CN105284617A - Efficient in-vitro propagation method for inducing root segment of kok-saghyz into seedling by one step - Google Patents
Efficient in-vitro propagation method for inducing root segment of kok-saghyz into seedling by one step Download PDFInfo
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- CN105284617A CN105284617A CN201510723035.2A CN201510723035A CN105284617A CN 105284617 A CN105284617 A CN 105284617A CN 201510723035 A CN201510723035 A CN 201510723035A CN 105284617 A CN105284617 A CN 105284617A
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Abstract
The invention relates to a quick propagation method for inducing the root segment of kok-saghyz which is a rubber production plant into a seedling by one step. The method comprises the following steps: (1) putting the root segment of the kok-saghyz into an MS culture medium, and performing one-step seedling development by inducing; (2) putting a germfree test-tube plantlet into the MS propagation culture medium for quick propagation culture on the test-tube plantlet, thus obtaining robust cluster buds or plants; (3) putting the robust cluster buds or plants into a 1/2 MS rooting culture medium for culture, thus obtaining a complete seedling with a root; (4) hardening the complete seedling with the root, then putting the seedling into a substrate, and transplanting the seedling into a field after the seedling grows for one month. The seedling obtained by the method is robust and high in survival rate, so that a large quantity of high-quality seedlings of the kok-saghyz can be provided within short time.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of Russian dandelion root segment one one-step inducing efficient in vifro culture method.
Background technology
Russian dandelion (
taraxacumkok-saghyzrodin) be composite family (Compositae) Dandelion perennial root herbaceous plant, profile and dandelion closely similar, be otherwise known as Russian dandelion, but its root contains natural rubber, is commonly called as Russian dandelion in China.In Russian dandelion, the 26S Proteasome Structure and Function of contained natural rubber is similar to paragutta, and the features such as (Russian dandelion root reach 6% ~ 28% containing natural rubber) that has that best in quality, industrial value is high and gel content is high, be the lactiferous plant having economic development future, there is very consequence in natural rubber production.
Russian dandelion happiness Cold and cool climate, is suitable at colder raw Alkalization Meadow or marsh aerial.Originate in the Tekes River basin of former Soviet Union mountain valley, Kazak republic Tianshan Mountains and Xinjiang, China.It is the important step setting up Russian dandelion regenerating system that Russian dandelion cultured in vitro obtains high frequency regeneration indefinite bud, is also one of precondition of the excellent new germ plasm of Russian dandelion genetic transformation and induction polyploid or new lines.In this research, using the Russian dandelion germ plasm resource introduced from Russia as test material, take root segment as explant, carry out induced bundle by organogenetic approach to sprout, filter out the best hormone concentration combination of applicable root segment direct differentiation and regeneration seedling, attempt the high frequency regenerating system setting up Russian dandelion, carry out breed improvement for utilizing gene engineering further and lay the foundation.
Summary of the invention
The object of this invention is to provide a kind of method of Russian dandelion forming seedling through one step culture Fast-propagation, it passes through with Russian dandelion root segment as explant, establish a kind of Fast-propagation being carried out Russian dandelion by organogenetic approach, the method significantly can simplify nursery program, shortens growing-seedling period, improves vegetative propagation coefficient, reduce variation frequency and easy and simple to handle, is the effective way realizing Russian dandelion high quality seedling factorial praluction.Meanwhile, can be the forming seedling through one step culture of similar plant, breed and take root integration breeding technical basis is provided.
A kind of Russian dandelion root segment forming seedling through one step culture efficient in vifro culture method of the present invention, comprises the following steps:
(1) root segment induction forming seedling through one step culture: Russian dandelion root segment section is inoculated in MS medium, within 20 ~ 30 days, obtain in vitro cuttings, containing NAA(methyl α-naphthyl acetate in its MS medium in the indoor cultivation of group training) 0 ~ 2.0mg/L, 6-BA(benzylaminopurine) 0 ~ 1.0mg/L, agar 4 ~ 7g/L, sucrose 30 ~ 40g/L.(2) test-tube plantlet Multiple Buds fast breeding is cultivated: by step (1) in the in vitro cuttings that obtains be placed in MS medium, within 10 ~ 25 days, obtain a large amount of in vitro cuttings in the indoor cultivation of group training, wherein in MS proliferated culture medium containing NAA0 ~ 3.0mg/L, 6-BA(benzylaminopurine) 0 ~ 1.0mg/L, agar 4 ~ 7g/L, sucrose 30 ~ 40g/L.(3) healthy and strong plant culture of rootage: by step (2) in the healthy and strong plant that obtains be placed in 1/2MS root media, within 10 ~ 20 days, obtain being with the whole plant of root in the indoor cultivation of group training, wherein in 1/2MS root media containing IAA(heteroauxin) 0 ~ 2.5mg/L, IBA(indolebutyric acid) 0 ~ 2.0mg/L, agar 4 ~ 6g/L, sucrose 30 ~ 40g/L.(4) hardening and transplanting: step at room temperature opens bottle cap after (3) obtaining the whole plant being with root, through hardening, takes out seedling after surface horny is formed, cleans root medium, be transplanted to immediately in matrix, transplant land for growing field crops after growing 20 ~ 30 days in matrix.
According to the further feature of the method for the invention, in described step (1), described medium is: add NAA0 ~ 2.0mg, 6-BA0 ~ 1.0mg, agar 4 ~ 7g, sucrose 30 ~ 40g in often liter of MS minimal medium.
According to the further feature of the method for the invention, in described step (1), adopt growth 20 ~ 30 days root segments to be explant, directly carry out aseptic inoculation.
According to the further feature of the method for the invention, in described step (2), described medium is: add the NAA of 0 ~ 3.0mg and the 6-BA of 0 ~ 1.0mg, agar 4 ~ 7g, sucrose 30 ~ 40g in often liter of MS minimal medium.
According to the further feature of the method for the invention, in described step (3), described medium is: containing IAA0 ~ 2.5mg, IBA0 ~ 2.0mg, agar 4 ~ 6g, sucrose 30 ~ 40g in often liter of 1/2MS root media.
According to the further feature of the method for the invention, in described step (3), the regrowth of the band root that clusters directly or the Cong Miao form be separated into 3 ~ 5 plant transplant.
Method according to claim 1, is characterized in that: in described step (4), transplanting medium adopt peat soil: perlite is 1:1 ~ 3:1(V/V) mixed-matrix.
According to the further feature of the method for the invention, in described step (4), the culture fluid after transplanting is running water or 1/4MS culture fluid.
Outstanding advantages of the present invention and effect:
1, the experiment proved that, adopt MS+0 ~ 2.0mg/LNAA+0 ~ 1.0mg/L6-BA+30 ~ 40g/L sucrose+4 ~ 7g/L agar as the inducing culture of rubber grass roots forming seedling through one step culture, outer value body induction is that the incidence of Multiple Buds reaches 91.2%, the strain of single outer value body average seedling 8 ~ 15, tissue culturing seedling's well-grown, basic maintenance original seed characteristic.
2, the experiment proved that, in the method for the invention, adopt Russian dandelion root segment as explant, the survival rate of explant is only 2.2% to 100%(pollution rate), inductivity is more than 88.7%, and explant directly can be induced and be differentiated multiple bud point.
3, the experiment proved that, in the method for the invention, adopt MS+0 ~ 3.0mg/LNAA+0 ~ 1.0mg/L6-BA+30 ~ 40g/L sucrose+4 ~ 7g/L agar as the proliferated culture medium of Russian dandelion Multiple Buds, proliferation times reaches as high as 15.3 times, test-tube plantlet growth is vigorous, petiole is sturdy, dark green leaf.
4, the experiment proved that, in the method for the invention, adopt 1/2MS+0 ~ 2.5mg/LIAA+0 ~ 2.0mg/LIBA+30 ~ 40g/L sucrose+4 ~ 7g/L agar as the root media of Russian dandelion aseptic seedling, the number of taking root of aseptic seedling is 1.0 ~ 1.2, root length is 8.0 ~ 11.9cm, rooting rate can reach more than 95.0%, and test-tube plantlet growing way is vigorous, thickening vanes.
5, the experiment proved that, in the method for the invention, through hardening, the tissue culturing seedling of clustering can directly or disperse to be transplanted in the mixed-matrix of [peat soil: perlite=1:1 ~ 3:1 (V/V)], after 30 days, the survival rate of tufted seedling is to 97.1%, and grows vigorous.
6, compared with existing propagation technique, technical method pole of the present invention significantly simplify Russian dandelion nursery program, shorten growing-seedling period, improve vegetative propagation coefficient, keep original seed characteristic, cultivation program is simple, and reaching the effect that namely explant induction obtains a large amount of seedling, is the effective way realizing Russian dandelion high quality seedling factorial praluction.
7, the impact that the germ contamination that the method for the invention overcomes explant without the need to task equipment causes nursery, easy and simple to handle.
In sum, the present invention with Russian dandelion root segment for material, study different combination of regulators formula to the impact of Russian dandelion Regeneration System, by screening and culturing base component, optimum culture condition, simplify tissue culture technique flow process, explore the forming seedling through one step culture quick-breeding method replacing vaccine program without traditional tissue cultures too many levels, establish the efficient of wild plant Russian dandelion rapid propagation in vitro, easy, stable practical technique system, reach and once inoculate, cultivate into healthy seedling, and significantly improve vegetative propagation coefficient, the factorial praluction of Russian dandelion commodity seedling can be directly applied to.
Accompanying drawing explanation
Fig. 1 is the present invention with rubber grass roots for explant carries out the technology path process flow chart of quick breeding method for tissue culture.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is further illustrated.
As shown in Figure 1, Russian dandelion root segment forming seedling through one step culture efficient in vifro culture method operation comprises the following steps:
(1) the induction of Russian dandelion in vitro cuttings: the aseptic Russian dandelion seedling getting healthy growth band root, cuts root segment, with sterilized filter paper suck dry moisture, then root segment is cut into root segment, is inoculated into by root segment in MS medium.In MS medium, NAA concentration is 0 ~ 2.0mg/L, 6-BA concentration is 0 ~ 1.0mg/L, and the concentration of sucrose and agar is respectively 30 ~ 40g/L and 4 ~ 7g/L.When cultivating 20 ~ 30 days, each explant induction is that the incidence of Multiple Buds reaches 91.2%, the strain of single outer value body average seedling 8 ~ 15, tissue culturing seedling's well-grown, basic maintenance original seed characteristic.
(2) test-tube plantlet Multiple Buds fast breeding is cultivated: by step (1) in the in vitro cuttings that obtains be placed in MS medium and cultivate.In MS proliferated culture medium, NAA concentration is 0 ~ 3.0mg/L, 6-BA concentration is 0 ~ 1.0mg/L, and the concentration of sucrose and agar is respectively 30 ~ 40g/L and 4 ~ 7g/L.When cultivating 10 ~ 25 days, height of seedling reaches as high as 3.9cm, and proliferation times reaches as high as 15.3 times, and test-tube plantlet growing way is vigorous, and petiole is sturdy, dark green leaf.
(3) healthy and strong plant culture of rootage: by step (2) in the healthy and strong plant that obtains be placed in 1/2MS root media and cultivate.Wherein in 1/2MS root media, IAA concentration is 0 ~ 2.5mg/L, IBA concentration is that the concentration of 0 ~ 2.0mg/L, sucrose and agar is respectively 30 ~ 40g/L and 4 ~ 6g/L.When cultivating 10 ~ 20 days, root number is 1.0 ~ 1.2, and root length can reach 8.0 ~ 11.9cm, and rooting rate can reach more than 95.0%, and test-tube plantlet growth is vigorous, and petiole is sturdy, thickening vanes.
hardening and transplanting: step at room temperature opens bottle cap after (3) obtaining the whole plant being with root, through hardening, take out seedling after surface horny is formed, clean root medium, be transplanted to immediately in matrix, transplant land for growing field crops after growing 20 ~ 30 days in matrix.After 30 days, the survival rate of regrowth is to 97.1%, and grows vigorous.
Claims (8)
1. a Russian dandelion root segment one one-step inducing seedling efficient in vifro culture method, is characterized in that, comprise the steps:
(1) the root segment induction seedling of Russian dandelion: aseptic for Russian dandelion root segment is inoculated in MS medium, within 20 ~ 30 days, obtain in vitro cuttings in the indoor cultivation of group training, wherein contain NAA0 ~ 2.0mg/L, 6-BA0 ~ 1.0mg/L, agar 4 ~ 7g/L, sucrose 30 ~ 40g/L in MS medium; (2) test-tube plantlet Multiple Buds fast breeding is cultivated: by step (1) in the in vitro cuttings that obtains be placed in MS medium, within 10 ~ 25 days, obtain a large amount of in vitro cuttings in the indoor cultivation of group training, wherein contain NAA0 ~ 3.0mg/L, 6-BA0 ~ 1.0mg/L, agar 4 ~ 7g/L, sucrose 30 ~ 40g/L in MS proliferated culture medium;
(3) healthy and strong plant culture of rootage: by step (2) in the healthy and strong plant that obtains be placed in 1/2MS root media, within 10 ~ 20 days, obtain in the indoor cultivation of group training the whole plant being with root, wherein contain IAA0 ~ 2.5mg/L, IBA0 ~ 2.0mg/L, agar 4 ~ 6g/L, sucrose 30 ~ 40g/L in 1/2MS root media;
(4) hardening and transplanting: step at room temperature opens bottle cap after (3) obtaining the whole plant being with root, through hardening, takes out seedling after surface horny is formed, cleans root medium, be transplanted to immediately in matrix, transplant land for growing field crops after growing 20 ~ 30 days in matrix.
2. method according to claim 1, is characterized in that, in described step (1), described medium is: add NAA0 ~ 2.0mg, 6-BA0 ~ 1.0mg, agar 4 ~ 7g, sucrose 30 ~ 40g in often liter of MS minimal medium.
3. method according to claim 1, is characterized in that: in described step (1), adopts growth 20 ~ 30 days sterilized roots to be explant, directly carries out aseptic inoculation.
4. method according to claim 1, is characterized in that: in described step (2), described medium is: add NAA0 ~ 3.0mg, 6-BA0 ~ 1.0mg, agar 4.5g, sucrose 30 ~ 40g in often liter of MS minimal medium.
5. method according to claim 1, is characterized in that: in described step (3), described medium is: containing IAA0 ~ 2.5mg, IBA0 ~ 2.0mg, agar 4.5g, sucrose 30 ~ 40g in often liter of 1/2MS root media.
6. method according to claim 1, is characterized in that: in described step (3), the regrowth clustered directly or the tufted seedling form be separated into 3 ~ 5 plant transplant.
7. method according to claim 1, is characterized in that: in described step (4), transplanting medium adopt peat soil: perlite is 1:1 ~ 3:1(V/V) mixed-matrix.
8. method according to claim 1, is characterized in that, in described step (4), the culture fluid after transplanting is running water or 1/4MS culture fluid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113149794A (en) * | 2021-01-13 | 2021-07-23 | 北京玲珑蒲公英科技发展有限公司 | Nutrient solution for improving transplanting survival rate of tissue culture seedlings of kochia scoparia and improving method thereof |
| CN113229125A (en) * | 2021-01-13 | 2021-08-10 | 北京玲珑蒲公英科技发展有限公司 | Method for inducing rooting of hydroponic rubber grass |
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| CN104604677A (en) * | 2015-01-04 | 2015-05-13 | 黑龙江省科学院自然与生态研究所 | Tissue culture propagation method for reducing tissue culture browning rate of traxacum koksaghyz |
| CN104782483A (en) * | 2015-03-27 | 2015-07-22 | 中国热带农业科学院橡胶研究所 | Method for inducing adventitious buds by using fibrous roots of kochia scoparia |
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| CN104604677A (en) * | 2015-01-04 | 2015-05-13 | 黑龙江省科学院自然与生态研究所 | Tissue culture propagation method for reducing tissue culture browning rate of traxacum koksaghyz |
| CN104782483A (en) * | 2015-03-27 | 2015-07-22 | 中国热带农业科学院橡胶研究所 | Method for inducing adventitious buds by using fibrous roots of kochia scoparia |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113149794A (en) * | 2021-01-13 | 2021-07-23 | 北京玲珑蒲公英科技发展有限公司 | Nutrient solution for improving transplanting survival rate of tissue culture seedlings of kochia scoparia and improving method thereof |
| CN113229125A (en) * | 2021-01-13 | 2021-08-10 | 北京玲珑蒲公英科技发展有限公司 | Method for inducing rooting of hydroponic rubber grass |
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