CN106718940A - A kind of tissue culture and rapid propagation method suitable for tung oil tree - Google Patents
A kind of tissue culture and rapid propagation method suitable for tung oil tree Download PDFInfo
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- 240000001866 Vernicia fordii Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 17
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- 238000003780 insertion Methods 0.000 claims abstract description 15
- 230000037431 insertion Effects 0.000 claims abstract description 15
- 239000002609 medium Substances 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 10
- 239000007921 spray Substances 0.000 claims abstract description 10
- 239000003921 oil Substances 0.000 claims abstract description 9
- 244000055346 Paulownia Species 0.000 claims abstract description 8
- 230000035755 proliferation Effects 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 3
- 238000005286 illumination Methods 0.000 claims description 27
- 239000006870 ms-medium Substances 0.000 claims description 24
- 229920001817 Agar Polymers 0.000 claims description 20
- 239000008272 agar Substances 0.000 claims description 20
- 241000206575 Chondrus crispus Species 0.000 claims description 16
- 230000003287 optical effect Effects 0.000 claims description 16
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 10
- 235000003704 aspartic acid Nutrition 0.000 claims description 10
- 150000001510 aspartic acids Chemical class 0.000 claims description 10
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 10
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 10
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000012549 training Methods 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 5
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 5
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 229960000304 folic acid Drugs 0.000 claims description 5
- 235000019152 folic acid Nutrition 0.000 claims description 5
- 239000011724 folic acid Substances 0.000 claims description 5
- 229960000367 inositol Drugs 0.000 claims description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 5
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 235000001968 nicotinic acid Nutrition 0.000 claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 5
- 239000011691 vitamin B1 Substances 0.000 claims description 5
- 239000011726 vitamin B6 Substances 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 239000011686 zinc sulphate Substances 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 4
- 239000007943 implant Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 241000196324 Embryophyta Species 0.000 description 5
- 238000012546 transfer Methods 0.000 description 3
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000028446 budding cell bud growth Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- ZMQAAUBTXCXRIC-UHFFFAOYSA-N safrole Chemical compound C=CCC1=CC=C2OCOC2=C1 ZMQAAUBTXCXRIC-UHFFFAOYSA-N 0.000 description 2
- 241001164374 Calyx Species 0.000 description 1
- 241000723346 Cinnamomum camphora Species 0.000 description 1
- 244000147058 Derris elliptica Species 0.000 description 1
- 241000221017 Euphorbiaceae Species 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- 244000057114 Sapium sebiferum Species 0.000 description 1
- 235000005128 Sapium sebiferum Nutrition 0.000 description 1
- 241000238370 Sepia Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 244000153888 Tung Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000021038 drupes Nutrition 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003864 humus Substances 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
It is applied to tung oil tree tissue culture and rapid propagation method the invention discloses one kind, including explant selection, explant treatment, initial bud inducement cultivation, Multiplying culture, strong bud culture and culture of rootage operation, collection semi-lignified, the paulownia spray of oil generation then of robust growth is used as explant, explant is pruned, sterilization obtains initial bud during initial bud inducement cultivation base is inoculated in after processing, initial bud is inoculated in proliferated culture medium by 34 cycle proliferation cultures again, form Multiple Buds, cultivated during Multiple Buds are accessed into strong buds medium, finally by root induction in the simple bud insertion root media of height >=2cm.Present invention reduces the growing-seedling period of tung oil tree, nursery material is saved, the problems such as nursery material requested is more, the cycle is long present in traditional seedling raising method are overcome, production cost is greatly reduced, production efficiency is improve, with preferable economic benefit, social benefit and ecological benefits.
Description
Technical field
The invention belongs to technical field of plant propagation, it is related to a kind of suitable for tung oil tree tissue culture and rapid propagation method.
Background technology
Tung oil tree（Vernicia fordii (Hemsl.) Airy Shaw）, Euphorbiaceae tung tree, category deciduous tree, bark
Grey, branch is sturdy, leaf oval, and flower monoecism, calyx outside is close by the micro- pubescence of sepia；Petal white, there is pale red
Vein, obovate, ovary is close by pubescence, and drupe is closely spherical, plants skin wooden.The flowering fruit bearing stage 3-9 months.It is distributed in Shaanxi, the river of China
South, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Hubei, Guangdong, Hainan, Guangxi, Sichuan, Guizhou, Yunnan etc. save
Area.Tung oil tree and oil tea, walnut, Chinese tallow tree simultaneously claim Chinese four big traditional oil trees.Tung oil tree is born in more than 1000 meters of area, happiness temperature
It is warm, avoid severe cold.18 DEG C of winter temperature drop is conducive to tung oil tree to grow, but being in less than -10 DEG C can cause jelly for a long time
Evil.It is suitable to be born in gentle slope and on the sunny side valley floor, basin and riverbed two sides tableland.Rich in humus, soil layer is deep, draining is good, neutral
To the most suitable tung oil tree growth of subacidity sandy loam.Tissue cultures can be by vegetative propagation, the good characteristic of hereditary seeds, can be short
Quick breeding obtains the tissue-cultured seedling of a large amount of high-quality in time, is promoted for high-quality introduces a collection and provides possibility.
The content of the invention
Bud growth coefficient is low during the purpose of the present invention is directed to tung oil tree tissue culture procedures, the problems such as slow-growing, there is provided
A kind of growth result is good, expand numerous fast tung oil tree spray tissue culture propagation method.
In order to realize foregoing invention purpose, technical scheme is as follows：
A kind of tissue culture and rapid propagation method suitable for tung oil tree, including explant selection, explant treatment, initial bud inducement cultivation, increasing
Culture, strong bud culture and culture of rootage operation are grown, collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant
Body, is pruned to explant, is inoculated in initial bud inducement cultivation base after sterilization treatment and obtains initial bud, then will be initial
Bud is inoculated in proliferated culture medium by 3-4 cycle proliferation culture, forms Multiple Buds, during Multiple Buds are accessed into strong buds medium
Culture, finally by root induction in the simple bud insertion root media of height >=2cm, concrete operation step is as follows：
（1）Explant is selected：Collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant；
（2）Explant treatment：Explant is placed under running water and rinses 10-15 min, then remove explant blade, then will be outer
Implant is immersed in the mercuric chloride solution of volumetric concentration 0.1%, controls soak time 15-20 min, finally cleans medicine with pure water
Thing, band at least one axillary bud is cut into by explant, stem section 1.5-3cm long, depending in stem section degree of lignification classified placing container,
It is standby；
（3）Initial bud inducement cultivation：By step（2）The explant for obtaining is cultivated in being inoculated in initial bud inducement cultivation base, culture
30-35 d, initial bud is sprouted, growth；
（4）Multiplying culture：Multiplying culture, 35-40 d switchings one are carried out during the initial bud of height >=1cm is accessed into proliferated culture medium
It is secondary, move in circles, through the 3-4 culture in cycle, form Multiple Buds；
（5）Strong bud culture：By step（4）The Multiple Buds for obtaining are cut, 4-5 bud/clump, in insertion strong buds medium, training
35-40 d are supported, strong bud is formed；
（6）Culture of rootage：By step（5）Root induction in the simple bud insertion root media of the height >=2cm for obtaining；Remaining budlet
Cong Zaici is inoculated in step（4）Proliferated culture medium in continue breed, then repeat step（5）, move in circles, obtain a large amount of
Strong bud.
The formula of above-described initial bud inducement cultivation base is：1/2 modified MS medium+6-BA 4.0-6.0 mg/L+
The mg/L+ folic acid 10mg/L+VB of NAA 2.0-3.0 mg/L+TDZ 1.0-2.0 mg/L+ZT 1.5-2.0 mg/L+ VC 152
The g/L of 15 mg/+ agar, 3.0 g/L+ carragheens 25.
The formula of above-described proliferated culture medium is：Improvement 1/2MS culture medium+6-BA 6.0-8.0 mg/L+NAA
2.0-3.0 mg/L+ZT 0.5-1.0 mg/L+VC 15 mg/L+VB2 The g/ of 15 mg+ aspartic acids, 20 mg/L+ agar 3.6
The g/L of L+ carragheens 30.
The formula of above-described strong buds medium is：Modified MS medium+6-BA 3.0-4.0 mg/L+NAA 1.0-
1.5 mg/L+VC 15 mg/L+VB2 The g/L of 15 mg+ aspartic acids, 20 mg/L+ agar, 3.5 g/L+ carragheens 25.
The formula of above-described root media is：1/2 modified MS medium+6-BA 1.5-2.5 mg/L+NAA
The g/L of 3.5 g/L+ carragheens of 1.0-1.5 mg/L+ agar 30.
The formula of above-described modified MS medium is：KNO3 660 mg·L-1；NH4NO3 850 mg·L-1；
CaCl2·2H2O 275 mg·L-1；MgSO4·7H2O 280 mg·L-1；Ca(NO3)2·4H2O 600 mg·L-1；KH2PO4
340 mg·L-1；MnSO4·H2O 22.3 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 105-1。
Above step（3）The cultivation control condition of described initial bud inducement cultivation is：Optical culture, illumination 500-1000
Lx, 20 ± 1 DEG C of cultivation temperature；Step（4）Described Multiplying culture, step（5）Described strong bud culture, step（6）Described
The cultivation control condition of culture of rootage is：Optical culture, illumination 2500-4000 lx, daily illumination 16 h, cultivation temperature 25-28
℃。
Relative to prior art, the present invention has the advantage that and has the beneficial effect that：
1st, the present invention using tung oil tree then give birth to spray as explant, by initial bud medium culture, initial bud sprout rate 90% with
On；By 3-4 cycle proliferation culture, Multiple Buds, bud growth coefficient 5/30d-7/30d are formed, then Multiple Buds are accessed into strong bud
Cultivated in culture medium, finally by root induction in the simple bud insertion root media of height >=2cm, so as to shorten the nursery of tung oil tree
In the cycle, nursery material is saved, overcome the problems such as nursery material requested is more, the cycle is long present in traditional seedling raising method, significantly
Production cost is reduced, production efficiency is improve.
2nd, by root induction in the simple bud insertion root media of height >=2cm, remaining budlet clump continues to breed training the present invention
Support so that material can multiple subculture, realize largely breeding, large-scale production is strengthened bud, realizes that the expansion of safrole type camphor tree is numerous.
3rd, the present invention is improved to MS minimal mediums, at the same emphasis have adjusted it is related first to the tung oil tree strong bud that sprouts
Element so that culture medium is more scientific, more targetedly, is more easy to generation, propagation and the strong bud for promoting tung oil tree bud to induce, effect is significant.
4th, operating process of the present invention is easy, and cultivation cycle is short, and subculture bud yield is big, and Functionality, quality and appealing design, and growth coefficient reaches 5
Tissue-cultured seedling industrialization technology requirement above, with good economic benefit, ecological benefits and social benefit.
Specific embodiment
With reference to specific embodiment, the present invention will be further described.
Embodiment 1：
（1）Explant is selected：Collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant；
（2）Explant treatment：Explant is placed in 10 min are rinsed under running water, then remove explant blade, then by explant
Body is immersed in the mercuric chloride solution of volumetric concentration 0.1%, controls the min of soak time 15, finally cleans medicine with pure water, will
Explant is cut into band at least one axillary bud, and stem section 1.5-3cm long is standby depending in stem section degree of lignification classified placing container；
（3）Initial bud inducement cultivation：By step（2）The explant for obtaining is cultivated in being inoculated in initial bud inducement cultivation base, is placed in
Optical culture, the lx of illumination 500, cultivate 30-35 d in the environment that 20 ± 1 DEG C of cultivation temperature, initial bud is sprouted, growth；Described is first
The beginning formula of bud inducement cultivation base is：The mg/ of 1/2 modified MS medium+6-BA, 4.0 mg/L+NAA, 2.0 mg/L+TDZ 1.0
The mg/L+ folic acid 10mg/L+VB of 1.5 mg/L+ VC of L+ZT 152 The g/L of 15 mg/+ agar, 3.0 g/L+ carragheens 25；
（4）Multiplying culture：Multiplying culture is carried out during the initial bud of height >=1cm is accessed into proliferated culture medium, optical culture, illumination is placed in
2500 lx, the h of daily illumination 16, in the environment of 25-28 DEG C of cultivation temperature, 35-40 d transfer once, move in circles, through 3-4
The culture in cycle, forms Multiple Buds；The formula of described proliferated culture medium is：The mg/L+ of 1/2 modified MS medium+6-BA 6.0
NAA 2.0 mg/L+ZT 0.5 mg/L+VC 15 mg/L+VB2 The g/L+ cards of 15 mg+ aspartic acids, 20 mg/L+ agar 3.6
Draw the g/L of glue 30；
（5）Strong bud culture：By step（4）The Multiple Buds for obtaining are cut, 4-5 bud/clump, in insertion strong buds medium, are put
In optical culture, the lx of illumination 2500, the h of daily illumination 16 cultivate 35-40 d in the environment of 25-28 DEG C of cultivation temperature, form strong
Bud；The formula of described strong buds medium is：The mg/ of 3.0 mg/L+NAA of modified MS medium+6-BA, 1.0 mg/L+VC 15
L+VB2 The g/L of 15 mg+ aspartic acids, 20 mg/L+ agar, 3.5 g/L+ carragheens 25；
（6）Culture of rootage：By step（5）In the simple bud insertion root media of the height >=2cm for obtaining, remaining budlet clump connects again
Plant in step（4）Proliferated culture medium in continue breed, then repeat step（5）, move in circles, obtain largely strong bud；It is raw
Root culture is placed in optical culture, the lx of illumination 2500, the h of daily illumination 16, root induction in the environment of 25-28 DEG C of cultivation temperature,
Rooting rate is 89%；The formula of described root media is：The mg/L+NAA 1.0 of 1/2 modified MS medium+6-BA 1.5
The g/L of 3.5 g/L+ carragheens of mg/L+ agar 30.
The formula of above-described modified MS medium is：KNO3 660 mg·L-1；NH4NO3 850 mg·L-1；
CaCl2·2H2O 275 mg·L-1；MgSO4·7H2O 280 mg·L-1；Ca(NO3)2·4H2O 600 mg·L-1；KH2PO4
340 mg·L-1；MnSO4·H2O 22.3 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 105-1。
Embodiment 2：
（1）Explant is selected：Collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant；
（2）Explant treatment：Explant is placed in 15 min are rinsed under running water, then remove explant blade, then by explant
Body is immersed in the mercuric chloride solution of volumetric concentration 0.1%, controls the min of soak time 20, finally cleans medicine with pure water, will
Explant is cut into band at least one axillary bud, and stem section 1.5-3cm long is standby depending in stem section degree of lignification classified placing container；
（3）Initial bud inducement cultivation：By step（2）The explant for obtaining is cultivated in being inoculated in initial bud inducement cultivation base, is placed in
Optical culture, the lx of illumination 1000, cultivate 30-35 d in the environment that 20 ± 1 DEG C of cultivation temperature, initial bud is sprouted, growth；Described
Initially the formula of bud inducement cultivation base is：The mg/L+TDZ 1.5 of 1/2 modified MS medium+6-BA, 5.0 mg/L+NAA 3.0
The mg/L+ folic acid 10mg/L+VB of 1.5 mg/L+ VC of mg/L+ZT 152 The g/L of 15 mg/+ agar, 3.0 g/L+ carragheens 25；
（4）Multiplying culture：Multiplying culture is carried out during the initial bud of height >=1cm is accessed into proliferated culture medium, optical culture, illumination is placed in
3000 lx, the h of daily illumination 16, in the environment of 25-28 DEG C of cultivation temperature, 35-40 d transfer once, move in circles, through 3-4
The culture in cycle, forms Multiple Buds；The formula of described proliferated culture medium is：The mg/L+ of 1/2 modified MS medium+6-BA 7.0
NAA 2.5 mg/L+ZT 1.0 mg/L+VC 15 mg/L+VB2 The g/L+ cards of 15 mg+ aspartic acids, 20 mg/L+ agar 3.6
Draw the g/L of glue 30；
（5）Strong bud culture：By step（4）The Multiple Buds for obtaining are cut, 4-5 bud/clump, in insertion strong buds medium, are put
In optical culture, the lx of illumination 3000, the h of daily illumination 16 cultivate 35-40 d in the environment of 25-28 DEG C of cultivation temperature, form strong
Bud；The formula of described strong buds medium is：The mg/ of 3.5 mg/L+NAA of modified MS medium+6-BA, 1.0 mg/L+VC 15
L+VB2 The g/L of 15 mg+ aspartic acids, 20 mg/L+ agar, 3.5 g/L+ carragheens 25；
（6）Culture of rootage：By step（5）In the simple bud insertion root media of the height >=2cm for obtaining, remaining budlet clump connects again
Plant in step（4）Proliferated culture medium in continue breed, then repeat step（5）, move in circles, obtain largely strong bud；It is raw
Root culture is placed in optical culture, the lx of illumination 3000, the h of daily illumination 16, root induction in the environment of 25-28 DEG C of cultivation temperature,
Rooting rate is 92%；The formula of described root media is：The mg/L+NAA 1.5 of 1/2 modified MS medium+6-BA 2.0
The g/L of 3.5 g/L+ carragheens of mg/L+ agar 30.
The formula of above-described modified MS medium is：KNO3 660 mg·L-1；NH4NO3 850 mg·L-1；
CaCl2·2H2O 275 mg·L-1；MgSO4·7H2O 280 mg·L-1；Ca(NO3)2·4H2O 600 mg·L-1；KH2PO4
340 mg·L-1；MnSO4·H2O 22.3 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 105-1。
Embodiment 3：
（1）Explant is selected：Collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant；
（2）Explant treatment：Explant is placed in 15 min are rinsed under running water, then remove explant blade, then by explant
Body is immersed in the mercuric chloride solution of volumetric concentration 0.1%, controls the min of soak time 20, finally cleans medicine with pure water, will
Explant is cut into band at least one axillary bud, and stem section 1.5-3cm long is standby depending in stem section degree of lignification classified placing container；
（3）Initial bud inducement cultivation：By step（2）The explant for obtaining is cultivated in being inoculated in initial bud inducement cultivation base, is placed in
Optical culture, the lx of illumination 1000, cultivate 30-35 d in the environment that 20 ± 1 DEG C of cultivation temperature, initial bud is sprouted, growth；Described
Initially the formula of bud inducement cultivation base is：The mg/L+TDZ 2.0 of 1/2 modified MS medium+6-BA, 6.0 mg/L+NAA 2.5
The mg/L+ folic acid 10mg/L+VB of 2.0 mg/L+ VC of mg/L+ZT 152 The g/L of 15 mg/+ agar, 3.0 g/L+ carragheens 25；
（4）Multiplying culture：Multiplying culture is carried out during the initial bud of height >=1cm is accessed into proliferated culture medium, optical culture, illumination is placed in
4000 lx, the h of daily illumination 16, in the environment of 25-28 DEG C of cultivation temperature, 35-40 d transfer once, move in circles, through 3-4
The culture in cycle, forms Multiple Buds；The formula of described proliferated culture medium is：The mg/L+ of 1/2 modified MS medium+6-BA 8.0
NAA 3.0 mg/L+ZT 1.0 mg/L+VC 15 mg/L+VB2 The g/L+ cards of 15 mg+ aspartic acids, 20 mg/L+ agar 3.6
Draw the g/L of glue 30；
（5）Strong bud culture：By step（4）The Multiple Buds for obtaining are cut, 4-5 bud/clump, in insertion strong buds medium, are put
In optical culture, the lx of illumination 4000, the h of daily illumination 16 cultivate 35-40 d in the environment of 25-28 DEG C of cultivation temperature, form strong
Bud；The formula of described strong buds medium is：The mg/ of 4.0 mg/L+NAA of modified MS medium+6-BA, 1.5 mg/L+VC 15
L+VB2 The g/L of 15 mg+ aspartic acids, 20 mg/L+ agar, 3.5 g/L+ carragheens 25；
（6）Culture of rootage：By step（5）In the simple bud insertion root media of the height >=2cm for obtaining, remaining budlet clump connects again
Plant in step（4）Proliferated culture medium in continue breed, then repeat step（5）, move in circles, obtain largely strong bud；It is raw
Root culture is placed in optical culture, the lx of illumination 4000, the h of daily illumination 16, root induction in the environment of 25-28 DEG C of cultivation temperature,
Rooting rate is 93%；；The formula of described root media is：The mg/L+NAA 1.5 of 1/2 modified MS medium+6-BA 2.5
The g/L of 3.5 g/L+ carragheens of mg/L+ agar 30.
The formula of above-described modified MS medium is：KNO3 660 mg·L-1；NH4NO3 850 mg·L-1；
CaCl2·2H2O 275 mg·L-1；MgSO4·7H2O 280 mg·L-1；Ca(NO3)2·4H2O 600 mg·L-1；KH2PO4
340 mg·L-1；MnSO4·H2O 22.3 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025
mg·L-1；H3BO3 6.2 mg·L-1；Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O
0.025 mg·L-1；Vitamin B1 1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；The mgL of nicotinic acid 0.5-1；Glycine
2.0 mg·L-1；The mgL of inositol 105-1。
Claims (7)
1. a kind of tissue culture and rapid propagation method suitable for tung oil tree, it is characterised in that：Selected including explant, explant is processed, initial
Bud inducement cultivation, Multiplying culture, strong bud culture and culture of rootage operation, collection semi-lignified, the oil generation paulownia then of robust growth
Spray is pruned to explant, is inoculated in initial bud inducement cultivation base after sterilization treatment and obtains just as explant
Beginning bud, then initial bud is inoculated in proliferated culture medium by 3-4 cycle proliferation culture, Multiple Buds are formed, Multiple Buds are connect
Enter culture in strong buds medium, finally by root induction in the simple bud insertion root media of height >=2cm, concrete operation step is such as
Under：
（1）Explant is selected：Collection semi-lignified, the paulownia spray of oil generation then of robust growth are used as explant；
（2）Explant treatment：Explant is placed under running water and rinses 10-15 min, then remove explant blade, then will be outer
Implant is immersed in the mercuric chloride solution of volumetric concentration 0.1%, controls soak time 15-20 min, finally cleans medicine with pure water
Thing, band at least one axillary bud is cut into by explant, stem section 1.5-3cm long, depending in stem section degree of lignification classified placing container,
It is standby；
（3）Initial bud inducement cultivation：By step（2）The explant for obtaining is cultivated in being inoculated in initial bud inducement cultivation base, culture
30-35 d, initial bud is sprouted, growth；
（4）Multiplying culture：Multiplying culture, 35-40 d switchings one are carried out during the initial bud of height >=1cm is accessed into proliferated culture medium
It is secondary, move in circles, through the 3-4 culture in cycle, form Multiple Buds；
（5）Strong bud culture：By step（4）The Multiple Buds for obtaining are cut, 4-5 bud/clump, in insertion strong buds medium, training
35-40 d are supported, strong bud is formed；
（6）Culture of rootage：By step（5）Root induction in the simple bud insertion root media of the height >=2cm for obtaining；Remaining budlet
Cong Zaici is inoculated in step（4）Proliferated culture medium in continue breed, then repeat step（5）, move in circles, obtain a large amount of
Strong bud.
2. a kind of tissue culture and rapid propagation method suitable for tung oil tree according to claim 1, it is characterised in that：Described initial bud
The formula of inducing culture is：1/2 modified MS medium+6-BA 4.0-6.0 mg/L+NAA 2.0-3.0 mg/L+TDZ
The mg/L+ folic acid 10mg/L+VB of 1.0-2.0 mg/L+ZT 1.5-2.0 mg/L+ VC 152 The g/L+ cards of 15 mg/+ agar 3.0
Draw the g/L of glue 25.
3. a kind of tissue culture and rapid propagation method suitable for tung oil tree according to claim 1, it is characterised in that：Described propagation training
Support base formula be：1/2 modified MS medium+6-BA 6.0-8.0 mg/L+NAA 2.0-3.0 mg/L+ZT 0.5-1.0
mg/L+VC 15 mg/L+VB2 The g/L of 15 mg+ aspartic acids, 20 mg/L+ agar, 3.6 g/L+ carragheens 30.
4. a kind of tissue culture and rapid propagation method suitable for tung oil tree according to claim 1, it is characterised in that：Described strong bud training
Support base formula be：The mg/L+VB of modified MS medium+6-BA 3.0-4.0 mg/L+NAA 1.0-1.5 mg/L+VC 152 15
The g/L of 20 mg/L+ agar of mg+ aspartic acids, 3.5 g/L+ carragheens 25.
5. a kind of tissue culture and rapid propagation method suitable for tung oil tree according to claim 1, it is characterised in that：Described training of taking root
Support base formula be：The g/L+ of 1/2 modified MS medium+6-BA 1.5-2.5 mg/L+NAA 1.0-1.5 mg/L+ agar 3.5
The g/L of carragheen 30.
6. according to a kind of any described tissue culture and rapid propagation methods suitable for tung oil tree of claim 2-5, it is characterised in that：Described
The formula of modified MS medium is：KNO3 660 mg·L-1；NH4NO3 850 mg·L-1；CaCl2·2H2O 275 mg·L-1；
MgSO4·7H2O 280 mg·L-1；Ca(NO3)2·4H2O 600 mg·L-1；KH2PO4 340 mg·L-1；MnSO4·H2O
22.3 mg·L-1；ZnSO4·7H2O 8.6 mg·L-1；CuSO4·5H2O 0.025 mg·L-1；H3BO3 6.2 mg·L-1；
Na2MoO4·2H2O 0.025 mg·L-1；KI 0.83 mg·L-1；CoCl2·6H2O 0.025 mg·L-1；Vitamin B1
1.0 mg·L-1；Vitamin B6 0.5 mg·L-1；The mgL of nicotinic acid 0.5-1；The mgL of glycine 2.0-1；Inositol 105
mg·L-1。
7. a kind of tissue culture and rapid propagation method suitable for tung oil tree according to claim 1, it is characterised in that：Step（3）It is described
The cultivation control condition of initial bud inducement cultivation be：Optical culture, illumination 500-1000 lx, 20 ± 1 DEG C of cultivation temperature；Step
（4）Described Multiplying culture, step（5）Described strong bud culture, step（6）The cultivation control condition of described culture of rootage is equal
For：Optical culture, illumination 2500-4000 lx, the h of daily illumination 16,25-28 DEG C of cultivation temperature.
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