CN109258470A - A kind of bush tissue culture and rapid propagation method - Google Patents

A kind of bush tissue culture and rapid propagation method Download PDF

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Publication number
CN109258470A
CN109258470A CN201811402016.XA CN201811402016A CN109258470A CN 109258470 A CN109258470 A CN 109258470A CN 201811402016 A CN201811402016 A CN 201811402016A CN 109258470 A CN109258470 A CN 109258470A
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bush
culture
transplanting
illumination
root
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林登淞
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林登淞
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants

Abstract

The invention discloses a kind of bush tissue culture and rapid propagation methods, and bush mainly uses the traditional approach such as cuttage, grafting and sowing to carry out nursery at present, and there are seedling cost height, and nursery stock growing way is bad, irregular, breeding potentiality fail the disadvantages of being not fully exerted.The present invention is using choiceness spray as explant; bush plant again in vitro is obtained by processes such as explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root generation, acclimatization and transplants; establish bush tissue culture rapid propagation technique system; the merit that can keep bush clone female parent is conducive to the large-scale production of bush choiceness nursery stock.

Description

A kind of bush tissue culture and rapid propagation method
Technical field
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, being related to a kind of bush tissue culture Quick-breeding method.
Background technique
Bush is leguminous plant, takes its dry duramen.General 5 ~ July cuts down tree, removes rough bark and sapwood, takes Qi Huanghong The heartwood of color or rufous, is dried.It is odorless, taste micro-puckery.With promoting circulation of blood dissolving stasis, swelling and pain relieving.For amenorrhoea, postpartum stasis abdomen Bitterly, chest and abdomen twinge, and injure fracture swelling and pain outside.Propulsion and merchantable timber price recently as Collective Forestry Property Rights System Reform it is upper Rise, the enthusiasm that self-employed tree cultivator manages bush increases, the continuous improvement of bush management level and breeding consciousness, to bush breeding strong sprout Demand increasingly increases, and therefore, how fast-propagation bush superior genotypes seem very necessary.Biotechnology in recent years Fast development, the especially foundation of tissue culture rapid propagation system provide certain technical support for the asexual breeding of forest.Utilize tissue Culture technique carries out rare and endangered species and excellent strain tree species breed, and has and accelerates breeding, shorten reproductive process, save sky Between, reduce labour, whole year production the features such as.And bush mainly uses the traditional approach such as cuttage, grafting and sowing to be educated at present Seedling, there are seedling cost height, and nursery stock growing way is bad, irregular, breeding potentiality fail the disadvantages of being not fully exerted, so that mesh The supply of the preceding high-quality strong sprout of bush is also unable to satisfy the demand in market.In order to meet market to the great demand of bush seedling, this hair It is bright using choiceness spray as explant, pass through explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root occur, hardening The processes such as transplanting obtain bush plant again in vitro, establish bush tissue culture rapid propagation technique system, can not only be effectively Overcome some defects in bush tradition nursery, and keeps maternal merit to rise the clone nursery stock bred out To certain effect, be conducive to the large-scale production of bush choiceness nursery stock.Documents 1, application number: CN201410170219, denomination of invention: the method for Karst region bush cultivation, including seed collecting and seed treatment, cultivating seedlings And the process that afforestation is fostered acquires seed and carries out seed treatment, be placed in be equipped with and educate by selecting bush select tree as seed collecting elite stand Seedling is cultivated in the container of seedling matrix, outplanting afforestation carries out tending management to new afforestation.This cultural method uses nonwoven Cloth Seedling bag cultivates seedling, is transplanted seedlings and is completely cured, differentiated control, full exposure hardening, is layered and discharges water-retaining agent and base manure, shape pruning And enter to clip the technical treatments such as terminal bud after autumn.The difference of documents and this case.
Summary of the invention
It is outer with choiceness spray the purpose of the present invention is to provide a kind of bush tissue culture and rapid propagation method, the present invention out Implant, by the processes such as explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root generation, acclimatization and transplants obtain bush from Body plant again, establishes bush tissue culture rapid propagation technique system, to realize the purpose of the present invention.
A kind of bush tissue culture and rapid propagation method of the invention, includes the following steps:
Step 1, explant acquisition and disinfection: bush stub base portion Morphological Characterization consistent current year raw the full of state of sprouting is taken Spray rinses 6h under flowing water as explant, is soaked in 3%~5% washing powder solution 13 minutes, is washed with banister brush 3%~5% Clothing powder solution gently washing material, then the form flushing 93min with tap water to drip, with distilled water flushing 8 times, in ultra-clean work Make in platform with 75%~80% alcohol solution dipping 63s, aseptic water washing 8 times, then with 0.8% mercuric chloride containing 0.08% Tween-20 Solution disinfection 18min, the moisture for blotting surface with aseptic filter paper after aseptic water washing 8 times are spare;
Step 2, Fiber differentiation: by treated that spray is cut into the stem section of about 1.8cm and is inoculated into induced medium through step (1) Inducing clumping bud culture is carried out, inoculation is placed on daily illumination 17 hours, intensity of illumination 2300lx, and being placed in cultivation temperature is 28 DEG C, statistics induction situation after relative air humidity is cultivated 33 days under conditions of being 75%~80%;Induced medium are as follows: MS+ 1.3mg/L NAA+8.3mg/L 6-BA+3.3mg/L KT+1.8g/L AC+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 3, adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing Culture is grown, inoculation is placed on daily illumination 17 hours, intensity of illumination 2400lx, and being placed in cultivation temperature is 25 DEG C, and air is opposite Humidity counts germinative number after cultivating 43 days under conditions of being 75%~80%;Proliferated culture medium are as follows: MS+0.8mg/L 2,4-D+ 5.3mg/L 6-BA+1.3mg/L NAA+1.8g/L AC+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 4, culture of rootage: the plant unrooted tender tip 6cm obtained in step (3) proliferation is cut and is seeded to from base portion and is taken root Root induction is carried out in culture medium, inoculation is placed on daily illumination 17 hours, intensity of illumination 3300lx, and being placed in cultivation temperature is 23~25 DEG C, relative air humidity counts situation of taking root after cultivating 33 days under conditions of being 75%~80%;Root media are as follows: White+1.3mg/L NAA+3.3mg/L IBA+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 5, acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often Temperature is opened bottle cap 6 days after lower 8 days, then washes away the culture medium being attached on seedling root system, soak in 1000 times of bacteria agent 13min is steeped, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag transplanting 1 Strain rooted seedling, is placed in the vinyl house inside holding moisturizing temperature control of the auto spraying with shading net at 15~35 DEG C, humidity It should be maintained at 75%~85% or so, avoid direct sunlight, transplanting counted survival rate after 33 days.Transplanting culture substrate is by mud Charcoal soil: vermiculite: pearl charcoal: pine=2:2:1:1 mixes matrix.
Compared with prior art the invention has the advantages that propulsion and merchantable timber recently as Collective Forestry Property Rights System Reform The rise of price, the enthusiasm that self-employed tree cultivator manages bush increase, and the continuous improvement of bush management level and breeding consciousness is good to bush The demand of kind strong sprout increasingly increases.And bush mainly uses the traditional approach such as cuttage, grafting and sowing to carry out nursery at present, exists Seedling cost is high, and nursery stock growing way is bad, irregular, breeding potentiality fail the disadvantages of being not fully exerted, so that current bush The supply of high-quality strong sprout is also unable to satisfy the demand in market.In order to meet market to the great demand of bush seedling, the present invention is with excellent Good clone spray is explant, passes through explant disinfection, Fiber differentiation, adventitious buds proliferation, adventitious root generation, acclimatization and transplants etc. Process obtains bush plant again in vitro, establishes bush tissue culture rapid propagation technique system, can keep bush clone Maternal merit, is conducive to the large-scale production of bush choiceness nursery stock.
Specific embodiment
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
(1) explant acquisition and disinfection: bush stub base portion Morphological Characterization consistent current year raw the full tender of state of sprouting is taken Branch is used as explant, rinses 5h under flowing water, is soaked in 3% washing powder solution 8min, gently with 3% washing powder solution of banister brush Washing material, then rinse 63min with tap water in the form dripped, with distilled water flushing 6 times, in superclean bench with 75% alcohol solution dipping 23s, aseptic water washing 8 times, then 8min is sterilized with 0.3% mercuric chloride solution containing 0.01% Tween-20, The moisture for blotting surface with aseptic filter paper after aseptic water washing 6 times is spare.
(2) Fiber differentiation: by treated that spray is cut into the stem section of about 1.8cm and is inoculated into Fiber differentiation through step (1) Base carries out inducing clumping bud culture.Inoculation is placed on daily illumination 15 hours, intensity of illumination 1800lx, and being placed in cultivation temperature is 23 DEG C, inductivity is up to 98% after relative air humidity is cultivated 33 days under conditions of being 75%.The induced medium are as follows: MS+ 0.5mg/L NAA+5.3mg/L 6-BA+1.8mg/L KT+1.1g/L AC+28g/L sucrose+5.3g/L agar, pH 5.6.
(3) adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing Grow culture.Inoculation is placed on daily illumination 15 hours, intensity of illumination 1800lx, and being placed in cultivation temperature is 23 DEG C, and air is opposite Humidity be 75% under conditions of cultivate 43 days after bud number 11 or more.The proliferated culture medium are as follows: MS+0.5mg/L 2,4-D+ 3.8mg/L 6-BA+0.8mg/L NAA+1.3g/L AC+ 28g/L sucrose+4.8g/L agar, pH 5.6.
(4) culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step (3) proliferation is cut and is seeded to from base portion Root induction is carried out in root media.Inoculation is placed on daily illumination 18 hours, intensity of illumination 2300lx, is placed in culture temperature Degree is 23 DEG C, and relative air humidity is taken root after cultivating 33 days under conditions of being 75% reaches 99% or more.The root media Are as follows: White+0.6mg/L NAA+1.8mg/L IBA+18g/L sucrose+4.1g/L agar, pH 5.6.
(5) acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often Temperature is opened bottle cap 2 days after lower 7 days, the culture medium being attached on seedling root system is then washed away, in 1000 times of bacteria agent 5min is impregnated, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag transplanting 1 plant of rooted seedling.The vinyl house inside holding moisturizing temperature for being placed in the auto spraying with shading net is controlled at 25 DEG C, and humidity is answered It is maintained at 75%~85% or so, avoids direct sunlight, survival rate reaches 93% or more after transplanting 33 days.The transplanting culture Matrix is by peat soil: vermiculite: pearl charcoal: pine=2:2:1:1 mixes matrix.
Embodiment 2:
(1) explant acquisition and disinfection: bush stub base portion Morphological Characterization consistent current year raw the full tender of state of sprouting is taken Branch is used as explant, rinses 6h under flowing water, is soaked in 3% washing powder solution 11min, gently with 4% washing powder solution of banister brush Washing material, then rinse 73min with tap water in the form dripped, with distilled water flushing 7 times, in superclean bench with 78% alcohol solution dipping 33s, aseptic water washing 8 times, then 10min is sterilized with 0.3% mercuric chloride solution containing 0.01% Tween-20, The moisture for blotting surface with aseptic filter paper after aseptic water washing 8 times is spare.
(2) Fiber differentiation: by treated that spray is cut into the stem section of about 1.8cm and is inoculated into Fiber differentiation through step (1) Base carries out inducing clumping bud culture.Inoculation is placed on daily illumination 16 hours, intensity of illumination 2300lx, and being placed in cultivation temperature is 25 DEG C, inductivity is up to 98.5% after relative air humidity is cultivated 33 days under conditions of being 75%.The induced medium are as follows: MS+ 0.6mg/LNAA+6.3mg/L 6-BA+2.3mg/L KT+1.3g/L AC+33g/L sucrose+5.3g/L agar, pH 5.6.
(3) adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing Grow culture.Inoculation is placed on daily illumination 16 hours, intensity of illumination 2800lx, and being placed in cultivation temperature is 25 DEG C, and air is opposite Humidity be 75% under conditions of cultivate 43 days after bud number 11 or more.The proliferated culture medium are as follows: MS+0.5mg/L 2,4-D+ 3.8mg/L 6-BA+0.8mg/L NAA+1.3g/L AC+ 28g/L sucrose+4.8g/L agar, pH 5.6.
(4) culture of rootage: the 2~3cm of plant unrooted tender tip obtained in step (3) proliferation is cut and is seeded to from base portion Root induction is carried out in root media.Inoculation is placed on daily illumination 16 hours, intensity of illumination 2300lx, is placed in culture temperature Degree is 23 DEG C, and relative air humidity is taken root after cultivating 33 days under conditions of being 75% reaches 96% or more.The root media Are as follows: White+1.1mg/L NAA+2.3mg/L IBA+26g/L sucrose+4.8g/L agar, pH 5.6.
(5) acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often Temperature is opened bottle cap 6 days after lower 7 days, the culture medium being attached on seedling root system is then washed away, in 1000 times of bacteria agent 10min is impregnated, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag is moved Plant 1 plant of rooted seedling.The vinyl house inside holding moisturizing temperature control of the auto spraying with shading net is placed at 25 DEG C, humidity It should be maintained at 75%~85% or so, avoid direct sunlight, survival rate reaches 98% or more after transplanting 33 days.The transplanting training Supporting matrix is by peat soil: vermiculite: pearl charcoal: pine=2:2:1:1 mixes matrix.

Claims (1)

1. a kind of bush tissue culture and rapid propagation method, it is characterised in that the following steps are included:
Step 1, explant acquisition and disinfection: bush stub base portion Morphological Characterization consistent current year raw the full of state of sprouting is taken Spray rinses 6h under flowing water as explant, is soaked in 3%~5% washing powder solution 13 minutes, is washed with banister brush 3%~5% Clothing powder solution gently washing material, then the form flushing 93min with tap water to drip, with distilled water flushing 8 times, in ultra-clean work Make in platform with 75%~80% alcohol solution dipping 63s, aseptic water washing 8 times, then with 0.8% mercuric chloride containing 0.08% Tween-20 Solution disinfection 18min, the moisture for blotting surface with aseptic filter paper after aseptic water washing 8 times are spare;
Step 2, Fiber differentiation: by treated that spray is cut into the stem section of about 1.8cm and is inoculated into induced medium through step (1) Inducing clumping bud culture is carried out, inoculation is placed on daily illumination 17 hours, intensity of illumination 2300lx, and being placed in cultivation temperature is 28 DEG C, statistics induction situation after relative air humidity is cultivated 33 days under conditions of being 75%~80%;Induced medium are as follows: MS+ 1.3mg/L NAA+8.3mg/L 6-BA+3.3mg/L KT+1.8g/L AC+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 3, adventitious buds proliferation: step (2) Fiber differentiation is obtained into Multiple Buds and is inoculated into proliferated culture medium progress adventitious bud increasing Culture is grown, inoculation is placed on daily illumination 17 hours, intensity of illumination 2400lx, and being placed in cultivation temperature is 25 DEG C, and air is opposite Humidity counts germinative number after cultivating 43 days under conditions of being 75%~80%;Proliferated culture medium are as follows: MS+0.8mg/L 2,4-D+ 5.3mg/L 6-BA+1.3mg/L NAA+1.8g/L AC+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 4, culture of rootage: the plant unrooted tender tip 6cm obtained in step (3) proliferation is cut and is seeded to from base portion and is taken root Root induction is carried out in culture medium, inoculation is placed on daily illumination 17 hours, intensity of illumination 3300lx, and being placed in cultivation temperature is 23~25 DEG C, relative air humidity counts situation of taking root after cultivating 33 days under conditions of being 75%~80%;Root media are as follows: White+1.3mg/L NAA+3.3mg/L IBA+33g/L sucrose+6.3g/L agar, pH 5.9;
Step 5, acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often Temperature is opened bottle cap 6 days after lower 8 days, then washes away the culture medium being attached on seedling root system, soak in 1000 times of bacteria agent 13min is steeped, then transplants into the container bag for filling designed transplanting culture substrate and is cultivated, each container bag transplanting 1 Strain rooted seedling, is placed in the vinyl house inside holding moisturizing temperature control of the auto spraying with shading net at 15~35 DEG C, humidity It should be maintained at 75%~85% or so, avoid direct sunlight, transplanting counts survival rate after 33 days, transplanting culture substrate is by mud Charcoal soil: vermiculite: pearl charcoal: pine=2:2:1:1 mixes matrix.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103907472A (en) * 2014-04-25 2014-07-09 广西壮族自治区林业科学研究院 Method for cultivating caesalpinia sappan linn in karst region
CN104663453A (en) * 2015-03-10 2015-06-03 朱海燕 Tissue culture and rapid propagation method for Cunninghamia lanceolate
CN106386478A (en) * 2016-08-28 2017-02-15 李志勇 Phoebe zhennan tissue culture rapid breeding method
CN108566886A (en) * 2017-03-14 2018-09-25 南京标科生物科技有限公司 A kind of rapid propagation method of bush plant regeneration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103907472A (en) * 2014-04-25 2014-07-09 广西壮族自治区林业科学研究院 Method for cultivating caesalpinia sappan linn in karst region
CN104663453A (en) * 2015-03-10 2015-06-03 朱海燕 Tissue culture and rapid propagation method for Cunninghamia lanceolate
CN106386478A (en) * 2016-08-28 2017-02-15 李志勇 Phoebe zhennan tissue culture rapid breeding method
CN108566886A (en) * 2017-03-14 2018-09-25 南京标科生物科技有限公司 A kind of rapid propagation method of bush plant regeneration

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ELIAS TERRA WERNER等: "Culture media, growth regulators and nitrogen sources in callus formation regulation of Brazilwood (Caesalpinia echinata Lam.)", 《ACTA BOTANICA BRASILICA》 *

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Application publication date: 20190125