CN106212288B - 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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- CN106212288B CN106212288B CN201610717249.3A CN201610717249A CN106212288B CN 106212288 B CN106212288 B CN 106212288B CN 201610717249 A CN201610717249 A CN 201610717249A CN 106212288 B CN106212288 B CN 106212288B
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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 a kind of method for tissue culture of Machilus pauhoi, belong to field of plant tissue culture technique；This method is to be handled, sterilized by explant, hormon type and concentration level ratio optimization, using Machilus pauhoi seed as explant, by adventitious bud inducing, bud Multiplying culture and culture of rootage, forms intact plant.The invention compensates for the blank that tissue-culturing rapid propagation is carried out using Machilus pauhoi select tree seed as explant, a kind of stabilization, quick Machilus pauhoi tissue culture propagation method are provided, the economic benefit to realize Machilus pauhoi elite germplasm Rapid Popularization, improving Machilus pauhoi artificial forest provides technical support.
Description
Technical field
The invention belongs to the technical field of tissue culture of plant, and in particular to the tissue culture propagation of Machilus pauhoi.
Background technology
Machilus pauhoi (Machilus pauhoi Kanehira) is subordinate to Lauraceae ((Lauraceae)) machilus
(Machilus), bearing tree height 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 is in provinces and regions such as Zhejiang, Anhui, Hunan, Jiangxi, Fujian, Guangxi, Guangdong and Hong Kong.Machilus pauhoi purposes is wide, can do
Face powder, adhesive extract essential oil, and tannin etc., complete stool utilization rate is high, is both commerical tree species and good economic tree, also may be used
For afforestation, there is important economic benefit and ecological benefits.In recent years, the domestic demand to Machilus pauhoi nursery stock is continuous
Increase, supply falls short of demand in market.
Machilus pauhoi is mainly bred by seeding and seedling raising at present, leads to the standing forest individual after Machilus pauhoi nursery stock and its afforestation
Differentiation is big, irregular, and then influences Afforestation Quality, yield and the economic benefit of artificial forest.And Machilus pauhoi is recalcitrant
Property seed, short life, hardly possible storage, " biennial bearing " phenomenon is again very serious, these problems seriously restrict 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.
Invention content
Based on this, it is necessary to for the blank for cultivating Machilus pauhoi using group culturation rapid propagating technology, provide a kind of Machilus pauhoi
Tissue culture propagation method.The present invention chooses that robust growth, tree-shaped be satisfactory, no disease and pests harm, and it is seed collecting elite stand to set high 10m or more, is adopted
Integrate mature seed as explant, by handling explant, minimal medium design, hormone kind, concentration and its proportioning it is excellent
Change, establish in vitro plant regeneration technique, reaches that explant Fiber differentiation pollution rate is low, the time is short, adventitious bud is more, rooting rate height etc.
Purpose, the breeding that required high quality seedling is cultivated for China Machilus pauhoi artificial forest provides technical support, to improving Machilus pauhoi people
Work woods economic benefit has extremely important meaning.
A kind of tissue culture propagation method of Machilus pauhoi of the present invention, steps are as follows：
(1) explant acquisition and processing：
Choose that robust growth, tree-shaped be satisfactory, no disease and pests harm, the Machilus pauhoi for setting high 10m or more is seed collecting elite stand, and seed is adopted
After collection, clear water impregnates 2~3d, rubs exosper with the hands, after rinsing well, picks out that kind of reality is full, the seed of no disease and pests harm.With
The GA of 1000mg/L3Solution impregnates seed 60min.
(2) explant surface sterilization：
Machilus pauhoi seed is carefully first peelled off into film quality endotesta (being careful not to injure embryo) on superclean bench, is added
Enter 0.1% mercuric chloride solution, impregnate 12min, constantly shakes therebetween, outwell mercuric chloride solution aseptic water washing 5~6 times.
(3) bud induces：
The seed disinfected is inoculated into inducing culture to cultivate, the formula of inducing culture is MS0In culture medium
Addition 25~40g/L of sucrose and carragheen 8g/L is made, until medium pH stops for 5.8-6.0.One seed of every bottle of inoculation.5~
After 7d, embryo starts to sprout, and seedling can reach 3~4cm high after one month.
(4) clump bud is proliferated：
The bud of 25~30d of culture is cut after step (3) is sprouted, and is cut to 2~3cm stem sections, is transferred in proliferated culture medium
Carry out Multiplying culture；First by stem section in addition AgNO3Preculture 10d, then turns in the proliferated culture medium of 0.5~2.0mg/L
It is connected to and is free of AgNO3Proliferated culture medium in cultivate 30d, evoking adventive bud forms sprouting clump.Proliferation culture medium formula is trained for MS
Addition 6-BA 1.0~3.0mg/L, IBA 0.01~0.1mg/L, 25~40g/L of sucrose and carragheen 8g/L in base is supported to be made,
Stop to medium pH for 5.8~6.0.It is 25 ± 2 DEG C that condition of culture, which is in temperature, illumination (60 μm of ol.m-2.s-1) time 12h/
d。
(5) culture of rootage：
Bud height is more than that the tender stem of 2cm is cut in the proliferation bud clump that step (4) obtains, and accesses root media；Culture of rootage
Based formulas is made by adding 0.1~0.5mg/L of IBA, 15~20g/L of sucrose and carragheen 8g/L in 1/2MS culture mediums, until training
Base pH is supported for 5.8~6.0 to stop.It is 25 ± 2 DEG C that condition of culture, which is in temperature, illumination (60 μm of ol.m-2.s-1) time 12h/d.
(6) it takes root transplantation of seedlings：The bottle seedling of taking root of culture of rootage 20d is moved on into adaptation 5~7d of external environment in greenhouse, then
By bottle cap from standard-sized sheet hardening 1d is partly reached, tissue-cultured seedling is carefully taken out, the culture medium sticked on root is cleaned, is transplanted to peat
Soil：Perlite=3：On 1 mixed-matrix, preceding 20d uses epiphragma moisturizing after transplanting, then opens film routinely seedling management,
The transplanting survival rate is higher than 90%.
The bud induced velocity of above-mentioned Machilus pauhoi tissue culture propagation, explant is fast；Inductivity is high, growth coefficient
Greatly, up to 3.52, rooting rate is up to 91%.By scale breeding, will be provided for China's Machilus pauhoi elite germplasm Rapid Popularization
Technical support provides high quality seedling guarantee to promote Machilus pauhoi artificial forest economic benefit, there is extremely important meaning.
Description of the drawings
Fig. 1 sprouts for select tree seed.
Fig. 2 is the bud clump of hypocotyl induced synthesis.
Fig. 3 is the proliferation bud in proliferated culture medium.
Fig. 4 is the rooted seedling in root media.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.The routine experimentation personnel of the technical field based on the contents of the disclosure of the present invention and the scope of each parameter,
It achieves the object of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition.
Embodiment one：
Explant acquires and processing：It chooses that robust growth, tree-shaped be satisfactory, no disease and pests harm, sets the Machilus pauhoi of high 10m or more
For seed collecting elite stand, after seed collection, clear water impregnates 2~3d, rubs exosper with the hands, and after rinsing well, it is full to pick out kind of reality, disease-free
The seed of insect pest.With the GA of 1000mg/L3Solution impregnates seed 60min.
Explant surface sterilization：Machilus pauhoi seed film quality endotesta is carefully first peelled off on superclean bench (to pay attention to
Not injure embryo), 0.1% mercuric chloride solution is added, impregnates 12min, constantly shakes therebetween, outwells mercuric chloride solution and rushed with sterile water
It washes 5~6 times.
Bud induces：The seed disinfected is inoculated into inducing culture MS0+ sucrose 30g/L+ carragheen 8g/L, until culture
Base pH stops for 5.8-6.0.One seed of every bottle of inoculation.After 5~7d, embryo starts to sprout, after one month seedling can reach 3~
4cm high.(Fig. 1)
Clump bud is proliferated：
Bud is cut, 2~3cm stem sections are cut to, is cultivated in proliferated culture medium of transferring.First in MS+6-BA 3.0mg/L+
IBA 0.01mg/L+AgNO3Preculture 10d on 1.0mg/L+ sucrose 30g/L+ carragheens 8g/L, then it is transferred to MS+6-BA
30d is cultivated in 3.0mg/L+IBA 0.01mg/L+ sucrose 30g/L+ carragheens 8g/L, it is 25 ± 2 DEG C that condition of culture, which is in temperature,
Illumination (60 μm of ol.m-2.s-1) time 12h/d.Growth coefficient is up to 3.52.(Fig. 2, Fig. 3).
Culture of rootage：
The healthy and strong effective bud that 2cm is chosen from proliferation bud clump, is transferred to 1/2MS+IBA 0.2mg/L+ sucrose 15g/L+ cards
It draws in the rooting induction culture medium of glue 8g/L and cultivates, it is 25 ± 2 DEG C that condition of culture, which is in temperature, illumination (60 μm of ol.m-2.s-1)
Time 12h/d.After 20d, the inductivity of adventitious root is 91%.(Fig. 4)
It takes root transplantation of seedlings：The bottle seedling of taking root of culture of rootage 20d is moved on into adaptation 5~7d of external environment in greenhouse, then will
Bottle cap carefully takes out tissue-cultured seedling from standard-sized sheet hardening 1d is partly reached, and cleans the culture medium sticked on root, is transplanted to peat
Soil：Perlite=3：On 1 mixed-matrix, preceding 20d uses epiphragma moisturizing after transplanting, then opens film routinely seedling management,
The transplanting survival rate is higher than 90%.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, protection scope of the present invention are limited by appended claims, any change on the basis of the claims in the present invention
All it is protection scope of the present invention.
Claims (4)
1. a kind of tissue culture propagation method of Machilus pauhoi, which is characterized in that include the following steps：
(1) explant acquisition and processing：It chooses that robust growth, tree-shaped be satisfactory, no disease and pests harm, sets the Machilus pauhoi of high 10m or more
For seed collecting elite stand, after seed collection, clear water impregnates 2~3d, rubs exosper with the hands, and after rinsing well, it is full to pick out kind of reality, disease-free
The seed of insect pest, with the GA of 1000mg/L3Solution impregnates seed 60min；
(2) explant surface sterilization：Machilus pauhoi seed is carefully first peelled off into film quality endotesta on superclean bench, is paid attention to not
Embryo is injured, 0.1% mercuric chloride solution is added, impregnates 12min, constantly shakes therebetween, outwells mercuric chloride solution aseptic water washing 5
~6 times；
(3) bud induces：The seed disinfected is inoculated into bud inducement cultivation base to cultivate, one seed of every bottle of inoculation；5d it
Afterwards, embryo starts to sprout；
(4) clump bud is proliferated：The bud of 25~30d of culture is cut after step (3) is sprouted, and is cut to 2~3cm stem sections, is transferred to proliferation
Multiplying culture is carried out in culture medium；First by stem section in addition AgNO3Preculture in the proliferated culture medium of 0.5~2.0mg/L
Then 10d is transferred to without AgNO3Proliferated culture medium in cultivate 30d, evoking adventive bud forms sprouting clump；
(5) culture of rootage：Bud height is more than that the tender stem of 2cm is cut in the proliferation bud clump that step (4) obtains, and accesses root media；
(6) it takes root transplantation of seedlings：The bottle seedling of taking root of culture of rootage 20d is moved on into adaptation 5~7d of external environment in greenhouse, then by group
Bottle cap is trained from standard-sized sheet hardening 1d is partly reached, tissue-cultured seedling is carefully taken out, the culture medium sticked on root is cleaned, is transplanted to peat soil：
Perlite=3：On 1 mixed-matrix, preceding 20d uses epiphragma moisturizing after transplanting, then opens film routinely seedling management；
Culture described in step (4) is first in 0.01~0.1mg/L+ of MS culture medium 1.0~3.0mg/L+IBA of+6-BA
AgNO3Preculture 10d on 0.5~2.0mg/L+ sucrose 25~40g/L+ carragheens 8g/L, then it is transferred to MS culture mediums+6-BA 1.0
30d is cultivated in~3.0mg/L+IBA 0.01~0.1mg/L+ sucrose 25~40g/L+ carragheens 8g/L.
2. a kind of tissue culture propagation method of Machilus pauhoi according to claim 1, which is characterized in that described in step (3)
Bud inducement cultivation base formula be MS0It adds 25~40g/L of sucrose and carragheen 8g/L in culture medium to be made, until medium pH
Stop for 5.8~6.0.
3. a kind of tissue culture propagation method of Machilus pauhoi according to claim 1, which is characterized in that step (5) described life
Root culture medium prescription is 0.1~0.5mg/L of addition IBA, 15~20g/L of sucrose and carragheen 8g/L systems in 1/2MS culture mediums
At until medium pH stops for 5.8~6.0.
4. a kind of tissue culture propagation method of Machilus pauhoi according to claim 1, which is characterized in that step (4) and step
(5) it is 25 ± 2 DEG C that the culture, which is in temperature, and intensity of illumination is 60 μm of ol.m-2.s-1, light application time is the culturing room of 12h/d
In cultivated.
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| CN106857251B (en) * | 2017-01-22 | 2018-10-09 | 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 |
| CN114651726B (en) * | 2022-05-05 | 2023-06-20 | 贵州大学 | Method for culturing aseptic seedlings of phoebe embryo |
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| JPH02207723A (en) * | 1989-02-06 | 1990-08-17 | Takasago Internatl Corp | Proliferation of plant |
| 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. |
| CN103444531A (en) * | 2013-08-28 | 2013-12-18 | 遵义县华富农业生物科技有限公司 | Rapid propagation method for tissue culture of phoebe zhennan |
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2016
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02207723A (en) * | 1989-02-06 | 1990-08-17 | Takasago Internatl Corp | Proliferation of plant |
| 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. |
| CN103444531A (en) * | 2013-08-28 | 2013-12-18 | 遵义县华富农业生物科技有限公司 | Rapid propagation method for tissue culture of phoebe zhennan |
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