CN104737906A - Building method of rosemarinus officinalis tissue culture system - Google Patents
Building method of rosemarinus officinalis tissue culture system Download PDFInfo
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Abstract
The invention discloses a building method of a rosemarinus officinalis tissue culture system. Rosemarinus is a lamiaceae rosmarinus perennial evergreen sub-shrub, of which a plant is enriched with multiple compounds such as phenol, ketone, acid, terpene and the like, has a high antioxidation function and has a good development prospect on anti-fatigue, anti-aging, anti-depression, anti-cancer and the like; therefore, the rosemarinus officinalis is a good economic plant. By utilizing a plant tissue culture technology, the reproduction speed and scale can be improved, so that the industrialized production of rosemarinus officinalis seedlings can be realized. With auxiliary bud stem sections as explants, induction, multiplication, rooting, hardening-seedling transplantation and the like are carried out to acquire a rosemarinus officinalis tissue culture regenerative plant, and a symmetrical rosemarinus officinalis tissue culture plant regeneration system is built.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to the method for building up of a kind of rosemary, Xue MingRosma rinus officinalis group training system.
Background technology
Rosemary, Xue MingRosma rinus officinalis (
rosemarinus officinalis) be Labiatae Rosmarinus perennial evergreen undershrub, originate in ring Mediterranean, present Europe, the U.S., all there is distribution in the areas such as China, and complete stool is rich in antioxidant content, mainly comprise carnosic acid, carnosol, rosmanol, ursolic acid, Rosmarinic acid etc., there is many-sided pharmacological actions such as anti-inflammatory, anti-bacteria and anti-virus, antitumor, immunological regulation, the generation of suppression uric acid, be mainly used in food preservation technology, the aspects such as cosmetics and medicine.Compared with traditional chemical preservative, Rosmarinus officinalis extract has cheap, the advantage such as highly effective and safe and abundant collection approach, especially on meat and the safety and sanitation of rich oil food and the application of preservation.
China starts late to rosemary, Xue MingRosma rinus officinalis introducing and planting, greatly about late 1970s and the initial stage eighties, starts to introduce as aromatic crop, and obtains successfully, at present in Beijing, all there is plantation on Guizhou, Xinjiang, the ground such as Yunnan.China scientist for rosemary, Xue MingRosma rinus officinalis further research oneself obtain certain achievement, but still there are improved seeds and introduce the relevant issues with domestication, for the variety development of suitable China most area ambient growth and rosemary, Xue MingRosma rinus officinalis the report of a large amount of Fast-propagation still less.Rosemary, Xue MingRosma rinus officinalis breeding comparatively difficulty, its main modes of reproduction comprises seminal propagation, cottage propagation and propagation by layering etc.When utilizing seminal propagation, there is difficult and that germination rate is low problem of germinateing in its seed, greatly reduce survival rate and planting percent, cost is higher, and income is few; When utilizing the mode of cuttage and press strip to breed, the branch of its cuttage is not easily taken root or comparatively slowly, is not easy to survive, also result in the problem of rosemary, Xue MingRosma rinus officinalis planting percent.Therefore, be badly in need of the rosemary, Xue MingRosma rinus officinalis tissue culture plants regenerating system of the system that establishes, to improve its reproduction speed and scale, thus realize the factorial praluction of rosemary, Xue MingRosma rinus officinalis seedling.
Summary of the invention
A kind of rosemary, Xue MingRosma rinus officinalis group is the object of the present invention is to provide out to train the method for building up of system, the present invention take stem with bud as explant, through inducing, breed, to take root and the step such as acclimatization and transplants successfully obtains rosemary, Xue MingRosma rinus officinalis Regenerated Plantlets, establish the rosemary, Xue MingRosma rinus officinalis tissue culture plants regenerating system of system, thus achieve object of the present invention.
The method for building up of a kind of rosemary, Xue MingRosma rinus officinalis group training system of the present invention, comprises the following steps:
(1) explant sterilization: gather the stem Duan Yi Cheongju water of returning and to rinse after 10 ~ 30min and to brush away impurity above gently with hairbrush, with the 10 ~ 30s that sterilizes in 70% ~ 80% ethanolic solution in superclean bench, afterwards with aseptic washing 3 ~ 5 times, again by 5% ~ 10% peace for rich people's solution rinsing 5 ~ 10min, with for subsequent use after sucking moisture with aseptic filter paper after aseptic water washing 4 ~ 6 times.
(2) Fiber differentiation: stem section step (1) disinfected is that a unit is cut into the long stem section of about 1.5cm by paired bud, cuts off brownization part in stem section bottom treatment process, is inoculated in inducing culture and carries out bud inducement cultivation.First full light culture 3 ~ 10 days under 25 ~ 28 DEG C of conditions after inoculation, be then placed in illumination every day 10 ~ 14 hours, intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until formation indefinite bud.
(3) Multiplying culture: step (2) is cultivated the bud seedling obtained, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, first full light culture 2 ~ 3 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 11 ~ 15 hours, intensity of illumination is 2000 ~ 3000lx, being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and 30 days subcultures once.
(4) culture of rootage: when the bud seedling of step (2) or (3) is grown to 1.5 ~ 2.5cm height, cut into simple bud and be inoculated on root media and carry out culture of rootage, first full light culture 2 ~ 3 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 ~ 16 hours, intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root.
(5) acclimatization and transplants: the blake bottle bottle cap growing to the rooting tube plantlet of 3 ~ 4cm is opened and is placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, plant by peat soil: in illumination every day 10 ~ 12 hours in the matrix that vermiculite=1:1 is mixed into, intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is 25 ~ 28 DEG C, air humidity is cultivate 5 ~ 7 days in the incubator of 80% ~ 90%, and then transplant planting is in large Tanaka.
Inducing culture described in above-mentioned steps (2) is: MS+0.2 ~ 1.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+0.01 ~ 0.2mg/L NAA+0.2 ~ 2.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (4) is: MS+0.5 ~ 3.0mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Advantage of the present invention is: rosemary, Xue MingRosma rinus officinalis (
rosemarinus officinalis) be Labiatae Rosmarinus perennial evergreen undershrub, its plant is rich in the multiple compounds such as phenol, ketone, acid, terpene, there is very strong antioxidation, antifatigue, anti-ageing, antidepression and anticancer etc. in there is good development prospect, be a kind of good economical plant.Current rosemary, Xue MingRosma rinus officinalis mainly carries out seedling breeding in modes such as seminal propagation, cuttage, press strips, there is the shortcomings such as reproduction coefficient is low, planting percent is low, cost is high, income is few, constrains the development of rosemary, Xue MingRosma rinus officinalis industry.Utilize plant tissue culture technique to improve its reproduction speed and scale, thus realize the factorial praluction of rosemary, Xue MingRosma rinus officinalis seedling.The present invention take stem with bud as explant, through inducing, breeding, take root and the step such as acclimatization and transplants successfully obtains rosemary, Xue MingRosma rinus officinalis Regenerated Plantlets, establishes the rosemary, Xue MingRosma rinus officinalis tissue culture plants regenerating system of system.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1
(1) explant sterilization: gather the stem Duan Yi Cheongju water of returning and to rinse after 10min and to brush away impurity above gently with hairbrush, with the 10s that sterilizes in 75% ethanolic solution in superclean bench, afterwards with aseptic washing 4 times, again by 5% peace for rich people's solution rinsing 5min, with for subsequent use after sucking moisture with aseptic filter paper after aseptic water washing 4 times.
(2) Fiber differentiation: stem section step (1) disinfected is that a unit is cut into the long stem section of about 1.5cm by paired bud, cuts off brownization part in stem section bottom treatment process, is inoculated in inducing culture and carries out bud inducement cultivation.First full light culture 3 days under 25 DEG C of conditions after inoculation, be then placed in illumination every day 10 hours, intensity of illumination is 2000lx, and being placed in cultivation temperature is cultivate under the condition of 25 DEG C until formation indefinite bud, and inductivity is 91.4%.Described inducing culture is: MS+0.5mg/L 6-BA+16g/L sucrose+3.8g/L agar, pH is 5.5.
(3) Multiplying culture: step (2) is cultivated the bud seedling obtained, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, first full light culture 2 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 12 hours, intensity of illumination is 2500lx, being placed in cultivation temperature is cultivate under the condition of 25 DEG C, and once, growth coefficient is 5.7 to 30 days subcultures.Described proliferated culture medium is: MS+0.1mg/L NAA+1.2mg/L 6-BA+23g/L sucrose+3.8g/L agar, pH is 5.5.
(4) culture of rootage: when the bud seedling of step (2) or (3) is grown to 1.5 ~ 2.5cm height, cut into simple bud and be inoculated on root media and carry out culture of rootage, first full light culture 2 days under 25 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 4000lx, and cultivation temperature is be cultured under the condition of 25 DEG C to take root.Described root media is: MS+2.0mg/L IBA+24g/L sucrose+4.2g/L agar, pH is 5.5.
(5) acclimatization and transplants: the blake bottle bottle cap growing to the rooting tube plantlet of 3 ~ 4cm is opened and is placed in natural lighting lower refining seedling after 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, plant by peat soil: in illumination every day 11 hours in the matrix that vermiculite=1:1 is mixed into, intensity of illumination is 3000lx, cultivation temperature is 25 DEG C, air humidity is cultivate 5 days in the incubator of 80%, and then transplant planting is in large Tanaka, and transplanting survival rate is 93.7%.
Embodiment 2
(1) explant sterilization: gather the stem Duan Yi Cheongju water of returning and to rinse after 12min and to brush away impurity above gently with hairbrush, with the 20s that sterilizes in 78% ethanolic solution in superclean bench, afterwards with aseptic washing 5 times, again by 5% peace for rich people's solution rinsing 8min, with for subsequent use after sucking moisture with aseptic filter paper after aseptic water washing 6 times.
(2) Fiber differentiation: stem section step (1) disinfected is that a unit is cut into the long stem section of about 1.5cm by paired bud, cuts off brownization part in stem section bottom treatment process, is inoculated in inducing culture and carries out bud inducement cultivation.First full light culture 4 days under 28 DEG C of conditions after inoculation, be then placed in illumination every day 12 hours, intensity of illumination is 2500lx, and being placed in cultivation temperature is cultivate under the condition of 28 DEG C until formation indefinite bud, and inductivity is 93.4%.Described inducing culture is: MS+1.0mg/L 6-BA+20g/L sucrose+4.5g/L agar, pH is 5.8.
(3) Multiplying culture: step (2) is cultivated the bud seedling obtained, cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture, first full light culture 3 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 3000lx, being placed in cultivation temperature is cultivate under the condition of 28 DEG C, and once, growth coefficient is 5.4 to 30 days subcultures.Described proliferated culture medium is: MS+0.5mg/L NAA+1.5mg/L 6-BA+25g/L sucrose+4.6g/L agar, pH is 5.8.
(4) culture of rootage: when the bud seedling of step (2) or (3) is grown to 1.5 ~ 2.5cm height, cut into simple bud and be inoculated on root media and carry out culture of rootage, first full light culture 2 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 15 hours, intensity of illumination is 4000lx, and cultivation temperature is be cultured under the condition of 25 DEG C to take root.Described root media is: MS+3.0mg/L IBA+24g/L sucrose+4.5g/L agar, pH is 5.5.
(5) acclimatization and transplants: the blake bottle bottle cap growing to the rooting tube plantlet of 3 ~ 4cm is opened and is placed in natural lighting lower refining seedling after 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, plant by peat soil: in illumination every day 11 hours in the matrix that vermiculite=1:1 is mixed into, intensity of illumination is 3000lx, cultivation temperature is 25 DEG C, air humidity is cultivate 5 days in the incubator of 80%, and then transplant planting is in large Tanaka, and transplanting survival rate is 95.8%.
Claims (4)
1. a method for building up for rosemary, Xue MingRosma rinus officinalis group training system, is characterized in that comprising the following steps:
(1) explant sterilization: gather the stem Duan Yi Cheongju water of returning and to rinse after 10 ~ 30min and to brush away impurity above gently with hairbrush; with the 10 ~ 30s that sterilizes in 70% ~ 80% ethanolic solution in superclean bench; afterwards with aseptic washing 3 ~ 5 times; again by 5% ~ 10% peace for rich people's solution rinsing 5 ~ 10min, with for subsequent use after sucking moisture with aseptic filter paper after aseptic water washing 4 ~ 6 times;
(2) Fiber differentiation: stem section step (1) disinfected is that a unit is cut into the long stem section of about 1.5cm by paired bud; cut off brownization part in stem section bottom treatment process; be inoculated in inducing culture and carry out bud inducement cultivation; first full light culture 3 ~ 10 days under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 10 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is cultivate until form indefinite bud under the condition of 25 ~ 28 DEG C;
(3) Multiplying culture: step (2) is cultivated the bud seedling obtained; cut bud from base portion and proceed to proliferated culture medium and carry out squamous subculture; first full light culture 2 ~ 3 days under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 11 ~ 15 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, and 30 days subcultures once;
(4) culture of rootage: when the bud seedling of step (2) or (3) is grown to 1.5 ~ 2.5cm height; cut into simple bud and be inoculated on root media and carry out culture of rootage; first full light culture 2 ~ 3 days under 25 ~ 28 DEG C of conditions after inoculation; then illumination every day is placed in 14 ~ 16 hours; intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root;
(5) acclimatization and transplants: the blake bottle bottle cap growing to the rooting tube plantlet of 3 ~ 4cm is opened and is placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, do not injure root as far as possible, plant by peat soil: in illumination every day 10 ~ 12 hours in the matrix that vermiculite=1:1 is mixed into, intensity of illumination is 3000 ~ 5000lx, and cultivation temperature is 25 ~ 28 DEG C, air humidity is cultivate 5 ~ 7 days in the incubator of 80% ~ 90%, and then transplant planting is in large Tanaka.
2. the method for building up of a kind of rosemary, Xue MingRosma rinus officinalis group training system according to claim 1, is characterized in that the inducing culture described in step (2) is: MS+0.2 ~ 1.0mg/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 rosemary, Xue MingRosma rinus officinalis group training system according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+0.01 ~ 0.2mg/L NAA+0.2 ~ 2.0mg/L 6-BA+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 rosemary, Xue MingRosma rinus officinalis group training system according to claim 1, is characterized in that the root media described in step (4) is: MS+0.5 ~ 3.0mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581473A (en) * | 2016-11-28 | 2017-04-26 | 吴福勤 | Rosemary antidepressant pharmaceutical composition |
| CN107494279A (en) * | 2017-10-16 | 2017-12-22 | 陈金水 | A kind of method for building up of crotons tissue culture system |
| CN107494276A (en) * | 2017-10-15 | 2017-12-22 | 陈金水 | A kind of method for building up of ilex pubescens tissue culture system |
| CN108308037A (en) * | 2018-05-08 | 2018-07-24 | 新疆维吾尔自治区药物研究所 | A kind of new tower flower quick breeding method for tissue culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH04104787A (en) * | 1990-08-23 | 1992-04-07 | Iseki & Co Ltd | Callus of rosemarinus officinalis l. |
| AU4734299A (en) * | 1999-03-17 | 2000-09-21 | University Of Massachusetts | Plant clonal lines and plants having elevated peroxidase activity |
-
2015
- 2015-03-05 CN CN201510097701.6A patent/CN104737906A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04104787A (en) * | 1990-08-23 | 1992-04-07 | Iseki & Co Ltd | Callus of rosemarinus officinalis l. |
| AU4734299A (en) * | 1999-03-17 | 2000-09-21 | University Of Massachusetts | Plant clonal lines and plants having elevated peroxidase activity |
Non-Patent Citations (1)
| Title |
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| 刘明家: "迷迭香组织培养及体内迷迭香酸和鼠尾草酸含量测定", 《东北林业大学硕士论文》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581473A (en) * | 2016-11-28 | 2017-04-26 | 吴福勤 | Rosemary antidepressant pharmaceutical composition |
| CN107494276A (en) * | 2017-10-15 | 2017-12-22 | 陈金水 | A kind of method for building up of ilex pubescens tissue culture system |
| CN107494279A (en) * | 2017-10-16 | 2017-12-22 | 陈金水 | A kind of method for building up of crotons tissue culture system |
| CN108308037A (en) * | 2018-05-08 | 2018-07-24 | 新疆维吾尔自治区药物研究所 | A kind of new tower flower quick breeding method for tissue culture |
| CN108308037B (en) * | 2018-05-08 | 2020-12-25 | 新疆维吾尔自治区药物研究所 | Ziziphora bungeana tissue culture rapid propagation method |
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Application publication date: 20150701 |