CN101595824A - Method with almug seed embryo rapid in-vitro seedling raising - Google Patents
Method with almug seed embryo rapid in-vitro seedling raising Download PDFInfo
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Abstract
With the method for almug seed embryo rapid in-vitro seedling raising, relate to the method for plant regeneration, specifically be method with almug seed embryo cultured in vitro fast seedling growing.It is to get the santal embryo to place on the synthetic medium, at 25 ± 2 ℃, aseptic dark culturing 10 days, increased illumination 16 hours on the 11st day, dark 8 hours, intensity of illumination is 2000lx, cultivated 10 days, again with the transplantation of seedlings of sprouting to sandy soil, 26 ± 2 ℃ of temperature, relative moisture 75 ± 5%, intensity of illumination be 1500～2000lx, illumination cultivated under the condition in 12 hours more than 30 days seedling, said synthetic medium is to add in the MS medium of 1L in the MS medium of the gibberellin of 2～6mg or 1L to add 2.0mg 6-benzylaminopurine.Beneficial effect of the present invention: shortened seedling raise period; The planting percent height, planting percent can reach 87%; Seedling germinates neat, is convenient to the large-scale production operation; Cost is low.
Description
Technical field
The present invention relates to the method for plant regeneration, specifically is the method for educating with the quick seedling of almug seed embryo cultured in vitro.
Technical background
Almug (Santalum album L.) is the Santalaceae aiphyllium, originates in India, Indonesia and Malaysia, and very high medical value and economic worth are arranged.Its trunk heartwood contains abundant volatile oil, is famous and precious traditional Chinese medicine, mainly contains the effect of warming middle-JIAO for easing the stomach, analgesic hemostatic, and the contained volatile oil purposes on medical industry of heartwood is very extensive.In addition, santal still is the valuable raw material during daily cosmetics is produced.Santal is an evergreen plant, also as the green tree species of subtropical zone, therefore, widelys popularize the santal plantation and is significant for solving domestic santal resource scarcity.
What the popularizing planting almug need solve is the source of seedling, the santal sapling is mainly by seminal propagation at present, owing to behind the santal seed maturity there is certain resting stage, the sand that needs half a year to one year before the sowing is hidden, sprout into seedling from being seeded into, generally need 2～3 months, and percentage of seedgermination is low, it is irregular to emerge, and is a big obstacle of santal seedling breeding.
Document " generation of santal somatic embryo and the research of plant regeneration " (food and medicine, 2008,10 (1): 35-37) reported the production of using modern biotechnology research and development almug seedling, its method is: the blade of using almug is at additional 1.1 μ mol/L TDZ (thidiazuron, N-phenyl-N '-1,2,3-thiadiazoles-5-urea) MS medium (Murashige and Skoog, 1962) the upward generation of inductor cell stage, again the somatic embryo that generates is transferred on the MS medium of gibberellin of additional 1.4 μ mol/L and cultivated, make it to develop into seedling.The cycle that this method is cultivated is long, cultivates from somatic embryo inducement to begin to need 3～4 months time to the aseptic seedling generation; The aseptic seedling that generates does not have root, and the sandalwood seedling of gained does not become seedling, can not apply.
CN101213936A discloses the method for the tissue culture quick breeding seedling of a kind of santal, but it is to carry out culture in vitro with the tender shoots of the almug more than 10 years, and its method needs four steps: the one, induce the cultivation that grows indefinite bud; The 2nd, clump bud breeding is cultivated; The 3rd, culture of rootage; The 4th, transplant.Complex steps becomes the seedling time long, and cost is high and in actual applications, success rate is very low, only is in theoretical research stage, is further improved on the method.
Therefore, seek a kind of practical technique fast culture santal sapling and have important and practical meanings for santal plant husbandry.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can be applied to produce in batches the method for almug seedling fast breeding.
To achieve the object of the present invention, the technical scheme that is adopted is: with the method for almug seed embryo rapid in-vitro seedling raising, it is to get the santal embryo to place on the synthetic medium, at 25 ± 2 ℃, aseptic dark culturing 10 days, increased illumination 16 hours on the 11st day, dark 8 hours, intensity of illumination is 2000lx, cultivated 10 days, again with the transplantation of seedlings of sprouting to sandy soil, 26 ± 2 ℃ of temperature, relative moisture 75 ± 5%, intensity of illumination is 1500～2000lx, cultivate under 12 hours conditions of illumination more than 30 days seedling, seedling can grow up to 15～20 centimeters high, and the sapling of well developed root system can be transplanted to the land for growing field crops.Said synthetic medium is to add in the MS medium of 1L in the MS medium of the gibberellin of 2～6mg or 1L to add 1.0～2.0mg 6-benzylaminopurine.
For germination rate and the survival rate that improves embryo, will remove the pericarp of meat as the ripening fruits of the almug of cultured in vitro, after cleaning, with concentration expressed in percentage by weight 0.1% HgCl
2Solution soaking 8～15 minutes, or 5～10% aqueous sodium hypochlorite solution soaked 15～20 minutes, or soaked 20～60 minutes with 0.5～1.0% liquor potassic permanganate, soak the back and take out the seed aseptic water washing, standby behind the removal moisture.
Beneficial effect of the present invention:
1. shortened seedling raise period: grow up to 15～20 centimeters high from beginning to be cultured to, can transplant the seedling in land for growing field crops, only need 50～60 days with embryo;
2. planting percent height, planting percent can reach 87%;
3. seedling germinates neatly, is convenient to the large-scale production operation;
4. cost is lower than tissue culture.
Further set forth technical scheme of the present invention below by embodiment
Embodiment
Embodiment 1 almug seed embryo exsomatizes and grows seedlings
1. materials and methods:
1.1 seed collection: between October～December, gather the age of tree and be about almug maturation (atropurpureus) fruit in 10 years, remove outer atropurpureus meat pericarp after, clean with clear water, dry standby.
The preparation of synthetic medium: press document Narayanaswami S, Norstog K.Plant embryo culture.Bot Rev[J], 1964,30:587-628 preparation MS medium, autoclaving is cooled to 50 ℃ after (0.1MPa, 121 ℃, 15 minutes), press, the MS medium of every 1L adds 4.8mg gibberellin.
1.2 embryo cultured in vitro: above-mentioned santal seed is divested red sclerotin mesocarp, take out seed, with concentration expressed in percentage by weight is after 10% aqueous sodium hypochlorite solution soaks 15 minutes, with aseptic water washing 5 times, blot surperficial excess moisture, under aseptic condition, be inoculated in (3 embryos of every bottle graft kind) on the synthetic medium, at 25 ± 1 ℃, dark culturing.Observed once record sprouting situation in per 5 days; Changed illumination cultivation (illumination 16 hours every days, 8 hours dark, intensity of illumination are 2000lx) on the 11st day over to, cultivate after 10 days most of embryo and all sprout, or grow up to seedling, can take out and transplant to sandy soil with 1～2 pair of leaf, 26 ± 2 ℃ of temperature, relative moisture 70～80% was cultivated 30 days under 12 hours the condition of how many illuminance illumination, the sandalwood seedling robust growth, well developed root system, height of seedling reach 15～20 centimeters, can become the cultivation and production seedling, add up the number of seedling amount simultaneously, calculate planting percent.
The results are shown in Table 1.
Result of the test:
Table 1 santal embryo culture in vitro becomes growth of cereal crop seedlings condition
Germination rate calculates: embryo quantity * 100% of the embryo quantity/inoculation of germination rate=sprouting
Planting percent calculates: embryo quantity * 100% of quantity/inoculation of planting percent=Cheng Miao
Embodiment 2 almug seed embryos grow seedlings with the medium of different hormone concentrations is stripped
1. materials and methods:
1.1 seed collection: with embodiment 1
Culture medium preparation: after embodiment 1 same procedure preparation MS medium and sterilization, MS medium interpolation 2.0mg or the preparation of 6.0mg gibberellin by every 1L are respectively experimental group 1,2.
1.2 embryo cultured in vitro:
Method is with embodiment 1.The seed of peelling off skin is with 0.1% mercuric chloride (HgCl
2) solution soaking 10 minutes.Processing method after the seed disinfection is with embodiment 1
Result: see Table 2
Table 2 santal embryo culture in vitro becomes growth of cereal crop seedlings condition
Embodiment 3 almug seed embryos exsomatize with other hormone culture-medium and grow seedlings
1. materials and methods:
1.1 seed collection: with embodiment 1
Culture medium preparation: with embodiment 1 same procedure preparation MS medium, press the consumption interpolation 6-benzylaminopurine (6-BA) of 2.0mg/L after, autoclaving (0.1MPa, 121 ℃, 15 minutes) again.
1.2 embryo cultured in vitro:
Method is with embodiment 1.
The results are shown in Table 3
Table 3 santal embryo is containing the one-tenth growth of cereal crop seedlings condition of cultivating on the synthetic medium of 6-BA
Embodiment 4, and seed compares with the seedling growth test of different sterilization methods
Materials and methods:
1.1 seed collection: with embodiment 1
Culture medium preparation: method is with embodiment 1
1.2 embryo cultured in vitro: method is with embodiment 1.Peel off the carrying out disinfection with following different disinfectant respectively of seed embryo of sclerderm: the A liquor natrii hypochloritis; The B mercuric chloride solution; The C liquor potassic permanganate; Processing method after the embryo sterilization and condition of culture are with embodiment 1.Result of the test sees Table 4
Table 4 adopts the result that grows seedlings of different sterilization methods
The calculating of pollution rate:
Bottle number * 100% of the bottle number/inoculation of pollution rate=sprouting
Planting percent calculates:
Embryo quantity * 100% of quantity/inoculation of planting percent=Cheng Miao
By above test as seen, it is feasible that the method for using almug seed embryo cultured in vitro carries out that santal grows seedlings, and can produce in batches.In the method, on the synthetic medium that adds gibberellin and 6-BA, carry out santal embryo cultured in vitro and can both successfully obtain to grow complete santal seedling.Wherein, add gibberellin the sprouting of santal embryo and the formation of seedling are all had obvious facilitation, application this method is carried out santal and is grown seedlings, from inoculation santal embryo, through 50～60 days, can obtain highly was 15～20 centimeters healthy and strong santal seedling, planting percent is 72～87%, compare with traditional seedling-cultivating method, shortened growing-seedling period greatly, thereby also shortened the production cycle of almug.
Claims (2)
1. method with almug seed embryo rapid in-vitro seedling raising, it is characterized in that: get the santal embryo and place on the synthetic medium, at 25 ± 2 ℃, aseptic dark culturing 10 days, increased illumination 16 hours on the 11st day, dark 8 hours, intensity of illumination is 2000lx, cultivated 10 days, again with the transplantation of seedlings of sprouting to sandy soil, 26 ± 2 ℃ of temperature, relative moisture 75 ± 5%, intensity of illumination is 1500～2000lx, cultivate under 12 hours conditions of illumination more than 30 days seedling, said synthetic medium is to add gibberellin 2～6mg or 6-benzylaminopurine 1.0～2.0mg in the MS medium of 1L.
2. according to the said method of claim 1, it is characterized in that: before getting the seed embryo of almug be 0.1% HgCl the seed concentration expressed in percentage by weight of almug with almug seed embryo rapid in-vitro seedling raising
2Aqueous solution soaking 5～15 minutes, or be that 5～15% aqueous sodium hypochlorite solution soaked 5～20 minutes with concentration expressed in percentage by weight, or be that 0.5～1.0% liquor potassic permanganate soaked 20～60 minutes with concentration expressed in percentage by weight, soak the back and take out the seed aseptic water washing, standby behind the removal moisture.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983557A (en) * | 2010-10-19 | 2011-03-09 | 廖俊杰 | In vitro quick breeding method of seedling stem of santal seed embryo |
| CN103039232A (en) * | 2012-12-18 | 2013-04-17 | 中国科学院华南植物园 | Industrial sandalwood planting forest land |
| CN103907470A (en) * | 2014-04-23 | 2014-07-09 | 南安聚华庄生态农业专业合作社 | Quick sandalwood cultivation method |
| CN104585038A (en) * | 2015-02-04 | 2015-05-06 | 中国科学院华南植物园 | Inducing method of sandalwood triploid plants |
| CN105580737A (en) * | 2016-03-21 | 2016-05-18 | 贵州师范学院 | Culture method for thesium chinense tissue culture vessel seedlings |
| CN106233951A (en) * | 2016-07-27 | 2016-12-21 | 阜南县创发工艺品有限公司 | A kind of efficient implantation methods of the purple willow for braiding |
| CN112690212A (en) * | 2021-01-07 | 2021-04-23 | 中国林业科学研究院热带林业研究所 | Tissue culture method of dalbergia odorifera |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106332724A (en) * | 2016-08-26 | 2017-01-18 | 李军 | Planting technology and planting methods for early bearing heartwood of algum |
-
2009
- 2009-07-01 CN CN2009100407089A patent/CN101595824B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983557A (en) * | 2010-10-19 | 2011-03-09 | 廖俊杰 | In vitro quick breeding method of seedling stem of santal seed embryo |
| CN101983557B (en) * | 2010-10-19 | 2012-10-03 | 东莞市睿绅生物技术有限公司 | In vitro quick breeding method of seedling stem of santal seed embryo |
| CN103039232A (en) * | 2012-12-18 | 2013-04-17 | 中国科学院华南植物园 | Industrial sandalwood planting forest land |
| CN103907470A (en) * | 2014-04-23 | 2014-07-09 | 南安聚华庄生态农业专业合作社 | Quick sandalwood cultivation method |
| CN103907470B (en) * | 2014-04-23 | 2015-08-19 | 南安聚华庄生态农业专业合作社 | The quick cultivating method of santal |
| CN104585038A (en) * | 2015-02-04 | 2015-05-06 | 中国科学院华南植物园 | Inducing method of sandalwood triploid plants |
| CN105580737A (en) * | 2016-03-21 | 2016-05-18 | 贵州师范学院 | Culture method for thesium chinense tissue culture vessel seedlings |
| CN106233951A (en) * | 2016-07-27 | 2016-12-21 | 阜南县创发工艺品有限公司 | A kind of efficient implantation methods of the purple willow for braiding |
| CN112690212A (en) * | 2021-01-07 | 2021-04-23 | 中国林业科学研究院热带林业研究所 | Tissue culture method of dalbergia odorifera |
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