CN104115750A - Method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation - Google Patents
Method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation Download PDFInfo
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Abstract
The invention discloses a method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation. The method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation comprises the following steps: 1) selecting an implant and carrying out pre-treatment; 2) peeling stem tips; 3) carrying out induction culture on the stem tips; 4) carrying out multiplication culture; 5) carrying out rooting culture; 6) culturing virus-free seedlings; and 7) detecting viruses and bacteria. The method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation has the advantages that a stem tip virus removal method, a high-temperature treatment virus removal method and a chemical reagent virus removal method are combined, a good virus removal effect in tissue culture and rapid propagation of prunus salicina seedling culture is realized, the obtained virus-free seedlings are subjected to virus detection, and results show that viruses of all the seedlings are successfully removed, and no prune dwarf virus (PDV) or prunus necrotic ringspot virus (PNRSV) infection is found. By utilizing the method disclosed by the invention for carrying out virus removal on prunus salicina, new virus-free prunus salicina seedlings are cultured through tissue culture, and then domestication and transplantation are carried out, so that the aims of increasing the yield and improve the fruit flavour are realized.
Description
Technical field
The present invention relates to the method for a kind of hollow plum tissue-culturing rapid propagation detoxification, belong to plant tissue culture fast breeding technique field and field of plant cell engineering technology.
Technical background
The viroses of plant are the Major Diseases in agricultural production, and the serious yield and quality harming the crops, once the viroses of plant show symptom, be treated very difficult.Sand hollow plum is Guizhou Province's characteristic fruit in county's sand town along the river, the time limit of plantation is very long, in the process of cultivation, subject to the harm of disease worm, the major way of breeding is at present all the method that adopts vegetative propagation grafting, causes fruit tree to accumulate virus, due to the harm of virus disease, have a strong impact on its fruit yield, quality, just do not making fruit fruit type, mouthfeel reduces, quality declines, yield reducation.We carry out Electronic Speculum detection to the hibernaculum gathering on county's sand town hollow plum nursery stock along the river, detected Prune dwarf virus (PDV), the rose mosaic virus (PNRSV) of the growth of main harm plum, this is 2 kinds of objects of generally acknowledging in the world in the main 3 kinds of viruses of harm Prunus.Because virus is day by day serious to plant hazard, research and the control of countries in the world to virus harm all very paid attention to, and obtained successful experience.Therefore, ensure fine quality and the output of this geographical indication characteristic fruit of sand hollow plum, necessary it be carried out to the research work of detoxification.
Detoxification generally adopts stem apex detoxify method, high temperature treatment detoxicity method and three kinds of modes of chemical reagent detoxicity method.
The cardinal principle of stem apex detoxify method is the skewness of virus in plant body, with plant position and age and different, wherein especially growing point (0.1-1.0mm region) band virus is minimum or be not with virus for stem apex part, and the closer to tip, virus concentration is lower, infects also lower.Because virus shifts with skeleton screen casing in host plant body, in shoot apical meristem, there is no fibrovascular system, viral movement difficulty, so, shoot apical meristem is very not low containing virion or virus concentration, and the transfer velocity of virus in host's shoot apical meristem lags behind the growth rate of stem apex, causes near the virus concentration of apical meristem low, even band virus, just can not obtain virus-free regeneration plant by stem apex cultured in vitro.The key of stem apex detoxify is stem apex size and virus elimination rate, because the success rate of detoxification is inversely proportional to the size of the shoot apical meristem that cuts, must cut enough little shoot apical meristem (0.2-0.5mm) in order to improve virus elimination rate.But too little shoot apical meristem regeneration plant is very difficult.Because regeneration capacity is directly proportional to merismatic size.
High temperature detoxification ratio juris is that high temperature can suppress viral propagation, slow down the diffusion velocity of virus in plant body, and high temperature has to many plants its growth of acceleration.For different plants, different cultivars and the different plant virus feature different to the sensitivity of temperature, to needing the plant of detoxification to carry out the heating power processing in uniform temperature and time range, make the growth rate of plant obviously exceed the speed that virus spreads in plant body, thereby make a part just at mushroom plant tissue, if terminal bud, tender tip be not slightly containing virus, not cutting and be inoculated on suitable medium containing virulent part, finally turn out nontoxic plant.The method of thermal treatment detoxification mainly contains 2 kinds: hot-water soak and high temperature air processing.For adopting stem apex to be difficult to remove viral plant, often adopt the method for high temperature treatment and stem apex combination, the shoot apical meristem size that combined with heat treatment is suitable is the key factor that obtains virus-free plant.But application thermal treatment detoxification also has certain limitation, and major defect is not to be that all virus is all to thermal treatment sensitivity.
Viral inhibitors is processed antiviral: chemical agent can remove plant virus to some extent, is mainly the metabolic way of utilizing antiviral chemical agent to suppress the synthetic of viral nucleic acid and protein or changing host.The poison-removing method that adopts viral inhibitors to combine with Shoot Tip Culture, can remove multiple virus relatively easily, and this method requires not tight to drawing materials.Inoculation stem apex can be greater than lmm, is easy to differentiation and seedling emergence, improves survival rate.At present mainly comprise purine and pyrimidine analogue, amino acid, antibiotic etc. for the antiviral agent of plant toxic, as 9-(2-hydroxyl dioxy) methyl guanine, 2. thiouracil, retrovir, 2,4-oxygen six hydrogen triazines (DHT) and similar purine bases metabolite ribavirin.Above-mentioned medicament can, suppressing to a certain degree in plant corpus or excised leaf inner virus synthetic, make its inactivation but still can not suppress virus completely.
The independent use of above several method has drawback, the more difficult removal of some virus, use separately wherein some methods to be difficult to succeed, according to our experience for many years, adopting the combine method processing of three-in-one detoxification of thermal treatment, antiviral agent and Shoot Tip Culture, is a kind of simple and efficient, efficient method.
Summary of the invention
The object of the invention is to, the method for a kind of hollow plum tissue-culturing rapid propagation detoxification is provided.Utilize the method to hollow lijin's detoxification treatment, cultivate the virus-free hollow plum sapling making new advances by group, then through domestication and transplanting, thereby reach increase output, improve the object of fruit mouthfeel.Concrete technical scheme is as follows:
A method for hollow plum tissue-culturing rapid propagation detoxification, comprises the steps:
1) plant choosing and pretreatment of body: the healthy and strong branch on sand hollow plum plant is cut, after processing with root-growing agent, cuttage is on treated artificial substratum, proceed in culturing room wait surviving after growing sprouting, cultivate 14-18 hour every day under 35-40 DEG C, 2000-3000Lux illumination condition, then under normal temperature, dark condition, cultivate 6-10 hour, cultivate altogether 28-35 days;
2) peeling off of stem apex: the step 1 of learning from else's experience) stem eye that pretreated hollow plum is sturdy, peel off blade, after rinsing well, carry out routine disinfection, then stem eye is placed under 40 times of anatomical lens, blade and leaf primordium are peeled off with nontoxic dissecting needle, isolate the meristematic tissue on stem eye top, then it is seeded in inducing culture fast;
3) induction of stem apex is cultivated: the stem apex of having inoculated in inducing culture is placed in and cultivates 14-18 hour every day under 22-28 DEG C, the illumination condition of 1500-2000Lux, until grow up to seedling;
4) propagation is cultivated: grow to 1-1.5cm when high until seedling, cut stem apex part, transfer on proliferated culture medium and continue to cultivate, condition of culture is: 32-38 DEG C of every day, 1500-2000Lux illumination 12-16 hour, and normal temperature is cultivated 8-12 hour; When height of seedling 2cm, the little seedling leaf of clip carries out virus and detects, and by detecting the virus-free seedling of rear confirmation, segment is transferred to and on proliferated culture medium, continued to be cultured to 10-20 times of quantity;
5) culture of rootage: by step 4) seedling that forms of propagation is divided into individual plant, transfers on root media, carries out at normal temperatures culture of rootage, growing up to the group training seedling of taking root;
6) cultivation of detoxic seedling: the seedling of having taken root is taken out in bottle, clean the medium of root, dry, then in middle antiviral agent, soak, the liver moss bag root of crossing by sterilization treatment, is planted in fry alms bowl or seedling-cultivating tray, be placed in the warm canopy with fly net and cultivate, after seedling grows into normal plant, clip branch cutting, the detoxic seedling of incubation production use;
7) viral Pathogen detection: adopt herbaceous plant instruction detection method or Electronic Speculum detection method to step 5) detoxic seedling detect.
Particularly, described step 1) in root-growing agent and processing method be: the aqueous solution of getting IBA and the each 25mg/L of NAA, soak the healthy and strong branch incision of hollow plum oral area 30min, artificial substratum consists of: perlite, vermiculite and loam are according to the ratio of mass ratio 3:3:4.
Particularly, described step 2) in the length of stem eye be 10-20mm, separating the merismatic length of stem apex is 0.5-1.5mm, when shoot apical meristem is cultivated, can be with 1-2 piece of spire to improve survival rate.
Particularly, step 2)-5) in each culture medium prescription as following table, pH value is 5.8:
The each medium constituent of table 1 and use scale
Particularly, described step 4) in stem apex size be 10-15mm;
Particularly, described step 5) in the culture of rootage time be 30-45d, the group of taking root training seedling is cultured to root number more than 3, length is more than 20mm.
Particularly, described step 6) in antiviral agent be the solution that contains IBA15-20mg/L, NAA15-20mg/L.
Particularly, described step 7) in herbaceous plant instruction detection method be:
Cucumber and elder brother's promise lamb's-quarters are planted respectively in advance in the matrix of sterilizing, 2 of the every little basin plantations of cucumber, one of the every little basin plantation of elder brother's promise lamb's-quarters, is cultured to multi-disc true leaf and grows stand-by.When inoculation, from treat measuring plants, getting 1～3g spire puts in mortar, add a small amount of water and equivalent 0.1mol/L phosphate buffer (pH is 7.0), wear into homogenate, draw a small amount of slurries and be coated in and scribble 1000 order emery grit parts, gently friction so that slurries can immerse blade table chrotoplast not damaged blade be degree.After approximately 5 minutes, rinse blade face with clear water, the indicator plant being vaccinated is placed in the greenhouse or culturing room's (15～25 DEG C) of anti-aphid guard.As the slurries of inoculation contain virus, there is visible symptom in indicator plant after a couple of days to several weeks.
Utilize the method for tissue-culturing rapid propagation of the present invention to hollow lijin's detoxification treatment, Japanese plum has following features: (1) virus removal seedling growing way is prosperous, and blade light is bud green, stretches nature, and resistance against diseases strengthens; (2) the individual plant number of blooming is many, and No. of inflorescences, fruit-setting rate on average increase approximately 50%, and without malformed fruit; (3) fruit outward appearance is good, and color and luster is vivid, evenly neat, and fruit is individual large, maximum single fruit weight 60-80 gram; (4) output is high, good in economic efficiency, virus removal fruit tree than former kind not the every strain of detoxification fruit tree can increase production 30-50%, have broad application prospects.
Embodiment:
Below adopt specific embodiment and experimental example, further prove effect of the present invention:
Embodiment 1
A method for hollow plum tissue-culturing rapid propagation detoxification, comprises the steps:
1) plant choosing and pretreatment of body: the healthy and strong branch on sand hollow plum plant is cut, after processing with root-growing agent, cuttage is on treated artificial substratum, proceed in culturing room wait surviving after growing sprouting, cultivate 16 hours every day under 38 DEG C, 2500Lux illumination condition, then under normal temperature, dark condition, cultivate 8 hours, cultivate altogether 30 days;
2) peeling off of stem apex: the step 1 of learning from else's experience) stem eye that pretreated hollow plum is sturdy, length is 15mm, peel off blade, after rinsing well, carry out routine disinfection, then stem eye is placed under 40 times of anatomical lens, with nontoxic dissecting needle, blade and leaf primordium is peeled off, isolate the meristematic tissue on stem eye top, length is 1.0mm, then it is seeded in inducing culture fast;
3) induction of stem apex is cultivated: the stem apex of having inoculated in inducing culture is placed in and cultivates 16 hours every day under 25 DEG C, the illumination condition of 2000Lux, until grow up to seedling;
4) propagation is cultivated: grow to 1-1.5cm when high until seedling, cut stem apex part, length is 10mm, transfers on proliferated culture medium and continues to cultivate, and condition of culture is: 35 DEG C of every days, 2000Lux illumination 14 hours, and normal temperature is cultivated 10 hours; When height of seedling 2cm, the little seedling leaf of clip carries out virus and detects, and by detecting the virus-free seedling of rear confirmation, segment is transferred to and on proliferated culture medium, continued to be cultured to 15 times of quantity;
5) culture of rootage: by step 4) seedling that forms of propagation is divided into individual plant, transfers on root media, and culture of rootage 30d at normal temperatures, growing up to the group training seedling of taking root, and root number is more than 3, and length is more than 20mm;
6) cultivation of detoxic seedling: the seedling of having taken root is taken out in bottle, clean the medium of root, dry, then in middle antiviral agent, soak, the liver moss bag root of crossing by sterilization treatment, is planted in fry alms bowl or seedling-cultivating tray, be placed in the warm canopy with fly net and cultivate, after seedling grows into normal plant, clip branch cutting, the detoxic seedling of incubation production use; Described antiviral agent is the solution that contains IBA15mg/L, NAA15mg/L;
7) viral Pathogen detection: adopt herbaceous plant instruction detection method to step 5) detoxic seedling detect.
Wherein, step 2)-5) in each medium composition and formula as table 1.
Embodiment 2
A method for hollow plum tissue-culturing rapid propagation detoxification, comprises the steps:
1) plant choosing and pretreatment of body: the healthy and strong branch on sand hollow plum plant is cut, after processing with root-growing agent, cuttage is on treated artificial substratum, proceed in culturing room wait surviving after growing sprouting, cultivate 14 hours every day under 40 DEG C, 3000Lux illumination condition, then under normal temperature, dark condition, cultivate 10 hours, cultivate altogether 28 days;
2) peeling off of stem apex: the step 1 of learning from else's experience) stem eye that pretreated hollow plum is sturdy, length is 10mm, peel off blade, after rinsing well, carry out routine disinfection, then stem eye is placed under 40 times of anatomical lens, with nontoxic dissecting needle, blade and leaf primordium is peeled off, isolate the meristematic tissue on stem eye top, length is 0.5mm, then it is seeded in inducing culture fast;
3) induction of stem apex is cultivated: the stem apex of having inoculated in inducing culture is placed in and cultivates 14 hours every day under 28 DEG C, the illumination condition of 2000Lux, until grow up to seedling;
4) propagation is cultivated: grow to 1-1.5cm when high until seedling, cut stem apex part, length is 15mm, transfers on proliferated culture medium and continues to cultivate, and condition of culture is: 38 DEG C of every days, 2000Lux illumination 12 hours, and normal temperature is cultivated 12 hours; When height of seedling 2cm, the little seedling leaf of clip carries out virus and detects, and by detecting the virus-free seedling of rear confirmation, segment is transferred to and on proliferated culture medium, continued to be cultured to 10 times of quantity;
5) culture of rootage: by step 4) seedling that forms of propagation is divided into individual plant, transfers on root media, and culture of rootage 40d at normal temperatures, growing up to the group training seedling of taking root, and root number is more than 3, and length is more than 20mm;
6) cultivation of detoxic seedling: the seedling of having taken root is taken out in bottle, clean the medium of root, dry, then in middle antiviral agent, soak, the liver moss bag root of crossing by sterilization treatment, is planted in fry alms bowl or seedling-cultivating tray, be placed in the warm canopy with fly net and cultivate, after seedling grows into normal plant, clip branch cutting, the detoxic seedling of incubation production use; Described antiviral agent is the solution that contains IBA20mg/L, NAA15mg/L;
7) viral Pathogen detection: adopt Electronic Speculum detection method to step 5) detoxic seedling detect.
Wherein, step 2)-5) in each medium composition and formula as table 1.
Embodiment 3 soluble concentrates
A method for hollow plum tissue-culturing rapid propagation detoxification, comprises the steps:
1) plant choosing and pretreatment of body: the healthy and strong branch on sand hollow plum plant is cut, after processing with root-growing agent, cuttage is on treated artificial substratum, proceed in culturing room wait surviving after growing sprouting, cultivate 18 hours every day under 35 DEG C, 2000Lux illumination condition, then under normal temperature, dark condition, cultivate 6 hours, cultivate altogether 35 days;
2) peeling off of stem apex: the step 1 of learning from else's experience) stem eye that pretreated hollow plum is sturdy, length is 20mm, peel off blade, after rinsing well, carry out routine disinfection, then stem eye is placed under 40 times of anatomical lens, with nontoxic dissecting needle, blade and leaf primordium is peeled off, isolate the meristematic tissue on stem eye top, length is 1.5mm, then it is seeded in inducing culture fast;
3) induction of stem apex is cultivated: the stem apex of having inoculated in inducing culture is placed in and cultivates 18 hours every day under 22 DEG C, the illumination condition of 1500Lux, until grow up to seedling;
4) propagation is cultivated: grow to 1-1.5cm when high until seedling, cut stem apex part, length is 12mm, transfers on proliferated culture medium and continues to cultivate, and condition of culture is: 32 DEG C of every days, 1500Lux illumination 16 hours, and normal temperature is cultivated 8 hours; When height of seedling 2cm, the little seedling leaf of clip carries out virus and detects, and by detecting the virus-free seedling of rear confirmation, segment is transferred to and on proliferated culture medium, continued to be cultured to 20 times of quantity;
5) culture of rootage: by step 4) seedling that forms of propagation is divided into individual plant, transfers on root media, and culture of rootage 45d at normal temperatures, growing up to the group training seedling of taking root, and root number is more than 3, and length is more than 20mm;
6) cultivation of detoxic seedling: the seedling of having taken root is taken out in bottle, clean the medium of root, dry, then in middle antiviral agent, soak, the liver moss bag root of crossing by sterilization treatment, is planted in fry alms bowl or seedling-cultivating tray, be placed in the warm canopy with fly net and cultivate, after seedling grows into normal plant, clip branch cutting, the detoxic seedling of incubation production use; Described antiviral agent is the solution that contains IBA15mg/L, NAA20mg/L;
7) viral Pathogen detection: adopt herbaceous plant instruction detection method or Electronic Speculum detection method to step 5) detoxic seedling detect.
Wherein, step 2)-5) in each medium composition and formula as table 1.
Comparative example 1: contrast with embodiment 1, preparation method is with embodiment 1, wherein by preparation process 1)-4) in high temperature light treatment scheme cancel.
Comparative example 2: contrast with embodiment 1, preparation method is with embodiment 1, wherein cancellation step 2)-5) in antiviral agent in each medium, cancel the use of 6-BA, NAA and ribavirin.
Experimental example:
The virus of harm plum growth is mainly Prune dwarf virus (PDV), rose mosaic virus (PNRSV), what the method for employing embodiment 1-3 and comparative example 1-2 was obtained grows seedlings, taking virus examination result, the growing way of growing seedlings, fruit-setting rate, fruit properties, rate of growth as index, to evaluate the detoxification efficiency of the inventive method.Result is as following table 2:
(1) virus removal seedling growing way is prosperous, and blade light is bud green, stretches nature, and resistance against diseases strengthens; (2) the individual plant number of blooming is many, and No. of inflorescences, fruit-setting rate on average increase approximately 50%, and without malformed fruit; (3) fruit outward appearance is good, and color and luster is vivid, evenly neat, and fruit is individual large, maximum single fruit weight 60-80 gram;
The contrast of table 2 detoxification efficiency
As can be seen from the above table, adopt poison-removing method of the present invention, stem apex detoxify, high temperature treatment detoxicity method and three kinds of modes of chemical reagent detoxicity method are combined, the tissue that sand hollow plum is grown seedlings is cultivated, fast breeding has good virus removal effect, the detoxic seedling obtaining detects through virus, all equal detoxification successes of growing seedlings, infect without Prune dwarf virus (PDV), rose mosaic virus (PNRSV).Compare with comparative example 2 (not using antiviral agent scheme) with comparative example 1 (not using high temperature illumination solution), the detoxification efficiency of hollow plum fruit tree seedling culture obviously improves, and in the planting process in later stage, there is clear superiority at aspects such as the growing way of growing seedlings, fruit-setting rate, fruit properties, ratio is detoxification fruit tree volume increase 30-50% not, than the fruit tree volume increase 20-25% of comparative example 1 and 2 method processing.Illustrate and adopt technical scheme of the present invention, improved the yield and quality of Lee fruit, have a good application prospect.
Claims (9)
1. a method for hollow plum tissue-culturing rapid propagation detoxification, comprises the steps:
1) plant choosing and pretreatment of body: the healthy and strong branch on sand hollow plum plant is cut, after processing with root-growing agent, cuttage is on treated artificial substratum, proceed in culturing room wait surviving after growing sprouting, cultivate 14-18 hour every day under 35-40 DEG C, 2000-3000Lux illumination condition, then under normal temperature, dark condition, cultivate 6-10 hour, cultivate altogether 28-35 days;
2) peeling off of stem apex: the step 1 of learning from else's experience) stem eye that pretreated hollow plum is sturdy, peel off blade, after rinsing well, carry out routine disinfection, then stem eye is placed under 40 times of anatomical lens, blade and leaf primordium are peeled off with nontoxic dissecting needle, isolate the meristematic tissue on stem eye top, then it is seeded in inducing culture fast;
3) induction of stem apex is cultivated: the stem apex of having inoculated in inducing culture is placed in and cultivates 14-18 hour every day under 22-28 DEG C, the illumination condition of 1500-2000Lux, until grow up to seedling;
4) propagation is cultivated: grow to 1-1.5cm when high until seedling, cut stem apex part, transfer on proliferated culture medium and continue to cultivate, condition of culture is: 32-38 DEG C of every day, 1500-2000Lux illumination 12-16 hour, and normal temperature is cultivated 8-12 hour; When height of seedling 2cm, the little seedling leaf of clip carries out virus and detects, and by detecting the virus-free seedling of rear confirmation, segment is transferred to and on proliferated culture medium, continued to be cultured to 10-20 times of quantity;
5) culture of rootage: by step 4) seedling that forms of propagation is divided into individual plant, transfers on root media, carries out at normal temperatures culture of rootage, growing up to the group training seedling of taking root;
6) cultivation of detoxic seedling: the seedling of having taken root is taken out in bottle, clean the medium of root, dry, then in middle antiviral agent, soak, the liver moss bag root of crossing by sterilization treatment, is planted in fry alms bowl or seedling-cultivating tray, be placed in the warm canopy with fly net and cultivate, after seedling grows into normal plant, clip branch cutting, the detoxic seedling of incubation production use;
7) viral Pathogen detection: adopt herbaceous plant instruction detection method or Electronic Speculum detection method to step 5) detoxic seedling detect.
2. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, it is characterized in that: described step 1) in root-growing agent and processing method be: the aqueous solution of getting IBA and the each 25mg/L of NAA, soak the healthy and strong branch incision of hollow plum oral area 30min, artificial substratum consists of: perlite, vermiculite and loam are according to the ratio of mass ratio 3:3:4.
3. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 2) in the length of stem eye be 10-20mm, separating the merismatic length of stem apex is 0.5-1.5mm.
4. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 2)-3) in inducing culture formula be: potassium nitrate KNO
3: 1900mg/L, ammonium nitrate NH
4nO
3: 1650mg/L, potassium dihydrogen phosphate KH
2pO
4: 170mg/L, magnesium sulfate MgSO
47H
2o:370mg/L, calcium chloride CaCl
22H
2o:440mg/L, potassium iodide KI:0.83mg/L, boric acid H
3bO
3: 6.2mg/L, manganese sulphate MnSO
44H
2o:22.3mg/L, zinc sulphate ZnSO
47H
2o:8.6mg/L, sodium molybdate Na
2moO
42H
2o:0.25mg/L, copper sulphate CuSO
45H
2o:0.025mg/L, cobalt chloride CoCl
26H
2o:0.025mg/L, disodium ethylene diamine tetraacetate Na
2eDTA:37.25mg/L, ferrous sulfate FeSO
27H
2o:27.85mg/L, inositol: 100mg/L, glycine: 2mg/L, thiamine hydrochloride VB
1: 0.1mg/L, puridoxine hydrochloride VB
6: 0.5mg/L, nicotinic acid: 0.5mg/L, 6-BA:0.5mg/L, NAA:0.2mg/L, ribavirin: 60mg/L, sucrose: 30g/L, agar: 6g/L; PH value is: 5.8.
5. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 4) in proliferation culture medium formula be: potassium nitrate KNO
3: 1900mg/L, ammonium nitrate NH
4nO
3: 1650mg/L, potassium dihydrogen phosphate KH
2pO
4: 170mg/L, magnesium sulfate MgSO
47H
2o:370mg/L, calcium chloride CaCl
22H
2o:440mg/L, potassium iodide KI:0.83mg/L, boric acid H
3bO
3: 6.2mg/L, manganese sulphate MnSO
44H
2o:22.3mg/L, zinc sulphate ZnSO
47H
2o:8.6mg/L, sodium molybdate Na
2moO
42H
2o:0.25mg/L, copper sulphate CuSO
45H
2o:0.025mg/L, cobalt chloride CoCl
26H
2o:0.025mg/L, disodium ethylene diamine tetraacetate Na
2eDTA:37.25mg/L, ferrous sulfate FeSO
27H
2o:27.85mg/L, inositol: 100mg/L, glycine: 2mg/L, thiamine hydrochloride VB
1: 0.1mg/L, puridoxine hydrochloride VB
6: 0.5mg/L, nicotinic acid: 0.5mg/L, 6-BA:1.0mg/L, NAA:0.5mg/L, ribavirin: 60mg/L, sucrose: 30g/L, agar: 6g/L; PH value is: 5.8.
6. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 5) in prescription of rooting medium be: potassium nitrate KNO
3: 1900mg/L, ammonium nitrate NH
4nO
3: 1650mg/L, potassium dihydrogen phosphate KH
2pO
4: 170mg/L, magnesium sulfate MgSO
47H
2o:370mg/L, calcium chloride CaCl
22H
2o:440mg/L, potassium iodide KI:0.83mg/L, boric acid H
3bO
3: 6.2mg/L, manganese sulphate MnSO
44H
2o:22.3mg/L, zinc sulphate ZnSO
47H
2o:8.6mg/L, sodium molybdate Na
2moO
42H
2o:0.25mg/L, copper sulphate CuSO
45H
2o:0.025mg/L, cobalt chloride CoCl
26H
2o:0.025mg/L, disodium ethylene diamine tetraacetate Na
2eDTA:37.25mg/L, ferrous sulfate FeSO
27H
2o:27.85mg/L, inositol: 100mg/L, glycine: 2mg/L, thiamine hydrochloride VB
1: 0.1mg/L, puridoxine hydrochloride VB
6: 0.5mg/L, nicotinic acid: 0.5mg/L, 6-BA:0.5mg/L, NAA:0.5mg/L, sucrose: 30g/L, agar: 6g/L; PH value is: 5.8.
7. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 4) in stem apex size be 10-15mm.
8. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 5) in the culture of rootage time be 30-45d, the group training seedling of taking root is cultured to root number more than 3, length is more than 20mm.
9. the method for hollow plum tissue-culturing rapid propagation as claimed in claim 1 detoxification, is characterized in that: described step 6) in antiviral agent be the solution that contains IBA15-20mg/L, NAA15-20mg/L.
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| CN104429942A (en) * | 2014-11-03 | 2015-03-25 | 中国农业科学院果树研究所 | Detoxification method combining heat treatment and chemical treatment for apple tissue culture seedlings |
| CN106613966A (en) * | 2016-11-22 | 2017-05-10 | 蚌埠市涂山绿园蔬菜科研专业合作社 | Detoxification method for potato seedlings |
| CN110384041A (en) * | 2019-05-23 | 2019-10-29 | 南京千亩休闲农业发展有限公司 | A kind of cultural method of sweet cherry rootstock asexual clonal detoxic seedling |
| CN115362935A (en) * | 2022-06-29 | 2022-11-22 | 贵州大学 | Isolated culture method of sand hollow plum |
| CN116076363A (en) * | 2023-01-10 | 2023-05-09 | 重庆市铜梁区果之王园艺研究院 | Culture medium for tissue culture seedlings of melissa plums and culture method for tissue culture seedlings of melissa plums |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429942A (en) * | 2014-11-03 | 2015-03-25 | 中国农业科学院果树研究所 | Detoxification method combining heat treatment and chemical treatment for apple tissue culture seedlings |
| CN106613966A (en) * | 2016-11-22 | 2017-05-10 | 蚌埠市涂山绿园蔬菜科研专业合作社 | Detoxification method for potato seedlings |
| CN110384041A (en) * | 2019-05-23 | 2019-10-29 | 南京千亩休闲农业发展有限公司 | A kind of cultural method of sweet cherry rootstock asexual clonal detoxic seedling |
| CN115362935A (en) * | 2022-06-29 | 2022-11-22 | 贵州大学 | Isolated culture method of sand hollow plum |
| CN116076363A (en) * | 2023-01-10 | 2023-05-09 | 重庆市铜梁区果之王园艺研究院 | Culture medium for tissue culture seedlings of melissa plums and culture method for tissue culture seedlings of melissa plums |
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