CN106212288A - A kind of tissue culture propagation method of Machilus pauhoi - Google Patents
A kind of tissue culture propagation method of Machilus pauhoi Download PDFInfo
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- CN106212288A CN106212288A CN201610717249.3A CN201610717249A CN106212288A CN 106212288 A CN106212288 A CN 106212288A CN 201610717249 A CN201610717249 A CN 201610717249A CN 106212288 A CN106212288 A CN 106212288A
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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
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Abstract
The present invention relates to the method for tissue culture of a kind of Machilus pauhoi, belong to field of plant tissue culture technique；The method is that hormon kind and concentration level ratio optimization, be outer implant with Machilus pauhoi seed by the process of outer implant, sterilization, cultivates and root culture through Induce aerosor, Shoot propagation, forms whole plant.It is the blank that outer implant carries out tissue-culturing rapid propagation that this invention compensate for Machilus pauhoi select tree seed, providing a kind of stable, quick Machilus pauhoi tissue culture propagation method, for realizing Machilus pauhoi elite germplasm Rapid Popularization, the economic benefit of raising Machilus pauhoi artificial forest provides technical support.
Description
Technical field
The invention belongs to the technical field of tissue culture of plant, be specifically related to the tissue culture propagation of Machilus pauhoi.
Background technology
Machilus pauhoi (Machilus pauhoi Kanehira) is subordinate to Lauraceae ((Lauraceae)) machilus
(Machilus) height of tree of, growing up is up to 20m, diameter of a cross-section of a tree trunk 1.3 meters above the ground 50cm.Machilus pauhoi is typical Subtropical Zone of East moist climate region seeds,
NATURAL DISTRIBUTION in Zhejiang, Anhui, Hunan, Jiangxi, Fujian, Guangxi, the provinces and regions such as Guangdong and Hong Kong.Machilus pauhoi purposes is wide, can do
XIANGFEN, binding agent, extract quintessence oil, tannin etc., Herb utilization rate is high, is commerical tree species, is again good economic tree, it is possible to
For afforestation, there are important economic benefit and ecological benefits.In recent years, the domestic demand to Machilus pauhoi nursery stock is continuous
Increasing, supply falls short of demand in market.
Machilus pauhoi is mainly bred by seeding and seedling raising at present, causes the standing forest after Machilus pauhoi nursery stock and afforestation thereof individual
Differentiation is big, uneven, and then has influence on the Afforestation Quality of artificial forest, yield and economic benefit.And Machilus pauhoi is recalcitrant
Property seed, the life-span is short, difficult storage, and " biennial bearing " phenomenon is the most serious, and these problems seriously govern actual production, therefore open
The tissue culture technique of exhibition Machilus pauhoi has important practical significance.
By literature search, any report about Machilus pauhoi tissue-culturing rapid propagation is had not yet to see.
Summary of the invention
Based on this, it is necessary to cultivate the blank of Machilus pauhoi for set of applications culturation rapid propagating technology, it is provided that a kind of Machilus pauhoi
Tissue culture propagation method.The present invention choose robust growth, tree-shaped satisfactory, without pest and disease damage, more than height of tree 10m be seed collecting elite stand, adopt
Collection mature seed is outer implant, is processed by external implant, minimal medium design, hormone kind, concentration and proportioning thereof excellent
Change, set up in vitro plant regeneration technique, reach that outer implant inducing culture pollution rate is low, the time is short, adventitious bud is many, rooting rate is high
Purpose, the breeding for China's required high quality seedling of Machilus pauhoi artificial forest cultivation provides technical support, to improving Machilus pauhoi people
Work woods economic benefit has earth shaking meaning.
A kind of tissue culture propagation method of the Machilus pauhoi of the present invention, step is as follows:
(1) outer implant collection and process:
Choose robust growth, tree-shaped satisfactory, without pest and disease damage, the Machilus pauhoi more than height of tree 10m is seed collecting elite stand, and seed is adopted
After collection, clear water soaks 2～3d, rubs episperm with the hands, after rinsing well, picks out kind of reality full, without the seed of pest and disease damage.With
The GA of 1000mg/L3Solution soaking seed 60min.
(2) outer planting surface sterilization:
Machilus pauhoi seed is first carefully peelled off film quality endotesta (being careful not to injure embryo) by superclean bench, adds
Enter 0.1% mercuric chloride solution, soak 12min, the most constantly shake, outwell mercuric chloride solution aseptic water washing 5～6 times.
(3) bud inducement:
The seed disinfected being inoculated into inducing culture cultivate, the formula of inducing culture is MS0In culture medium
Add sucrose 25～40g/L and carrageenan 8g/L to make, be that 5.8-6.0 stops to medium pH.Every one seed of bottle graft kind.5～
After 7d, embryo starts to sprout, and after one month, seedling can reach 3～4cm height.
(4) clump Shoot propagation:
The bud cultivating 25～30d after step (3) being sprouted cuts, and is cut to 2～3cm stem sections, is transferred in proliferated culture medium
Carry out enrichment culture；First stem section is being added AgNO3Preculture 10d in the proliferated culture medium of 0.5～2.0mg/L, then turns
Receive without AgNO3Proliferated culture medium in cultivate 30d, evoking adventive bud, formed sprouting clump.Proliferation culture medium formula is MS training
Supporting and add 6-BA 1.0～3.0mg/L, IBA 0.01～0.1mg/L in base, sucrose 25～40g/L and carrageenan 8g/L is made,
It is 5.8～6.0 only to medium pH.Condition of culture is to be 25 ± 2 DEG C in temperature, illumination (60 μm ol.m-2.s-1) time 12h/
d。
(5) root culture:
In the propagation bud clump that step (4) obtains, the bud height tender stem more than 2cm cuts, and accesses root media；Root culture
Based formulas is that in 1/2MS culture medium, interpolation IBA 0.1～0.5mg/L, sucrose 15～20g/L and carrageenan 8g/L are made, to training
Supporting base pH is 5.8～6.0 only.Condition of culture is to be 25 ± 2 DEG C in temperature, illumination (60 μm ol.m-2.s-1) time 12h/d.
(6) take root transplantation of seedlings: a bottle Seedling of taking root of root culture 20d moves on to adapt in greenhouse external environment 5～7d, then
By tissue culture bottle lid from partly reaching standard-sized sheet seedling exercising 1d, tissue cultured seedling is carefully taken out, clean and stick at the culture medium on root, be transplanted to peat
Soil: on perlite=3:1 mixed-matrix, after transplanting, front 20d uses epiphragma moisturizing, then opens thin film seedling management routinely,
This transplanting survival rate is higher than 90%.
Above-mentioned Machilus pauhoi tissue culture propagation, the bud inducement speed of outer implant is fast；Inductivity is high, growth coefficient
Greatly, reaching 3.52, rooting rate is up to 91%.By scale breeding, will provide for China's Machilus pauhoi elite germplasm Rapid Popularization
Technical support, provides high quality seedling guarantee for promoting Machilus pauhoi artificial forest economic benefit, has earth shaking meaning.
Accompanying drawing explanation
Fig. 1 is select tree seed germination.
Fig. 2 is the bud clump of hypocotyl induced synthesis.
Fig. 3 is the propagation bud in proliferated culture medium.
Fig. 4 is the Seedling of taking root in root media.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.The routine experimentation personnel of described technical field according to above present disclosure and the taken scope of each parameter,
Realize the purpose of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition.
Embodiment one:
Outer implant collection and process: choose robust growth, tree-shaped satisfactory, without pest and disease damage, the Machilus pauhoi more than height of tree 10m
For seed collecting elite stand, after seed collection, clear water soaks 2～3d, rubs episperm with the hands, after rinsing well, picks out kind of reality full, anosis
The seed of insect pest.With the GA of 1000mg/L3Solution soaking seed 60min.
Sterilizing in outer planting surface: Machilus pauhoi seed is first carefully peelled off on superclean bench film quality endotesta and (notes
Do not injure embryo), add 0.1% mercuric chloride solution, soak 12min, the most constantly shake, outwell mercuric chloride solution sterilized water and rush
Wash 5～6 times.
Bud inducement: the seed disinfected is inoculated into inducing culture MS0+ sucrose 30g/L+ carrageenan 8g/L, to cultivating
Base pH is that 5.8-6.0 stops.Every one seed of bottle graft kind.After 5～7d, embryo starts to sprout, after one month seedling can reach 3～
4cm is high.(Fig. 1)
Clump Shoot propagation:
Bud is cut, is cut to 2～3cm stem sections, proliferated culture medium of transferring is cultivated.First at MS+6-BA 3.0mg/L+
IBA 0.01mg/L+AgNO3Preculture 10d on 1.0mg/L+ sucrose 30g/L+ carrageenan 8g/L, then proceed to MS+6-BA
Cultivating 30d in 3.0mg/L+IBA 0.01mg/L+ sucrose 30g/L+ carrageenan 8g/L, condition of culture is to be 25 ± 2 DEG C in temperature,
Illumination (60 μm ol.m-2.s-1) time 12h/d.Growth coefficient is up to 3.52.(Fig. 2, Fig. 3).
Root culture:
From propagation bud clump, choose the healthy and strong effectively bud of 2cm, be transferred to 1/2MS+IBA 0.2mg/L+ sucrose 15g/L+ card
Drawing in the root induction culture medium of glue 8g/L and cultivate, condition of culture is to be 25 ± 2 DEG C in temperature, illumination (60 μm ol.m-2.s-1)
Time 12h/d.After 20d, the inductivity of adventitious root is 91%.(Fig. 4)
Take root transplantation of seedlings: a bottle Seedling of taking root of root culture 20d moves on to adapt in greenhouse external environment 5～7d, then will
Tissue cultured seedling, from partly reaching standard-sized sheet seedling exercising 1d, is carefully taken out by tissue culture bottle lid, cleans and sticks at the culture medium on root, is transplanted to peat
Soil: on perlite=3:1 mixed-matrix, after transplanting, front 20d uses epiphragma moisturizing, then opens thin film seedling management routinely,
This transplanting survival rate is higher than 90%.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limiting, protection scope of the present invention is limited by appended claims, any change on the basis of the claims in the present invention
It it is all protection scope of the present invention.
Claims (5)
1. the tissue culture propagation method of a Machilus pauhoi, it is characterised in that comprise the following steps:
(1) outer implant collection and process: choose robust growth, tree-shaped satisfactory, without pest and disease damage, the Machilus pauhoi more than height of tree 10m
For seed collecting elite stand, after seed collection, clear water soaks 2～3d, rubs episperm with the hands, after rinsing well, picks out kind of reality full, anosis
The seed of insect pest, with the GA of 1000mg/L3Solution soaking seed 60min；
(2) outer planting surface sterilization: first Machilus pauhoi seed is carefully peelled off film quality endotesta on superclean bench, note not
Embryo to be injured, adds 0.1% mercuric chloride solution, soaks 12min, the most constantly shakes, outwell mercuric chloride solution aseptic water washing 5
～6 times；
(3) bud inducement: the seed disinfected is inoculated into bud inducement culture medium and cultivates, one seed of every bottle graft kind；5～7d
Afterwards, embryo starts to sprout；
(4) clump Shoot propagation: after step (3) being sprouted, the bud of cultivation 25～30d cuts, and is cut to 2～3cm stem sections, is transferred to propagation
Culture medium carries out enrichment culture；First stem section is being added AgNO3Preculture in the proliferated culture medium of 0.5～2.0mg/L
10d, is then transferred to without AgNO3Proliferated culture medium in cultivate 30d, evoking adventive bud, formed sprouting clump；
(5) root culture: in the propagation bud clump that step (4) obtains, the bud height tender stem more than 2cm cuts, and accesses root media；
(6) take root transplantation of seedlings: a bottle Seedling of taking root of root culture 20d moves on to adapt in greenhouse external environment 5～7d, then by tissue culture bottle
Tissue cultured seedling, from partly reaching standard-sized sheet seedling exercising 1d, is carefully taken out by lid, cleans and sticks at the culture medium on root, is transplanted to peat soil: Margarita
On rock=3:1 mixed-matrix, after transplanting, front 20d uses epiphragma moisturizing, then opens thin film seedling management routinely.
The tissue culture propagation method of a kind of Machilus pauhoi the most according to claim 1, it is characterised in that described in step (3)
The formula of bud inducement culture medium be MS0Culture medium is added sucrose 25～40g/L and carrageenan 8g/L make, to medium pH
Stop for 5.8-6.0.
The tissue culture propagation method of a kind of Machilus pauhoi the most according to claim 1, it is characterised in that described in step (4)
Cultivation is first at MS culture medium+6-BA 1.0～3.0mg/L+IBA 0.01～0.1mg/L+AgNO30.5～2.0mg/L+ sugarcane
Sugar 25～40g/L+ carrageenan 8g/L on preculture 10d, then proceed to MS culture medium+6-BA 1.0～3.0mg/L+IBA 0.01～
0.1mg/L+ sucrose 25～40g/L+ carrageenan 8g/L cultivates 30d.
The tissue culture propagation method of a kind of Machilus pauhoi the most according to claim 1, it is characterised in that step (5) described life
Root culture medium prescription is to add IBA 0.1～0.5mg/L, sucrose 15～20g/L and carrageenan 8g/L system in 1/2MS culture medium
Become, be 5.8～6.0 only to medium pH.
The tissue culture propagation method of a kind of Machilus pauhoi the most according to claim 1, it is characterised in that step (4) and step
(5) described cultivation is to be 25 ± 2 DEG C in temperature, and intensity of illumination is 60 μm ol.m-2.s-1, light application time is the culturing room of 12h/d
In cultivate.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106857251A (en) * | 2017-01-22 | 2017-06-20 | 浙江农林大学 | A kind of Phoebe bournei somatic embryo and adventitious bud inducing method |
| CN108605633A (en) * | 2018-05-04 | 2018-10-02 | 永嘉县原野园林工程有限公司 | A kind of nanmu tissue culture and rapid propagation method |
| CN114651726A (en) * | 2022-05-05 | 2022-06-24 | 贵州大学 | Method for culturing nanmu seed embryo aseptic seedlings |
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| CN101595845A (en) * | 2009-07-21 | 2009-12-09 | 福建农林大学 | Stripped embryo culture of euscaphis konishii and plant regeneration method |
| CN101911912A (en) * | 2010-08-24 | 2010-12-15 | 上海世华生物工程有限公司 | Method for rapidly propagating jatrohpa curcas L. |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106857251A (en) * | 2017-01-22 | 2017-06-20 | 浙江农林大学 | A kind of Phoebe bournei somatic embryo and adventitious bud inducing method |
| CN108605633A (en) * | 2018-05-04 | 2018-10-02 | 永嘉县原野园林工程有限公司 | A kind of nanmu tissue culture and rapid propagation method |
| CN114651726A (en) * | 2022-05-05 | 2022-06-24 | 贵州大学 | Method for culturing nanmu seed embryo aseptic seedlings |
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