CN104719158A - Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants - Google Patents
Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants Download PDFInfo
- Publication number
- CN104719158A CN104719158A CN201510101660.3A CN201510101660A CN104719158A CN 104719158 A CN104719158 A CN 104719158A CN 201510101660 A CN201510101660 A CN 201510101660A CN 104719158 A CN104719158 A CN 104719158A
- Authority
- CN
- China
- Prior art keywords
- medium
- callus
- tissue culture
- aseptic
- sucrose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention provides a method for rapidly establishing a medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants. The method mainly comprises the following steps: performing selection, disinfection and soaking on a medium-sized Chinese pennisetum herb raw material; performing inoculation, culture and differentiation on callus tissues; performing rooting culture on tissue culture seedlings; and performing acclimatization and transplanting. By adopting the method provided by the invention, an indoor rapid tissue culture regeneration system of medium-sized Chinese pennisetum herb wild species can be rapidly established, and the indoor rapid tissue culture regeneration system is an important foundation for further researching a molecular mechanism with low seed setting and germination rate; and moreover, the method is simple to operate and easy to complete.
Description
Technical field
The present invention relates to the method for tissue culture of a Plants, being specifically related to a kind of take seed as the method setting up medium-sized pearl millet Tissue Culture Regeneration System fast of explant.
Background technology
Medium-sized pearl millet (
pennisetum longissimumvar.intermedium) be grass family Pennisetum Perennual wild herb, in Yunnan, all there is distribution on Sichuan, Guizhou, the ground such as Hunan.Medium-sized pearl millet stem stalk is more sturdy, well developed root system, wide adaptability, regeneration capacity is strong, grass yield is high, nutrient component is relatively high, and it has very strong disease resistance, drought resisting, moisture-proof and Saline alkali tolerance, is the important species carrying out the important mechanism such as high quality forage popularization and research gramineous plants salt tolerant, drought resisting, high yield.But, due to its seed-setting rate and germination rate all lower at northern area, limit its northern area promote speed.Although carry out breeding can implement with trophosome, too large for the large-area popularization difficulty that operates.Although also have the relevant method of being carried out tissue cultures by trophosome, as (number of patent applications: the method for tissue culture 200910049855.2) being established a kind of pennisetum setaceum ' Rubrum ' by young shoot as explant such as Chen Jianhuas at present.Although can carry out the fast-propagation of pearl millet tissue indoor, the mode of tissue culture propagating wastes time and energy, and fundamentally can not solve the problem that medium-sized pearl millet expands numerous popularization in a large number, improves its seed-setting and germination rate is only the key point of dealing with problems.But at present also do not study clear for its seed-setting and the low molecule mechanism of germination rate, wherein important problem is exactly the basic condition of its molecular mechanism research---and take seed as the pearl millet tissue culture regeneration system of explant, also do not set up, greatly limit carrying out of correlative study, and under traditional culture dish sealing training mode callus and atomization slow, lacking a kind of at present take seed as the method setting up medium-sized pearl millet Tissue Culture Regeneration System fast of explant.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of take seed as the method setting up medium-sized pearl millet Tissue Culture Regeneration System fast of explant, for research improve its seed ripening rate and sprouting, promote that it provides technical support in the popularization of northern area.
Of the present invention a kind of take seed as the method setting up medium-sized pearl millet Tissue Culture Regeneration System fast of explant, step is as follows:
(1) medium-sized pearl millet is raw-material chooses, sterilizes and soaks;
(2) inoculation of callus and cultivation: after the sterilization of step (1) being soaked, seed is inoculated in callus inducing medium, aseptic and under the environment ventilated shading cultivate, form callus; The formula of described callus inducing medium is: MS medium+1 ~ 3wt% sucrose+0.5 ~ 1.0wt% agar+2 ~ 5 mg/L 2,4-D+1 ~ 2 mg/L KT, and pH is 5-6; Can form callus after 7 ~ 14 days under these conditions, callus rate can reach more than 95%, and can be used for squamous subculture.
(3) differentiation of callus: be inoculated in differential medium by the callus that step (2) obtains, in aseptic and illumination cultivation in the environment ventilated, obtains growing blade and with the tissue of root hair; Wherein, the formula of described differential medium is: MS medium+1 ~ 3wt% sucrose+0.5 ~ 1.0wt% agar+0.5 ~ 1.5 mg/L 6-BA+0.2 ~ 0.5 mg/L NAA, and pH is 5 ~ 6; The parameter of described illumination cultivation is: intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 ~ 26 DEG C; Hormone combination in above-mentioned medium can promote that the cell in callus terminates Dedifferentiation, restarts the differentiation to each tissue fast.And be the differentiation promoting bud point fast, start photosynthesis, change condition of culture into illumination cultivation; Cultivate and can occur bud point after 7 ~ 14 days, can grow blade and Gen Mao after 25 ~ 45 days, differentiation rate can reach more than 75%.
(4) plantlet in vitro culture of rootage: step (3) is differentiated blade and tissue with root hair moves in root media, in aseptic and illumination cultivation in the environment ventilated, obtains the plantlet in vitro of well developed root system; Wherein, the formula of described root media is: 1/2MS medium+2.0 ~ 5.0wt% sucrose+1.0 ~ 3.0wt% agar, and pH is 5 ~ 6; The parameter of described illumination cultivation is: intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 ~ 26 DEG C; After cultivating 14 ~ 30 days under these conditions, its rooting rate can reach more than 80%, and its well developed root system, plantlet in vitro grows fine;
(5) hardening and transplanting: carry out hardening when plantlet in vitro grows to 8 ~ 10cm, is transplanted afterwards and is normally cultivated.Survival rate more than 90% after transplanting, completing whole take seed as the foundation of the medium-sized pearl millet Tissue Culture Regeneration System of explant.
Preferably, described in step (1) soak be by sterilization after medium-sized pearl millet seed put into aseptic deionized water, be placed on superclean bench and carry out under aseptic condition, described soak time is 8 ~ 12 hours.
Preferably, described in step (2) ~ (4) aseptic and cultivate as cultivating in the band culture dish of fungi-proofing ventilated membrane or tissue culture bottle in the environment ventilated.
Because traditional callus and plantlet in vitro normally carry out sealed, sterile cultivation by sealed membrane in culture dish, CO in ware after sealing
2content consumes gradually along with tissue growth and can not get circulation exchange, limits the growth rate of callus to a certain extent.Therefore the condition of culture in step described in the present invention (2) ~ (4), except described light and temperature, no matter callus or plantlet in vitro, all need aseptic and grow under the condition of ventilating.
Further, described in step (2), the formula of callus inducing medium is: MS medium+3.0% sucrose+0.7wt% agar+3.0mg/L 2,4-D+2.0mg/L KT, pH is 5.8.
Further, described in step (3), the formula of differential medium is: MS medium+3.0wt% sucrose+0.7wt% agar+1.5mg/L 6-BA+0.5mg/L NAA, pH is 5.8.
Further, described in step (4), the formula of described root media is: 1/2MS medium+5.0wt% sucrose+1.0wt% agar, pH is 5.8.
The indoor rapid tissue that method provided by the invention can set up medium-sized pearl millet wild species fast cultivates regenerating system, be that next step studies the important foundation of its seed-setting and the low molecule mechanism of germination rate, and the method is simple to operate, has been easy to.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the effect diagram that illumination and dark condition cultivate to medium-sized pearl millet seed inoculated and cultured healing rate;
Fig. 2 is that illumination and dark condition cultivate the effect diagram medium-sized pearl millet seed inoculated and cultured being gone out to the more time;
Fig. 3 is that sealing tape ventilated membrane training method is to the effect diagram of healing rate, differentiation rate, rooting rate after medium-sized pearl millet seed inoculated and cultured;
Fig. 4 is the callus growth figure of application method of the present invention medium-sized pearl millet seed inoculated and cultured after 14 days;
Fig. 5 is the growth figure of the application medium-sized pearl millet Calli Differentiation of method of the present invention after 25 days;
Fig. 6 is the growth figure of application method of the present invention medium-sized pearl millet plantlet in vitro culture of rootage after 40 days;
Fig. 7 is that the medium-sized pearl millet training tissue culture seedling of application method of the present invention cultivates the growth figure after 3 days.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is commercially available.
embodiment 1
Of the present invention a kind of be that the medium-sized pearl millet tissue culture regeneration method step of explant is as follows with seed:
(1) raw-material choosing prepares and sterilization
The seed tap water choosing mature and plump is placed in superclean bench for several times, with the alcohol disinfecting 3min of 75%, the clorox (NaClO) of 5% is sterilized 10min, soaks 8 hours imbibitions after aseptic water washing 3 ~ 5 times on superclean bench with aseptic deionized water.
(2) inoculation of callus and cultivation
By the seed in step (1) with MS medium+1.0% sucrose+0.5% agar+2.0mg/L 2,4-D+1.0mg/L KT, and pH value is adjusted to 6.0 as the inducing culture of the callus of medium-sized pearl millet wild species, temperature 24 DEG C.Then the seed after sterilization is inoculated on above-mentioned medium, based on the Experimental comparison of illumination and dark culturing, adopt the shading training method that healing rate is higher, 20 seeds inoculated by the culture dish of the fungi-proofing ventilated membrane of each band, callus can be formed after 14 days, callus rate can reach 95%, and can be used for squamous subculture.Culture dish with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro are aseptic and grow under the condition of ventilating.
(3) differentiation of callus
By the callus in step (2) with MS medium+1.0% sucrose+0.5% agar+0.5mg/L 6-BA+0.2mg/L NAA, and pH value is adjusted to 6.0 as the differential medium of the callus of medium-sized pearl millet wild species.Hormone combination in this medium can promote that the cell in callus terminates Dedifferentiation, restarts the differentiation to each tissue fast.And be the differentiation promoting bud point fast, start photosynthesis, change condition of culture into illumination cultivation, wherein intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 DEG C.Can occur bud point after cultivating 14 days with the culture dish of fungi-proofing ventilated membrane, can grow blade and Gen Mao after 45 days, differentiation rate can reach 76%.
(4) plantlet in vitro culture of rootage
Blade will be differentiated in step (3) and to move in the tissue culture bottle of the fungi-proofing ventilated membrane of band on 1/2MS medium+2.0% sucrose+2.0% agar medium with the tissue of root hair, do not add any hormone, pH value is adjusted to 6.0, illumination cultivation conditional synchronization rapid (3), temperature 24 DEG C, cultivate its rooting rate after 25 days and can reach 80%, and its well developed root system, plantlet in vitro grows fine.
Condition of culture in step (2) ~ (4), except described light and temperature, no matter callus or plantlet in vitro, all need aseptic and grow under the condition of ventilating.
(5) hardening and transplanting
When plantlet in vitro grows to 8 ~ 10cm, open bottle cap hardening one week, can be transplanted in ordinary nutritional soil afterwards and normally cultivate.Watered a water every 2 days, keep water-retaining quantity among field of soil to reach 75%, after transplanting, survival rate can reach 95%, and completing whole take seed as the foundation of the medium-sized pearl millet tissue culture regeneration method of explant.
embodiment 2
Of the present invention a kind of be that the medium-sized pearl millet tissue culture regeneration method step of explant is as follows with seed:
(1) raw-material choosing prepares and sterilization
The seed tap water choosing mature and plump is placed in superclean bench for several times, with the alcohol disinfecting 3min of 75%, the clorox (NaClO) of 5% is sterilized 10min, soaks 8 hours imbibitions after aseptic water washing 3 ~ 5 times on superclean bench with aseptic deionized water.
(2) inoculation of callus and cultivation
By the seed in step (1) with MS medium+2.0% sucrose+1.0% agar+5.0mg/L 2,4-D+1.5mg/L KT, and pH value is adjusted to 5 as the inducing culture of the callus of medium-sized pearl millet wild species, temperature 26 DEG C.Then the seed after sterilization is inoculated on above-mentioned medium, based on the Experimental comparison of illumination and dark culturing, the shading that healing rate is higher is adopted to cultivate, 20 seeds inoculated by the culture dish of the fungi-proofing ventilated membrane of each band, callus can be formed after 11 days, callus rate can reach 98%, and can be used for squamous subculture.Culture dish with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro are aseptic and grow under the condition of ventilating.
(3) differentiation of callus
By the callus in step (2) with MS medium+2.0% sucrose+1.0% agar+1.0mg/L 6-BA+0.3mg/L NAA, and pH value is adjusted to 5.0 as the differential medium of the callus of medium-sized pearl millet wild species.Hormone combination in this medium can promote that the cell in callus terminates Dedifferentiation, restarts the differentiation to each tissue fast.And be the differentiation promoting bud point fast, start photosynthesis, change condition of culture into illumination cultivation, wherein intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 26 DEG C.Can occur bud point after cultivating 12 days with the culture dish of fungi-proofing ventilated membrane, can grow blade and Gen Mao after 30 days, differentiation rate can reach 82%.
(4) plantlet in vitro culture of rootage
Blade will be differentiated in step (3) and to move in the tissue culture bottle of the fungi-proofing ventilated membrane of band on 1/2MS medium+4.0% sucrose+3.0% agar medium with the tissue of root hair, do not add any hormone, pH value is adjusted to 5.0, illumination cultivation conditional synchronization rapid (3), cultivate its rooting rate after 30 days and can reach 92%, and its well developed root system, plantlet in vitro grows fine.Tissue culture bottle formula with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro grow under the condition aseptic and ventilation.
Condition of culture in step (2) ~ (4), except described light and temperature, no matter callus or plantlet in vitro, all need aseptic and grow under the condition of ventilating.
(5) hardening and transplanting
When plantlet in vitro grows to 8 ~ 10cm, open bottle cap hardening one week, can be transplanted in ordinary nutritional soil afterwards and normally cultivate.Watered a water every 2 days, keep water-retaining quantity among field of soil to reach 75%, after transplanting, survival rate can reach 95%, and completing whole take seed as the foundation of the medium-sized pearl millet tissue culture regeneration method of explant.
embodiment 3
Of the present invention a kind of be that the medium-sized pearl millet tissue culture regeneration method step of explant is as follows with seed:
(1) raw-material choosing prepares and sterilization
The seed tap water choosing mature and plump is placed in superclean bench for several times, with the alcohol disinfecting 3min of 75%, the clorox (NaClO) of 5% is sterilized 10min, soaks 12 hours imbibitions after aseptic water washing 3 ~ 5 times on superclean bench with aseptic deionized water.
(2) inoculation of callus and cultivation
By the seed in step (1) with MS medium+3.0% sucrose+0.7% agar+3.0mg/L 2,4-D+2.0mg/L KT, and pH value is adjusted to 5.8 as the inducing culture of the callus of medium-sized pearl millet wild species, temperature 24 DEG C.Then the seed after sterilization is inoculated on above-mentioned medium, based on the Experimental comparison of illumination and dark culturing, adopt the shading training method that healing rate is higher, 20 seeds inoculated by the culture dish of the fungi-proofing ventilated membrane of each band, callus can be formed after 7 days, callus rate can reach 95%, and can be used for squamous subculture.Culture dish with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro are aseptic and grow under the condition of ventilating.
(3) differentiation of callus
By the callus in step (2) with MS medium+3.0% sucrose+0.7% agar+1.0mg/L 6-BA+0.5mg/L NAA, and pH value is adjusted to 5.8 as the differential medium of the callus of medium-sized pearl millet wild species, hormone combination in this medium can promote that the cell in callus terminates Dedifferentiation, restarts the differentiation to each tissue fast.And be the differentiation promoting bud point fast, start photosynthesis, change condition of culture into illumination cultivation, wherein intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 DEG C.Can occur bud point after cultivating 7 days with the culture dish of fungi-proofing ventilated membrane, can grow blade and Gen Mao after 40 days, differentiation rate is 75%.
(4) plantlet in vitro culture of rootage
Blade will be differentiated in step (3) and to move in the tissue culture bottle of the fungi-proofing ventilated membrane of band on 1/2MS medium+3.0% sucrose+1.0% agar medium with the tissue of root hair, do not add any hormone, pH is 5.8, illumination cultivation conditional synchronization rapid (3), cultivate its rooting rate after 20 days and can reach 85%, and its well developed root system, plantlet in vitro grows fine.Tissue culture bottle formula with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro grow under the condition aseptic and ventilation.
Condition of culture in step (2) ~ (4), except described light and temperature, no matter callus or plantlet in vitro, all need aseptic and grow under the condition of ventilating.
(5) hardening and transplanting
When plantlet in vitro grows to 8 ~ 10cm, open bottle cap hardening one week, can be transplanted in ordinary nutritional soil afterwards and normally cultivate.Watered a water every 2 days, keep water-retaining quantity among field of soil to reach 70%, after transplanting, survival rate can reach 90%, and completing whole take seed as the foundation of the medium-sized pearl millet tissue culture regeneration method of explant.
embodiment 4
Of the present invention a kind of be that the medium-sized pearl millet tissue culture regeneration method step of explant is as follows with seed:
(1) raw-material choosing prepares and sterilization
The seed tap water choosing mature and plump is placed in superclean bench for several times, with the alcohol disinfecting 3min of 75%, the clorox (NaClO) of 5% is sterilized 10min, soaks 10 hours imbibitions after aseptic water washing 3 ~ 5 times on superclean bench with aseptic deionized water.
(2) inoculation of callus and cultivation
By the seed in step (1) with MS medium+3.0% sucrose+0.7% agar+3.0mg/L 2,4-D+1.0mg/L KT hormone, and pH value is adjusted to 5.8 as the inducing culture of the callus of medium-sized pearl millet wild species, temperature 24 DEG C.Then the seed after sterilization is inoculated on above-mentioned medium, based on the Experimental comparison of illumination and dark culturing, adopt the shading training method that healing rate is higher, 20 seeds inoculated by the culture dish of the fungi-proofing ventilated membrane of each band, callus can be formed after 10 days, callus rate can reach 96%, and can be used for squamous subculture.Culture dish with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro grow under the condition aseptic and ventilation.
(3) differentiation of callus
By the callus in step (2) with MS medium+3.0% sucrose+0.7% agar+1.5mg/L 6-BA+0.5mg/L NAA, and pH value is adjusted to 5.8 as the differential medium of the callus of medium-sized pearl millet wild species.Condition of culture changes illumination cultivation into, and wherein intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 DEG C.Can occur bud point after cultivating 10 days with the culture dish of fungi-proofing ventilated membrane, can grow blade and Gen Mao after 25 days, differentiation rate can reach 85%.
(4) plantlet in vitro culture of rootage
Blade will be differentiated in step (3) and train on medium with the group in the tissue culture bottle of the fungi-proofing ventilated membrane of tissue immigration band of root hair, group training culture medium prescription is: 1/2MS medium+5.0% sucrose+1.0% agar medium, do not add any hormone, pH is 5.8, illumination cultivation conditional synchronization rapid (3), cultivate its rooting rate after 14 days and can reach 89%, and its well developed root system, plantlet in vitro grows fine.Blake bottle with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro grow under the condition aseptic and ventilation.
(5) hardening and transplanting
When plantlet in vitro grows to 8 ~ 10cm, open bottle cap hardening one week, can be transplanted in ordinary nutritional soil afterwards and normally cultivate.Watered a water every 2 days, keep water-retaining quantity among field of soil to reach 70%, after transplanting, survival rate can reach 90%, and completing whole take seed as the foundation of the medium-sized pearl millet tissue culture regeneration method of explant.
The present embodiment extends Callus formation time and bud point divergaence time compared with embodiment 3, but improves differentiation rate, shorten blade and Gen Mao and root growth time, and rooting rate increases.
embodiment 5
Of the present invention a kind of be that the medium-sized pearl millet tissue culture regeneration method step of explant is as follows with seed:
(1) raw-material choosing prepares and sterilization
The seed tap water choosing mature and plump is placed in superclean bench for several times, with the alcohol disinfecting 3min of 75%, the clorox (NaClO) of 5% is sterilized 10min, soaks 8 hours imbibitions after aseptic water washing 3 ~ 5 times on superclean bench with aseptic deionized water.
(2) inoculation of callus and cultivation
By the seed in step (1) with MS medium+3.0% sucrose+0.7% agar+3.0mg/L 2,4-D+2.0mg/L KT, and pH value is adjusted to 5.8 as the inducing culture of the callus of medium-sized pearl millet wild species, temperature 24 DEG C.Then the seed after sterilization is inoculated on above-mentioned medium, based on the Experimental comparison of illumination and dark culturing, the shading that healing rate is higher is adopted to cultivate, 20 seeds inoculated by the culture dish of the fungi-proofing ventilated membrane of each band, callus can be formed after 7 days, callus rate can reach 95%, and can be used for squamous subculture.
(3) differentiation of callus
By the callus in step (2) with MS medium+3.0% sucrose+0.7% agar+1.5mg/L 6-BA+0.5mg/L NAA, and pH value is adjusted to 5.8 as the differential medium of the callus of medium-sized pearl millet wild species.Condition of culture changes illumination cultivation into, and wherein intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 DEG C.Can occur bud point after cultivating 9 days with the culture dish of fungi-proofing ventilated membrane, can grow blade and Gen Mao after 25 days, differentiation rate can reach 88%.
(4) plantlet in vitro culture of rootage
Blade will be differentiated in step (3) and to move in the tissue culture bottle of the fungi-proofing ventilated membrane of band on 1/2MS medium+5.0% sucrose+1.0% agar medium with the tissue of root hair, do not add any hormone, pH value is adjusted to 5.8, illumination cultivation conditional synchronization rapid (3), cultivate its rooting rate after 18 days and can reach 90%, and its well developed root system, plantlet in vitro grows fine.Tissue culture bottle formula with fungi-proofing ventilated membrane can ensure that callus and plantlet in vitro grow under the condition aseptic and ventilation.
Condition of culture in step (2) ~ (4), except described light and temperature, no matter callus or plantlet in vitro, all need aseptic and grow under the condition of ventilating.
(5) hardening and transplanting
When plantlet in vitro grows to 8 ~ 10cm, open bottle cap hardening one week, can be transplanted in ordinary nutritional soil afterwards and normally cultivate.Watered a water every 2 days, keep water-retaining quantity among field of soil to reach 75%, after transplanting, survival rate can reach 94%, and completing whole take seed as the foundation of the medium-sized pearl millet tissue culture regeneration method of explant.
The present embodiment in conjunction with the embodiments 3 and embodiment 4, obtains the shorter Callus formation time and organizes divergaence time, be advanced by Gen Mao and root growth time, and rooting rate obtaining increase.For optimum embodiment of the present invention.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. be the method setting up medium-sized pearl millet Tissue Culture Regeneration System fast of explant with seed, it is characterized in that: step is as follows:
(1) medium-sized pearl millet is raw-material chooses, sterilizes and soaks;
(2) inoculation of callus and cultivation: after the sterilization of step (1) being soaked, seed is inoculated in callus inducing medium, aseptic and under the environment ventilated shading cultivate, form callus; The formula of described callus inducing medium is: MS medium+1 ~ 3wt% sucrose+0.5 ~ 1.0wt% agar+2 ~ 5 mg/L 2,4-D+1 ~ 2 mg/L KT, and pH is 5 ~ 6;
(3) differentiation of callus: be inoculated in differential medium by the callus that step (2) obtains, in aseptic and illumination cultivation in the environment ventilated, obtains growing blade and with the tissue of root hair; Wherein, the formula of described differential medium is: MS medium+1 ~ 3wt% sucrose+0.5 ~ 1.0wt% agar+0.5 ~ 1.5 mg/L 6-BA+0.2 ~ 0.5 mg/L NAA, and pH is 5 ~ 6; The parameter of described illumination cultivation is: intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 ~ 26 DEG C;
(4) plantlet in vitro culture of rootage: step (3) is differentiated blade and tissue with root hair moves in root media, in aseptic and illumination cultivation in the environment ventilated, obtains the plantlet in vitro of well developed root system; Wherein, the formula of described root media is: 1/2MS medium+2.0 ~ 5.0wt% sucrose+1.0 ~ 3.0wt% agar, and pH is 5-6; The parameter of described illumination cultivation is: intensity of illumination is 50 μm of ol/ (m
2s), the photoperiod is 16h/d, temperature 24 ~ 26 DEG C;
(5) hardening and transplanting: carry out hardening when plantlet in vitro grows to 8 ~ 10cm, is transplanted afterwards and is normally cultivated.
2. method according to claim 1, is characterized in that: described in step (1) soak be by sterilization after medium-sized pearl millet seed put into aseptic deionized water, be placed on superclean bench and carry out under aseptic condition, described soak time is 8 ~ 12 hours.
3. method according to claim 1, is characterized in that: aseptic and cultivate as cultivating in the band culture dish of fungi-proofing ventilated membrane or tissue culture bottle in the environment ventilated described in step (2) ~ (4).
4., according to the arbitrary described method of claim 1-3, it is characterized in that: described in step (2), the formula of callus inducing medium is: MS medium+3.0% sucrose+0.7wt% agar+3.0mg/L 2,4-D+2.0mg/L KT, pH is 5.8.
5., according to the arbitrary described method of claim 1-3, it is characterized in that: described in step (3), the formula of differential medium is: MS medium+3.0wt% sucrose+0.7wt% agar+1.5mg/L 6-BA+0.5mg/L NAA, pH is 5.8.
6., according to the arbitrary described method of claim 1-3, it is characterized in that: described in step (4), the formula of root media is: 1/2MS medium+5.0wt% sucrose+1.0wt% agar, pH is 5.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510101660.3A CN104719158A (en) | 2015-03-09 | 2015-03-09 | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510101660.3A CN104719158A (en) | 2015-03-09 | 2015-03-09 | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104719158A true CN104719158A (en) | 2015-06-24 |
Family
ID=53444033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510101660.3A Pending CN104719158A (en) | 2015-03-09 | 2015-03-09 | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104719158A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105248280A (en) * | 2015-10-30 | 2016-01-20 | 王晓翔 | One-step seedling culturing tissue culture and rapid propagation method of pennisetum hydridum |
| CN106332781A (en) * | 2016-09-29 | 2017-01-18 | 遵义市龙驰生物科技有限公司 | High-efficiency expanding propagation method for crossbred Chinese pennisetum |
| CN109900533A (en) * | 2019-02-26 | 2019-06-18 | 齐鲁师范学院 | A kind of home position observation method of plant root hair |
| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
| CN115362939A (en) * | 2022-10-27 | 2022-11-22 | 西南林业大学 | Tissue culture method and application of pennisetum setosum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869054A (en) * | 2009-04-23 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method for pennisetum setaceum 'Rubrum' |
| CN101953304A (en) * | 2010-10-19 | 2011-01-26 | 赵国平 | Pennisetum americanum rapid propagation method and pennisetum americanum rapid virus-free propagation method |
| CN102204468A (en) * | 2011-05-13 | 2011-10-05 | 中国农业科学院兰州畜牧与兽药研究所 | Sexual propagation cultivation technique for medium-sized herb of Chinese pennisetum |
-
2015
- 2015-03-09 CN CN201510101660.3A patent/CN104719158A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869054A (en) * | 2009-04-23 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method for pennisetum setaceum 'Rubrum' |
| CN101953304A (en) * | 2010-10-19 | 2011-01-26 | 赵国平 | Pennisetum americanum rapid propagation method and pennisetum americanum rapid virus-free propagation method |
| CN102204468A (en) * | 2011-05-13 | 2011-10-05 | 中国农业科学院兰州畜牧与兽药研究所 | Sexual propagation cultivation technique for medium-sized herb of Chinese pennisetum |
Non-Patent Citations (3)
| Title |
|---|
| S AROCKIASAMY ET.AL.,: "Efficient protocols for in vitro regeneration of Pennisetum glaucum(L)Br.", 《INDIAN JOURNAL OF EXPERIMENTAL BIOLOGY》 * |
| 张晓莹等: "外源激素对紫穗狼尾草愈伤组织诱导及分化的影响", 《草业科学》 * |
| 龚束芳等: "狼尾草成熟种子的无菌播种与组织培养", 《植物生理学通讯》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105248280A (en) * | 2015-10-30 | 2016-01-20 | 王晓翔 | One-step seedling culturing tissue culture and rapid propagation method of pennisetum hydridum |
| CN106332781A (en) * | 2016-09-29 | 2017-01-18 | 遵义市龙驰生物科技有限公司 | High-efficiency expanding propagation method for crossbred Chinese pennisetum |
| CN109900533A (en) * | 2019-02-26 | 2019-06-18 | 齐鲁师范学院 | A kind of home position observation method of plant root hair |
| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
| CN115362939A (en) * | 2022-10-27 | 2022-11-22 | 西南林业大学 | Tissue culture method and application of pennisetum setosum |
| CN115362939B (en) * | 2022-10-27 | 2023-01-24 | 西南林业大学 | Tissue culture method and application of pennisetum setosum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102301951B (en) | Method for rapidly propagating roots of subprostrate sophora by tissue culture | |
| CN103931497B (en) | A kind of method improving dragon fruit plantlet in vitro planting percent | |
| CN101507415B (en) | In-vitro culture method of antlerpilose grass | |
| CN102845313A (en) | Method for quickly in-vitro actinidia kolomikta propagating | |
| CN104719158A (en) | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants | |
| CN104186351A (en) | Tissue culture method of strawberries | |
| CN104041412A (en) | Rapid propagation method for tissue culture of Guizhou hemiboea cavaleriei | |
| CN103392597A (en) | Tissue culture method of North American begonia | |
| CN104126506A (en) | Tissue culture method of America Lagerstroemia indica Red Rocket | |
| CN103430845A (en) | Strawberry tissue culturing method | |
| CN107211891B (en) | Konjak callus direct seeding and rapid propagation technology | |
| CN101897297B (en) | Two-step tissue culture quick propagation method for hemerocallis | |
| CN101803571A (en) | Tissue culture rapid propagation method of Rhizoma Typhonii Flagelliformis | |
| CN103444536B (en) | Method for reducing tissue culture production cost of hosta plantagineu | |
| CN102907326A (en) | Tissue culture propagation method for Medicagao Sativa L. | |
| CN106973796A (en) | A kind of tissue cultivating and seedling method of Idesia polycarpa | |
| CN105475129A (en) | Tissue-culture rapid propagation method for arundina graminifolia | |
| CN110558233A (en) | Method for disinfecting explant of stem segment with buds of black tiger and method for directly inducing aseptic buds to rapidly proliferate | |
| CN104488723A (en) | Tissue-culture and rapid-propagation method of epimedium koreanum nakai | |
| CN106665367B (en) | A kind of Golden Bell Tree quick breeding method for tissue culture | |
| CN109452170A (en) | A kind of method of sword-like iris seed root evoked callus culture | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN103477976A (en) | Stem tissue culture seedling method of dendrobium candidum | |
| CN101965799A (en) | Tissue culture quick propagation method for Opisthopappus Shih | |
| CN101518205B (en) | Sundew asepsis seeding reproduction method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150624 |