CN106135005A - A kind of little Semen Pini Tissue Culture Regeneration System construction method - Google Patents
A kind of little Semen Pini Tissue Culture Regeneration System construction method Download PDFInfo
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- CN106135005A CN106135005A CN201610732230.6A CN201610732230A CN106135005A CN 106135005 A CN106135005 A CN 106135005A CN 201610732230 A CN201610732230 A CN 201610732230A CN 106135005 A CN106135005 A CN 106135005A
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- culture
- semen pini
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- little semen
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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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- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses a kind of little Semen Pini Tissue Culture Regeneration System construction method, obtained the technical method of strain stabilization seeds Seedling by Fast Asexual Propagation Technique.The present invention is outer implant with little Semen Pini tree immature embryo, little Semen Pini tree Tissue Culture Regeneration System is constructed through seed disinfection, embryo callus induction, differentiation culture, root culture, seedling exercising and transplanting and other steps, both solved the problem that little Semen Pini tree high quality seedling lacks, can be again to carry out little Semen Pini tree large area expanding species transformation from now on to provide firm guarantee.
Description
Technical field
The invention belongs to economy of forestry plant field, a kind of little Semen Pini Tissue Culture Regeneration System construction method.
Background technology
Oil is a kind of non-renewable resources, and the petroleum resources of underground, ground one day, the world can use up exhaustion, and this is also
It is strategic materials, is well-known.Therefore, develop tidewater in the world and generate electricity, wind power generation etc., and at plant refining art
Still in the developmental research stage.Little Pinus tabuliformis is also little Semen Pini, little strobile, fruit, can refine diesel oil, and oil yield, more than 30%, is a kind of
Thick raw thick long, that easily pipe is easily planted trees.
Summary of the invention
It is an object of the invention to provide a kind of by disinfection, induce, break up, cultivate, seedling exercising, method for transplanting composition
Regenerating system, obtains a kind of survival rate high, the little strobile tree of Miao Zhuan, root fat tree cyclopentadienyl, it is achieved thereby that the present invention.
The technical solution of the present invention is such, a kind of little Semen Pini Tissue Culture Regeneration System construction method, and it is special
Levy and be to comprise the following steps:
(1) seed disinfection: selection initial stage maturation tea remains blue or green little Semen Pini fruit and puts into the detergent water soaking of intermediate concentration
32min, after cleaning, tap water rinses 2.5h, takes out sterilized seed, and in superclean bench, strip off episperm, first with 75%
With aseptic washing 6 times after ethanol disinfection 32s, then with 0.1% mercuric chloride solution sterilization 30min, with standby after aseptic water washing 8 times；
(2) embryo callus induction: 0.6cm × 0.6cm × 0.6cm inoculation will be cut into by immaturity embryo after step (1) processes
On inducing culture, first full light culture 46 days, then illumination every day 15 hours under the conditions of 29 DEG C, intensity of illumination is
3200lx, until induced synthesis embryo callus；
(3) differentiation culture: step (2) is cultivated the greeny Cotyledon Callus obtained and proceeds to carry out on division culture medium
Differentiation culture, first full light culture 15 days, then illumination every day 18 hours under the conditions of 29 DEG C after inoculation, intensity of illumination is
5500lx, is placed under conditions of cultivation temperature is 29 DEG C and cultivates until forming Multiple Buds；
(4) root culture: the Multiple Buds of height about 3.6cm step (3) process obtained is cut and is inoculated into root media
On carry out root culture, first full light culture 15 days, then illumination every day 18 hours under the conditions of 29 DEG C after inoculation, intensity of illumination is
3200lx, cultivation temperature is cultivated to taking root under conditions of being 29 DEG C；
(5) acclimatization and transplants: after the rooting tube plantlet of high about 9cm step (4) obtained removes bottle cap natural lighting lower refining seedling 8 days,
Test tube Seedling is taken out from culture bottle, washes root culture medium off, plant in the substrate being mixed into by peat soil and yellow sand mud and determine
Plant in big Tanaka.
Inducing culture described in step (2) is: ER+5-BA4mg/L+NAA0.6mg/L+GA36 mg/L+ sucrose 32g/L
+ agar 3.2g/L, pH are 5.9.
Division culture medium described in step (3) is: ER+5-BA6mg/L+IBA0.6mg/L+ sucrose 32g/L+ agar 6.5g/
L, pH are 5.9.
Root media described in step (4) is: 1/2MS+IBA1.0mg/L+GGR2.2mg/L+KT3.5mg/L+ sucrose
32g/L+ agar 3.0g/L, pH are 5.9.
The present invention prominent effect compared with prior art is: technically reliable, ripe, and process route is simple, processing ease,
Low cost, the nursery stock plantation survival rate of formation is up to more than 90%, solves the problem that seedling is rare well, for forestry from now on
Economic development provides powerful guarantee.
Detailed description of the invention
Following example are to further illustrate the present invention, are not limitations of the present invention.
Embodiment 1:
(1) seed disinfection: select to be in the initial stage dark green fruit that comes to the ripening period and put into neutral concentration detergent water soaking
20min, after outwash, tap water rinses half an hour, takes out kind of a fruit, in superclean bench, strip off episperm, first disappears with 70% ethanol
With aseptic washing 2 times after poison 3s, then sterilize 15min with 0.1% mercuric chloride solution, with standby after aseptic water washing 3 times, inoculate one week
Rear statistics pollution rate can as little as 2%.
(2) embryo callus induction: 0.6cm × 0.6cm × 0.6cm will be cut into be inoculated into by embryo after step (1) processes
On inducing culture, first full light culture 30 days, then illumination every day 8 hours under the conditions of 26 DEG C, intensity of illumination is 1600x, directly
To induced synthesis embryo callus, callus induction rate is 65%.Described inducing culture is ER+5-BA1.0mg/L+
NAA0.6mg/L+GA36mg/L+ sucrose 12g/L+ agar 3.2g/L, pH is 5.5.
(3) differentiation culture: step (2) is cultivated the greeny Cotyledon Callus obtained and proceeds on division culture medium
Carrying out differentiation culture, first full light culture 6 days, then illumination every day 10 hours under the conditions of 26 DEG C after inoculation, intensity of illumination is
3500lx, cultivation temperature be 26 DEG C under conditions of cultivate until formed Multiple Buds, described division culture medium is ER+5-
BA1.0mg/L+IBA0.2mg/L+ sucrose 10g/L+ agar 3.2g/L, pH is 5.5.
(4) root culture: the Multiple Buds of the height about 2.6～3.8cm step (3) process obtained is cut and is inoculated into life
Root culture is carried out, first full light culture 6 days, then illumination every day 12 hours, light under the conditions of 26 DEG C after inoculation in root culture medium
Being 3000lx according to intensity, cultivation temperature is cultivated to taking root under conditions of being 26 DEG C, and rooting rate is 90%.Described root media
For 1/2MS+IBA0.1mg/L+GGR1.0mg/L+KT1.2mg/L+ sucrose 15g/L+ agar 3.2g/L, pH is 5.5.
(5) acclimatization and transplants: high about 8.5～the rooting tube plantlet of 9cm that step (4) are obtained go under bottle cap natural lighting
After seedling exercising 8 days, test tube Seedling is taken out from culture bottle, wash root culture medium off, plant into being mixed into by peat soil and yellow sand mud
In substrate and be colonizated in big Tanaka, transplanting survival rate is up to 92%.
Embodiment 2:
(1) seed disinfection: select 1 year half crude fruit, in detergent water soaking 16min, after outwash, tap water rinses 1.5h, takes
Go out the most immature seed, in superclean bench, strip off episperm, first with after 75% ethanol disinfection 9s with aseptic washing 4 times,
Again with 0.1% mercuric chloride solution sterilization 11min, with standby after aseptic water washing 5 times, adding up pollution rate after inoculating one week can as little as 6%.
(2) embryo callus induction: 0.4cm × 0.4cm × 0.4cm will be cut into by immaturity embryo after step (1) processes
Being inoculated on inducing culture, first full light culture 32 days, then illumination every day 11 hours under the conditions of 26 DEG C, intensity of illumination is
1600x, until induced synthesis embryo callus, callus induction rate is 65%.Described inducing culture is ER+8-
BA1.0mg/L+NAA0.3mg/L+GA34mg/L+ sucrose 15g/L+ agar 3.6g/L, pH is 5.9.
(3) differentiation culture: the greeny Cotyledon Callus that step (2) cultivation obtains is proceeded to division culture medium enterprising
Row differentiation culture, first full light culture 8 days, then illumination every day 11 hours under the conditions of 26 DEG C after inoculation, intensity of illumination is
2500lx, cultivation temperature is cultivated until forming Multiple Buds under conditions of being 26 DEG C.Described division culture medium is ER+8-
BA1.0mg/L+IBA0.2mg/L+ sucrose 15g/L+ agar 3.0g/L, pH is 5.9.
(4) root culture: the Multiple Buds of height about 3.6cm step (3) process obtained is cut and is inoculated into training of taking root
Supporting and carry out root culture on base, first full light culture 8 days under the conditions of 26 DEG C after inoculation, then illumination every day 12 hours, illumination is strong
Degree is 3000lx, and cultivation temperature is cultivated to taking root under conditions of being 26 DEG C, and rooting rate is 85%.Described root media is 1/
2MS+IBA0.5mg/L+GGR2.0mg/L+KT1.2mg/L+ sucrose 15g/L+ agar 3.0g/L, pH value is 5.9.
(5) acclimatization and transplants: the rooting tube plantlet of high about 9cm step (4) obtained removes bottle cap natural lighting lower refining seedling 8 days
After, test tube Seedling is taken out from culture bottle, washes root culture medium off, plant in the substrate being mixed into by peat soil and yellow sand mud also
Being colonizated in big Tanaka, transplanting survival rate is up to more than 90%.
Claims (4)
1. one kind little Semen Pini Tissue Culture Regeneration System construction method, it is characterised in that comprise the following steps:
(1) seed disinfection: selection initial stage maturation tea remains blue or green little Semen Pini fruit and puts into the detergent water soaking of intermediate concentration
32min, after cleaning, tap water rinses 2.5h, takes out sterilized seed, and in superclean bench, strip off episperm, first with 75%
With aseptic washing 6 times after ethanol disinfection 32s, then with 0.1% mercuric chloride solution sterilization 30min, with standby after aseptic water washing 8 times；
(2) embryo callus induction: 0.6cm × 0.6cm × 0.6cm inoculation will be cut into by immaturity embryo after step (1) processes
On inducing culture, first full light culture 46 days, then illumination every day 15 hours under the conditions of 29 DEG C, intensity of illumination is
3200lx, until induced synthesis embryo callus；
(3) differentiation culture: step (2) is cultivated the greeny Cotyledon Callus obtained and proceeds to carry out on division culture medium
Differentiation culture, first full light culture 15 days, then illumination every day 18 hours under the conditions of 29 DEG C after inoculation, intensity of illumination is
5500lx, is placed under conditions of cultivation temperature is 29 DEG C and cultivates until forming Multiple Buds；
(4) root culture: the Multiple Buds of height about 3.6cm step (3) process obtained is cut and is inoculated into root media
On carry out root culture, first full light culture 15 days, then illumination every day 18 hours under the conditions of 29 DEG C after inoculation, intensity of illumination is
3200lx, cultivation temperature is cultivated to taking root under conditions of being 29 DEG C；
(5) acclimatization and transplants: after the rooting tube plantlet of high about 9cm step (4) obtained removes bottle cap natural lighting lower refining seedling 8 days,
Test tube Seedling is taken out from culture bottle, washes root culture medium off, plant in the substrate being mixed into by peat soil and yellow sand mud and determine
Plant in big Tanaka.
One the most according to claim 1 little Semen Pini Tissue Culture Regeneration System construction method, it is characterised in that step
(2) inducing culture described in is: ER+5-BA4mg/L+NAA0.6mg/L+GA36 mg/L+ sucrose 32g/L+ agar 3.2g/L,
PH is 5.9.
One the most according to claim 1 little Semen Pini Tissue Culture Regeneration System construction method, it is characterised in that step (3)
Described division culture medium is: ER+5-BA6mg/L+IBA0.6mg/L+ sucrose 32g/L+ agar 6.5g/L, pH is 5.9.
One the most according to claim 1 little Semen Pini Tissue Culture Regeneration System construction method, it is characterised in that step (4)
Described root media is: 1/2MS+IBA1.0mg/L+GGR2.2mg/L+KT3.5mg/L+ sucrose 32g/L+ agar 3.0g/
L, pH are 5.9.
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| CN201610732230.6A CN106135005A (en) | 2016-08-26 | 2016-08-26 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
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| CN201610732230.6A CN106135005A (en) | 2016-08-26 | 2016-08-26 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109275573A (en) * | 2018-11-29 | 2019-01-29 | 广西玉林市千力农业科技有限公司 | A kind of macleaya cordata Tissue Culture Regeneration System construction method |
| CN109302988A (en) * | 2018-11-29 | 2019-02-05 | 广西玉林玖旺农业科技有限公司 | A kind of walnut tissue culture regenerating system construction method |
| CN109380115A (en) * | 2018-11-22 | 2019-02-26 | 张世燊 | A kind of fructus alpiniae oxyphyllae Tissue Culture Regeneration System construction method |
| CN109380085A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of coffee senna Tissue Culture Regeneration System construction method |
| CN109380117A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of Rubus parvifolius Tissue Culture Regeneration System construction method |
| CN109463278A (en) * | 2018-11-29 | 2019-03-15 | 广西玉林玖旺农业科技有限公司 | A kind of semen momordicae Tissue Culture Regeneration System construction method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109380115A (en) * | 2018-11-22 | 2019-02-26 | 张世燊 | A kind of fructus alpiniae oxyphyllae Tissue Culture Regeneration System construction method |
| CN109380085A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of coffee senna Tissue Culture Regeneration System construction method |
| CN109380117A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of Rubus parvifolius Tissue Culture Regeneration System construction method |
| CN109275573A (en) * | 2018-11-29 | 2019-01-29 | 广西玉林市千力农业科技有限公司 | A kind of macleaya cordata Tissue Culture Regeneration System construction method |
| CN109302988A (en) * | 2018-11-29 | 2019-02-05 | 广西玉林玖旺农业科技有限公司 | A kind of walnut tissue culture regenerating system construction method |
| CN109463278A (en) * | 2018-11-29 | 2019-03-15 | 广西玉林玖旺农业科技有限公司 | A kind of semen momordicae Tissue Culture Regeneration System construction method |
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Application publication date: 20161123 |