CN102487818B - Method for improving kenaf cotyledon adventitious bud inductivity - Google Patents
Method for improving kenaf cotyledon adventitious bud inductivity Download PDFInfo
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- CN102487818B CN102487818B CN 201110372472 CN201110372472A CN102487818B CN 102487818 B CN102487818 B CN 102487818B CN 201110372472 CN201110372472 CN 201110372472 CN 201110372472 A CN201110372472 A CN 201110372472A CN 102487818 B CN102487818 B CN 102487818B
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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 cultivation.
Background technology
Bluish dogbane (kenaf,
Hibiscus cannabinus L.) have fiber production height, wide adaptability, strong stress resistance, drought-enduring, Salt And Alkali Tolerance, anti-extensive, fast-growing and an easy characteristic such as cultivation.It not only is recognized 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 and the cultivation gross area and gross output occupy first place in the world, and its biological yield is 3-4 times of forest, CO
2Absorbing capacity is 4-5 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 is cultivated and is being made some progress aspect the researchs such as Fast-propagation, anther culture, protoplast cultivation, somatic embryo generation, organ generation.But these technology are all not too improved and are ripe, not yet form ripe, stable, the efficient regenerative system of a cover, and this also becomes, and the bluish dogbane transgenic breeding lags behind and the bottleneck of restriction development.So set up ripe, stable, the efficient bluish dogbane regenerating system of a cover, significant to the transgenic breeding of bluish dogbane.
In the bluish dogbane tissue is cultivated, utilize the various plants hormone to carry out the research of adventitious bud proliferation though have.Such as TDZ, NAA, 6-BA, 2,4-D, IBA, KT etc., but less about the research of other regulatory factors.In tissue was cultivated, the rate of increase of explant indefinite bud often became the standard of passing judgment on a group training system quality.The plant of cultured in vitro can produce and distribute ethene, and the accumulation of ethene in culture vessel can affect growth and the propagation of culture, AgNO
3In Ag
+By being incorporated into competitively the ethane cyclic amp receptor protein on the cell membrane, thus the activity of ethene suppressing.The present invention is directed to AgNO
3Impact in the bluish dogbane adventitious bud inducing is studied, and this still belongs to the first in the bluish dogbane tissue is cultivated.
Summary of the invention
The object of the present invention is to provide a kind of method that improves the kenaf cotyledon adventitious bud induction frequency.
Purpose of the present invention is achieved through the following technical solutions:
The method of raising kenaf cotyledon adventitious bud inducing of the present invention is carried out adventitious bud inducing, propagation take kenaf seeds aseptic seedling cotyledon as explant material, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The concrete steps of described method comprise: kenaf seeds is after sterilization, aseptic process, on the MS medium, carry out light, the dark cultivation, at light, dark incubation time is respectively 14h and 10h loops, 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
2Rectangular being seeded on the adventitious bud induction culture base cultivate 30-40d, will induce again cotyledon after the cultivation to transfer and in the adventitious bud proliferation medium, carry out the propagation of indefinite bud.
The indefinite bud that produces after the propagation is used for inducing plant regeneration, method of operating is: when indefinite bud grows to 3-4cm, indefinite bud is cut down from explant, what be divided into individual plant is inoculated in the adventitious bud rooting medium without offspring: MS+0.05mg/L NAA, induce the generation root, can form complete plant.
The preparation of described 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) solution divides and 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, then divide to install to be the adventitious bud induction culture base in the aseptic blake bottle.
The preparation of described adventitious bud proliferation medium 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) solution divides and 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, then divide to install to be the adventitious bud proliferation medium in the aseptic blake bottle.
The preparation of described adventitious bud rooting medium 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) solution is divided be filled in the conical flask, add agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to about 60 ℃, in superclean bench, operate, its minute installed to be the adventitious bud rooting medium in the aseptic blake bottle.
In the medium, described TDZ is a kind of artificial synthetic, efficient plant growth regulator, and its effect is similar to the basic element of cell division, can induce ethene to produce; Described NAA is a kind of growth hormone for Plant Tissue Breeding; 6-BA is phytocytomine, and IBA is auximone; LH is lactoalbumin hydrolysate.
Remarkable advantage of the present invention:
The present invention utilizes AgNO
3Improve the method for kenaf cotyledon adventitious bud induction frequency, formation that can the establishment callus in the Induction Process of indefinite bud, and shorten time of 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, affect 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 shorten 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 rear AgNO
3Effect study accordingly and AgNO
3May become a kind of effective means that is widely used in the plant tissue culture method improvement.
In the tissue of other crops is cultivated, to AgNO
3Research less, utilize to add debita spissitudo AgNO among the present invention
3Improved the inductivity of kenaf cotyledon indefinite bud, this belongs to pioneering in the crudefiber crop tissue is cultivated, and also can be the bluish dogbane gene transformation a kind of quick, efficient, stable regenerative system is provided, thus the efficient of raising bluish dogbane transgenic breeding.AgNO
3Utilization in the bluish dogbane tissue is cultivated equally also can be generalized to the research of the tissue cultivation of other bast fiber crops, and this method can change the bast fiber crop tissue and cultivate the difficult and low problem of inductivity of 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 after the kenaf seeds sterilization;
Fig. 2 is that kenaf cotyledon is inoculated in inducing culture;
Fig. 3 is that the cotyledon evoking adventive bud produces the bud point;
Fig. 4 cotyledon differentiates indefinite bud;
Fig. 5 is that indefinite bud carries out culture of rootage;
Fig. 6 forms whole plant after the culture of rootage.
Embodiment
The present invention is further illustrated below by embodiment, and 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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 the indefinite spore induction of bluish dogbane by 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 Kenaf Cultivars good fortune red 992 that University Of Agriculture and Forestry In Fujian preserves behind the 7d, light, dark cultivation in the MS medium after the routine sterilization, light, dark incubation time is respectively 14h and 10h loops, 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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The result shows that carry out the indefinite spore induction of bluish dogbane by 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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The result shows that carry out the indefinite spore induction of bluish dogbane by 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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The result shows that carry out the indefinite spore induction of bluish dogbane by 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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The result shows that carry out the indefinite spore induction of bluish dogbane by 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 superclean bench in too
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) will induce cotyledon after the cultivation to transfer and carry out the propagation of indefinite bud in the adventitious bud proliferation medium, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
The result shows that carry out the indefinite spore induction of bluish dogbane by present embodiment, the adventitious bud induction frequency of cotyledon is 5.71%.
Draw by above embodiment, the indefinite spore induction of embodiment 2 kenaf cotyledons is the highest, has reached 85%.This kind method can improve the efficient that the bluish dogbane tissue is cultivated greatly, for the bluish dogbane transgenic breeding is had laid a good foundation.
Claims (6)
1. method that improves the kenaf cotyledon adventitious bud induction frequency, it is characterized in that, carry out adventitious bud inducing, propagation take kenaf seeds aseptic seedling cotyledon as explant material, 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 take light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is cultivated as 20000LX, temperature as 26 ± 1 ℃ condition.
2. the method for raising kenaf cotyledon adventitious bud induction frequency according to claim 1, it is characterized in that, the concrete steps of described method comprise: kenaf seeds is after sterilization, aseptic process, on the MS medium, carry out light, the dark cultivation, at light, dark incubation time is respectively 14h and 10h loops, intensity of illumination is 20000LX, temperature is to cultivate 7-10d under 26 ± 1 ℃ the condition, then complete cotyledon is cut into (3-5) * (3-5) mm
2Rectangular being seeded on the adventitious bud induction culture base cultivate 30-40d, will induce again cotyledon after the cultivation to transfer and in the adventitious bud proliferation medium, carry out the propagation of indefinite bud.
3. the method for raising kenaf cotyledon adventitious bud induction frequency according to claim 2, it is characterized in that, the indefinite bud that produces after the propagation is used for inducing plant regeneration, method of operating is: when indefinite bud grows to 3-4cm, indefinite bud is cut down from explant, what be divided into individual plant is inoculated in the adventitious bud rooting medium without offspring: MS+0.05mg/L NAA, induce the generation root, and can form complete plant.
4. the method for raising kenaf cotyledon adventitious bud induction frequency according to claim 1 is characterized in that, the preparation of described 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;
(3) solution divides and is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to 50-60 ℃, in superclean bench, operate, add AgNO
3, then divide to install to be the adventitious bud induction culture base in the aseptic blake bottle.
5. the method for raising kenaf cotyledon adventitious bud induction frequency according to claim 1 is characterized in that, the preparation of described adventitious bud proliferation medium 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) solution divides and is filled in the conical flask, adds agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to 60 ℃, in superclean bench, operate, add lactoalbumin hydrolysate LH, then divide to install to be the adventitious bud proliferation medium in the aseptic blake bottle.
6. the method for raising kenaf cotyledon adventitious bud induction frequency according to claim 1 is characterized in that, the preparation of described adventitious bud rooting medium 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) solution is divided be filled in the conical flask, add agar powder;
(4) 0.1Mpa, 121 ℃ of autoclaving 20min;
(5) be cooled to 60 ℃, in superclean bench, operate, its minute installed to be the adventitious bud rooting medium in the aseptic blake bottle.
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| CN 201110372472 CN102487818B (en) | 2011-11-22 | 2011-11-22 | Method for improving kenaf cotyledon adventitious bud inductivity |
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|---|---|---|---|
| CN 201110372472 CN102487818B (en) | 2011-11-22 | 2011-11-22 | Method for improving kenaf cotyledon adventitious bud inductivity |
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| CN102487818B true CN102487818B (en) | 2013-10-23 |
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| 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 |
| CN114467756B (en) * | 2022-03-07 | 2023-01-10 | 福建农林大学 | Kenaf tissue culture method using cotyledon petiole as explant |
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