CN106386496A - Primary culture shoot induction method for lithocarpus elmerrillii - Google Patents

Primary culture shoot induction method for lithocarpus elmerrillii Download PDF

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Publication number
CN106386496A
CN106386496A CN201610900768.3A CN201610900768A CN106386496A CN 106386496 A CN106386496 A CN 106386496A CN 201610900768 A CN201610900768 A CN 201610900768A CN 106386496 A CN106386496 A CN 106386496A
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explant
wanning
bud
culture
shoot
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CN201610900768.3A
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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 CN201610900768.3A priority Critical patent/CN106386496A/en
Publication of CN106386496A publication Critical patent/CN106386496A/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 primary culture shoot induction method for lithocarpus elmerrillii. The method is characterized in that a basal coppice shoot of lithocarpus elmerrillii is taken as explant and subjected to aseptic treatment, a high-quality primary culture shoot induction medium is inoculated with the explant, the explant is cultured under the conditions of proper temperature, humidity and illumination intensity for induction of germination. With the adoption of the method, the problems of browning, vitrification and the like of the explant during shoot induction are solved, the shoot induction rate, the germination index and the shoot growth condition are increased, a large number of shoots with good quality are obtained, the enrichment culture requirement is met, and construction of a rapid propagation system for tissue culture of lithocarpus elmerrillii is promoted.

Description

A kind of Wanning Ke's Initial culture bud abductive approach
Technical field
The present invention relates to Wanning Ke's vegetative propagation technique, a kind of especially Wanning Ke's Initial culture bud abductive approach.
Background technology
Wanning Ke（Lithocarpus elmerrillii）, it is Fagaceae（Fagaceae）Lithocarpus（Lithocarpus）Tall Wood, up to 25 meters, current growth has longitudinal furrow rib, no hair, crineous after doing, 2 or 3 years raw branch scattered canescence hole skins, perula semicircle Shape or wealthy triangle are glossy glossy after doing.The thin keratin of leaf, oblong or rare ovate-elliptic, long 10-17 centimetre, Wide 3-6 centimetre, top is tapering, base portion gradually long and narrow point, and along the downward near base portion of petiole, full edge, lateral vein every side 9-12 bar, in nearly leaf At edge, sharp turn is disappeared upwards, and offshoot is very thin, and blade back has the wax squama layer of consolidation, carries canescence after doing；Petiole is long 2-2.5 centimetre. Single of female flower scattered on rachis；Infructescence axle thick 6-7 millimeter, acorn-cup consor is in the top of infructescence axle, shallow bowl type, high 6-10 milli Rice, wide 17-25 millimeter, top is very thin, gradually substantially thickens to bottom, and wooden, base portion is narrow, in very short handle, encloses Nut base portion, squamella triangle, it is close to shell wall, the imbricate arrangement on top, very little, the usual adhesion of bottom is circlewise；Hard The nearly spheroidal of fruit and top is sharp, or for oblate, high 20-25 millimeter, high 25-30 millimeter, no hair, Chestnut, have vertical when knowing well Crackle, areola is located at bottom, deep about 2 millimeters, footpath 14-16 millimeter, and Guo Bi top thickness reaches 2.5 millimeters.The fruiting period 9-10 month.Produce Hainan The middle and south（Hang Luoshan）.It is born in height above sea level 500-750 rice evergreen broadleaf forest.
At present, Wanning Ke resource is still in wild distribution, but the demand with protection Plant Diversity, scale people Work cultivation Wanning Ke's resource is imperative.Tissue culture technique is a kind of fast asexual propagation technology, selects Wanning Ke You respectable family System or clone are material, breed nursery stock by method for tissue culture, both can meet the quantitative requirement in production, and can protect again Hold maternal character.In tissue culture procedures, Initial culture is the committed step that restriction tissue cultures are smoothed out, and is directed to In the acquisition of explant, the sterilization of explant, culture medium selection and incubation, culture body brown stain, nursery stock vitrifying etc. are important Problem.
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, 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 Wanning Ke's Initial culture bud abductive approach of the foundation of Wanning Ke's tissue-culturing quick-propagation system.
To achieve these goals, technical scheme is as follows：
A kind of Wanning Ke's Initial culture bud abductive approach, is explant using Wanning Ke's base portion coppice shoot, 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 selects：The coppice shoot selecting the semi-lignified of Wanning Ke's base portion sprouting is explant；
（2）Explant sterilization and inoculation：Wanning Ke's 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 conditions.
Above-described its material content of bud inducement cultivation base is：1/3～1/2 improvement WPM culture medium+6-KT 1.0～ 2.0mg/L+ vitamin C 10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.
Above-described improvement WPM nutrient media components 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)2·4H2O 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, puridoxine 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 Wanning Ke's coppice shoot needle, are placed in triangular flask, plus Enter the liquid detergent solution that volumetric concentration is 1 ~ 5 %, bottleneck is sealed using gauze, is placed in the shaking table supernatant that rotating speed is 200 r/min It is placed under flowing water flushing 1 ~ 2 h after washing 10 ~ 15 min, then use sterile water wash 2 times, explant is gone on superclean bench With volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min；It is cut into a length of 3.5 after adopting aseptic water washing 8 ~ 10 times again ~ The stem section of 4.0 cm length is inoculated in bud inducement cultivation base, 2 stem sections of every bottle of inoculation.
Above step（3）Described suitable indoor conditions is：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 medium culture base as minimal medium, and emphasis have adjusted and ten thousand simultaneously Ning Ke sprout correlation trace element and organic principle so that culture medium is more scientific, more targetedly, effectively overcome Wanning The vitrification phenomenon easily occurring in Ke's tissue cultures bud Induction Process, is more easy to promote the generation of Wanning Ke's bud induction, promotes and sprout The speed of growth of bud 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 conditions 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 Wanning Ke's base portion sprouting, a length of 6 ~ 7cm, the coppice shoot of the semi-lignified of diameter 0.2～0.3 cm as explant.Will Wanning Ke's coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 1 %, seals bottle using gauze Mouthful, it is placed in be placed in after cleaning 10 min on the shaking table that rotating speed is 200 r/min and under flowing water, rinses 2 h, then use sterile water wash 2 Secondary, explant is gone to volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min is used on superclean bench；Adopt aseptic again Water is cut into a length of 3.5 cm length stem section after rinsing 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-KT 1.0mg/L+ vitamin C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components 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)2·4H2O 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, puridoxine 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 Wanning Ke's base portion sprouting, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of diameter 0.2～0.3 cm as explant.Will Wanning Ke's coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 2%, seals bottle using gauze Mouthful, it is placed in be placed in after cleaning 10 min on the shaking table that rotating speed is 200 r/min and under flowing water, rinse 1.5 h, then clear with sterilized water Wash 2 times, explant is gone to volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min is used on superclean bench；Adopt again no Bacterium water is cut into a length of 3.5 cm length stem section after rinsing 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-KT 1.5mg/L+ vitamin C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components 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)2·4H2O 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, puridoxine 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 Wanning Ke's base portion sprouting, a length of 7 ~ 8cm, the coppice shoot of the semi-lignified of diameter 0.3～0.4 cm as explant.Will Wanning Ke's coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 4%, seals bottle using gauze Mouthful, it is placed in be placed in after cleaning 15 min on the shaking table that rotating speed is 200 r/min and under flowing water, rinses 1 h, then use sterile water wash 2 Secondary, explant is gone to volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min is used on superclean bench；Adopt aseptic again Water is cut into a length of 4.0 cm length stem section after rinsing 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-KT 1.5mg/L+ vitamin C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components 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)2·4H2O 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, puridoxine 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 Wanning Ke's base portion sprouting, a length of 8 ~ 9cm, the coppice shoot of the semi-lignified of diameter 0.3～0.4 cm as explant.Will Wanning Ke's coppice shoot needle cuts off, and is placed in triangular flask, the liquid detergent solution adding volumetric concentration to be 5 %, seals bottle using gauze Mouthful, it is placed in after cleaning 15 min on the shaking table that rotating speed is 200 r/min and is placed in flushing 1h under flowing water, then use sterile water wash 2 Secondary, explant is gone to volumetric concentration 0.1% mercuric chloride solution soaking disinfection 10 min is used on superclean bench；Adopt aseptic again Water is cut into a length of 4.0 cm length stem section after rinsing 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-KT 2.0mg/L+ vitamin C10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.Wherein improvement WPM nutrient media components 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)2·4H2O 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, puridoxine 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 Wanning Ke's Initial culture bud abductive approach it is characterised in that：It is explant using Wanning Ke's base portion coppice shoot, 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 selects：The coppice shoot selecting the semi-lignified of Wanning Ke's base portion sprouting is explant；
（2）Explant sterilization and inoculation：Wanning Ke's 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 conditions.
2. a kind of Wanning Ke's Initial culture bud abductive approach according to claim 1 it is characterised in that：Described bud induction Its material content of culture medium is：1/3～1/2 improvement WPM culture medium+6-KT 1.0～2.0mg/L+ vitamin C 10mg/L+ lactose 5.0g/L+ sucrose 20.0g/L.
3. a kind of Wanning Ke's 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：NH4NO3850 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, ZnSO4·7H2O 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, Puridoxine hydrochloride 1.0 mg/L, thiamine hydrochloride 2.0 mg/L, glycine 2.0 mg/L.
4. a kind of Wanning Ke's Initial culture bud abductive approach according to claim 1 it is characterised in that：Described coppice shoot is A length of 6 ~ 9cm, the coppice shoot of the semi-lignified of diameter 0.2～0.4 cm.
5. a kind of Wanning Ke's Initial culture bud abductive approach according to claim 1 it is characterised in that：Step（2）Described Sterilization and inoculation method be that Wanning Ke's coppice shoot needle is cut off, be placed in triangular flask, add volumetric concentration be 1 ~ 5 % wash clean Smart solution, seals bottleneck using gauze, is placed in and is placed under flowing water after cleaning 10 ~ 15 min on the shaking table that rotating speed is 200 r/min Rinse 1 ~ 2 h, then use sterile water wash 2 times, explant is gone to and on superclean bench, uses volumetric concentration 0.1% mercury chloride molten Liquid soaking disinfection 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 and lures Lead in culture medium, 2 stem sections of every bottle of inoculation.
6. a kind of Wanning Ke's Initial culture bud abductive approach according to claim 1 it is characterised in that：Step（3）Described Suitable indoor conditions be：Illumination 500～1000 lx, illumination 8～12h/d is switched to after light culture 10d；Cultivation temperature 20 ± 1 DEG C, humidity is 50 ~ 55 %.
CN201610900768.3A 2016-10-17 2016-10-17 Primary culture shoot induction method for lithocarpus elmerrillii Pending CN106386496A (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: 20170215