CN104756869A - Method for preparation of pinus massoniana lamb superior individual sterile explant and induction of initial bud - Google Patents
Method for preparation of pinus massoniana lamb superior individual sterile explant and induction of initial bud Download PDFInfo
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- CN104756869A CN104756869A CN201510163793.3A CN201510163793A CN104756869A CN 104756869 A CN104756869 A CN 104756869A CN 201510163793 A CN201510163793 A CN 201510163793A CN 104756869 A CN104756869 A CN 104756869A
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- masson pine
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- 235000011609 Pinus massoniana Nutrition 0.000 title claims abstract description 54
- 241000018650 Pinus massoniana Species 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 235000019687 Lamb Nutrition 0.000 title abstract 5
- 230000006698 induction Effects 0.000 title abstract 4
- 241000196324 Embryophyta Species 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 14
- 230000001939 inductive effect Effects 0.000 claims description 13
- 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
- 238000007796 conventional method Methods 0.000 claims description 12
- 238000005286 illumination Methods 0.000 claims description 12
- 239000006870 ms-medium Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 238000011010 flushing procedure Methods 0.000 claims description 9
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 8
- 230000035584 blastogenesis Effects 0.000 claims description 8
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 8
- 239000006013 carbendazim Substances 0.000 claims description 8
- 230000000249 desinfective effect Effects 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 230000004083 survival effect Effects 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 229920000136 polysorbate Polymers 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
- 229920001817 Agar Polymers 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
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 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
- 229960003512 nicotinic acid Drugs 0.000 claims description 6
- 235000001968 nicotinic acid Nutrition 0.000 claims description 6
- 239000011664 nicotinic acid Substances 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims description 4
- 239000003899 bactericide agent Substances 0.000 claims description 4
- 238000001228 spectrum Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 2
- 238000011017 operating method Methods 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 5
- 239000000645 desinfectant Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000007654 immersion Methods 0.000 description 5
- 235000005205 Pinus Nutrition 0.000 description 3
- 241000218602 Pinus <genus> Species 0.000 description 3
- 235000011613 Pinus brutia Nutrition 0.000 description 3
- 239000005842 Thiophanate-methyl Substances 0.000 description 3
- RIOXQFHNBCKOKP-UHFFFAOYSA-N benomyl Chemical compound C1=CC=C2N(C(=O)NCCCC)C(NC(=O)OC)=NC2=C1 RIOXQFHNBCKOKP-UHFFFAOYSA-N 0.000 description 3
- MITFXPHMIHQXPI-UHFFFAOYSA-N benzoxaprofen Natural products N=1C2=CC(C(C(O)=O)C)=CC=C2OC=1C1=CC=C(Cl)C=C1 MITFXPHMIHQXPI-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical group COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 3
- 241000218641 Pinaceae Species 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001954 sterilising effect 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
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
- 241000779819 Syncarpia glomulifera Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
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- 239000000835 fiber Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000008601 oleoresin Substances 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
- 238000011160 research Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003892 spreading Methods 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
- 230000002792 vascular Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for preparation of a pinus massoniana lamb superior individual sterile explant and induction of an initial bud. The method comprises the following steps: grafting by selecting a sprout twig with a newly grown tender top in the current year of an adult superior individual as a scion and taking a pinus massoniana lamb tissue culture seedling as a stock, performing disinfection treatment on the sprout twig by adopting a series of disinfectants to match for disinfection, and putting the explant scion in an optimized induction culture medium to perform initial bud induction to obtain the sterile explant and the pinus massoniana lamb initial bud with rapid growth and vigorous vitality. By adopting the method disclosed by the invention, the technical problems of difficulty in disinfection of the explant, browning, low incidence of the initial bud, poor activity of a bud shoot and the like in tissue culture of the pinus massoniana lamb adult superior individual are solved, the incidence of the initial bud and the activity of the bud shoot are improved, the robust explant which is sterile above 90% can be obtained, and better economic benefits and social benefits are obtained.
Description
Technical field
The invention belongs to technical field of plant propagation, relate to tissue cultivating and seedling technology, especially a kind of masson pine fine individual plant aseptic explant preparation and initial bud inducement method.
Background technology
Masson pine (
pinus massonianalamb.) belong to Pinaceae (Pinaceae) abieteae (Pinoideae) Pinus (
pinusl.) two vascular bundle pine subgenus (
subgen Pinus) plant, be the dual-purpose seeds of Subtropic of China area peculiar native soil afforestation fat material.The seeds high as a kind of comprehensive utilization value, promotion prospect is wide, 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 the main industrial cut stock seeds of China and Tree Species as Bio-energy, greatly develops Masson Pine Plantation necessary.
Guangxi, at the collection of masson pine germ plasm resource, seed selection and store method, belongs to antecedent in the whole nation.Since " six or five " National Key Research Programs, a large amount of masson pine choiceness of seed selection.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 the industrialization of masson pine clone.
The seeds of horse hair Pinus tissue cultures difficulty, especially with adult maternal plant stem section bud for explant carry out group training cultivate time, because Oleoresin Contents is high, explant sterilization difficulty, and in initial bud inducement is cultivated, the bottleneck problems such as easy brownization, initial bud incidence are low, bud seedling does not extend, poor activity, seriously limit the propagation and employment of masson pine choiceness.
Summary of the invention
The present invention is directed to that existing masson pine grows up that explant sterilization difficulty in excellent strain tissue cultures, brownization, initial bud incidence are low, the technical problem of bud seedling poor activity etc., provide a kind of masson pine fine individual plant aseptic explant to prepare and the method for initial bud inducement.
To achieve these goals, technical scheme of the present invention is as follows:
The preparation of a kind of masson pine fine individual plant aseptic explant and initial bud inducement method, comprise that explant prepares, explant is disinfected, initial bud inducement, obtains aseptic explant and growth is fast, the initial bud of masson pine that flushes, and its operating procedure is as follows:
(1) explant prepares:
Adopt the masson pine plantlet in vitro of semi-annual to be stock, excellent masson pine elite stand of selecting to grow up newly takes out the tender tip then as scion, carries out grafting by conventional method, and cutting grafting leading shoot after surviving, to urge blastogenesis long, and choosing the long rudiment bar of 3.0-5.0 cm is explant;
(2) explant is disinfected:
After explant is clean with aseptic water washing, being placed in volumetric concentration is that the wide-spectrum bactericide of 0.1-0.3 % soaks 0.5-1 h, it is soak 1-2 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 10-20 min in the liquor natrii hypochloritis of 5-8 %, take out and be put in after hydrogen peroxide that volumetric concentration is 10-25 % and 5-10 drip in the mixed liquor of tween and stir and soak 20-30 min, finally with aseptic washing 3-4 time, each flushing 3-5 min;
(3) initial bud inducement: the explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 0.5-2.0 mgL
-1+ Meta-Topolin(MT) 1.0-3.0 mgL
-1+ KT 1.0-3.0 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.1-0.3 % wide-spectrum bactericide; Initial bud inducement cultivation cycle is: 2-3 month, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is :≤500 lx, the initial bud of masson pine obtain robust growth, flushing.
Consisting of 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.
In explant set-up procedure described in above step (1), after scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel and take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, complete grafting.
In explant set-up procedure described in above step (1), after grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % carbendazim by conventional method, adopt down in fair weather the rudiment bar that flushes after 1-2 day for subsequent use as explant.
In contrast to prior art, advantage of the present invention and good effect as follows:
1, the explant that the present invention selects is for stock with masson pine plantlet in vitro, this anvil grafting that tender scion is a little carried out newly is taken out in excellent strain of growing up then, because masson pine plantlet in vitro compares general seedling, well developed root system, with luxuriant foliage and spreading branches in leafy profusion, cane is sturdy, it is vigorous to nourish and grow, grafting wound healing is fast, compatibility good, survival rate is high, removes the master of grafting survival seedling after a while, and bud seedling germination rate is high, growth is fast, rudiment bar juvenile form degree for explant is high, drastically increases the validity of explant.
2, the present invention's mode of disinfecting of adopting the broad spectrum antimicrobicide of certain concentration and disinfecting time, alcohol, clorox, hydrogen peroxide and tween to match successively, aseptic explant acquisition rate is high, reaches more than 90%.
3, the present invention uses and optimizes inducing culture and training method, and Brown is few, and initial bud incidence is high, and bud seedling extends soon, flushes, and has good economic benefit and social benefit.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1:
The preparation of a kind of masson pine fine individual plant aseptic explant and initial bud inducement method, adopt semi-annual masson pine plantlet in vitro to be stock, selects newly to take out the tender tip then as scion from the state-run masson pine excellent strain GL38 that grows up that sends in the superior stand of forest farm, Yangshan in Guangxi.After scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel and take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, carry out grafting by conventional method.After grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % carbendazim by conventional method, adopt down in fair weather after 1-2 day and flush, the long long rudiment bar for 3.0-5.0 cm is for subsequent use as explant.
After explant is clean with aseptic water washing, being placed in volumetric concentration is that the carbendazim solution of 0.1 % soaks 1 h, it is soak 1 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 20 min in the liquor natrii hypochloritis of 5 %, take out after stirring immersion 30 min in the mixed liquor being put in hydrogen peroxide that volumetric concentration is 10 % and 5 tweens, finally with aseptic washing 3 times, each flushing 3 min.
Explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 0.5 mgL
-1+ Meta-Topolin(MT) 1.0 mgL
-1+ KT 1.0 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.1 % carbendazim.Consisting of 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.
Initial bud inducement cultivation cycle is: 90 d, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is: 400 lx, and the initial bud of masson pine obtain robust growth, flushing, it is 90.8% that initial bud obtains bud rate.
Embodiment 2:
The preparation of a kind of masson pine fine individual plant aseptic explant and initial bud inducement method, adopt semi-annual masson pine plantlet in vitro to be stock, selects newly to take out the tender tip then as scion from the state-run masson pine excellent strain GL38 that grows up that sends in the superior stand of forest farm, Yangshan in Guangxi.After scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel and take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, carry out grafting by conventional method.After grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % carbendazim solution by conventional method, adopt down in fair weather after 1-2 day and flush, the long long rudiment bar for 3.0-5.0 cm is for subsequent use as explant.
After explant is clean with aseptic water washing, be placed in carbendazim solution immersion 0.5 h that volumetric concentration is 0.3 %, it is soak 1 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 15 min in the liquor natrii hypochloritis of 6 %, take out after stirring immersion 30 min in the mixed liquor being put in hydrogen peroxide that volumetric concentration is 15 % and 10 tweens, finally with aseptic washing 4 times, rinse 3min at every turn.
Explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 1.0 mgL
-1+ Meta-Topolin(MT) 2.0 mgL
-1+ KT 1.5 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.3 % carbendazim.Consisting of 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.
Initial bud inducement cultivation cycle is: 65 d, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is :≤500 lx.The initial bud of masson pine obtain robust growth, flushing, it is 91.5% that initial bud obtains bud rate.
Embodiment 3:
The preparation of a kind of masson pine fine individual plant aseptic explant and initial bud inducement method, adopt semi-annual masson pine plantlet in vitro to be stock, selects newly to take out the tender tip then as scion from the state-run masson pine excellent strain GL105 that grows up that sends in the superior stand of forest farm, Yangshan in Guangxi.After scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel and take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, carry out grafting by conventional method.After grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % thiophanate methyl solution by conventional method, adopt down in fair weather after 1-2 day and flush, the long long rudiment bar for 3.0-5.0 cm is for subsequent use as explant.
After explant is clean with aseptic water washing, being placed in volumetric concentration is that the thiophanate methyl solution of 0.2 % soaks 1 h, it is soak 2 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 15 min in the liquor natrii hypochloritis of 8 %, take out after stirring immersion 25 min in the mixed liquor being put in hydrogen peroxide that volumetric concentration is 20 % and 8 tweens, finally with aseptic washing 4 times, rinse 5min at every turn.
Explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 1.5 mgL
-1+ Meta-Topolin(MT) 2.5 mgL
-1+ KT 2.0 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.2 % thiophanate methyl.Consisting of 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.
Initial bud inducement cultivation cycle is: 84 d, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is: 400 lx, and the initial bud of masson pine obtain robust growth, flushing, it is 90.3% that initial bud obtains bud rate.
Embodiment 4:
The preparation of a kind of masson pine fine individual plant aseptic explant and initial bud inducement method, adopt semi-annual masson pine plantlet in vitro to be stock, selects newly to take out the tender tip then as scion from the state-run masson pine excellent strain GL68 that grows up that sends in the superior stand of forest farm, Yangshan in Guangxi.After scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel and take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, carry out grafting by conventional method.After grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % benomyl solution by conventional method, adopt down in fair weather after 1-2 day and flush, the long long rudiment bar for 3.0-5.0 cm is for subsequent use as explant.
After explant is clean with aseptic water washing, being placed in volumetric concentration is that the benomyl solution of 0.3 % soaks 0.5 h, it is soak 2 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 10 min in the liquor natrii hypochloritis of 8 %, take out be put in volumetric concentration be 25% hydrogen peroxide and 10 tweens mixed liquor in stir immersion 20 min after, finally with aseptic washing 4 times, rinse 5min at every turn.
Explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 2.0 mgL
-1+ Meta-Topolin(MT) 3.0 mgL
-1+ KT 3.0 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.2 % benomyl.Consisting of 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.
Initial bud inducement cultivation cycle is: 60 d, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is: 400 lx, and the initial bud of masson pine obtain robust growth, flushing, it is 94.5% that initial bud obtains bud rate.
Claims (4)
1. a masson pine fine individual plant aseptic explant preparation and initial bud inducement method, it is characterized in that: comprise that explant prepares, explant disinfects, initial bud inducement, obtain aseptic explant and grow the initial bud of masson pine that is fast, that flush, its operating procedure is as follows:
(1) explant prepares:
Adopt the masson pine plantlet in vitro of semi-annual to be stock, excellent masson pine elite stand of selecting to grow up newly takes out the tender tip then as scion, carries out grafting by conventional method, and cutting grafting leading shoot after surviving, to urge blastogenesis long, and choosing the long rudiment bar of 3.0-5.0 cm is explant;
(2) explant is disinfected:
After explant is clean with aseptic water washing, being placed in volumetric concentration is that the wide-spectrum bactericide of 0.1-0.3 % soaks 0.5-1 h, it is soak 1-2 min in 70 % alcohol that taking-up explant is placed on volumetric concentration, moving into volumetric concentration is again soak 10-20 min in the liquor natrii hypochloritis of 5-8 %, take out and be put in after hydrogen peroxide that volumetric concentration is 10-25 % and 5-10 drip in the mixed liquor of tween and stir and soak 20-30 min, finally with aseptic washing 3-4 time, each flushing 3-5 min;
(3) initial bud inducement: the explant after disinfecting is cut into 1.0 cm long after, be inoculated in inducing culture; Described inducing culture is: modified MS medium+Thidiazuron (TDZ) 0.5-2.0 mgL
-1+ Meta-Topolin(MT) 1.0-3.0 mgL
-1+ KT 1.0-3.0 mgL
-1+ glucose 30000 mgL
-1+ agar 3500 mgL
-1+ 0.1-0.3 % wide-spectrum bactericide; Initial bud inducement cultivation cycle is: 2-3 month, temperature: 20 ± 1 DEG C, and the illumination cultivation time is: 16 h, and intensity of illumination is :≤500 lx, the initial bud of masson pine obtain robust growth, flushing.
2. masson pine fine individual plant aseptic explant preparation according to claim 1 and initial bud inducement method, is characterized in that: consisting of 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.
3. masson pine fine individual plant aseptic explant preparation according to claim 1 and initial bud inducement method, it is characterized in that: in the explant set-up procedure described in step (1), after scion is cut from adult excellent masson pine elite stand, put into the incubator that ice bag is housed after wrapping with wet towel to take back and be placed in 4 DEG C of refrigerator cold-storages, in 24 h, complete grafting.
4. masson pine fine individual plant aseptic explant preparation according to claim 1 and initial bud inducement method, it is characterized in that: in the explant set-up procedure described in step (1), after grafting survival, destroying grafting leading shoot, to urge blastogenesis long, until rudiment bar reach 3.0-5.0 cm long time, spray volumetric concentration 0.1 % carbendazim by conventional method, adopt down in fair weather the rudiment bar that flushes after 1-2 day for subsequent use as explant.
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| CN105052742A (en) * | 2015-08-14 | 2015-11-18 | 广西壮族自治区林业科学研究院 | Method for breeding pinus massoniana good group asexual tissue culture seedlings |
| CN105393917A (en) * | 2015-11-30 | 2016-03-16 | 北京林业大学 | Rapid propagation method of lonicera oblata |
| CN106665361A (en) * | 2017-02-14 | 2017-05-17 | 唐春艳 | Method for collecting syzygium buxifolium tissue culture initial bud |
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| CN106718938A (en) * | 2017-02-14 | 2017-05-31 | 唐春艳 | A kind of acquisition methods of the initial bud of tree of heaven |
| CN106879463A (en) * | 2017-02-14 | 2017-06-23 | 唐春艳 | Red cone aseptic explant is prepared and initial bud abductive approach |
| CN106888966A (en) * | 2017-02-14 | 2017-06-27 | 唐春艳 | Red skin Qinggang aseptic explant is prepared and initial bud abductive approach |
| CN107616096A (en) * | 2017-11-08 | 2018-01-23 | 南京林业大学 | A kind of aseptic pine seedlings cultural method |
| CN108308030A (en) * | 2018-04-04 | 2018-07-24 | 中国热带农业科学院南亚热带作物研究所 | A method of promoting avocado stem section evoked callus and sterile bud |
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| CN105052742A (en) * | 2015-08-14 | 2015-11-18 | 广西壮族自治区林业科学研究院 | Method for breeding pinus massoniana good group asexual tissue culture seedlings |
| CN105052742B (en) * | 2015-08-14 | 2017-05-10 | 广西壮族自治区林业科学研究院 | Method for breeding pinus massoniana good group asexual tissue culture seedlings |
| CN105393917A (en) * | 2015-11-30 | 2016-03-16 | 北京林业大学 | Rapid propagation method of lonicera oblata |
| CN106665361A (en) * | 2017-02-14 | 2017-05-17 | 唐春艳 | Method for collecting syzygium buxifolium tissue culture initial bud |
| CN106718937A (en) * | 2017-02-14 | 2017-05-31 | 唐春艳 | The method that Qinggang tree obtains aseptic explant preparation and initial bud |
| CN106718938A (en) * | 2017-02-14 | 2017-05-31 | 唐春艳 | A kind of acquisition methods of the initial bud of tree of heaven |
| CN106879463A (en) * | 2017-02-14 | 2017-06-23 | 唐春艳 | Red cone aseptic explant is prepared and initial bud abductive approach |
| CN106888966A (en) * | 2017-02-14 | 2017-06-27 | 唐春艳 | Red skin Qinggang aseptic explant is prepared and initial bud abductive approach |
| CN107616096A (en) * | 2017-11-08 | 2018-01-23 | 南京林业大学 | A kind of aseptic pine seedlings cultural method |
| CN108308030A (en) * | 2018-04-04 | 2018-07-24 | 中国热带农业科学院南亚热带作物研究所 | A method of promoting avocado stem section evoked callus and sterile bud |
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