CN105145350B - A kind of preparation method and application of solid-liquid two-phase PEG culture mediums - Google Patents
A kind of preparation method and application of solid-liquid two-phase PEG culture mediums Download PDFInfo
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Abstract
The invention provides a kind of preparation method and application of solid-liquid two-phase PEG culture mediums, it is to configure solidified MS media in blake bottle bottom, solid gel is cooled into after autoclaving, vertically remove fraction solids gel, the solid gel of half volume is preferably removed along blake bottle diameter, vacancy is formed in blake bottle bottom, the MS fluid nutrient mediums containing PEG are added in the vacancy, the volume of solid gel of the volume of the MS fluid nutrient mediums containing PEG of the addition with removing is identical.The culture medium of the present invention is made up of solid phase MS culture mediums with liquid phase P EG nutrient solution two parts, and liquid PEG horizontal directions, which are penetrated into, accesses potato test tube seedling stem section on solid gel, solid-phase culture base, carry out drought resistance measure；With the features such as making is simple, required time is short, stem section inoculation facilitates.
Description
Technical field
The invention belongs to plant drought resistance research field, and in particular to a kind of preparation method of solid-liquid two-phase PEG culture mediums
And application.
Background technology
Potato is the fourth-largest cereal crops in the world, is also the potential staple food grain of China, and with vegetables, the spy of grain dual-purpose
Point.The growth of potato needs large quantity of moisture, and water shortage influences very big on it with salt stress, and the environment stress such as arid is potato raw
The important restriction factor of production, the inverse Journal of Sex Research such as its drought resisting has important value.
Bansal etc. (1991) has found that (polyethylene glycol, molecular weight is 6000) solution to potato leaf disk to PEG6000
Resistant expression it is consistent with the drought resistance of donor kind, macromolecule PEG can as potato drought resistant selective agent and measure
Agent.In fact, macromolecule PEG does not enter cell, it is the optimal selective agent that the plant drought resistance including potato is determined,
Using most universal.The small-molecule substances such as mannitol also can be as osmotic stress agent, but enters cell interior, or even participates in being metabolized,
Its effect can not be compared with PEG.
Using potato test tube seedling as material, based assays drought resistance can be cultivated using liquid PEG, but using liquid PEG trainings
Support having the disadvantage that for base：1) it is difficult material to be tested is kept upright, the particularly small material of volume.2) in liquid PEG,
Whether the physiological reaction under whether plant coerces the physiological reaction of simulation drought stress with soil drought is consistent, there is no research.
Drought resistance is tested using the MS solid mediums containing PEG, not limited by stem section size, test tube seedling stem section can all be protected
Hold upright, cauline leaf and root can normal growths, it can be seen that the overall process that stem section grows under stress conditions, drought resistance feature performance is filled
Point.In solid PEG, test tube seedling is to simulation drought stress and the physiological reaction of salt stress and the physiological reaction one under soil drought stress
Cause, as a result more convincingness.
The drought resistance for having two kinds of solid PEG culture mediums to have been used to potato test tube seedling in the prior art is determined：
The first：Ordinary solid PEG culture mediums, are that PEG is added in common MS culture mediums, are cooled to after autoclaving
For solid PEG culture mediums, such a culture medium has preparation method simple, the characteristics of material inoculation facilitates.But if culture medium
Middle PEG concentration can suppress the formation of agar gel more than 8%, that is to say, that can not directly be made by this method highly concentrated
Spend solid PEG culture mediums.And on low concentration ordinary solid PEG culture mediums, it is impossible to show to take effect to effect substantially from stress, directly
To the complete performance of complete inhibition test tube seedling growth, the requirement for determining potato test tube seedling drought resistance is not reached much, particularly
Do not reach the requirement of drought resistance screening varieties, therefore the application of ordinary solid PEG culture mediums is received and greatly limited.
Second：Vertical osmosis solid PEG culture mediums.Van der Weele etc. (2000) report a kind of new side
Method：Add isometric PEG solution on solid MS flat boards, by 24h osmotic equilibriums, just can be obtained containing not after outwelling liquid and drying
With concentration PEG solid medium.Its principle is that the PEG solution on upper strata is penetrated into lower floor's solid MS gels, is referred to as vertical infiltration
Method, the method is the classical way of compounding high concentration PEG solid mediums.But the shortcoming of vertical osmosis solid PEG culture mediums
Substantially：1) culture medium making step is complicated, and required time is up to 24h or longer；2) easily there is the pollution of culture medium；3)PEG
Concentration successively decrease in gradient from top to bottom in solid gel distribution, the PEG concentration of test tube seedling root impression is uneven；4) cultivate
The single of base, which makes, takes that super-clean bench space is big, and holding time is long, it is impossible to do with.
Both at home and abroad, the research of potato drought resistant mechanism and drought resistance improvement is also weaker, this and potato drought resistant
Research method validity is poor, and particularly test tube seedling drought resistance assay method not enough simplicity has direct relation.Directly with vertical infiltration
Seldom, such as Anithakumari (2011) uses vertical osmosis to the report of method solid PEG culture mediums research test tube seedling drought resistance
PEG solid mediums (time of penetration 3d) are made, and have carried out the drought resistance of potato test tube seedling and are determined；Deng Zhen etc. (2014) is used
The PEG solid mediums (time of penetration 48h) that vertical osmosis makes, determine the growth indexes such as 11 kind plant heights, and right
Relative indicatrix value has carried out clustering, has no that this kind of culture medium is used for other reports of potato test tube seedling study on drought resistance.
For potato test tube seedling, have successively by the use of the low concentration PEG solid mediums less than 8% or with mannitol as
The report that drought resistance is studied in agent is determined, even transgenic seedling directly determines its drought resistance without solid PEG, this is all highly concentrated
Spend ordinary solid PEG using limited, vertical osmosis it is inconvenient for use caused by.Therefore solid PEG culture mediums are improved
It is necessary.
The content of the invention
To solve high concentration ordinary solid PEG using limited, the vertical awkward technical problem of osmosis, the present invention is special
A kind of solid-liquid two-phase PEG culture mediums of horizontal permeation are not devised.
A kind of preparation method of solid-liquid two-phase PEG culture mediums of the present invention, is in the configuration solid MS cultures of blake bottle bottom
Solid gel is cooled into after base, autoclaving, fraction solids gel is vertically removed, sky is formed in blake bottle bottom
Lack, the vacancy add the MS fluid nutrient mediums containing PEG, the volume of the MS fluid nutrient mediums containing PEG of the addition with
The volume of the solid gel of removal is identical.
It is preferred that, the fraction solids gel that vertically removes is the solid that half volume is removed along blake bottle diameter
Gel.
It is preferred that, PEG bulking value specific concentration is 0-25% in the MS fluid nutrient mediums containing PEG.
It is preferred that, sucrose containing 2-4% in the MS culture mediums, 0.6-1.5% agar powders are w/v.
The solid-liquid two-phase PEG culture mediums that the preparation method of the present invention is obtained are applied to the study on drought resistance of plant.
Further, the solid-liquid two-phase PEG culture mediums of the present invention are applied to different Potato Cultivars test tube seedling drought resistances
Measure.
Further, the solid-liquid two-phase PEG culture mediums of the present invention are applied to different Potato Cultivars transgenosis test tube seedlings
The measure of drought resistance.
Further, the solid-liquid two-phase PEG culture mediums of the present invention are obtained into test tube applied to different Potato Cultivars mutagenesis
The measure of seedling drought resistance.
Further, the application process of solid-liquid two-phase PEG culture mediums of the invention, is vertically moved in blake bottle
Go out after fraction solids gel, test tube seedling is accessed in solidified MS media, on-position is apart from solid-liquid two-phase boundary line 0.3-
0.6cm, then adds the MS fluid nutrient mediums containing PEG.
Compared with prior art, advantages and positive effects of the present invention are：The preparation method simplicity of the present invention is, it is necessary to the time
It is short, can be with making with of operating parameter is easily replicated；Material inoculation is convenient, can first access material, rear injection PEG solution；It is difficult
Pollute；No matter explant size, its position is fixed, posture upright, can be observed to grow overall process；PEG liquid is along level
Direction is permeated, and PEG is without top-down concentration gradient in gel, and drought resistance measurement result has more convincingness；PEG liquid phases are in training
Support existence time in base long, its coercion more continuous and effective；PEG valid density is high (more than 8%) in solid gel, can be with
It was observed that from low to high under various concentrations PEG stress, complete performance of the explant growth by stress feature；Different cultivars can be met
The need for drought resistance is determined with high drought resistance variety evaluation, screening, meet mutation breeding and determined with transgenic breeding medium drought resistant
The need for identification and screening with high drought resistance material.
Brief description of the drawings
At the solid-liquid two-phase PEG culture medium top views of Fig. 1 present invention, × signal inoculation material；
The solid-liquid two-phase PEG culture medium side views of Fig. 2 present invention；
Double rich No. 5 Drought Resistance of Variety are determined in Fig. 3 embodiments 1；
Fig. 4 embodiments 1 ancient name for China ripple base of a fruit Drought Resistance of Variety is determined；
The central Atlantic Drought Resistance of Variety of Fig. 5 embodiments 1 is determined；
Gram new 18 Drought Resistance of Variety is determined in Fig. 6 embodiments 1；
PEG coerces the influence to plant height in Fig. 7 embodiments 1；
PEG coerces the influence to root length in Fig. 8 embodiments 1；
PEG coerces the influence to radical in Fig. 9 embodiments 1；
PEG coerces the influence to fresh weight in Figure 10 embodiments 1；
PEG coerces the influence to dry weight in Figure 11 embodiments 1；
The measure of transfer-gen plant drought resistance in Figure 12 embodiments 2；
The measure of adjoining tree drought resistance in Figure 13 embodiments 2；
PEG coerces the influence to plant height in Figure 14 embodiments 2；
PEG coerces the influence to fresh weight in Figure 15 embodiments 2；
The measure of transplanted seedling drought resistance, wherein A in Figure 16 embodiments 2：Before Osmotic treatment；B：Osmotic treatment 20 days；C：Pour
The 5th day after water；D：The 18th day after watering；1-3：Transfer-gen plant；4-5：Compare (non-transfer-gen plant).
Embodiment
Technical scheme is described in further detail with reference to embodiment.The present invention is previously mentioned
Ratio, it is if without particularly marking, be defined with weight/volume (%, W/V), i.e., solid containing how many grams in 100mL liquid
Body.
The preparation method of the solid-liquid two-phase PEG culture mediums of the present invention, prepares contain 3% sucrose, 0.8% agar powder, pH first
For 5.8 solidified MS media, packing 50mL, high pressure in each 230mL glass blake bottle (diameter 6.5cm, height 9.5cm)
30-40min formation solid gels are cooled down after sterilizing.Gel of walking half vertically is cut along blake bottle diameter, and in vacancy addition etc.
PEG concentration is 0-25% (W/V) in the MS nutrient solutions containing PEG of volume, the MS fluid nutrient mediums containing PEG, obtains this
The solid-liquid two-phase PEG culture mediums (as shown in Figure 1) of invention.The culture medium is respectively accounted for by solid phase MS gels and liquid phase P EG solution compositions
The half of culture medium cumulative volume, is a kind of equal volume PEG culture mediums of solid-liquid two.
The present invention solid-liquid two-phase PEG culture mediums be horizontal permeation method solid PEG culture mediums, including solid phase MS culture mediums and
Liquid phase P EG nutrient solution two parts, liquid PEG horizontal directions, which are penetrated into, can access potato test tube on solid gel, solid-phase culture base
Seedling stem section, carries out drought resistance measure；The culture medium has that ordinary solid culture matrix manufacturing is simple, required time is short, stem section inoculation
The features such as facilitating, its application is by with the very big potentiality for promoting potato drought resistant Journal of Sex Research.
The application method of the solid-liquid two-phase PEG culture mediums of the present invention, is vertically to remove part admittedly in blake bottle
After body gel, test tube seedling is accessed in solidified MS media, on-position is apart from solid-liquid two-phase boundary line 0.3-0.6cm, preferably
For 0.5cm, the MS fluid nutrient mediums containing PEG are then added.In making and inoculation step, solid-liquid two-phase culture medium determination method
Using very convenient.Compared with vertical osmosis, simplify operation sequence, reduce manufacture difficulty, be that one kind is significantly improved, should
The application of method will directly facilitate the fast development of the research of potato drought resistant.
Using the solid-liquid two-phase culture medium of the present invention, stem section is accessed after solid phase, and it grows overall process and entered under PEG stress
OK, characteristicness is more representative, can disclose under Series P EG concentration, the pedigree performance rule of the multiple parameters such as plant height.
The solid-liquid two-phase PEG culture mediums of the present invention can apply to the drought resisting research of the plants such as potato, specific research bag
Include the measure 1) to different Potato Cultivars test tube seedling drought resistances；2) survey of different Potato Cultivars transgenosis test tube seedling drought resistances
It is fixed；3) different Potato Cultivars mutagenesis obtain the measure of test tube seedling drought resistance.
Embodiment 1
Different Potato Cultivars test tube seedling drought resistances can be applied to below by experimental verification solid-liquid two-phase PEG culture mediums
The measure of difference.
Take solid phase MS culture medium of the potato of four different cultivars in the solid-liquid two-phase PEG culture mediums of the present invention enterprising
Row culture, configures several groups of MS fluid nutrient mediums containing PEG, wherein PEG concentration be respectively 0%, 5%, 10%, 15%, 20%,
25%, the growth conditions of test tube seedling are observed.The potato of the four strains is respectively double rich No. 5, Xia Bodi, great Xi
Ocean and grams new 18.Specifically condition of culture is：Using robust growth, potato test tube seedling highly consistent with growing way as material, clip
Stem sections of the length about 2cm containing terminal bud is explant, and 3 stem sections are accessed in every bottle of solid-phase culture base, are arranged in rows, positional distance
Solid-liquid two-phase boundary line 0.5cm.Blake bottle ventilated membrane or bottle cap are sealed, and condition of culture is：25 DEG C, illumination 16h/d, illumination is strong
Spend 2000Lx.Culture 4 weeks, observes the growing state of plant.Plant is taken out, root agar gel is washed away with water, surface water is blotted
Point, determine and weigh total dry weight after being dried at individual plant plant height, root length, radical and fresh weight, last 65 DEG C.
It is as shown in Figures 3 to 6 the morphologic observation result of 4 kind test tube seedlings, it can be seen that with liquid phase medium
The growth of test tube seedling is significantly suppressed on the rise of PEG concentration, solid phase gel, is shown as plant height and is gradually become short, and root length becomes
Short, increment diminishes, growth potential dies down, and shows that PEG can be penetrated into effectively in solid phase gel, and the growth of test tube seedling is coerced to PEG
Compel reaction sensitive.Be in the range of 0% to 25% in PEG concentration, the growths of 4 kind test tube seedlings from unaffected (0%) to
Growth retardation (25%), systematic performance has reached the requirement that drought resistance is determined, has overcome low concentration PEG solid mediums
Disclose hypodynamic defect.For same kind, it can substantially disclose PEG actions concentration, act on obvious concentration and complete
The full concentration for suppressing growth.Observation also shows that cauline leaf is different to PEG susceptibilitys from the growth of root (such as Fig. 3), on solid medium
Show abundant growth information.
Such as Fig. 7 to Figure 11 show the measurement result of the growth indexes of 4 kind test tube seedlings, can more accurately embody plant height
With the changing rule (such as Fig. 7, Fig. 8) of root length；With the raising of PEG concentration, the growth indexes such as radical, fresh weight and dry weight are also showed
Go out the changing rule (Fig. 9 to Figure 11) progressively reduced, also can as study on drought resistance index.Wherein plant height, radical and fresh weight
It is sensitive to PEG, and root length is eased up with dry weight variation tendency, the sensitiveness to PEG is poor (Fig. 7 to Figure 11).
The measurement result of morphologic observation and growth indexes can disclose the difference of multi-parameter between kind, solid-liquid two-phase PEG trainings
Supporting base has the ability for distinguishing different cultivars test tube seedling PEG Stress of Susceptibility, that is to say, that resist with potato test tube seedling is disclosed
The ability of non-irrigated sex differernce.When PEG concentration is 5%, the growth of double rich No. 5 is substantially suppressed, and plant height is reduced to control treatment
55% (Fig. 3, the Fig. 7) of (no PEG), and the growth of other kinds is normal.When PEG concentration is 10%, the growth of 4 kind test tube seedlings
It is suppressed, but suppressed degree is different.When PEG concentration is 15%, Xia Bodi growth is a greater impact (Fig. 4, Fig. 7
To Figure 11)；When PEG concentration is 20%, Xia Bodi stops growing (Fig. 4)；When PEG concentration reaches 25%, double rich No. 5 and grams new 18
Also stop growing (Fig. 3, Fig. 6), and now there be increment in the Atlantic Ocean.Table 1 summarizes the degree that test tube seedling growth is coerced by PEG,
Initial analysis shows that the order of different cultivars drought resistance size is：The Atlantic Ocean>Grams new 18>Double rich No. 5>Xia Bodi.
The degree that the growth of the different cultivars test tube seedling of table 1 is coerced by PEG
| PEG concentration | Double rich No. 5 | Xia Bodi | The Atlantic Ocean | Grams new 18 |
| 0% | Normally | Normally | Normally | Normally |
| 5% | + | Normally | Normally | Normally |
| 10% | + | + | + | + |
| 15% | + | +++ | ++ | ++ |
| 20% | +++ | Do not grow | ++ | +++ |
| 25% | Do not grow | Do not grow | +++ | Do not grow |
Note："+" represents suppressed degree, and plus sige to be represented to suppress more obvious
Embodiment 2
The solid-liquid two-phase PEG culture mediums of the present invention can also be applied to the measure of transgenosis test tube seedling drought resistance.
In PEG simulating droughts measure, converted with Porphyra yezoensis trehalose-6-phosphate trehalose synthetase gene (TPS)
The test tube seedling that western auspicious (Desiree) kind in potato bottom is obtained is material, and non-transgenosis test tube seedling is control, with consolidating for the present invention
Liquid two-phase PEG culture mediums carry out coercing cultivation, cultivate 4 weeks, the growing state of observation plant and determine plant height, it is root length, radical, fresh
The parameter such as weight and dry weight.During water shortage drought stress is determined, transgenosis and non-transgenosis Desiree potato test tube seedlings are uncapped domestication
One week, and transplant into the dixie cup containing Nutrition Soil.When plant height reaches 20cm, the drought stress that do not water of 20 days is carried out, then
Rewater, the upgrowth situation of observed and recorded plant.
Measurement result shows, on solid-liquid two-phase PEG culture mediums, and the suppressed degree of plant strain growth is different, transfer-gen plant
Upgrowth situation be better than control (Figure 12, Figure 13).When PEG concentration is 5%, transfer-gen plant and the plant height and fresh weight difference that compare
Up to the pole level of signifiance, when PEG concentration is 10%, difference reaches the level of signifiance (Figure 14, Figure 15), shows the anti-PEG sides of body of transfer-gen plant
The ability of compeling is higher than adjoining tree.
In the drought stress do not watered, during Osmotic treatment 20 days, transfer-gen plant wilting degree is less than control (Figure 16-B)；
5 days after watering, plant wilting degree loses alleviation, the trend (Figure 16-C) for still having exacerbation；Aquaponic is poured to 18 days, transgenosis is planted
Strain grows young leaves, top gradually greening, and the withered degree of adjoining tree continues to aggravate (Figure 16-D)；As a result transfer-gen plant is shown
Drought resistance is apparently higher than control.
The raising of the anti-PEG stress ability of transfer-gen plant is improved with its drought resistance to be consistent, and it is suitable to further demonstrate that PEG
The measure agent of potato test tube seedling study on drought resistance, solid-liquid two-phase PEG culture mediums are effectively used for potato test tube seedling drought resisting
The measure of property.
The solid-liquid two-phase culture medium of the present invention can meet the drought resistance comparative study of paired material.Transfer-gen plant with it is right
According in, mutant materials and the contrast of donor drought resistance, the solid medium that alternative vertical osmosis is prepared obtains rapidly result,
Early screening and identification are realized, the effect of ordinary solid culture medium is reached.
The culture medium is applied not only to potato test tube seedling Identification of Drought, it can also be used to the test tube seedling in other plant source,
Particularly asexually propagated plant, extensively, application potential is larger for suitable material, with the huge applications for promoting plant drought Journal of Sex Research
Potentiality.
The present invention has prepared solid-liquid two-phase PEG culture mediums using the method for routine operation, and is successfully applied to potato
Drought resistance is determined to distinguish and determined with transfer-gen plant drought resistance between kind, and its preparation method has very big plasticity, changes culture
Doleiform shape can improve its producing efficiency using particular manufacturing craft, and optimize measure effect, potential to progressively reach ordinary solid training
Support the convenience and applicability of base.
Above example is only several in several preferred embodiment of the invention, it is noted that the invention is not restricted to
Above-described embodiment；For the person of ordinary skill of the art, still can be to the technical scheme described in previous embodiment
Modify, or equivalent substitution is carried out to which part technical characteristic；And these modifications or substitutions, do not make relevant art side
The essence of case departs from the spirit and scope of claimed technical solution of the invention.
Claims (7)
1. a kind of application of solid-liquid two-phase PEG culture mediums, it is characterised in that the preparation method of the solid-liquid two-phase PEG culture mediums
It is to be cooled into solid gel after preparing solidified MS media, autoclaving in blake bottle bottom, one is removed along blake bottle diameter
The solid gel of half volume, vacancy is formed in blake bottle bottom, and the MS fluid nutrient mediums containing PEG, institute are added in the vacancy
The volume for stating the MS fluid nutrient mediums containing PEG of addition is identical with the volume of the solid gel of removal；The solid-liquid two-phase PEG trainings
Support the study on drought resistance that base is applied to plant.
2. the application of solid-liquid two-phase PEG culture mediums according to claim 1, it is characterised in that the MS liquid containing PEG
PEG bulking value specific concentration is 0-25% in culture medium.
3. the application of solid-liquid two-phase PEG culture mediums according to claim 1, it is characterised in that contain in the MS culture mediums
2-4% sucrose, 0.6-1.5% agar powders are w/v.
4. the application of solid-liquid two-phase PEG culture mediums according to claim 1, it is characterised in that applied to different potatos
The measure of kind test tube seedling drought resistance.
5. the application of solid-liquid two-phase PEG culture mediums according to claim 1, it is characterised in that applied to different potatos
The measure of kind transgenosis test tube seedling drought resistance.
6. the application of solid-liquid two-phase PEG culture mediums according to claim 1, it is characterised in that applied to different potatos
Kind mutagenesis obtains the measure of test tube seedling drought resistance.
7. the application of the solid-liquid two-phase PEG culture mediums according to claim any one of 4-6, it is characterised in that in blake bottle
In vertically remove after fraction solids gel, by test tube seedling access solidified MS media in, on-position is apart from solid-liquid two
Intersecting boundary line 0.3-0.6cm, then adds the MS fluid nutrient mediums containing PEG.
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