CN105379627A - Sequoia sempervirens in vitro rooting culture method - Google Patents
Sequoia sempervirens in vitro rooting culture method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to a sequoia sempervirens in vitro rooting culture method. The culture method comprises the steps of selecting a current-year sequoia sempervirens branch as a material, through disinfecting and washing, growing the branch into an aseptic seedling on an MS (Murashige and Skoog) medium, then using a terminal bud or an axillary bud as an explant and performing multiplication culture on the explant in a multiplying culture medium, performing rooting culture on the multiplied aseptic seedling, wherein a rooting medium is 1/4MS plus 0.1 to 1.0mg/L indolebutyric acid plus 30g/L saccharose plus 7g/L agar powder, and finally, when 3 to 6 strips of roots grow out from a base part, and the average value of main roots reaches 4.5 to 6cm, finally performing hardening-seedling and transplanting on a tissue culture seedling with the root system length being 3 to 6cm. According to the culture method provided by the invention, the steps are easy to operate, by bringing the step of seedling strengthening in sequoia sempervirens in vitro rooting ahead of rooting culture, the rate of survival of the tissue culture seedling after transplanting is effectively improved, and the rooting medium is improved, so the rooting efficiency during sequoia sempervirens in vitro culture can be accelerated.
Description
Technical field
The present invention relates to a kind of sequoia sempervirens isolated rooting culture method, belong to China fir and cultivate propagation technique field.
Background technology
Sequoia sempervirens originates in California, United States state seashore, introduces a fine variety to Hangzhou China when being visited China by Nixon the earliest.Sequoia sempervirens belongs to especially big arbor, can up to 110 meters in original producton location.Sequoia sempervirens tree performance is grand, the close life of branches and leaves, and growth rapidly.Be applicable to isolated planting or mass-planting in lakeside, waterside, lawn, view is beautiful, the avenue system on both sides of the road of Ye Keyan garden, and grandeur is very excellent Landscape Tree Species.
But because sequoia sempervirens cuttage survival rate is low, method for tissue culture is taken root the rare report of situation of stage difficulty, domestic breeding, therefore overcomes sequoia sempervirens Cultivating techniques difficulty, improves cultured in vitro efficiency, significant to enriching of Chinese ornamental plant.
Summary of the invention
The object of the invention is to solve sequoia sempervirens cuttage survival rate low, tissue cultures is taken root stage more difficult problem, provides that a kind of method is easy is easy to operation, and rooting rate is very fast, the sequoia sempervirens isolated rooting culture method that survival rate is higher.
The present invention adopts following technical scheme: a kind of sequoia sempervirens isolated rooting culture method, comprises the steps:
(1) acquisition of sterilizable material: choosing sequoia sempervirens branch raw is then material, be inoculated in MS medium blot excessive moisture with aseptic filter paper after sterilization, cleaning after, after 20 ~ 22 days, axillalry bud grows aseptic seedling;
(2) Multiplying culture: using aseptic seedling as material, cut terminal bud or axillalry bud as explant, be seeded on proliferated culture medium, described proliferated culture medium be MS+0.1 ~ 1.0mg/L6-benzyl aminoadenine+0.1 ~ 1.0mg/L methyl α-naphthyl acetate+30g/L sucrose+7g/L agar powder, pH5.5 ~ 6, cultivate 25 ~ 30 day time;
(3) in strong sprout: after the aseptic seedling of propagation being cut, move into strong seedling culture base, strong seedling culture base is MS+0.1 ~ 0.4 methyl α-naphthyl acetate, PH5.5 ~ 6, and cultivate 2 ~ 3 weeks, temperature is 16 ~ 25 DEG C;
(4) preparation of root media: the macroelement in MS medium, trace element, inositol, organic consumption are reduced to 1/4 of conventional amount used respectively, is mixed with 1/4MS medium;
(5) culture of rootage: by the tissue culture plant inoculation after strong sprout on root media, root media is 1/4MS+0.1 ~ 1.0mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder, and pH5.5 ~ 6, after 12 ~ 16 days, base portion grows 3 ~ 6 main roots, and main root length average reaches 4.5 ~ 6cm;
(6) hardening and transplanting: will highly at 2 ~ 5cm, root length carries out hardening at the plantlet in vitro of 3 ~ 6cm, open bottle cap 3 ~ 7 days, it is made to adapt to external environment, wash the root media of root after 3 ~ 7 days off, soak 1 ~ 10min with the carbendazim of 800 ~ 1000 times, transplant in booth, overlay film moisturizing, appropriate shading.
Further, in described step (4), the condition of culture temperature of root media is 20 ~ 24 DEG C.
Further, sterilization in described step (1), cleaning method are: with the alcohol disinfecting 10 ~ 40s of 70 ~ 75% on superclean bench, with aseptic water washing 3 ~ 5 times, then to sterilize 10 ~ 30min with liquor natrii hypochloritis, with aseptic water washing 4 ~ 6 times.
Further, described macroelement is potassium nitrate, ammonium nitrate, epsom salt, potassium dihydrogen phosphate and calcium chloride dihydrate.
Further, described trace element is four water manganese sulphates, white vitriol, boric acid, potassium iodide, seven water sodium molybdates, cupric sulfate pentahydrate and CoCL2 6H2O.
Further, described organic matter is glycine, puridoxine hydrochloride, Tyiamine Hd element and nicotinic acid.
Cultural method of the present invention is simple, step is easy to operation, before the step in strong sprout advances to culture of rootage in sequoia sempervirens off-body kidney, effectively raises the survival rate after plantlet in vitro transplanting, root media is improved, the efficiency of taking root during sequoia sempervirens cultured in vitro can be accelerated.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment one: a kind of sequoia sempervirens isolated rooting culture method, comprises the steps:
(1) acquisition of sterilizable material: choosing sequoia sempervirens branch raw is then material, with the alcohol disinfecting 20s of 75% on superclean bench, with aseptic water washing 3 times, then to sterilize 10min with liquor natrii hypochloritis, with aseptic water washing 4 times, then be inoculated into after blotting excessive moisture with aseptic filter paper in MS medium, after 20 days, axillalry bud grows aseptic seedling;
(2) Multiplying culture: using aseptic seedling as material, cut terminal bud or axillalry bud as explant, be seeded on proliferated culture medium, proliferated culture medium be MS+0.1mg/L6-benzyl aminoadenine+0.1mg/L methyl α-naphthyl acetate+30g/L sucrose+7g/L agar powder, pH5.5, cultivate 30 day time, temperature is at 16 DEG C;
(3) in strong sprout: after the aseptic seedling of propagation being cut, move into strong seedling culture base, strong seedling culture base is MS+0.1 methyl α-naphthyl acetate, PH6, cultivates 2 weeks, temperature 16 DEG C.
(4) preparation of root media: by macroelement potassium nitrate, ammonium nitrate, epsom salt, potassium dihydrogen phosphate and calcium chloride dihydrate in MS medium, trace element four water manganese sulphates, white vitriol, boric acid, potassium iodide, seven water sodium molybdates, cupric sulfate pentahydrate and CoCL2 6H2O, the consumption of inositol, organic matter glycine, puridoxine hydrochloride, Tyiamine Hd element and nicotinic acid reduces to 1/4 of conventional amount used respectively, the 1/4MS medium after composition improvement;
(5) culture of rootage: by the tissue culture plant inoculation after strong sprout in step (3) on root media, root media is 1/4MS+0.1mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder, pH5.5, the temperature of culture of rootage is 20 DEG C, and after 16 days, base portion grows 3 main roots;
(6) hardening and transplanting: will highly at 2cm, root length carries out hardening at the plantlet in vitro of 3cm, opens bottle cap 3 days, make it adapt to external environment, after 3 days, wash the root media of root off, soak 10min with the carbendazim of 800 times, transplant in booth, overlay film moisturizing, appropriate shading.
Embodiment two: a kind of sequoia sempervirens isolated rooting culture method, comprises the steps:
(1) acquisition of sterilizable material: choosing sequoia sempervirens branch raw is then material, with the alcohol disinfecting 40s of 75% on superclean bench, with aseptic water washing 4 times, then to sterilize 20min with liquor natrii hypochloritis, with aseptic water washing 5 times, then aseptic filter paper is inoculated into after blotting excessive moisture in MS medium, and after 21 days, axillalry bud grows aseptic seedling;
(2) Multiplying culture: using aseptic seedling as material, cut terminal bud or axillalry bud as explant, be seeded on proliferated culture medium, described proliferated culture medium be MS+1.0mg/L6-benzyl aminoadenine+1.0mg/L methyl α-naphthyl acetate+30g/L sucrose+7g/L agar powder, pH5.8, cultivate 28 day time, temperature is at 20 DEG C;
(3) in strong sprout: after the aseptic seedling of propagation being cut, move into strong seedling culture base, strong seedling culture base is MS+0.4 methyl α-naphthyl acetate, PH5.8, cultivates 3 weeks, temperature 25 DEG C;
(4) preparation of root media: by macroelement potassium nitrate, ammonium nitrate, epsom salt, potassium dihydrogen phosphate and calcium chloride dihydrate in MS medium, trace element four water manganese sulphates, white vitriol, boric acid, potassium iodide, seven water sodium molybdates, cupric sulfate pentahydrate and CoCL2 6H2O, the consumption of inositol, organic matter glycine, puridoxine hydrochloride, Tyiamine Hd element and nicotinic acid reduces to 1/4 of conventional amount used respectively, the 1/4MS medium after composition improvement;
(5) culture of rootage: by the tissue culture plant inoculation after strong sprout in step (3) on root media, macroelement in MS medium, trace element, inositol, organic consumption are kept to 1/4 of original specified volume respectively, 1/4MS medium after composition improvement, root media is 1/4MS+1.0mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder, pH5.8, the temperature of culture of rootage is 22 DEG C, and after 15 days, base portion grows 6 main roots;
(6) hardening and transplanting: will highly at 5cm, root length carries out hardening at the plantlet in vitro of 6cm, opens bottle cap 7 days, make it adapt to external environment, after 7 days, wash the root media of root off, soak 10min with the carbendazim of 800 times, transplant in booth, overlay film moisturizing, appropriate shading.
Embodiment three: a kind of sequoia sempervirens isolated rooting culture method, comprises the steps:
(1) acquisition of sterilizable material: choosing sequoia sempervirens branch raw is then material, with the alcohol disinfecting 10s of 70% on superclean bench, with aseptic water washing 5 times, then to sterilize 30min with liquor natrii hypochloritis, with aseptic water washing 6 times, then be inoculated into after blotting excessive moisture with aseptic filter paper in MS medium, after 22 days, axillalry bud grows aseptic seedling;
(2) Multiplying culture: using aseptic seedling as material, cut terminal bud or axillalry bud as explant, be seeded on proliferated culture medium, described proliferated culture medium be MS+0.5mg/L6-benzyl aminoadenine+0.15mg/L methyl α-naphthyl acetate+30g/L sucrose+7g/L agar powder, pH6, cultivate 25 day time, temperature is at 18 DEG C;
(3) in strong sprout: after the aseptic seedling of propagation being cut, move into strong seedling culture base, strong seedling culture base is MS+0.34 methyl α-naphthyl acetate, PH5.5, cultivates 3 weeks, temperature 17 DEG C;
(4) preparation of root media: by macroelement potassium nitrate, ammonium nitrate, epsom salt, potassium dihydrogen phosphate and calcium chloride dihydrate in MS medium, trace element four water manganese sulphates, white vitriol, boric acid, potassium iodide, seven water sodium molybdates, cupric sulfate pentahydrate and CoCL2 6H2O, the consumption of inositol, organic matter glycine, puridoxine hydrochloride, Tyiamine Hd element and nicotinic acid reduces to 1/4 of conventional amount used respectively, the 1/4MS medium after composition improvement;
(5) culture of rootage: by the tissue culture plant inoculation after strong sprout in step (3) on root media, macroelement in MS medium, trace element, inositol, organic consumption are kept to 1/4 of original specified volume respectively, 1/4MS medium after composition improvement, root media is 1/4MS+0.8mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder, pH5.5, after 16 days, base portion grows 5 main roots;
(6) hardening and transplanting: will highly at 4cm, root length carries out hardening at the plantlet in vitro of 3cm, opens bottle cap 4 days, make it adapt to external environment, after 4 days, wash the root media of root off, soak 4min with the carbendazim of 1000 times, transplant in booth, overlay film moisturizing, appropriate shading.
Adopt the isolated rooting culture method shown in embodiment two sequoia sempervirens is cultivated, adopt different types of root media, the rootage duration of sequoia sempervirens and the result of rooting rate as shown in table 1.
Table 1
As seen from the results in Table 1, adopt different root medias, the rootage duration of sequoia sempervirens and rooting rate have larger difference, when adopting the root media of 1/2MS+0.1 indolebutyric acid, the longest needs of rootage duration 2 months, and rooting rate is only 10%, adopt the root media 1/4MS+1.0mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder after improvement when cultivating in embodiment two, rootage duration shortens greatly only needs 21 days and rooting rate reaches 93%.
Claims (6)
1. a sequoia sempervirens isolated rooting culture method, is characterized in that: comprise the steps:
(1) acquisition of sterilizable material: choosing sequoia sempervirens branch raw is then material, be inoculated in MS medium blot excessive moisture with aseptic filter paper after sterilization, cleaning after, after 20 ~ 22 days, axillalry bud grows aseptic seedling;
(2) Multiplying culture: using aseptic seedling as material, cut terminal bud or axillalry bud as explant, be seeded on proliferated culture medium, described proliferated culture medium be MS+0.1 ~ 1.0mg/L6-benzyl aminoadenine+0.1 ~ 1.0mg/L methyl α-naphthyl acetate+30g/L sucrose+7g/L agar powder, pH5.5 ~ 6, cultivate 25 ~ 30 day time;
(3) in strong sprout: after the aseptic seedling of propagation being cut, move into strong seedling culture base, strong seedling culture base is MS+0.1 ~ 0.4 methyl α-naphthyl acetate, pH5.5 ~ 6, and cultivate 2 ~ 3 weeks, temperature is 16 ~ 25 DEG C;
(4) preparation of root media: the macroelement in MS medium, trace element, inositol, organic consumption are reduced to 1/4 of conventional amount used respectively, is mixed with 1/4MS medium;
(5) culture of rootage: by the tissue culture plant inoculation after strong sprout on root media, root media is 1/4MS+0.1 ~ 1.0mg/L indolebutyric acid+30g/L sucrose+7g/L agar powder, and pH5.5 ~ 6, after 12 ~ 16 days, base portion grows 3 ~ 6 main roots, and main root length average reaches 4.5 ~ 6cm;
(6) hardening and transplanting: will highly at 2 ~ 5cm, root length carries out hardening at the plantlet in vitro of 3 ~ 6cm, open bottle cap 3 ~ 7 days, it is made to adapt to external environment, wash the root media of root after 3 ~ 7 days off, soak 1 ~ 10min with the carbendazim of 800 ~ 1000 times, transplant in booth, overlay film moisturizing, appropriate shading.
2. sequoia sempervirens isolated rooting culture method as claimed in claim 1, is characterized in that: in described step (4), the condition of culture temperature of root media is 20 ~ 24 DEG C.
3. sequoia sempervirens isolated rooting culture method as claimed in claim 1, it is characterized in that: the sterilization in described step (1), cleaning method are: with the alcohol disinfecting 10 ~ 40s of 70 ~ 75% on superclean bench, with aseptic water washing 3 ~ 5 times, then to sterilize 10 ~ 30min with liquor natrii hypochloritis, with aseptic water washing 4 ~ 6 times.
4. sequoia sempervirens isolated rooting culture method as claimed in claim 1, is characterized in that: described macroelement is potassium nitrate, ammonium nitrate, epsom salt, potassium dihydrogen phosphate and calcium chloride dihydrate.
5. sequoia sempervirens isolated rooting culture method as claimed in claim 1, is characterized in that: described trace element is four water manganese sulphates, white vitriol, boric acid, potassium iodide, seven water sodium molybdates, cupric sulfate pentahydrate and CoCL2 6H2O.
6. sequoia sempervirens isolated rooting culture method as claimed in claim 1, is characterized in that: described organic matter is glycine, puridoxine hydrochloride, Tyiamine Hd element and nicotinic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106359093A (en) * | 2016-08-31 | 2017-02-01 | 李军 | Establishing method for redwood tissue culture rapid propagation system |
| CN107996409A (en) * | 2018-01-25 | 2018-05-08 | 上海为绿景观建设有限公司 | A kind of stem apex sterilization method and sequoia sempervirens cultural method |
| CN115152352A (en) * | 2022-07-25 | 2022-10-11 | 黑龙江省农业科学院大庆分院 | Industrial hemp sterile seedling breeding method |
| CN117617124A (en) * | 2024-01-23 | 2024-03-01 | 云南林业职业技术学院 | North American sequoia tissue culture rapid propagation method and application |
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| CN102613083A (en) * | 2012-04-08 | 2012-08-01 | 徐州工程学院 | North American redwood tissue cultivation method |
| CN103070077A (en) * | 2013-02-05 | 2013-05-01 | 石林红杉农林科技开发有限公司 | Method for obtaining sequoia sempervirens seedlings by cell mass culture combined with tissue culture |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102613083A (en) * | 2012-04-08 | 2012-08-01 | 徐州工程学院 | North American redwood tissue cultivation method |
| CN103070077A (en) * | 2013-02-05 | 2013-05-01 | 石林红杉农林科技开发有限公司 | Method for obtaining sequoia sempervirens seedlings by cell mass culture combined with tissue culture |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106359093A (en) * | 2016-08-31 | 2017-02-01 | 李军 | Establishing method for redwood tissue culture rapid propagation system |
| CN107996409A (en) * | 2018-01-25 | 2018-05-08 | 上海为绿景观建设有限公司 | A kind of stem apex sterilization method and sequoia sempervirens cultural method |
| CN115152352A (en) * | 2022-07-25 | 2022-10-11 | 黑龙江省农业科学院大庆分院 | Industrial hemp sterile seedling breeding method |
| CN117617124A (en) * | 2024-01-23 | 2024-03-01 | 云南林业职业技术学院 | North American sequoia tissue culture rapid propagation method and application |
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