CN105165627A - Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain - Google Patents
Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain Download PDFInfo
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Abstract
The invention discloses a tissue culture disinfection and sterilization formula of ormosia henryi prain and a tissue culture method of the ormosia henryi prain. Seed seedling stems are disinfected by 70% ethanol for 30 seconds, sterilized by 0.1% mercury bichloride for 8 minutes, disinfected by the 70% ethanol for 30 seconds and treated by 0.5ml/L of a plant tissue culture antibacterial agent to achieve the best disinfection effect; the optimal collection time of seedling explants is April; the stems are immersed by the 70% ethanol for 30 seconds, immersed by a mixed solution (containing 50mg/L rifampicin and 50mg/L chloramphenicol) for 30 minutes and immersed by 0.1% mercury bichloride for 8 minutes to achieve the best disinfection effect; and leaves are immersed by the 70% ethanol for 30 seconds, immersed by the mixed solution (containing 50mg/L rifampicin and 50mg/L chloramphenicol) for 30 minutes and immersed by 0.1% mercury bichloride for 6 minutes to achieve the best disinfection effect. Leaves of grown plants are relatively suitable for collecting in April, and are immersed by the 70% ethanol for 30 seconds, immersed by the mixed solution (containing 50mg/L rifampicin and 50mg/L chloramphenicol) for 30 minutes and immersed by 0.1% mercury bichloride for 12 minutes. According to the tissue culture disinfection and sterilization formula and the tissue culture method, seed seedlings, seedlings and grown plant tissues of ormosia henryi prain explants are disinfected by different disinfection methods, and the relatively good survival rate can be obtained; and sources of ormosia henryi prain tissue culture multiplication materials are greatly expanded, and the tissue culture effect of the ormosia henryi prain can be greatly improved by controlling disinfection time.
Description
Technical field
The invention belongs to technical field of tissue culture, be specifically related to a kind of rosewood group training sterilization formula, also relate to a kind of rosewood tissue culture method.
Background technology
Rosewood (
ormosiahenryiprain) be pulse family Ormosia aiphyllium, have another name called rosewood, smelly paulownia bavin, belong to national secondary national key protected plant, high 5-13 rice, imparipinnate leaf, Long Circle or oval lanceolar, the four seasons are evergreen, without phenomenon of obviously falling leaves; Sprig and the close raw lark pubescence of blade back; The florescence 6-7 month, yellow-white, total shape titbit; Pod is flat, is pitchy time ripe, and seed is red.Rosewood timber is fine and close, texture is beautiful, glossy, corrosion-resistant, Cutting free, tangent plane are smooth, is Precious Timber Species, and again because its bark viridescent is smooth, tree performance grace becomes high-quality Landscape Trees, is suitable as street tree and garden cultivation; Its root, branch, leaf or rare medicinal herbs, dispelling wind-evil and transforming calculus, detoxification and removing stasis.
Current rosewood is based on seminal propagation, but because rosewood seed skin is coated with wax coat, obstruction Seed imbibes germinates, thus under natural conditions, seed maturity is difficult to nature sprouting after being scattered, adopt 85 DEG C of hot water carry out artificial treatment to seed coat or carry out broken skin process during artificial seeding, its germination rate can be significantly improved.Because rosewood Wild plant is rare, solid difficulty and ripening rate is low, seminal propagation coefficient is low.Tissue cultures has the advantage that reproduction speed is fast, reproduction coefficient is large, characters of progenies is neat.Thus set up rosewood tissue culture quick breeding system, expanding propagation coefficient, to the protection of rosewood wild resource and artificial culture significant.
In tissue culture procedures, the sterilization of explant is extremely important step, selects suitable explant and suitable explant disinfection method to be the successful prerequisites of tissue cultures.Existing rosewood tissue culture method is with rosewood axenic germination seedling for explant carries out, by sprouting the sterile seedlings that obtains after rosewood seed disinfection as explant in gnotobasis, through step structure group training systems such as breeding, take root.But due to the restriction of rosewood seed resource, the acquisition of explant still has certain limitation.If the plant of nature germination and growth taken from by rosewood group training Initial culture material, because its coefficient that carries disease germs is very high, group training process China and foreign countries implant body pollution is serious, and survival rate is extremely low.Only filter out more effective sterilization method, just can carry out further Study on tissue culture.
At document " tissue cultures of rosewood and Fast-propagation (Yao Jun etc., Plant Physiology Communications, 2007, the band base of leaf section adopting seed asepsis sprouting to obtain 01:123-124) " carries out tissue cultures and Fast-propagation research, in document " induction of rosewood plumular axis callus and plant regeneration (Koryo etc., Agriculture of Anhui science, 2009, the plumular axis then adopting rosewood seed asepsis sprouting to obtain 33:16271-16273) " carries out tissue cultures and the induction of follow-up cold resistance, the explant that two people adopt all derives from the material of seed asepsis sprouting, propagating materials is still limited.About rosewood nature germinated plantlet and adult plants explant acquisition group training successful story, there is not been reported, and its reason is that nature germinated plantlet and adult plants explant endophyte are many, aseptic culture Establishing difficulty.
Summary of the invention
The technical problem to be solved in the present invention is: provide a kind of rosewood group to train sterilization formula and tissue culture method, be intended to solve seriously polluted in rosewood Explants In Tissue Culture, survival rate is extremely low and be difficult to induction problem, to overcome the deficiency of prior art problem.
Technical scheme of the present invention: a kind of rosewood group training sterilization formula, comprises 70% alcohol, 0.1% mercuric chloride, 70% alcohol and add 0.5ml/L Plant Tissue Breeding antibacterial agent (PPM) in inducing culture.
A kind of rosewood group training sterilization formula, comprises 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
A kind of rosewood group training sterilization formula, comprises 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
A kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt the seedling age rosewood seedling stem explants of 10 days;
(2) clean: adopt washing agent to soak 15 minutes, with running water 30 minutes;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 1, adopt 70% alcohol, 0.1% mercuric chloride and 70% alcohol-pickled successively, its soak time is respectively 0.5 minute, 8 minutes and 0.5 minute, disinfects all to use aseptic water washing 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element, and in inducing culture, add 0.5ml/L Plant Tissue Breeding antibacterial agent (PPM);
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
A kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt the band top leaf stem section of raw rosewood sowing seedling then, stem segment with lateral bud or leaf explant, acquisition time is April;
(2) clean: adopt washing agent to soak 30 minutes, with running water 1 hour;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 1, adopt the mercuric chloride immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% successively, 0.5 minute, 30 minutes and 6 minutes are respectively for its soak time of blade, be respectively 0.5 minute, 30 minutes and 8 minutes for its soak time of stem section, disinfect at every turn and all use aseptic water washing explant 4-5 time afterwards;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
A kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt the sheet of leave then on perennial rosewood adult plants, acquisition time is April;
(2) clean: adopt washing agent to soak 1 hour, with running water 2 hours;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 1, adopt the immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride successively, its soak time is respectively 0.5 minute, 30 minutes and 12 minutes, disinfects all to use aseptic water washing explant 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
Preferably, above-mentioned MS medium component is as follows:
Potassium nitrate KNO
31900mg/L
Ammonium nitrate NH
4nO
31650mg/L
Epsom salt MgSO
4.7H
2o370mg/L
Potassium dihydrogen phosphate KH
2pO
4170mg/L
Calcium chloride dihydrate CaC
l2.2H
2o440mg/L
Four water manganese sulphate MnSO
4.4H
2022.3mg/L
White vitriol ZnSO
4.7H
2o8.60mg/L
Boric acid H
3bO
36.20mg/L
Potassium iodide KI0.83mg/L
Sodium Molybdate Dihydrate NaMoO
4.2H
2o0.25mg/L
Cupric sulfate pentahydrate CuSO
4.5H
2o0.025mg/L
CoCL2 6H2O CoC
l2.6H
2o0.025mg/L
Disodium ethylene diamine tetraacetate Na
2-EDTA37.30mg/L
Four aqueous ferrous sulfate FeSO
4.4H
2o27.80mg/L
Glycine 2.00mg/L
Thiamine hydrochloride 0.10mg/L
Puridoxine hydrochloride 0.50mg/L
Nicotinic acid 0.50mg/L
Inositol 100mg/L;
Above-mentioned supplementary element is 6-BA(6-benzamido group purine, 6-Benzylaminopurine) 2.0mg/L, NAA(methyl α-naphthyl acetate, I-NaphthyIacetic) 1.0mg/L, agar 8g/L, sucrose 30g/L.
Beneficial effect of the present invention: compared with prior art, the present invention has following effect:
(1) the present invention adopts 70% alcohol, 0.1% mercuric chloride, 70% alcohol and add 0.5ml/L Plant Tissue Breeding antibacterial agent (PPM) in increment medium and carries out disinfection to seedling, greatly can reduce pollution rate, by Contamination rate control 6.67%, thus greatly improve the group training survival rate of seedling, reach 93.33%, inductivity reaches 100%, group training Be very effective.
(2) the present invention adopts 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1% mercuric chloride soaks the stem section of 8min to seedling and carries out disinfection and and adopt 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1% mercuric chloride soaks the blade of 6min to seedling and carries out disinfection, greatly can both reduce pollution rate, the former and the latter by Contamination rate control 0, greatly improve the group training survival rate of seedling explants, the former survival rate is to 33.3%, the latter's survival rate is to 100%, the former arrives 60% by inductivity, the latter's inductivity arrives 64.29%, group training Be very effective.
(3) the present invention adopts 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1% mercuric chloride soak 12min to rosewood adult plants then leave sheet carry out disinfection, can greatly reduce pollution rate by Contamination rate control 66.67%, thus greatly improve the group training survival rate of adult plants explant, reach 33.33%, blade inductivity reaches 80%, stem section inductivity reaches 20%, and group training effect is more remarkable.
(4) the present invention adopts the seedling of rosewood explant, seedling and adult plants blade to carry out group training, greatly extend the source of tissue culture propagation material, efficiently solve in prior art the limitation that rosewood seed germination seedling can only be adopted to carry out organizing training, and also substantially increase survival rate and the inductivity of group training.Due to seedling explant and raw seedling explants is comparatively young tender then, overlong time can cause explant dead, and too short meeting causes Disinfection Effect poor, and therefore, the sterilization soak time in employing the present invention can improve Disinfection Effect greatly.
(5) sterilised formula of the present invention's employing is simple, but disinfective action well can be played, soak time be set according to specific circumstances in the tissue cultures of rosewood and add Plant Tissue Breeding antibacterial agent, serving extraordinary Disinfection Effect and group training effect.
Embodiment
Now in conjunction with following example, the present invention is further explained.
Embodiment 1: a kind of rosewood group training sterilization formula, comprises 70% alcohol, 0.1% mercuric chloride, 70% alcohol and add 0.5ml/L Plant Tissue Breeding antibacterial agent in increment medium.
Embodiment 2: a kind of rosewood group training sterilization formula, comprises 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
Embodiment 3: a kind of rosewood group training sterilization formula, comprises 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
Embodiment 4: a kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt the seedling age rosewood seedling stem explants of 10 days;
(2) clean: adopt washing agent to soak 15 minutes, with running water 30 minutes;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, embodiment 1 one kinds of rosewood group training sterilization formulas are adopted to carry out disinfection, adopt 70% alcohol, 0.1% mercuric chloride and 70% alcohol-pickled successively, its soak time is respectively 0.5 minute, 8 minutes and 0.5 minute, disinfects all to use aseptic water washing 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element, and in inducing culture, add 0.5ml/L Plant Tissue Breeding antibacterial agent (PPM);
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
Embodiment 5: a kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt the band top leaf stem section of raw rosewood sowing seedling then, stem segment with lateral bud or leaf explant, acquisition time is April;
(2) clean: adopt washing agent to soak 30 minutes, with running water 1 hour;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt embodiment 2 one kinds of rosewood group training sterilization formulas, adopt the mercuric chloride immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% successively, 0.5 minute, 30 minutes and 6 minutes are respectively for its soak time of blade, be respectively 0.5 minute, 30 minutes and 8 minutes for its soak time of stem section, disinfect at every turn and all use aseptic water washing explant 4-5 time afterwards;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
Embodiment 6: a kind of rosewood tissue culture method, the method comprises the following steps:
(1) collection of explant: adopt perennial rosewood adult plants leave sheet then, acquisition time is April;
(2) clean: adopt washing agent to soak 1 hour, with running water 2 hours;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, embodiment 3 one kinds of rosewood group training sterilization formulas are adopted to carry out disinfection, adopt the immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride successively, its soak time is respectively 0.5 minute, 30 minutes and 12 minutes, disinfects all to use aseptic water washing explant 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
As preferably, in above-described embodiment 4-6, MS medium component is as follows:
Potassium nitrate KNO
31900mg/L
Ammonium nitrate NH
4nO
31650mg/L
Epsom salt MgSO
4.7H
2o370mg/L
Potassium dihydrogen phosphate KH
2pO
4170mg/L
Calcium chloride dihydrate CaC
l2.2H
2o440mg/L
Four water manganese sulphate MnSO
4.4H
2022.3mg/L
White vitriol ZnSO
4.7H
2o8.60mg/L
Boric acid H
3bO
36.20mg/L
Potassium iodide KI0.83mg/L
Sodium Molybdate Dihydrate NaMoO
4.2H
2o0.25mg/L
Cupric sulfate pentahydrate CuSO
4.5H
2o0.025mg/L
CoCL2 6H2O CoC
l2.6H
2o0.025mg/L
Disodium ethylene diamine tetraacetate Na
2-EDTA37.30mg/L
Four aqueous ferrous sulfate FeSO
4.4H
2o27.80mg/L
Glycine 2.00mg/L
Thiamine hydrochloride 0.10mg/L
Puridoxine hydrochloride 0.50mg/L
Nicotinic acid 0.50mg/L
Inositol 100mg/L;
Supplementary element is 6-BA(6-benzamido group purine, 6-Benzylaminopurine) 2.0mg/L, NAA(methyl α-naphthyl acetate, I-NaphthyIacetic) 1.0mg/L, agar 8g/L, sucrose 30g/L.
Following comparative's test further illustrates the invention process and beneficial effect thereof.
1 materials and methods
1.1 experiment material
Carry out the disinfection experiment of seedling stem section, seedling stem section, adult plants stem section and tender leaf respectively.Seedling explant takes from the seedling sprouting 10d in greenhouse, and clip 3cm long shoot section does explant; Seedling explants takes from nursery raw tree seedling then, respectively at 4,8, stem section that December, clip band terminal bud was about 6cm does explant; Adult plants explant takes from nursery life in 7 years and farm 20 years raw rosewood band terminal bud branches, respectively at 4,8, clip current growth in December is about 20cm semi-lignified branch and tests.Various stem section is cut into 1.5cm long band terminal bud or stem segment with lateral bud, the sheet of leave then on seedling and adult plants stem section is cut into 1cm × 1cm size and is used as explant.
1.2 sterilization method
The seedling explant of collection, seedling explants, adult plants explant are soaked 15min, 30min with washing agent, the seedling explant of collection, seedling explants, adult plants explant are soaked 15min, 30min, 1h with washing agent respectively respectively, and scrub gently with toothbrush, adult plants explant need remove surperficial fine hair, and material rinses 30min, 1h, 2h respectively under flowing water.Medicament surface sterilization is carried out afterwards in superclean bench.All material first uses 70% alcohol-pickled 30s, with after aseptic water washing 2-3 time respectively the measure of employing table 1 disinfect further.All aseptic water washing 4-5 time is used after each process.
1.3 inoculation method
On superclean bench, adopt increment culture medium inoculated by after explant process, increment culture medium prescription is MS+6-BA2.0mg/L+NAA1.0mg/L+ agar 8g/L+ sucrose 30g/L, pH5.8-6.0, often kind of each process inoculation of material 15 bottles, every bottle of explant.Inoculation is cultivated under being placed on cultivation temperature (25 ± 2) DEG C, intensity of illumination 3000lx, light application time 12h/ days.
1.4 result statistical methods
After inoculation, every day observes material contamination situation, and record, result computing formula is as follows
Pollution rate=(polluting number/inoculation number) × 100%
Survival rate=(survival number/inoculation number) × 100%
Inductivity=(successfully inducing number/survival number) × 100%
Sterilizing effective value=(the uncontaminated rate of 1-) × survival rate × inductivity (decimal is multiplied) * 100%.
2. results and analysis
2.1 seedling stem section Analysis of Disinfection Effects
As shown in Table 2, after soaking, then use the secondary sterilization process of 70% alcohol-pickled 30s (No. 3, No. 4) can reduce the pollution rate of seedling stem section with 0.1% mercuric chloride, same process is lower is better than 6min with 0.1% mercuric chloride process 8min.And under same treatment condition, No. 4 process explant pollution rates adding PPM in the medium significantly reduce, survival rate significantly improves, and improves 26.66% than No. 3 process.Therefore, to seedling stem Duan Eryan, soak 8min+70% alcohol-pickled 30s+PPM Disinfection Effect with 70% alcohol-pickled 30s+0.1% mercuric chloride best.No matter disinfect any, the explant induction rate preserved all reaches 100%.
2.2 seedling explants Analysis of Disinfection Effects
2.2.1 blade
For seedling leaves explant, identical disinfect condition under, the seedling leaves pollution rate that April gathers and survival rate relatively low (table 3), but inductivity is higher, between 50%-100%.Although the blade survival rate that August and December gather is high, inductivity is 0.With (100mg/L benomyl+100mg/L streptomycin) No. 30min(11-13, mixed liquid dipping process) and (50mg/L rifampin+50mg/L chloramphenicol) No. 30min(14-16, mixed liquid dipping process) all effectively can reduce the pollution of blade in April, its uncontaminated rate is 100%, higher than No. 9, No. 10 process, but its survival rate can reduce.In No. 11-16 process, the difference on effect that antibiotic consumption produces is not obvious, and explant survival rate, inductivity all increase with the mercuric chloride processing time and reduce, best with No. 14 treatment effects, survival rate, inductivity are respectively 33.33%, 60%, and sterilizing effective value reaches 19.99%.Therefore, gathering seedling leaves in April and do explant, with 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) to soak 6min Disinfection Effect best for mixed liquid dipping 30min+0.1% mercuric chloride.
2.2.2 stem section
Test shows (table 4), and the pollution rate of stem section is generally: > in April > in August December, but the pollution rate difference that great majority are disinfected is not obvious, and the pollution rate mean value of No. 9-16 process relatively.Compared with sterilizing with blade, the sterilization method of No. 9-16 process all can produce better effects to stem section, and visible seedling stem explants requires do not have blade strict to month of drawing materials.Explant survival rate then shows as: > in December > in August April.This may be younger tender relevant with seedling stem section in April; Inductivity is then the highest with April, average inductivity 70.86%, and December takes second place, average inductivity 58.67%, the stem section in minimum is August, average inductivity 55.71%.Explant survival rate, inductivity all increase with the mercuric chloride processing time and reduce.From material effective value, the material of No. 9, No. 10, No. 15 process to different month is all relatively good.Although it should be noted that the explant material effective value in August and December is higher than April, less and easy brownization of callus that explant induction produces.The stem section of suggestion collection in April is disinfected with No. 10, and namely 70% alcohol-pickled 30s+0.1% mercuric chloride soaks in 6min+ medium and adds PPM; Stem section No. 15 process that August, December gather, namely use 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1% mercuric chloride immersion 8min Disinfection Effect is better.
2.3 adult plants explants
2.3.1 blade
Test shows (table 5), and from pollution rate, > in August > in December April, the blade in August carries disease germs many, sterilizing difficulty.Compared with seedling leaves, adult plants blade sterilization difficulty, even the leaf explant that April gathers, pollution rate is also more than 60%, and after adding PPM0.5ml/L in medium, sterilization effect is still very poor.In 4 process, No. 8 treatment effects are better, and after adding antibacterial agent process, pollution rate is 66.67%, and survival rate is 33.33%, inductivity 80%, and sterilizing effective value is 8.89%.Therefore, be explant with adult plants blade, with April gather, with 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1% mercuric chloride soak 12min sterilize and inducing effect best.
2.3.2 stem section
Adult plants stem explants is disinfected than its blade more difficult (table 6), and the stem section that April, December gather is carried disease germs less relatively, but pollution rate is lower than 60%, and carrying disease germs at most of August, no matter which kind of sterilization treatment is all polluted.Adult plants stem section Disinfection Effect is with capturing material in December, and better with No. 7 process Disinfection Effects, pollution rate is 63.67%, survival rate is 33.33%, but from inductivity, with capturing material in April, add No. PPM0.5ml/L(6 process in the medium) effect better, inductivity is up to 100%.From sterilizing effective value, No. 6 process and No. 7 treatment effects all poor, adult plants sterile system foundation need further exploration.
3. brief summary and discussion
In plant tissue culture course, the foundation of aseptic culture system is the important step obtaining plantlet in vitro.Show using the rosewood blade of Different growth phases, stem section as the disinfection test result of explant, seedling explant is to add No. PPM0.5ml/l(4 process in 70% ethanol disinfection 30s+0.1% mercuric chloride sterilizing 8min+70% ethanol disinfection 30s+ medium) Disinfection Effect best.Seedling explants is better with collection effect in April, seedling stem section 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1%Hgcl soaks No. 8min(15 process) Disinfection Effect is best, seedling leaves 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1%Hgcl soaks No. 6min(14 process) Disinfection Effect is best.Adult plants explant blade gathered better with April, and best sterilization method is: 70% alcohol-pickled 30s+(50mg/L rifampin+50mg/L chloramphenicol) mixed liquid dipping 30min+0.1%Hgcl soaks No. 12min(8 process).
The crucial part of explant sterilization is the selected of suitable sterilization method and disinfecting time, and the suitable sterilization method of disinfectant combination and the foundation of determination to the aseptic rapid propagation system of plant of suitable disinfecting time all have important function.Judging that whether certain sterilization method is suitable, take survival rate as Main Basis, and integrated survey pollution rate and inductivity.Research finds, adopts antibacterial agent (50mg/L rifampin+50mg/L chloramphenicol) mixed liquor and (100mg/L benomyl+100mg/L streptomycin) mixed liquid dipping all can play certain sterilization effect in the sterilization of rosewood explant.Add the plant tissue culture antibacterial agent (PPM) of low concentration (0.5ml/L) in the medium, under the prerequisite not affecting inductivity, can significantly promote uncontaminated rate and the survival rate of explant.
Different biopsy method position and the sterilization complexity of time to explant of drawing materials have extreme influence.The tissue cultures of time to most plants of drawing materials all has a certain impact.Research finds, the Disinfection Effect of drawing materials in April and December is better than the rosewood explant of drawing materials August, and this may be cause mushroom activity to rise because temperature after April rises.In addition, because plant growth during April is more vigorous, the explant induction better effects if of therefore now drawing materials.Study discovery, select young leaflet tablet and band to push up leaf stem section during sterilization better than bottom stem section Disinfection Effect, because its growth time is not long, the attachments such as miscellaneous bacteria are less, and thus sterilizing is easier to simultaneously.
Claims (7)
1. a rosewood group training sterilization formula, is characterized in that: comprise 70% alcohol, 0.1% mercuric chloride, 70% alcohol and in proliferated culture medium, add 0.5ml/L Plant Tissue Breeding antibacterial agent.
2. a rosewood group training sterilization formula, is characterized in that: comprise 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
3. a rosewood group training sterilization formula, is characterized in that: comprise 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride.
4. a rosewood tissue culture method, is characterized in that: the method comprises the following steps:
(1) collection of explant: adopt the seedling age rosewood seedling stem explants of 10 days;
(2) clean: adopt washing agent to soak 15 minutes, with running water 30 minutes;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 1, adopt 70% alcohol, 0.1% mercuric chloride and 70% alcohol-pickled successively, its soak time is respectively 0.5 minute, 8 minutes and 0.5 minute, disinfects all to use aseptic water washing 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element, and in inducing culture, add 0.5ml/L Plant Tissue Breeding antibacterial agent;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
5. a rosewood tissue culture method, is characterized in that: the method comprises the following steps:
(1) collection of explant: adopt the band top leaf stem section of raw rosewood sowing seedling then, stem segment with lateral bud or leaf explant, acquisition time is April;
(2) clean: adopt washing agent to soak 30 minutes, with running water 1 hour;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 2, adopt the mercuric chloride immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% successively, 0.5 minute, 30 minutes and 6 minutes are respectively for its soak time of blade, be respectively 0.5 minute, 30 minutes and 8 minutes for its soak time of stem section, disinfect at every turn and all use aseptic water washing explant 4-5 time afterwards;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
6. a rosewood tissue culture method, is characterized in that: the method comprises the following steps:
(1) collection of explant: adopt the sheet of leave then on perennial rosewood adult plants, acquisition time is April;
(2) clean: adopt washing agent to soak 1 hour, with running water 2 hours;
(3) sterilize: the explant cleaned in step (2) is taken in aseptic working platform, adopt a kind of rosewood group training sterilization formula according to claim 3, adopt the immersion of 70% alcohol, 50mg/L rifampin and 50mg/L chloramphenicol mixed liquor and 0.1% mercuric chloride successively, its soak time is respectively 0.5 minute, 30 minutes and 12 minutes, disinfects all to use aseptic water washing explant 4-5 time afterwards at every turn;
(4) inoculate: be seeded on inducing culture by the explant after cleaning in step (3), described inducing culture comprises MS medium and supplementary element;
(5) cultivate: put in incubator by the explant be placed in inducing culture in step (4) and cultivate, employing condition of culture is: temperature 23-27 DEG C, light intensity 2500-3000lx, daylight 12 hours.
7. a kind of rosewood tissue culture method as described in as arbitrary in claim 4-6, is characterized in that: described MS minimal medium composition is as follows:
Potassium nitrate KNO
31900mg/L
Ammonium nitrate NH
4nO
31650mg/L
Epsom salt MgSO
4.7H
2o370mg/L
Potassium dihydrogen phosphate KH
2pO
4170mg/L
Calcium chloride dihydrate CaCl
2.2H
2o440mg/L
Four water manganese sulphate MnSO
4.4H
2022.3mg/L
White vitriol ZnSO
4.7H
2o8.60mg/L
Boric acid H
3bO
36.20mg/L
Potassium iodide KI0.83mg/L
Sodium Molybdate Dihydrate NaMoO
4.2H
2o0.25mg/L
Cupric sulfate pentahydrate CuSO
4.5H
2o0.025mg/L
CoCL2 6H2O CoCl
2.6H
2o0.025mg/L
Disodium ethylene diamine tetraacetate Na
2-EDTA37.30mg/L
Four aqueous ferrous sulfate FeSO
4.4H
2o27.80mg/L
Glycine 2.00mg/L
Thiamine hydrochloride 0.10mg/L
Puridoxine hydrochloride 0.50mg/L
Nicotinic acid 0.50mg/L
Inositol 100mg/L;
Described inducing culture supplementary element is 6-BA2.0mg/L, NAA1.0mg/L, agar 8g/L, sucrose 30g/L.
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| CN116814479A (en) * | 2023-06-08 | 2023-09-29 | 贵州大学 | Bacillus GM-12-PT and method for promoting germination of Pterocarpus marsupium seeds by using same |
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