CN105409708A - Evaluation method of cold resistance of germplasm resources of Vitis amurensis in dormant period - Google Patents
Evaluation method of cold resistance of germplasm resources of Vitis amurensis in dormant period Download PDFInfo
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- CN105409708A CN105409708A CN201510814158.7A CN201510814158A CN105409708A CN 105409708 A CN105409708 A CN 105409708A CN 201510814158 A CN201510814158 A CN 201510814158A CN 105409708 A CN105409708 A CN 105409708A
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- 240000002503 Vitis amurensis Species 0.000 title claims abstract description 101
- 238000011156 evaluation Methods 0.000 title abstract description 13
- 235000004283 Vitis amurensis Nutrition 0.000 title abstract description 5
- 238000007710 freezing Methods 0.000 claims abstract description 31
- 230000008014 freezing Effects 0.000 claims abstract description 31
- 238000004043 dyeing Methods 0.000 claims description 81
- 238000000034 method Methods 0.000 claims description 64
- 230000000284 resting effect Effects 0.000 claims description 25
- 238000012545 processing Methods 0.000 claims description 23
- 230000008569 process Effects 0.000 claims description 17
- 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 description 13
- 239000000975 dye Substances 0.000 claims description 12
- 241000219095 Vitis Species 0.000 claims description 10
- 231100000518 lethal Toxicity 0.000 claims description 10
- 230000001665 lethal effect Effects 0.000 claims description 10
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 6
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 6
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 6
- 238000004364 calculation method Methods 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000010792 warming Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 abstract description 11
- 230000000007 visual effect Effects 0.000 abstract description 2
- 239000012192 staining solution Substances 0.000 abstract 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 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 abstract 1
- 238000011010 flushing procedure Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 abstract 1
- 238000007447 staining method Methods 0.000 abstract 1
- 239000002023 wood Substances 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000005034 decoration Methods 0.000 description 10
- 238000005259 measurement Methods 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 238000012797 qualification Methods 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 235000009392 Vitis Nutrition 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 238000000611 regression analysis Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 240000004859 Gamochaeta purpurea Species 0.000 description 2
- 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
- 238000009835 boiling Methods 0.000 description 2
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- 239000011121 hardwood Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 1
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- 241000221785 Erysiphales Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 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
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- 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 invention provides an evaluation method of cold resistance of germplasm resources of Vitis amurensis in a dormant period. The evaluation method includes following steps: peeling a Vitis amurensis branch in the dormant period after being frozen at different freezing temperatures, placing the branch in a triphenyl tetrazoliu chloride staining solution, and disposing the solution in a constant-temperature environment for staining treatment; flushing away the staining solution on the surface of the branch, determining staining level according to staining area of a wood portion of the branch, and calculating staining index of the branch according to the staining level; determining low-temperature semi-lethal temperature according to the staining index corresponding to the freezing temperature of the branch. The lower the low-temperature semi-lethal temperature is, the higher the cold resistance of germplasms of Vitis amurensis is. A TTC staining method is adopted, thereby being slightly influenced by outside factors and visual in result, so that the evaluation method is simple, convenient and easy to implement and accurate and reliable.
Description
Technical field
The present invention relates to seeds cold hardness evaluation field, in particular to cold resistant authentication method of a kind of V. amurensis germ plasm resource resting stage.
Background technology
V. amurensis (VitisamurensisRupr.) originates in Northeast China and Korea, the Far East Area of the former Soviet Union.Also there is distribution in the Daqunshan Mountains of natural distribution area mainly to the east of the mountain area in Changbai Mountain, Jilin Province, Wandashan Mountains, Heilongjiang Province, the Xiaoxinanlin Mountains, the north, Liaoning Province, upper level, Wulanchabu League of Inner Mongolia alliance of China's V. amurensis.It is the primary raw material that northeast makes grape wine, and its wine is ruby, bright-coloured transparent, has unique local flavor, the dark welcome by consumer.
V. amurensis is a kind the most cold-resistant in Vitis, the severe cold of complicated and confused ability-40 DEG C, and root system can resistance to-14 ~-16 DEG C of low temperature, also has stronger resistivity to white rot, powdery mildew, anthracnose and black soya bean are sick etc.Therefore, it is the most important resource of the outer Cold-resistant Grape Breeding of Present Domestic.
The cold resistance of V. amurensis has higher heritability, its cold tolerance significant difference of different wild grape resources, and the screening that the index that V. amurensis cold resistance is correlated with fast, reliably can be V. amurensis cold hardness evaluation and cold-resistant kind provides important evidence.But the method being applicable to V. amurensis cold hardness evaluation is at present still not clear, understands not enough to the cold resistance power of separate sources V. amurensis kind matter, have impact on the development and utilization of cold resistance germ plasm resource.
The strong and weak the most frequently used method of existing qualification Vitis cold resistance is conductance method, but we find due to CO in temperature, soak time and air in practical measurement
2the factor such as dissolving all can the electric conductivity value of practical measurement be had an impact, cause measurement result repeatability poor, qualification result is inaccurate.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide cold resistant authentication method of a kind of V. amurensis germ plasm resource resting stage.For during current By Means of Electrical Conductivity Vitis cold resistance due to CO in temperature, soak time and air
2the factor such as dissolving cause measurement result repeatability poor, the inaccurate problem of qualification result, the present invention adopts TTC decoration method to identify the cold resistance of V. amurensis germ plasm resource resting stage.Because TTC decoration method carries out dyeing process rear observation branch xylem staining conditions by the V. amurensis branch after freezing processing being put into chloro triphenyltetrazolium chloride dyeing liquor, affect less in mensuration process by extraneous factor, therefore, the method is adopted to measure V. amurensis germ plasm resource resting stage cold resistance simple and easy to do, accurately and reliably.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of V. amurensis germ plasm resource resting stage cold resistant authentication method, comprises the steps:
(1) by the V. amurensis branch of resting stage de-epithelization after freezing processing under different cryogenic temperature, put into chloro triphenyltetrazolium chloride dyeing liquor, be placed in isoperibol and carry out dyeing process;
(2) wash away the dyeing liquor on V. amurensis branch surface, determine according to the stained area of V. amurensis branch xylem the rank that dyes, and the Di of grape branch of coming out of retirement and taking up an official post according to dyeing level calculation;
(3) the Di determination half lethal low temperature corresponding to the cryogenic temperature of V. amurensis branch.
Chloro triphenyltetrazolium chloride (i.e. TTC) is a kind of developer, can reduce by the enzyme system in the activated cell of tool, form red water-fast three benzene base first Za (TTF).The present invention adopts TTC decoration method to identify the cold resistance of V. amurensis germ plasm resource resting stage, the V. amurensis branch after freezing processing is carried out under different cryogenic temperatures de-epithelization after freezing processing, puts into TTC dyeing liquor and dye.
After dyeing, the red area of V. amurensis branch xylem is larger, then higher in the go down the hill dyeing rank of grape branch of this cryogenic temperature, Di is larger.By determining the half lethal low temperature LT of this V. amurensis kind matter after calculating the Di corresponding to different cryogenic temperature
50in order to weigh the cold resistance of V. amurensis kind matter.LT
50less, show that the cold resistance of this V. amurensis kind matter is better.
Owing to adopting TTC decoration method to affect less in mensuration process by extraneous factor, and visual result, therefore, adopt the method qualification V. amurensis germ plasm resource resting stage cold resistance simple and easy to do, accurately and reliably.
Preferably, in step (2), the described staining conditions according to V. amurensis branch xylem determines that the step of dyeing rank specifically comprises:
After the rip cutting of V. amurensis branch, determine according to the stained area of V. amurensis branch vertical section xylem the rank that dyes.
At present, about in the research of TTC Determination Staining shoot tissue vigor, be all judge by observing the painted depth degree of branch cross section clip xylem.In practical operation, the red TTF that dyeing generates piles up outside branch cross section clip of being everlasting, and causes xylem hyperchromasia to produce observation error; And adopt free-hand slicing method to cut the very difficult operation of branch cross section thin slice; The range estimation of xylem painted depth degree is also difficult to accurate judgement.In the present invention, improve TTC decoration method, dyeing look-out station changes the xylem of branch vertical section into, longitudinally can cut with sharp cutter, easy and simple to handle.
Meanwhile, vertical section xylem is regular rectangular shape substantially, is conducive to stained area estimation, and make dyeing gradational boundary know and can follow, to overcome the deficiency of conventional method, result more accurately and reliably.
For the ease of dyeing, improve Color, preferably, in step (1), describedly after freezing processing after de-epithelization, also to comprise the steps: before putting into chloro triphenyltetrazolium chloride dyeing liquor under different cryogenic temperature
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, further preferably, in described chloro triphenyltetrazolium chloride dyeing liquor, the number of branch section is 10 ~ 15.
Similarly, for the ease of dyeing, improve Color, further preferably, in step (1), the temperature of described dyeing process is 28 ~ 32 DEG C, dyeing time 18 ~ 24h, and carries out under lucifuge condition.
Preferably, in step (1), the concentration of described chloro triphenyltetrazolium chloride dyeing liquor is 0.5 ~ 1.0%.
The collocation method of chloro triphenyltetrazolium chloride dyeing liquor used is: the triphenyltetrazolium chloride taking 0.5 ~ 1.0g, with deionized water dissolving and constant volume in 100mL volumetric flask, prepare rear lucifuge, room temperature preservation.
Dehydrase content in Different Crop tissue is different, and active different, the dyeing condition thus needed is also different.In the present invention, screened suitable TTC dyeing condition, comprise dyeing branch segment length, stin of thickness, dyeing temperature, dyeing time, these parameters act synergistically, and jointly improve Color.
Preferably, in step (1), the step of described freezing processing under different cryogenic temperature specifically comprises:
Freezing 12 ~ 15h at the V. amurensis branch of resting stage is placed in-20 ~-50 DEG C, after temperature is increased to 20 ~ 30 DEG C, and keep 4-6h at this temperature.
Preferably, the step of described often group freezing processing under different cryogenic temperature specifically comprises:
According to the cooling rate of 4 ~ 5 DEG C/h, the temperature of environment residing for V. amurensis branch is down to design temperature, keeps 12 ~ 13h at this temperature, after be warming up to 20 ~ 30 DEG C gradually, keep 4 ~ 5h at this temperature.
By the V. amurensis branch after above-mentioned freezing processing, during in order to carry out cold hardness evaluation, its result is more accurate.
Preferably, in step (2), the defining method of described dyeing rank is:
When xylem vertical section has the area of more than 85% to take on a red color, then described dyeing rank is 4 grades; When xylem vertical section has the area of 46% ~ 85% to take on a red color, then described dyeing rank is 3 grades; When xylem vertical section has the area of 20% ~ 45% to take on a red color, then described dyeing rank is 2 grades; When xylem vertical section has the area of less than 20% to take on a red color, then described dyeing rank is 1 grade.
Further preferably, in step (2), described Di=Σ (dyeing rank × branch hop count)/(branch section sum × the highest dyeing rank).
The Di determined by said method can reflect the cold resistance of different V. amurensis kind more truly, can be used for distinguishing the cold resistance between different V. amurensis kind.
Compared with prior art, beneficial effect of the present invention is:
(1) for during current By Means of Electrical Conductivity Vitis cold resistance due to CO in temperature, soak time and air
2the factor such as dissolving cause measurement result repeatability poor, the inaccurate problem of qualification result, the present invention adopts chloro triphenyltetrazolium chloride decoration method to identify the cold resistance of V. amurensis germ plasm resource resting stage.Because TTC decoration method carries out dyeing process rear observation branch xylem staining conditions by the V. amurensis branch after freezing processing being put into chloro triphenyltetrazolium chloride dyeing liquor, affect less in mensuration process by extraneous factor, therefore, the method is adopted to measure V. amurensis germ plasm resource resting stage cold resistance simple and easy to do, accurately and reliably.
(2) current, about in the research of TTC Determination Staining shoot tissue vigor, be all judge by observing the painted depth degree of branch cross section clip xylem.In practical operation, redness three benzene base first Za (TTF) that dyeing generates is piled up outside branch cross section clip of being everlasting, and causes xylem hyperchromasia to produce observation error; And adopt free-hand cross section method to cut the very difficult operation of thin slice; The range estimation of xylem painted depth degree is also difficult to accurate judgement.In the present invention, improve TTC decoration method, dyeing look-out station changes the xylem of branch vertical section into, longitudinally can cut with sharp cutter, easy and simple to handle.Meanwhile, vertical section xylem is regular rectangular shape substantially, is conducive to stained area estimation, and make dyeing gradational boundary know and can follow, to overcome the deficiency of conventional method, result more accurately and reliably.
(3) in the present invention, screened suitable TTC dyeing condition, comprise dyeing branch segment length, stin of thickness, dyeing temperature, dyeing time, these parameters act synergistically, and jointly improve Color.
(4) operating condition of the present invention is not harsh, in operating process, TTC concentration can at 0.5 ~ 1.0% (W (g)/V (mL)), without the need to preparing especially accurately, stained area is easy to estimation, each step is simple to operate, to instrument, reagent etc. require lower, can apply in V. amurensis kind matter resting stage cold hardness evaluation.
(5) the invention provides Di and half lethal low temperature as the cold resistant identification of indicator of V. amurensis germ plasm resource, accurately can distinguish the cold resistance of different V. amurensis kind.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is the schematic diagram of V. amurensis branch coloured differently rank;
Fig. 2 is the matched curve of " two rich " and " northern ice is red " obtained according to assay method of the present invention;
Fig. 3 is correlation analysis result figure between the half lethal low temperature that obtains of assay method of the present invention and cold-resisting index average membership.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment 1
The V. amurensis germ plasm resource resting stage cold resistant assay method that the present embodiment provides, comprises the steps:
The freezing processing of V. amurensis branch:
The V. amurensis branch of " two rich " this germ plasm resource is selected to carry out freezing processing.By the V. amurensis branch of this germ plasm resource resting stage respectively at-25 DEG C ,-30 DEG C ,-35 DEG C ,-40 DEG C ,-45 DEG C, at-50 DEG C, carry out freezing processing.V. amurensis branch is placed 10h under above-mentioned 6 cryogenic temperatures, after be warming up to 20 DEG C after place 3h again.
Mid-January the V. amurensis branch of resting stage used gather the V. amurensis kind matter Dormant hardwood annual, maturity is consistent, is cut into the branch section of long 20 ~ 25cm, after atoleine sealing, loads the valve bag of sealing, preserve in-15 DEG C of refrigerators.
The dyeing process of freezing branch:
By the V. amurensis branch de-epithelization through above-mentioned freezing processing, the V. amurensis branch of de-epithelization is put into the chloro triphenyltetrazolium chloride dyeing liquor that concentration is 1.5%, dye in the light protected environment at 20 DEG C 15h.
V. amurensis shoot tissue vitality test:
By the V. amurensis branch deionized water rinsing after above-mentioned dyeing process, with blade by branch crosscut, observe the stained area of cross section xylem, determine the rank that dyes, go out Di according to dyeing level calculation.
According to the Di often organizing cryogenic temperature and correspondence thereof, combine the Logistic equation model temperature flex point improved with nonlinear regression analysis, utilize DPS9.0 software analysis to determine the half lethal low temperature of each germ plasm resource branch.
In the present embodiment, the defining method of the rank that dyes is: when xylem vertical section has the area of more than 85% to take on a red color, then described dyeing rank is 4 grades; When xylem vertical section has the area of 46%-85% to take on a red color, then described dyeing rank is 3 grades; When xylem vertical section has the area of 20%-45% to take on a red color, then described dyeing rank is 2 grades; When xylem vertical section has the area of less than 20% to take on a red color, then described dyeing rank is 1 grade.
The computing formula of Di is: Di=Σ (dyeing rank × branch hop count)/(branch section sum × the highest dyeing rank).
Embodiment 2
In the present embodiment provides V. amurensis germ plasm resource resting stage cold resistant assay method, the freezing processing of V. amurensis branch, the dyeing treatment step of freezing branch are all identical with embodiment 1.
Unlike in the step of V. amurensis shoot tissue vitality test, in the present embodiment,
V. amurensis branch water after above-mentioned dyeing process is rinsed, with blade by branch rip cutting, observes the stained area of vertical section xylem, determine the rank that dyes, go out Di according to dyeing level calculation.
Embodiment 3
The V. amurensis germ plasm resource resting stage cold resistant authentication method that the present embodiment provides, comprises the steps:
The freezing processing of V. amurensis branch:
The V. amurensis branch of " two rich " this germ plasm resource is selected to carry out freezing processing.By the V. amurensis branch of this germ plasm resource resting stage respectively at-20 DEG C ,-25 DEG C ,-30 DEG C ,-35 DEG C ,-40 DEG C ,-45 DEG C, freezing processing at-50 DEG C.Environment residing for V. amurensis branch is down to the cryogenic temperature of setting according to 4 DEG C/h cooling rate, and places 12h under corresponding cryogenic temperature, after be warming up to 20 DEG C after place 4h again.
Mid-January the V. amurensis branch of resting stage used gather the V. amurensis kind matter Dormant hardwood annual, maturity is consistent, is cut into the branch section of long 20 ~ 25cm, after atoleine sealing, loads the valve bag of sealing, preserve in-15 DEG C of refrigerators.
The dyeing process of freezing branch:
By the V. amurensis branch de-epithelization through above-mentioned freezing processing, the V. amurensis branch of de-epithelization is cut into the branch section of 0.3 ~ 0.5cm, often organize freezing processing be all cut into 10 branch sections, after put into the tetrazole dyeing liquor that concentration is 0.5%, dye in the light protected environment at 28 DEG C 18h.
V. amurensis shoot tissue vitality test:
V. amurensis branch section water after above-mentioned dyeing process is rinsed, with blade by branch section rip cutting, observes the stained area of vertical section xylem, determine the rank that dyes, go out Di according to dyeing level calculation.
According to the Di often organizing cryogenic temperature and correspondence thereof, combine the Logistic equation model temperature flex point improved with nonlinear regression analysis, utilize DPS9.0 software analysis to determine the half lethal low temperature of each germ plasm resource branch.
In the present embodiment, the defining method of the rank that dyes is: when xylem vertical section has the area of more than 85% to take on a red color, then described dyeing rank is 4 grades; When xylem vertical section has the area of 46%-85% to take on a red color, then described dyeing rank is 3 grades; When xylem vertical section has the area of 20%-45% to take on a red color, then described dyeing rank is 2 grades; When xylem vertical section has the area of less than 20% to take on a red color, then described dyeing rank is 1 grade.
The computing formula of Di is: Di=Σ (dyeing rank × branch hop count)/(branch section sum × the highest dyeing rank).
In embodiment 3, often the Di of group corresponding to cryogenic temperature is as shown in table 1.And to go down the hill the activity of grape branch in order to the Di obtained under verifying each cryogenic temperature accurately can reflect this cryogenic temperature, under the application have detected respectively and often organizes cryogenic temperature, branch germination rate, TTC percent reduction, the SDA of " two rich " V. amurensis branch, its result is all in table 1.
Di, germination rate, TTC percent reduction and SDA that table 1 cryogenic temperature is corresponding
As can be seen from the result of table 1, along with the reduction of cryogenic temperature, the Di of V. amurensis branch reduces gradually, and this point also meets general knowledge: when V. amurensis branch is freezing at a lower temperature, the number of its competent cell is then fewer.
And the branch germination rate detected, TTC percent reduction and SDA and Di are in extremely significantly positive correlation (as shown in table 2), show that cold hardness evaluation method provided by the invention can the freeze injury change procedure of real simulation plant after enduring cold.
Correlation analysis figure between table 2 Di and germination rate, TTC percent reduction, SDA
After the authentication method provided through embodiment 3 detects and obtains the Di of V. amurensis kind " two rich " under different cryogenic temperature, nonlinear regression analysis is adopted to combine the Logistic equation model temperature flex point improved, the half lethal low temperature of " two rich " is-38.63 DEG C to utilize DPS9.0 software analysis to determine, its fit equation is y=0.9887/ (1+e
(-11.5911-0.3002x)), degree of fitting is 0.9987.
Experimental example 1
In order to verify that the V. amurensis cold resistance of different germ plasm resource can make a distinction by cold resistance detection method provided by the invention, adopt the assay method that embodiment 3 provides, also have detected the V. amurensis of 39 kind matter except " two rich " this kind respectively, comprise " Zuo Shanyi ", " two red ", " northern ice is red ", " Zuo Youhong ", " 75047 ", " 73099 ", " 84005 " etc. at-20 DEG C,-25 DEG C,-30 DEG C,-35 DEG C,-40 DEG C,-45 DEG C, the Di of-50 DEG C, and determine its half lethal low temperature according to its result.Its result is as shown in table 3.
The Measurement of Cold Resistance result of the V. amurensis of the different germ plasm resource of table 3
As seen from Figure 3, for 40 parts of V. amurensis kind matter LT of examination
50value there are differences, and can different V. amurensis germ plasm resource cold resistance power be made a distinction accordingly.Wherein " 75047 ", " 74003-1 ", " 73099 ", " 73068 ", " 84005 " are high cold-resistant kind of matter, and " 73134 ", " Zuo Youhong ", " northern ice is red " are low cold-resistant kind of matter, and all the other are medium cold-resistant kind of matter.
For " two rich " and " northern ice is red ", traditionally both cold resistances cannot make a distinction by conductance method, and by assay method provided by the invention, both cold resistances obviously different (see table 3 and Fig. 2), can make a distinction by both fit equation and matched curve.
In addition, cold-resisting index average membership also can carry out overall merit as V. amurensis cold resistance comprehensive evaluation index to V. amurensis germ plasm resource cold resistance.In order to verify whether accurately and reliably authentication method provided by the invention further, in the present invention, the V. amurensis 40 parts of kind matter LT tried to achieve by the TTC decoration method improved
50value and its cold-resisting index average membership are in extremely significantly positive correlation, and correlation coefficient square value is 0.7794 (see Fig. 3), and both explanations cold hardness evaluation result is consistent.Therefore, the TTC decoration method of improvement can as a kind of short-cut method of V. amurensis germ plasm resource resting stage cold hardness evaluation, and qualification result accurately and reliably.
Experimental example 2
Adopt the cold resistance of conductance method qualification V. amurensis germ plasm resource resting stage.Its method is as follows:
Relative conductivity measures: the wild grape resource branch getting each low temperature treatment, remove epidermis, avoid eye, be cut into the thin slice of 2 ~ 3mm, mix, accurately take 0.50g and put into 10ml graduated centrifuge tube, add 8ml deionized water, bleed after 40min, shake up, measure its just electric conductivity value R with DDS-308A type conductivity gauge (thunder magnetic)
1, then test tube is placed in boiling water water-bath, boiling water bath 1h, afterwards after left at room temperature 8h, then survey its whole electric conductivity value R
2, calculate relative conductivity.Each process repetition 3 times.Relative conductivity computing formula: relative conductivity R (%)=(R
1/ R
2) × 100%.
Half lethal low temperature (LT
50) determine: the Di of each low temperature treatment branch in the curvilinear motion of " S " type with the decline for the treatment of temperature, combines the Logistic equation model temperature flex point improved, utilizes DPS9.0 software analysis to determine the LT of various matter branch with nonlinear regression analysis
50.
The data that the data of By Means of Electrical Conductivity measure with the present invention compared, its result is as shown in table 4.
The Comparative result of table 4 two kinds of assay methods
As can be seen from the testing result of table 4, adopt the V. amurensis branch LT of By Means of Electrical Conductivity
50mean value be-28.86 DEG C, the present invention measure LT
50mean value be-36.80 DEG C.And illustrated by the measurement result of table 2, the V. amurensis branch under-35 DEG C of process still can rudiment, the LT that conductance method is determined
50value mean value is only-28.86 DEG C, so when adopting the cold resistance of By Means of Electrical Conductivity V. amurensis germ plasm resource, measurement result is inaccurate.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. V. amurensis germ plasm resource resting stage a cold resistant authentication method, it is characterized in that, comprise the steps:
(1) by the V. amurensis branch of resting stage de-epithelization after freezing processing under different cryogenic temperature, put into chloro triphenyltetrazolium chloride dyeing liquor, be placed in isoperibol and carry out dyeing process;
(2) wash away the dyeing liquor on V. amurensis branch surface, determine according to the stained area of V. amurensis branch xylem the rank that dyes, and the Di of grape branch of coming out of retirement and taking up an official post according to dyeing level calculation;
(3) the Di determination half lethal low temperature corresponding to the cryogenic temperature of V. amurensis branch.
2. authentication method according to claim 1, is characterized in that, in step (2), the described stained area according to V. amurensis branch xylem determines that the step of dyeing rank specifically comprises:
After the rip cutting of V. amurensis branch, determine according to the stained area of V. amurensis branch vertical section xylem the rank that dyes.
3. authentication method according to claim 2, is characterized in that, in step (1), describedly after freezing processing after de-epithelization, also to comprise the steps: before putting into chloro triphenyltetrazolium chloride dyeing liquor under different cryogenic temperature
The V. amurensis branch of de-epithelization is cut into the branch section of 0.3 ~ 0.5cm.
4. authentication method according to claim 3, is characterized in that, the number of described branch section is 10 ~ 15.
5. authentication method according to claim 3, is characterized in that, in step (1), the temperature of described dyeing process is 28 ~ 32 DEG C, dyeing time 18 ~ 24h, and carries out under lucifuge condition.
6. authentication method according to claim 3, is characterized in that, in step (1), the concentration of described chloro triphenyltetrazolium chloride dyeing liquor is 0.5 ~ 1.0%.
7. authentication method according to claim 1, is characterized in that, in step (1), the step of described freezing processing under different cryogenic temperature specifically comprises:
Freezing 12 ~ 15h at the V. amurensis branch of resting stage is placed in-20 ~-50 DEG C, after temperature is increased to 20 ~ 30 DEG C, and keep 4 ~ 6h at this temperature.
8. authentication method according to claim 7, is characterized in that, the step of described freezing processing under different cryogenic temperature specifically comprises:
According to the cooling rate of 4 ~ 5 DEG C/h, the temperature of environment residing for V. amurensis branch is down to design temperature, keeps 12 ~ 13h at this temperature, after be warming up to 20 ~ 30 DEG C gradually, keep 4 ~ 5h at this temperature.
9. the authentication method according to any one of claim 1 ~ 8, is characterized in that, in step (2), the defining method of described dyeing rank is:
When xylem vertical section has the area of more than 85% to take on a red color, then described dyeing rank is 4 grades; When xylem vertical section has the area of 46% ~ 85% to take on a red color, then described dyeing rank is 3 grades; When xylem vertical section has the area of 20% ~ 45% to take on a red color, then described dyeing rank is 2 grades; When xylem vertical section has the area of less than 20% to take on a red color, then described dyeing rank is 1 grade.
10. authentication method according to claim 9, is characterized in that, in step (2), and described Di=Σ (dyeing rank × branch hop count)/(branch section sum × the highest dyeing rank).
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