CN106613948A - An initial culture bud inducing method for lithocarpus carolinae - Google Patents
An initial culture bud inducing method for lithocarpus carolinae Download PDFInfo
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- CN106613948A CN106613948A CN201610903545.2A CN201610903545A CN106613948A CN 106613948 A CN106613948 A CN 106613948A CN 201610903545 A CN201610903545 A CN 201610903545A CN 106613948 A CN106613948 A CN 106613948A
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- bud
- explant
- red heart
- initial culture
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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
An initial culture bud inducing method for lithocarpus carolinae is disclosed. The method is characterized in that lithocarpus carolinae root coppice shoots are adopted as explants, and the explants are subjected to aseptic processing, then inoculated to a high-quality initial culture bud inducing medium, and cultured under conditions including a proper temperature, proper humidity and proper illumination intensity to induce bud germination. The method overcomes problems such as explant browning and vitrification in a bud inducing process, increases the bud inducing rate, the budding index and bud growth statuses, and can obtain a high number of high-quality buds, thus meeting requirements of multiplication culture and promoting establishment of a tissue culture rapid multiplication system for the lithocarpus carolinae.
Description
Technical field
The present invention relates to red heart Ke's vegetative propagation technique, especially a kind of red heart Ke Initial culture bud abductive approach.
Background technology
Red heart Ke（Lithocarpus carolinae(Skan)Rehd）, it is Fagaceae（Fagaceae）Lithocarpus
（Lithocarpus）Arbor, high about 20 meters, 40 centimetres of the diameter of a cross-section of a tree trunk 1.3 meters above the ground, top perula triangular shape long lanceolar, current growth has vertical rib, does
Black dull brown afterwards, biennial branch has scattered hole skin, and branch, perula, leaf are without hair.Leaf ground paper matter, Long Circle, dilute length of falling ovate is ellipse
Circle, long 13-18 centimetre, wide 4-6 centimetre, the short point of top shape of tail, base portion wedge point, middle arteries are flat in blade face microprotrusion, or epimere,
, per side 15-20 bars, sharp turn is upwards, gradually very thin and disappear at the increment of the leaf margin that goes directly, or nearly leaf margin, there is starlike at arteries and veins for lateral vein
Little feathering, leaf margin hypomere full edge, epimere bores dentation, and two sides is homochromy, crineous after doing；Petiole is long 1.5-2 centimetre.Acorn-cup is per 3 or 5
Individual cluster, ripe acorn-cup shallow discoid, high 10-15 millimeters, wide 30-40 millimeters, base portion has very short handle, and shell wall top is relatively thin, under
Portion is thick about 2.5 millimeters, encloses nut lower half, crineous after doing, slightly glossy, squamella width triangle, and base portion assumes diamond in shape, in
Centre ridge rib shape is longitudinally thickened, and is close to, imbricate arrangement；Nut is oblate, high 24-30 millimeters, wide 40-45 millimeters, fruit wall thickness 6-
10 millimeters, top flat, but central authorities are slightly concave sunken, crineous, without hair, slightly gloss, areola depth 2-4 millimeters, bore 25-30 millis
Rice, uneven, middle body slightly swells.The fruiting period 9-10 month.Red heart Ke produces south of Yunnan to the southeast（Mojiang, screen side, gold
It is flat）.In being born in height above sea level 1500-2000 rice mountain region shaw.
At present, red heart Ke resource is still in wild distribution, but with keeping the demand of Plant Diversity, scale people
Work cultivation red heart Ke's resource is imperative.Tissue culture technique is a kind of fast asexual propagation technology, selects red heart 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 red heart Ke's Initial culture bud abductive approach of the foundation of red heart Ke's tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows：
A kind of red heart Ke Initial culture bud abductive approach, adopts red heart 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 red heart Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Red heart 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-BA 1.0～
2.0mg/L+ activated carbon 10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media componentses and envelope-bulk to weight ratio are：NH4NO3850 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 red heart 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 using 1/3～1/2 improvement WPM medium culture bases as minimal medium, while emphasis have adjusted with it is red
Heart Ke sprout correlation trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcomes red heart
The vitrification phenomenon easily occurred in Ke's tissue cultures bud Induction Process, is more easy to the generation for promoting red heart 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：
Red heart 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
Red heart 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-BA 1.0mg/L+ activated carbons
10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 850
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：
Red heart 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
Red heart 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-BA 1.5mg/L+ activated carbons
10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 850
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：
Red heart 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
Red heart 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-BA 1.5mg/L+ activated carbons
10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 850
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：
Red heart 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
Red heart 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-BA 2.0mg/L+ activated carbons
10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 850
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 red heart Ke Initial culture bud abductive approach, it is characterised in that：Adopt red heart 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 red heart Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Red heart 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 red heart 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-BA 1.0～2.0mg/L+ activated carbon 10mg/L+ lactose
5.0g/L+ sucrose 20.0g/L.
3. a kind of red heart 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：NH4NO3850 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 red heart 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 red heart 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 red heart 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 red heart 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577952A (en) * | 2012-02-10 | 2012-07-18 | 中国林业科学研究院亚热带林业研究所 | Tissue culturing method for quercus virginiana |
| 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 |
-
2016
- 2016-10-17 CN CN201610903545.2A patent/CN106613948A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577952A (en) * | 2012-02-10 | 2012-07-18 | 中国林业科学研究院亚热带林业研究所 | Tissue culturing method for quercus virginiana |
| 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 |