CN106472316A - A kind of Quercus acutissima initial culture bud abductive approach - Google Patents
A kind of Quercus acutissima initial culture bud abductive approach Download PDFInfo
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- CN106472316A CN106472316A CN201610903544.8A CN201610903544A CN106472316A CN 106472316 A CN106472316 A CN 106472316A CN 201610903544 A CN201610903544 A CN 201610903544A CN 106472316 A CN106472316 A CN 106472316A
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- explant
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- quercus acutissima
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- 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
- A01H4/008—Methods for regeneration to complete plants
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- 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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Abstract
The invention discloses a kind of Quercus acutissima initial culture bud abductive approach it is characterised in that：It is explant using Quercus acutissima base portion coppice shoot, after explant aseptic process, explant is inoculated on high-quality initial culture bud inducement cultivation base, is cultivated under the conditions of being placed in suitable temperature, humidity and intensity of illumination, the sprouting of induced bud.The present invention overcomes in bud Induction Process the problems such as Explant browning and vitrification, improve bud induction rate, sprout index and sprout growth situation, acquisition quantity is many, the measured rudiment of matter, thus meeting the needs of enrichment culture, promote the foundation of Quercus acutissima tissue-culturing quick-propagation system.
Description
Technical field
The present invention relates to Quercus acutissima vegetative propagation technique, especially a kind of Quercus acutissima initial culture bud abductive approach.
Background technology
Quercus acutissima（Quercus acutissima Carruth）Also known as Qinggang, Oak Tree, it is Fagaceae（Fagaceae）Oak
Belong to（Quercus）Arbor, up to 30 meters, the diameter of a cross-section of a tree trunk 1.3 meters above the ground reaches 1 meter, the dark-grey brown of bark, deep lobe.It is born in the mountain of height above sea level 60-2200 rice
Ground tailo, becomes small pieces pure forest or mixed forest, positive light, likes moist climate.Cold-resistant, drought-resistant barren, not water-fast wet, not salt tolerant
Alkali, grows best on moistening fertile deep, well-drained neutrality is to subacidity sandy loam, and impeded drainage or chaor are unsuitable
Plantation.Mix friendship with other seeds and can form good form, deep-rootedness, rudiment power is strong, but intolerant to transplanting.Antipollution, antidusting soil,
Wind loading rating is all stronger.Life-span length, up to 500～600 years.Produce Liaoning, Hebei, Shanxi, Shandong, Jiangsu, Anhui, Zhejiang, river
The provinces and regions such as west, Fujian, Henan, Hubei, Hunan, Guangdong, Hainan, Guangxi, Sichuan, Guizhou, Yunnan.
At present, the common planting patternss of Quercus acutissima are afforestation seeding and planting by stock.Tissue culture technique is a kind of quick nothing
Sexual reproduction technology, selects Quercus acutissima superior families or clone to be material, breeds nursery stock by method for tissue culture, both can expire
The quantitative requirement that foot produces, can keep maternal character again.In tissue culture procedures, initial culture is that restriction tissue culture is smooth
The committed step carrying out, has been directed to train in acquisition, the sterilization of explant, culture medium selection and the incubation of explant
The major issues such as foster body brown stain, nursery stock vitrification.
Content of the invention
It is an object of the invention to provide one kind can overcome in bud Induction Process the problems such as Explant browning and vitrification,
Improve bud induction rate, sprout index and sprout growth situation, acquisition quantity is many, the measured rudiment of matter, thus meeting propagation training
Foster needs, promote the Quercus acutissima initial culture bud abductive approach of the foundation of Quercus acutissima tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows：
A kind of Quercus acutissima initial culture bud abductive approach, is explant using Quercus acutissima base portion coppice shoot, after explant aseptic process,
Explant is inoculated on high-quality initial culture bud inducement cultivation base, under the conditions of being placed in suitable temperature, humidity and intensity of illumination
Cultivated, the sprouting of induced bud；Its operating procedure is,
（1）Explant selects：The coppice shoot selecting the semi-lignified of Quercus acutissima base portion sprouting is explant；
（2）Explant sterilization and inoculation：Quercus acutissima coppice shoot is cleaned through liquid detergent, flowing water rinses and chemical reagent processes sterilization
Afterwards, cut into stem section to access in bud inducement cultivation base；
（3）Bud inducement cultivation：Postvaccinal explant is placed in culture under suitable indoor conditionss.
Above-described its material content of bud inducement cultivation base is：1/3～1/2 improvement WPM culture medium+6-BA 1.0～
2.0mg/L+ vitamin C 10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media components and envelope-bulk to weight ratio are：NH4NO3780 mg/L, CaCl2·2H2O
192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O 180 mg/L, KI 0.83
Mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L, Na2MoO4·2H2O 0.25
Mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O 27.8 mg/L, inositol 85 mg/
L, nicotinic acid 1.0 mg/L, pyridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L, glycine 2.0 mg/L.
Above-described coppice shoot is a length of 6 ~ 9cm, the coppice shoot of the semi-lignified of diameter 0.2～0.4 cm.
Above step（2）Described sterilization and inoculation method are to cut off Quercus acutissima coppice shoot needle, are placed in triangular flask, add
Volumetric concentration is the liquid detergent solution of 1 ~ 5 %, seals bottleneck using gauze, is placed in cleaning on the shaking table that rotating speed is 200 r/min
It is placed under flowing water flushing 1 ~ 2 h after 10 ~ 15 min, then use sterile water wash 2 times, explant is gone to and uses on superclean bench
Volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；It is cut into a length of 3.5 ~ 4.0 after adopting aseptic water washing 8 ~ 10 times again
The stem section of cm length is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Above step（3）Described suitable indoor conditionss are：Illumination 500～1000 lx, illumination 8 is switched to after light culture 10d
～12h/d；20 ± 1 DEG C of cultivation temperature, humidity is 50 ~ 55 %.
The present invention has the advantage that and has the beneficial effect that：
1st, the present invention adopts 1/3～1/2 improvement WPM culture medium culturing base as minimal medium, and emphasis have adjusted and fiber crops simultaneously
Oak sprout correlation trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcome Quercus acutissima group
Knit the vitrification phenomenon easily occurring in culture bud Induction Process, be more easy to promote the generation of Quercus acutissima bud induction, promote the life of rudiment
Long speed and robustness.
2nd, the present invention is using the sturdy coppice shoot of semi-lignified of base portion length 6 ~ 9 cm length as explant it is ensured that explant is stronger
Meristematic capacity, promote bud induction；The present invention carries out cleaning after explant collection, sterilizes, and effectively reduces the dirt of explant
Dye rate, improves bud ratio.
3rd, postvaccinal explant is placed in temperature by the present invention is 20 ± 1 DEG C of degree, and humidity is 50 ~ 55%, and illumination is by light culture
Go to culture under the indoor conditionss that intensity is 500～1000 lx afterwards, temperature and humidity is low, the also not high interior of intensity of illumination
Condition, advantageously reduces vitrified generation in bud Induction Process.
Specific embodiment
With reference to specific embodiment, the present invention will be further described.
Embodiment 1：
Select the sprouting of Quercus acutissima base portion, a length of 6 ~ 7cm, the coppice shoot of the semi-lignified of diameter 0.2～0.3 cm as explant.By fiber crops
Oak coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 1 %, seals bottleneck using gauze, puts
In rotating speed be 200 r/min shaking table on clean 10 min after be placed under flowing water and rinse 2 h, then use sterile water wash 2 times, will
Explant goes to uses volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min on superclean bench；Adopt aseptic water washing again
The stem section being cut into a length of 3.5 cm length after 8 ~ 10 times is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/3 improvement WPM culture medium+6-BA 1.0mg/L+ vitamin
C10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are：NH4NO3780
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, inositol 100 mg/L, nicotinic acid 1.0 mg/L, pyridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L are sweet
Propylhomoserin 2.0 mg/L.
Postvaccinal explant is placed in 20 ± 1 DEG C of temperature, humidity switchs to illumination 500 for after 50 % interior light culture 10d
Lx, is cultivated under conditions of illumination 12h/d, the sprouting of induced bud.
Embodiment 2：
Select the sprouting of Quercus acutissima base portion, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of diameter 0.2～0.3 cm as explant.By fiber crops
Oak coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 2%, seals bottleneck using gauze, puts
In rotating speed be 200 r/min shaking table on clean 10 min after be placed under flowing water and rinse 1.5 h, then use sterile water wash 2 times,
Explant is gone to volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min is used on superclean bench；Sterilized water is adopted to rush again
The stem section being cut into a length of 3.5 cm length after washing 8 ~ 10 times is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/3 improvement WPM culture medium+6-BA 1.5mg/L+ vitamin
C10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are：NH4NO3780
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, inositol 100 mg/L, nicotinic acid 1.0 mg/L, pyridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L are sweet
Propylhomoserin 2.0 mg/L.
Postvaccinal explant is placed in 20 ± 1 DEG C of temperature, humidity switchs to illumination for after 50 % interior light culture 10d
500lx, is cultivated under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 3：
Select the sprouting of Quercus acutissima base portion, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of diameter 0.3～0.4 cm as explant.By fiber crops
Oak coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 4%, seals bottleneck using gauze, puts
In rotating speed be 200 r/min shaking table on clean 15 min after be placed under flowing water and rinse 1 h, then use sterile water wash 2 times, will
Explant goes to uses volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min on superclean bench；Adopt aseptic water washing again
The stem section being cut into a length of 4.0 cm length after 8 ~ 10 times is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/2 improvement WPM culture medium+6-BA 1.5mg/L+ vitamin
C10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are：NH4NO3780
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, inositol 100 mg/L, nicotinic acid 1.0 mg/L, pyridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L are sweet
Propylhomoserin 2.0 mg/L.
Postvaccinal explant is placed in 20 ± 1 DEG C of temperature, humidity switchs to illumination 1000 for after 55 % interior light culture 10d
Lx, is cultivated under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 4：
Select the sprouting of Quercus acutissima base portion, a length of 8 ~ 9cm, the coppice shoot of the semi-lignified of diameter 0.3～0.4 cm as explant.By fiber crops
Oak coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 5 %, seals bottleneck using gauze, puts
In rotating speed be 200 r/min shaking table on clean 15 min after be placed under flowing water flushing 1h, then use sterile water wash 2 times, will
Explant goes to uses volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min on superclean bench；Adopt aseptic water washing again
The stem section being cut into a length of 4.0 cm length after 8 ~ 10 times is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/2 improvement WPM culture medium+6-BA 2.0mg/L+ vitamin
C10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are：NH4NO3780
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, inositol 100 mg/L, nicotinic acid 1.0 mg/L, pyridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L are sweet
Propylhomoserin 2.0 mg/L.
Postvaccinal explant is placed in 20 ± 1 DEG C of temperature, humidity switchs to illumination 1000 for after 55 % interior light culture 10d
Lx, is cultivated under conditions of illumination 8h/d, the sprouting of induced bud.
Claims (6)
1. a kind of Quercus acutissima initial culture bud abductive approach it is characterised in that：It is explant using Quercus acutissima base portion coppice shoot, by explant
After body aseptic process, explant is inoculated on high-quality initial culture bud inducement cultivation base, is placed in suitable temperature, humidity and light
Cultivated according under strength condition, the sprouting of induced bud；Its operating procedure is,
（1）Explant selects：The coppice shoot selecting the semi-lignified of Quercus acutissima base portion sprouting is explant；
（2）Explant sterilization and inoculation：Quercus acutissima coppice shoot is cleaned through liquid detergent, flowing water rinses and chemical reagent processes sterilization
Afterwards, cut into stem section to access in bud inducement cultivation base；
（3）Bud inducement cultivation：Postvaccinal explant is placed in culture under suitable indoor conditionss.
2. a kind of Quercus acutissima initial culture bud abductive approach according to claim 1 it is characterised in that：Described bud induction training
Its material content of foster base is：1/3～1/2 improvement WPM culture medium+6-BA 1.0～2.0mg/L+ vitamin C 10mg/L+ agar
5.0g/L+ sucrose 20.0g/L.
3. a kind of Quercus acutissima initial culture bud abductive approach according to claim 2 it is characterised in that：Described improvement WPM
Nutrient media components and envelope-bulk to weight ratio are：NH4NO3780 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300
Mg/L, KH2PO4270 mg/L, Ca (NO3)2·4H2O 180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·
H2O 22.4 mg/L, ZnSO4·7H2O 8.6 mg/L, Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/
L, Na2EDTA 37.3 mg/L, FeSO4·7H2O 27.8 mg/L, inositol 100 mg/L, nicotinic acid 1.0 mg/L, hydrochloric acid pyrrole is trembled
Alcohol 1.0 mg/L, thiamine hydrochloride 2.0 mg/L, glycine 2.0 mg/L.
4. a kind of Quercus acutissima initial culture bud abductive approach according to claim 1 it is characterised in that：Described coppice shoot is long
Coppice shoot for 6 ~ 9cm, the semi-lignified of diameter 0.2～0.4 cm.
5. a kind of Quercus acutissima initial culture bud abductive approach according to claim 1 it is characterised in that：Step（2）Described
Sterilization and inoculation method are to cut off Quercus acutissima coppice shoot needle, are placed in triangular flask, and the liquid detergent adding volumetric concentration to be 1 ~ 5 % is molten
Liquid, seals bottleneck using gauze, is placed under flowing water and rinses 1 after being placed in cleaning 10 ~ 15 min on the shaking table that rotating speed is 200 r/min
~ 2 h, then use sterile water wash 2 times, explant is gone to and uses volumetric concentration 0.1% mercuric chloride solution to soak on superclean bench
Sterilize 10 min；The stem section being cut into a length of 3.5 ~ 4.0 cm length after adopting aseptic water washing 8 ~ 10 times again is inoculated in bud inducement cultivation
In base, 2 stem sections of every bottle of inoculation.
6. a kind of Quercus acutissima initial culture bud abductive approach according to claim 1 it is characterised in that：Step（3）Described
Suitably indoor conditionss are：Illumination 500～1000 lx, illumination 8～12h/d is switched to after light culture 10d；20 ± 1 DEG C of cultivation temperature,
Humidity is 50 ~ 55 %.
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