CN106172017A - A kind of cork oak initial culture bud inducement method - Google Patents

A kind of cork oak initial culture bud inducement method Download PDF

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Publication number
CN106172017A
CN106172017A CN201610900779.1A CN201610900779A CN106172017A CN 106172017 A CN106172017 A CN 106172017A CN 201610900779 A CN201610900779 A CN 201610900779A CN 106172017 A CN106172017 A CN 106172017A
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Prior art keywords
cork oak
outer implant
bud inducement
bud
culture
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CN201610900779.1A
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Chinese (zh)
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黄文卫
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Liuzhou Tinkling Of Pieces Of Jade Leads To Science And Technology Ltd Co
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Liuzhou Tinkling Of Pieces Of Jade Leads To Science And Technology Ltd Co
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Priority to CN201610900779.1A priority Critical patent/CN106172017A/en
Publication of CN106172017A publication Critical patent/CN106172017A/en
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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
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • 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 kind of cork oak initial culture bud inducement method, it is characterized in that: using cork oak base portion coppice shoot is outer implant, after outer implant aseptic process, outer implant is inoculated in high-quality initial culture bud inducement culture medium, cultivate under the conditions of being placed in suitable temperature, humidity and intensity of illumination, the sprouting of induced bud.The present invention overcomes the problem such as Explant browning and vitrification during bud inducement, improving bud induction rate, sprout index and sprout growth situation, acquisition quantity is many, the measured rudiment of matter, thus meet the needs of enrichment culture, promote the foundation of cork oak tissue-culturing quick-propagation system.

Description

A kind of cork oak initial culture bud inducement method
Technical field
The present invention relates to cork oak vegetative propagation technique, especially a kind of cork oak initial culture bud inducement method.
Background technology
Cork oak (Quercus variabilis Bl) have another name called cork oak, rough bark Qinggang, hemp oak, be Fagaceae (Fagaceae) oak belongs to arbor, and tree crown is the most avette, and trunk is many, taupe, deep lobe, and phellem layer spy is thick.Sprig light brown Color, staminate inflorescence is born in annual shoot bottom, the life of female flower list or twin and annual shoot axil.May at florescence；Really 9-10 in the next year month becomes Ripe.It is Chinese important commerical tree species.Cork oak happiness light, is often born in mountain region tailo, but treelet is to have preferably lateral shelter.To gas Wait, the strong adaptability of soil.It is resistant to the low temperature of-20 DEG C, in the acidity of pH4-8, neutrality and calacareous soil, all has growth, also Drought-resistant, barren and with loam deep, fertile, the most moistening and well-drained and sandy loam optimum, be weak to hydrops. Produce Liaoning, Hebei, Shanxi, Shaanxi, Gansu, Shandong, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Henan, Hubei, Hunan, The provinces and regions such as Guangdong, Guangxi, Sichuan, Guizhou, Yunnan.The tailo of height above sea level less than 800 meters is generally born in by North China, and southwest can Reach height above sea level 2000-3000 rice.
At present, the planting type that cork oak is common is afforestation seeding.Tissue culture technique is a kind of fast asexual propagation skill Art, selecting cork oak superior families or clone is material, breeds nursery stock by method for tissue culture, both can meet production On quantitative requirement, maternal character can be kept again.In tissue culture procedures, initial culture restricts tissue culture and is smoothed out Committed step, has been directed to cultivate body in the acquisition of outer implant, the sterilization of outer implant, culture medium selection and incubation brown The major issues such as change, nursery stock vitrification.
Summary of the invention
It is an object of the invention to provide one and can overcome the problem such as Explant browning and vitrification during bud inducement, Improving bud induction rate, sprout index and sprout growth situation, acquisition quantity is many, the measured rudiment of matter, thus meets propagation training The needs supported, promote the cork oak initial culture bud inducement method of the foundation of cork oak tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows:
A kind of cork oak initial culture bud inducement method, using cork oak base portion coppice shoot is outer implant, by the aseptic place of outer implant After reason, outer implant is inoculated in high-quality initial culture bud inducement culture medium, is placed in suitable temperature, humidity and intensity of illumination bar Cultivate under part, the sprouting of induced bud；Its operating procedure is,
(1) outer implant selects: the coppice shoot selecting the semi-lignified of cork oak base portion sprouting is outer implant；
(2) sterilization of outer implant and inoculation: cleaned through liquid detergent by cork oak coppice shoot, flowing water rinses and chemical reagent processes sterilization After, cut into stem section and access in bud inducement culture medium；
(3) bud inducement is cultivated: postvaccinal outer implant is placed under suitable indoor conditions cultivation.
Above-described its material content of bud inducement culture medium is: 1/3～1/2 improvement WPM culture medium+6-BA 1.0～ 2.0mg/L+ vitamin C 10mg/L+ glucose 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media components and envelope-bulk to weight ratio be: NH4NO3850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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 the coppice shoot of the semi-lignified of a length of 6 ~ 9cm, diameter 0.2～0.4 cm.
Sterilization and inoculation method described in above step (2) are to be cut off by cork oak coppice shoot needle, are placed in triangular flask, add Enter the liquid detergent solution that volumetric concentration is 1 ~ 5 %, use gauze to seal bottleneck, be placed in the shaking table supernatant that rotating speed is 200 r/min Wash 10 ~ 15 min and be placed under flowing water flushing 1 ~ 2 h, then use sterile water wash 2 times, outer implant is forwarded on superclean bench With volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；Be cut into after using aseptic water washing 8 ~ 10 times more a length of 3.5 ~ The stem section of 4.0 cm length is inoculated in bud inducement culture medium, 2 stem sections of every bottle graft kind.
Suitable indoor conditions described in above step (3) is: transfer illumination 500～1000 lx, illumination 8 after light culture 10d to ～12h/d；Cultivation temperature 20 ± 1 DEG C, humidity is 50 ~ 55 %.
The present invention has the advantage that and has the beneficial effect that:
1, the present invention uses 1/3～1/2 improvement WPM culture medium culturing base as minimal medium, and emphasis have adjusted and bolt simultaneously Skin oak sprouts relevant trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcomes cork The vitrification phenomenon easily occurred during oak tissue culture bud inducement, is more easy to promote the generation of cork oak bud inducement, promotes and sprout The speed of growth of bud and robustness.
2, the present invention is using the sturdy coppice shoot of semi-lignified of base portion length 6 ~ 9 cm length as outer implant, it is ensured that outer implant is stronger Meristematic capacity, promote bud induction；Carry out after present invention implant collection outside cleaning, sterilizing, effectively reduce the dirt of outer implant Dye rate, improves bud ratio.
3, postvaccinal outer implant is placed in temperature degree of being 20 ± 1 DEG C by the present invention, and humidity is 50 ~ 55%, and illumination is by light culture After forward under the indoor conditions that intensity is 500～1000 lx cultivate, temperature and humidity is on the low side, the indoor that intensity of illumination is the highest Condition, vitrified generation during advantageously reducing bud inducement.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1:
Select the coppice shoot of semi-lignified of the sprouting of cork oak base portion, a length of 6 ~ 7cm, diameter 0.2～0.3 cm as outer implant.Will Cork oak coppice shoot needle cuts off, and is placed in triangular flask, adds the liquid detergent solution that volumetric concentration is 1 %, uses gauze to seal bottle Mouthful, it is placed on the shaking table that rotating speed is 200 r/min and cleans 10 min and be placed under flowing water and rinse 2 h, then by sterile water wash 2 Secondary, outer implant is forwarded on superclean bench with volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；Use aseptic again Water is cut into the stem section of a length of 3.5 cm length and is inoculated in bud inducement culture medium after rinsing 8 ~ 10 times, 2 stem sections of every bottle graft kind.
Described its material content of bud inducement culture medium is: 1/3 improvement WPM culture medium+6-BA 1.0mg/L+ vitamin C10mg/L+ glucose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are: NH4NO3 850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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, glycine 2.0 mg/L.
Postvaccinal outer implant is placed in temperature 20 ± 1 DEG C, and humidity is to transfer illumination 500 to after 50 % indoor light culture 10d Lx, cultivates under conditions of illumination 12h/d, the sprouting of induced bud.
Embodiment 2:
Select the coppice shoot of semi-lignified of the sprouting of cork oak base portion, a length of 7 ~ 8cm, diameter 0.2～0.3 cm as outer implant.Will Cork oak coppice shoot needle cuts off, and is placed in triangular flask, adds the liquid detergent solution that volumetric concentration is 2%, uses gauze to seal bottle Mouthful, it is placed on the shaking table that rotating speed is 200 r/min and cleans 10 min and be placed under flowing water and rinse 1.5 h, then clear with sterilized water Wash 2 times, outer implant is forwarded on superclean bench with volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；Use nothing again Bacterium water is cut into the stem section of a length of 3.5 cm length and is inoculated in bud inducement culture medium after rinsing 8 ~ 10 times, 2 stem sections of every bottle graft kind.
Described its material content of bud inducement culture medium is: 1/3 improvement WPM culture medium+6-BA 1.5mg/L+ vitamin C10mg/L+ glucose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are: NH4NO3 850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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, glycine 2.0 mg/L.
Postvaccinal outer implant is placed in temperature 20 ± 1 DEG C, and humidity is to transfer illumination to after 50 % indoor light culture 10d 500lx, cultivates under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 3:
Select the coppice shoot of semi-lignified of the sprouting of cork oak base portion, a length of 7 ~ 8cm, diameter 0.3～0.4 cm as outer implant.Will Cork oak coppice shoot needle cuts off, and is placed in triangular flask, adds the liquid detergent solution that volumetric concentration is 4%, uses gauze to seal bottle Mouthful, it is placed on the shaking table that rotating speed is 200 r/min and cleans 15 min and be placed under flowing water and rinse 1 h, then by sterile water wash 2 Secondary, outer implant is forwarded on superclean bench with volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；Use aseptic again Water is cut into the stem section of a length of 4.0 cm length and is inoculated in bud inducement culture medium after rinsing 8 ~ 10 times, 2 stem sections of every bottle graft kind.
Described its material content of bud inducement culture medium is: 1/2 improvement WPM culture medium+6-BA 1.5mg/L+ vitamin C10mg/L+ glucose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are: NH4NO3 850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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, glycine 2.0 mg/L.
Postvaccinal outer implant is placed in temperature 20 ± 1 DEG C, and humidity is to transfer illumination 1000 to after 55 % indoor light culture 10d Lx, cultivates under conditions of illumination 10h/d, the sprouting of induced bud.
Embodiment 4:
Select the coppice shoot of semi-lignified of the sprouting of cork oak base portion, a length of 8 ~ 9cm, diameter 0.3～0.4 cm as outer implant.Will Cork oak coppice shoot needle cuts off, and is placed in triangular flask, adds the liquid detergent solution that volumetric concentration is 5 %, uses gauze to seal bottle Mouthful, it is placed on the shaking table that rotating speed is 200 r/min and cleans 15 min and be placed under flowing water flushing 1h, then by sterile water wash 2 Secondary, outer implant is forwarded on superclean bench with volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；Use aseptic again Water is cut into the stem section of a length of 4.0 cm length and is inoculated in bud inducement culture medium after rinsing 8 ~ 10 times, 2 stem sections of every bottle graft kind.
Described its material content of bud inducement culture medium is: 1/2 improvement WPM culture medium+6-BA 2.0mg/L+ vitamin C10mg/L+ glucose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components and envelope-bulk to weight ratio are: NH4NO3 850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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, glycine 2.0 mg/L.
Postvaccinal outer implant is placed in temperature 20 ± 1 DEG C, and humidity is to transfer illumination 1000 to after 55 % indoor light culture 10d Lx, cultivates under conditions of illumination 8h/d, the sprouting of induced bud.

Claims (6)

1. a cork oak initial culture bud inducement method, it is characterised in that: using cork oak base portion coppice shoot is outer implant, passes through After outer implant aseptic process, outer implant is inoculated in high-quality initial culture bud inducement culture medium, is placed in suitable temperature, humidity Cultivate with under the conditions of intensity of illumination, the sprouting of induced bud；Its operating procedure is,
(1) outer implant selects: the coppice shoot selecting the semi-lignified of cork oak base portion sprouting is outer implant；
(2) sterilization of outer implant and inoculation: cleaned through liquid detergent by cork oak coppice shoot, flowing water rinses and chemical reagent processes sterilization After, cut into stem section and access in bud inducement culture medium；
(3) bud inducement is cultivated: postvaccinal outer implant is placed under suitable indoor conditions cultivation.
A kind of cork oak initial culture bud inducement method the most according to claim 1, it is characterised in that: described bud inducement Its material content of culture medium is: 1/3～1/2 improvement WPM culture medium+6-BA 1.0～2.0mg/L+ vitamin C 10mg/L+ Fructus Vitis viniferae Sugar 5.0g/L+ sucrose 20.0g/L.
A kind of cork oak initial culture bud inducement method the most according to claim 2, it is characterised in that: described improvement WPM nutrient media components and envelope-bulk to weight ratio be: NH4NO3850 mg/L, CaCl2·2H2O 192 mg/L, MgSO4·7H20 320 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, ZnSO4·7H2O 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, glycine 2.0 mg/L.
A kind of cork oak initial culture bud inducement method the most according to claim 1, it is characterised in that: described coppice shoot is A length of 6 ~ 9cm, the coppice shoot of semi-lignified of diameter 0.2～0.4 cm.
A kind of cork oak initial culture bud inducement method the most according to claim 1, it is characterised in that: step (2) is described Sterilization and inoculation method be that cork oak coppice shoot needle is cut off, be placed in triangular flask, add volumetric concentration be that washing of 1 ~ 5 % is clean Essence solution, uses gauze to seal bottleneck, is placed on the shaking table that rotating speed is 200 r/min cleaning 10 ~ 15 min and is placed under flowing water Rinse 1 ~ 2 h, then use sterile water wash 2 times, outer implant is forwarded on superclean bench molten with volumetric concentration 0.1% mercuric chloride Liquid soaking disinfection 10 min；It is cut into the stem section of a length of 3.5 ~ 4.0 cm length after using aseptic water washing 8 ~ 10 times again to be inoculated in bud and lure Lead in culture medium, 2 stem sections of every bottle graft kind.
A kind of cork oak initial culture bud inducement method the most according to claim 1, it is characterised in that: step (3) is described Suitable indoor conditions be: transfer illumination 500～1000 lx, illumination 8～12h/d after light culture 10d to；Cultivation temperature 20 ± 1 DEG C, humidity is 50 ~ 55 %.
CN201610900779.1A 2016-10-17 2016-10-17 A kind of cork oak initial culture bud inducement method Pending CN106172017A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘淑莹: "《增强自主创新能力 促进吉林经济发展》", 31 October 2006 *
宋敏: "栓皮栎离体培养再生植株的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
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