CN105409708B - A kind of identification method of V. amurensis germ plasm resource rest period winter resistance - Google Patents
A kind of identification method of V. amurensis germ plasm resource rest period winter resistance Download PDFInfo
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- CN105409708B CN105409708B CN201510814158.7A CN201510814158A CN105409708B CN 105409708 B CN105409708 B CN 105409708B CN 201510814158 A CN201510814158 A CN 201510814158A CN 105409708 B CN105409708 B CN 105409708B
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- 240000002503 Vitis amurensis Species 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 50
- 238000004043 dyeing Methods 0.000 claims abstract description 86
- 238000012545 processing Methods 0.000 claims abstract description 35
- 238000010186 staining Methods 0.000 claims abstract description 34
- 238000007710 freezing Methods 0.000 claims abstract description 27
- 230000008014 freezing Effects 0.000 claims abstract description 27
- 241000219095 Vitis Species 0.000 claims abstract description 17
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 13
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 13
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 13
- XXKXGOJPDAHNGV-UHFFFAOYSA-M [Cl-].Cl[N+]=1N(N(N(C=1)C1=CC=CC=C1)C1=CC=CC=C1)C1=CC=CC=C1 Chemical compound [Cl-].Cl[N+]=1N(N(N(C=1)C1=CC=CC=C1)C1=CC=CC=C1)C1=CC=CC=C1 XXKXGOJPDAHNGV-UHFFFAOYSA-M 0.000 claims abstract description 11
- 231100000518 lethal Toxicity 0.000 claims abstract description 11
- 230000001665 lethal effect Effects 0.000 claims abstract description 11
- 238000004364 calculation method Methods 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 3
- 239000000975 dye Substances 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 150000003536 tetrazoles Chemical class 0.000 claims description 2
- 235000015110 jellies Nutrition 0.000 claims 1
- 239000008274 jelly Substances 0.000 claims 1
- 238000005034 decoration Methods 0.000 abstract description 11
- 230000000007 visual effect Effects 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000011156 evaluation Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 238000004040 coloring Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 4
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 4
- 235000009392 Vitis Nutrition 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 238000000611 regression analysis Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 206010050808 Hyperchromasia Diseases 0.000 description 2
- 235000017190 Vitis vinifera subsp sylvestris Nutrition 0.000 description 2
- 244000237969 Vitis vulpina Species 0.000 description 2
- 235000017242 Vitis vulpina Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000005059 dormancy Effects 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
- YJTKZCDBKVTVBY-UHFFFAOYSA-N 1,3-Diphenylbenzene Chemical group C1=CC=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 YJTKZCDBKVTVBY-UHFFFAOYSA-N 0.000 description 1
- MARUHZGHZWCEQU-UHFFFAOYSA-N 5-phenyl-2h-tetrazole Chemical compound C1=CC=CC=C1C1=NNN=N1 MARUHZGHZWCEQU-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000221785 Erysiphales Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 235000004283 Vitis amurensis Nutrition 0.000 description 1
- MOFINMJRLYEONQ-UHFFFAOYSA-N [N].C=1C=CNC=1 Chemical class [N].C=1C=CNC=1 MOFINMJRLYEONQ-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- -1 chloro triphenyltetrazol Azoles Chemical class 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000009746 freeze damage Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000010979 ruby Substances 0.000 description 1
- 229910001750 ruby Inorganic materials 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/02—Cultivation of hops or vines
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Botany (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Soil Sciences (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of identification methods of V. amurensis germ plasm resource rest period winter resistance, include the following steps：By the V. amurensis branch of rest period under different cryogenic temperatures de-epithelization after freezing processing, be put into chloro triphenyltetrazolium chloride dyeing liquor, be placed in isoperibol and carry out dyeing processing；The dyeing liquor for washing away V. amurensis branch surface determines dyeing rank according to V. amurensis branch xylem stained area, and is come out of retirement and taken up an official post the staining index of grape branch according to dyeing level calculation；Staining index corresponding to the cryogenic temperature of V. amurensis branch determines half lethal low temperature.Half lethal low temperature is lower, shows that the winter resistance of the V. amurensis germplasm is better.It is smaller due to being influenced by extraneous factor in continuous mode using TTC decoration methods, and visual result, it is therefore, simple and easy to do using this method identification V. amurensis germ plasm resource rest period winter resistance, accurately and reliably.
Description
Technical field
The present invention relates to seeds cold hardness evaluation fields, anti-in particular to a kind of V. amurensis germ plasm resource rest period
Cold identification method.
Background technology
The V. amurensis northeast originating in China (Vitis amurensis Rupr.) and Korea, the Far East Area of the former Soviet Union.China mountain
The natural distribution area of grape mainly Jilin Province Changbai Mountain, Wandashan Mountains, Heilongjiang Province, the Xiaoxinanlin Mountains, Liaoning Province the north mountain
Area, upper level, the Daqunshan Mountains to the east of Wulanchabu League of Inner Mongolia alliance are also distributed.It is the primary raw material that northeast makes grape wine,
Its wine is ruby, bright-coloured transparent, has unique flavor, welcomed by consumers.
V. amurensis is one kind of most cold-resistant in Vitis, and branches and tendrils are resistant to -40 DEG C of severe cold, and root system is resistant to -14~-16 DEG C
Low temperature also has stronger resistivity to white rot, powdery mildew, anthracnose and black soya bean disease etc..Therefore, it is current domestic and international Portugal
The most important resource of grape cold-tolerance breeding.
The winter resistance of V. amurensis have higher genetic force, its cold tolerance significant difference of different wild grape resources, soon
Fast, the reliable relevant index of V. amurensis winter resistance can be provided for the screening of V. amurensis cold hardness evaluation and cold-resistant kind it is important according to
According to.But it is still not clear at present suitable for the method for V. amurensis cold hardness evaluation, it is strong to the winter resistance of separate sources V. amurensis germplasm
Weak understanding is insufficient, affects the development and utilization of winter resistance germ plasm resource.
The strong and weak most common method of existing identification Vitis winter resistance is conductance method, however, we are in practical measurement
It was found that due to CO in temperature, soaking time and air2The factors such as dissolving the electric conductivity value of practical measurement can all be had an impact,
Cause measurement result less reproducible, qualification result is inaccurate.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of identification methods of V. amurensis germ plasm resource rest period winter resistance.For current
Due to CO in temperature, soaking time and air when Means of Electrical Conductivity Vitis winter resistance2The factors such as dissolving cause to measure and tie
Fruit is less reproducible, the problem of qualification result inaccuracy, and the present invention identifies V. amurensis germ plasm resource suspend mode using TTC decoration methods
The winter resistance of phase.Since TTC decoration methods are by the way that the V. amurensis branch after freezing processing is put into chloro triphenyltetrazolium chloride dye
Carry out observing branch xylem staining conditions after dyeing processing in color liquid, in continuous mode by extraneous factor influenced compared with
It is small, it is therefore, simple and easy to do using this method measurement V. amurensis germ plasm resource rest period winter resistance, accurately and reliably.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of identification method of V. amurensis germ plasm resource rest period winter resistance, includes the following steps：
(1) by the V. amurensis branch of rest period under different cryogenic temperatures de-epithelization after freezing processing, be put into chloro three
In phenyl tetrazole dyeing liquor, it is placed in isoperibol and carries out dyeing processing；
(2) dyeing liquor for washing away V. amurensis branch surface is determined according to the stained area of V. amurensis branch xylem and is dyed
Rank, and come out of retirement and taken up an official post the staining index of grape branch according to dyeing level calculation；
(3) staining index corresponding to the cryogenic temperature of V. amurensis branch determines half lethal low temperature.
Chloro triphenyltetrazolium chloride (i.e. TTC) is a kind of color developing agent, can be by the enzyme system institute in active cell
Reduction forms red San Ben Ji Jia Za (TTF) not soluble in water.The present invention identifies that V. amurensis germplasm provides using TTC decoration methods
The winter resistance of source rest period is peelled off after the V. amurensis branch after freezing processing is carried out freezing processing under different cryogenic temperatures
Epidermis is put into TTC dyeing liquors and is dyed.
The red area of V. amurensis branch xylem is bigger after dyeing, then the dyeing for grape branch of going down the hill in the cryogenic temperature
Rank is higher, and staining index is bigger.By determining the V. amurensis germplasm after calculating the staining index corresponding to different cryogenic temperatures
Half lethal low temperature LT50To weigh the winter resistance of V. amurensis germplasm.LT50It is smaller, show the cold-resistant of the V. amurensis germplasm
Property is better.
It is smaller due to being influenced by extraneous factor in continuous mode using TTC decoration methods, and therefore visual result uses
This method identifies that V. amurensis germ plasm resource rest period winter resistance is simple and easy to do, accurately and reliably.
Preferably, in step (2), described the step of determining dyeing rank according to the staining conditions of V. amurensis branch xylem
It specifically includes：
By V. amurensis branch it is longitudinal sectional after, according to the stained area of V. amurensis branch vertical section xylem determine dyeing rank.
It is by observing branch cross section clip wood currently, in being studied about TTC Determination Staining shoot tissue vigor
Matter portion colours depth degree to be judged.In practical operation, the red TTF of generation is dyed often outside the clip of branch cross section
Side is accumulated, and xylem hyperchromasia is caused to generate observation error；And branch cross section thin slice is cut using free-hand slicing method and is difficult
Operation；Xylem coloring depth degree range estimation is also difficult to accurate judgement.In the present invention, TTC decoration methods, dyeing observation portion are improved
Position is changed to the xylem of branch vertical section, can longitudinally be cut with sharp cutter, easy to operate.
Meanwhile vertical section xylem is in regular rectangular shape substantially, is conducive to stained area estimation so that dyeing gradational boundary is clear
Chu can follow, as a result more accurate and reliable to overcome the shortcomings of conventional method.
For the ease of dyeing, coloring is improved, it is preferable that described under different cryogenic temperatures at freezing in step (1)
Further include following steps before being put into chloro triphenyltetrazolium chloride dyeing liquor after reason after de-epithelization：
The V. amurensis branch of de-epithelization is cut into the branch section of 0.3~0.5cm.
In order to ensure the accuracy of result, it is further preferred that branch section in the chloro triphenyltetrazolium chloride dyeing liquor
Number be 10~15.
Similarly, for the ease of dyeing, coloring is improved, it is further preferred that in step (1), the dyeing processing
Temperature be 28~32 DEG C, dyeing time 18~for 24 hours, and being carried out under the conditions of being protected from light.
Preferably, in step (1), a concentration of the 0.5~1.0% of the chloro triphenyltetrazolium chloride dyeing liquor.
The configuration method of chloro triphenyltetrazolium chloride dyeing liquor used is：Weigh the chlorinated triphenyl base four of 0.5~1.0g
Nitrogen azoles, with deionized water dissolving and constant volume is in 100mL volumetric flasks, is protected from light after preparing, room temperature preservation.
Dehydrogenase content in Different Crop tissue is different, and activity is different, thus the dyeing condition needed is also different.This hair
In bright, suitable TTC dyeing conditions, including dyeing branch segment length, stin of thickness, dyeing temperature, dyeing time have been screened, this
A little parameter synergistic effects, improve coloring jointly.
Preferably, described to be specifically included the step of freezing processing under different cryogenic temperatures in step (1)：
The V. amurensis branch of rest period is placed at -20~-50 DEG C 12~15h of freezing, after raise the temperature to 20~30
DEG C, and 4-6h is kept at this temperature.
Preferably, it is specifically included the step of freezing processing under different cryogenic temperatures for described every group：
The temperature of V. amurensis branch local environment is down to set temperature according to the cooling rate of 4~5 DEG C/h, in this temperature
Degree is lower to keep 12~13h, after be gradually heating to 20~30 DEG C, keep 4~5h at this temperature.
By the V. amurensis branch after above-mentioned freezing processing, when carrying out cold hardness evaluation, result is more accurate.
Preferably, in step (2), the determination method of the dyeing rank is：
When xylem vertical section has 85% or more area to take on a red color, then the dyeing rank is 4 grades；When xylem is longitudinal sectional
Face has 46%~85% area to take on a red color, then the dyeing rank is 3 grades；When xylem vertical section has 20%~45% face
Product takes on a red color, then the dyeing rank is 2 grades；When xylem vertical section has 20% area below to take on a red color, then the dyeing
Rank is 1 grade.
It is further preferred that in step (2), the staining index=Σ (dyeing rank × branch hop count)/(branch section sum ×
Highest dyes rank).
The staining index determined by the above method can more truly reflect the winter resistance of different V. amurensis kinds, can
For distinguishing the winter resistance between different V. amurensis kinds.
Compared with prior art, beneficial effects of the present invention are：
(1) due to CO in temperature, soaking time and air when being directed to current Means of Electrical Conductivity Vitis winter resistance2It is molten
The factors such as solution cause measurement result less reproducible, the problem of qualification result inaccuracy, and the present invention uses chloro triphenyltetrazol
Azoles decoration method identifies the winter resistance of V. amurensis germ plasm resource rest period.Since TTC decoration methods are by will be after freezing processing
V. amurensis branch is put into chloro triphenyltetrazolium chloride dyeing liquor carry out dyeing processing after observation branch xylem staining conditions i.e.
Can, it is influenced by extraneous factor in continuous mode smaller, therefore, V. amurensis germ plasm resource rest period cold-resistant is measured using this method
Property is simple and easy to do, accurately and reliably.
(2) currently, in being studied about TTC Determination Staining shoot tissue vigor, cut by observing branch cross section
Mouth xylem colours depth degree to be judged.In practical operation, the red San Ben Ji Jia Za (TTF) for dyeing generation often exists
Accumulation on the outside of the clip of branch cross section, causes xylem hyperchromasia to generate observation error；And it is cut using unarmed cross section method
Thin slice is difficult operation；Xylem coloring depth degree range estimation is also difficult to accurate judgement.In the present invention, TTC decoration methods are improved, are contaminated
Color look-out station is changed to the xylem of branch vertical section, can longitudinally be cut with sharp cutter, easy to operate.Meanwhile vertical section
Xylem is in regular rectangular shape substantially, is conducive to stained area estimation so that dyeing gradational boundary understands and can follow, to overcome tradition side
The deficiency of method, it is as a result more accurate and reliable.
(3) in the present invention, suitable TTC dyeing conditions, including dyeing branch segment length, stin of thickness, dyeing have been screened
Temperature, dyeing time, these parameters synergistic effect, improve coloring jointly.
(4) operating condition of the invention is not harsh, and TTC concentration is in 0.5~1.0% (W (g)/V (mL)) in operating process
Can, be not necessarily to particularly accurate preparation, stained area is easy to estimate, and each step operation is simple, to instrument, reagent etc. require compared with
It is low, it can be promoted and applied in V. amurensis germplasm rest period cold hardness evaluation.
(5) mirror the present invention provides staining index and half lethal low temperature as V. amurensis germ plasm resource winter resistance
Determine index, the winter resistance of different V. amurensis kinds can be accurately distinguished.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the schematic diagram that V. amurensis branch difference dyes rank；
Fig. 2 is the matched curve of " double rich " and " northern ice is red " that are obtained according to assay method of the present invention；
Fig. 3 correlations between the obtained half lethal low temperature of assay method of the present invention and cold- resisting index average membership
Analysis result figure.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The assay method of V. amurensis germ plasm resource rest period winter resistance provided in this embodiment, includes the following steps：
The freezing processing of V. amurensis branch：
The V. amurensis branch of " double rich " this germ plasm resource is selected to carry out freezing processing.By the mountain of the germ plasm resource rest period
Grape branch carries out freezing processing at -25 DEG C, -30 DEG C, -35 DEG C, -40 DEG C, -45 DEG C, -50 DEG C respectively.By V. amurensis branch
Place 10h under above-mentioned 6 cryogenic temperatures, after be warming up to 20 DEG C after place 3h again.
The V. amurensis branch of rest period used is to acquire V. amurensis germplasm annual, that maturity is consistent mid-January to stop
Dormancy phase branch is cut into the branch section of long 20~25cm and is packed into the valve bag of sealing after atoleine sealing, is protected in -15 DEG C of refrigerators
It deposits.
Freeze the dyeing processing of branch：
By the V. amurensis branch de-epithelization Jing Guo above-mentioned freezing processing, the V. amurensis branch of de-epithelization is put into concentration
For in 1.5% chloro triphenyltetrazolium chloride dyeing liquor, 15h is dyed in the light protected environment at 20 DEG C.
V. amurensis shoot tissue vitality test：
By above-mentioned dyeing, treated that V. amurensis branch is rinsed with deionized water, with blade that branch is crosscutting, and observation is crosscutting
The stained area of face xylem determines dyeing rank, goes out staining index according to dyeing level calculation.
According to every group of cryogenic temperature and its corresponding staining index, improved Logistic is combined with nonlinear regression analysis
Equation model temperature inflection point analyzes the half lethal low temperature for determining each germ plasm resource branch using DPS9.0 softwares.
In the present embodiment, the determination method for dyeing rank is：When xylem vertical section has 85% or more area in red
Color, then the dyeing rank is 4 grades；When xylem vertical section has the area of 46%-85% to take on a red color, then the dyeing rank is
3 grades；When xylem vertical section has the area of 20%-45% to take on a red color, then the dyeing rank is 2 grades；When xylem vertical section
There is 20% area below to take on a red color, then the dyeing rank is 1 grade.
The calculation formula of staining index is：Staining index=Σ (dyeing rank × branch hop count)/(branch section sum × highest dye
Color rank).
Embodiment 2
In the assay method of V. amurensis germ plasm resource rest period winter resistance provided in this embodiment, the freezing of V. amurensis branch
Processing, the dyeing processing step of freezing branch are identical with embodiment 1.
Unlike the V. amurensis shoot tissue vitality test the step of in, in the present embodiment,
By above-mentioned dyeing, treated that V. amurensis branch is rinsed with water, with blade that branch is longitudinal sectional, and observation vertical section is wooden
The stained area in portion determines dyeing rank, goes out staining index according to dyeing level calculation.
Embodiment 3
The identification method of V. amurensis germ plasm resource rest period winter resistance provided in this embodiment, includes the following steps：
The freezing processing of V. amurensis branch：
The V. amurensis branch of " double rich " this germ plasm resource is selected to carry out freezing processing.By the mountain of the germ plasm resource rest period
The grape branch freezing processing at -20 DEG C, -25 DEG C, -30 DEG C, -35 DEG C, -40 DEG C, -45 DEG C, -50 DEG C respectively.By V. amurensis branch
Local environment is down to the cryogenic temperature of setting according to 4 DEG C/h cooling rates, and places 12h under corresponding cryogenic temperature, after
4h is placed again after being warming up to 20 DEG C.
The V. amurensis branch of rest period used is to acquire V. amurensis germplasm annual, that maturity is consistent mid-January to stop
Dormancy phase branch is cut into the branch section of long 20~25cm and is packed into the valve bag of sealing after atoleine sealing, is protected in -15 DEG C of refrigerators
It deposits.
Freeze the dyeing processing of branch：
By the V. amurensis branch de-epithelization Jing Guo above-mentioned freezing processing, the V. amurensis branch of de-epithelization is cut into 0.3~
The branch section of 0.5cm, every group of freezing processing are cut into 10 branch sections, after be put into a concentration of 0.5% tetrazole dyeing liquor,
18h is dyed in light protected environment at 28 DEG C.
V. amurensis shoot tissue vitality test：
By above-mentioned dyeing, treated that V. amurensis branch section is rinsed with water, with blade that branch section is longitudinal sectional, observes vertical section
The stained area of xylem determines dyeing rank, goes out staining index according to dyeing level calculation.
According to every group of cryogenic temperature and its corresponding staining index, improved Logistic is combined with nonlinear regression analysis
Equation model temperature inflection point analyzes the half lethal low temperature for determining each germ plasm resource branch using DPS9.0 softwares.
In the present embodiment, the determination method for dyeing rank is：When xylem vertical section has 85% or more area in red
Color, then the dyeing rank is 4 grades；When xylem vertical section has the area of 46%-85% to take on a red color, then the dyeing rank is
3 grades；When xylem vertical section has the area of 20%-45% to take on a red color, then the dyeing rank is 2 grades；When xylem vertical section
There is 20% area below to take on a red color, then the dyeing rank is 1 grade.
The calculation formula of staining index is：Staining index=Σ (dyeing rank × branch hop count)/(branch section sum × highest dye
Color rank).
In embodiment 3, the staining index corresponding to every group of cryogenic temperature is as shown in table 1.And in order to verify each freezing temperature
Can the lower staining index obtained of degree accurately reflect the cryogenic temperature and go down the hill the activity of grape branch, and the application has detected often respectively
It organizes under cryogenic temperature, the branch germination rate of " double rich " V. amurensis branch, TTC reduction rates, succinate dehydrogenase activity, result is equal
It is shown in Table 1.
The corresponding staining index of 1 cryogenic temperature of table, germination rate, TTC reduction rates and succinate dehydrogenase activity
As the reduction of cryogenic temperature, the staining index of V. amurensis branch continuously decrease it can be seen from the result of table 1,
This point also complies with common sense：When V. amurensis branch freezes at a lower temperature, the number of competent cell is then fewer.
And branch germination rate, TTC reduction rates and the succinate dehydrogenase activity and staining index detected is in extremely notable positive
It closes (as shown in table 2), shows that cold hardness evaluation method provided by the invention being capable of freeze injury change of the real simulation plant after enduring cold
Change process.
Correlation analysis figure between 2 staining index of table and germination rate, TTC reduction rates, succinate dehydrogenase activity
The identification method provided by embodiment 3 detects to obtain dye of the V. amurensis kind " double rich " under different cryogenic temperatures
After colour index, improved Logistic equation models temperature inflection point is combined using nonlinear regression analysis, utilizes DPS9.0 softwares
Analysis determines that the half lethal low temperature of " double rich " is -38.63 DEG C, and fit equation is y=0.9887/ (1+e(-11.5911-0.3002x)), degree of fitting 0.9987.
Experimental example 1
It can be by the V. amurensis winter resistance area of different germ plasm resources in order to verify winter resistance detection method provided by the invention
It separates, the assay method provided using embodiment 3 also has detected the mountain of 39 germplasm in addition to " double rich " this kind respectively
Grape, including " Zuo Shanyi ", " double red ", " northern ice is red ", " Zuo Youhong ", " 75047 ", " 73099 ", " 84005 " etc. be at -20 DEG C, -
25 DEG C, -30 DEG C, -35 DEG C, -40 DEG C, -45 DEG C, -50 DEG C of staining index, and its low temperature semilethal temperature is determined according to its result
Degree.The results are shown in Table 3 for it.
The Measurement of Cold Resistance result of the V. amurensis of the different germ plasm resources of table 3
As seen from Figure 3, for 40 parts of V. amurensis germplasm LT of examination50Value has differences, can be accordingly by different V. amurensis
Germ plasm resource winter resistance power distinguishes.Wherein " 75047 ", " 74003-1 ", " 73099 ", " 73068 ", " 84005 " are height
Cold-resistant germplasm, " 73134 ", " Zuo Youhong ", " northern ice is red " are low cold-resistant germplasm, remaining is medium cold-resistant germplasm.
By taking " double rich " and " northern ice is red " as an example, the winter resistance of the two can not be distinguished, and led to according to traditional conductance method
Assay method provided by the invention is crossed, the fit equation of the two and matched curve are significantly different (being shown in Table 3 and Fig. 2), can be by two
The winter resistance of person distinguishes.
In addition, cold- resisting index average membership can also be used as V. amurensis winter resistance comprehensive evaluation index to V. amurensis germplasm
Resource winter resistance carries out overall merit.It is whether accurate and reliable in order to further verify identification method provided by the invention, the present invention
In, 40 parts of germplasm LT of V. amurensis for being acquired with improved TTC decoration methods50Value is with its cold- resisting index average membership in extremely significantly just
Correlation, related coefficient square value are 0.7794 (see Fig. 3), illustrate that the two cold hardness evaluation result is consistent.Therefore, improved TTC
Decoration method can be as a kind of short-cut method of V. amurensis germ plasm resource rest period cold hardness evaluation, and qualification result is accurate and reliable.
Experimental example 2
The winter resistance of V. amurensis germ plasm resource rest period is identified using conductance method.Its method is as follows：
Relative conductivity measures：It takes the wild grape resource branch of each low-temperature treatment, removes epidermis, avoid bud eye, it is cut into 2~
The thin slice of 3mm is uniformly mixed, accurately weighs 0.50g and be put into 10ml graduated centrifuge tubes, adds 8ml deionized waters, is evacuated 40min
Afterwards, it shakes up, its first electric conductivity value R is measured with DDS-308A types conductivity gauge (thunder magnetic)1, then test tube is placed in boiling water water-bath, it boils
Water-bath 1h after standing 8h at room temperature later, then surveys its whole electric conductivity value R2, calculate relative conductivity.Each processing is repeated 3 times.Phase
To Conductivity Calculation formula：Relative conductivity R (%)=(R1/R2) × 100%.
Half lethal low temperature (LT50) determine：The staining index of each low-temperature treatment branch is in " S " with the decline for the treatment of temperature
Type curvilinear motion combines improved Logistic equation models temperature inflection point with nonlinear regression analysis, utilizes DPS9.0 softwares
Analysis determines the LT of each germplasm branch50。
By the data of Means of Electrical Conductivity compared with the data that the present invention measures, the results are shown in Table 4.
The Comparative result of 4 two kinds of assay methods of table
Using the V. amurensis branch LT of Means of Electrical Conductivity it can be seen from the testing result of table 450Average value be -28.86
DEG C, the LT that the present invention measures50Average value be -36.80 DEG C.And illustrated by the measurement result of table 2, the mountain under -35 DEG C of processing
Grape branch remains able to rudiment, the LT that conductance method determines50It is only -28.86 DEG C to be worth average value, so using Means of Electrical Conductivity mountain
When the winter resistance of Grape Germplasm resource, measurement result is inaccurate.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (7)
1. a kind of identification method of V. amurensis germ plasm resource rest period winter resistance, which is characterized in that include the following steps：
(1) by the V. amurensis branch of rest period under different cryogenic temperatures de-epithelization after freezing processing, be put into chloro triphenyl
In tetrazole dyeing liquor, it is placed in isoperibol and carries out dyeing processing；
(2) dyeing liquor for washing away V. amurensis branch surface determines dyeing rank according to the stained area of V. amurensis branch xylem,
And the staining index for grape branch of being come out of retirement and taken up an official post according to dyeing level calculation；
(3) staining index corresponding to the cryogenic temperature of V. amurensis branch determines half lethal low temperature；
In step (2), described the step of determining dyeing rank according to the stained area of V. amurensis branch xylem, specifically includes：
By V. amurensis branch it is longitudinal sectional after, according to the stained area of V. amurensis branch vertical section xylem determine dyeing rank；
In step (2), the determination method of the dyeing rank is：
When xylem vertical section has the area more than 85% to take on a red color, then the dyeing rank is 4 grades；When xylem vertical section has
46%~85% area takes on a red color, then the dyeing rank is 3 grades；It is in when xylem vertical section has 20%~45% area
Red, then the dyeing rank is 2 grades；When xylem vertical section has 20% area below to take on a red color, then the dyeing rank
It is 1 grade；
In step (2), staining index=Σ (dyeing rank × branch hop count)/(branch section sum × highest dyes rank).
2. identification method according to claim 1, which is characterized in that described cold under different cryogenic temperatures in step (1)
Further include following steps before being put into chloro triphenyltetrazolium chloride dyeing liquor after jelly processing after de-epithelization：
The V. amurensis branch of de-epithelization is cut into the branch section of 0.3~0.5cm.
3. identification method according to claim 2, which is characterized in that the number of the branch section is 10~15.
4. identification method according to claim 2, which is characterized in that in step (1), the temperature of the dyeing processing is 28
~32 DEG C, dyeing time 18~for 24 hours, and carried out under the conditions of being protected from light.
5. identification method according to claim 2, which is characterized in that in step (1), the chloro triphenyltetrazolium chloride dye
A concentration of the 0.5~1.0% of color liquid.
6. identification method according to claim 1, which is characterized in that described cold under different cryogenic temperatures in step (1)
The step of freezing processing specifically includes：
The V. amurensis branch of rest period is placed at -20~-50 DEG C 12~15h of freezing, after raise the temperature to 20~30 DEG C,
And 4~6h is kept at this temperature.
7. identification method according to claim 6, which is characterized in that the step of the freezing processing under different cryogenic temperatures
Suddenly it specifically includes：
The temperature of V. amurensis branch local environment is down to set temperature according to the cooling rate of 4~5 DEG C/h, at this temperature
Keep 12~13h, after be gradually heating to 20~30 DEG C, at this temperature keep 4~5h.
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