CN107006371B - A kind of walnut tissue culture and rapid propagation method for eliminating tissue-cultured seedling yellow - Google Patents
A kind of walnut tissue culture and rapid propagation method for eliminating tissue-cultured seedling yellow Download PDFInfo
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- CN107006371B CN107006371B CN201710311958.6A CN201710311958A CN107006371B CN 107006371 B CN107006371 B CN 107006371B CN 201710311958 A CN201710311958 A CN 201710311958A CN 107006371 B CN107006371 B CN 107006371B
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- 235000009496 Juglans regia Nutrition 0.000 title claims abstract description 54
- 235000020234 walnut Nutrition 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 16
- 240000007049 Juglans regia Species 0.000 title description 42
- 230000012010 growth Effects 0.000 claims abstract description 22
- 238000009395 breeding Methods 0.000 claims abstract description 16
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 230000001488 breeding effect Effects 0.000 claims abstract description 14
- 241000758789 Juglans Species 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 13
- 239000007921 spray Substances 0.000 claims abstract description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 11
- 241000607479 Yersinia pestis Species 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 8
- 230000006698 induction Effects 0.000 claims abstract description 6
- 230000008569 process Effects 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 238000005286 illumination Methods 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 230000006872 improvement Effects 0.000 claims description 10
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 claims description 5
- 238000013475 authorization Methods 0.000 claims description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000006870 ms-medium Substances 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- IXVMHGVQKLDRKH-QHBHMFGVSA-N 24-epi-brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-QHBHMFGVSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims 2
- 239000004471 Glycine Substances 0.000 claims 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims 1
- 229930003451 Vitamin B1 Natural products 0.000 claims 1
- ICSSIKVYVJQJND-UHFFFAOYSA-N calcium nitrate tetrahydrate Chemical compound O.O.O.O.[Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ICSSIKVYVJQJND-UHFFFAOYSA-N 0.000 claims 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims 1
- 239000000413 hydrolysate Substances 0.000 claims 1
- 229960000367 inositol Drugs 0.000 claims 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 claims 1
- 229960003512 nicotinic acid Drugs 0.000 claims 1
- 235000001968 nicotinic acid Nutrition 0.000 claims 1
- 239000011664 nicotinic acid Substances 0.000 claims 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 claims 1
- 229960003495 thiamine Drugs 0.000 claims 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims 1
- 235000010374 vitamin B1 Nutrition 0.000 claims 1
- 239000011691 vitamin B1 Substances 0.000 claims 1
- 235000019158 vitamin B6 Nutrition 0.000 claims 1
- 239000011726 vitamin B6 Substances 0.000 claims 1
- 229940011671 vitamin b6 Drugs 0.000 claims 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims 1
- 230000004069 differentiation Effects 0.000 abstract description 8
- 238000012258 culturing Methods 0.000 abstract description 4
- 208000006278 hypochromic anemia Diseases 0.000 abstract description 3
- 230000000384 rearing effect Effects 0.000 abstract description 3
- 230000009467 reduction Effects 0.000 abstract description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 8
- 229940088594 vitamin Drugs 0.000 description 8
- 229930003231 vitamin Natural products 0.000 description 8
- 235000013343 vitamin Nutrition 0.000 description 8
- 239000011782 vitamin Substances 0.000 description 8
- 150000003722 vitamin derivatives Chemical class 0.000 description 8
- 230000008901 benefit Effects 0.000 description 5
- 235000006040 Prunus persica var persica Nutrition 0.000 description 4
- 240000005809 Prunus persica Species 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004161 plant tissue culture Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 244000144725 Amygdalus communis Species 0.000 description 1
- 235000011437 Amygdalus communis Nutrition 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 244000226021 Anacardium occidentale Species 0.000 description 1
- 244000147058 Derris elliptica Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000758791 Juglandaceae Species 0.000 description 1
- 235000013757 Juglans Nutrition 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 235000020226 cashew nut Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- ZOOODBUHSVUZEM-UHFFFAOYSA-N ethoxymethanedithioic acid Chemical compound CCOC(S)=S ZOOODBUHSVUZEM-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 239000012991 xanthate Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of walnut tissue culture and rapid propagation methods for eliminating tissue-cultured seedling yellow, including propagation material selection, explant processing, the induction of initial bud and Multiplying culture process；The walnut spray tip of acquisition current growth stalwartness and no disease and pests harm is as propagation material, be trimmed to stem section as explant to branch, it induces initial bud to occur to being inoculated in initial bud inducement cultivation base after the processing of explant sterilization, then obtains the tissue culture subculture bud of no aetiolation after initial bud is inoculated in proliferated culture medium.The present invention obtain aseptic seedling on the basis of explore walnut tissue-culturing rapid propagation seedling raising process in squamous subculture when there is chlorosis yellow, the normal differentiation and growth of walnut tissue-cultured seedling are seriously affected, it proposes a solution so as to cause the problem of growth coefficient reduction, eliminate walnut tissue-cultured seedling aetiolation, energy normal differentiation and growth, breeding coefficient is improved, inhereditary feature is stablized, so that walnut tissue-cultured seedling be enable to reach the standard of industrial seedling rearing.
Description
Technical field
The invention belongs to field of plant tissue culture technique, are related to a kind of walnut tissue-culturing rapid propagation side for eliminating tissue-cultured seedling yellow
Method.
Background technique
Walnut(Juglans regia L.)Gu claims English walnut, belongs to Juglandaceae, and juglans originates in W. Asia and Asia
Western part, China are one of walnut source areas.Since walnut is important nut and woody oil tree species, there is very high economic valence
Value, occupies first of the big dry fruit in the world four (walnut, almond, cashew nut, fibert), is existed with its very high economic, ecology and social benefit
Cultivation extensively all over the world.Walnut forms many high yields, high-quality and disease resistance in cultivated since long and artificial selection course
Good excellent Walnut Cultivars, in order to be able to maintain the merit of these kinds, offspring's breeding of walnut is mainly using grafting
With the vegetative propagation means of cuttage, but use propagation by grafiting, it is low that there are breeding coefficients, and speed is slow, and survival rate is unstable, by fringe bar
The unfavorable factors such as quantity limitation；Using cuttage breeding method, since, there are a large amount of tannin inhibiting substances, cuttage is not in walnut body
It easily takes root, expanding rapidly for the excellent variety for resulting in walnut new in this way is restricted.Plant tissue culture quick-breeding method is also current
One of main method of asexual propagation of seedlings, its main feature is that accelerating while regeneration plant can be made to keep maternal inheritance stability
Reproduction speed, several sterile budlets can breed million plants or more of nursery stock in 1 year, and the production cycle greatly shortens, can compared with
A large amount of high quality seedlings are cultivated in short time, and can establish large-scale production.Due to the superiority of plant tissue culture technology, China and foreign countries are learned
Person carries out the research of tissue rapid propagation technical aspect to walnut since late 1960s and makes some progress, but core
Some key technologies in peach tissue culture squamous subculture all fail to be solved very well for many years, as walnut easily goes out in squamous subculture
Existing chlorosis yellow, xanthate ratio are up to 95% or more, the normal differentiation and growth of walnut tissue-cultured seedling have been seriously affected, so as to cause increasing
It grows coefficient to substantially reduce, so that walnut tissue-cultured seedling industrial seedling rearing is difficult to realize always.
Summary of the invention
The invention proposes lose when one kind overcomes squamous subculture in walnut tissue-culturing rapid propagation seedling raising process in the prior art
Greenish-yellowization has seriously affected the normal differentiation and growth of walnut tissue-cultured seedling, so as to cause the deficiency that growth coefficient reduces, proposes
One kind can eliminate walnut tissue-cultured seedling aetiolation, energy normal differentiation and growth, improve breeding coefficient, stablize disappearing for inhereditary feature
Except the walnut tissue culture and rapid propagation method of tissue-cultured seedling yellow.
To achieve the goals above, technical scheme is as follows:
A kind of walnut tissue culture and rapid propagation method for eliminating tissue-cultured seedling yellow, including propagation material selection, explant handle, are initial
Bud induction and Multiplying culture process；The walnut spray tip of acquisition current growth stalwartness and no disease and pests harm is as propagation material, to branch
Item be trimmed to stem section as explant, induces being inoculated in initial bud inducement cultivation base after the processing of explant sterilization
Initial bud occurs, then the tissue culture subculture bud of no aetiolation is obtained after initial bud is inoculated in proliferated culture medium；Primary operational
Steps are as follows:
(1) propagation material selects: improved variety of walnut clone, the age of tree 5~8 that selection passes through national or provincial breeding authorization
Year life is scion-seed tree；In the top acquisition current growth stalwartness of scion-seed tree tree crown periphery and the walnut spray of no disease and pests harm
The tip, the excellent propagation material as walnut tissue culture；
(2) explant is handled: the collected walnut spray tip being cut into and only retains the stem of terminal bud or 2~3cm long with axillary bud
Duan Zuowei explant impregnates 30 s of sterilizing to explant with 75% ethanol solution of volumetric concentration, by explant after distilled water flushing 2 times
Body is placed in super-clean bench, then with volumetric concentration 0.1%HgCl2Solution carries out sterilization treatment 8~10 to explant in oscillating fashion
Min is finally used aseptic water washing 5 times；
(3) initial bud induction: the explant that step (2) processing obtains is inoculated in induced medium, temperature is placed in
25 ± 2 DEG C, 2000~3500LX of intensity of illumination, initial bud is induced in the environment of 10~14 h/d of illumination to occur；
(4) Multiplying culture: being inoculated in proliferated culture medium for the initial bud that step (3) obtain, be placed in 25 ± 2 DEG C of temperature,
2000~3500LX of intensity of illumination carries out Multiplying culture in the environment of 10~14 h/d of illumination, obtains the tissue culture of no aetiolation
Subculture bud.
The raw material components and mass content of induced medium described in above step (2) are as follows: improvement MS+6-BA 0.5
mg·L-130000 mg L of+sucrose-1300 mg L of+lactoalbumin hydrolysate-10.2 mg L of+epi-brassinolide-1+ agar 3000
mg•L-1。
The raw material components and mass content of proliferated culture medium described in above step (3) are as follows: improvement MS+6-BA 1.0~
2.0 mg·L-130000 mg L of+sucrose-1300~500 mg L of+lactoalbumin hydrolysate-1+ epi-brassinolide 0.3~0.5
mg•L-13000 mg L of+agar-1。
The composition of above-described modified MS medium are as follows: KNO3 1600 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2 350 mg·L-1；MgSO4.7H2O 370 mg·L-1；Ca(NO3)2. 4 H2O 100 mg•L-1；KH2PO3 270 mg•
L-1；MnSO4.4 H2O 22.3 mg•L-1；ZnSO4.7 H2O 10.6 mg•L-1；CuSO4.5 H2O 0.025 mg•L-1；H3BO3
8.2 mg•L-1；Na2MoO4.2 H2O 0.25 mg•L-1；KI 0.83 mg•L-1；CoCl2.6 H2O 0.025 mg•L-1；Na2•
EDTA 37.3 mg•L-1；FeSO4.7 H2O 27.8 mg•L-1；0.4 mg L of vitamin B1-1；0.5 mg L of vitamin B6-1；2.0 mg L of niacin-1；1.0 mg L of glycine-1；120 mg L of inositol-1。
Compared with the existing technology, the present invention have the advantage that and the utility model has the advantages that
1, the present invention improves the content and ratio of the nutriment of traditional MS minimal medium, is added to the newborn egg of hydrolysis
It is existing to completely eliminate the yellow that walnut tissue-cultured seedling occurs during tissue culture for white and two kinds of chemical additives of epi-brassinolide
As enabling tissue-cultured seedling normal differentiation and growth, proliferation times increase 3~4.5 times, cultivate the good of a large amount of high-quality health in a short time
Kind nursery stock, with good economic efficiency, social benefit and ecological benefits.
2, the present invention obtain aseptic seedling on the basis of explore walnut tissue-culturing rapid propagation seedling raising process in squamous subculture when go out
Existing chlorosis yellow, has seriously affected the normal differentiation and growth of walnut tissue-cultured seedling, mentions so as to cause the problem of growth coefficient reduction
Solution out eliminates walnut tissue-cultured seedling aetiolation, energy normal differentiation and growth, improves breeding coefficient, stablizes inhereditary feature,
To enable walnut tissue-cultured seedling to reach the standard of industrial seedling rearing.
Specific embodiment
The present invention will be further described combined with specific embodiments below.
Embodiment 1:
In the continuous weather to clear up 2 days or more, age of tree life in 5~8 years is selected, by the core of national or provincial breeding authorization
Peach breeding clone is as scion-seed tree；Top acquisition current growth in scion-seed tree tree crown periphery is healthy and strong and no disease and pests harm
The walnut spray tip, the excellent propagation material as walnut tissue culture.
The collected walnut spray tip is cut into and only retains the stem section of terminal bud or 2~3cm long with axillary bud as explant, is used
75% ethanol solution of volumetric concentration impregnates 30 s of sterilizing to explant, and explant is placed in super-clean bench after distilled water flushing 2 times, then
With volumetric concentration 0.1%HgCl2Solution carries out 8~10 min of sterilization treatment to explant in oscillating fashion, finally with sterile
Water rinses 5 times.
The explant that sterilization is handled is inoculated in induced medium, is placed in 25 ± 2 DEG C of temperature, illumination is strong
2000~2500LX is spent, initial bud is induced to occur in the environment of 14 h/d of illumination.The raw material components of the induced medium and
Mass content are as follows: improvement 0.5 mgL of MS+6-BA-130000 mg L of+sucrose-1300 mg L of+lactoalbumin hydrolysate-1+ table oil
0.2 mg L of rape lactone-13000 mg L of+agar-1。
Axillary bud starts to sprout after cultivating 8 d, culture 35 d buds grow tall 3~5 cm when, bud is cut from former stem section, be inoculated with
In proliferated culture medium, the raw material components and mass content of proliferated culture medium are as follows: improvement 1.0~2.0 mgL of MS+6-BA-1+ sugarcane
30000 mg L of sugar-1300 mg L of+lactoalbumin hydrolysate-10.3 mg L of+epi-brassinolide-13000 mg L of+agar-1, and
25 ± 2 DEG C of temperature, 2000~2500LX of intensity of illumination are placed in, Multiplying culture is carried out in the environment of 14 h/d of illumination, promotes culture
The axillary bud and lateral bud of object are sprouted.About 35d switching is primary, cultivates 35 d, and average bud is up to 4.2 cm, the dark green no Huang of nursery stock leaf color
Change, growth coefficient 3.8.
The composition of above-described modified MS medium are as follows: KNO3 1600 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2 350 mg·L-1；MgSO4.7H2O 370 mg·L-1；Ca(NO3)2. 4 H2O 100 mg•L-1；KH2PO3 270 mg•
L-1；MnSO4.4 H2O 22.3 mg•L-1；ZnSO4.7 H2O 10.6 mg•L-1；CuSO4.5 H2O 0.025 mg•L-1；H3BO3
8.2 mg•L-1；Na2MoO4.2 H2O 0.25 mg•L-1；KI 0.83 mg•L-1；CoCl2.6 H2O 0.025 mg•L-1；Na2•
EDTA 37.3 mg•L-1；FeSO4.7 H2O 27.8 mg•L-1；0.4 mg L of vitamin B1-1；0.5 mg L of vitamin B6-1；2.0 mg L of niacin-1；1.0 mg L of glycine-1；120 mg L of inositol-1。
Embodiment 2:
In the continuous weather to clear up 2 days or more, age of tree life in 5~8 years is selected, by the core of national or provincial breeding authorization
Peach breeding clone is as scion-seed tree；Top acquisition current growth in scion-seed tree tree crown periphery is healthy and strong and no disease and pests harm
The walnut spray tip, the excellent propagation material as walnut tissue culture.
The collected walnut spray tip is cut into stem section of the 2~3cm long with axillary bud as explant, with volumetric concentration 75%
Ethanol solution impregnates 30 s of sterilizing to explant, explant is placed in super-clean bench after distilled water flushing 2 times, then use volumetric concentration
0.1%HgCl2Solution carries out 8~10 min of sterilization treatment to explant in oscillating fashion, finally uses aseptic water washing 5 times.
The explant that sterilization is handled is inoculated in induced medium, is placed in 25 ± 2 DEG C of temperature, illumination is strong
2500~3000LX is spent, initial bud is induced to occur in the environment of 12 h/d of illumination.The raw material components of the induced medium and
Mass content are as follows: improvement 0.5 mgL of MS+6-BA-130000 mg L of+sucrose-1300 mg L of+lactoalbumin hydrolysate-1+ table oil
0.2 mg L of rape lactone-13000 mg L of+agar-1。
Axillary bud starts to sprout after cultivating 8 d, culture 35 d buds grow tall 3~5 cm when, bud is cut from former stem section, be inoculated with
In proliferated culture medium, the raw material components and mass content of proliferated culture medium are as follows: improvement 1.5 mgL of MS+6-BA-1+ sucrose
30000 mg•L-1400 mg L of+lactoalbumin hydrolysate-10.4 mg L of+epi-brassinolide-13000 mg L of+agar-1, juxtaposition
Multiplying culture is carried out in 25 ± 2 DEG C of temperature, 2500~3000LX of intensity of illumination, the environment of 12 h/d of illumination, promotes culture
Axillary bud and lateral bud sprout.About 35d switching is primary, cultivates 35 d, and average bud is up to 3.8 cm, the dark green no yellow of nursery stock leaf color,
Growth coefficient 4.5.
The composition of above-described modified MS medium are as follows: KNO3 1600 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2 350 mg·L-1；MgSO4.7H2O 370 mg·L-1；Ca(NO3)2. 4 H2O 100 mg•L-1；KH2PO3 270 mg•
L-1；MnSO4.4 H2O 22.3 mg•L-1；ZnSO4.7 H2O 10.6 mg•L-1；CuSO4.5 H2O 0.025 mg•L-1；H3BO3
8.2 mg•L-1；Na2MoO4.2 H2O 0.25 mg•L-1；KI 0.83 mg•L-1；CoCl2.6 H2O 0.025 mg•L-1；Na2•
EDTA 37.3 mg•L-1；FeSO4.7 H2O 27.8 mg•L-1；0.4 mg L of vitamin B1-1；0.5 mg L of vitamin B6-1；2.0 mg L of niacin-1；1.0 mg L of glycine-1；120 mg L of inositol-1。
Embodiment 3:
In the continuous weather to clear up 2 days or more, age of tree life in 5~8 years is selected, by the core of national or provincial breeding authorization
Peach breeding clone is as scion-seed tree；Top acquisition current growth in scion-seed tree tree crown periphery is healthy and strong and no disease and pests harm
The walnut spray tip, the excellent propagation material as walnut tissue culture.
The collected walnut spray tip is cut into stem section of the 2~3cm long with axillary bud as explant, with volumetric concentration 75%
Ethanol solution impregnates 30 s of sterilizing to explant, explant is placed in super-clean bench after distilled water flushing 2 times, then use volumetric concentration
0.1%HgCl2Solution carries out 8~10 min of sterilization treatment to explant in oscillating fashion, finally uses aseptic water washing 5 times.
The explant that sterilization is handled is inoculated in induced medium, is placed in 25 ± 2 DEG C of temperature, illumination is strong
3000~3500LX is spent, initial bud is induced to occur in the environment of 10 h/d of illumination.The raw material components of the induced medium and
Mass content are as follows: improvement 0.5 mgL of MS+6-BA-130000 mg L of+sucrose-1300 mg L of+lactoalbumin hydrolysate-1+ table oil
0.2 mg L of rape lactone-13000 mg L of+agar-1。
Axillary bud starts to sprout after cultivating 8 d, culture 35 d buds grow tall 3~5 cm when, bud is cut from former stem section, be inoculated with
In proliferated culture medium, the raw material components and mass content of proliferated culture medium are as follows: improvement 2.0 mgL of MS+6-BA-1+ sucrose
30000 mg•L-1500 mg L of+lactoalbumin hydrolysate-10.5 mg L of+epi-brassinolide-13000 mg L of+agar-1, juxtaposition
Multiplying culture is carried out in 25 ± 2 DEG C of temperature, 3000~3500LX of intensity of illumination, the environment of 10 h/d of illumination, promotes culture
Axillary bud and lateral bud sprout.About 35d switching is primary, cultivates 35 d, and average bud is up to 4.1 cm, the dark green no yellow of nursery stock leaf color,
Growth coefficient 4.3.
The composition of above-described modified MS medium are as follows: KNO3 1600 mg·L-1；NH4NO3 1200 mg·L-1；
CaCl2 350 mg·L-1；MgSO4.7H2O 370 mg·L-1；Ca(NO3)2. 4 H2O 100 mg•L-1；KH2PO3 270 mg•
L-1；MnSO4.4 H2O 22.3 mg•L-1；ZnSO4.7 H2O 10.6 mg•L-1；CuSO4.5 H2O 0.025 mg•L-1；H3BO3
8.2 mg•L-1；Na2MoO4.2 H2O 0.25 mg•L-1；KI 0.83 mg•L-1；CoCl2.6 H2O 0.025 mg•L-1；Na2•
EDTA 37.3 mg•L-1；FeSO4.7 H2O 27.8 mg•L-1；0.4 mg L of vitamin B1-1；0.5 mg L of vitamin B6-1；2.0 mg L of niacin-1；1.0 mg L of glycine-1；120 mg L of inositol-1。
Claims (1)
1. a kind of walnut tissue culture and rapid propagation method for eliminating tissue-cultured seedling yellow, it is characterised in that: including propagation material selection, explant
Processing, the induction of initial bud and Multiplying culture process；The walnut spray tip for acquiring current growth stalwartness and no disease and pests harm is used as breeding
Material be trimmed to stem section as explant to branch, trains to initial bud induction is inoculated in after the processing of explant sterilization
It supports and initial bud is induced to occur in base, then obtain the tissue culture subculture of no aetiolation after initial bud is inoculated in proliferated culture medium
Bud；Main operational steps are as follows:
(1) propagation material selects: selection passes through national level or the improved variety of walnut clone of provincial breeding authorization, age of tree life in 5~8 years
It is scion-seed tree；The walnut spray tip of current growth stalwartness and no disease and pests harm is acquired in the top of scion-seed tree tree crown periphery,
Excellent propagation material as walnut tissue culture；
(2) explant is handled: the collected walnut spray tip being cut into and only retains the stem section work of terminal bud or 2~3cm long with axillary bud
For explant, sterilizing 30s is impregnated to explant with 75% ethanol solution of volumetric concentration, sets explant after distilled water flushing 2 times
In super-clean bench, then with volumetric concentration 0.1%HgCl2Solution carries out 8~10min of sterilization treatment to explant in oscillating fashion,
Finally use aseptic water washing 5 times；
(3) initial bud induction: the explant that step (2) processing obtains is inoculated in induced medium, temperature 25 ± 2 is placed in
DEG C, 2000~3500LX of intensity of illumination induces in the environment of 10~14h/d of illumination initial bud to occur；
(4) Multiplying culture: the initial bud that step (3) obtain is inoculated in proliferated culture medium, is placed in 25 ± 2 DEG C of temperature, illumination
2000~3500LX of intensity carries out Multiplying culture in the environment of 10~14h/d of illumination, obtains the tissue culture subculture of no aetiolation
Bud；
The raw material components and mass content of induced medium described in step (3) are as follows: improvement MS+6-BA 0.5mgL-1+ sucrose
30000mg·L-1+ lactoalbumin hydrolysate 300mgL-1+ epi-brassinolide 0.2mgL-1+ agar 3000mgL-1；
The raw material components and mass content of proliferated culture medium described in step (4) are as follows: improvement 1.0~2.0mgL of MS+6-BA-1+
Sucrose 30000mgL-1300~500mgL of+lactoalbumin hydrolysate-10.3~0.5mgL of+epi-brassinolide-1+ agar
3000mg·L-1；
The composition of modified MS medium described in step (3) and (4) are as follows: KNO3 1600mg·L-1；NH4NO3 1200mg·L-1；
CaCl2 350mg·L-1；MgSO4.7H2O 370mg·L-1；Ca(NO3)2.4H2O 100mg·L-1；KH2PO3 270mg·L-1；
MnSO4.4H2O 22.3mg·L-1；ZnSO4.7H2O 10.6mg·L-1；CuSO4.5H2O 0.025mg·L-1；H3BO3
8.2mg·L-1；Na2MoO4.2H2O 0.25mg·L-1；KI 0.83mg·L-1；CoCl2.6H2O 0.025mg·L-1；Na2·
EDTA 37.3mg·L-1；FeSO4.7H2O 27.8mg·L-1；Vitamin B1 0.4mgL-1；Vitamin B6 0.5mgL-1；
Niacin 2.0mgL-1；Glycine 1.0mgL-1；Inositol 120mgL-1。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101297637A (en) * | 2008-06-18 | 2008-11-05 | 中国科学院新疆理化技术研究所 | Plant tissue culture method for thinly-skinned walnut |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101297637A (en) * | 2008-06-18 | 2008-11-05 | 中国科学院新疆理化技术研究所 | Plant tissue culture method for thinly-skinned walnut |
Non-Patent Citations (2)
| Title |
|---|
| Walnut(Juglans regia L.)micropropagation;M.A.Revilla等;《Annals of forest science》;19891231;第149-151页 |
| 薄皮核桃组织培养与快速繁殖;苗玉清等;《新疆农业科学》;20101231;第47卷(第3期);1.2 方法，2 结果与分析，表3 |
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Application publication date: 20170804 Assignee: Guangxi Shengjin Agricultural Technology Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION FORESTRY Research Institute Contract record no.: X2023980045763 Denomination of invention: A Rapid Propagation Method for Walnut Tissue Culture to Eliminate Yellowing of Tissue Culture Seedlings Granted publication date: 20190301 License type: Common License Record date: 20231106 |