CN106665360A - Method for cultivating virus-free seedlings of rhizoma atractylodis macrocephalae via two-step processes - Google Patents
Method for cultivating virus-free seedlings of rhizoma atractylodis macrocephalae via two-step processes Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for cultivating virus-free seedlings by the aid of vegetative bodies of rhizoma atractylodis macrocephalae via two-step processes. The method includes firstly, establishing aseptic cultivation systems by the aid of terminal buds of the rhizoma atractylodis macrocephalae, to be more specific, disinfecting the terminal buds of the rhizoma atractylodis macrocephalae, then inoculating the terminal buds in primary cultivation media, and carrying out multiplication cultivation allowing continuously transferring the terminal buds so as to establish the aseptic cultivation systems with the terminal buds of the rhizoma atractylodis macrocephalae; secondly, carrying out virus-free cultivation on stem tips of aseptic seedlings, to be more specific, acquiring the aseptic seedlings from the established aseptic cultivation systems, stripping the stem tips with the sizes ranging from 0.3mm to 0.5 mm, inoculating the stem tips to induction cultivation media, detecting viruses by the aid of DAS-ELISA (double antibody sandwich-enzyme linked immuno sorbent assay) processes, discarding seedlings with the viruses, continuing to cultivate the seedlings (namely, the virus-free seedlings) without the viruses, transferring the virus-free seedlings into multiplication cultivation media, carrying out multiplication cultivation, transferring the virus-free seedlings to rooting cultivation media, carrying out rooting cultivation and exercising and transplanting rooted seedlings. The viruses are removed from a part of bud seedlings grown by the induction cultivation media. The method for cultivating the virus-free seedlings of the rhizoma atractylodis macrocephalae via the two-step processes has the advantages that damage to the stem tips due to disinfectants can be prevented, the small stem tips can be cultivated to develop into seedlings, and accordingly the virus-free seedlings can be obtained.
Description
Technical field
The invention belongs to plant toxic and group culturation rapid propagating technology field, in particular relate to one kind and passed through using bighead atractylodes rhizome trophosome
The method of two stage culture detoxic seedling.
Background technology
The bighead atractylodes rhizome（Atractylodes macrocephala Koidz）It is perennial feverfew, using wide, consumption is big,
There is artificial cultivation in the whole nation more.But pest and disease damage is serious in bighead atractylodes rhizome cultivation, the special effect agent that wherein virosis is not prevented and treated, generally
Prevention and control are carried out using the method for detoxification.
The testing result of the embryonal connective tissue without virus of cotyledon was once removed in laboratory according to the bighead atractylodes rhizome, by embryonal connective tissue culture
Successfully obtain detoxic seedling and apply for patent of invention（Patent No. ZL201410041675.0）, the cultural method need to utilize seed,
Cultivated with the embryonal connective tissue in seed.
Such problem is sometimes run into production practices：Field finds the excellent plant of growth performance, it is desirable to by nothing
The offspring that sexual reproduction obtains the plant is used to produce, and this is accomplished by being cultivated using trophosome.Stem apex detoxification is to be widely used
One of detoxification means, experiment finds that the stem apex that 0.3mm ~ 0.5mm is stripped after the bighead atractylodes rhizome material for picking up from field is sterilized is trained
Support, survival rate is 0%；The stem apex for stripping 0.8mm ~ 1mm is cultivated, survival rate 25%, DAS-ELISA detection displays PVX, TMV
Removal efficiency is that 0%, CMV removal efficiencies only have 12.5%.Illustrate that smaller stem apex fails to cultivate successfully, though larger stem apex can be induced
Bud, but viral removal efficiency is low, it is difficult to obtain detoxic seedling.Smaller stem apex（0.3mm~0.5mm）The reason for culture can not be survived has can
Can be due to having damaged stem apex to a certain extent to disinfecting for stem apex before culture, caused by declining its viability.
The content of the invention
Present invention aim at a set of method that virus-free culture is carried out using bighead atractylodes rhizome trophosome is set up, for bighead atractylodes rhizome routine stem
Point culture is difficult to obtain the problem of detoxic seedling, and the present inventor devises the method by two stage culture bighead atractylodes rhizome detoxic seedling：I.e.
One step：Asepsis training department is set up using bighead atractylodes rhizome terminal bud；Second step：Taking aseptic seedling from the asepsis training department set up carries out stem apex training
Detoxification is supported, culture materials is removed sterilisation step from, it is ensured that its viability, it is possible to making smaller Shoot Tip Culture success, obtained
Preferable detoxification efficiency.
The purpose of the present invention and the technical problem underlying of solution are realized using following technical scheme：A kind of two stage culture
The method of bighead atractylodes rhizome detoxic seedling, including following operating process：
1. asepsis training department is set up using bighead atractylodes rhizome terminal bud：
1）Bighead atractylodes rhizome terminal bud Initial culture：Choose field growing and show excellent bighead atractylodes rhizome plant, cut the 2cm long shoots comprising terminal bud
Section, removes blade, is rinsed 30 minutes with being put into after detergent washes clean in flowing water, standby；The material strips that will be got ready enter ultra-clean
Workbench, cuts terminal bud, and bubble 30s is invaded with 75% alcohol, and then aseptic water washing 3 times soaks 15min, phase with 1% sodium hypochlorite
Between stir, with aseptic water washing 5 times, filter paper blots surface moisture, terminal bud is inoculated in and fills Initial culture base：MS+6-BA
The flat based tubes of 0.5mg/l+NAA0.1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, often 1 terminal bud of pipe, is placed in illumination 12h/
My god, light intensity 2000lx is cultivated in the culturing room that 21 ± 2 DEG C of temperature, daily observation, finds that part explant has pollution feelings after 7 days
Condition, clears out of culturing room by the culture test tube of pollution in time, and rear portion exceptionally implant starts to turn green within 10 days, browning partly occurs and shows
As turn green terminal bud around gradually expanded by the culture of 15 days, and base portion can form light green callus, after 30 days gradually
Differentiate 3cm ~ 4 cm sprouts；
2）Increment culture：The sprout that Initial culture is obtained for 30 days is transferred on superclean bench fills proliferated culture medium：MS+
In the blake bottle of 6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, every bottle connects 5 young plants,
It is incubated at illumination 12h/ days, light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；After 40 days, count average proliferation multiplying power is
4.6；
2. aseptic seedling stem apex virus-free culture：
1）Stem tip induction seedling culture：Take aseptic seedling from the asepsis training department set up, on superclean bench with scalpel and
Tweezers ecto-entad successively peels off cauline leaf and spire, until the apical meristem of semi-round ball is fully exposed, cuts 0.3mm
The stem apex of ~ 0.5mm sizes, is inoculated into and fills inducing culture：1/2MS+6-BA0.5g/l+NAA0.1g/l+ sucrose 30g/l+ fine jades
In the blake bottle of cosmetics 5.5g/l, every bottle connects 3 stem apexs, is incubated at illumination 12h/ days, light intensity 2000lx, 21 ± 2 DEG C of temperature
In culturing room, timing daily is observed, and stem apex expands growth after 15 days, and the bud point projection of green occurs in shoot tip meristem after 20 days,
Bud point grows tall and grows up after 25 days, begins to differentiate into bud, can be differentiated to form 1cm ~ 3cm sprouts high after 40 days, planting percent 90%,
Some is stripped of virus, that is, the desired detoxic seedling of the present invention in the sprout of formation；
2）Detoxic seedling is screened：After Shoot Tip Culture 45 days, the sprout that aseptic seedling Shoot Tip Culture is obtained is inoculated in proliferated culture medium：MS
Proliferation of propagation is carried out in+6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, after 40 days
Propagation seedling is removed from blake bottle, clip 0.2g cauline leafs carry out Viral diagnosis；
Testing result shows that the stem apex for stripping 0.3mm ~ 0.5mm sizes using aseptic seedling is cultivated, and the removal efficiency to PVX is
73.3%, the removal efficiency to TMV is 66.7%, and the removal efficiency to CMV is 61.5%, and the seedling containing virus is abandoned, by without virus
Seedling, i.e. detoxic seedling continue to cultivate；
3）, detoxic seedling Multiplying culture：
Detoxic seedling is transferred on superclean bench fills proliferated culture medium：MS+ 6-BA 1mg/l+NAA0.4mg/l+
In the blake bottle of IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, every bottle connects 5 young plants, is incubated at illumination 12h/ days, light intensity
2000lx, in the culturing room that 21 ± 2 DEG C of temperature；
4）, detoxic seedling culture of rootage：
Root media is transferred to after the tufted seedling of Multiplying culture to 4cm is divided into individual plant on superclean bench：1/2MS+
In NAA0.4mg/l+IBA0.6mg/l+ sucrose 20g/l+ agar powders 5.5g/l, every bottle connects 5 young plants, is incubated at illumination 12h/ days,
Light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；Counted after 40 days, rooting rate 100% ,/plant of number 10.8 of averagely taking root；
5）, detoxic seedling acclimatization and transplantses：
Culture of rootage 40d is taken, every plant of Gen Shuo≤8, the detoxic seedling of root more than 1cm long carry out practicing transplantation of seedlings, blake bottle from group
Between training room moves to buffering, normal temperature closes bottle hardening a 3d, half-open lid 3d, full open end 3d；Complete hardening after with tweezers by bighead atractylodes rhizome seedling from
Blake bottle is removed, and is put into clear water, cleans root agar, is transplanted in matrix, pours permeable, daily morning and evening blade face watering, to protect
Moistening is held, watering number of times is reduced after one week；After one month, the seedling of transplant survival starts young leaves long, survival rate 93%.
Above-mentioned method, in the step 12）Increment culture：Treat that seedling covers with one bottle, continue to turn Multiplying culture, 1 bottle turns
10 bottles are connect, Multiplying culture is continuously transferred, thus set up bighead atractylodes rhizome terminal bud asepsis training department.
Above-mentioned method, in the step 22）Detoxic seedling is screened：Method for detecting virus uses Enzyme-linked Immunosorbent Assay skill
Art-double-antibody method DAS-ELISA, step is as follows：
A, preparation wet box：3 ~ 4 hygenic towelettes are put into crisper, for moisturizing and lucifuge；
B, preparation capture antibody-solutions：According to 1:200 dilution ratio is diluted to capture antibody, and dilution is PH9.6's
Carbonate coating buffer solution；
C, coating plate：Coated antibody in ELISA Plate, 100 μ L capture antibody-solutions are added per hole；
D, incubation plate:ELISA Plate is put into wet box, is placed in overnight incubation in 4 °C of refrigerators, and standing time is no more than 24h；
E, board-washing：Next day, solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse
3 times；
F, sample preparation：Seedling to be detected is removed from blake bottle, clip 0.2g cauline leafs are placed in mortar, add the general of 2ml to carry
Take buffer solution GEB and be ground to homogenate shape；1000rmp is centrifuged 10min, takes supernatant standby；
G, sample-adding：Positive control, negative control, sample solution are separately added into washed ELISA Plate, 100 μ L are added per hole,
Blank control wells add the GEB of 100 μ L；
H, incubation plate：ELISA Plate is put into wet box, 2h is incubated in 37 °C of incubators；
I, the enzyme-linked antibody-solutions of preparation：According to 1:200 dilution ratio is diluted to enzyme-linked antibody, and dilution is PH9.6's
Carbonate coating buffer solution；
J, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 7 times；
K, enzyme-added len antibody：The 100 enzyme-linked antibody-solutions of μ L are added in plate per hole；
L, incubation plate：ELISA Plate is put into wet box and is incubated 2h in 37 °C；
M, preparation PNPP solution：Need to be now with the current, by PNPP powder buffer solutions, final concentration of 1mg/ml；
N, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 8 times；
O, plus PNPP solution：100 μ LPNPP solution are added in plate per hole；
P, incubation plate：ELISA Plate is put into wet box and is incubated 1h in 37 °C, notes lucifuge；
Q, result detection：On ELIASA, at 405nm, the OD values of each reacting hole are surveyed after being returned to zero with blank control wells, if greatly
Then it is the positive in 2 times of negative control OD value.
Above-mentioned method, in the step 23）Detoxic seedling Multiplying culture：Treat that seedling covers with one bottle, continue to turn Multiplying culture,
General 1 bottle is transferred 10 bottles, and Multiplying culture is continuously transferred.
Above-mentioned method, in the step 25）Detoxic seedling acclimatization and transplantses：The matrix is fertile soil, vermiculite, pearl
Rock, ratio is 3：1：1.
The present invention has clear advantage and beneficial effect compared with prior art.From above technical scheme, this hair
It is bright that damage of the disinfectant to stem apex is dexterously avoided by two stage culture bighead atractylodes rhizome detoxic seedling, the viability of stem apex is saved,
Make smaller Shoot Tip Culture success, obtain preferable detoxification efficiency.The first step sets up sterile culture system using bighead atractylodes rhizome terminal bud, to push up
Bud is explant, has filtered out suitable disinfection way, obtains Initial culture base and proliferation culture medium formula, establishes terminal bud
Sterile culture system；Second step aseptic seedling Shoot Tip Culture detoxification, stem apex is taken with aseptic seedling（0.3mm~0.5mm）Carry out induction point
Change, orthogonality analysis method has screened suitable Fiber differentiation based formulas and prescription of rooting medium.By above-mentioned two steps culture, set up
For the virus-free culture method of bighead atractylodes rhizome trophosome, removing PVX, TMV, CMV tri- kinds of tissue-cultured seedling of virus are obtained, be in production
Application in practice is laid a good foundation.
Specific embodiment
It is specifically real to the method according to two stage culture bighead atractylodes rhizome detoxic seedling proposed by the present invention below in conjunction with preferred embodiment
Mode, feature and its effect are applied, is described in detail as follows.
A kind of method of two stage culture bighead atractylodes rhizome detoxic seedling, including following operating process：
1. asepsis training department is set up using bighead atractylodes rhizome terminal bud：
1）Bighead atractylodes rhizome terminal bud Initial culture：Choose field growing and show excellent bighead atractylodes rhizome plant, cut the about 2cm long shoots comprising terminal bud
Section, removes blade, is rinsed 30 minutes with being put into after detergent washes clean in flowing water, standby.The material strips that will be got ready enter ultra-clean
Workbench, cuts terminal bud, and bubble 30s is invaded with 75% alcohol, and then aseptic water washing 3 times soaks 15min, phase with 1% sodium hypochlorite
Between stir, with aseptic water washing 5 times, filter paper blots surface moisture, terminal bud is inoculated in and fills Initial culture base（MS+6-BA
0.5mg/l+NAA0.1mg/l+ sucrose 30g/l+ agar powders 5.5g/l）Flat based tubes, often 1 terminal bud of pipe, is placed in illumination 12h/
My god, light intensity 2000lx is cultivated in the culturing room that 21 ± 2 DEG C of temperature, daily observation, finds that part explant has pollution feelings after 7 days
Condition, clears out of culturing room by the culture test tube of pollution in time, and rear portion exceptionally implant starts to turn green within 10 days, browning partly occurs and shows
As turn green terminal bud around gradually expanded by the culture of 15 days, and base portion can form light green callus, after 30 days gradually
Differentiate 3cm ~ 4 cm sprouts；
2）Increment culture：The sprout that Initial culture is obtained for 30 days is transferred on superclean bench fills proliferated culture medium（MS+
6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l）Blake bottle in, every bottle connects 5 young plants,
It is incubated at illumination 12h/ days, light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；After 40 days, count average proliferation multiplying power is
4.6.Treat that seedling covers with one bottle, can continue to turn Multiplying culture, general 1 bottle switchable 10 bottles, Multiplying culture can continuously transfer, thus
Set up bighead atractylodes rhizome terminal bud asepsis training department.
2. aseptic seedling stem apex virus-free culture：
1）Stem tip induction seedling culture：Take aseptic seedling from the asepsis training department set up, on superclean bench with scalpel and
Tweezers ecto-entad successively peels off cauline leaf and spire, until the apical meristem of semi-round ball is fully exposed, cuts 0.3mm
The stem apex of ~ 0.5mm sizes, is inoculated into and fills inducing culture（1/2MS+6-BA0.5g/l+NAA0.1g/l+ sucrose 30g/l+ fine jades
Cosmetics 5.5g/l）Blake bottle in, every bottle connects 3 stem apexs, is incubated at illumination 12h/ days, light intensity 2000lx, 21 ± 2 DEG C of temperature
In culturing room, timing daily is observed, and 15 days or so stem apexs expand growth, and the bud point that green occurs in shoot tip meristem after 20 days is dashed forward
Rise, bud point is grown tall and grows up after 25 days, begins to differentiate into bud, and 1cm ~ 3cm sprouts high, planting percent can be differentiated to form after 40 days
90%, some is stripped of virus, that is, the desired detoxic seedling of the present invention in the sprout of formation；
2）Detoxic seedling is screened：After Shoot Tip Culture 45 days, the sprout that aseptic seedling Shoot Tip Culture is obtained is inoculated in proliferated culture medium（MS
+ 6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l）Proliferation of propagation is carried out, will after 40 days
Propagation seedling is removed from blake bottle, and clip 0.2g cauline leafs carry out Viral diagnosis, using DAS-ELISA（Enzyme-linked Immunosorbent Assay technology-bis-
Anti- sandwich method）, step is as follows：
A, preparation wet box：3 ~ 4 hygenic towelettes are put into crisper, for moisturizing and lucifuge.
B, preparation capture antibody-solutions：According to 1:200 dilution ratio is diluted to capture antibody, and dilution is
The carbonate coating buffer solution of PH9.6.
C, coating plate：Coated antibody in ELISA Plate, 100 μ L capture antibody-solutions are added per hole.
D, incubation plate:ELISA Plate is put into wet box, is placed in overnight incubation in 4 °C of refrigerators, and standing time is no more than 24h.
E, board-washing：Next day, solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent,
Rinse 3 times.
F, sample preparation：Seedling to be detected is removed from blake bottle, clip 0.2g cauline leafs are placed in mortar, add leading to for 2ml
Homogenate shape is ground to Extraction buffer GEB.1000rmp is centrifuged 10min, takes supernatant standby.
G, sample-adding：Positive control, negative control, sample solution are separately added into washed ELISA Plate, 100 are added per hole
μ L, blank control wells add the GEB of 100 μ L.
H, incubation plate：ELISA Plate is put into wet box, 2h is incubated in 37 °C of incubators.
I, the enzyme-linked antibody-solutions of preparation：According to 1:200 dilution ratio is diluted to enzyme-linked antibody, and dilution is
The carbonate coating buffer solution of PH9.6.
J, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 7
It is secondary.
K, enzyme-added len antibody：The 100 enzyme-linked antibody-solutions of μ L are added in plate per hole.
L, incubation plate：ELISA Plate is put into wet box and is incubated 2h in 37 °C.
M, preparation PNPP solution：Need to be now with the current, by PNPP powder buffer solutions, final concentration of 1mg/ml.
N, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 8
It is secondary.
O, plus PNPP solution：100 μ LPNPP solution are added in plate per hole.
P, incubation plate：ELISA Plate is put into wet box and is incubated 1h in 37 °C, notes lucifuge.
Q, result detection：On ELIASA, at 405nm, the OD values of each reacting hole are surveyed after being returned to zero with blank control wells,
If being the positive more than 2 times of negative control OD value.
Testing result shows that the stem apex for stripping 0.3mm ~ 0.5mm sizes using aseptic seedling is cultivated, the removing to PVX
Rate is 73.3%, and the removal efficiency to TMV is 66.7%, and the removal efficiency to CMV is 61.5%, the seedling containing virus is abandoned, without virus
Seedling（That is detoxic seedling）Continue to cultivate.
3）, detoxic seedling Multiplying culture：
Detoxic seedling is transferred on superclean bench fills proliferated culture medium（MS+ 6-BA 1mg/l+NAA0.4mg/l+
IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l）Blake bottle in, every bottle connects 5 young plants, is incubated at illumination 12h/ days, light intensity
2000lx, in the culturing room that 21 ± 2 DEG C of temperature；Treat that seedling covers with one bottle, can continue to turn Multiplying culture, general 1 bottle switchable 10 bottles,
Multiplying culture can continuously transfer.
4）, detoxic seedling culture of rootage：
Root media is transferred to after the tufted seedling of Multiplying culture to 4cm or so is divided into individual plant on superclean bench（1/2MS
+ NAA0.4mg/l+IBA0.6mg/l+ sucrose 20g/l+ agar powders 5.5g/l）In, every bottle connects 5 young plants, is incubated at illumination 12h/
My god, light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；Counted after 40 days, rooting rate 100%, number 10.8 of averagely taking root/
Strain.
5）, detoxic seedling acclimatization and transplantses：
Culture of rootage 40d is taken, every plant of Gen Shuo≤8, the detoxic seedling of root more than 1cm long carry out practicing transplantation of seedlings, blake bottle from group
Between training room moves to buffering, normal temperature closes bottle hardening a 3d, half-open lid 3d, full open end 3d.Complete hardening after with tweezers by bighead atractylodes rhizome seedling from
Blake bottle is removed, and is put into clear water, cleans root agar, is transplanted in matrix（Fertile soil, vermiculite, perlite ratio are 3：1：1）
In, pour permeable, daily morning and evening blade face watering reduces watering number of times to keep moistening, after one week.After one month, transplant survival
Seedling starts young leaves long, survival rate 93%.
Wherein, bighead atractylodes rhizome terminal bud disinfection way experiment：Disinfectant is done with liquor natrii hypochloritis, liquor natrii hypochloritis's concentration is selected
With two factors of process time, 3 concentration levels and 3 process time levels are set respectively, design 9 groups of experiments, every group of experiment
Except mode of disinfecting is different, remaining treatment is all identical.Explant is invaded into bubble 30s with 75% alcohol, after aseptic water washing 3 times, respectively
Group is processed according to the sterilization method described in table 1, and after the completion of sterilization, with aseptic water washing 5 times, filter paper blots the moisture on surface, will
Terminal bud is inoculated in the flat based tubes for filling MS culture mediums, and every group 20 bottles, every bottle of 1 terminal bud starts daily observation, and do after 3 days
Record easy to remember, in time the culture test tube isolation processing of microbiological contamination, after being inoculated with 20 days, statistics pollution rate, melting brown rate and survival rate, as a result
It is shown in Table 1.
Explant sum × 100% of pollution rate=pollution number/access culture medium
Explant sum × 100% of melting brown rate=browning number/access culture medium
Explant sum × 100% of survival rate=turn green number/access culture medium
In this sterilizing test, preferably, survival rate has 50% to the effect under the conditions of experiment 5 and experiment 7.7 pollution rates are tested relatively to test
5 low but melting brown rates are high compared with testing 5, and inventor selects the relatively low mode of experiment 5 of decontaminant concentration, to preserve training to greatest extent
Support the viability of material.Therefore the suitable disinfection way of bighead atractylodes rhizome terminal bud is：75% alcohol 30s+1% sodium hypochlorite 15min.
Wherein, terminal bud Initial culture base formula test：The formula of bighead atractylodes rhizome Initial culture base, selection 6-BA, NAA two because
Element, sets 3 concentration levels respectively, designs 9 groups of experiments（It is shown in Table 2）, each group experiment except plant growth substance contained by culture medium not
With outer, remaining processing mode is all identical, the culture medium based on MS, every group of culture medium sucrose containing 30g/L and 5.5g/L agar
Powder, pH value is 5.8.The terminal bud after sterilization is inoculated into the Initial culture base described in table 2 on superclean bench, every group 20
Bottle, every bottle of 1 terminal bud starts daily observation after 3 days, and makes a record, in time the culture test tube isolation processing of microbiological contamination.Connect
After planting 30 days, planting percent is counted, analyze result of the test（The results are shown in Table 2）.
Planting percent=number of seedling/（Access the explant sum-pollution number of culture medium）×100%
As can be seen from Table 2, preferably, bighead atractylodes rhizome planting percent is up to 88% for effect of the bighead atractylodes rhizome terminal bud culture under the conditions of experiment 3.Therefore at the beginning of the bighead atractylodes rhizome
Foster appropriate media formula of being commissioned to train is：MS+6-BA 0.5mg/l+NAA0.1mg/l+ sucrose 30g/l+ agar powders 5.5g/l.
Wherein, proliferation culture medium formula experiment：Selection tri- factors of 6-BA, NAA, IBA, set three concentration water respectively
It is flat, 9 groups of experiments of orthogonal design（It is shown in Table 3）, each group experiment except contained by culture medium plant growth substance difference in addition to, remaining processing mode
All same, with MS as minimal medium, each group sucrose containing 30g/L and 5.5g/L agar powders, pH value is 5.8.In ultra-clean work
The seedling that Initial culture is obtained is transferred in the proliferated culture medium described in table 3 on platform, every group 6 bottles, every bottle connects 5 young plants, i.e., every group
30 young plants are connect altogether, and inoculation counts propagation multiplying power, analyzes result of the test after 40 days（The results are shown in Table 3 and table 4）.
Total seedling number × 100% of the proliferation times=sum that sprouts/access culture medium；
As can be seen from Table 3, preferably, proliferation times are 4.6 to effect of the bighead atractylodes rhizome Multiplying culture under the conditions of experiment 3.Range analysis table
It is bright（It is shown in Table 4）, preferable concentration proportioning is A1B3C3, therefore bighead atractylodes rhizome Multiplying culture appropriate media formula is：MS+6-BA1mg/l+
NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l.
Wherein, aseptic seedling Stem tip induction seedling culture medium prescription experiment：Selection minimal medium, 6-BA, NAA tri- because
Element, sets 3 concentration levels, 9 groups of experiments of orthogonal test respectively（It is shown in Table 5）, each to process in addition to culture medium difference, remaining treatment side
Formula all same.Every group of culture medium sucrose containing 30g/L and 5.5g/L agar powders, pH value is 5.8.Will be aseptic on superclean bench
Seedling is taken out from blake bottle, and cauline leaf and spire are successively peelled off with scalpel and tweezers ecto-entad, until the top point of semi-round ball
Raw tissue is fully exposed, and cuts the stem apex of 0.3mm ~ 0.5mm sizes, is inoculated into the inducing culture described in table 5, every group
10 bottles, every bottle connects 3 stem apexs, i.e., every group and connects 30 stem apexs altogether, after being inoculated with 45 days, counts planting percent, analyzes result of the test（It is shown in Table
5 and table 6）.
Planting percent=number of seedling/（Access the explant sum-pollution number of culture medium）×100%；
As seen from Table 5, effect of the aseptic seedling Stem tip induction under the conditions of experiment 4 is preferable, planting percent 90%.Range analysis（It is shown in Table 6）
Show, three kinds of preferable concentration proportionings of factor are A2B1C2, therefore aseptic seedling Stem tip induction appropriate media is：1/2MS+6-
BA0.5g/l+NAA0.1g/l+ sucrose 30g/l+ agar powders 5.5g/l.
Wherein, aseptic seedling stem apex detoxification efficiency detection：The seedling that aseptic seedling stem apex virus-free culture is obtained is numbered, is bred
Viral diagnosis are carried out using DAS-ELISA after expansion is numerous, virus elimination rate is calculated, 7 are the results are shown in Table,
Virus elimination rate=detoxic seedling number/seedling number × 100% containing virus；
There are 15 plants to contain CMV containing TMV, 13 plants containing PVX, 21 plants in table 7 before the non-detoxification of 24 young plants, 4 plants contain PVX after detoxification, and 11 plants take off
Except PVX, PVX removal efficiencies are 73.3%；7 plants contain TMV, 14 plants of removing TMV, and TMV removal efficiencies are 66.7%；5 plants contain CMV, 8 plants of removings
CMV, CMV removal efficiency are 61.5%.Removing 17 plants of the seedling of virus of tri- kinds of PVX, TMV, CMV is obtained, total virus elimination rate is 71%, illustrates nothing
The less stem apex of vaccine can reach preferably virus removal effect after culture.
Present invention aseptic seedling stem apex makees explant, eliminates sterilisation step, it is to avoid damage of the disinfectant to stem apex, into
Work(will smaller stem apex（0.3mm~0.5mm）Sprout is turned out, planting percent 90%, DAS-ELISA detections show that PVX removal efficiencies are
73.3%；TMV removal efficiencies are 66.7%；CMV removal efficiencies are 61.5%, and removing tri- kinds of viruses of PVX, TMV, CMV are obtained after screening
Tissue-cultured seedling.
The above, is only presently preferred embodiments of the present invention, and any formal limitation is not made to the present invention, is appointed
What any is simply repaiied without departing from technical solution of the present invention content according to what technical spirit of the invention was made to above example
Change, equivalent variations and modification, still fall within the range of technical solution of the present invention.
Claims (5)
1. a kind of method of two stage culture bighead atractylodes rhizome detoxic seedling, it is characterised in that：Including following operating process：
1. asepsis training department is set up using bighead atractylodes rhizome terminal bud：
1）Bighead atractylodes rhizome terminal bud Initial culture：Choose field growing and show excellent bighead atractylodes rhizome plant, cut the 2cm long shoots comprising terminal bud
Section, removes blade, is rinsed 30 minutes with being put into after detergent washes clean in flowing water, standby；The material strips that will be got ready enter ultra-clean
Workbench, cuts terminal bud, and bubble 30s is invaded with 75% alcohol, and then aseptic water washing 3 times soaks 15min, phase with 1% sodium hypochlorite
Between stir, with aseptic water washing 5 times, filter paper blots surface moisture, terminal bud is inoculated in and fills Initial culture base：MS+6-BA
The flat based tubes of 0.5mg/l+NAA0.1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, often 1 terminal bud of pipe, is placed in illumination 12h/
My god, light intensity 2000lx is cultivated in the culturing room that 21 ± 2 DEG C of temperature, daily observation, finds that part explant has pollution feelings after 7 days
Condition, clears out of culturing room by the culture test tube of pollution in time, and rear portion exceptionally implant starts to turn green within 10 days, browning partly occurs and shows
As turn green terminal bud around gradually expanded by the culture of 15 days, and base portion can form light green callus, after 30 days gradually
Differentiate 3cm ~ 4 cm sprouts；
2）Increment culture：The sprout that Initial culture is obtained for 30 days is transferred on superclean bench fills proliferated culture medium：MS+
In the blake bottle of 6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, every bottle connects 5 young plants,
It is incubated at illumination 12h/ days, light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；After 40 days, statistical average propagation multiplying power；
2. aseptic seedling stem apex virus-free culture：
1）Stem tip induction seedling culture：Take aseptic seedling from the asepsis training department set up, on superclean bench with scalpel and
Tweezers ecto-entad successively peels off cauline leaf and spire, until the apical meristem of semi-round ball is fully exposed, cuts 0.3mm
The stem apex of ~ 0.5mm sizes, is inoculated into and fills inducing culture：1/2MS+6-BA0.5g/l+NAA0.1g/l+ sucrose 30g/l+ fine jades
In the blake bottle of cosmetics 5.5g/l, every bottle connects 3 stem apexs, is incubated at illumination 12h/ days, light intensity 2000lx, 21 ± 2 DEG C of temperature
In culturing room, timing daily is observed, and stem apex expands growth after 15 days, and the bud point projection of green occurs in shoot tip meristem after 20 days,
Bud point is grown tall and grows up after 25 days, begins to differentiate into bud, and 1cm ~ 3cm sprouts high, the sprout of formation can be differentiated to form after 40 days
In some be stripped of virus；
2）Detoxic seedling is screened：After Shoot Tip Culture 45 days, the sprout that aseptic seedling Shoot Tip Culture is obtained is inoculated in proliferated culture medium：MS
Proliferation of propagation is carried out in+6-BA 1mg/l+NAA0.4mg/l+IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, after 40 days
Propagation seedling is removed from blake bottle, clip 0.2g cauline leafs carry out Viral diagnosis；Seedling containing virus is abandoned, by without virus
Seedling, i.e. detoxic seedling continue to cultivate；
3）, detoxic seedling Multiplying culture：
Detoxic seedling is transferred on superclean bench fills proliferated culture medium：MS+ 6-BA 1mg/l+NAA0.4mg/l+
In the blake bottle of IBA1mg/l+ sucrose 30g/l+ agar powders 5.5g/l, every bottle connects 5 young plants, is incubated at illumination 12h/ days, light intensity
2000lx, in the culturing room that 21 ± 2 DEG C of temperature；
4）, detoxic seedling culture of rootage：
Root media is transferred to after the tufted seedling of Multiplying culture to 4cm is divided into individual plant on superclean bench：1/2MS+
In NAA0.4mg/l+IBA0.6mg/l+ sucrose 20g/l+ agar powders 5.5g/l, every bottle connects 5 young plants, is incubated at illumination 12h/ days,
Light intensity 2000lx, in the culturing room that 21 ± 2 DEG C of temperature；Rooting rate is counted after 40 days；
5）, detoxic seedling acclimatization and transplantses：
Culture of rootage 40d is taken, every plant of Gen Shuo≤8, the detoxic seedling of root more than 1cm long carry out practicing transplantation of seedlings, blake bottle from group
Between training room moves to buffering, normal temperature closes bottle hardening a 3d, half-open lid 3d, full open end 3d；Complete hardening after with tweezers by bighead atractylodes rhizome seedling from
Blake bottle is removed, and is put into clear water, cleans root agar, is transplanted in matrix, pours permeable, daily morning and evening blade face watering, to protect
Moistening is held, watering number of times is reduced after one week；After one month, the seedling of transplant survival starts young leaves long, survival rate 93%.
2. the method for two stage culture bighead atractylodes rhizome detoxic seedling as claimed in claim 1, it is characterised in that：In the step 12）
Increment culture：Treat that seedling covers with one bottle, continue to turn Multiplying culture, 1 bottle is transferred 10 bottles, and Multiplying culture is continuously transferred, and thus sets up white
Art terminal bud asepsis training department.
3. the method for two stage culture bighead atractylodes rhizome detoxic seedling as claimed in claim 1, it is characterised in that：In the step 22）
Detoxic seedling is screened：Method for detecting virus uses Enzyme-linked Immunosorbent Assay technology-double-antibody method DAS-ELISA, and step is as follows：
A, preparation wet box：3 ~ 4 hygenic towelettes are put into crisper, for moisturizing and lucifuge；
B, preparation capture antibody-solutions：According to 1:200 dilution ratio is diluted to capture antibody, and dilution is PH9.6's
Carbonate coating buffer solution；
C, coating plate：Coated antibody in ELISA Plate, 100 μ L capture antibody-solutions are added per hole；
D, incubation plate:ELISA Plate is put into wet box, is placed in overnight incubation in 4 °C of refrigerators, and standing time is no more than 24h；
E, board-washing：Next day, solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, moistened
Wash 3 times；
F, sample preparation：Seedling to be detected is removed from blake bottle, clip 0.2g cauline leafs are placed in mortar, add the general of 2ml to carry
Take buffer solution GEB and be ground to homogenate shape；1000rmp is centrifuged 10min, takes supernatant standby；
G, sample-adding：Positive control, negative control, sample solution are separately added into washed ELISA Plate, 100 μ L are added per hole,
Blank control wells add the GEB of 100 μ L；
H, incubation plate：ELISA Plate is put into wet box, 2h is incubated in 37 °C of incubators；
I, the enzyme-linked antibody-solutions of preparation：According to 1:200 dilution ratio is diluted to enzyme-linked antibody, and dilution is PH9.6's
Carbonate coating buffer solution；
J, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 7 times；
K, enzyme-added len antibody：The 100 enzyme-linked antibody-solutions of μ L are added in plate per hole；
L, incubation plate：ELISA Plate is put into wet box and is incubated 2h in 37 °C；
M, preparation PNPP solution：Need to be now with the current, by PNPP powder buffer solutions, final concentration of 1mg/ml；
N, board-washing：Solution in ELISA Plate hole is fallen, reacting hole is filled up with PBST cleaning solutions, then dried to the greatest extent, rinse 8 times；
O, plus PNPP solution：100 μ LPNPP solution are added in plate per hole；
P, incubation plate：ELISA Plate is put into wet box and is incubated 1h in 37 °C, notes lucifuge；
Q, result detection：On ELIASA, at 405nm, the OD values of each reacting hole are surveyed after being returned to zero with blank control wells, if greatly
Then it is the positive in 2 times of negative control OD value.
4. the method for two stage culture bighead atractylodes rhizome detoxic seedling as claimed in claim 1, it is characterised in that：In the step 23）
Detoxic seedling Multiplying culture：Treat that seedling covers with one bottle, continue to turn Multiplying culture, general 1 bottle is transferred 10 bottles, and Multiplying culture can continuously turn
Connect.
5. the method for two stage culture bighead atractylodes rhizome detoxic seedling as claimed in claim 1, it is characterised in that：In the step 25）
Detoxic seedling acclimatization and transplantses：The matrix is fertile soil, vermiculite, perlite, and ratio is 3：1：1.
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| 孙慧生: "《马铃薯生产技术百问百答》", 31 October 2005, 中国农业出版社 * |
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