CN103329803B - 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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- 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
- 239000000835 fiber Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 235000011609 Pinus massoniana Nutrition 0.000 description 5
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Classifications
-
- 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
-
- 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 technical field of plant propagation, relate to tissue cultivating and seedling technology, especially a kind of Southern Pine grove bud inducement and subculture method.
Background technology
The seeds high as a kind of comprehensive utilization value, promotion prospect is wide, pine tree 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 to peter out etc. between intensification of contradictions, 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, pine tree, as the main industrial cut stock seeds of China and Tree Species as Bio-energy, greatly develops Larix gmelini plantations necessary.
China, since " six or five " National Key Research Programs, has achieved comparatively quantum jump in pine tree fine-variety breeding and Fast-growing Cultivation of Cinnamomun etc.Wherein, masson pine of one's native land (
pinus massonianan) and introduce wet-land pine tree (
p.Elliottii), torch pine (
p.taedai), pinus caribaea (
p.caribaea), wet add pine (
p.Elliottii × P.caribaea) etc. be the main afforestation Pinus seeds of south China.Modern forestry is particular about fast growing, high-quality and efficient, and for reaching re-set target, the most effective approach realizes choiceness nursery stock afforestation.The use of plant tissue culture technology is the effective way realizing pine tree choiceness nursery stock fast breeding.
At present, it is more difficult that pine tissues is cultivated, and the bud that induction produces not easily breaks up 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 influence later stage simple bud and to take root power.
Summary of the invention
It is more difficult to the object of the invention is for existing pine tissues cultivation, induced bundle bud not easily breaks up elongation, and growth coefficient is low, and subculture cycle is long, the technical problem that subculture bud seedling vigor is on the low side, second-rate, provides a kind of method being suitable for south China pine tree clump bud inducement and squamous subculture.
The present invention is achieved in that
A kind of Southern Pine grove bud inducement and subculture method, comprise explant selection and process, inducing clumping bud, Multiple Buds extend, subculture bud is cultivated, and obtain scale tissue cultures south pine tree subculture bud, its operating procedure is as follows:
(1) explant is selected and process
After pine tree seed disinfection, aseptically carry out seed germination, then intercepting the long hypocotylar germ free apical bud of band 0.5 cm is that explant carries out tissue cultures;
(2) inducing clumping bud
Aseptic explant is seeded in inducing culture and carries out clump bud inducement, cultivation cycle: 20-26 d, cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 0-16 h, intensity of illumination: 0-2000 lx, obtain Multiple Buds;
(3) Multiple Buds extends
The Multiple Buds induced is carried out in elongation medium elongation to cultivate, cultivation cycle: 14-28 d, cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, obtain elongation bud of growing thickly;
(4) subculture bud is cultivated
Be inoculated in subculture multiplication medium by the single terminal bud cutting elongation bud and breed, cultivation cycle: 14-21 d, cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, obtains subculture bud I; Subculture bud I is repeated step (3) to extend, single cut elongation subculture bud I and be divided into terminal bud and band lateral bud bud section, then in step (4) subculture multiplication medium, continue propagation obtain subculture bud II, subculture bud II is repeated step (3) and step (4) again, obtain new proliferation and subculture bud; Repetitive cycling step (3) and (4), thus realize scale proliferation and subculture group and train southern pine tree subculture bud.
Above-described subculture bud I selects singlely to cut water stain-free sample, obtain without the long terminal bud of 2 cm that extends bud of growing thickly that vitrification phenomenon, growth vigor are vigorous carries out breeding.
Above-described subculture bud II selects shoot proliferation bud subculture bud I to carry out being stretched to 3-5 cm, and be divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section is carried out shoot proliferation and obtains.
It is repetitive cycling subculture Shoot propagation, Multiple Buds elongation process 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, 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, pH value is 5.6-6.0.
Above-described subculture multiplication 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, pH value is 5.6-6.0.
Basic composition is of above-described modified MS medium: KNO
31650 mgL
-1; NH
4nO
3600 mgL
-1; CaCl
22H
2o 260 mgL
-1; MgSO
47H
2o 370 mgL
-1; Ca (NO
3)
24H
2o 400 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.
The present invention has the following advantages relative to existing technology:
1, the present invention adopts and optimizes different ratio medium and training method, and clump bud induction rate reaches more than 97%, and bud seedling extends soon, flushes.
2, squamous subculture of the present invention repeats squamous subculture mode by extending and breeding two sections, carry out N time to cultivate, subculture bud output increases substantially, growth coefficient reaches more than 6.8, for Pinus seeds fast growing, high-quality and efficient afforestation provide choiceness nursery stock, there is good economic benefit and social benefit.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
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 the Pinus Massoniana Lamb In Seed Orchard of the ancient fluffy fine provenance construction in Xincheng, Guangxi.
2, experimental technique
(1) aseptic explant is selected and aseptic process
By the cone of newly gathering first airing under sunshine, after synphyllodium opens, shake off out seed gently, with plastic sack seed installed that to be placed in 4 DEG C of refrigerator cold-storages for subsequent use.The refrigeration seed of a week is taken out from refrigerator, 15 min are soaked with 0.5% liquor potassic permanganate, then through running water 5 min, after deionized water soaks 12 h, 0.1% mercuric chloride is sterilized 40 min, aseptic water washing 5 times, is directly sowed at and carries out seed germination in the sand matrix of autoclave sterilization process.Choose the modified MS medium that robust growth germination seed seedling terminal bud (retaining the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out aseptic explant screening.
Basic composition is of described modified MS medium: KNO
31650 mgL
-1; NH
4nO
3600 mgL
-1; CaCl
22H
2o 260 mgL
-1; MgSO
47H
2o 370 mgL
-1; Ca (NO
3)
24H
2o 400 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) inducing clumping bud
Aseptic explant is shifted out from 0 hormone improvement MS minimal medium, cuts away its hypocotyl base portion until about 0.5 cm is long, be then inoculated into improvement MS+6-BA 3.0 mgL
-1+ NAA 0.05 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1medium on carry out Fiber differentiation.Condition of culture is 24 ± 1 DEG C, and illumination 0-2000 lx(first week is light culture), illumination every day 16 h, pH value is 5.8.After cultivating 18 d, average clump bud induction rate reaches 97.4%.
(3) Multiple Buds extends
The budlet that grows thickly induced is transferred to improvement MS++NAA 0.25 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1in elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8.Extend cultivation 22 d, clump bud height 3-5 cm.
(4) subculture bud is cultivated
The simple bud of plant height 2-3 cm in Multiple Buds is cut, 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 Shoot propagation.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8, shoot proliferation cycle 16-21 d.
Shoot proliferation bud is put in step (3) elongation medium, to promote that shoot proliferation bud extends.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8.Extend and cultivate 24-28 d, shoot proliferation bud height 3-5 cm.After elongation subculture bud being divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section, repeat step (4) shoot proliferation and the elongation of step (3) clump bud.Squamous subculture 5 times, average proliferation coefficient 6.8.
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 the Pinus Massoniana Lamb In Seed Orchard of the fine provenance constructions such as Ningming of Guangxi portiatree.
2, experimental technique
(1) aseptic explant is selected and aseptic process
By the cone of newly gathering first airing under sunshine, after synphyllodium opens, shake off out seed gently, with plastic sack seed installed that to be placed in 4 DEG C of refrigerator cold-storages for subsequent use.The refrigeration seed of a week is taken out from refrigerator, 15 min are soaked with 0.5% liquor potassic permanganate, then through running water 5 min, after deionized water soaks 12 h, 0.1% mercuric chloride is sterilized 40 min, aseptic water washing 5 times, is directly sowed at and carries out seed germination in the sand matrix of autoclave sterilization process.Choose the modified MS medium that robust growth germination seed seedling terminal bud (retaining the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out 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, cuts away its hypocotyl base portion until about 0.5 cm is long, be then inoculated into improvement MS+6-BA 4.0 mgL
-1+ NAA 0.1 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1medium on carry out Fiber differentiation.Condition of culture is 24 ± 1 DEG C, and illumination 0-2000 lx(first week is light culture), illumination every day 16 h, pH value is 5.8.After cultivating 25 d, average clump bud induction rate reaches 98.1%.
(3) Multiple Buds extends
The budlet that grows thickly induced is transferred to improvement MS++NAA 0.35 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1in elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8.Extend cultivation 25 d, clump bud height 3-5 cm.
(4) subculture bud is cultivated
The simple bud of plant height 2-3 cm in Multiple Buds is cut, 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 Shoot propagation.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8, shoot proliferation cycle 20 d.
Shoot proliferation bud is put in step (3) elongation medium, to promote that shoot proliferation bud extends.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8.Extend cultivation 26 d, shoot proliferation bud height 3-5 cm.After elongation subculture bud being divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section, repeat step (4) shoot proliferation and the elongation of step (3) clump bud.Squamous subculture 4 times, average proliferation coefficient 7.2.
embodiment 3
1, experiment material
With wet-land pine tree, ripe cone seed germination germ free apical bud is for explant then, and cone picks up from No. 1, state-owned Bobai forest farm, Bobai County, Guangxi District superior families Bobai, No. 2, Bobai.
2, experimental technique
(1) aseptic explant is selected and aseptic process
By the cone of newly gathering first airing under sunshine, after synphyllodium opens, shake off out seed gently, with plastic sack seed installed that to be placed in 4 DEG C of refrigerator cold-storages for subsequent use.The refrigeration seed of a week is taken out from refrigerator, 15 min are soaked with 0.5% liquor potassic permanganate, then through running water 5 min, after deionized water soaks 12 h, 0.1% mercuric chloride is sterilized 40 min, aseptic water washing 5 times, is directly sowed at and carries out seed germination in the sand matrix of autoclave sterilization process.Choose the modified MS medium that robust growth germination seed seedling terminal bud (retaining the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out 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, cuts away its hypocotyl base portion until about 0.5 cm is long, be then inoculated into improvement MS+6-BA 2.0-3.5 mgL
-1+ NAA 0.05-0.08 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1medium on carry out Fiber differentiation.Condition of culture is 24 ± 1 DEG C, illumination 2000 lx, illumination every day 16 h, and pH value is 5.6.After cultivating 24-26 d, average clump bud induction rate reaches 97.0%.
3) Multiple Buds extends
The budlet that grows thickly induced is transferred to improvement MS+NAA 0.3-0.4 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1in elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.6.Extend and cultivate 24-28 d, clump bud height 3-5 cm.
4) subculture bud is cultivated
The simple bud of plant height 2-3 cm in Multiple Buds is cut, 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 Shoot propagation.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.6, shoot proliferation cycle 15-20 d.
Shoot proliferation bud is put in step (3) elongation medium, to promote that shoot proliferation bud extends.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 2000 lx, pH value is 5.8.Extend and cultivate 23-28 d, shoot proliferation bud height 3-5 cm.After elongation subculture bud being divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section, repeat step (4) shoot proliferation and the elongation of step (3) clump bud.Squamous subculture 4 times, average proliferation coefficient 7.5.
embodiment 4
1, experiment material
Ripe cone seed germination germ free apical bud is for explant then to add pine to wet, and seed directly introduces fine germplasm resources from Australia.
2, experimental technique
(1) aseptic explant is selected and aseptic process
Take out being placed in 4 DEG C of refrigerator cold-storages seed of a week for subsequent use from refrigerator, 15 min are soaked with 0.5% liquor potassic permanganate, then through running water 5 min, after deionized water soaks 12 h, 0.1% mercuric chloride is sterilized 40 min, aseptic water washing 5 times, is directly sowed at and carries out seed germination in the sand matrix of autoclave sterilization process.Choose the modified MS medium that robust growth germination seed seedling terminal bud (retaining the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out 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, cuts away its hypocotyl base portion until about 0.5 cm is long, be then inoculated into improvement MS+6-BA 2.5 mgL
-1+ NAA 0.065 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1medium on carry out Fiber differentiation.Condition of culture is 24 ± 1 DEG C, illumination 1500-2000 lx, illumination every day 16 h, and pH value is 6.0.After cultivating 24 d, average clump bud induction rate reaches 98.1%.
(3) Multiple Buds extends
The budlet that grows thickly induced is transferred to improvement MS+ active carbon 3000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1in elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, pH value is 6.0.Extend cultivation 26 d, clump bud height 3-5 cm.
(4) subculture bud is cultivated
The simple bud of plant height 2-3 cm in Multiple Buds is cut, 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 Shoot propagation.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 18 h, intensity of illumination: 1500-2000 lx, pH value is 6.0, shoot proliferation cycle 20 d.
Shoot proliferation bud is put in step (3) elongation medium, to promote that shoot proliferation bud extends.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, pH value is 6.0.Extend cultivation 28 d, shoot proliferation bud height 3-5 cm.After elongation subculture bud being divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section, repeat step (4) shoot proliferation and the elongation of step (3) clump bud.Squamous subculture 8 times, average proliferation coefficient 8.2.
embodiment 5
1, experiment material
Ripe cone seed germination germ free apical bud is for explant then to add pine to wet, and seed directly introduces fine germplasm resources from Australia.
2, experimental technique
(1) aseptic explant is selected and aseptic process
Take out being placed in 4 DEG C of refrigerator cold-storages seed of a week for subsequent use from refrigerator, 15 min are soaked with 0.5% liquor potassic permanganate, then through running water 5 min, after deionized water soaks 12 h, 0.1% mercuric chloride is sterilized 40 min, aseptic water washing 5 times, is directly sowed at and carries out seed germination in the sand matrix of autoclave sterilization process.Choose the modified MS medium that robust growth germination seed seedling terminal bud (retaining the long hypocotyl of 1 cm) is inoculated into 0 hormone, carry out 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, cuts away its hypocotyl base portion until about 0.5 cm is long, be then inoculated into improvement MS+6-BA 3.0 mgL
-1+ NAA 0. 08 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1medium on carry out Fiber differentiation.Condition of culture is 24 ± 1 DEG C, illumination 1500-2000 lx, illumination every day 16 h, and pH value is 6.0.After cultivating 26 d, average clump bud induction rate reaches 98.7%.
(3) Multiple Buds extends
The budlet that grows thickly induced is transferred to improvement MS+ active carbon 5000 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1in elongation medium, to promote the elongation growth of culture.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, pH value is 6.0.Extend cultivation 27 d, clump bud height 3-5 cm.
(4) subculture bud is cultivated
The simple bud of plant height 2-3 cm in Multiple Buds is cut, 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 Shoot propagation.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, pH value is 6.0, shoot proliferation cycle 19 d.
Shoot proliferation bud is put in step (3) elongation medium, to promote that shoot proliferation bud extends.Cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, pH value is 6.0.Extend cultivation 27 d, shoot proliferation bud height 3-5 cm.After elongation subculture bud being divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section, repeat step (4) shoot proliferation and the elongation of step (3) clump bud.Squamous subculture 8 times, average proliferation coefficient 8.6.
Claims (4)
1. Southern Pine grove bud inducement and a subculture method, is characterized in that: comprise that explant is selected to extend with process, inducing clumping bud, Multiple Buds, subculture bud cultivates, acquisition scale tissue cultures southern pine tree subculture bud, and its operating procedure is as follows:
(1) explant is selected and process: after pine tree seed disinfection, aseptically carry out seed germination, and then intercepting the long hypocotylar germ free apical bud of band 0.5 cm is that explant carries out tissue cultures;
(2) inducing clumping bud: aseptic explant is seeded in inducing culture and carries out clump bud inducement, cultivation cycle: 20-26 d, cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 0-16 h, intensity of illumination: 0-2000 lx, obtains Multiple Buds;
(3) Multiple Buds extends: the Multiple Buds induced is carried out in elongation medium elongation and cultivate, cultivation cycle: 14-28 d, cultivation temperature: 24 ± 1 DEG C, the illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, obtain elongation bud of growing thickly;
(4) subculture bud is cultivated: be inoculated in subculture multiplication medium by the single terminal bud cutting elongation bud and breed, cultivation cycle: 14-21 d, cultivation temperature: 24 ± 1 DEG C, illumination cultivation time: 16 h, intensity of illumination: 1500-2000 lx, obtains subculture bud I; Subculture bud I is repeated step (3) to extend, single cut elongation subculture bud I and be divided into terminal bud and band lateral bud bud section, then in step (4) subculture multiplication medium, continue propagation obtain subculture bud II, subculture bud II is repeated step (3) and step (4) again, obtain new proliferation and subculture bud; Repetitive cycling step (3) and (4), thus realize scale proliferation and subculture group and train southern pine tree subculture bud;
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, pH value is 5.6-6.0;
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, pH value is 5.6-6.0;
Described subculture multiplication medium is: modified MS medium+6-BA 1.0-3.0 mgL
-1+ KT0-1.0 mgL
-1+ NAA 0.1-0.3 mgL
-1+ sucrose 30000 mgL
-1+ agar 3500 mgL
-1, pH value is 5.6-6.0;
Consisting of of described modified MS medium: KNO3 1650 mgL
-1; NH4NO3 600 mgL
-1; CaCl22H2O 260 mgL
-1; MgSO47H2O 370 mgL
-1; Ca (NO3) 24H2O 400 mgL
-1; KH2PO4 170 mgL
-1; MnSO4H2O 22.3 mgL
-1; ZnSO47H2O 8.6 mgL
-1; CuSO45H2O 0.025 mgL
-1; H3BO3 6.2 mgL
-1; Na2MoO42H2O 0.025 mgL
-1; KI 0.83 mgL
-1; CoCl26H2O 0.025 mgL
-1; Vitamin B1 1.0 mgL
-1; Vitamin B6 0.5 mgL
-1; Nicotinic acid 0.5 mgL
-1; Glycine 2.0 mgL
-1; Inositol 200 mgL
-1.
2. Southern Pine grove bud inducement according to claim 1 and subculture method, is characterized in that: described subculture bud I selects singlely to cut water stain-free sample, obtain without the long terminal bud of 2 cm that extends bud of growing thickly that vitrification phenomenon, growth vigor are vigorous carries out breeding.
3. Southern Pine grove bud inducement according to claim 1 and subculture method, it is characterized in that: described subculture bud II selects shoot proliferation bud subculture bud I to carry out being stretched to 3-5 cm, and be divided into the long terminal bud of 2 cm and 1 cm long band lateral bud bud section is carried out shoot proliferation and obtains.
4. Southern Pine grove bud inducement according to claim 1 and subculture method, is characterized in that: it is repetitive cycling subculture Shoot propagation, Multiple Buds elongation process that described subculture bud is cultivated, and carries out N time and cultivates.
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| CN103636506B (en) * | 2013-12-24 | 2015-06-10 | 黑龙江省林业科学研究所 | method for performing plant culture by utilizing shepherdia argentea caulicle regenerated plant induction culture medium and |
| 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 |
| 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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