CN104663455A - Method for establishing aquilaria sinensis tissue culture regeneration system - Google Patents
Method for establishing aquilaria sinensis tissue culture regeneration system Download PDFInfo
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Abstract
The invention discloses a method for establishing an aquilaria sinensis tissue culture regeneration system. Aquilaria sinensis belongs to thymelaeaceae aquilaria perennial aiphyllium, is a precious medicinal tree in China, and has relatively high economic values and biological values of gardens and greening. The method comprises the following steps: by taking an annual terminal bud of aquilaria sinensis as an explant, performing callus induction culture, differentiation, multiplication culture, rooting culture, and acclimatization and transplant, thereby successfully obtaining an aquilaria sinensis in-vitro plant. An aquilaria sinensis tissue culture rapid propagation technique system is established, and a contribution to clone breeding, effective aquilaria sinensis resource protection, tissue culture seedling production acceleration and market requirement meeting can be made.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of method for building up of buta-buta Tissue Culture Regeneration System.
Background technology
Buta-buta (
aquilaria sinensis) also known as suspension culture of Aquilaria sinensis, tabernaemontanus bulrush is fragrant, daughter is fragrant, for Thymelaeceae agalloch eaglewood belongs to perennial evergreen arbor, be China peculiar and the medicinal plant of preciousness and aromatic plant, Ye Shi China produces unique plant resources of agalloch eaglewood.Sporadicly be distributed in the ground such as Guangdong, Guangxi, Fujian, Hainan, Taiwan at present.Buta-buta not only has high medicinal and economic worth, and tree-like attractive in appearance, tree performance is graceful, flourishing, the four seasons are evergreen, is the Landscape Trees that a kind of ornamental value is quite high.In recent years because people ask for the immoderate of agalloch eaglewood, cause buta-buta natural forest to be seriously damaged, natural forest updating ability is weak in addition; domestic buta-buta wild resource is closely exhausted; now only have fragmentary scattered distribution, current resources situation allows of no optimist, and has been listed in national secondary Top-rated protected wild plants.
In recent years, buta-buta forest plantation obtains fast development, but the general seed seedling that adopts is afforested.Because genetic variation is large, output differs greatly.In addition, buta-buta wild select tree ripening rate is low, not easily obtains a large amount of high quality seed, and seed should not be preserved, easy devitalization, makes the process being managed and develop forest plantation by sexual propagation become slow and difficult.Adopt Plant Tissue Breeding propagation technique, maternal merit can be retained to a certain extent, increase reproduction ability and the quantity of breeding, become one of effective measures of protection and breeding rare tree.Therefore, by plant tissue culture technique, the not enough problem of seed effectively can be solved, for buta-buta resource conservation with open up new approach, also for buta-buta Clone Breeding provides a kind of technological means.The present invention gives birth to terminal bud for explant then with buta-buta; the in vitro plant again of buta-buta is have successfully been obtained by processes such as induction of callus, differentiation, Multiplying culture, culture of rootage, acclimatization and transplantses; set up buta-buta tissue culture rapid propagation technique system; for Clone Breeding and available protecting buta-buta resource; acceleration plantlet in vitro is produced, and meets the need of market and contributes.
Summary of the invention
The object of the present invention is to provide out a kind of method for building up of buta-buta Tissue Culture Regeneration System, the present invention gives birth to terminal bud for explant then with buta-buta, the in vitro plant again of buta-buta is have successfully been obtained by processes such as induction of callus, differentiation, Multiplying culture, culture of rootage, acclimatization and transplantses, set up buta-buta tissue culture rapid propagation technique system, thus achieve object of the present invention.
The method for building up of a kind of buta-buta Tissue Culture Regeneration System of the present invention, comprises the following steps:
(1) explant sterilization: choose growth vigorous, healthy and strong, without the terminal bud of 2 years raw Hainan provenance buta-buta nursery stocks of damage by disease and insect.With the cleaning of washing powder water 10 ~ 30min, the running water 30 ~ 60min of 3%.In superclean bench, with 70% ~ 80% alcohol solution dipping 20s ~ 60s, aseptic water washing 4 ~ 6 times, then with 0.1% ~ 0.2% mercuric chloride solution sterilization 5 ~ 15min, then use aseptic water washing explant to non-foam.The explant disinfected is placed in the dish being covered with aseptic filter paper, the moisture on blotting material surface.
(2) callus induction: the terminal bud that step (1) disinfects is cut away the part that end contacts with liquid, then be cut into the segment of about 3.0cm and be inoculated into inducing culture and carry out induction of callus.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 30 days.
(3) differentiation is cultivated: the block of step (2) induce the callus that obtains to take out to be cut into diameter to be about 0.5cm is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up differentiation situation afterwards in 30 days.
(4) Multiplying culture: the indefinite bud that selecting step (3) differentiation cultivation obtains is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% after 30 days to add up proliferative conditions.
(5) culture of rootage: cut from base portion the healthy and strong indefinite bud obtained step (4) Multiplying culture and be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling to move under normal temperature after 4 ~ 5 days, open bottle cap 2 ~ 3 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, after 14 days, completely de-bag, transplants and adds up survival rate after 30 days.
Inducing culture described in above-mentioned steps (2) is: MS+0.05 ~ 0.2mg/L NAA+0.5 ~ 2.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (3) is: MS+0.1 ~ 1.0mg/L 6-BA+0.01 ~ 0.1mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (4) is: MS+0.1 ~ 1.0mg/L KT+0.01 ~ 0.1mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (5) is: 1/2MS+1.0 ~ 2.0mg/L ABT
5+ 0.01 ~ 0.1mg/L NAA+0.1 ~ 1.0mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar+1 ~ 3g/L peptone+50 ~ 100ml/L coconut milk, pH is 5.4 ~ 5.8.
Advantage of the present invention is: buta-buta (
aquilaria sinensis) for Thymelaeceae agalloch eaglewood belongs to perennial evergreen arbor, be the Medicinal species of the peculiar preciousness of China, there is higher economic worth and the ecological value such as gardens, greening.Because of excessive collection agalloch eaglewood, wild buta-buta is tending towards in imminent danger, has been put into national Precious, Rare, Endangered three-level protective plant and national secondary Top-rated protected wild plants register.Therefore, in order to protect buta-buta seeds better, the tissue-culturing rapid propagation system of research and Erecting and improving has important practical significance.The present invention gives birth to terminal bud for explant then with buta-buta; the in vitro plant again of buta-buta is have successfully been obtained by processes such as induction of callus, differentiation, Multiplying culture, culture of rootage, acclimatization and transplantses; set up buta-buta tissue culture rapid propagation technique system; for Clone Breeding and available protecting buta-buta resource; acceleration plantlet in vitro is produced, and meets the need of market and contributes.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1
(1) explant sterilization: choose growth vigorous, healthy and strong, without the terminal bud of 2 years raw Hainan provenance buta-buta nursery stocks of damage by disease and insect.With the cleaning of washing powder water 10min, the running water 30min of 3%.In superclean bench, with 70% alcohol solution dipping 40s, aseptic water washing 6 times, then with 0.1% mercuric chloride solution sterilization 15min, then use aseptic water washing explant to non-foam.The explant disinfected is placed in the dish being covered with aseptic filter paper, the moisture on blotting material surface.
(2) callus induction: the terminal bud that step (1) disinfects is cut away the part that end contacts with liquid, then be cut into the segment of about 3.0cm and be inoculated into inducing culture and carry out induction of callus.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate inductivity after 30 days under the condition of 75% be 86.67%.Described inducing culture is: MS+0.08mg/L NAA+1.2mg/L 6-BA+25g/L sucrose+4.5g/L agar, pH is 5.5.
(3) differentiation is cultivated: the block of step (2) induce the callus that obtains to take out to be cut into diameter to be about 0.5cm is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate differentiation rate after 30 days under the condition of 75% be 90%.Described differential medium is: MS+0.3mg/L 6-BA+0.05mg/LNAA+30g/L sucrose+3.8g/L agar, pH is 5.5.
(4) Multiplying culture: the indefinite bud that selecting step (3) differentiation cultivation obtains is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate growth coefficient after 30 days under the condition of 75% be 10.5.Described proliferated culture medium is: MS+0.8mg/L KT+0.06mg/LNAA+18g/L sucrose+4.5g/L agar, pH is 5.5.
(5) culture of rootage: cut from base portion the healthy and strong indefinite bud obtained step (4) Multiplying culture and be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that under the condition of 75%, cultivation 30 days rooting rates are 73.4%.Described root media is: 1/2MS+1.3mg/L ABT
5+ 0.05mg/L NAA+0.7mg/L IBA+20g/L sucrose+4.7g/L agar+1.5g/L peptone+70ml/L coconut milk, pH is 5.5.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling moves to normal temperature after lower 4 days, open bottle cap 2 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplanting survival rate after 30 days is 87.4%.
Embodiment 2
(1) explant sterilization: choose growth vigorous, healthy and strong, without the terminal bud of 2 years raw Hainan provenance buta-buta nursery stocks of damage by disease and insect.With the cleaning of washing powder water 15min, the running water 50min of 3%.In superclean bench, with 75% alcohol solution dipping 30s, aseptic water washing 6 times, then with 0.1% mercuric chloride solution sterilization 10min, then use aseptic water washing explant to non-foam.The explant disinfected is placed in the dish being covered with aseptic filter paper, the moisture on blotting material surface.
(2) callus induction: the terminal bud that step (1) disinfects is cut away the part that end contacts with liquid, then be cut into the segment of about 3.0cm and be inoculated into inducing culture and carry out induction of callus.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 1500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate inductivity after 30 days under the condition of 75% be 83.12%.Described inducing culture is: MS+0.1mg/L NAA+1.5mg/L 6-BA+30g/L sucrose+5.5g/L agar, pH is 5.7.
(3) differentiation is cultivated: the block of step (2) induce the callus that obtains to take out to be cut into diameter to be about 0.5cm is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate differentiation rate after 30 days under the condition of 75% be 86.2%.Described differential medium is: MS+0.5mg/L 6-BA+0.1mg/LNAA+30g/L sucrose+4.8g/L agar, pH is 5.7.
(4) Multiplying culture: the indefinite bud that selecting step (3) differentiation cultivation obtains is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate growth coefficient after 30 days under the condition of 75% be 8.3.Described proliferated culture medium is: MS+1.0mg/L KT+0.03mg/LNAA+25g/L sucrose+4.5g/L agar, pH is 5.7.
(5) culture of rootage: cut from base portion the healthy and strong indefinite bud obtained step (4) Multiplying culture and be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 2500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that under the condition of 75%, cultivation 30 days rooting rates are 68.9%.Described root media is: 1/2MS+1.5mg/L ABT
5+ 0.06mg/L NAA+1.0mg/L IBA+20g/L sucrose+5.0g/L agar+2.0g/L peptone+100ml/L coconut milk, pH is 5.7.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling moves to normal temperature after lower 4 days, open bottle cap 2 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplanting survival rate after 30 days is 83.9%.
Claims (5)
1. a method for building up for buta-buta Tissue Culture Regeneration System, is characterized in that comprising the following steps:
(1) explant sterilization: choose growth vigorous, healthy and strong, without the terminal bud of 2 years raw Hainan provenance buta-buta nursery stocks of damage by disease and insect; with the washing powder water cleaning 10 ~ 30min of 3%; running water 30 ~ 60min; in superclean bench; with 70% ~ 80% alcohol solution dipping 20s ~ 60s; aseptic water washing 4 ~ 6 times; again with 0.1% ~ 0.2% mercuric chloride solution sterilization 5 ~ 15min; then use aseptic water washing explant to non-foam; the explant disinfected is placed in the dish being covered with aseptic filter paper, the moisture on blotting material surface;
(2) callus induction: the terminal bud that step (1) disinfects is cut away the part that end contacts with liquid; be cut into the segment of about 3.0cm again and be inoculated into inducing culture and carry out induction of callus; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 30 days;
(3) differentiation is cultivated: the block of step (2) induce the callus that obtains to take out to be cut into diameter to be about 0.5cm is seeded to differential medium and carries out differentiation cultivation; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up differentiation situation afterwards in 30 days;
(4) Multiplying culture: the indefinite bud that selecting step (3) differentiation cultivation obtains is seeded to proliferated culture medium and carries out squamous subculture; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% after 30 days to add up proliferative conditions;
(5) culture of rootage: cut from base portion the healthy and strong indefinite bud obtained step (4) Multiplying culture and be seeded in root media and carry out root induction; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days;
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling to move under normal temperature after 4 ~ 5 days, open bottle cap 2 ~ 3 days, then the medium being attached to shoot root and fastening is washed away, transplant to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplant and with 0.3% potassium permanganate solution, matrix spray is sterilized and added a cover film for first 5 days, film is opened after 3 days, stir matrix, transplant water of matrix being drenched for first 1 day, need by matrix compacting during transplanting, and carry out individual plant bagging, sooner or later spraying, guarantee growing environment is moist, transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplant and add up survival rate after 30 days.
2. the method for building up of a kind of buta-buta Tissue Culture Regeneration System according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+0.05 ~ 0.2mg/L NAA+0.5 ~ 2.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. the method for building up of a kind of buta-buta Tissue Culture Regeneration System according to claim 1, it is characterized in that the differential medium described in step (3) is: MS+0.1 ~ 1.0mg/L 6-BA+0.01 ~ 0.1mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. the method for building up of a kind of buta-buta Tissue Culture Regeneration System according to claim 1, it is characterized in that the proliferated culture medium described in step (4) is: MS+0.1 ~ 1.0mg/L KT+0.01 ~ 0.1mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
5. the method for building up of a kind of buta-buta Tissue Culture Regeneration System according to claim 1, is characterized in that the root media described in step (5) is: 1/2MS+1.0 ~ 2.0mg/L ABT
5+ 0.01 ~ 0.1mg/L NAA+0.1 ~ 1.0mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar+1 ~ 3g/L peptone+50 ~ 100ml/L coconut milk, pH is 5.4 ~ 5.8.
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| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN105010142A (en) * | 2015-07-14 | 2015-11-04 | 中国林业科学研究院热带林业研究所 | Vietnamese Aquilaria agallocha Roxb tissue culture method |
| CN107517743A (en) * | 2017-09-16 | 2017-12-29 | 韦志雄 | A kind of process of the exposed grafting of agalloch eaglewood chess nanmu tree |
| CN107691225A (en) * | 2017-11-14 | 2018-02-16 | 广西壮族自治区药用植物园 | The rapid propagation method of buta-buta callus seedling |
| CN111149674A (en) * | 2020-02-28 | 2020-05-15 | 科稷达隆生物技术有限公司 | Culture method for improving transplanting survival rate of plant tissue culture seedlings |
| CN114831026A (en) * | 2022-05-23 | 2022-08-02 | 中国热带农业科学院热带作物品种资源研究所 | Method for efficiently propagating agilawood tissue culture seedlings by utilizing sterile micro-cutting rooting |
| CN116584392A (en) * | 2023-06-09 | 2023-08-15 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN105010142A (en) * | 2015-07-14 | 2015-11-04 | 中国林业科学研究院热带林业研究所 | Vietnamese Aquilaria agallocha Roxb tissue culture method |
| CN107517743A (en) * | 2017-09-16 | 2017-12-29 | 韦志雄 | A kind of process of the exposed grafting of agalloch eaglewood chess nanmu tree |
| CN107691225A (en) * | 2017-11-14 | 2018-02-16 | 广西壮族自治区药用植物园 | The rapid propagation method of buta-buta callus seedling |
| CN111149674A (en) * | 2020-02-28 | 2020-05-15 | 科稷达隆生物技术有限公司 | Culture method for improving transplanting survival rate of plant tissue culture seedlings |
| CN114831026A (en) * | 2022-05-23 | 2022-08-02 | 中国热带农业科学院热带作物品种资源研究所 | Method for efficiently propagating agilawood tissue culture seedlings by utilizing sterile micro-cutting rooting |
| CN114831026B (en) * | 2022-05-23 | 2023-06-16 | 中国热带农业科学院热带作物品种资源研究所 | Method for efficiently propagating agilawood tissue culture seedlings by utilizing sterile micro-cuttage rooting |
| CN116584392A (en) * | 2023-06-09 | 2023-08-15 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
| CN116584392B (en) * | 2023-06-09 | 2023-12-08 | 中国林业科学研究院热带林业研究所 | Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil |
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Application publication date: 20150603 |