CN103749310B - Method for promoting pinus massoniana tissue-cultured subcultured bud to root - Google Patents
Method for promoting pinus massoniana tissue-cultured subcultured bud to root Download PDFInfo
- Publication number
- CN103749310B CN103749310B CN201410041856.3A CN201410041856A CN103749310B CN 103749310 B CN103749310 B CN 103749310B CN 201410041856 A CN201410041856 A CN 201410041856A CN 103749310 B CN103749310 B CN 103749310B
- Authority
- CN
- China
- Prior art keywords
- bud
- mgl
- subculture
- medium
- root
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000011609 Pinus massoniana Nutrition 0.000 title claims abstract description 46
- 241000018650 Pinus massoniana Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000001737 promoting effect Effects 0.000 title abstract description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 31
- 229930006000 Sucrose Natural products 0.000 claims abstract description 31
- 239000005720 sucrose Substances 0.000 claims abstract description 31
- 239000002689 soil Substances 0.000 claims abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000003415 peat Substances 0.000 claims abstract description 5
- 239000010451 perlite Substances 0.000 claims abstract description 5
- 235000019362 perlite Nutrition 0.000 claims abstract description 5
- 239000010455 vermiculite Substances 0.000 claims abstract description 5
- 235000019354 vermiculite Nutrition 0.000 claims abstract description 5
- 229910052902 vermiculite Inorganic materials 0.000 claims abstract description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 4
- 239000011159 matrix material Substances 0.000 claims abstract description 3
- 239000006870 ms-medium Substances 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 23
- 230000006698 induction Effects 0.000 claims description 19
- 230000035755 proliferation Effects 0.000 claims description 17
- 238000012549 training Methods 0.000 claims description 16
- 230000035584 blastogenesis Effects 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 13
- 238000005286 illumination Methods 0.000 claims description 10
- 239000012877 elongation medium Substances 0.000 claims description 9
- 238000005520 cutting process Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 230000003203 everyday effect Effects 0.000 claims description 5
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 5
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000003020 moisturizing effect Effects 0.000 claims description 5
- 230000007226 seed germination Effects 0.000 claims description 5
- 239000007921 spray Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 3
- 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 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 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 3
- 229960000367 inositol Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 3
- 230000001351 cycling effect Effects 0.000 claims description 2
- 238000011017 operating method Methods 0.000 claims description 2
- 230000003252 repetitive effect Effects 0.000 claims description 2
- 238000002054 transplantation Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 11
- 230000004083 survival effect Effects 0.000 abstract description 9
- 230000008929 regeneration Effects 0.000 abstract description 6
- 238000011069 regeneration method Methods 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract 3
- QOPBEBWGSGFROG-UHFFFAOYSA-N 2-(1h-indol-2-yl)acetic acid Chemical class C1=CC=C2NC(CC(=O)O)=CC2=C1 QOPBEBWGSGFROG-UHFFFAOYSA-N 0.000 abstract 1
- LBLSRDDHGILUJH-UHFFFAOYSA-N 4-(1h-indol-2-yl)butanoic acid Chemical class C1=CC=C2NC(CCCC(=O)O)=CC2=C1 LBLSRDDHGILUJH-UHFFFAOYSA-N 0.000 abstract 1
- 229930192334 Auxin Natural products 0.000 abstract 1
- 150000000996 L-ascorbic acids Chemical class 0.000 abstract 1
- 239000002363 auxin Substances 0.000 abstract 1
- 238000011419 induction treatment Methods 0.000 abstract 1
- 229920001817 Agar Polymers 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 230000006872 improvement Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 235000009430 Thespesia populnea Nutrition 0.000 description 3
- 244000299492 Thespesia populnea Species 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 235000005205 Pinus Nutrition 0.000 description 2
- 241000218602 Pinus <genus> Species 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
- 241000779819 Syncarpia glomulifera Species 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 239000001739 pinus spp. Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 description 1
- 229940036248 turpentine Drugs 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a method for promoting a pinus massoniana tissue-cultured subcultured bud to root. The method comprises the following steps: selecting the robust and half lignified pinus massoniana tissue-cultured subcultured bud with a seedling height of 2-3 cm; by adopting different culture media in a two-step rooting method, inoculating the subcultured bud in a 1/2 modified MS culture medium IV that contains indoleacetic acids, indolebutyric acids and sucrose; conducting induction treatment on an adventitious root primordium 21-28 d; transplanting the subcultured bud to a 1/2 modified MS culture medium V that contains activated carbon or ascorbic acids, and sucrose, without auxins, in order to promote the adventitious root to express and extend; transplanting a rooted regeneration plant to a mixed matrix that contains yellow soil or peat soil, vermiculite and perlite for cultivating. According to the invention, the method can tremendously promote and improve the rooting condition of the subcultured bud, the rooting rate of the pinus massoniana subcultured bud is above 93.1%, and the highest rooting rate is as high as 97.6%; the root system quality is higher; the survival rate of transplanting is above 85.1%, and the highest survival rate of transplanting is as high as 93.0%; the method has good economic benefits and social benefits.
Description
Technical field
The invention belongs to technical field of plant propagation, relate to tissue cultivating and seedling technology, especially a kind of promotion masson pine group training subculture blastogenesis method for root.
Background technology
Masson pine (Pinus massonianan) originates in China, is one of the important species of the important green barren hill of south China, paper making raw material woods and resin tapping woods, natural land woods, very wide in China's distribution.The seeds high as a kind of comprehensive utilization value, promotion potential is large, masson pine not only can be used to production three plate, papermaking and chemical fibre industry manufacture, also can be used to produce rosin, the turpentine wet goods raw material of industry.In recent years, the life requirement growing along with people and the non-renewable resources such as timber resource shortage and oil such as to be petered out at the intensification of contradictions, and ecological, economic and social sustainable development is more and more subject to social concerns., the problem such as resource security and social safety peaceful based on China's ecology, masson pine, as main one of the industrial cut stock seeds and Tree Species as Bio-energy of China, greatly develops Masson Pine Plantation necessary.
China, since " six or five " National Key Research Programs, has achieved comparatively quantum jump in masson pine fine-variety breeding and Fast-growing Cultivation of Cinnamomun etc.At present, masson pine breeding is mainly based on the sexual propagation of seed orchard and seed production stand.But sexual propagation also exists the problem of differentiation, genetic improvement gain can not be improved to greatest extent, and vegetative propagation can overcome the above-mentioned deficiency of sexual propagation.But the conventional genderless propagation technique of masson pine (cuttage and grafting) is still difficult at present meets the demand of afforesting, and constrains the process of Fine Variety Breeding of Pinus massoniana.Modern forestry is particular about fast growing, high-quality and efficient, and the use of plant tissue culture technology is the effective way realizing masson pine choiceness nursery stock fast breeding.
Horse hair Pinus tissue cultures take root comparatively difficulty seeds.Numerous scholar's research showed in the past, and carrying out taking root with the masson pine simple bud of Initial culture can obtain ideal rooting rate usually, and after subculture, its simple bud rooting rate is obviously on the low side.As with masson pine mature seed for explant is originated, simple bud after the Initial culture of clump bud inducement and elongation directly takes root by Wu little Qin etc. (2009) and season Kong Shu etc. (2010), and its rooting rate is respectively 85% and 92.5%; And Xue Ying (2006) is taken root getting simple bud after squamous subculture, its rooting rate is only 26.7%.Realize masson pine plantlet in vitro factorial praluction, subculture blastogenesis root difficulty bottleneck factor must be broken through.
Summary of the invention
Object of the present invention trains the low present situation of subculture bud rooting rate mainly for masson pine group, for realizing the large-scale production of masson pine plantlet in vitro, providing a kind of and promoting masson pine group training subculture blastogenesis method for root.
The present invention is achieved in that
A kind of promotion masson pine group training subculture blastogenesis method for root, comprise the two step rooting of clump bud inducement, the elongation of clump bud, squamous subculture, root induction, adventitious root extension, test-tube seedling transplanting, obtain masson pine group training subculture blastogenesis offspring, its operating procedure is as follows:
(1) clump bud inducement: after masson pine seed being sterilized, aseptically carry out seed germination, then intercepting the long hypocotylar germ free apical bud of band 0.5cm is explant, in inducing culture I, carry out clump bud inducement;
(2) clump bud extends: the Multiple Buds induced is carried out in elongation medium II elongation and cultivate;
(3) squamous subculture: the single terminal bud cutting elongation bud is inoculated in subculture multiplication medium III and breeds, obtain subculture bud I; Subculture bud I is repeated step (2) to extend, single cut elongation subculture bud I and be divided into terminal bud and band lateral bud bud section, then in step (3) subculture multiplication medium, continue propagation obtain subculture bud II, subculture bud II is repeated step (2) and step (3) again, obtain new proliferation and subculture bud; Repetitive cycling step (2) and (3), until obtain a large amount of enough culture of rootage subculture buds;
(4) root induction: the single subculture bud cutting robust growth, semi-lignified, high 2-3cm, is seeded in 1/2 modified MS medium IV containing heteroauxin (IAA), indolebutyric acid (IBA), sucrose, induction process adventitious root primordia 21-28d;
(5) adventitious root extension: will have in 1/2 modified MS medium V that the visible adventive root explant of naked eyes proceeds to containing active carbon (AC) or ascorbic acid (Vc), sucrose, and promote expression and the elongation of adventive root;
(6) test-tube seedling transplanting: be cultivate in the mixing light ground mass of 1: 1: 1 in yellow soil or peat soil, vermiculite and perlite volume ratio by the Transplantation of Regenerated Plantlets of having taken root, water spray moisturizing.
Above-described its material content of medium I is: 6-BA2.5-4.0mgL
-1, NAA0.1-0.3mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium II is: NAA0.25-0.4mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium III is: ZT1.0-3.0mgL
-1, KT0-1.0mgL
-1, NAA0-0.3mgL
-1, sucrose 30000mgL
-1and modified MS medium.
Above-described its material content of medium IV is: IAA1.0-2.5mgL
-1, IBA0.1-0.5mgL
-1, sucrose 1.0-2.0% and 1/2 modified MS medium.
Above-described its material content of medium V is: AC3000-5000mgL
-1or Vc10-30mgL
-1, sucrose 2.0% and 1/2 modified MS medium.
Basic composition is of above-described modified MS medium: KNO
31580mgL
-1; NH
4nO
3800mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o200mgL
-1; KH
2pO
4170mgL
-1; MnSO
4h
2o22.3mgL
-1; ZnSO
47H
2o8.6mgL
-1; CuSO
45H
2o0.025mgL
-1; H
3bO
36.2mgL
-1; Na
2moO
42H
2o0.025mgL
-1; KI0.83mgL
-1; CoCl
26H
2o0.025mgL
-1; Cobastab
11.0mgL
-1; Cobastab
60.5mgL
-1; Nicotinic acid 0.5mgL
-1; Glycine 2.0mgL
-1; Inositol 200mgL
-1.
Above-described cultivation controlled condition is: cultivation temperature 20-30 DEG C, illumination 1500-2000lx, illumination every day 12-16h.
The present invention has the following advantages relative to existing technology:
Promotion masson pine group training subculture blastogenesis method for root of the present invention, select the masson pine group training subculture bud of robust growth, semi-lignified and height of seedling 2-3cm, adopt different medium with two step rooting, first induction process 21-28d days, then be forwarded to and there is no growth hormone, but contain in the medium of active carbon or ascorbic acid, greatly promote and improve subculture blastogenesis root situation.Adopt this rooting method, masson pine subculture bud rooting rate reaches more than 93.1%, and the highest rooting rate reaches 97.6%, and root system quality is higher, and transplanting survival rate reaches more than 85.1%, and most high-servival rate reaches 93.0%, has good economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 is that masson pine group of the present invention training is taken root seedling;
Fig. 2 is masson pine group of the present invention training transplanted seedling.
Embodiment
The following examples can make the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.
embodiment 1
1, experiment material
With masson pine, ripe cone seed germination germ free apical bud is for explant then, and cone picks up from No. 3, High yielding excellent masson pine family Ningming in Ningming of Guangxi portiatree provenance construction masson pine seed production stand.
2, experimental technique
(1) clump bud inducement
Aseptic explant is inoculated into improvement MS+6-BA3.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on carry out Fiber differentiation.After cultivating 26d, average clump bud induction rate reaches 99.1%.
Basic composition is of described modified MS medium: KNO
31580mgL
-1; NH
4nO
3800mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o200mgL
-1; KH
2pO
4170mgL
-1; MnSO
4h
2o22.3mgL
-1; ZnSO
47H
2o8.6mgL
-1; CuSO
45H
2o0.025mgL
-1; H
3bO
36.2mgL
-1; Na
2moO
42H
2o0.025mgL
-1; KI0.83mgL
-1; CoCl
26H
2o0.025mgL
-1; Cobastab
11.0mgL
-1; Cobastab
60.5mgL
-1; Nicotinic acid 0.5mgL
-1; Glycine 2.0mgL
-1; Inositol 200mgL
-1.
(2) clump bud extends
The budlet that grows thickly induced is transferred to improvement MS+NAA0.25mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 27d, clump bud height 3-5cm.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, is inoculated into modified MS medium+ZT2.0mgL
-1+ KT1.0mgL
-1+ NAA0.2mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture Shoot propagation 23d.
Shoot proliferation bud is put in step (2) elongation medium, to promote that shoot proliferation bud extends.Extend and cultivate 28d, shoot proliferation bud height 3-5cm.After elongation subculture bud being divided into the long terminal bud of 2cm and 1cm long band lateral bud bud section, repeat step (3) shoot proliferation and the elongation of step (2) clump bud.Squamous subculture 6 times, average proliferation coefficient 6.4, obtain large quantities of can for subculture bud of taking root.
(4) root induction
The single subculture bud cutting robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA2.5mgL
-1+ IBA0.1mgL
-1the former base of inducing adventitious root in+sucrose 1.0% medium.After induction 25d, root induction rate reaches 97.6%.
(5) adventitious root extension
1/2 modified MS medium+AC5000mgL is proceeded to by obviously having the visible adventive root explant of naked eyes
-1promote adventive root to express in+sucrose 2.0% medium and extend, adding up adventive root incidence after 28d, record mean elements also observes root system quality (between rhizome callus a situation arises).Concrete data are in table 1.
(6) test-tube seedling transplanting
Cleaned by regeneration plant root agar, transplant and cultivate in yellow soil, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in table 1.
The condition of culture of said process: temperature 20-30 DEG C, illumination 1500-2000lx, illumination every day 12-16h.
embodiment 2
1, experiment material
With masson pine, ripe cone seed germination germ free apical bud is for explant then, and cone picks up from No. 4, High yielding excellent masson pine family Ningming in Ningming of Guangxi portiatree provenance construction masson pine seed production stand.
2, experimental technique
(1) clump bud inducement
Aseptic explant is inoculated into improvement MS+6-BA4.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on carry out Fiber differentiation.After cultivating 26d, average clump bud induction rate reaches 98.4%.
The basic composition of described modified MS medium is with embodiment 1.
(2) clump bud extends
The budlet that grows thickly induced is transferred to improvement MS+NAA0.3mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 28d, clump bud height 3-5cm.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, is inoculated into modified MS medium+ZT2.5mgL
-1+ KT0.5mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture Shoot propagation 26d.
Shoot proliferation bud is put in step (2) elongation medium, to promote that shoot proliferation bud extends.Extend and cultivate 25d, shoot proliferation bud height 3-5cm.After elongation subculture bud being divided into the long terminal bud of 2cm and 1cm long band lateral bud bud section, repeat step (3) shoot proliferation and the elongation of step (2) clump bud.Squamous subculture 5 times, average proliferation coefficient 6.0.
(4) root induction
The single subculture bud cutting robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA1.5mgL
-1+ IBA0.3mgL
-1the former base of inducing adventitious root in+sucrose 1.0% medium.After induction 22d, root induction rate reaches 93.1%.
(5) adventitious root extension
1/2 modified MS medium+Vc10mgL is proceeded to by obviously having the visible adventive root explant of naked eyes
-1promote adventive root to express in+sucrose 2.0% medium and extend, adding up adventive root incidence after 28d, record mean elements also observes root system quality (between rhizome callus a situation arises).Concrete data are in table 1.
(6) test-tube seedling transplanting
Cleaned by regeneration plant root agar, transplant and cultivate in yellow soil, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in table 1.
The condition of culture of said process: temperature 20-30 DEG C, illumination 1500-2000lx, illumination every day 12-16h.
embodiment 3
1, experiment material
With masson pine, ripe cone seed germination germ free apical bud is for explant then, and cone picks up from No. 7, High yielding excellent masson pine family Ningming in Ningming of Guangxi portiatree provenance construction masson pine seed production stand.
2, experimental technique
(1) clump bud inducement
Aseptic explant is inoculated into improvement MS+6-BA4.0mgL
-1+ NAA0.1mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1medium on carry out Fiber differentiation.After cultivating 26d, average clump bud induction rate reaches 99.0%.
The basic composition of described modified MS medium is with embodiment 1.
(2) clump bud extends
The budlet that grows thickly induced is transferred to improvement MS+NAA person and says 0.4mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1in elongation medium, to promote the elongation growth of culture.Extend and cultivate 28d, clump bud height 3-5cm.
(3) subculture bud is cultivated
The simple bud of plant height 2-3cm in Multiple Buds is cut, is inoculated into modified MS medium+ZT2.5mgL
-1+ KT0.5mgL
-1+ NAA0.25mgL
-1+ sucrose 30000mgL
-1+ agar 3500mgL
-1on carry out subculture Shoot propagation 26d.
Shoot proliferation bud is put in step (2) elongation medium, to promote that shoot proliferation bud extends.Extend and cultivate 25d, shoot proliferation bud height 3-5cm.After elongation subculture bud being divided into the long terminal bud of 2cm and 1cm long band lateral bud bud section, repeat step (3) shoot proliferation and the elongation of step (2) clump bud.Squamous subculture 5 times, average proliferation coefficient 6.6.
(4) root induction
The single subculture bud cutting robust growth, semi-lignified, high 2-3cm, is inoculated into 1/2 modified MS medium+IAA1.0mgL
-1+ IBA0.5mgL
-1the former base of inducing adventitious root in+sucrose 2.0% medium.After induction 28d, root induction rate reaches 95.1%.
(5) adventitious root extension
1/2 modified MS medium+AC3500mgL is proceeded to by obviously having the visible adventive root explant of naked eyes
-1promote adventive root to express in+sucrose 2.0% medium and extend, adding up adventive root incidence after 28d, record mean elements also observes root system quality (between rhizome callus a situation arises).Concrete data are in table 1.
(6) test-tube seedling transplanting
Cleaned by regeneration plant root agar, transplant and in peat soil, vermiculite and perlite volume ratio be: cultivate in the mixed-matrix of 1: 1: 1, water spray moisturizing, added up transplanting survival rate after one month.Concrete data are in table 1.
The condition of culture of said process: temperature 20-30 DEG C, illumination 1500-2000lx, illumination every day 12-16h.
In above embodiment 1 to 3, masson pine group training subculture blastogenesis root and transplant survival situation, as shown in Table 1 below.
Table 1 masson pine group training subculture blastogenesis root and transplant survival situation
Show from table 1 result, adopt the present invention to make masson pine each family plantlet in vitro subculture bud rooting rate reach more than 93.1%, the highest rooting rate reaches 97.6%, and more than every plant average root of hair 2-3 bar, and the callus of plantlet in vitro base portion is less, connects closely between rhizome.Transplant after to yellow soil or peat soil, perlite, vermiculite mixing light ground mass, regeneration plant well-grown, after one month, transplanting survival rate is more than 85.1%, and most high-servival rate reaches 93.0%.The present invention solves the problem of masson pine group training subculture blastogenesis root difficulty preferably, for factorial praluction masson pine tissue culture regeneration plant provides group training subculture blastogenesis method for root.
Claims (2)
1. one kind promotes masson pine group training subculture blastogenesis method for root, it is characterized in that: the two step rooting comprising clump bud inducement, the elongation of clump bud, squamous subculture, root induction, adventitious root extension, test-tube seedling transplanting, obtain masson pine group training subculture blastogenesis offspring, its operating procedure is as follows:
Clump bud inducement: after masson pine seed being sterilized, aseptically carry out seed germination, then intercepting the long hypocotylar germ free apical bud of band 0.5 cm is explant, in inducing culture I, carry out clump bud inducement;
Clump bud extends: the clump bud induced is carried out in elongation medium II elongation and cultivate;
Squamous subculture: the single terminal bud cutting elongation bud is inoculated in subculture multiplication medium III and breeds, obtain subculture bud I; Subculture bud I is repeated step (2) to extend, single cut elongation subculture bud I and be divided into terminal bud and band lateral bud bud section, then in step (3) subculture multiplication medium III, continue propagation obtain subculture bud II, subculture bud II is repeated step (2) and step (3) again, obtain new proliferation and subculture bud; Repetitive cycling step (2) and (3), until obtain a large amount of enough culture of rootage subculture buds;
Root induction: the single subculture bud cutting robust growth, semi-lignified, high 2-3 cm, is seeded in 1/2 modified MS medium IV containing heteroauxin (IAA), indolebutyric acid (IBA), sucrose, induction process adventitious root primordia 21-28 d;
Adventitious root extension: proceeded to by the explant with the visible adventive root of naked eyes in 1/2 modified MS medium V containing active carbon (AC) or ascorbic acid (Vc), sucrose, promotes expression and the elongation of adventive root;
Test-tube seedling transplanting: by the Transplantation of Regenerated Plantlets of having taken root in yellow soil, or be: peat soil: vermiculite: perlite=1: cultivate in the mixed-matrix of 1: 1, water spray moisturizing;
Described its material content of inducing culture I is: 6-BA 2.5-4.0 mgL
-1, NAA 0.1-0.3 mgL
-1, sucrose 30000 mgL
-1and modified MS medium;
Described its material content of elongation medium II is: NAA 0.25-0.4 mgL
-1, sucrose 30000 mgL
-1and modified MS medium;
Described its material content of subculture multiplication medium III is: ZT 1.0-3.0 mgL
-1, KT 0.5-1.0 mgL
-1, NAA 0-0.3 mgL
-1, sucrose 30000 mgL
-1and modified MS medium;
Described its material content of medium IV is: IAA 1.0-2.5 mgL
-1, IBA 0.1-0.5 mgL
-1, sucrose 1.0-2.0% and 1/2 modified MS medium;
Described its material content of medium V is: AC 3000-5000 mgL
-1or Vc 10-30 mgL
-1, sucrose 2.0% and 1/2 modified MS medium;
Basic composition is of described modified MS medium: KNO
31580 mgL
-1; NH
4nO
3800 mgL
-1; CaCl
22H
2o 260 mgL
-1; MgSO
47H
2o 370 mgL
-1; Ca (NO
3)
24H
2o 200 mgL
-1; KH
2pO
4170 mgL
-1; MnSO
4h
2o 22.3 mgL
-1; ZnSO
47H
2o 8.6 mgL
-1; CuSO
45H
2o 0.025 mgL
-1; H
3bO
36.2 mgL
-1; Na
2moO
42H
2o 0.025 mgL
-1; KI 0.83 mgL
-1; CoCl
26H
2o 0.025 mgL
-1; Cobastab
11.0 mgL
-1; Cobastab
60.5 mgL
-1; Nicotinic acid 0.5 mgL
-1; Glycine 2.0 mgL
-1; Inositol 200 mgL
-1.
2. one according to claim 1 promotes masson pine group training subculture blastogenesis method for root, it is characterized in that: cultivating controlled condition is: cultivation temperature 20-30 DEG C, illumination 1500-2000 lx, illumination every day 12-16 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410041856.3A CN103749310B (en) | 2013-11-27 | 2014-01-28 | Method for promoting pinus massoniana tissue-cultured subcultured bud to root |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310617394.0 | 2013-11-27 | ||
| CN2013106173940 | 2013-11-27 | ||
| CN201310617394.0A CN103609450A (en) | 2013-11-27 | 2013-11-27 | Method for accelerating rooting of masson pine tissue culture secondary sprout |
| CN201410041856.3A CN103749310B (en) | 2013-11-27 | 2014-01-28 | Method for promoting pinus massoniana tissue-cultured subcultured bud to root |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103749310A CN103749310A (en) | 2014-04-30 |
| CN103749310B true CN103749310B (en) | 2015-04-08 |
Family
ID=50160187
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310617394.0A Pending CN103609450A (en) | 2013-11-27 | 2013-11-27 | Method for accelerating rooting of masson pine tissue culture secondary sprout |
| CN201410041856.3A Expired - Fee Related CN103749310B (en) | 2013-11-27 | 2014-01-28 | Method for promoting pinus massoniana tissue-cultured subcultured bud to root |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310617394.0A Pending CN103609450A (en) | 2013-11-27 | 2013-11-27 | Method for accelerating rooting of masson pine tissue culture secondary sprout |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN103609450A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663445A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | In-vitro rapid propagation technology of high lipid-producing pinus elliottii |
| CN104756869B (en) * | 2015-04-09 | 2016-04-20 | 广西壮族自治区林业科学研究院 | The preparation of masson pine fine individual plant aseptic explant and initial bud inducement method |
| CN105359948B (en) * | 2015-09-11 | 2018-08-28 | 中国林业科学研究院林业研究所 | Hybrid larch micro cuttage method for culturing seedlings |
| CN105746288B (en) * | 2016-04-15 | 2019-05-28 | 中国林业科学研究院热带林业研究所 | A kind of method for building up of cassie Asia pine cuttage nursery system |
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN108967195B (en) * | 2018-08-07 | 2021-07-02 | 广西壮族自治区林业科学研究院 | Culture method for proliferation and rejuvenation of tissue culture subculture bud of pinus massoniana |
| CN111616050B (en) * | 2020-05-19 | 2022-11-15 | 广西壮族自治区林业科学研究院 | Method for improving rooting stability of superior tree subculture buds of high-yield masson pine |
| CN111657150B (en) * | 2020-07-24 | 2021-07-27 | 广西壮族自治区林业科学研究院 | Method for improving rooting capacity of masson pine based on endogenous hormone |
| CN113142053B (en) * | 2021-04-08 | 2023-02-24 | 中南林业科技大学 | Culture method for promoting induced proliferation differentiation and effective elongation of masson pine cluster buds |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101584297B (en) * | 2008-11-19 | 2012-05-30 | 南京林业大学 | Tissue culture and regenerated plant in vitro mycorrhization method for pinus massoniana |
| CN103461121B (en) * | 2013-09-04 | 2014-11-26 | 广西壮族自治区林业科学研究院 | Ex-vitro rooting method for tissue culture seedlings of pinus massoniana |
-
2013
- 2013-11-27 CN CN201310617394.0A patent/CN103609450A/en active Pending
-
2014
- 2014-01-28 CN CN201410041856.3A patent/CN103749310B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103609450A (en) | 2014-03-05 |
| CN103749310A (en) | 2014-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103749310B (en) | Method for promoting pinus massoniana tissue-cultured subcultured bud to root | |
| CN103461121B (en) | Ex-vitro rooting method for tissue culture seedlings of pinus massoniana | |
| CN102870680B (en) | Efficient rapid propagation technique appropriate for detoxified rabbiteye blueberries | |
| CN110583482B (en) | High-efficiency in-vitro regeneration method for larch needles | |
| CN109618932B (en) | Method for inducing adventitious buds of rhododendron dauricum and regenerating plants | |
| CN103229720A (en) | Method for regenerating plant from arundo donax linn callus | |
| CN105028209A (en) | Method for improving rooting of vaccinium ashei tissue culture seedlings | |
| CN102283128B (en) | Method for overcoming vitrification phenomenon of sisal hemp tissue culture seedlings | |
| CN108142283B (en) | Tissue culture rapid propagation method of Acer catalpa Maxim | |
| CN102217538B (en) | Method for treating walnut tissue culture stem segments inside test tubes and rooting outside test tubes | |
| CN103609453A (en) | Construction method of in vitro regeneration system of tea tree | |
| CN103340153A (en) | Tissue culture method taking masson pine in-vitro mature embryo as explant | |
| CN103329803B (en) | Tufted-bud induction and subculture method for southern pine trees | |
| CN104094848B (en) | The method of the induction of tung oil tree hypocotyledonery axis callus and highly efficient regeneration plant | |
| CN103283504B (en) | Method for grafting pear polyploidy test-tube plantlet outside test tube | |
| CN113207690A (en) | Efficient one-step regeneration method taking paper mulberry root as explant | |
| CN105145363A (en) | Method for significantly improving fir tissue culture production emergence rate | |
| CN104686327A (en) | Method for establishing tissue culture rapid propagation system of single-leaf robinia. pseudoacacia | |
| CN101836589B (en) | Method of rapid propagation of populus | |
| CN114431154B (en) | Method for asexual propagation through acer nikoense dormant buds | |
| CN106857255B (en) | A kind of culture medium of dragon fruit tissue cultures | |
| CN105052742B (en) | Method for breeding pinus massoniana good group asexual tissue culture seedlings | |
| CN101707981A (en) | Rubber tree cotyledon embryo high-efficiency embryonic callus induction and regeneration method | |
| CN112273229B (en) | One-step seedling method for hydrangea | |
| CN108112479A (en) | A kind of stem section of papaya sprout Bud Differentiation vacantly plants leaf promoting root growth method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150408 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |