CN106613946A - Primary culture bud inducing method of lithocarpus oleaefolius - Google Patents
Primary culture bud inducing method of lithocarpus oleaefolius Download PDFInfo
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- CN106613946A CN106613946A CN201610900766.4A CN201610900766A CN106613946A CN 106613946 A CN106613946 A CN 106613946A CN 201610900766 A CN201610900766 A CN 201610900766A CN 106613946 A CN106613946 A CN 106613946A
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- bud
- explant
- olive leaf
- culture
- coppice shoot
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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
-
- 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
Abstract
The invention discloses a primary culture bud inducing method of lithocarpus oleaefolius. The primary culture bud inducing method is characterized by being prepared by applying root coppice shoot of lithocarpus oleaefolius as an explant; after the asepsis of the explant, inoculating the explant to an inducing culture base of a high-quality primary culture bud; and culturing the explant under suitable temperature, humidity and light intensity, and inducing the bud germination. The method overcomes the problems of explant brown stain, vitrification and other problems during the bud inducing process, improves the bud inducing rate, budding rate and bud growth status, and acquires buds with large amount and high quality, and thereby meeting the demand of multiplication culture and promoting the setup of a rapid propagation system of the lithocarpus oleaefolius tissue culture.
Description
Technical field
The present invention relates to olive leaf Ke's vegetative propagation technique, especially a kind of olive leaf Ke Initial culture bud abductive approach.
Background technology
Lan Yeke（Lithocarpus oleaefoliusA. Camus）Also known as olive leaf lithocarpus glaber, olive leaf C.-glauca, it is Fagaceae
（Fagaceae）Lithocarpus（Lithocarpus）Arbor, high 8～15 meters, perula is several without hair, annual shoot, petiole, blade back and inflorescence
By caducous brown rust or the long pubescence of brown color, the dun black of biennial branch, hole skin is very little, is difficult to examine and sees for axle.Bark dull gray
To grey black, do not ftracture, the bark that about 13 meters of the height of tree is thick about 8 millimeters, and endothelium reddish tan, ridge rib is significantly projected.Fruit is containing shallow lake
Powder is 50%～60%.It is distributed in Jiangxi, Fujian, Hunan, Guizhou Si Sheng south, Guangdong, Guangxi.It is born in 500～1200 meters of height above sea level
In the shaw of mountain region.
At present, olive leaf Ke resource is still in wild distribution, but with keeping the demand of Plant Diversity, scale people
Work cultivation olive leaf Ke's resource is imperative.Tissue culture technique is a kind of fast asexual propagation technology, selects olive leaf Ke You respectable family
System or clone are material, and by method for tissue culture nursery stock is bred, and can both meet the quantitative requirement in production, can be protected again
Hold maternal character.In tissue culture procedures, Initial culture is to restrict the committed step that tissue cultures are smoothed out, and is directed to
It is important body brown stain, nursery stock vitrifying etc. to be cultivated in the acquisition of explant, the sterilization of explant, culture medium selection and incubation
Problem.
The 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 vitrifying,
Raising bud induction rate, sprout index and sprout growth situation, acquisition quantity is more, the measured rudiment of matter, so as to meet propagation training
Foster needs, promote olive leaf Ke's Initial culture bud abductive approach of the foundation of olive leaf Ke's tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows：
A kind of olive leaf Ke Initial culture bud abductive approach, adopts olive leaf Ke's base portion coppice shoot for explant, by the aseptic place of explant
After reason, explant is inoculated on high-quality Initial culture bud inducement cultivation base, is placed in suitable temperature, humidity and intensity of illumination bar
Cultivated under part, the sprouting of induced bud；Its operating procedure is,
（1）Explant is selected：The coppice shoot for selecting the semi-lignified of olive leaf Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Olive leaf Ke coppice shoot is cleaned through liquid detergent, flowing water is rinsed 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 under suitable indoor conditions and is cultivated.
Above-described its material content of bud inducement cultivation base is：1/3～1/2 improvement WPM culture mediums+6-KT 1.0～
2.0mg/L+ activated carbon 10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media componentses and envelope-bulk to weight ratio are：NH4NO3820 mg/L, CaCl2·2H2O
192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2The mg/L of O 180, 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, FeSO4·7H2The mg/L of O 27.8, the mg/ of inositol 85
L, the mg/L of nicotinic acid 1.0, the mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0, the mg/L of glycine 2.0.
Above-described coppice shoot is a length of 6 ~ 9cm, the coppice shoot of the semi-lignified of the cm of diameter 0.2～0.4.
Above step（2）Described sterilization and inoculation method is to cut off olive leaf Ke's coppice shoot needle, in being placed in triangular flask, plus
Enter the liquid detergent solution that volumetric concentration is 1 ~ 5 %, bottleneck is sealed using gauze, be placed in the shaking table supernatant that rotating speed is 200 r/min
Wash and 1 ~ 2 h of flushing is placed under flowing water after 10 ~ 15 min, then with sterile water wash 2 times, explant is gone on superclean bench
With the min of 0.1% mercuric chloride solution soaking disinfection of volumetric concentration 10；Again using being cut into a length of 3.5 after aseptic water washing 8 ~ 10 times ~
The stem section of 4.0 cm length is inoculated in bud inducement cultivation base, 2 stem sections of per bottle of inoculation.
Above step（3）Described suitable indoor conditions is：Switch to the lx of illumination 500～1000, illumination 8 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 improves WPM medium culture bases as minimal medium using 1/3～1/2, while emphasis have adjusted and olive
Ye Ke sprout correlation trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcomes olive leaf
The vitrification phenomenon easily occurred in Ke's tissue cultures bud Induction Process, is more easy to the generation for promoting olive leaf Ke bud to induce, and promotes and sprouts
The speed of growth and robustness of bud.
2nd, the present invention is using the sturdy coppice shoot of semi-lignified of the cm length of base portion length 6 ~ 9 as explant, it is ensured that explant is stronger
Meristematic capacity, promote bud induction；The present invention is cleaned after explant collection, is sterilized, and effectively reduces the dirt of explant
Dye rate, improves bud ratio.
3rd, postvaccinal explant is placed in temperature to spend 20 ± 1 DEG C by the present invention, and humidity is 50 ~ 55%, and illumination is by light culture
Go to afterwards and cultivated under the indoor conditions that intensity is 500～1000 lx, 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：
Olive leaf Ke base portion sprouting, a length of 6 ~ 7cm, the coppice shoot of the semi-lignified of the cm of diameter 0.2～0.3 are selected as explant.Will
Olive leaf Ke's coppice shoot needle cuts off, and in being placed in triangular flask, the liquid detergent solution for adding volumetric concentration to be 1 % seals bottle using gauze
Mouthful, it is placed on the shaking table that rotating speed is 200 r/min to clean to be placed in after 10 min under flowing water and rinses 2 h, then with sterile water wash 2
It is secondary, explant is gone to and use on superclean bench the min of 0.1% mercuric chloride solution soaking disinfection of volumetric concentration 10；Again using aseptic
Water is cut into the stem section of a length of 3.5 cm length and is inoculated in bud inducement cultivation base after rinsing 8 ~ 10 times, 2 stem sections of per bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/3 improvement WPM culture medium+6-KT 1.0mg/L+ activated carbons
10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 820
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2The mg/L of O 8.6,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, the mg/L of inositol 100, the mg/L of nicotinic acid 1.0, the mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0 are sweet
The mg/L of propylhomoserin 2.0.
Postvaccinal explant is placed in into 20 ± 1 DEG C of temperature, humidity is to switch to illumination 500 after light culture 10d in 50 % rooms
Lx, is cultivated under conditions of illumination 12h/d, the sprouting of induced bud.
Embodiment 2：
Olive leaf Ke base portion sprouting, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of the cm of diameter 0.2～0.3 are selected as explant.Will
Olive leaf Ke's coppice shoot needle cuts off, and in being placed in triangular flask, the liquid detergent solution for adding volumetric concentration to be 2% seals bottle using gauze
Mouthful, it is placed on the shaking table that rotating speed is 200 r/min to clean to be placed in after 10 min under flowing water and rinses 1.5 h, it is then clear with sterilized water
Wash 2 times, explant is gone to and use on superclean bench the min of 0.1% mercuric chloride solution soaking disinfection of volumetric concentration 10；Nothing is adopted again
Bacterium water is cut into the stem section of a length of 3.5 cm length and is inoculated in bud inducement cultivation base after rinsing 8 ~ 10 times, 2 stem sections of per bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/3 improvement WPM culture medium+6-KT 1.5mg/L+ activated carbons
10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 820
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2The mg/L of O 8.6,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, the mg/L of inositol 100, the mg/L of nicotinic acid 1.0, the mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0 are sweet
The mg/L of propylhomoserin 2.0.
Postvaccinal explant is placed in into 20 ± 1 DEG C of temperature, humidity is to switch to illumination after light culture 10d in 50 % rooms
500lx, is cultivated under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 3：
Olive leaf Ke base portion sprouting, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of the cm of diameter 0.3～0.4 are selected as explant.Will
Olive leaf Ke's coppice shoot needle cuts off, and in being placed in triangular flask, the liquid detergent solution for adding volumetric concentration to be 4% seals bottle using gauze
Mouthful, it is placed on the shaking table that rotating speed is 200 r/min to clean to be placed in after 15 min under flowing water and rinses 1 h, then with sterile water wash 2
It is secondary, explant is gone to and use on superclean bench the min of 0.1% mercuric chloride solution soaking disinfection of volumetric concentration 10；Again using aseptic
Water is cut into the stem section of a length of 4.0 cm length and is inoculated in bud inducement cultivation base after rinsing 8 ~ 10 times, 2 stem sections of per bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/2 improvement WPM culture medium+6-KT 1.5mg/L+ activated carbons
10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 820
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2The mg/L of O 8.6,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, the mg/L of inositol 100, the mg/L of nicotinic acid 1.0, the mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0 are sweet
The mg/L of propylhomoserin 2.0.
Postvaccinal explant is placed in into 20 ± 1 DEG C of temperature, humidity is to switch to illumination 1000 after light culture 10d in 55 % rooms
Lx, is cultivated under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 4：
Olive leaf Ke base portion sprouting, a length of 8 ~ 9cm, the coppice shoot of the semi-lignified of the cm of diameter 0.3～0.4 are selected as explant.Will
Olive leaf Ke's coppice shoot needle cuts off, and in being placed in triangular flask, the liquid detergent solution for adding volumetric concentration to be 5 % seals bottle using gauze
Mouthful, it is placed on the shaking table that rotating speed is 200 r/min to clean to be placed in after 15 min under flowing water and rinses 1h, then with sterile water wash 2
It is secondary, explant is gone to and use on superclean bench the min of 0.1% mercuric chloride solution soaking disinfection of volumetric concentration 10；Again using aseptic
Water is cut into the stem section of a length of 4.0 cm length and is inoculated in bud inducement cultivation base after rinsing 8 ~ 10 times, 2 stem sections of per bottle of inoculation.
Described its material content of bud inducement cultivation base is：1/2 improvement WPM culture medium+6-KT 2.0mg/L+ activated carbons
10mg/L+ agar 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 820
Mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2O
180 mg/L, KI 0.83 mg/L, H3BO36.2 mg/L, MnSO4·H2O 22.4 mg/L, ZnSO4·7H2The mg/L of O 8.6,
Na2MoO4·2H2O 0.25 mg/L, CuSO4·5H2O 0.25 mg/L, Na2EDTA 37.3 mg/L, FeSO4·7H2O
27.8 mg/L, the mg/L of inositol 100, the mg/L of nicotinic acid 1.0, the mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0 are sweet
The mg/L of propylhomoserin 2.0.
Postvaccinal explant is placed in into 20 ± 1 DEG C of temperature, humidity is to switch to illumination 1000 after light culture 10d in 55 % rooms
Lx, is cultivated under conditions of illumination 8h/d, the sprouting of induced bud.
Claims (6)
1. a kind of olive leaf Ke Initial culture bud abductive approach, it is characterised in that：Adopt olive leaf Ke's base portion coppice shoot for explant, pass through
After explant aseptic process, explant is inoculated on high-quality Initial culture bud inducement cultivation base, is placed in suitable temperature, humidity
Cultivated with the conditions of intensity of illumination, the sprouting of induced bud；Its operating procedure is,
（1）Explant is selected：The coppice shoot for selecting the semi-lignified of olive leaf Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Olive leaf Ke coppice shoot is cleaned through liquid detergent, flowing water is rinsed 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 under suitable indoor conditions and is cultivated.
2. a kind of olive leaf Ke Initial culture bud abductive approach according to claim 1, it is characterised in that：Described bud induction
Culture medium its material content is：1/3～1/2 improvement WPM culture medium+6-KT 1.0～2.0mg/L+ activated carbon 10mg/L+ agar
5.0g/L+ sucrose 20.0g/L.
3. a kind of olive leaf Ke Initial culture bud abductive approach according to claim 2, it is characterised in that：Described improvement
WPM nutrient media componentses and envelope-bulk to weight ratio are：NH4NO3820 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20
300 mg/L, KH2PO4250 mg/L, Ca (NO3)2·4H2The mg/L of O 180, 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, FeSO4·7H2The mg/L of O 27.8, the mg/L of inositol 100, the mg/L of nicotinic acid 1.0,
The mg/L of puridoxine hydrochloride 1.0, the mg/L of thiamine hydrochloride 2.0, the mg/L of glycine 2.0.
4. a kind of olive leaf Ke Initial culture bud abductive approach according to claim 1, it is characterised in that：Described coppice shoot is
The coppice shoot of a length of 6 ~ 9cm, the semi-lignified of the cm of diameter 0.2～0.4.
5. a kind of olive leaf Ke Initial culture bud abductive approach according to claim 1, it is characterised in that：Step（2）It is described
Sterilization and inoculation method be cut off olive leaf Ke's coppice shoot needle, in being placed in triangular flask, add volumetric concentration to wash clean for 1 ~ 5 %
Smart solution, using gauze bottleneck is sealed, and is placed on the shaking table that rotating speed is 200 r/min and is placed under flowing water after 10 ~ 15 min of cleaning
1 ~ 2 h is rinsed, then with sterile water wash 2 times, explant is gone to molten with the mercury chloride of volumetric concentration 0.1% on superclean bench
The min of liquid soaking disinfection 10；It is inoculated in bud using the stem section that a length of 3.5 ~ 4.0 cm length is cut into after aseptic water washing 8 ~ 10 times again to lure
In leading culture medium, 2 stem sections of per bottle of inoculation.
6. a kind of olive leaf Ke Initial culture bud abductive approach according to claim 1, it is characterised in that：Step（3）It is described
Suitable indoor conditions be：Switch to the lx of illumination 500～1000,8～12h/d of illumination after light culture 10d；Cultivation temperature 20 ± 1
DEG C, humidity is 50 ~ 55 %.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283598A (en) * | 2013-05-23 | 2013-09-11 | 广西壮族自治区林业科学研究院 | Primary fir wood culture bud induction method |
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- 2016-10-17 CN CN201610900766.4A patent/CN106613946A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283598A (en) * | 2013-05-23 | 2013-09-11 | 广西壮族自治区林业科学研究院 | Primary fir wood culture bud induction method |
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Application publication date: 20170510 |