CN103329803A - Tufted-bud induction and subculture method for southern pine trees - Google Patents
Tufted-bud induction and subculture method for southern pine trees Download PDFInfo
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- CN103329803A CN103329803A CN2013102585443A CN201310258544A CN103329803A CN 103329803 A CN103329803 A CN 103329803A CN 2013102585443 A CN2013102585443 A CN 2013102585443A CN 201310258544 A CN201310258544 A CN 201310258544A CN 103329803 A CN103329803 A CN 103329803A
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000005018 Pinus echinata Nutrition 0.000 title claims abstract description 17
- 241001236219 Pinus echinata Species 0.000 title claims abstract description 17
- 235000017339 Pinus palustris Nutrition 0.000 title claims abstract description 17
- 230000006698 induction Effects 0.000 title abstract description 10
- 229920001817 Agar Polymers 0.000 claims abstract description 24
- 239000008272 agar Substances 0.000 claims abstract description 24
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 5
- 108010079058 casein hydrolysate Proteins 0.000 claims abstract description 3
- 238000005286 illumination Methods 0.000 claims description 52
- 230000035755 proliferation Effects 0.000 claims description 39
- 239000006870 ms-medium Substances 0.000 claims description 23
- 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 description 21
- 229930006000 Sucrose Natural products 0.000 claims description 21
- 239000005720 sucrose Substances 0.000 claims description 21
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims description 17
- 235000011613 Pinus brutia Nutrition 0.000 claims description 17
- 241000018646 Pinus brutia Species 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000012877 elongation medium Substances 0.000 claims description 14
- 230000001939 inductive effect Effects 0.000 claims description 13
- 230000007226 seed germination Effects 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 230000001351 cycling effect Effects 0.000 claims description 4
- 230000003252 repetitive effect Effects 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 238000011017 operating method Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000004017 vitrification Methods 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract 6
- 238000002474 experimental method Methods 0.000 description 10
- 229940088597 hormone Drugs 0.000 description 10
- 239000005556 hormone Substances 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 6
- 235000011609 Pinus massoniana Nutrition 0.000 description 5
- 241000018650 Pinus massoniana Species 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000003203 everyday effect Effects 0.000 description 5
- 230000035784 germination Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 229960002523 mercuric chloride Drugs 0.000 description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 5
- 239000004576 sand Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 235000005205 Pinus Nutrition 0.000 description 3
- 241000218602 Pinus <genus> Species 0.000 description 3
- 241001223353 Pinus caribaea Species 0.000 description 3
- 238000005057 refrigeration Methods 0.000 description 3
- 241000142776 Pinus elliottii Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000012549 training Methods 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
- 239000002028 Biomass Substances 0.000 description 1
- 235000009324 Pinus caribaea Nutrition 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
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- 239000000835 fiber Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000001739 pinus spp. Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 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
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tufted-bud induction and subculture method for southern pine trees. The method comprises the following steps of: using an apical bud of an aseptic seedling with a hypocotyl which is 0.5cm long as an explant, performing induction culture for 20-26 days by an improved MS culture medium, 2.5-4.0 mg/L 6-BA, 0.05-0.1 mg/L NAA, 30,000 mg/L sugar crane, 3,500 mg/L agar and 500 mg/L casein hydrolysate, then performing cluster bud extending and mulitiplication culture by a culture medium comprising the improved MS culture medium, 0.25-0.4 mg/L NAA or 3,000-5,000 mg/L active carbon, 30,000 mg/L sugar crane and 3,500 mg/L agar and a culture medium comprising the improved MS culture medium, 1.0-3.0 mg/L 6-BA, 0-1.0 mg/L KT, 0-0.3 mg/L NAA, 30,000 mg/L sugar crane and 3,500 mg/L agar, wherein the proportions of components of the culture media are different; repeating the subculture by the culture mode for N times, and obtaining large-scale transgenerational buds. According to the method, the cluster bud induction rate is over 97 percent; the bud seedlings are high in extending rate and super-vital; the bud yield is high; the multiplication coefficient is more than 6.8; high-quality and high-efficiency southern pine seedling forestation can be supplied; and the method has high economical and social benefits.
Description
Technical field
The invention belongs to the plant propagation technical field, relate to the tissue cultivating and seedling technology, especially a kind of southern pine tree clump bud is induced and the subculture cultural method.
Background technology
As a kind of comprehensive utilization value height, seeds that promotion prospect is wide, pine tree not only can be used to produce three plates, papermaking and chemical fibre industry manufacturing, also can be used to produce rosin, the turpentine wet goods raw material of industry.In recent years, along with the growing life requirement of people and non-renewable resources such as timber resource shortage and oil peter out etc. between intensification of contradictions, ecological, economic and social sustainable development more and more is subjected to social concerns.Problems such as, resource security peaceful based on China's ecology and social safety, pine tree is as China main industrial cut stock seeds and biomass energy seeds, and it is necessary to greatly develop pine tree forest plantation.
China has obtained than quantum jump at aspects such as pine tree fine-variety breeding and fast growing culture techniques since the tackling key problem of " six or five " national science and technology.Wherein, masson pine of one's native land (
Pinus massonianan) and the wet-land pine tree introduced (
P.Elliottii), torch pine (
P.taedai), the pinus caribaea (
P.caribaea), wet add pine (
P.Elliottii * P.caribaea) wait the southern main afforestation Pinus seeds into China.Modern forestry is particular about fast growing, high-quality and efficient, desires to reach re-set target, and valid approach is to realize choiceness nursery stock afforestation.The use of plant tissue culture technology is the effective way that realizes pine tree choiceness nursery stock fast breeding.
At present, the pine tree tissue is cultivated difficult, induce the bud of generation to be difficult for the differentiation elongation, and growth coefficient is low, subculture cycle is long, and the subculture bud seedling vigor of large-scale production is on the low side, second-rate, and then badly influences the later stage simple bud power of taking root.
Summary of the invention
It is difficult to the objective of the invention is to cultivate at existing pine tree tissue, the induced bundle bud is difficult for the differentiation elongation, and growth coefficient is low, and subculture cycle is long, on the low side, the second-rate technical problem of subculture bud seedling vigor provides a kind of China's south pine tree clump bud that is suitable for to induce and the subculture cultured method.
The present invention is achieved in that
A kind of southern pine tree clump bud is induced and the subculture cultural method, comprises explant selection and processing, inducing clumping bud, the bud of growing thickly elongation, the cultivation of subculture bud, obtains the scale tissue and cultivates southern pine tree subculture bud, and its operating procedure is as follows:
(1) explant is selected and is handled
After the pine tree seed disinfection, under aseptic condition, carry out seed germination, intercepting then and being with the long hypocotylar aseptic seedling terminal bud of 0.5 cm is that explant is organized cultivation;
(2) inducing clumping bud
Aseptic explant is seeded in carries out clump bud in the inducing culture and induce cultivation cycle: 20-26 d, cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 0-16 h, intensity of illumination: 0-2000 lx obtains to grow thickly bud;
(3) grow thickly bud elongation
The bud of growing thickly that induces is extended cultivation, cultivation cycle in elongation medium: 14-28 d, cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx obtains to grow thickly the elongation bud;
(4) the subculture bud is cultivated
The terminal bud of single cutting-out elongation bud is inoculated in the shoot proliferation medium breeds cultivation cycle: 14-21 d, cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx obtains subculture bud I; Subculture bud I repeating step (3) is extended, the subculture bud I of single cutting-out elongation also is divided into terminal bud and band lateral bud bud section, in step (4) shoot proliferation medium, continue propagation then and obtain subculture bud II, with subculture bud II repeating step (3) and step (4) again, obtain new proliferation and subculture bud; Repetitive cycling step (3) and (4), thus realization scale proliferation and subculture group is trained southern pine tree subculture bud.
Above-described subculture bud I is that the long terminal bud of 2 cm of selecting for use single cutting-out not have the vigorous elongation bud of growing thickly of water stain sample, no vitrification phenomenon, growth vigor is bred and got.
Above-described subculture bud II is to select for use shoot proliferation bud subculture bud I to be stretched to 3-5 cm, and is divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section and carries out shoot proliferation and get.
It is repetitive cycling subculture bud propagation, the bud elongation process of growing thickly that above-described subculture bud is cultivated, and carries out N time and cultivates.
Above-described inducing culture is: modified MS medium+6-BA 2.5-4.0 mgL
-1+ NAA 0.05-0.1 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1+ caseinhydrolysate 500 mgL
-1, the pH value is 5.6-6.0.
Above-described elongation medium is: modified MS medium+NAA 0.25-0.4 mgL
-1Or active carbon 3000-5000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1, the pH value is 5.6-6.0.
Above-described shoot proliferation medium is: modified MS medium+6-BA 1.0-3.0 mgL
-1+ KT 0-1.0 mgL
-1+ NAA 0-0.3 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1, the pH value is 5.6-6.0.
Basic composition is of above-described modified MS medium: KNO
31650 mgL
-1NH
4NO
3600 mgL
-1CaCl
22H
2O 260 mgL
-1MgSO
47H
2O 370 mgL
-1Ca (NO
3)
24H
2O 400 mgL
-1KH
2PO
4170 mgL
-1MnSO
4H
2O 22.3 mgL
-1ZnSO
47H
2O 8.6 mgL
-1CuSO
45H
2O 0.025 mgL
-1H
3BO
36.2 mgL
-1Na
2MoO
42H
2O 0.025 mgL
-1KI 0.83 mgL
-1CoCl
26H
2O 0.025 mgL
-1Cobastab
11.0 mgL
-1Cobastab
60.5 mgL
-1Nicotinic acid 0.5 mgL
-1Glycine 2.0 mgL
-1Inositol 200 mgL
-1
The present invention has the following advantages with respect to existing technology:
1, the present invention adopts and optimizes different proportioning medium and training method, and the clump bud induction rate reaches more than 97%, and the bud seedling extends soon, flushes.
2, subculture cultivation of the present invention is by elongation and breeds two sections and repeat the subculture training methods, carrying out N time cultivates, subculture bud output increases substantially, growth coefficient reaches more than 6.8, for Pinus seeds fast growing, high-quality and efficient afforestation provide the choiceness nursery stock, have favorable economic benefit and social benefit.
Embodiment
The present invention will be further described below in conjunction with embodiment.
Embodiment 1
1, experiment material
With masson pine then ripe cone seed germination aseptic seedling terminal bud be explant, cone picks up from the masson pine seed garden of the ancient fluffy fine provenance construction in Xincheng, Guangxi.
2, experimental technique
(1) aseptic explant is selected and aseptic process
With the cone of newly gathering airing under sunshine earlier, treat to shake off out seed gently after synphyllodium opens, with plastic sack seed is installed and place 4 ℃ of refrigerator cold-storages standby.The seed in one week of refrigeration is taken out from refrigerator, soak 15 min with 0.5% liquor potassic permanganate, wash 5 min through flowing water then, after deionized water soaks 12 h, 0.1% mercuric chloride, 40 min that sterilize, aseptic water washing 5 times directly is sowed in the sand matrix that autoclave sterilization is handled and carries out seed germination.Choose the modified MS medium that robust growth germination seed seedling terminal bud (keeping the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out the aseptic explant screening.
Basic composition is of described modified MS medium: KNO
31650 mgL
-1NH
4NO
3600 mgL
-1CaCl
22H
2O 260 mgL
-1MgSO
47H
2O 370 mgL
-1Ca (NO
3)
24H
2O 400 mgL
-1KH
2PO
4170 mgL
-1MnSO
4H
2O 22.3 mgL
-1ZnSO
47H
2O 8.6 mgL
-1CuSO
45H
2O 0.025 mgL
-1H
3BO
36.2 mgL
-1Na
2MoO
42H
2O 0.025 mgL
-1KI 0.83 mgL
-1CoCl
26H
2O 0.025 mgL
-1Cobastab
11.0 mgL
-1Cobastab
60.5 mgL
-1Nicotinic acid 0.5 mgL
-1Glycine 2.0 mgL
-1Inositol 200 mgL
-1
(2) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, and it is long until about 0.5 cm to cut away its hypocotyl base portion, is inoculated into improvement MS+6-BA 3.0 mgL then
-1+ NAA 0.05 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1Medium on induce cultivation.Condition of culture is 24 ± 1 ℃, and first week of illumination 0-2000 lx(is dark the cultivation), illumination every day 16 h, the pH value is 5.8.After cultivating 18 d, average clump bud induction rate reaches 97.4%.
(3) grow thickly bud elongation
The budlet that grows thickly that induces is transferred to improvement MS++NAA 0.25 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On the elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8.22 d are cultivated in elongation, the high 3-5 cm of clump bud.
(4) the subculture bud is cultivated
The simple bud of plant height 2-3 cm that will grow thickly in the bud is cut, and is inoculated into modified MS medium+6-BA 1.5 mgL
-1+ KT 0.8 mgL
-1+ NAA 0.2 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On carry out subculture bud propagation.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8, shoot proliferation cycle 16-21 d.
The shoot proliferation bud is put on step (3) elongation medium, to promote the elongation of shoot proliferation bud.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8.24-28 d, the high 3-5 cm of shoot proliferation bud are cultivated in elongation.After will extending the subculture bud and being divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section, repeating step (4) shoot proliferation and the elongation of step (3) clump bud.Subculture is cultivated 5 times, average growth coefficient 6.8.
Embodiment 2
1, experiment material
With masson pine then ripe cone seed germination aseptic seedling terminal bud be explant, cone picks up from the masson pine seed garden of fine provenance such as Ningming, Guangxi portiatree construction.
2, experimental technique
(1) aseptic explant is selected and aseptic process
With the cone of newly gathering airing under sunshine earlier, treat to shake off out seed gently after synphyllodium opens, with plastic sack seed is installed and place 4 ℃ of refrigerator cold-storages standby.The seed in one week of refrigeration is taken out from refrigerator, soak 15 min with 0.5% liquor potassic permanganate, wash 5 min through flowing water then, after deionized water soaks 12 h, 0.1% mercuric chloride, 40 min that sterilize, aseptic water washing 5 times directly is sowed in the sand matrix that autoclave sterilization is handled and carries out seed germination.Choose the modified MS medium that robust growth germination seed seedling terminal bud (keeping the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out the aseptic explant screening.The basic composition of described modified MS medium is with embodiment 1.
(2) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, and it is long until about 0.5 cm to cut away its hypocotyl base portion, is inoculated into improvement MS+6-BA 4.0 mgL then
-1+ NAA 0.1 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1Medium on induce cultivation.Condition of culture is 24 ± 1 ℃, and first week of illumination 0-2000 lx(is dark the cultivation), illumination every day 16 h, the pH value is 5.8.After cultivating 25 d, average clump bud induction rate reaches 98.1%.
(3) grow thickly bud elongation
The budlet that grows thickly that induces is transferred to improvement MS++NAA 0.35 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On the elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8.25 d are cultivated in elongation, the high 3-5 cm of clump bud.
(4) the subculture bud is cultivated
The simple bud of plant height 2-3 cm that will grow thickly in the bud is cut, and is inoculated into modified MS medium+6-BA 3.0 mgL
-1+ KT 1.0 mgL
-1+ NAA 0.3 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On carry out subculture bud propagation.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8, shoot proliferation cycles 20 d.
The shoot proliferation bud is put on step (3) elongation medium, to promote the elongation of shoot proliferation bud.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8.26 d, the high 3-5 cm of shoot proliferation bud are cultivated in elongation.After will extending the subculture bud and being divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section, repeating step (4) shoot proliferation and the elongation of step (3) clump bud.Subculture is cultivated 4 times, average growth coefficient 7.2.
Embodiment 3
1, experiment material
With wet-land pine tree then ripe cone seed germination aseptic seedling terminal bud be explant, No. 2, cone picks up from state-owned Bobai forest farm, Bobai County, Guangxi district superior families Bobai No. 1, Bobai.
2, experimental technique
(1) aseptic explant is selected and aseptic process
With the cone of newly gathering airing under sunshine earlier, treat to shake off out seed gently after synphyllodium opens, with plastic sack seed is installed and place 4 ℃ of refrigerator cold-storages standby.The seed in one week of refrigeration is taken out from refrigerator, soak 15 min with 0.5% liquor potassic permanganate, wash 5 min through flowing water then, after deionized water soaks 12 h, 0.1% mercuric chloride, 40 min that sterilize, aseptic water washing 5 times directly is sowed in the sand matrix that autoclave sterilization is handled and carries out seed germination.Choose the modified MS medium that robust growth germination seed seedling terminal bud (keeping the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out the aseptic explant screening.The basic composition of described modified MS medium is with embodiment 1.
2) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, and it is long until about 0.5 cm to cut away its hypocotyl base portion, is inoculated into improvement MS+6-BA 2.0-3.5 mgL then
-1+ NAA 0.05-0.08 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1Medium on induce cultivation.Condition of culture is 24 ± 1 ℃, illumination 2000 lx, and illumination every day 16 h, the pH value is 5.6.After cultivating 24-26 d, average clump bud induction rate reaches 97.0%.
3) grow thickly bud elongation
The budlet that grows thickly that induces is transferred to improvement MS+NAA 0.3-0.4 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On the elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.6.24-28 d is cultivated in elongation, the high 3-5 cm of clump bud.
4) the subculture bud is cultivated
The simple bud of plant height 2-3 cm that will grow thickly in the bud is cut, and is inoculated into modified MS medium+6-BA 2.0-3.0 mgL
-1+ KT 0.6-1.0 mgL
-1+ NAA 0.15-0.25 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On carry out subculture bud propagation.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.6, shoot proliferation cycle 15-20 d.
The shoot proliferation bud is put on step (3) elongation medium, to promote the elongation of shoot proliferation bud.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, the pH value is 5.8.23-28 d, the high 3-5 cm of shoot proliferation bud are cultivated in elongation.After will extending the subculture bud and being divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section, repeating step (4) shoot proliferation and the elongation of step (3) clump bud.Subculture is cultivated 4 times, average growth coefficient 7.5.
Embodiment 4
1, experiment material
With wet add pine then ripe cone seed germination aseptic seedling terminal bud be explant, seed is directly to introduce fine germplasm resources from Australia.
2, experimental technique
(1) aseptic explant is selected and aseptic process
To place the seed in 4 ℃ of standby weeks of refrigerator cold-storage to take out from refrigerator, soak 15 min with 0.5% liquor potassic permanganate, wash 5 min through flowing water then, after deionized water soaks 12 h, 0.1% mercuric chloride, 40 min that sterilize, aseptic water washing 5 times directly is sowed in the sand matrix that autoclave sterilization is handled and carries out seed germination.Choose the modified MS medium that robust growth germination seed seedling terminal bud (keeping the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out the aseptic explant screening.The basic composition of described modified MS medium is with embodiment 1.
(2) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, and it is long until about 0.5 cm to cut away its hypocotyl base portion, is inoculated into improvement MS+6-BA 2.5 mgL then
-1+ NAA 0.065 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1Medium on induce cultivation.Condition of culture is 24 ± 1 ℃, illumination 1500-2000 lx, and illumination every day 16 h, the pH value is 6.0.After cultivating 24 d, average clump bud induction rate reaches 98.1%.
(3) grow thickly bud elongation
The budlet that grows thickly that induces is transferred to improvement MS+ active carbon 3000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On the elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0.26 d are cultivated in elongation, the high 3-5 cm of clump bud.
(4) the subculture bud is cultivated
The simple bud of plant height 2-3 cm that will grow thickly in the bud is cut, and is inoculated into modified MS medium+6-BA 1.5 mgL
-1+ NAA 0.1 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On carry out subculture bud propagation.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 18 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0, shoot proliferation cycles 20 d.
The shoot proliferation bud is put on step (3) elongation medium, to promote the elongation of shoot proliferation bud.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0.28 d, the high 3-5 cm of shoot proliferation bud are cultivated in elongation.After will extending the subculture bud and being divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section, repeating step (4) shoot proliferation and the elongation of step (3) clump bud.Subculture is cultivated 8 times, average growth coefficient 8.2.
Embodiment 5
1, experiment material
With wet add pine then ripe cone seed germination aseptic seedling terminal bud be explant, seed is directly to introduce fine germplasm resources from Australia.
2, experimental technique
(1) aseptic explant is selected and aseptic process
To place the seed in 4 ℃ of standby weeks of refrigerator cold-storage to take out from refrigerator, soak 15 min with 0.5% liquor potassic permanganate, wash 5 min through flowing water then, after deionized water soaks 12 h, 0.1% mercuric chloride, 40 min that sterilize, aseptic water washing 5 times directly is sowed in the sand matrix that autoclave sterilization is handled and carries out seed germination.Choose the modified MS medium that robust growth germination seed seedling terminal bud (keeping the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out the aseptic explant screening.The basic composition of described modified MS medium is with embodiment 1.
(2) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, and it is long until about 0.5 cm to cut away its hypocotyl base portion, is inoculated into improvement MS+6-BA 3.0 mgL then
-1+ NAA 0. 08 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1Medium on induce cultivation.Condition of culture is 24 ± 1 ℃, illumination 1500-2000 lx, and illumination every day 16 h, the pH value is 6.0.After cultivating 26 d, average clump bud induction rate reaches 98.7%.
(3) grow thickly bud elongation
The budlet that grows thickly that induces is transferred to improvement MS+ active carbon 5000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On the elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0.27 d are cultivated in elongation, the high 3-5 cm of clump bud.
(4) the subculture bud is cultivated
The simple bud of plant height 2-3 cm that will grow thickly in the bud is cut, and is inoculated into modified MS medium+6-BA 2.0 mgL
-1+ NAA 0.2 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1On carry out subculture bud propagation.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0, shoot proliferation cycles 19 d.
The shoot proliferation bud is put on step (3) elongation medium, to promote the elongation of shoot proliferation bud.Cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, the pH value is 6.0.27 d, the high 3-5 cm of shoot proliferation bud are cultivated in elongation.After will extending the subculture bud and being divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section, repeating step (4) shoot proliferation and the elongation of step (3) clump bud.Subculture is cultivated 8 times, average growth coefficient 8.6.
Claims (8)
1. a southern pine tree clump bud is induced and the subculture cultural method, it is characterized in that: comprise explant selection and processing, inducing clumping bud, the bud of growing thickly elongation, the cultivation of subculture bud, obtain the scale tissue and cultivate southern pine tree subculture bud that its operating procedure is as follows:
(1) explant is selected and handled: after the pine tree seed disinfection, carry out seed germination under aseptic condition, intercepting then and being with the long hypocotylar aseptic seedling terminal bud of 0.5 cm is that explant is organized cultivation;
(2) inducing clumping bud: aseptic explant is seeded in carries out clump bud in the inducing culture and induce cultivation cycle: 20-26 d, cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 0-16 h, intensity of illumination: 0-2000 lx obtains to grow thickly bud;
(3) grow thickly bud elongation: the bud of growing thickly that will induce extends cultivation, cultivation cycle in elongation medium: 14-28 d, and cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx obtains to grow thickly the elongation bud;
(4) the subculture bud is cultivated: the terminal bud of single cutting-out elongation bud is inoculated in the shoot proliferation medium breeds, cultivation cycle: 14-21 d, cultivation temperature: 24 ± 1 ℃, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx obtains subculture bud I; Subculture bud I repeating step (3) is extended, the subculture bud I of single cutting-out elongation also is divided into terminal bud and band lateral bud bud section, in step (4) shoot proliferation medium, continue propagation then and obtain subculture bud II, with subculture bud II repeating step (3) and step (4) again, obtain new proliferation and subculture bud; Repetitive cycling step (3) and (4), thus realization scale proliferation and subculture group is trained southern pine tree subculture bud.
2. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, it is characterized in that: described subculture bud I is that the long terminal bud of 2 cm of selecting for use single cutting-out not have the vigorous elongation bud of growing thickly of water stain sample, no vitrification phenomenon, growth vigor is bred and got.
3. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, it is characterized in that: described subculture bud II is to select for use shoot proliferation bud subculture bud I to be stretched to 3-5 cm, and is divided into the long band of the long terminal bud of 2 cm and 1 cm lateral bud bud section and carries out shoot proliferation and get.
4. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, it is characterized in that: it is repetitive cycling subculture bud propagation, the bud elongation process of growing thickly that described subculture bud is cultivated, and carries out N time and cultivates.
5. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, it is characterized in that: described inducing culture is modified MS medium+6-BA 2.5-4.0 mgL
-1+ NAA 0.05-0.1 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1+ caseinhydrolysate 500 mgL
-1, the pH value is 5.6-6.0.
6. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, and it is characterized in that: described elongation medium is: modified MS medium+NAA 0.25-0.4 mgL
-1Or active carbon 3000-5000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1, the pH value is 5.6-6.0.
7. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, and it is characterized in that: described shoot proliferation medium is: modified MS medium+6-BA 1.0-3.0 mgL
-1+ KT 0-1.0 mgL
-1+ NAA 0-0.3 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1, the pH value is 5.6-6.0.
8. southern pine tree clump bud according to claim 1 is induced and the subculture cultural method, it is characterized in that: basic composition is of described modified MS medium: KNO
31650 mgL
-1NH
4NO
3600 mgL
-1CaCl
22H
2O 260 mgL
-1MgSO
47H
2O 370 mgL
-1Ca (NO
3)
24H
2O 400 mgL
-1KH
2PO
4170 mgL
-1MnSO
4H
2O 22.3 mgL
-1ZnSO
47H
2O 8.6 mgL
-1CuSO
45H
2O 0.025 mgL
-1H
3BO
36.2 mgL
-1Na
2MoO
42H
2O 0.025 mgL
-1KI 0.83 mgL
-1CoCl
26H
2O 0.025 mgL
-1Cobastab
11.0 mgL
-1Cobastab
60.5 mgL
-1Nicotinic acid 0.5 mgL
-1Glycine 2.0 mgL
-1Inositol 200 mgL
-1
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| CN113142053A (en) * | 2021-04-08 | 2021-07-23 | 中南林业科技大学 | Culture method for promoting induced proliferation differentiation and effective elongation of masson pine cluster buds |
| CN117243125A (en) * | 2023-10-26 | 2023-12-19 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Tissue culture method of coastal pine |
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