CN103535280B - A kind of water oak tissue culture propagation - Google Patents

A kind of water oak tissue culture propagation Download PDF

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CN103535280B
CN103535280B CN201310519136.9A CN201310519136A CN103535280B CN 103535280 B CN103535280 B CN 103535280B CN 201310519136 A CN201310519136 A CN 201310519136A CN 103535280 B CN103535280 B CN 103535280B
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water
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shoots
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CN103535280A (en
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黄利斌
张敏
陈智敏
窦全琴
董筱昀
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Jiangsu Forestry Academy
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Abstract

The invention discloses a kind of water oak new varieties tissue culture propagation, comprise selection, disinfect, Shoots in vitro cultivation, strong seedling culture, Multiplying culture, culture of rootage, transplant, survive sequence of operations; Cultivate in the medium that body embryonal induction, propagation, maturation, sprouting are cultivated.It is high that the present invention has reproduction coefficient, and repoductive time is not subject to seasonal restrictions, and draws materials little to parent injury, without damage by disease and insect puzzlement, in water oak nursery stock production and scientific research, all have important using value.

Description

A kind of water oak tissue culture propagation
Technical field
The present invention relates to the mating system of water oak, specifically relate to the method for tissue culture of water oak.
Background technology
Water oak ( quercus nigra) be Fagaceae oak kind, originate in southeastern US, mainly be distributed in subtropics coastal plain and the Mississippi middle and lower reaches alluvial plain of southeastern US, all have distribution on low ground and height above sea level 450 meters of, happiness sun, strong adaptability, soil is required not tight, both drought-resistant barren, more water-fast wet again, cold-resistant, be excellent use material and Afforestation Landscape seeds.But due to water oak shorter in the time of introducing a fine variety of China, and it is real comparatively slow to yield positive results, and solid biennial bearing phenomenon is obvious, and seed source is few, and the survival rate of cuttage and grafting is all very low, brings difficulty to breeding in a large number of seedling.Tissue culture technique is one of main contents of current forestry biomass technology, and its main application is large-scale breeding and the molecular breeding of forest.The advantage of this technology is that reproductive efficiency is high, takes up room little, is not subject to the restriction of season and external environment, needs fertile material few, can provide a large amount of high quality seedling in a short time.
Summary of the invention
Goal of the invention: the object of the invention is, for the comparatively outmoded problem of the existing propagation technique of water oak, to provide a kind of method for tissue culture of water oak breeding, its object is to improve its reproduction coefficient to adapt to the demand that it introduces a fine variety popularization and Landscape Application.
Technical scheme: a kind of water oak tissue culture propagation provided by the invention, its operating procedure comprises as follows:
(1) selection: gather seed in October Mo on the 10 years unboiled water roburs introduced a fine variety, adopt water seaoning removing cur, the seed selected is placed in sand, vegetable particles fertilizer, reed charcoal, careless carbon mix matrix cultivate, the tender stem of excellent seedling of growing directly from seeds of water intaking oak seed growing is inoculation material;
(2) disinfect: by tender for seedling stem disinfection;
(3) Shoots in vitro is cultivated: aseptically, explant after sterilization in step (2) is cut off the wound of contact sterilizing liquid, 1 ~ 2cm stem with bud is stayed to be seeded in Primary culture base, then being placed on common fluorescent lamp is under the environment of light source, and wherein intensity of illumination is 1600 ~ 2500lx, and every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is 50% ~ 65%, cultivates 22 ~ 25 days, and differentiation grows up to Shoots in vitro;
(4) strong seedling culture: because the seedling of just generation induction is directly bred very easily withered because being subject to the impact of recalcitrant phenomenon, therefore first strong seedling culture is carried out, by the Shoots in vitro obtained in step (3), shear with after the stem section of 1 ~ 2 axillalry bud or terminal bud, on ultra-clean workbench and under aseptic condition, be inoculated in and contain in the glass blake bottle of strong seedling culture base through what sterilize, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out strong seedling culture, next step is entered after 15 ~ 18 days,
(5) Multiplying culture: the Shoots in vitro that will grow up to from step (4), shear with after the stem section of 1 ~ 2 axillalry bud or terminal bud, on ultra-clean workbench and under aseptic condition, transfer in containing in the glass blake bottle of proliferated culture medium through what sterilize, then being placed on common fluorescent lamp is under the environment of light source, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out Multiplying culture to bud seedling, after 15 ~ 18 days, growth coefficient is 2.45 ~ 4;
(6) culture of rootage: by the test-tube plantlet obtained in step (5), remove base portion tissue and partial blade, stay 3 ~ 4 blades, be inoculated under aseptic condition and contain in the glass blake bottle of root media through what sterilize, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, and temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carries out culture of rootage and goes to containing continuation cultivation in the WPM medium of hormone after 15 days; After 15 ~ 20 days, rooting rate reaches 50%;
(7) transplant, survive: by bottle seedling of taking root obtained in step (6), take out and clean, be transplanted in the matrix containing peat and yellow soil, wherein the volume ratio of peat and yellow soil is 1:1, then waters permeable, use covered rearing with plastic film vessel port, keep temperature 25 ~ 30 DEG C, relative moisture 75% ~ 85%, cultivates 30 ~ 35 days, young leaves launches completely, and transplanting survival rate is 80% ~ 83%.
Further, the process of disinfecting in described step (2) comprises: the explant chosen cuts into the long segment of 3 ~ 4cm, 5 min are soaked with washing powder solution, tap water 10 times, proceeds to superclean bench, puts into sterile beaker, alcohol with 70% carries out surface sterilization 30 s, aseptic water washing 3 times, then sterilize 4.5 minutes with the mercuric chloride solution that concentration is 0.1%, finally use aseptic water washing 6 times.
Further, the sand in described step (1), vegetable particles fertilizer, reed charcoal, careless carbon mix volume ratio are 4:1:1:1.
Further, the peat in described step (7) and the volume ratio of yellow soil are 1:1 ~ 1.5.
Further, the Primary culture based formulas in described step (3) is: WPM minimal medium, 0.01 mg. L -16-BA, 0.02mg. L -1nAA, 30g.L -1sucrose, 6.5 g.L -1carragheen; The pH value of described Primary culture base controls 5.7 ~ 5.8.
Further, the strong seedling culture based formulas in described step (5) is: WPM minimal medium, 0.01mg. L -16-BA, 0.02 mg. L -1nAA, 0.5 mg. L -1zeatin, 30g.L -1sucrose, 6.5 g.L -1carragheen; Described strong seedling culture based formulas pH value controls 5.7 ~ 5.8.
Further, the proliferation culture medium formula of described step (6) is: WPM minimal medium, 0. 1mg. L -16-BA, 0.02 mg. L -1nAA, 30g.L -1sucrose, 6.5 g.L -1carragheen; Proliferated culture medium pH value controls 5.7 ~ 5.8.
Further, the prescription of rooting medium in described step (7) is: WPM minimal medium, 1.5mg.L-1IBA, 30g.L -1sucrose, 6.5 g.L -1carragheen, the pH value of described root media is 5.7 ~ 5.8.
Further, described WPM minimal medium and the concrete formula of woody plant tissure medium as follows:
Ammonium nitrate NH4NO3 400 mg/L
Nitrate of lime Ca (NO 3) 24H 2o 556 mg/L
Calcium chloride CaCl 22H 2o 96 mg/L
Magnesium sulfate MgSO 47H 2o 370 mg/L
Potassium dihydrogen phosphate KH 2pO 4170 mg/L
Potassium sulfate K 2sO4 990 mg/L
Disodium ethylene diamine tetraacetate Na 2eDTA 37.3 mg/L
Ferrous sulfate FeSO 47H 2o 27.8 mg/L
Manganese sulphate MnSO 4h 2o 22.3 mg/L
Zinc sulphate ZnSO 47H 2o 8.6 mg/L
Boric acid H 3bO 36.2 mg/L
Sodium molybdate Na 2moO 42H 2o 0.25 mg/L
Copper sulphate CuSO 45H 2o 0.025 mg/L
Cobalt chloride CoCl 2.6 20.025 mg/L
Inositol C 6h 12o 62H 2o 100 mg/L
Glycine NH 2cH 2cOOH 2 mg/L
Thiamine hydrochloride C 12h 17clOS2HCl 1 mg/L
Puridoxine hydrochloride C 8h 10nO 5p 0.5 mg/L
Nicotinic acid NC 5h 4cOOH 0.5 mg/L.
Beneficial effect: the present invention has the following advantages in terms of existing technologies:
(1) a kind of water oak method for tissue culture of the present invention is for seed propagation and other vegetative propagations, by suitable operation and suitable medium, have easy and simple to handle, saving occupation of land space, repoductive time is not subject to seasonal restrictions, its germination rate is high, reaches more than 90%.
(2) the present invention draws materials to parent fanout free region, and without damage by disease and insect puzzlement, reproduction coefficient is high, can keep the advantage of the merit of former plant, in water oak nursery stock production and scientific research, all have important using value.
(3) the present invention is compared with conventional tissue culture propagation method, because the water oak seedling of just generation induction is directly bred very easily withered because being subject to the impact of recalcitrant phenomenon, strong seedling culture is put forward previous step by this law in operation, carries out before proliferation-inducing, effectively ensure that and is proliferated into motility rate and value-added coefficient.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
The formula of the minimal medium that following instance is used is as follows:
Bud inducement minimal medium is WPM medium, and wherein, the component of macroelement is as follows with the working concentration of its correspondence: ammonium nitrate (NH 4nO 3) 400 mg/L, nitrate of lime (Ca (NO 3) 24H 2o) 556 mg/L, calcium chloride (CaCl 22H 2o) 96 mg/L, magnesium sulfate (MgSO 47H 2o) 370 mg/L, potassium dihydrogen phosphate (KH 2pO 4) 170 mg/L, potassium sulfate (K 2sO 4) 990 mg/L;
The component of trace element is as follows with the working concentration of its correspondence: manganese sulphate (MnSO 4h 2o) 22.3 mg/L, zinc sulphate (ZnSO 47H 2o) 8.6 mg/L, boric acid (H 3bO 3) 6.2 mg/L, sodium molybdate (Na 2moO 42H 2o) 0.25 mg/L, copper sulphate (CuSO 45H 2o) 0.025 mg/L, copper sulphate (CuSO 45H 2o) 0.025 mg/L, cobalt chloride (CoCl 2.6 2) 0.025 mg/L;
The component of molysite is as follows with the working concentration of its correspondence: disodium ethylene diamine tetraacetate (Na 2eDTA) 37.3 mg/L, ferrous sulfate (FeSO 47H 2o) 27.8 mg/L;
The component of organic principle is as follows with the concentration of its correspondence: inositol 100 mg/L, glycine (Gly) 2 mg/L, thiamine hydrochloride (VB 1) 1 mg/L, puridoxine hydrochloride (VB 6) 0.5 mg/L, nicotinic acid (VPP) 0.5 mg/L.
Utilize above-mentioned minimal medium configuration inducing culture, strong seedling culture base, proliferated culture medium and root media.
Wherein, inducing culture: WPM minimal medium+6-BA(0.05 mg. L -1)+NAA (0.02 mg. L -1)+sucrose (30g.L -1)+carragheen (6.5 g.L -1), pH value is 5.7 ~ 5.8.
Strong seedling culture base is: WPM minimal medium+6-BA (0.1 mg. L -1)++sucrose (25g.L -1)+carragheen (6.5 g.L- 1)+active carbon (1g.L -1), pH value is 5.7 ~ 5.8.
Proliferated culture medium: WPM minimal medium+6-BA (0.1 mg. L -1)+NAA (0.02 mg. L -1)+sucrose (30g.L -1)+carragheen (6.5 g.L -1), pH value is 5.7 ~ 5.8.
Described root media is: WPM minimal medium+IBA (2.5mg.L-1)+sucrose (30g.L -1)+carragheen (6.5 g.L -1), pH value is 5.7 ~ 5.8.
By in the above-mentioned medium implantation glass bottle configured, through autoclave sterilization 20 minutes, stand-by, wherein, temperature was 120-125 DEG C, and pressure is 1.1 KG/CM 2.
The present embodiment selection: state is introduced water oak Provenance seed and carried out breeding and afforestation test from Texas, U.S. original producton location, Tennessee, Louisiana and Arkansas etc., respectively in forest-science academy of Jiangsu Province, Yixing City forest farm, the ground such as Hui Shan forest park, Wuxi construction provenance test woods, select the Comprehensive Traits such as the height of tree, the diameter of a cross-section of a tree trunk 1.3 meters above the ground, hat width, branch, resistance and show No. 9903 excellent, water oak Louisiana provenance, being defined as fine provenance, is material therefor of the present invention.
A kind of water oak tissue culture propagation, comprises the following steps:
(1) selection: gather seed in October Mo on the 10 years unboiled water roburs introduced a fine variety, adopt water seaoning removing cur, the seed selected is placed in sand, vegetable particles fertilizer, reed charcoal, careless carbon mix matrix cultivate, sand in described step (1), vegetable particles fertilizer, reed charcoal, careless carbon mix volume ratio are 4:1:1:1, and the tender stem of excellent seedling of growing directly from seeds of water intaking oak seed growing is inoculation material;
(2) disinfect: by tender for seedling stem disinfection;
(3) Shoots in vitro is cultivated: aseptically, explant after sterilization in step (2) is cut off the wound of contact sterilizing liquid, staying 1 ~ 2cm stem with bud to be seeded in contains in the glass blake bottle of Primary culture base through what sterilize, then being placed on common fluorescent lamp is under the environment of light source, and wherein intensity of illumination is 1600 ~ 2500lx, and every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is 50% ~ 65%, cultivates 25 days, and differentiation grows up to Shoots in vitro;
(4) strong seedling culture: because the seedling of just generation induction is directly bred very easily withered because being subject to the impact of recalcitrant phenomenon, therefore first strong seedling culture is carried out, by the Shoots in vitro obtained in step (3), shear with after the stem section of 1 ~ 2 axillalry bud or terminal bud, on ultra-clean workbench and under aseptic condition, be inoculated in and contain in the glass blake bottle of strong seedling culture base through what sterilize, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out strong seedling culture, next step is entered after 18 days,
(5) Multiplying culture: the Shoots in vitro that will grow up to from step (4), shear with after the stem section of 1 ~ 2 axillalry bud or terminal bud, on ultra-clean workbench and under aseptic condition, transfer in containing in the glass blake bottle of proliferated culture medium through what sterilize, then being placed on common fluorescent lamp is under the environment of light source, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out Multiplying culture to bud seedling, growth coefficient is 2.45 ~ 4;
(6) culture of rootage: by the test-tube plantlet obtained in step (5), remove base portion tissue and partial blade, stay 3 ~ 4 blades, on ultra-clean workbench and under aseptic condition, be inoculated in and contain in the glass blake bottle of root media through what sterilize, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out culture of rootage go to after 15 days not containing hormone WPM medium in continue cultivation after 18 days rooting rate 50%;
(7) transplant, survive: by bottle seedling of taking root obtained in step (6), take out and clean, be transplanted in the matrix containing peat and yellow soil, wherein the volume ratio of peat and yellow soil is 1:1, then waters permeable, use covered rearing with plastic film vessel port, keep temperature 25 ~ 30 DEG C, relative moisture 75% ~ 85%, cultivates 30 ~ 35 days, young leaves launches completely, and transplanting survival rate is 80% ~ 83%.
Can draw from upper table: the present embodiment tissue culture method effectively can utilize seed, germination rate can reach more than 90%, relative to and conventional seed germination need higher accumulated temperature, nursery germination percent only reaches 45.1% ~ 70%, other vegetative propagation is as cuttage, propagation by grafiting, and survival rate is all not high.Adopt spray from 1 ~ 2 year treelet to carry out the summer and insert, survival rate is 20 ~ 30%; Do stock with 1 year raw Quercus acutissima, scion grafting in spring motility rate is 13.3 ~ 15.0%.In a growth cycle that is 6 months, the reproduction coefficient of tissue culture method is 8 ~ 10 times of cottage propagation.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (4)

1. a water oak tissue culture propagation, is characterized in that: comprise the following steps:
(1) selection: gather seed in October Mo on the 10 years unboiled water roburs introduced a fine variety, adopt water seaoning removing cur, the seed selected is placed in sand, vegetable particles fertilizer, reed charcoal, careless carbon mix matrix cultivate, the tender stem of excellent seedling of growing directly from seeds of water intaking oak seed growing is inoculation material; Described sand, vegetable particles fertilizer, reed charcoal, careless carbon mix volume ratio are 4:1:1:1;
(2) disinfect: by tender for seedling stem disinfection;
(3) Shoots in vitro is cultivated: aseptically, explant after sterilization in step (2) is cut off the wound of contact sterilizing liquid, staying 1 ~ 2cm stem with bud to be seeded in contains in the glass blake bottle of Primary culture base through what sterilize, then being placed on common fluorescent lamp is under the environment of light source, humidity is 50% ~ 65%, cultivate 22 ~ 25 days, differentiation grows up to Shoots in vitro; Described Primary culture based formulas is: WPM minimal medium, 0.01mgL -16-BA, 0.02mgL -1nAA, 30gL -1sucrose, 6.5gL -1carragheen; The pH value of described Primary culture base controls 5.7 ~ 5.8;
(4) strong seedling culture: by the Shoots in vitro obtained in step (3), shear with after the stem section of 1 ~ 2 axillalry bud or terminal bud, transfer in containing in the glass blake bottle of strong seedling culture base through what sterilize, humidity is under the condition of 50% ~ 65%, carry out strong seedling culture, after 15 ~ 18 days, enter next step; Described strong seedling culture based formulas is: WPM minimal medium, 0.01mgL -16-BA, 0.02mgL -1nAA, 0.5mgL -1zeatin, 30gL -1sucrose, 6.5gL -1carragheen; Described strong seedling culture based formulas pH value controls 5.7 ~ 5.8;
(5) Multiplying culture: the Shoots in vitro that will grow up to from step (4), shears with after the stem section of 1 ~ 2 axillalry bud or terminal bud, transfers in proliferated culture medium, humidity 50% ~ 65%; Multiplying culture 15 ~ 20 days; Described proliferation culture medium formula is: WPM minimal medium, 0.1mgL -16-BA, 0.02mgL -1nAA, 30gL -1sucrose, 6.5gL -1carragheen; Proliferated culture medium pH value controls 5.7 ~ 5.8;
(6) culture of rootage: by the test-tube plantlet obtained in step (5), remove base portion tissue and partial blade, stay 3 ~ 4 blades, aseptically, be inoculated in and contain in the glass blake bottle of root media through what sterilize, be 1600 ~ 2500lx in intensity of illumination, every day, light application time was 16 hours, temperature is (25 ± 2) DEG C, humidity is under the condition of 50% ~ 65%, carry out culture of rootage go to after 15 days containing hormone WPM medium in do not continue cultivation 15 ~ 20 days; Described prescription of rooting medium is: WPM minimal medium, 1.5mgL -1iBA, 30gL -1sucrose, 6.5gL -1carragheen, the pH value of described root media is 5.7 ~ 5.8;
(7) transplant, survive: by bottle seedling of taking root obtained in step (6), take out and clean, be transplanted in the matrix containing peat and yellow soil, then water permeable, use covered rearing with plastic film vessel port, keep temperature 25 ~ 30 DEG C, relative moisture 75% ~ 85%, cultivates 30 ~ 35 days;
Described WPM minimal medium and the concrete formula of woody plant tissure medium as follows:
2. water oak tissue culture propagation according to claim 1, it is characterized in that: the condition of culture in described step (3), step (4), step (5), step (6) is: intensity of illumination is 1600 ~ 2500lx, every day, light application time was 16h, and temperature is (25 ± 2) DEG C.
3. water oak tissue culture propagation according to claim 1, it is characterized in that: the process of disinfecting in described step (2) comprises: the explant chosen cuts into the long segment of 3 ~ 4cm, 5min is soaked with washing powder solution, tap water 10 times, proceeds to superclean bench, puts into sterile beaker, alcohol with 70% carries out surface sterilization 30s, aseptic water washing 3 times, then sterilize 4.5 minutes with the mercuric chloride solution that concentration is 0.1%, finally use aseptic water washing 6 times.
4. water oak tissue culture propagation according to claim 1, is characterized in that: the peat in described step (7) and the volume ratio of yellow soil are 1:1 ~ 1:1.5.
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CN111034522B (en) * 2019-12-11 2022-03-18 辽宁省蚕业科学研究所 Transplanting and domesticating method for quercus acutissima tissue culture seedlings
CN115380829B (en) * 2022-09-29 2023-09-01 广州百德园艺有限公司 Oak She Hujue stolon tissue culture method
CN115735774B (en) * 2022-12-10 2024-03-26 河北省洪崖山国有林场(河北雄安新区白洋淀上游规模化林场) Tissue culture method of Quercus mongolica

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