CN106613947A - Primarily-cultured bud inducing method of Lithocarpus craibianus - Google Patents

Primarily-cultured bud inducing method of Lithocarpus craibianus Download PDF

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Publication number
CN106613947A
CN106613947A CN201610900769.8A CN201610900769A CN106613947A CN 106613947 A CN106613947 A CN 106613947A CN 201610900769 A CN201610900769 A CN 201610900769A CN 106613947 A CN106613947 A CN 106613947A
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explant
bud
dead ears
culture
coppice shoot
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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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    • 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

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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses a primarily cultured bud inducing method of Lithocarpus craibianus. The method is characterized in that a stump plant of the stem base of Lithocarpus craibianus is taken as an explant, a primarily cultured bud inducing medium is inoculated with the explant after the explant is treated aseptically and placed under the proper temperature, humidity and illumination intensity conditions for culture, and germination of buds is induced. By means of the method, the problems that the explant browns, vitrifies and the like in the bud inducing process are solved, the bud inducing rate is increased, the budding index and the sprout growth condition are improved, and a large number of high-quality buds are obtained, so that the demand of multiplication culture is met, and establishment of a rapid propagation system of tissue culture of Lithocarpus craibianus is promoted.

Description

A kind of dead ears Ke Initial culture bud abductive approach
Technical field
The present invention relates to dead ears Ke's vegetative propagation technique, especially a kind of dead ears Ke Initial culture bud abductive approach.
Background technology
Dead ears Ke（Lithocarpus craibianus Barn.）, it is Fagaceae（Fagaceae）Lithocarpus （Lithocarpus）Arbor, arbor, up to 20 meters.Annual shoot, blade back and female inflorescence axle have brown color or canescence wax squama Layer.Leaf keratin, avette or ovate-elliptic is long 12-19 centimetre, sometimes shorter or longer, wide 4-7 centimetre, and dilute wider, top is long Point, the short point of base portion to wide wedge shape, full edge, the hypomere of middle arteries is thick on blade face, and vein there are about 8-12 bars, often recessed in channel-shaped is split on blade face Fall into, offshoot does not show or mays be seen indistinctly, often there is glossy gloss on blade face after tender leaf is dry, biennial leaf is then grey dark；Petiole is long 1-2.5 li Rice.Tassel locusta armpit is given birth to, dilute for panicle, up to 15 centimetres；The top of female inflorescence often raw minority male flower, up to 30 lis Rice, 3-5-7 collection of female flower generates cluster, the long 1-1.2 millimeters of style.Acorn-cup spheroidal is slightly flat, and top is in often mamillary short projection, Footpath 15-20 millimeters, full bag nut occasionally has top cracking and warp, therefore nut part is exposed, squamella triangle, apex point Shape, fits perfectly in shell wall, imbricate arrangement, clearly, slightly long and narrow at the top of acorn-cup and to curved under acorn-cup oral area, by yellowish-brown, Fine platy, the wax squama being slightly close to；The nearly spheroidal of nut, footpath 13-18 millimeters, by very sparse thin volt hair, the hair at top is closeer, fruit Navel is raised, accounts for the 1/3 of nut area.The month at florescence 8-9, fruit same period next year is ripe.Produce South-west Sichuan, south of Yunnan and the west and south Various places.In being born in 1 500-2,700 meters of mountain region shaws, it is more common in and be relatively dried hillside fields, Chang Yin is cut down, therefore generates shrub shape, Usually about 10 meters of dungarunga.Laos, Northern Thailand also have.
At present, dead ears Ke resource is still in wild distribution, but with keeping the demand of Plant Diversity, scale people Work cultivation dead ears Ke's resource is imperative.Tissue culture technique is a kind of fast asexual propagation technology, selects dead ears 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 dead ears Ke's Initial culture bud abductive approach of the foundation of dead ears Ke's tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows：
A kind of dead ears Ke Initial culture bud abductive approach, adopts dead ears 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 dead ears Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Dead ears 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+ vitamin C 10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media componentses and envelope-bulk to weight ratio are：NH4NO3810 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 dead ears 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 vain Fringe Ke sprout correlation trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcomes dead ears The vitrification phenomenon easily occurred in Ke's tissue cultures bud Induction Process, is more easy to the generation for promoting dead ears 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：
Dead ears 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 Dead ears 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+ vitamins C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 810 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：
Dead ears 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 Dead ears 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+ vitamins C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 810 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：
Dead ears 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 Dead ears 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+ vitamins C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 810 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：
Dead ears 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 Dead ears 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+ vitamins C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improve WPM nutrient media componentses and envelope-bulk to weight ratio is：NH4NO3 810 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 dead ears Ke Initial culture bud abductive approach, it is characterised in that：Adopt dead ears 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 dead ears Ke base portion sprouting is explant；
（2）Explant is sterilized and is inoculated with：Dead ears 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 dead ears 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+ vitamin C 10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.
3. a kind of dead ears 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：NH4NO3810 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 dead ears 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 dead ears 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 dead ears 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 dead ears 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 %.
CN201610900769.8A 2016-10-17 2016-10-17 Primarily-cultured bud inducing method of Lithocarpus craibianus Pending CN106613947A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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