CN105993955B - A kind of false-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings - Google Patents
A kind of false-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings Download PDFInfo
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- CN105993955B CN105993955B CN201610377608.5A CN201610377608A CN105993955B CN 105993955 B CN105993955 B CN 105993955B CN 201610377608 A CN201610377608 A CN 201610377608A CN 105993955 B CN105993955 B CN 105993955B
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses be related to a kind of false-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings.It is using false-yellowflower milkwort root or herb tender stem as explant, after sterilized, evoking adventive bud in the primary culture medium of 2.0 mg/L+NAA of MS+6 BA, 0.05 mg/L is inoculated in occur, then adventitious bud is seeded in the proliferation and root media of additional variety classes and concentration exogenous hormone and carries out proliferation and culture of rootage, and rooting tube plantlet is transplanted in hardening matrix hardening.The false-yellowflower milkwort root or herb rapid propagation in vitro system that the present invention establishes, convenient material drawing, stability is good, and repeatability is high, for quickly the breeding and solving its wild resource scarcity of realization false-yellowflower milkwort root or herb elite germplasm in the upper present situation providing method that supply falls short of demand of production.
Description
Technical field
The present invention relates to false-yellowflower milkwort root or herb rapid propagation in vitro technical fields, more particularly to false-yellowflower milkwort root or herb rapid propagation in vitro nursery
Method.
Background technology
False-yellowflower milkwort root or herb (Polygala fallax Hemsl. it is big that) alias chrysanthemum is joined, chrysanthemum hangs water lotus, kwan-yin string, chrysanthemum
Radix Polygalae etc., category Polygalaceae (Polygalaceae) rogation flower (Polygala) plant [1].It is distributed mainly on Fujian, the lake in China
The ground such as south, Guangxi [2-4].It is a kind of more rare Chinese medicine, all herbal medicine, nature and flavor are sweet, slight bitter, put down.There is tonifying Qi and blood, is good for
Spleen dampness removing, the function of promoting blood circulation for regulating menstruation.It is precious jade, seedling, the common folk medicine of Zhuan Deng ethnic groups [5].False-yellowflower milkwort root or herb is as one
There is the situation that supply falls short of demand again and again as medicinal material market is continuously increased its demand in the kind valuable ingredients of traditional Chinese medicine, some areas are even
It is in the presence of in short supply.Wild resource is constantly mined utilization, has been difficult to meet the current market demand.False-yellowflower milkwort root or herb is to plant
Son breeding is more, but fertility is weak [6].Therefore false-yellowflower milkwort root or herb this valuable source is developed and be made good use of, must just be solved
Certainly its fast numerous problem.And tissue culture technique is the most effectual way for expanding rapidly seedling quantity in a short time.Related chrysanthemum falls
The rapid propagation in vitro of water lotus is studied, and there is relevant report [7-9] in rarely seen country, but has certain office in terms of stability and repeatability
It is sex-limited.Therefore, establish that stability is good, the high false-yellowflower milkwort root or herb rapid propagation in vitro technology of repeatability is expected to realize that false-yellowflower milkwort root or herb is excellent
The quick breeding of breeding matter is of great significance to solving its wild resource scarcity in the upper present situation that supply falls short of demand of production.
In the prior art, application No. is 201210147606.9, a kind of entitled tissue cultures of false-yellowflower milkwort root or herb are numerous
It grows method and has been disclosed for a kind of tissue culture propagation method for Polygala fallax Hemsl, include the following steps：
（1）Explant acquires and disinfection：Take false-yellowflower milkwort root or herb current year green tape axillary bud tender stem segments as explant, it is clean with washing
10 min of smart immersion treatment, stem section surface is gently scrubbed with banister brush, and flowing water rinses 30 min, is placed on superclean bench, cuts
At the stem section with 1-2 internode, the KMnO for being 0.1% with mass concentration4 Immersion treatment 5min, aseptic water washing 2 times, volume
A concentration of 75% 20 s of ethyl alcohol immersion treatment, then with mass concentration be 0.1% 10 min of mercuric chloride immersion treatment, sterile water punching
It washes 6 times, obtains sterilizable material；
（2）The induction of adventitious bud：Above-mentioned sterilizable material is inoculated on adventitious bud induction culture base, in temperature 25 ± 1
DEG C, false-yellowflower milkwort root or herb adventitious bud, Fiber differentiation 30 days are induced under conditions of 11 h/d of light application time, 3000 LX of intensity of illumination；
（3）Adventitious bud proliferation culture：The false-yellowflower milkwort root or herb adventitious bud induced is forwarded in adventitious bud proliferation culture medium
The Multiplying culture of adventitious bud is carried out, condition of culture is：25 ± 1 DEG C of temperature, 11 h/d of light application time, 3000 LX of intensity of illumination,
Multiplying culture 25 days；
（4）Adventitious bud rooting culture：The adventitious bud of Multiplying culture is transferred in adventitious bud rooting culture medium and carries out training of taking root
It supports, condition of culture is：25 ± 1 DEG C of temperature, 11 h/d of light application time, 3000 LX of intensity of illumination, culture of rootage can obtain for 25 days
Obtain tissue-cultured seedling；
（5）Hardening and transplanting：Greenhouse is moved it to when tissue-cultured seedling 80% grows root restriction and carries out hardening, by bottle after 7 days
Seedling gently presss from both sides out, cleans root culture medium, and 2~3 min of mancozeb immersion treatment for being 0.1% with mass concentration dries in the shade,
After moisture on plant to be planted limb dries in the shade well, it is transplanted into the transplanting for having used the potassium permanganate that mass concentration is 0.1% to sterilize in advance
In matrix, keep 25 ± 1 DEG C of environment temperature, spraying and moisturizing that air humidity is made to be maintained at 85-95%, covering shading rate 70%
Sunshade net, the wide-spectrum bactericide of sprinkling in every 7 days, pours one time twice MS a great number of elements solution when growing new root after 30 days.
The patent is by taking with the current year raw tender band axillary bud of children（It does not sprout）Stem section is sterilized to obtain sterile material as explant
After material, proliferation by evoking adventive bud, then through adventitious bud takes root to obtain tissue-cultured seedling, and tissue-cultured seedling is planted by domestication
Train seedling, from explant be induced to obtain survival false-yellowflower milkwort root or herb cultivation seedling only need 110 days, greatly speed up false-yellowflower milkwort root or herb
Reproduction speed.The inductivity of false-yellowflower milkwort root or herb adventitious bud is 95.1 in the invention, while the growth coefficient of adventitious bud monthly is
6.12, rooting rate 95.67%, transplanting survival rate is up to 92.6%, therefore, can provide a large amount of false-yellowflower milkwort root or herb in a short time
Seedling is cultivated, certainly solves that current false-yellowflower milkwort root or herb wild resource is limited, leads to resource critical shortage, while being its high quality seedling
Large-scale production so that Resource Cultivation with using laying the foundation.But the invention has one in terms of stability and repeatability
Fixed limitation.Therefore, establish that stability is good, the high false-yellowflower milkwort root or herb rapid propagation in vitro technology of repeatability is expected to realize that chrysanthemum falls
The quick breeding of water lotus elite germplasm has important meaning to solving its wild resource scarcity in the upper present situation that supply falls short of demand of production
Justice.
Invention content
In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides false-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings,
Purpose is with 75% alcohol disinfecting 10s, and 0.1% mercuric chloride disinfection 15min obtains preferable Disinfection Effect, pollution rate down to
3.15%；Culture medium suitable for false-yellowflower milkwort root or herb proliferation is MS+6-BA 2.0mg/L+NAA 0.10mg/L, and growth coefficient is
5.50；In the root media of 0.1 mg/L+NAA 0.3mg/L+0.3g/L activated carbons of 1/2MS+IBA, rooting rate can
Up to 96%；The optimum substrate of false-yellowflower milkwort root or herb test tube seedling hardening is red soil：Fertile soil：Perlite=1:1:1, survival rate 94.5%,
It has been successfully established false-yellowflower milkwort root or herb rapid propagation in vitro technical system.
The technical solution adopted in the present invention is：False-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings, includes the following steps：
Step 1, material obtain：Current year green tape has the false-yellowflower milkwort root or herb branch of tender stem；
Step 2, pretreatment：Explant rinses 3-5 min in liquid detergent water, then rinses 25-35 with flowing water
Min simultaneously trims long or excessive branch in cleaning process, facilitates subsequent disinfection；
Step 3, explant disinfection：Pretreated chrysanthemum water lotus branch in step 2 is taken, with 75% alcohol disinfecting 10-30s,
It is again 10-20min with 0.1% mercuric chloride disinfecting time；
Step 4 induces axillary bud：Material after will be sterilized in step 3, which is cut into, is about stem sections of the 2cm with 1-2 axil,
It inserts in 2.0 mg/L+NAA of culture medium MS+6-BA, 0.05 mg/L vertically, adds 30.0g/L sucrose, 5.0g/L agar, pH
5.7, explant culture 12-18d can induce out axillary bud at axil, can be grown to 2-3cm high after 30d；
Step 5, Multiplying culture：The axillary bud induced in interception above-mentioned steps four is inoculated in proliferated culture medium, is increased
Culture is grown, 20-25d cultivates a generation；
Step 6, culture of rootage：The healthy and strong test tube seedling obtained in selecting step five cuts the long 2cm in top and accesses training of taking root
It supports in base and carries out culture of rootage, test tube seedling can take root after 20d；
Step 7, acclimatization and transplants：It will take root in step 6 good, the test tube seedling of robust growth moves to general from tissue culture room
Bottle cap, is then opened 1/3 aperture, hardening 2d finally takes off bottle cap completely by logical laboratory or greenhouse, in-bottle seeding 5d
It opens, after hardening 1d, by tissue-cultured seedling from transplanting to matrix in tissue culture bottle, Small plastic shed is used in combination to cover；During hardening, it is ensured that base
The enough moisture of matter, and take shading measure；It is i.e. viable after 25d；Above step four, five and six is inoculated with work ultra-clean
It is operated in workbench, the culture of test tube seedling carries out in tissue cultures room, and condition of culture is：In 25 DEG C of temperature, illumination is strong
1200-1500Lx is spent, is carried out under conditions of periodicity of illumination 12h/d.
Further, step 7 acclimatization and transplants：Using red soil, fertile soil and perlite as raw material, will take root good, growth is strong
Strong test tube seedling moves to common lab or greenhouse from tissue culture room, then bottle cap is opened 1/3 by in-bottle seeding 5d
Aperture, hardening 2d；Finally bottle cap is opened completely, after hardening 1d, by tissue-cultured seedling from transplanting to matrix in tissue culture bottle, is used in combination small
Arched shed covers.
Further, it is made using red soil, perlite and fertile soil cooperation in step 7 and practices seedling grafting matrix.
Further, the hardening matrix in step 7 acclimatization and transplants is red soil：Fertile soil：Perlite=1:1:1 or humic
Soil：Perlite=1:One kind in 1 or full fertile soil.
Further, 75% alcohol disinfecting time was 10s in step 1, and 0.1% mercuric chloride is 15min, and the disinfection of explant is imitated
Fruit is preferable.
Further, the proliferated culture medium of step 5 is MS+6-BA 1.5mg/L+NAA 0.05mg/L.
Further, the prescription of rooting medium described in step 6 is 0.1 mg/L+ NAA of 1/2MS+IBA, 0.3 mg/L
+ 0.2 g/L activated carbons.
Compared with prior art, the beneficial effects of the invention are as follows：Using false-yellowflower milkwort root or herb tender stem as explant, through distinct methods
After disinfection, it is inoculated in the culture medium of 2.0 mg/L+NAA of MS+6-BA, 0.05 mg/L and induces axillary bud, then by axillary bud
The culture medium for being seeded in additional variety classes and concentration exogenous hormone is cultivated, and according to being proliferated and situation of taking root, screening is suitable
False-yellowflower milkwort root or herb proliferation and root media；Then by rooting tube plantlet transplanting in different matrix, according to survival rate, really
Its fixed best hardening matrix.Preferable Disinfection Effect is obtained, is polluted with 75% alcohol disinfecting 10s, 0.1% mercuric chloride disinfection 15min
Rate is down to 3.15%；Culture medium suitable for false-yellowflower milkwort root or herb proliferation is MS+6-BA 2.0mg/L+NAA 0.10mg/L, growth coefficient
It is 5.50；In the root media of 0.1 mg/L+NAA 0.3mg/L+0.3g/L activated carbons of 1/2MS+IBA, rooting rate
Up to 96%；The optimum substrate of false-yellowflower milkwort root or herb test tube seedling hardening is red soil：Fertile soil：Perlite=1:1:1, survival rate is
94.5%.In the present invention, 100-105d is only needed from the false-yellowflower milkwort root or herb seedling for obtaining and surviving that is induced to of explant, and taking root
General management afterwards, without being poured with twice of MS a great number of elements solution.The numerous of false-yellowflower milkwort root or herb is greatly accelerated in this way
Speed is grown, and this method stability and repeatability are high.Therefore, a large amount of false-yellowflower milkwort root or herb cultivation can be provided in a short time
Seedling, certainly solves that current false-yellowflower milkwort root or herb wild resource is limited, leads to resource critical shortage, can meet its factory's metaplasia
Production.
Description of the drawings
Fig. 1 is the photo of the one embodiment in false-yellowflower milkwort root or herb axillary bud deriving stage；
Fig. 2 is the photo of the one embodiment in false-yellowflower milkwort root or herb Multiplying culture stage；
Fig. 3 is the photo of the one embodiment in false-yellowflower milkwort root or herb culture of rootage stage；
Fig. 4 is the photo of one embodiment that false-yellowflower milkwort root or herb transplants the hardening stage.
Specific implementation mode
In order to deepen the understanding of the present invention, present invention will be further explained below with reference to the attached drawings and examples, the implementation
Example is only used for explaining the present invention, does not constitute and limits to protection scope of the present invention.
Embodiment carries out such as using step and design parameter needed for false-yellowflower milkwort root or herb rapid propagation in vitro Establishing as object
Lower experiment
1 materials and methods
1.1 test material
Test material is the branch that current year green tape has tender stem, is derived from Qingliu County of Sanming, Fujian Province city.
1.2 test method
1.2.1 pretreatment
Explant rinses 3 ~ 5 min in liquid detergent water, then rinses 30 min with flowing water.To long in cleaning process
Or excessive branch is trimmed, and subsequent disinfection is facilitated.
1.2.2 explant disinfection and axillary bud deriving
Take above-mentioned pretreated material, carried out disinfection with the mercuric chloride of 75% alcohol and 0.1%, be arranged different disinfecting times with
Determine the disinfection scheme suitable for false-yellowflower milkwort root or herb（Table 1）.It is cut into after material is sterilized and is about stem sections of the 2cm with 1-2 axil, erected
Straight cutting adds 30.0g/L sucrose, 5.0g/L agar, pH in 2.0 mg/L+NAA of culture medium MS+6-BA, 0.05 mg/L
5.7（Similarly hereinafter）.4 sections of explant is placed in total 12 processing of experiment, each 20 bottles of processing, often processing.It is induced at stem section axil
Adventitious bud, then be used as Multiplying culture material.It is as shown in Figure 1 the one embodiment in false-yellowflower milkwort root or herb axillary bud deriving stage
Photo.
1 false-yellowflower milkwort root or herb explant of table sterilizes scheme
| Number | 75% alcohol（s） | 0.1% mercuric chloride（min） |
| 1 | 10 | 5 |
| 2 | 20 | 5 |
| 3 | 30 | 5 |
| 4 | 10 | 10 |
| 5 | 20 | 10 |
| 6 | 30 | 10 |
| 7 | 10 | 15 |
| 8 | 20 | 15 |
| 9 | 30 | 15 |
| 10 | 10 | 20 |
| 11 | 20 | 20 |
| 12 | 30 | 20 |
1.2.3 Multiplying culture
The axillary bud induced in interception 1.2.2, about 1cm long are respectively basic culture medium with MS and WPM, are swashed using identical
Plain type and concentration proportioning design scheme carry out the screening of false-yellowflower milkwort root or herb proliferated culture medium.Experimental design such as table 2.Experiment is altogether
4 sections of material is placed in 18 processing of meter and 1 control, each 20 bottles of processing, often processing.Fig. 2 is false-yellowflower milkwort root or herb Multiplying culture rank
The photo of one embodiment of section.
2 false-yellowflower milkwort root or herb proliferated culture medium scheme of table
| Processing | 6-BA（mg/L） | TDZ（mg/L） | NAA（mg/L） |
| 1 | 1.0 | 0 | 0.05 |
| 2 | 1.0 | 0 | 0.10 |
| 3 | 1.0 | 0 | 0.20 |
| 4 | 1.5 | 0 | 0.05 |
| 5 | 1.5 | 0 | 0.10 |
| 6 | 1.5 | 0 | 0.20 |
| 7 | 2.0 | 0 | 0.05 |
| 8 | 2.0 | 0 | 0.10 |
| 9 | 2.0 | 0 | 0.20 |
| 10 | 0 | 0.01 | 0.05 |
| 11 | 0 | 0.01 | 0.10 |
| 12 | 0 | 0.01 | 0.20 |
| 13 | 0 | 0.03 | 0.05 |
| 14 | 0 | 0.03 | 0.10 |
| 15 | 0 | 0.03 | 0.20 |
| 16 | 0 | 0.05 | 0.05 |
| 17 | 0 | 0.05 | 0.10 |
| 18 | 0 | 0.05 | 0.20 |
| CK | 0 | 0 | 0 |
1.2.4 culture of rootage
Two kinds of hormone various concentrations of experimental design IBA, NAA are matched with different amounts of activated carbon, are added to 1/2MS culture mediums
In（Table 3）.The healthy and strong test tube seedling through Multiplying culture is chosen, top is cut and is about in 2cm access culture mediums.At experiment is 9 total
4 sections of material is placed in reason and 1 control, each 20 bottles of processing, often processing.It is illustrated in figure 3 the false-yellowflower milkwort root or herb culture of rootage stage
One embodiment photo.
3 false-yellowflower milkwort root or herb root media scheme of table
| Processing | IBA(mg/L) | NAA(mg/L) | Activated carbon（g/L) |
| 1 | 0.1 | 0.1 | 0.1 |
| 2 | 0.3 | 0.1 | 0.2 |
| 3 | 0.5 | 0.1 | 0.3 |
| 4 | 0.1 | 0.3 | 0.2 |
| 5 | 0.3 | 0.3 | 0.3 |
| 6 | 0.5 | 0.3 | 0.1 |
| 7 | 0.1 | 0.5 | 0.3 |
| 8 | 0.3 | 0.5 | 0.1 |
| 9 | 0.5 | 0.5 | 0.2 |
| CK | 0 | 0 | 0 |
1.2.5 acclimatization and transplants
Using red soil, fertile soil and perlite as raw material, 6 kinds of different matrix, proportion design such as table 4 are configured.It will take root
Well, the test tube seedling of robust growth moves to common lab or greenhouse, in-bottle seeding 5d, then by bottle from tissue culture room
Lid opens 1/3 aperture, and hardening 2d finally opens bottle cap completely, and after hardening 1d, tissue-cultured seedling is transplanted from tissue culture bottle to base
In matter, Small plastic shed is used in combination to cover.During hardening, it is ensured that the enough moisture of matrix, and take certain shading measure.Such as figure
4 show the photo of the one embodiment in false-yellowflower milkwort root or herb transplanting hardening stage.
4 false-yellowflower milkwort root or herb transplanting medium scheme of table
| Number | Matrix | Matrix feature |
| 1 | Full red soil | Water-retaining property preferably, poor air permeability, organic matter it is less |
| 2 | Red soil：Fertile soil=1:1 | Water-retaining property is preferable, gas permeability is general, organic matter is abundant |
| 3 | Red soil：Perlite=1:1 | Water-retaining property preferably, good permeability, organic matter it is less |
| 4 | Red soil：Fertile soil：Perlite=1:1:1 | Water-retaining property is preferable, gas permeability is preferable, organic matter is abundant |
| 5 | Fertile soil：Perlite=1:1 | Water-retaining property is poor, good permeability, organic matter are abundant |
| 6 | Full fertile soil | Water-retaining property is general, good permeability, organic matter are abundant |
1.2.6 condition of culture
Above-mentioned interior culture is carried out under conditions of 25 DEG C, intensity of illumination 1200Lx, periodicity of illumination 12h/d of temperature
's.
1.3 data analysis
Test data is for statistical analysis using SPSS17.0 softwares.
As a result with analysis
2.1 explants sterilize
False-yellowflower milkwort root or herb is after difference is disinfected, and as a result such as table 5, in being handled at 12 kinds, highest pollution rate is processing
1, pollution rate 45.71%, pollution rate it is minimum be processing 8, pollution rate 2.47%.Between disinfecting time processing 7,8,9 and 10
Without significant difference, pollution rate is relatively low（Respectively 3.15%, 2.47%, 2.66% and 3.52%）.Illustrate, in this 4 kinds of conditions for sterilization
Under, it can get good Disinfection Effect.But in processing 7, the relatively processing 8,9 and 10 of explant growing state will be got well.Therefore recognize
For processing 7（75% alcohol 10s, 0.1% mercuric chloride 15min）For the most ideal method of false-yellowflower milkwort root or herb explant disinfection.Initial culture
12-18d or so can induce out adventitious bud at axil, can be grown to 2-3cm high after 30d.
5 difference of table is disinfected effect and is compared
| Processing | Pollution rate (%) | Growing state |
| 1 | 45.71a | Well |
| 2 | 36.18b | Well |
| 3 | 35.84b | It is good a small amount of dead |
| 4 | 12.39c | Preferably |
| 5 | 9.27d | Well |
| 6 | 9.51d | It is good a small amount of dead |
| 7 | 3.15f | Preferably |
| 8 | 2.47f | It is good a small amount of dead |
| 9 | 2.66f | It is general a small amount of dead |
| 10 | 3.52f | It is general a small amount of dead |
| 11 | 6.03e | Poor mortality |
| 12 | 5.23e | Poor mortality |
Note：It is notable with column data difference lowercase letter indication difference in table（P ＜ 0.05）, similarly hereinafter.
2.2 Multiplying culture
In different proliferated culture mediums, the cultivation effect such as table 6 of false-yellowflower milkwort root or herb axillary bud is not added in the control of hormone
Growth coefficient is minimum, and respectively 2.08 and 1.89, illustrate that the addition of exogenous hormone can significantly improve the proliferation of false-yellowflower milkwort root or herb
Coefficient.It is up to 5.50 with the growth coefficient that MS is processing 4 in basic culture medium in the processing for being added to exogenous hormone, and
There were significant differences with other each processing.The growth coefficient minimum 2.29 of processing 11；It is to handle 6 in basic culture medium with WPM
Growth coefficient be up to 4.13, and there were significant differences with other each processing.The growth coefficient minimum 1.39 of processing 10.Cause
This, false-yellowflower milkwort root or herb adventitious bud optimum multiplication medium is MS+6-BA 1.5mg/L+NAA 0.05mg/L.
Influence of 6 different culture media of table to false-yellowflower milkwort root or herb adventitious bud proliferation
2.4 culture of rootage
The rooting efficiency of false-yellowflower milkwort root or herb is different under the conditions of different culture of rootage（Such as table 7）.Rooting rate it is minimum processing be
It is not added with the control group CK of any hormone, is 21.0%, the rooting rate in remaining 1-9 processing experiments is 50.0% or more, explanation
The addition of exogenous hormone is conducive to false-yellowflower milkwort root or herb and takes root.Rooting rate up to handles 4, up to 96.0%, and take root item number and root long
Also the highest in all processing.Comprehensive analysis thinks, handles 4（1/2MS+IBA 0.1 mg/L+ NAA 0.3 mg/L+0.2 g/
L activated carbons）For the best root media of false-yellowflower milkwort root or herb.
The influence that 7 different culture media of table takes root to false-yellowflower milkwort root or herb
2.5 acclimatization and transplants
As shown in Table 8, there were significant differences for rooting rate of the different transplanting mediums to false-yellowflower milkwort root or herb, survival rate it is minimum be
Processing 1, survival rate is only 66.7%, in the matrix of processing 6, the survival rate highest of false-yellowflower milkwort root or herb test tube seedling（It is 95.2%）,
And difference is not notable between processing 4 and 5（Respectively 94.5% and 94.9%）, so, the matrix of this 3 processing is suitable for chrysanthemum
The transplanting hardening of omei meadowrue herb.But the guarantor that a certain proportion of red soil helps to improve matrix is added at originally from management and matrix
It is aqueous, moisturizing number is reduced, the cost of matrix can also be reduced.Therefore, suggest in actual production cultivation red using processing 4
Earth：Fertile soil：Perlite=1:1:1 substrate composition, survival rate is up to 94.5%.
Influence of 8 different substrates of table to false-yellowflower milkwort root or herb hardening
| Number | Matrix feature | Survival rate（%） |
| 1 | Water-retaining property preferably, poor air permeability, organic matter it is less | 66.7d |
| 2 | It is aqueous preferably, gas permeability is general, organic matter is abundant | 75.4c |
| 3 | Water-retaining property preferably, good permeability, organic matter it is less | 87.6b |
| 4 | It is aqueous preferably, gas permeability is preferable, organic matter is abundant | 94.5a |
| 5 | Water-retaining property is poor, good permeability, organic matter are abundant | 94.9a |
| 6 | Water-retaining property is general, good permeability, organic matter are abundant | 95.2a |
3 discuss
Test result shows that MS culture mediums are suitble to false-yellowflower milkwort root or herb explant axillary bud deriving, Luo Wanye etc. [8], Li Cuilan
Deng [9] when establishing false-yellowflower milkwort root or herb rapid propagation in vitro technical system, same culture medium has also been selected.And Liu Xiufang etc. [7] is then
It is basic culture medium with 1/2MS, additional and the identical hormone kind of this experiment and proportioning induce false-yellowflower milkwort root or herb stem section
Culture, also achieves comparatively ideal effect.MS is to balance preferable, the strong basal medium of buffer capacity [10] between a kind of element,
1/2MS only halves a great number of elements.At axillary bud deriving initial stage, a certain amount of energy and nutrition are stored in explant in itself,
Even these energy and nutrition are enough or required beyond induction axillary bud, and therefore, the selection of such basis culture lures axillary bud
Lead influence and it is little.In addition, the genotype difference of the condition and materials when culture can also have a certain impact.
It is found when Multiplying culture, is whether the training of basic culture medium with MS or through WPM with increasing for 6-BA concentration
In the scheme of supporting, the growth coefficient of adventitious bud is in rising trend.But it is most appropriate when with 1.5 mg/L, it is opened more than then growth coefficient
Begin to decline, and vitrification phenomenon occurs.The result of study of this and Liu Xiufang etc. [7] and to congener Shanxi production Radix Polygalae carry out
The result obtained when proliferation test is consistent [11].Under general feelings, the action intensity of TDZ is greater than 6-BA.And test result table
It is bright, the TDZ of 0.01,0.03,0.05 mg/L is added in two kinds of basal mediums, cultivation effect is significantly not so good as the effect of 6-BA
Fruit.This may be relatively low related with the concentration of the TDZ added.
In culture of rootage, the auxin for adding concentration range reasonable has taking root using false-yellowflower milkwort root or herb sprout.But
No matter the culture medium of any type hormone combination will produce a certain amount of callus.And such case can be with growth
The raising of plain concentration and aggravate.During experiment, often along with the generation of more callus while high rooting rate.Flower
Yellow omei meadowrue herb culture of rootage test result shows to add a certain amount of activated carbon in root media, can preferably inhibit to be cured
The formation of injured tissue, in later stage hardening, root is not easy to disconnect, and is conducive to improve survival rate when transplanting hardening.This and Li Cui
The result of study of orchid etc. [9] is consistent.
It has been reported and thinks, peat soil：Yellow soil：Perlite=2:1:1 is the hardening matrix of more suitable false-yellowflower milkwort root or herb,
Transplanting survival rate is up to 92.6% [7].The display of this test result, red soil：Fertile soil：Perlite=1:1:1, fertile soil：Perlite=
1:1, the survival rate of three kinds of substrate combinations of full fertile soil is above 94%.However, in the matrix of full red soil, after transplanting a couple of days, seedling
The wooden root blackening is simultaneously gradually rotted, final mortality.Supposition is that matrix gas permeability is too poor, and root anaerobic respiration is caused to generate wine
Essence then causes root to poison.It is more studies have shown that the water conservation of test tube seedling transplanting survival rate and matrix itself, fertilizer conservation, gas permeability
It is closely related with self stability.It is therefore desirable to which hardening matrix wants loose ventilative, and there is certain retention ability [12-15].
What the embodiment of the present invention was announced is preferred embodiment, and however, it is not limited to this, the ordinary skill people of this field
Member understands the spirit of the present invention easily according to above-described embodiment, and makes different amplification and variation, but as long as not departing from this
The spirit of invention, all within the scope of the present invention.
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[5] the new medical college Chinese medicine dictionaries in Jiangsu:The Shanghai volume two [M]:Shanghai Science Press, 1986,2079.
[6] Zhang Hangying, Zheng Keli, it is tall and erect bright blue, wait medicinal plant false-yellowflower milkwort root or herb progress [J] Sanming College to learn
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[7] Liu Xiufang, the woods Culture Revolution, Su Minghua wait false-yellowflower milkwort root or herbs（Polygala fallaxHemsl）Tissue-culturing rapid propagation skill
Art research [J] seeds, 2012,31 (2):57-59,63.
[8] Luo Wanye, Wei Jinqiu, Cai Meiling wait the agrotechnical clothes of false-yellowflower milkwort root or herb tissue-culturing rapid propagation Study on Seedling Cultivation Technique [J]
Business, 2012,29 (12):1335-1336.
[9] Li Cuilan, Su Yuqin, Mo Yanlan wait the foundation of false-yellowflower milkwort root or herbs tissue culturing system to study [J] Modern Agricultures
Industry science and technology, 2012 (13):77,79..
[10] Beijing Gong Zhenhui, Shen book emerging Plant Tissue Breeding [M]:Chemical Industry Press, 2007,36.
[11] Shanxi production Radix Polygalae seed of chatting about recklessly sprouts, tissue cultures and the Taiyuan root Research of microstructure [D]:University Of Shanxi,
2008.
[12] Xin Peiyao, Liu Yan, Li Genqian wait the transplantation technique of the Yunnan poplar tissue-cultured seedling to study the Central-South forestry science and technologies of [J] big
Learn journal:Natural science edition, 2012,32 (2):23-25.
[13] rainbow is worn, Ma Shaoying, Li Sheng, Su Liwei, Shi Zhenzhen, Su Lirong, Hu Cuizhen V. amurensis ' double red ' and ' double
It is excellent ' tube rapid propagation research [J] Gansu Agriculture Universities journal, 2014,49 (4):63-68,72.
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News, 2015 (6):26-28.
Claims (1)
1. a kind of false-yellowflower milkwort root or herb rapid propagation in vitro method for culturing seedlings, which is characterized in that include the following steps：
Step 1, material obtain：Current year green tape has the false-yellowflower milkwort root or herb branch of tender stem；
Step 2, pretreatment：Explant rinses 3-5 min in liquid detergent water, then rinses 25-35 min with flowing water,
Long or excessive branch is trimmed simultaneously in cleaning process, facilitates subsequent disinfection；
Step 3, explant disinfection：Pretreated false-yellowflower milkwort root or herb branch in step 2 is taken, with 75% alcohol disinfecting 10s, then is used
The mercuric chloride of 0 .1% sterilizes, time 15min；
Step 4 induces axillary bud：Material after will be sterilized in step 3 is cut into stem sections of the long 2cm with 1-2 axil, vertical to insert
In culture medium MS+6-BA2.0mg/L+NAA0.05 mg/L, additional 30 .0g/L sucrose, 5 .0g/L agar, 5 .7 of pH, outside
Implant culture 12-18d induces axillary bud at axil, can be grown to 2-3cm high after 30d；
Step 5, Multiplying culture：The axillary bud induced in interception above-mentioned steps four is inoculated in proliferated culture medium, false-yellowflower milkwort root or herb
Proliferated culture medium is 0 .05mg/L of MS+6-BA 1.5mg/L+NAA, carries out Multiplying culture, and 20-25d cultivates a generation；
Step 6, culture of rootage：The healthy and strong test tube seedling obtained in selecting step five cuts the long 2cm accesses root media in top
Middle carry out culture of rootage, rooting of vitro seedling after 20d；
Step 7, acclimatization and transplants：It will take root in step 6 good, the test tube seedling of robust growth moves to common reality from tissue culture room
Room or greenhouse are tested, then bottle cap is opened 1/3 aperture by in-bottle seeding 5d, hardening 2d finally opens bottle cap completely,
After hardening 1d, by tissue-cultured seedling from transplanting to matrix in tissue culture bottle, Small plastic shed is used in combination to cover；During hardening, it is ensured that matrix
Enough moisture, and take shading measure；It is i.e. viable after 25d；
Above step four, five and six is inoculated with work and operates in superclean bench, and the culture of test tube seedling is in tissue culture room
Interior progress, condition of culture are：It is carried out under conditions of 25 DEG C, intensity of illumination 1200-1500Lx, periodicity of illumination 12h/d of temperature
's；
Matrix in step 7 acclimatization and transplants is red soil：Fertile soil：Perlite=1:1:1 or fertile soil：Perlite=1:1 or complete is rotten
Grow one kind in soil；
Prescription of rooting medium described in step 6 is that 0 .1 mg/L+ NAA of 1/2MS+IBA, 0 .3 mg/L+0 .2 g/L live
Property charcoal.
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| CN102640708A (en) * | 2012-05-14 | 2012-08-22 | 地缘（厦门）生物科技有限公司 | Tissue culture propagation method for Polygala fallax Hemsl |
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| DE102007063133A1 (en) * | 2007-12-24 | 2009-06-25 | Sasol Germany Gmbh | Process for producing wax in water Dispersions from self-emulsifying gel concentrates |
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| CN102640708A (en) * | 2012-05-14 | 2012-08-22 | 地缘（厦门）生物科技有限公司 | Tissue culture propagation method for Polygala fallax Hemsl |
| CN104996298A (en) * | 2015-07-06 | 2015-10-28 | 三明学院 | Method for cultivating polygala fallax hemsl tissue culture seedlings based on multiple internodal stem segments integration |
| CN105532458A (en) * | 2015-12-29 | 2016-05-04 | 李永华 | Tissue culture method of polygala crotalarioides |
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