CN105052742A - Method for breeding pinus massoniana good group asexual tissue culture seedlings - Google Patents
Method for breeding pinus massoniana good group asexual tissue culture seedlings Download PDFInfo
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- CN105052742A CN105052742A CN201510502243.XA CN201510502243A CN105052742A CN 105052742 A CN105052742 A CN 105052742A CN 201510502243 A CN201510502243 A CN 201510502243A CN 105052742 A CN105052742 A CN 105052742A
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- 235000011609 Pinus massoniana Nutrition 0.000 title claims abstract description 61
- 241000018650 Pinus massoniana Species 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000009395 breeding Methods 0.000 title claims abstract description 21
- 230000001488 breeding effect Effects 0.000 title claims abstract description 21
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 72
- 239000001963 growth medium Substances 0.000 claims abstract description 18
- 230000035784 germination Effects 0.000 claims abstract description 16
- 230000000392 somatic effect Effects 0.000 claims abstract description 13
- 238000005286 illumination Methods 0.000 claims description 30
- 239000006870 ms-medium Substances 0.000 claims description 30
- 229930006000 Sucrose Natural products 0.000 claims description 24
- 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 24
- 239000005720 sucrose Substances 0.000 claims description 24
- 239000012877 elongation medium Substances 0.000 claims description 22
- 238000000338 in vitro Methods 0.000 claims description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- 230000003203 everyday effect Effects 0.000 claims description 15
- 239000012869 germination medium Substances 0.000 claims description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 12
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 claims description 12
- 238000005520 cutting process Methods 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 230000004069 differentiation Effects 0.000 claims description 7
- BUDWTFCZGZYQHZ-UHFFFAOYSA-N 3-[(7h-purin-6-ylamino)methyl]phenol Chemical compound OC1=CC=CC(CNC=2C=3NC=NC=3N=CN=2)=C1 BUDWTFCZGZYQHZ-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 6
- 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 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 6
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- 239000008103 glucose Substances 0.000 claims description 6
- 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 6
- 229960000367 inositol Drugs 0.000 claims description 6
- 239000008101 lactose Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
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- 239000011664 nicotinic acid Substances 0.000 claims description 6
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for breeding pinus massoniana good group asexual tissue culture seedlings. The method comprises the following processes of somatic embryo germination, somatic embryo seedling elongation, multiplication culture, clumpy bud elongation and rooting culture, to be specific, mature pinus massoniana somatic embryos are placed in a germination culture medium and then transferred to an elongation culture medium for culture after germinating into bud seedlings, after bud seedlings elongate, root systems are cut off, the bud seedlings are transferred to multiplication culture medium for clumpy bud induction, induced clumpy buds are transferred to an elongation culture medium for elongation, and after bud seedlings are 2-3 cm in height, bud seedlings are cut off separately and then placed in a rooting culture medium for rooting treatment, so that pinus massoniana good group asexual tissue culture seedlings are obtained. By adopting the method for efficiently breeding pinus massoniana good group asexual tissue culture seedlings, lots of high-quality pinus massoniana excellent tissue culture seedlings can be rapidly and efficiently obtained, the problems that somatic embryo seedlings are relatively poor in root system and poor in growth in somatic embryogenesis, and bud seedlings are low in reproduction rate and long in breeding cycle in organogenesis are solved and obvious economic and social benefits are realized.
Description
Technical field
The invention belongs to technical field of plant propagation, relate to tissue cultivating and seedling technology, especially a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro.
Background technology
Masson pine (
pinusmassonianan) originate in China, be 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 genetic variation and genetic differentiation, genetic improvement gain can not be improved to greatest extent, and vegetative propagation can overcome the above-mentioned deficiency of sexual propagation.
Body embryo occurs and organ is two kinds of asexual tissue culture technology approach that the current whole world is commonly used the most.Wherein, body embryo generation technique content is high, reproduction speed is fast, it is mainly explant with immature zygotic embryos, and cultivate phorozoon embryo seedling by the body embryo culture technique of series, this technology energy obtains a large amount of nursery stock within a short period of time, but because body embryo seedling root system development is more weak, and its growth is higher to requirement of nitrogen, therefore poor to the adaptive capacity of environment, survival rate is not high.Organ generation technique is more easily grasped, and comparatively conventional in production, wherein " with the numerous bud of bud " tissue-culturing rapid propagation mode is particularly common, it is flourishing that this technology cultivates seedling root, and bud seedling is healthy and strong, and environmental suitability is strong, but because its breeding rate is lower, subculture cycle is long, and production cost is higher.
The seeds of horse hair Pinus tissue cultures difficulty.Numerous domestic scholar occurs masson pine body embryo and organ generation technique has carried out large quantifier elimination, wherein masson pine body embryo generation technique mainly concentrates on body embryonal induction, propagation and the research of maturation culture aspect, significant results with regard to body embryo germination and conversion aspect are still fresh rare, its reason be mainly body embryo seedling sprout and conversion ratio not high, root system is of poor quality, obtain seedling rate on the low side, technology application is not strong.And pass through the masson pine group culturation rapid propagating technology of adventitious organogenesis, because subculture Shoot propagation coefficient is on the low side, cultivation period is long, seriously constrains the fast breeding of masson pine plantlet in vitro.
Summary of the invention
Object of the present invention is low mainly for masson pine body embryo seedling germination rate, root system is of poor quality, the present situations such as, the squamous subculture cycle low by the masson pine tissue culture propagation efficiency of adventitious organogenesis is long, deficient for solving masson pine breeding, realize masson pine excellent colony asxualization plantlet in vitro wide popularization and application, a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro is provided.
The present invention is achieved in that
A kind of method of breeding masson pine excellent colony asxualization plantlet in vitro, comprise body embryo germination, body embryo seedling extends, Multiplying culture, the operation of the elongation of clump bud and culture of rootage, fully-developed masson pine somatic embryo is placed in germination medium, proceed to elongation medium after sprouting into bud seedling to cultivate, after bud seedling extends, cut away root system to proceed to proliferated culture medium and carry out clump bud inducement, induced bundle bud is proceeded to elongation medium extend, when bud seedling reaches 3-5cm height, single for bud seedling cutting is placed in root media and carries out rooting treatment, obtain the excellent population groups of masson pine and train seedling of taking root, its operating procedure is as follows:
(1) body embryo germination: by White-opalescent, has wax gloss, and the ripe masson pine somatic embryo developing into cotyledon period is placed in germination medium sprouts;
(2) body embryo seedling extends: wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out elongation cultivation;
(3) Multiplying culture: when body embryo seedling is stretched to 3-5cm height, directly puts into proliferated culture medium after body embryo seedling is cut root system and carries out clump bud inducement;
(4) clump bud extends: the clump bud induced is proceeded to elongation medium and carry out elongation cultivation;
(5) culture of rootage: the single masson pine bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage, obtains the excellent population groups of masson pine and trains seedling of taking root.
Its material content of above-described germination medium is: active carbon 10gL
-1, glucose 20gL
-1, lactose 5gL
-1, sucrose 5gL
-1with 1/2 modified MS medium.
Its material content of above-described elongation medium is: active carbon 5gL
-1, sucrose 30gL
-1and modified MS medium.
Its material content of above-described proliferated culture medium is: 6-BA0-1.0mgL
-1, Thidiazuron (TDZ) 1.0-3.0mgL
-1, Meta-Topolin(MT) 1.0-2.0mgL
-1, sucrose 30gL
-1and modified MS medium.
Its material content of above-described root media is: ABT
61.0-2.0mgL
-1, IAA0.5-1.0mgL
-1, coconut milk 10mLL
-1, active carbon 1.0-2.5gL
-1, sucrose 10gL
-1with 1/2 modified MS medium.
Basic composition is of above-described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
The cultivation controlled condition of the body embryo germination described in above step (1) is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C; Body embryo seedling elongation described in step (2), the Multiplying culture described in step (3), the clump bud described in step (4) are extended, the cultivation controlled condition of culture of rootage described in step (5) is: light is cultivated, illumination 1500-2000lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
The present invention has the following advantages relative to existing technology:
1, invention creates the raising technology that a kind of high effect culture masson pine excellent colony asxualization plantlet in vitro merged mutually occurs to occur with organ body embryo, a large amount of high-quality masson pine excellent colonies plantlet in vitro can be obtained rapidly, efficiently, efficiently solve the problems such as body embryo shoot root system is more weak, growth is poor and in organ generation, seedling and bud proliferation rate is low, the breeding cycle is long in the generation of body embryo, there is significant novelty and progressive.
2, the technology of the present invention is passed through, masson pine body embryo seedling germination rate is up to more than 97%, and by the culture of rootage of adventitious organogenesis, rooting rate reaches 95%, and well developed root system, bud seedling is healthy and strong, flush, and survival rate is high, and the cultivating seedlings cycle obviously shortens, Be very effective, solve the problem of masson pine breeding scarcity, realize masson pine excellent colony asxualization plantlet in vitro wide popularization and application, there is good economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 is the masson pine embryogenic cell line flushed.
Fig. 2 is the somatic embryo that masson pine embryogenic cell line is formed after maturation culture.
Fig. 3 is the masson pine body embryo seedling produced after body embryo germination.
Fig. 4 is that the masson pine group training formed after culture of rootage is taken root seedling.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
embodiment 1:
Breed a method for masson pine excellent colony asxualization plantlet in vitro, with masson pine superior families unmature subleaf zygotic embryo in early stage for explant, obtain by body embryo the masson pine embryogenic cell line that flushes in a large number.Wherein, by the White-opalescent that embryogenic cell line SE-3 produces after maturation culture, there is wax gloss, and the mature somatic embryo developing into cotyledon period is placed in germination medium sprouts; Described germination medium is: 1/2 modified MS medium+active carbon 10gL
-1+ glucose 20gL
-1+ lactose 5gL
-1+ sucrose 5gL
-1.Cultivation controlled condition is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C.Cultivate 1 week, germination rate reaches 97.4%.
Wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out body embryo seedling elongation cultivation; Described elongation medium is: modified MS medium+active carbon 5gL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation after 40 days, when body embryo seedling is stretched to 3-5cm height, directly puts into proliferated culture medium after body embryo seedling is cut root system and carry out clump bud inducement Multiplying culture; Described proliferated culture medium is: modified MS medium+Thidiazuron (TDZ) 2.0mgL
-1+ Meta-Topolin(MT) 1.5mgL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Multiplying culture 30 days, proceeds to the clump bud induced in elongation medium described above and carries out clump bud elongation cultivation.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation 45 days, the single bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage; Described root media is 1/2 modified MS medium+ABT
61.0mgL
-1+ IAA1.0mgL
-1+ coconut milk 10mLL
-1+ active carbon 1.0gL
-1+ sucrose 10gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.Culture of rootage 28 days, rooting rate reaches 93%.
Basic composition is of above-described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
embodiment 2:
Breed a method for masson pine excellent colony asxualization plantlet in vitro, with masson pine superior families unmature subleaf zygotic embryo in early stage for explant, obtain by body embryo the masson pine embryogenic cell line that flushes in a large number.Wherein, by the White-opalescent that embryogenic cell line SE-3 produces after maturation culture, there is wax gloss, and the mature somatic embryo developing into cotyledon period is placed in germination medium sprouts; Described germination medium is: 1/2 modified MS medium+active carbon 10gL
-1+ glucose 20gL
-1+ lactose 5gL
-1+ sucrose 5gL
-1.Cultivation controlled condition is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C.Cultivate 2 weeks, germination rate reaches 98.1%.
Wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out body embryo seedling elongation cultivation; Described elongation medium is: modified MS medium+active carbon 5gL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation when within 40 days, body embryo seedling is stretched to 3-5cm height, directly put into proliferated culture medium after body embryo seedling is cut root system and carry out clump bud inducement Multiplying culture; Described proliferated culture medium is: modified MS medium+6-BA0.5mgL
-1+ Thidiazuron (TDZ) 1.5mgL
-1+ Meta-Topolin(MT) 2.0mgL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Multiplying culture 25 days, proceeds to the clump bud induced in elongation medium described above and carries out clump bud elongation cultivation.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation 45 days, the single bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage; Described root media is 1/2 modified MS medium+ABT
62.0mgL
-1+ IAA0.5mgL
-1+ coconut milk 10mLL
-1+ active carbon 1.5gL
-1+ sucrose 10gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.Culture of rootage 35 days, rooting rate 95%.
Basic composition is of above-described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
embodiment 3:
Breed a method for masson pine excellent colony asxualization plantlet in vitro, with masson pine superior families unmature subleaf zygotic embryo in early stage for explant, obtain by body embryo the masson pine embryogenic cell line that flushes in a large number.Wherein, by the White-opalescent that embryogenic cell line SE-3 produces after maturation culture, there is wax gloss, and the mature somatic embryo developing into cotyledon period is placed in germination medium sprouts; Described germination medium is: 1/2 modified MS medium+active carbon 10gL
-1+ glucose 20gL
-1+ lactose 5gL
-1+ sucrose 5gL
-1.Cultivation controlled condition is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C.Cultivate 3 weeks, germination rate reaches 98.6%.
Wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out body embryo seedling elongation cultivation; Described elongation medium is: modified MS medium+active carbon 5gL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation when within 40 days, body embryo seedling is stretched to 3-5cm height, directly put into proliferated culture medium after body embryo seedling is cut root system and carry out clump bud inducement Multiplying culture; Described proliferated culture medium is: modified MS medium+6-BA1.0mgL
-1+ Thidiazuron (TDZ) 1.0mgL
-1+ Meta-Topolin(MT) 1.0mgL
-1+ sucrose 30gL
-1.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Multiplying culture 35 days, proceeds to the clump bud induced in elongation medium described above and carries out clump bud elongation cultivation.Cultivation controlled condition is: light is cultivated, illumination 1500lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Extend cultivation 50 days, the single bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage; Described root media is 1/2 modified MS medium+ABT
61.5mgL
-1+ IAA1.0mgL
-1+ coconut milk 10mLL
-1+ active carbon 2.5gL
-1+ sucrose 10gL
-1.Culture of rootage 30 days, rooting rate reaches 91%.
The cultivation controlled condition of the elongation of above-mentioned body embryo seedling, Multiplying culture, the elongation of clump bud, process of rooting culture is: light is cultivated, illumination 2000lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Basic composition is of above-described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
embodiment 4:
Breed a method for masson pine excellent colony asxualization plantlet in vitro, with masson pine superior families unmature subleaf zygotic embryo in early stage for explant, obtain by body embryo the masson pine embryogenic cell line that flushes in a large number.Wherein, by the White-opalescent that embryogenic cell line SE-9 produces after maturation culture, there is wax gloss, and the mature somatic embryo developing into cotyledon period is placed in germination medium sprouts; Described germination medium is: 1/2 modified MS medium+active carbon 10gL
-1+ glucose 20gL
-1+ lactose 5gL
-1+ sucrose 5gL
-1.Cultivation controlled condition is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C.Cultivate 1 week, germination rate reaches 97.0%.
Wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out body embryo seedling elongation cultivation; Described elongation medium is: modified MS medium+active carbon 5gL
-1+ sucrose 30gL
-1.
Extend cultivation when within 45 days, body embryo seedling is stretched to 3-5cm height, directly put into proliferated culture medium after body embryo seedling is cut root system and carry out clump bud inducement Multiplying culture; Described proliferated culture medium is: modified MS medium+6-BA0.5mgL
-1+ Thidiazuron (TDZ) 3.0mgL
-1+ Meta-Topolin(MT) 1.5mgL
-1+ sucrose 30gL
-1.
Multiplying culture 35 days, proceeds to the clump bud induced in elongation medium described above and carries out clump bud elongation cultivation.
Extend cultivation 55 days, the single bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage; Described root media is 1/2 modified MS medium+ABT
61.5mgL
-1+ IAA0.8mgL
-1+ coconut milk 10mLL
-1+ active carbon 2.0gL
-1+ sucrose 10gL
-1.Culture of rootage 35 days, rooting rate reaches 92%.
The cultivation controlled condition of the elongation of above-mentioned body embryo seedling, Multiplying culture, the elongation of clump bud, process of rooting culture is: light is cultivated, illumination 2000lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
Basic composition is of above-described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
Claims (7)
1. breed the method for masson pine excellent colony asxualization plantlet in vitro for one kind, it is characterized in that: comprise body embryo germination, body embryo seedling extends, Multiplying culture, the operation of the elongation of clump bud and culture of rootage, fully-developed masson pine somatic embryo is placed in germination medium, proceed to elongation medium after sprouting into bud seedling to cultivate, after bud seedling extends, cut away root system to proceed to proliferated culture medium and carry out clump bud inducement, induced bundle bud is proceeded to elongation medium extend, when bud seedling reaches 2-3cm height, single for bud seedling cutting is placed in root media and carries out rooting treatment, obtain masson pine population groups and train seedling of taking root, its operating procedure is as follows:
(1) body embryo germination: by White-opalescent, has wax gloss, and the ripe masson pine somatic embryo developing into cotyledon period is placed in germination medium sprouts;
(2) body embryo seedling extends: wait that sprouting body embryo seedling leaf turns green, hypocotyl base portion reddens, and after root, stem and leaf differentiation, goes in elongation medium and carries out elongation cultivation;
(3) Multiplying culture: when body embryo seedling is stretched to 3-5cm height, directly puts into proliferated culture medium after body embryo seedling is cut root system and carries out clump bud inducement;
(4) clump bud extends: the clump bud induced is proceeded to elongation medium and carry out elongation cultivation;
(5) culture of rootage: the single masson pine bud seedling cutting robust growth, semi-lignified, high 2-3cm, is seeded in root media, carries out culture of rootage, obtains masson pine population groups and trains seedling of taking root.
2. a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro according to claim 1, is characterized in that: its material content of described germination medium is: active carbon 10gL
-1, glucose 20gL
-1, lactose 5gL
-1, sucrose 5gL
-1with 1/2 modified MS medium.
3. a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro according to claim 1, is characterized in that: its material content of described elongation medium is: active carbon 5gL
-1, sucrose 30gL
-1and modified MS medium.
4. a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro according to claim 1, is characterized in that: its material content of described proliferated culture medium is: 6-BA0-1.0mgL
-1, Thidiazuron (TDZ) 1.0-3.0mgL
-1, Meta-Topolin(MT) 1.0-2.0mgL
-1, sucrose 30gL
-1and modified MS medium.
5. a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro according to claim 1, is characterized in that: its material content of described root media is: ABT
61.0-2.0mgL
-1, IAA0.5-1.0mgL
-1, coconut milk 10mLL
-1, active carbon 1.0-2.5gL
-1, sucrose 10gL
-1with 1/2 modified MS medium.
6., according to Claims 2 or 3 or 4 or 5 arbitrary a kind of described methods of breeding masson pine excellent colony asxualization plantlet in vitro, it is characterized in that: basic composition is of described modified MS medium: KNO
31050mgL
-1; NH
4nO
3850mgL
-1; CaCl
22H
2o260mgL
-1; MgSO
47H
2o370mgL
-1; Ca (NO
3)
24H
2o600mgL
-1; KH
2pO
4340mgL
-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 100mgL
-1.
7. a kind of method of breeding masson pine excellent colony asxualization plantlet in vitro according to claim 1, is characterized in that: the cultivation controlled condition of the body embryo germination described in step (1) is: light culture, unglazed photograph, cultivation temperature 20 ± 1 DEG C; Body embryo seedling elongation described in step (2), the Multiplying culture described in step (3), the clump bud described in step (4) are extended, the cultivation controlled condition of culture of rootage described in step (5) is: light is cultivated, illumination 1500-2000lx, illumination every day 16h, cultivation temperature 20-28 DEG C.
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| CN111616050A (en) * | 2020-05-19 | 2020-09-04 | 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305833A (en) * | 2019-07-10 | 2019-10-08 | 广西壮族自治区林业科学研究院 | A kind of suspension culture method of masson pine cells,primordial proliferation |
| CN110305833B (en) * | 2019-07-10 | 2022-04-01 | 广西壮族自治区林业科学研究院 | Suspension culture method for proliferation of embryonic cells of pinus massoniana |
| CN111616050A (en) * | 2020-05-19 | 2020-09-04 | 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 |
| WO2021232507A1 (en) * | 2020-05-19 | 2021-11-25 | 广西壮族自治区林业科学研究院 | Method for improving rooting stability of subculture buds of high-fat-yield pinus massoniana superior tree |
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