CN106376461A - Rooting culture and hardening seedling method of cunninghamia lanceolata tissue-cultured seedling - Google Patents
Rooting culture and hardening seedling method of cunninghamia lanceolata tissue-cultured seedling Download PDFInfo
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- CN106376461A CN106376461A CN201610755011.XA CN201610755011A CN106376461A CN 106376461 A CN106376461 A CN 106376461A CN 201610755011 A CN201610755011 A CN 201610755011A CN 106376461 A CN106376461 A CN 106376461A
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- hardening
- culture
- seedling
- rootage
- plantlet
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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
-
- 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 relates to a rooting culture and hardening seedling method of a cunninghamia lanceolata tissue-cultured seedling. The method comprises steps of selection of tissue-cultured bud seedlings, rooting and inoculation, rooting culture, rooting and cap-sealed hardening seedling, and uncapped hardening seedling. The method combines rooting with hardening seedling, short-period rooting culture is carried out in a culture room, and rooting and cap-sealed hardening seedling and uncapped hardening seedling culture are carried out, so that the tissue-cultured seedling is subjected to rooting and hardening seedling at the same time. Time for rooting and hardening seedling of the cunninghamia lanceolata tissue-cultured seedlings is obviously shortened. Compared with a conventional method, the method allows the seedling culture period to be obviously shortened, provides suitable root length, and obviously improves the seedling culture efficiency. Energy and expense saving is achieved through natural temperature control and light control. The problems of high cost and low survival rate of transplanting in a production process of a cunninghamia lanceolata tissue-cultured seedling are solved. The method is beneficial for establishing an excellent asexual seedling culture base for cunninghamia lanceolata and provides superior seedlings for forestry production.
Description
Technical field
The present invention relates to agricultural technology field, the method for more particularly to a kind of China fir plantlet in vitro culture of rootage and hardening.
Background technology
China fir (Cunninghamia Lanceolata) is Taxodiaceae (Taxodiaceae), Cunninghamia aiphyllium seeds,
Growth is fast, material is good, is one of important fast-growing commodity commerical tree species of south China.Plant Tissue Breeding is Fast-propagation plant
The important method of new varieties, while keeping kind excellent hereditary capacity, can breed high quality seedling in a short time in a large number for life
Produce application, be greatly facilitated the improved variety process of Industry plantation.China Fir Study on tissue culture last decade quickly grows,
Set up China fir tissue culture production line on Fujian, Guangdong and other places, but prior art generally existing production cost is high, nursery effect is low asks
Topic, not yet has the method for tissue culture of high-efficiency and economic to set up high quality seedling production base as support, adds existing breeding garden yield
Deficiency, supply falls short of demand to lead to Cunninghamia Lanceolata Plantations breeding nursery stock, produces very different with kind of hereditary quality, how economical, quickly numerous
Educate breeding of new generation extremely urgent.Simple dependence China fir existing tissue culture seedling rooting and hardening technology carry out the seedling-wood breeding cycle
Long, transplanting survival rate is low, has no related shortening China fir tissue culture seedling rooting and the method for hardening time report.
Content of the invention
Based on this, the invention provides a kind of method of China fir plantlet in vitro culture of rootage and hardening, the method can shorten
China fir tissue culture seedling rooting and the time of hardening, shorten the seedling-wood breeding cycle, and the transplanting survival rate of nursery stock can be improved.
Concrete technical scheme is as follows.
A kind of China fir plantlet in vitro culture of rootage and the method for hardening, comprise the following steps：
(1) Tube plantlets are selected：The tissue culture bud more than 3cm for the length is selected in the China fir Tube plantlets carry out Multiplying culture
Seedling is as the material of culture of rootage；
(2) take root inoculation：The part of the top tip of Tube plantlets described in clip down long 2.5cm～3.0cm, insertion is equipped with life
In the blake bottle of root culture medium, seal；
(3) culture of rootage：The Tube plantlets inoculated are carried out culture of rootage, cultivation temperature is 25 ± 5 DEG C, relative humidity
For 50%～60%, light culture carries out optical culture 15～20 days after 3～5 days again, the intensity of illumination of optical culture be 1500Lx～
3000Lx, light application time is daily 12h～16h；
(4) take root seal cap hardening：The nursery stock being spread in a single layer after step (3) culture of rootage, makes every bottle seedling wood have
Natural light irradiation, carries out covering hardening 10～15 days, so that nursery stock is taken root hardening, the temperature of capping hardening is 20～35
℃；
(5) uncap hardening：When capping hardening makes the root system of nursery stock grow to length for 0.5cm～1.0cm, open blake bottle
Lid, is filled to water surface elevation in blake bottle and exceedes media surface 0.5cm～1.0cm, and carry out uncapping hardening 2～5 days, opens
The temperature of lid hardening is 20～35 DEG C, and illumination condition is natural lighting.
Wherein in some embodiments, the time of inoculation of taking root described in step (2) is 2 months August～next year.
Wherein in some embodiments, described root media is containing 0.5～0.8mg/L methyl α-naphthyl acetate, 0.1～0.2mg/
The 1/2MS culture medium of L indolebutyric acid, 24～26g/L sucrose and 6～8g/L agar.
Wherein in some embodiments, the volume of described blake bottle is 220～260mL, a height of 85～95mm, bore are 60
～64mm, inoculates 12～15 plants of Tube plantlets in each blake bottle.
Wherein in some embodiments, described culture of rootage is carried out in Sterile culture room, daily with uviol lamp to aseptic
Culturing room sterilizes 2～3 times, 25～35min every time.
Wherein in some embodiments, described capping hardening and hardening of uncapping are entered all in hot house or in glass room
OK.
Wherein in some embodiments, when the hardening time being 9～November, add a cover 70%～75% shading net, and beat
Door window makes cross-ventilation, by hardening temperature control between 30 ± 5 DEG C；When the hardening time being 2 months December～next years, remove
Shading net, and close the doors and windows, by hardening temperature control between 25 ± 5 DEG C.
Wherein in some embodiments, described uncap in experienced seedling step, open culture bottle cover time be afternoon 4:00
After.
Wherein in some embodiments, the method for described China fir plantlet in vitro culture of rootage and hardening also includes transplantation of seedlings
Step：By the nursery stock that takes root after hardening of uncapping, take out from blake bottle, the culture medium cleaning up nursery stock base portion entered after the same day
Row is transplanted；The matrix transplanted is yellow soil.
Wherein in some embodiments, the time that fine day is transplanted is afternoon 4：After 00, the time of overcast and rainy transplanting is complete
My god.
The method of the China fir plantlet in vitro culture of rootage of the present invention and hardening has advantages below and beneficial effect：
The present inventor determines the side of a kind of China fir plantlet in vitro culture of rootage and hardening by lot of experiments
Method, the method operation is fairly simple, will take root and combine with hardening, and first carrying out short-term culture of rootage, to treat that nursery stock base portion has been formed swollen
During the root system of swollen callus or small particles sample of just having emerged, that is, carry out seal cap hardening culture of taking root, make plantlet in vitro
Take root one side hardening；During culture of rootage, light culture is combined with optical culture, and during hardening, capping hardening is combined with hardening of uncapping；And
Rationally control each parameter in culture of rootage and hardening step further, make the method can significantly shorten China fir tissue culture seedling rooting
With the time of hardening so that growing-seedling period is than conventional method, and notable shortening, nursery effect significantly improve, can effectively solving existing
China fir plantlet in vitro production process in high cost, the problem of low transplanting survival rate, contribute to excellent Chinese fir clone nursery base
Foundation, for production of forestry provide high quality seedling.
In addition, the method employs nature temperature control control light measure to hardening environment, that is, utilize natural lighting and air humidity,
Be spread in a single layer nursery stock, makes every bottle seedling wood have uniform natural light irradiation, adds a cover 75% shading net, and beat on hot house top
Door window keeps cross-ventilation thus controlling temperature, for comparing more existing employing air-conditioning and light temperature control control light, more energy-conservation
Section, environmental protection.
Specific embodiment
Below in conjunction with specific embodiment, the China fir plantlet in vitro culture of rootage of the present invention and the method for hardening are carried out further
Detailed description.
Embodiment 1
Cultivate 6 Clones of Cunninghamia Lanceolatas in Guangdong Academy of Forestry, the nursery stock that takes root amounts to 6,000 plants, transplant after hardening
5,000 plants of nursery stock.The China fir plantlet in vitro culture of rootage that the present embodiment adopts and the method for hardening are specific as follows：
(1) Tube plantlets for culture of rootage are selected：On September 15th, 2015, is carrying out the China fir tissue culture of Multiplying culture
In sprout, the sprout that growth selection is healthy and strong, leaf color is dark green, length is more than 3cm carries out culture of rootage；
(2) take root inoculation：On superclean bench, the top tip that clip is used for the Tube plantlets of culture of rootage is down long
The part of 2.5cm～3.0cm, timely insertion has been got ready in the glass blake bottle of root media, every bottle of inoculation nursery stock 12～15
Strain, covered plastic closure, the volume of blake bottle is 240mL, a height of 90mm, bore are 62mm.The consisting of of root media
1/2MS+0.5～0.8mg/L methyl α-naphthyl acetate (NAA)+0.1～0.2mg/L indolebutyric acid (IBA)+25g/L sucrose+7g/L agar；
(3) culture of rootage：On September 17th, 2015, puts into the blake bottle having inoculated Tube plantlets in Sterile culture room to enter
Row culture of rootage, at 25 ± 2 DEG C, relative humidity controls 50%～60% culturing room's temperature control, keeps indoor environment cleaning,
And After Hours open the aseptic 30min that culturing room is sterilized of uviol lamp respectively at daily noon and evening staff；First to group
Training sprout carries out the light culture of 5 days, then proceeds by within 22nd the optical culture of 15 days by a definite date, the light of optical culture in September in 2015
It is 3000Lx according to intensity, light application time is daily 16h, when nursery stock base portion has formed the callus of swelling or little Bai of just having emerged
During the root system of point sample, you can carry out seal cap hardening culture of taking root；
(4) take root seal cap hardening：On October 7th, 2015, the nursery stock after culture of rootage is moved to from Sterile culture room and moulds
Carry out, in material booth, seal cap hardening treatment of taking root, using natural lighting and air humidity, be spread in a single layer nursery stock, make every bottle seedling wood
There is natural light irradiation；75% shading net, for preventing temperature of shed too high, is added a cover on hot house top, and opens door and window and protect
Hold cross-ventilation, temperature control is in (30 ± 5) DEG C；
(5) uncap hardening：On October 27th, 2015, during the long 0.5cm～1.0cm of seedling root, afternoon 4:After 00, open
The lid of blake bottle, injects running water, and the water surface exceedes media surface 0.5cm～1.0cm, and carry out uncapping hardening, hardening of uncapping
Temperature and the same step of humid control (4)；
(6) go out transplantation of seedlings：On October 30th, 2015, at the cloudy day, the nursery stock that takes root after hardening of uncapping is taken out from blake bottle,
Transplanted after the culture medium cleaning up nursery stock base portion, transplanting medium is yellow soil, average transplanting survival rate is 82%.This reality
Apply example take root hardening situation and its effect refers to table 1.
Comparative example 1
Taking clone C7 as a example, the tradition that this comparative example adopts is taken root and hardening off method comprises the following steps：
(1) in the Clones of Cunninghamia Lanceolata Tube plantlets carrying out Multiplying culture, growth selection is healthy and strong, leaf color is dark green, length is super
The sprout crossing 3cm carries out culture of rootage；
(2) with the step (2) in embodiment 1；
(3) blake bottle having inoculated Tube plantlets is put into and in Sterile culture room, carry out culture of rootage, cultivate room temperature control
At 25 ± 2 DEG C, relative humidity controls 50%～60% system, and opposite offspring first carries out the light culture of 5 days, is then scheduled to last
The optical culture of 30 days, intensity of illumination 3000Lx, the daily 16h of light application time, as long more than the 0.5cm of seedling root, you can carry out
Hardening；
(4) nursery stock having taken root is moved to hot house inner cover hardening 15 days from Sterile culture room, seedling root is long
1.5cm more than；
(5) fine day afternoon 4:After 00 or cloudy, the nursery stock that takes root after hardening is taken out from blake bottle, cleans up seedling
Transplanted after the culture medium of wooden base portion, transplanting medium is yellow soil, average transplanting survival rate 50%.The refining of taking root of this comparative example
Growth of cereal crop seedlings condition refers to table 1.
The implementation result of the method for table 1 China fir plantlet in vitro culture of rootage and hardening
From 2010, inventor carried out extensive Clones of Cunninghamia Lanceolata plantlet in vitro experimental study, amount within 6 years breed asexual
It is 10, nearly 1,500,000 plants of tissue-culture container seedling, take root about 100,000 plants of nursery stock, average rooting rate 89%, and transplant survival nursery stock is less than 50,000
Strain, average transplanting survival rate is less than 50%, and nursery stock rootage duration is 25～45 days, and the hardening time is 15～20 days, takes root plus refining
Seedling flow process takes total 40～65 days, breeds cost about 2.0 yuan/plant.Finally work out the plantlet in vitro culture of rootage of the present embodiment
With the method for hardening, cultivate China fir tissue culture and take root nearly 30,000 plants of seedling, average rooting rate 92%, transplanting survival rate improves to 82%, raw
Root adds hardening flow process and foreshortens to 30～45 days, and seedling cost is down to about 1.5 yuan/plant.This method except shorten cultivation period in addition to, to refining
Seedling environment employs nature temperature control control light measure, that is, utilize natural lighting and air humidity, be spread in a single layer nursery stock, makes every bottle seedling wood
Have uniform natural light irradiation, 75% shading net added a cover on hot house top, and open door and window keep cross-ventilation thus
Control temperature, for comparing more existing employing air-conditioning and light temperature control control light, more energy-saving prop up, environmental protection.By the method
It is applied in excellent Chinese fir clonal tissue culture production line, be that good base has been established in production and the popularization and application of China fir high quality seedling
Plinth.
Each technical characteristic of embodiment described above can arbitrarily be combined, for making description succinct, not to above-mentioned reality
The all possible combination of each technical characteristic applied in example is all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all it is considered to be the scope of this specification record.
Embodiment described above only have expressed the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
Say, without departing from the inventive concept of the premise, some deformation can also be made and improve, these broadly fall into the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of method of China fir plantlet in vitro culture of rootage and hardening is it is characterised in that comprise the following steps：
(1) Tube plantlets are selected：The Tube plantlets that length is more than 3cm are selected to make in the China fir Tube plantlets carry out Multiplying culture
Material for culture of rootage；
(2) take root inoculation：The part of the top tip of Tube plantlets described in clip down long 2.5cm～3.0cm, insertion is equipped with training of taking root
In the blake bottle of foster base, seal；
(3) culture of rootage：The Tube plantlets inoculated are carried out culture of rootage, cultivation temperature is 25 ± 5 DEG C, relative humidity is
50%～60%, light culture carries out optical culture 15～20 days after 3～5 days again, the intensity of illumination of optical culture be 1500Lx～
3000Lx, light application time is daily 12h～16h；
(4) take root seal cap hardening：The nursery stock being spread in a single layer after step (3) culture of rootage, makes every bottle seedling wood have nature
Light irradiation, carries out covering hardening 10～15 days, so that nursery stock is taken root hardening, and the temperature of capping hardening is 20～35 DEG C；
(5) uncap hardening：When capping hardening makes the root system of nursery stock grow to length for 0.5cm～1.0cm, open the lid of blake bottle
Son, is filled to water surface elevation in blake bottle and exceedes media surface 0.5cm～1.0cm, and carry out uncapping hardening 2～5 days, uncaps
The temperature of hardening is 20～35 DEG C, and illumination condition is natural lighting.
2. the method for China fir plantlet in vitro culture of rootage according to claim 1 and hardening is it is characterised in that step (2) institute
The time stating inoculation of taking root is 2 months August～next year.
3. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that described life
Root culture medium is containing 0.5～0.8mg/L methyl α-naphthyl acetate, 0.1～0.2mg/L indolebutyric acid, 24～26g/L sucrose and 6～8g/L
The 1/2MS culture medium of agar.
4. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that described training
The volume of foster bottle is 220～260mL, a height of 85～95mm, bore are 60～64mm, inoculates Tube plantlets 12 in each blake bottle
～15 plants.
5. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that described life
Root culture is carried out in Sterile culture room, with uviol lamp, Sterile culture room is sterilized 2～3 times daily, 25～35min every time.
6. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that described envelope
Lid hardening and hardening of uncapping are carried out all in hot house or in glass room.
7. the method for China fir plantlet in vitro culture of rootage according to claim 6 and hardening is it is characterised in that work as the hardening time
During for 9～November, adding a cover 70%～75% shading net, and open door and window makes cross-ventilation, by hardening temperature control 30 ± 5
Between DEG C；When the hardening time being 2 months December～next year, remove shading net, and close the doors and windows, by hardening temperature control 25 ±
Between 5 DEG C.
8. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that described open
Lid is practiced in seedling step, and the time opening culture bottle cover is afternoon 4:After 00.
9. the method for China fir plantlet in vitro culture of rootage according to claim 1 and 2 and hardening is it is characterised in that also include
The step going out transplantation of seedlings：By the nursery stock that takes root after hardening of uncapping, take out from blake bottle, clean up the culture medium of nursery stock base portion
Transplanted after the same day；The matrix transplanted is yellow soil.
10. the method for China fir plantlet in vitro culture of rootage according to claim 9 and hardening is it is characterised in that fine day is transplanted
Time be afternoon 4：After 00, the time of overcast and rainy transplanting is whole day.
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Cited By (3)
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| CN107197746A (en) * | 2017-07-27 | 2017-09-26 | 广东省林业科学研究院 | A kind of mating system of China fir field excellent resources |
| CN109348953A (en) * | 2018-10-18 | 2019-02-19 | 南阳师范学院 | A kind of Kiwi berry culture of rootage and acclimatization and transplants method |
| CN113826547A (en) * | 2021-02-01 | 2021-12-24 | 广东省林业科学研究院 | Method for recycling fir tissue culture polluted adventitious buds |
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| CN109348953A (en) * | 2018-10-18 | 2019-02-19 | 南阳师范学院 | A kind of Kiwi berry culture of rootage and acclimatization and transplants method |
| CN113826547A (en) * | 2021-02-01 | 2021-12-24 | 广东省林业科学研究院 | Method for recycling fir tissue culture polluted adventitious buds |
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