high pressure 0.12～0.15Mpa, solid → at transfer room superclean bench inoculation individual plant, aseptic seedling → proceeding to culturing room carries out aseptic culture for sterilizing 15～20 minutes → be cooled to;
Culturing room's condition: 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 2000～3500lx, note the regular sterilization of culturing room.
3. described its tissue-culturing rapid propagation processing step of seedless golden jujube tissue-culturing rapid propagation technique is as follows:
A, first culture
In 3～April, clip is given birth to high-quality, sturdy root turion bar then, and in culturing room, water planting is cultivated; 26～30 ℃ of cultivation temperature, light application time 14～18hd
-1, intensity of illumination 1500～3500lx, grow the water planting bud of 1.5～2cm stalwartness in 8～10 days; Clip water planting bud, as explant, under aseptic condition, is first used 70～75% alcohol surface sterilization 30～60s, aseptic water washing 1～2 time; Put into again 0.1% mercuric chloride solution (adding several Tween 80s) and soak 8～10min, during constantly rock, then use aseptic water washing 5～6 times, cut the contacted wound of liquid; The water planting bud of 1.5～2.5cm is proceeded to 0.3～0.7cm in the first culture base of high-temperature sterilization, carry out aseptic culture in culturing room;
B, subculture are cultivated
The plants stems section of first culture is transferred into the subculture medium through high-temperature sterilization, 26～30 ℃ of cultivation temperature, intensity of illumination 2000～3500lx, light application time 14～18hd
-1, subculture cycle 35～40 days;
C, culture of rootage
The plants stems section that subculture is cultivated is transferred into the root media through high-temperature sterilization, 26～30 ℃ of cultivation temperature, intensity of illumination 2000～3500lx, light application time 14～18hd
-1;
The transplanting of D, rooting tube plantlet
Transplant in first 5～10 days and open gradually bottleneck, the air humidity in blake bottle is reduced gradually, test-tube plantlet progressively adapts to external environment; Take out gently test-tube plantlet with tweezers during transplanting, clean the root medium, be transplanted in the seedling-cultivating tray (4cm * 4cm) or pot for growing seedlings (d>=4cm) that matrix is housed, matrix is the peat composed of rotten mosses+rural area soil+vermiculite (1～2:1～2:2～4), and the front matrix of transplanting is 800～1000 times of carbendazim or 0.1%KMnO for palpus
4spray disinfectant, water permeablely immediately after transplanting, and use the plastic foil moisturizing, relative moisture 90～98%, 26～28 ℃ of temperature; Ventilate gradually after one week, finally remove plastic foil, within every 7～10 days, spray one time of nutrition liquid, after planting, within 30～35 days, carry out outdoor transplanting.
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| CN103766220A (en) * | 2014-01-30 | 2014-05-07 | 北京林业大学 | Method for establishing efficient regeneration system of Chahu date |
| CN104082146A (en) * | 2014-07-14 | 2014-10-08 | 北京林业大学 | Method for inducing polyploid through wild jujube adventitious buds |
| CN105993945A (en) * | 2016-05-24 | 2016-10-12 | 象州县科学技术局 | Tissue culture rapid propagation method of Zaocuiwang jujubes |
| CN106386477A (en) * | 2016-05-24 | 2017-02-15 | 象州县科学技术局 | Tissue culture and rapid propagation method for Ziziphus jujube Mill.cv.Jinsi 4 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766220A (en) * | 2014-01-30 | 2014-05-07 | 北京林业大学 | Method for establishing efficient regeneration system of Chahu date |
| CN104082146A (en) * | 2014-07-14 | 2014-10-08 | 北京林业大学 | Method for inducing polyploid through wild jujube adventitious buds |
| CN104082146B (en) * | 2014-07-14 | 2016-04-27 | 北京林业大学 | A kind of method of wild jujube indefinite bud approach induction polyploid |
| CN105993945A (en) * | 2016-05-24 | 2016-10-12 | 象州县科学技术局 | Tissue culture rapid propagation method of Zaocuiwang jujubes |
| CN106386477A (en) * | 2016-05-24 | 2017-02-15 | 象州县科学技术局 | Tissue culture and rapid propagation method for Ziziphus jujube Mill.cv.Jinsi 4 |
| CN105993945B (en) * | 2016-05-24 | 2018-04-17 | 象州县科学技术局 | A kind of tissue culture and rapid propagation method of morning crisp king's jujube |
| CN106386477B (en) * | 2016-05-24 | 2018-05-29 | 象州县科学技术局 | A kind of tissue culture and rapid propagation method of Jujube 4 |
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