CN102487818A - Method for improving kenaf cotyledon adventitious bud inductivity - Google Patents
Method for improving kenaf cotyledon adventitious bud inductivity Download PDFInfo
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- CN102487818A CN102487818A CN2011103724726A CN201110372472A CN102487818A CN 102487818 A CN102487818 A CN 102487818A CN 2011103724726 A CN2011103724726 A CN 2011103724726A CN 201110372472 A CN201110372472 A CN 201110372472A CN 102487818 A CN102487818 A CN 102487818A
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- bluish dogbane
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Abstract
The present invention provides a method for improving kenaf cotyledon adventitious bud inductivity. According to the method, aseptic seedling cotyledons of the kenaf seeds are adopted as the explant material to carry out adventitious bud induction and proliferation, wherein the culture medium of the adventitious bud induction comprises: 0.1-3 mg/L of MS+, 0.01-1 mg/L of TDZ, 0.1-1 mg/L of NAA+, and 0.1-1 mg/L of AgNO3, and the culture medium of the adventitious bud proliferation comprises: 2 mg/L of MS+, 0.1 mg/L of 6-BA, 100 mg/L of IBA+, and LH. With the technology of the present invention, the time for induction of the adventitious bud of the kenaf cotyledon can be shortened, the adventitious bud inductivity is improved, the good basis is established for the transgenic research of the kenaf, and the genetic breeding efficiency is improved.
Description
Technical field
The invention belongs to the cell engineering field, be specifically related to the abductive approach of cotyledon indefinite bud in a kind of bluish dogbane tissue culture.
Background technology
Bluish dogbane (kenaf,
Hibiscus cannabinus L.) have fiber production height, wide adaptability, strong stress resistance, drought-enduring, salt tolerant alkali, anti-extensive, speed is given birth to and be prone to characteristic such as cultivation.It is not only generally acknowledged it is the paper making raw material of alternative wood pulp, also is the essential industry raw material crop of traditional bast fibre spinning.China's bluish dogbane breeding unit yield occupies first place in the world with the cultivation gross area and gross output, and its biological yield is 3-4 a times of forest, CO
2Absorbing capacity is 4-5 a times of forest, and dry-matter accumulation can reach 22.5t/ha in 4 months growth cycles, and the deoxygenation amount is 26557.5Kg/ha, CO
2Absorptive amount reaches 36517.8Kg/ha, is described as 21 century development low-carbon economy, the dominant crop of preserving the ecological environment, the extremely attention of international community and favor.
The development in over one hundred year of Plant Tissue Breeding process has become indispensable technology in the bioscience, and it has important scientific meaning to improving crop breeding or genetic transformation efficiency.China's crudefiber crop tissue culture is obtaining certain progress aspect the researchs such as breeding fast, anther culture, protoplast cultivation, somatic embryo generation, organ generation.But these technology are all not too improved and are ripe, do not form ripe, stable, the regenerative system efficiently of a cover as yet, and this also becomes, and the bluish dogbane transgenic breeding lags behind and the bottleneck of restriction development.So set up ripe, stable, the bluish dogbane regenerating system efficiently of a cover, significant to the transgenic breeding of bluish dogbane.
In the bluish dogbane tissue culture, utilize the various plants hormone to carry out the research of adventitious bud proliferation though have.Like TDZ, NAA, 6-BA, 2,4-D, IBA, KT etc., but less about the research of other regulatory factors.In tissue culture, the rate of increase of explant indefinite bud often becomes the good and bad standard of group training system of passing judgment on.The plant of cultured in vitro can produce and distribute ethene, and the accumulation of ethene in culture vessel can influence the growth and the propagation of culture, AgNO
3In Ag
+Through being incorporated into the ethane cyclic amp receptor protein on the cell membrane competitively, thus the activity of ethene suppressing.The present invention is directed to AgNO
3Influence in the bluish dogbane adventitious bud inducing is studied, and this still belongs to the first in the bluish dogbane tissue culture.
Summary of the invention
The object of the present invention is to provide a kind of method that improves bluish dogbane cotyledon adventitious bud induction frequency.
The object of the invention is realized through following technical scheme:
The method of raising bluish dogbane cotyledon adventitious bud inducing of the present invention is that explant material carries out adventitious bud inducing, propagation with kenaf seeds aseptic seedling cotyledon, and the adventitious bud induction culture base is: MS+0.1-3mg/L TDZ+ 0.01-1mg/L NAA+0.1-3mg/L AgNO
3, the adventitious bud induction culture base is: MS+2mg/L 6-BA+0.1mg/L IBA+100mg/L LH.The condition of culture of above adventitious bud inducing, propagation all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The concrete steps of said method comprise: kenaf seeds is after sterilization, aseptic process; On the MS medium, carry out light, the dark cultivation; Be respectively 14h and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is to cultivate 7-10d under 26 ± 1 ℃ the condition at light, dark incubation time, then complete cotyledon cut into 3-5 * 3-5mm
2Rectangular being seeded on the adventitious bud induction culture base cultivate 30-40d, the cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud again.
The indefinite bud that the propagation back produces is used to induce plant regeneration; Method of operating is: when indefinite bud grows to 3-4cm; Indefinite bud is cut down from explant; The no offspring that is divided into individual plant is inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, and can form complete plant.
The preparation of said adventitious bud induction culture base may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add TDZ, NAA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(4) the solution branch is filled in the conical flask, adds agar powder;
(5) 0.1Mpa, 121 ℃ of autoclaving 20min;
(6) be cooled to 50-60 ℃, in superclean bench, operate, add AgNO
3, divide to install to then to be the adventitious bud induction culture base in the aseptic blake bottle.
Said adventitious bud proliferation culture medium preparation may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add 6-BA, IBA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(3) the solution branch is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to about 60 ℃, in superclean bench, operate, add lactoalbumin hydrolysate LH, divide to install to then to be the adventitious bud proliferation medium in the aseptic blake bottle.
Said adventitious bud rooting culture medium preparation may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add NAA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(3) the solution branch is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to about 60 ℃, in superclean bench, operate, its branch is installed to be the adventitious bud rooting medium in the aseptic blake bottle.
In the medium, described TDZ, be a kind of synthetic, plant growth regulator efficiently, its effect is similar to the basic element of cell division, can induce ethene to produce; Described NAA is a kind of growth hormone that is used for Plant Tissue Breeding; 6-BA is a phytocytomine, and IBA is an auximone; LH is a lactoalbumin hydrolysate.
Remarkable advantage of the present invention:
The present invention utilizes AgNO
3Improve the method for bluish dogbane cotyledon adventitious bud induction frequency, in the formation that can effectively suppress callus in the process of inducing of indefinite bud, and the time of shortening adventitious bud proliferation.The rate of increase of indefinite bud is not adding AgNO
3The time, follow a large amount of callus to produce during adventitious bud proliferation, influence the growth of indefinite bud, and make troubles to experimental implementation; In addition, its generation time is long, and adventitious bud induction frequency is 64%.Add AgNO
3After almost do not have callus to produce, the adventitious bud proliferation time shortened for 2 weeks, adventitious bud induction frequency brings up to 85%.
The result shows interpolation AgNO
3Propagation to the bluish dogbane indefinite bud has obvious facilitation, and it significantly improves the inductivity of bluish dogbane indefinite bud, reaches 85%.This explanation is trained in the research work in the group of other crops in the future, also can be to adding back AgNO
3Effect study and AgNO accordingly
3Possibly become a kind of effective means adaptable across the plant tissue culture method improvement.
In the tissue culture of other crops, to AgNO
3Research less, utilize to add debita spissitudo AgNO among the present invention
3Improved the inductivity of bluish dogbane cotyledon indefinite bud, this belongs to initiative in the crudefiber crop tissue culture, also can be the conversion of bluish dogbane foreign gene a kind of quick, efficient, stable regenerative system is provided, thereby improve the efficient of bluish dogbane transgenic breeding.AgNO
3Utilization in the bluish dogbane tissue culture equally also can be generalized to the research of the tissue culture of other bast fiber crops, and this method can change the difficult and low problem of inductivity of bast fiber crop tissue culture adventitious bud inducing.In addition, the optimization of crudefiber crop tissue culturing system there is important facilitation, a new research thinking, AgNO is provided also for other crops
3Might become the key factor that improves and improve other crop tissue culture techniques.This possible realization will make Plant Tissue Breeding move towards a renewal, higher platform, promotes plant tissue culture technique further to improve and development.
Description of drawings
Fig. 1 is inoculated in the MS medium for after the kenaf seeds sterilization;
Fig. 2 is inoculated in inducing culture for the bluish dogbane cotyledon;
Fig. 3 produces the bud point for the cotyledon evoking adventive bud;
Fig. 4 cotyledon differentiates indefinite bud;
Fig. 5 carries out culture of rootage for indefinite bud;
Fig. 6 forms whole plant for after the culture of rootage.
Embodiment
Through embodiment the present invention is further described below, its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention:
Embodiment 1:
(1) will cultivate bluish dogbane aseptic seedling cotyledon behind the 7-10d, in superclean bench, be cut into 3 * 4mm
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate 30-40d, the adventitious bud induction culture base is: MS+0.1mg/L TDZ+0.01mg/L NAA+ 0.1mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 3.03%.
Embodiment 2:
(1) will cultivate the bluish dogbane aseptic seedling cotyledon (seed of the bluish dogbane kind good fortune red 992 that University Of Agriculture and Forestry In Fujian preserves behind the 7d; Light, dark cultivation in the MS medium after conventional sterilization; Light, dark incubation time are respectively 14h and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is 26 ± 1 ℃), in superclean bench, be cut into 3 * 5mm
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate about 30d, the adventitious bud induction culture base is: MS+0.1mg/L TDZ+0.1mg/L NAA+ 1.0mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
Above condition of culture all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 85%.
Embodiment 3:
(1) will cultivate bluish dogbane aseptic seedling cotyledon behind the 8d, in superclean bench, be cut into 4 * 5mm
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate about 30d, the adventitious bud induction culture base is: MS+1mg/L TDZ+0.1mg/L NAA+3mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
Above condition of culture all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 25%.
Embodiment 4:
(1) will cultivate bluish dogbane aseptic seedling cotyledon behind the 9d, in superclean bench, be cut into 3-5 * 3-5mm
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate about 35d, the adventitious bud induction culture base is: MS+1mg/L TDZ+1mg/L NAA+0 .1mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
Above condition of culture all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 43.9%.
Embodiment 5:
(1) will cultivate bluish dogbane aseptic seedling cotyledon behind the 10d, in superclean bench, be cut into 3-5 * 3-5mm
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate about 40d, the adventitious bud induction culture base is: MS+3mg/L TDZ+0.1mg/L NAA+ 0.1mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
Above condition of culture all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 40%.
Embodiment 6:
(1) will cultivate bluish dogbane aseptic seedling cotyledon behind the 8d, be cut into 2 * 3mm in too in superclean bench
2Rectangular, be inoculated in the adventitious bud induction culture base and cultivate about 30d, the adventitious bud induction culture base is: MS+3mg/L TDZ+1mg/L NAA+ 1mg/L AgNO
3
(2) cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA.
(3) when indefinite bud grows to 3-4cm, it is cut apart be inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
Above condition of culture all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
The result shows that carry out inducing of bluish dogbane indefinite bud through present embodiment, the adventitious bud induction frequency of cotyledon is 5.71%.
Draw through above embodiment, inducing of embodiment 2 bluish dogbane cotyledon indefinite buds is the highest, has reached 85%.This kind method can improve the efficient of bluish dogbane tissue culture greatly, for the bluish dogbane transgenic breeding is had laid a good foundation.
Claims (6)
1. method that improves bluish dogbane cotyledon adventitious bud induction frequency; It is characterized in that; With kenaf seeds aseptic seedling cotyledon is that explant material carries out adventitious bud inducing, propagation, and the adventitious bud induction culture base is: MS+0.1-3mg/L TDZ+ 0.01-1mg/L NAA+0.1-3mg/L AgNO
3, the adventitious bud proliferation medium is: MS+2mg/L 6-BA+0.1mg/L IBA+100mg/L LH; The condition of culture of above adventitious bud inducing, propagation all is respectively 14h with light, dark incubation time and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is that 26 ± 1 ℃ condition is cultivated.
2. the method for raising bluish dogbane cotyledon adventitious bud induction frequency according to claim 1; It is characterized in that; The concrete steps of said method comprise: kenaf seeds is after sterilization, aseptic process; On the MS medium, carry out light, the dark cultivation, be respectively 14h and the 10h circulation is carried out, intensity of illumination is 20000LX, temperature is to cultivate 7-10d under 26 ± 1 ℃ the condition, then complete cotyledon is cut into 3-5 * 3-5mm at light, dark incubation time
2Rectangular being seeded on the adventitious bud induction culture base cultivate 30-40d, the cotyledon behind the inducing culture is transferred in the adventitious bud proliferation medium, carry out the propagation of indefinite bud again.
3. the method for raising bluish dogbane cotyledon adventitious bud inducing according to claim 2; It is characterized in that the indefinite bud that the propagation back produces is used to induce plant regeneration, method of operating is: when indefinite bud grows to 3-4cm; Indefinite bud is cut down from explant; The no offspring that is divided into individual plant is inoculated in the adventitious bud rooting medium: MS+0.05mg/L NAA, induce the generation root, and can form complete plant.
4. the method for raising bluish dogbane cotyledon adventitious bud induction frequency according to claim 1 is characterized in that the preparation of said adventitious bud induction culture base may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add TDZ, NAA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(4) the solution branch is filled in the conical flask, adds agar powder;
(5) 0.1Mpa, 121 ℃ of autoclaving 20min;
(6) be cooled to 50-60 ℃, in superclean bench, operate, add AgNO
3, divide to install to then to be the adventitious bud induction culture base in the aseptic blake bottle.
5. the method for raising bluish dogbane cotyledon adventitious bud induction frequency according to claim 1 is characterized in that said adventitious bud proliferation culture medium preparation may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add 6-BA, IBA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(3) the solution branch is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to about 60 ℃, in superclean bench, operate, add lactoalbumin hydrolysate LH, divide to install to then to be the adventitious bud proliferation medium in the aseptic blake bottle.
6. the method for raising bluish dogbane cotyledon adventitious bud induction frequency according to claim 1 is characterized in that said adventitious bud rooting culture medium preparation may further comprise the steps:
(1) macroelement among the MS, trace element, molysite, organic matter and sucrose are mixed;
(2) add NAA, use the distilled water constant volume, the pH value transfers to 5.8-6.0 with 1mol/L NaOH;
(3) the solution branch is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to about 60 ℃, in superclean bench, operate, its branch is installed to be the adventitious bud rooting medium in the aseptic blake bottle.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103155870A (en) * | 2012-12-27 | 2013-06-19 | 河南农业大学 | Method for improving inducing, growing and regeneration of sweet wormwood multiple shoots by silver nitrate |
| CN104285817A (en) * | 2014-11-03 | 2015-01-21 | 杨业容 | Rapid propagation method for tissue culture of jute |
| CN114467756A (en) * | 2022-03-07 | 2022-05-13 | 福建农林大学 | Kenaf tissue culture method using cotyledon petiole as explant |
-
2011
- 2011-11-22 CN CN 201110372472 patent/CN102487818B/en not_active Expired - Fee Related
Non-Patent Citations (4)
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| 刘恒蔚等: "红麻子叶和真叶不定芽直接诱导的研究", 《湖南农业大学学报(自然科学版)》 * |
| 潘昌立，等: "苎麻体细胞植株再生及其影响因素的研究", 《中国麻作》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103155870A (en) * | 2012-12-27 | 2013-06-19 | 河南农业大学 | Method for improving inducing, growing and regeneration of sweet wormwood multiple shoots by silver nitrate |
| CN104285817A (en) * | 2014-11-03 | 2015-01-21 | 杨业容 | Rapid propagation method for tissue culture of jute |
| CN114467756A (en) * | 2022-03-07 | 2022-05-13 | 福建农林大学 | Kenaf tissue culture method using cotyledon petiole as explant |
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