CN101953304A - Pennisetum americanum rapid propagation method and pennisetum americanum rapid virus-free propagation method - Google Patents
Pennisetum americanum rapid propagation method and pennisetum americanum rapid virus-free propagation method Download PDFInfo
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Abstract
The invention relates to a pennisetum americanum rapid propagation method and a pennisetum americanum rapid virus-free propagation method, which comprise the steps of preparation and sterilization of explants, inoculation, one-time seedling survival culture, root taking culture, seedling sorting and transplanting, virus-free detection and the like. The established pennisetum americanum virus-free tissue culture rapid propagation technology system has good virus-free effect with the root taking rate up to 100% and the transplanting survival rate of more than 95%. The system is applicable to industrial seedling culture, can be used for commercial production and is applicable to the industrial rapid propagation of pennisetum americanum sprouts, and thus, the lands used by seed propagation by the traditional method are greatly saved. The methods provide a powerful guaranty for large-scale industrial seedling culture.
Description
Technical field
The invention belongs to field of plant tissue culture technique, more specifically relate to the method for the quick breeding of a kind of American pennisetum alopecuroides group training detoxification Cheng Miao.
Background technology
Pearl millet is country's authorization kind, has the advantages that grass yield height, strong stress resistance, quality better, anti-cradling and do not have obvious sick worm harm the time of infertility, is high-quality green fodder of herbvore livestock and poultry and desirable water and soil conservation crop.In recent years, along with hybrid Chinese pennisetum high-quality paper pulp, medium density fiberboard production technology and constantly perfect as the biomass energy transformation technology, the demand of hybrid Chinese pennisetum is increased sharply.But when the kind large scale application, find that hybrid Chinese pennisetum has a certain proportion of assorted strain, obviously influence heterotic performance and utilization, it is relevant with purity to trace it to its cause.Pearl millet often suffers virus infraction, makes the product qualitative change bad, and output descends, and has also quickened deterioration of variety simultaneously.Relevant American pennisetum alopecuroides technology rapid detoxification propagation technique is not seen formal report as yet.
Tissue culture is a kind of propagation technique efficiently, adopts young tender lobus cardiacus or shoot apical meristem to cultivate, and also is the effective way that obtains anosis seedling.Traditional American pennisetum alopecuroides tissue culture and rapid propagation method, the subculture number of callus induction and differentiation is many, and aberration rate is higher, complex operation, pollution probability is big; Axillalry bud is cultivated and the stem apex vegetative cone is cultivated and can be reduced aberration rate effectively, but need the experience seedling and grow thickly that seedling is induced, subculture repeatedly such as enrichment culture and culture of rootage.Above-mentioned two approach will carry out the cultivation of some months at least to material usually, but the ability differentiation and seedling emergence, length consuming time, efficient is low.Therefore, press for the improvement innovation on the technical method,, improve American pennisetum alopecuroides group training efficient, realize that for real GAP cultivation and large-scale production provide technology platform, have bigger application prospect with the consumption that economizes on resources
Summary of the invention
In view of this, technical problem to be solved by this invention is: encroached on by disease at pearl millet in the prior art, make the product qualitative change bad, output descends, the shortcoming of deterioration of variety, provide a kind of and can breed American pennisetum alopecuroides in a large number, fast, can remove institute's poison and through special detection, to guarantee nontoxic detoxification, group training and the matching method that detects in spite of illness again.American pennisetum alopecuroides is realized from the tender lobus cardiacus of children and stem apex evoked callus to being divided into complete or semi-holonomic seedling, reach rapidly and efficiently, simplified control reduces aberration rate, save the purpose of cost, for a large amount of American pennisetum alopecuroides tissue cultural seedlings of free of producing lay a good foundation.
In order to achieve the above object, the present invention adopts following technical scheme to realize:
A kind of American pennisetum alopecuroides method for quickly breeding comprises drawing materials and sterilize, inoculating and carry out once-seedling forming cultivation, culture of rootage, acclimatization and transplants step of explant, it is characterized in that:
(1) drawing materials and sterilization steps of explant: get American pennisetum alopecuroides stem apex top or young tender lobus cardiacus, as the detoxication and tissue culture explant; Be to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 5～10min with mass concentration again, use aseptic water washing then 3～5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1～2cm, be inoculated on the inducing culture, place 20-25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20～23 ℃, humidity are 60%～65%, intensity of illumination is 70～80 μ mol cm-2s-1, illumination every day 12～14h be 1～1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, is that to be cultured to the long length of aseptic blastogenesis under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be that 2～5cm is little seedling in temperature;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be 6～8cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2-3cm, carry out acclimatization and transplants.
A kind of American pennisetum alopecuroides rapid detoxification propagation method, what comprise explant draws materials and sterilizes, inoculates steps such as carrying out once-seedling forming cultivation, culture of rootage, acclimatization and transplants, it is characterized in that:
(1) drawing materials and sterilization steps of explant: get American pennisetum alopecuroides stem apex top or young tender lobus cardiacus, as the detoxication and tissue culture explant; Be to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 5～10min with mass concentration again, use aseptic water washing then 3～5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1～2cm, be inoculated on the inducing culture, place 20-25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20～23 ℃, humidity are 60%～65%, intensity of illumination is 70～80 μ mol cm-2s-1, illumination every day 12～14h be 1～1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, is that to be cultured to the long length of aseptic blastogenesis under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be that 2～5cm is little seedling in temperature;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be 6～8cm in temperature.
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2-3cm, carry out acclimatization and transplants;
(7) virus ELISA detects: mosaic virus (the fritillary mosaicVirus that the field diseased plant is entrained, FMV) and Y virus (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into the virus-specific antiserum, the American pennisetum alopecuroides group being trained into seedling carry out the virus ELISA detection.
Wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also comprise the carragheen of IAA, 3000-4000mg/L of KT, 0.03～0.05mg/L of 0.3～0.5mg/L and mass concentration and be 1.5～2.0% white granulated sugar, the medium pH value is 5.0～5.5; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and also to comprise the NAA of 6-BA, 0.01～0.05mg/L of 0.05～0.1mg/L and mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), medium and also comprise the NAA of 0.5-1.5mg/L, and the IBA of 1-2.5mg/L and mass concentration are 2.0～2.5% white granulated sugar, the medium pH value is 5.6～6.0; The matrix of taking root in the step (6) is that 50% turfy soil, 25% vermiculite and 25% perlite are formed by mass ratio, and the water content of substrate of taking root is 30%～40%.
As shown from the above technical solution, the invention has the beneficial effects as follows:
Utilize tender lobus cardiacus of American pennisetum alopecuroides children and stem apex to produce a spot of embryo callus and be divided into regeneration plant, avoided callus to add in numerous process effectively, the appearance of variation has kept the kind of original material better;
American pennisetum alopecuroides virus (FMV and FVY) coat protein gene RT-PCR amplification, clone, prokaryotic expression and the high sensitivity ELISA detection architecture set up, use by this detection technique, can guarantee virus-free existence in the detoxification American pennisetum alopecuroides, propagate for further controlling virus disease, the American pennisetum alopecuroides variety rejuvenation is laid a good foundation;
The American pennisetum alopecuroides detoxication and tissue culture rapid propagation technical system of setting up, detoxification efficiency is good, its rooting rate reaches 100%, transplanting survival rate reaches more than 95%, be fit to factorial seedling growth, but commercialization production is applicable to that the batch production of American pennisetum alopecuroides seedling breeds fast, thereby has saved numerous kind of land used of conventional method greatly.For a large amount of factorial seedling growths, provide sound assurance.
Embodiment
In order to make those skilled in the art can further understand feature of the present invention and technology contents, the present invention is described in further detail by following examples.
Embodiment one
(1) drawing materials and sterilization steps of explant: May, fine weather is got American pennisetum alopecuroides stem apex top, as the detoxication and tissue culture explant; Being to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, using aseptic water washing then 3 times, is 0.1% mercuric chloride solution sterilization 5min again with mass concentration, uses aseptic water washing then 3 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1cm, be inoculated on the once-seedling forming medium, the mode that keeps stem section morphology upper end to make progress is vertically inserted the stem section in the medium, place 20 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20 ℃, humidity are 60%%, intensity of illumination is 70 μ mol cm-2s-1, illumination every day 12h be 1cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, in temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 25 ℃, humidity are 70%, intensity of illumination is 40 μ mol cm-2s-1, illumination every day 16h be 2cm, is little seedling;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25 ℃, humidity are 70%, intensity of illumination is 40 μ mol cm-2s-1, illumination every day 16h be 4cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2cm, carry out acclimatization and transplants; With become around the seedling take root the matrix compacting and on take root stromal surface and little branches and leaves face water spray slightly, be covered with preservative film then and cultivating indoor cultivation; Water 5 time/week in the mode of spraying every day, takes off film 6min ventilation every day sooner or later, and striping after 7 days was cultivated 7 days;
(7) virus ELISA detects: mosaic virus (the fritillary mosaicVirus that the field diseased plant is entrained, FMV), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into the virus-specific antiserum, the American pennisetum alopecuroides group being trained into seedling carry out the virus ELISA detection.
Wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also to comprise the carragheen of IAA, 3000mg/L of KT, 0.03mg/L of 0.3mg/L and mass concentration be 1.5% white granulated sugar, and the pH value is 5.0; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and also to comprise the NAA of 6-BA, 0.01mg/L of 0.05mg/L and mass concentration be 2.0% sucrose, and the pH value is 5.6; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.0% sucrose, and the pH value is 5.6; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), and also comprises the NAA of 0.5mg/L, and the IBA of 1mg/L and mass concentration are 2.0% white granulated sugar, and the pH value is 5.6; The matrix of taking root in the step (6) is that 50% turfy soil, 25% vermiculite and 25% perlite are formed by mass ratio, and the water content of substrate of taking root is 30%.
Embodiment two
(1) drawing materials and sterilization steps of explant: October, fine weather is got the tender lobus cardiacus of American pennisetum alopecuroides children, as the detoxication and tissue culture explant; Being to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, using aseptic water washing then 5 times, is 0.1% mercuric chloride solution sterilization 10min again with mass concentration, uses aseptic water washing then 5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 2cm, be inoculated on the once-seedling forming medium, the mode that keeps stem section morphology upper end to make progress is vertically inserted the stem section in the inducing culture, place 25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 23 ℃, humidity are 65%, intensity of illumination is 80 μ mol cm-2s-1, illumination every day 14h be 1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, in temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 28 ℃, humidity are 80%, intensity of illumination is 60 μ mol cm-2s-1, illumination every day 18h be 5cm, is little seedling;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 28 ℃, humidity are 80%, intensity of illumination is 60 μ mol cm-2s-1, illumination every day 18h be 6cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 2 axillalry buds and length be the stem section of 2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 3cm, carry out acclimatization and transplants; With become around the seedling take root the matrix compacting and on take root stromal surface and little branches and leaves face water spray slightly, be covered with preservative film then and cultivating indoor cultivation; Water 3 time/week in the mode of spraying every day, takes off film 6-10min ventilation every day sooner or later, and striping after 10 days was cultivated 10 days;
(7) virus ELISA detects: the Y virus that the field diseased plant is entrained (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into the virus-specific antiserum, the American pennisetum alopecuroides group being trained into seedling carry out the virus ELISA detection.
Wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also to comprise the carragheen of IAA, 4000mg/L of KT, 0.05mg/L of 0.5mg/L and mass concentration be 2.0% white granulated sugar, and the pH value is 5.5; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and the NAA and the mass concentration that also comprise 6-BA, the 0.05mg/L of 0.1mg/L be the sucrose of 2.5 %, and the pH value is 6.0; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.5% sucrose, and the pH value is 6.0; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), and also comprises the NAA of 1.5mg/L, and the IBA of 1-2.5mg/L and mass concentration are 2.5% white granulated sugar, and the pH value is 6.0; The matrix of taking root in the step (6) is that 50% turfy soil, 25% vermiculite and 25% perlite are formed by mass ratio, and the water content of substrate of taking root is 30%～40%.
Adopt technical scheme of the present invention, can obtain having the bottle seedling of the about 3-5cm of plant height of a small amount of root system about 40-50 days, about 50-70 days, obtain the complete seedling of American pennisetum alopecuroides of band root.One section American pennisetum alopecuroides taper can breed 100-200 strain seedling through 50-70 days time, utilized the present invention to simplify the program that the American pennisetum alopecuroides tissue cultivating seedling is produced, and saved cost, and the planting percent height has significantly shortened production cycle of American pennisetum alopecuroides tissue cultivating seedling.
But the above only is a preferable possible embodiments of the present invention, is not in order to limiting to claim of the present invention, thus the equivalent structure that all utilizations description of the present invention is done change, all in like manner within the scope of the present invention.
Claims (5)
1. an American pennisetum alopecuroides method for quickly breeding comprises drawing materials and sterilize, inoculating and carry out once-seedling forming cultivation, culture of rootage, acclimatization and transplants step of explant, it is characterized in that:
(1) drawing materials and sterilization steps of explant: get American pennisetum alopecuroides stem apex top or young tender lobus cardiacus, as the detoxication and tissue culture explant; Be to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 5～10min with mass concentration again, use aseptic water washing then 3～5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1～2cm, be inoculated on the inducing culture, place 20-25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20～23 ℃, humidity are 60%～65%, intensity of illumination is 70～80 μ mol cm-2s-1, illumination every day 12～14h be 1～1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, is that to be cultured to the long length of aseptic blastogenesis under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be that 2～5cm is little seedling in temperature;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be 6～8cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2-3cm, carry out acclimatization and transplants.
2. a kind of American pennisetum alopecuroides method for quickly breeding according to claim 1, it is characterized in that: wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also comprise the carragheen of IAA, 3000-4000mg/L of KT, 0.03～0.05mg/L of 0.3～0.5mg/L and mass concentration and be 1.5～2.0% white granulated sugar, the medium pH value is 5.0～5.5; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and also to comprise the NAA of 6-BA, 0.01～0.05mg/L of 0.05～0.1mg/L and mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), medium and also comprise the NAA of 0.5-1.5mg/L, and the IBA of 1-2.5mg/L and mass concentration are 2.0～2.5% white granulated sugar, the medium pH value is 5.6～6.0; The matrix of taking root in the step (6) is that 50% turfy soil, 25% vermiculite and 25% perlite are formed by mass ratio, and the water content of substrate of taking root is 30%～40%.
3. American pennisetum alopecuroides rapid detoxification propagation method, what comprise explant draws materials and sterilizes, inoculates steps such as carrying out once-seedling forming cultivation, culture of rootage, acclimatization and transplants, detoxification detection, it is characterized in that:
(1) drawing materials and sterilization steps of explant: get American pennisetum alopecuroides stem apex top or young tender lobus cardiacus, as the detoxication and tissue culture explant; Be to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 5～10min with mass concentration again, use aseptic water washing then 3～5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1～2cm, be inoculated on the inducing culture, place 20-25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20～23 ℃, humidity are 60%～65%, intensity of illumination is 70～80 μ mol cm-2s-1, illumination every day 12～14h be 1～1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, is that to be cultured to the long length of aseptic blastogenesis under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be that 2～5cm is little seedling in temperature;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be 6～8cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2-3cm, carry out acclimatization and transplants;
(7) virus ELISA detects: mosaic virus (the fritillary mosaicVirus that the field diseased plant is entrained, FMV) and Y virus (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into the virus-specific antiserum, the American pennisetum alopecuroides group being trained into seedling carry out the virus ELISA detection.
4. a kind of American pennisetum alopecuroides rapid detoxification propagation method according to claim 3, it is characterized in that: wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also comprise the carragheen of IAA, 3000-4000mg/L of KT, 0.03～0.05mg/L of 0.3～0.5mg/L and mass concentration and be 1.5～2.0% white granulated sugar, the medium pH value is 5.0～5.5; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and also to comprise the NAA of 6-BA, 0.01～0.05mg/L of 0.05～0.1mg/L and mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), medium and also comprise the NAA of 0.5-1.5mg/L, and the IBA of 1-2.5mg/L and mass concentration are 2.0～2.5% white granulated sugar, the medium pH value is 5.6～6.0; The matrix of taking root in the step (6) is that 50% turfy soil, 25% vermiculite and 25% perlite are formed by mass ratio, and the water content of substrate of taking root is 30%～40%.
5. American pennisetum alopecuroides rapid detoxification propagation method, what comprise explant draws materials and sterilizes, inoculates steps such as carrying out once-seedling forming cultivation, culture of rootage, acclimatization and transplants, it is characterized in that:
(1) drawing materials and sterilization steps of explant: get American pennisetum alopecuroides stem apex top or young tender lobus cardiacus, as the detoxication and tissue culture explant; Be to be 75% alcohol solution dipping 60s again with another part mass concentration behind 75% the alcohol solution dipping 30s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 5～10min with mass concentration again, use aseptic water washing then 3～5 times;
(2) inducing culture step: under aseptic condition, the explant that disinfects is cut into the stem section that 1 terminal bud or axillalry bud and length are 1～2cm, be inoculated on the inducing culture, place 20-25 ℃ dark condition to cultivate 5 days down, change illumination condition then over to and cultivate, temperature is that to be cultured to the long length of aseptic blastogenesis under the condition that 20～23 ℃, humidity are 60%～65%, intensity of illumination is 70～80 μ mol cm-2s-1, illumination every day 12～14h be 1～1.5cm;
(3) successive transfer culture step: the aseptic bud of hyperplasia is transferred together in subculture medium, is that to be cultured to the long length of aseptic blastogenesis under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be that 2～5cm is little seedling in temperature;
(4) strong seedling culture step: each little seedling is inoculated in the strong seedling culture base, is that to be cultured to little branch growth length under the condition that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ mol cm-2s-1, illumination every day 16～18h be 6～8cm in temperature;
(5) culture of rootage step: will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, and be inoculated in root media then piecemeal and carry out culture of rootage;
(6) acclimatization and transplants step: culture of rootage 30 days and root system are reached the complete seedling of 2-3cm, carry out acclimatization and transplants; With around the seedling take root the matrix compacting and on take root stromal surface and little branches and leaves face water spray slightly, be covered with preservative film then and cultivating indoor cultivation; Water 5 time/week in the mode of spraying every day, takes off film 6min ventilation every day sooner or later, and striping after 7 days was cultivated 7 days;
(7) virus ELISA detects: mosaic virus (the fritillary mosaicVirus that the field diseased plant is entrained, FMV) and Y virus (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into the virus-specific antiserum, the American pennisetum alopecuroides group being trained into seedling carry out the virus ELISA detection;
Wherein inducing culture is to be minimal medium with improvement 1/2MS solid culture medium in the step (2), and also comprise the carragheen of IAA, 3000-4000mg/L of KT, 0.03～0.05mg/L of 0.3～0.5mg/L and mass concentration and be 1.5～2.0% white granulated sugar, the medium pH value is 5.0～5.5; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step (3), and also to comprise the NAA of 6-BA, 0.01～0.05mg/L of 0.05～0.1mg/L and mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; The strong seedling culture base is to be minimal medium with the solid medium of MS in the step (4), and also to comprise mass concentration be 2.0～2.5% sucrose, and the medium pH value is 5.6～6.0; Root media is to be minimal medium with the solid medium of improvement 1/4MS in the step (5), medium and also comprise the NAA of 0.5-1.5mg/L, and the IBA of 1-2.5mg/L and mass concentration are 2.0～2.5% white granulated sugar, the medium pH value is 5.6～6.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104719158A (en) * | 2015-03-09 | 2015-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants |
| 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 |
| CN107155875A (en) * | 2017-07-03 | 2017-09-15 | 中国科学院亚热带农业生态研究所 | A kind of allopolyploid induction production method of hybrid Chinese pennisetum |
| CN112655556A (en) * | 2020-12-22 | 2021-04-16 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture detoxification and rapid propagation method of wangcao |
| CN114931095A (en) * | 2022-05-25 | 2022-08-23 | 四川农业大学 | Method for regenerating plant by tissue culture of mature embryo of pennisetum americanum |
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2010
- 2010-10-19 CN CN2010105110654A patent/CN101953304A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104719158A (en) * | 2015-03-09 | 2015-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants |
| 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 |
| CN107155875A (en) * | 2017-07-03 | 2017-09-15 | 中国科学院亚热带农业生态研究所 | A kind of allopolyploid induction production method of hybrid Chinese pennisetum |
| CN112655556A (en) * | 2020-12-22 | 2021-04-16 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture detoxification and rapid propagation method of wangcao |
| CN114931095A (en) * | 2022-05-25 | 2022-08-23 | 四川农业大学 | Method for regenerating plant by tissue culture of mature embryo of pennisetum americanum |
| CN114931095B (en) * | 2022-05-25 | 2023-02-07 | 四川农业大学 | Method for regenerating plant by tissue culture of mature embryo of pennisetum americanum |
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