CN102650622B - Early evaluating method for tea tree winter resistance - Google Patents
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Abstract
The invention belongs to the field of botany, and particularly relates to an early evaluating method for tea tree winter resistance. The soluble sugar (mainly cane sugar) content is one important physiological basis for the strength of the tea tree winter resistance and one important presentation of the low temperature adaptability of the tea tree variety; and as the temperature is gradually lowered with the approaching of the winter, the cane sugar content of the tea tree leaves is gradually increased, however the tea tree variety with high winter resistance has higher-increased cane sugar content than the tea tree variety with low winter resistance. The early evaluating method for the tea tree winter resistance evaluates the winter resistance of the tea tree by measuring the cane sugar content in the tee tree leaves, so as to evaluate and select the tee tree variety with high winter resistance and provide basis for variety introduction for the tea tree.
Description
Technical field
The invention belongs to the botany field, relate to a kind of tea tree winter resistance authentication method, especially a kind of cane sugar content carries out the method that winter resistance is identified in the tea leaf by measuring.
Background technology
China is the original producton location of tea tree, it also is the country of planting tea, tea making in the world the earliest, drinking tea, the artificial cultivate agrocybe of China has more than 3000 year history, tealeaves is China's " state's drink ", be Tea Production and consumption big country, China's tea tree planting area was 1,800,000 hectares in 2009, and output surpasses 1,300,000 tons, all occupies first place in the world.Tealeaves also is universally acknowledged most important natural health-care beverage, and the whole world has 60 countries to plant tea, and the country of import tealeaves spreads all over more than 150 countries and regions, and more than 20 hundred million people grow tender of tea-drinking.Along with the variation of healthy beverage consumption idea, the consumption of tealeaves and trade prospect are more wide.Tea Production is being occupied critical role aspect China's agricultural economy, rural development and the new socialist countryside construction, and especially in a lot of mountain areas, hills, its economic status is more outstanding.
Low temperature is to influence distribute important ecological factor with plantation of tea tree, and wind freezes, frost, ice and snow freeze in most of tea district's spring of China, autumn, winter and often takes place, and causes tea tree all to freeze to death or serious frostbite, causes the significantly underproduction, quality to reduce.The winter resistance of tea tree has higher genetic force, its cold tolerance significant difference of different tea tree breeds, and the screening that the index that the tea tree winter resistance is relevant fast, reliably can be the evaluation of tea tree winter resistance and cold-resistant kind provides important evidence.Mainly be from the following aspects the winter resistance of tea tree to be studied at present:
1, natomical leaf structure and tea tree winter resistance
There are some researches show: ratio and the winter resistance of the ratio of blade epicuticle thickness, palisade tissue thickness, palisade tissue thickness and blade gross thickness, palisade tissue and spongy tissue thickness are intensity positive correlation.The actual winter resistance performance of this index and growth of tea plant is contradictory sometimes, not accurate enough and reliable.
2, tissue free water and bound water content and tea tree winter resistance
The blade free water is different with the irreducible water role, and the relative content of irreducible water is higher, when the relative content of free water is low, is conducive to freeze proof.Someone thinks, the leaf tissue free water content, organizes bound water content ratio between two value can be used as to weigh the cold resistant physical signs of tea tree breed.This index is not done deeply, systematic research, also is in the theoretical supposition stage.
3, cell liquid concentration and tea tree winter resistance
The relation of cell liquid concentration reflection cell inclusion and moisture ratio, cell liquid concentration height, soluble sugar and Protein content height, intracellular bound water content is just high, and free water content then reduces relatively, and frost resistance is just strong.The refractive index of leaf juice has reflected the concentration of cell liquid, cell liquid concentration height, and the refractive index of juice is big.Therefore, the someone thinks that the leaf juice refractive index can be used as tea tree winter resistance identification of indicator.This index is not done deeply yet, systematic research, also is in the theoretical supposition stage.
4, conductivity and resistance value and tea tree winter resistance
Have result of study to show, not only the degree that increases of membrane permeability is less for the kind that winter resistance is strong or the lighter that is injured, and the variation of saturating property can reverse, and is easy to recover normal, so resistance value is big.Otherwise, the kind that winter resistance is weak or the not only membrane permeability increase of severe patient of being injured, and this variation is irreversible, can not recover normal, so that damage even death.Someone thinks that blade resistance value and conductivity can be used as a physical and chemical index of tea tree winter resistance power.Conductance method suffered influence factor in practical measurement is more, CO in temperature, soak time and the air
2Dissolving etc. all can the electric conductivity value of practical measurement be exerted an influence, so make that measurement result repeatability is relatively poor.
5, MDA (MDA) content and tea tree winter resistance
Have research to think, after Different Tea Varieties suffered freeze injury, its mda content changed and there are differences, and the protective capability that accumulates the bright tissue of multilist more is more weak, and cell membrane wrecks more serious, and winter resistance is also just more weak.But MDA content just there are differences under different low temperature, and then difference is not remarkable between each kind.
6, protectiveness enzyme and tea tree winter resistance
Plant balance of intracellular free-radical generating and elimination under cryogenic conditions can be destroyed, and the film system of the radical pair plant of accumulation damages, and makes film fat produce peroxidation, causes the 26S Proteasome Structure and Function of cell membrane to be damaged.And the generation of free radical and eliminate by the system of the protective enzyme in the cell and control, protection system comprises reducing substanceses such as enzyme such as SOD (superoxide dismutase), CAT (hydrogen peroxidase), POX (peroxidase), glutathione peroxidase and Ve, Va, Vc, ubiquinone, carotenoid.These enzymes are subjected to the direct or indirect influence of low temperature and activity change take place in the tea tree body, and the someone thinks, can utilize the vigor of enzyme to make tea tree winter resistance identification of indicator.The actual winter resistance performance of this index and growth of tea plant is contradictory, not accurate enough and reliable, also need do deep, systematic research.
Summary of the invention
The present invention is in order to solve the deficiency that prior art exists, provide a kind of and identify tea tree winter resistance (cane sugar content and the positive correlation of tea tree winter resistance) by measuring in the tea leaf cane sugar content, introduce a fine variety foundation is provided for identifying and screen the strong tea tree breed of winter resistance and tea tree.
The early stage authentication method of a kind of tea tree winter resistance is identified the tea tree winter resistance by cane sugar content in the mensuration tea leaf, and described sucrose sugar content assaying method comprises following steps:
A. sample extracting solution preparation
1) gets the ripe fresh leaves 25g of tea tree, be cut into 0.2-0.5cm
2Fragment;
2) blade after step 1) is shredded adds in the pure water, under the air-proof condition, and lixiviate 1h in 90 ℃ of water-baths; Blade and pure water lixiviate number percent are 1: 10
3) with step 2) in leaching liquor take out and to be cooled to room temperature, change the centrifugal 15min of 5000rpm in the centrifuge tube over to, supernatant is collected in impurity elimination;
4) with the supernatant concentration in the step 3) to 1/10 of original volume, obtain filtered fluid through 0.45 μ m water system membrane filtration, described filtered fluid is sample extracting solution.
B. cane sugar content is measured
1) chromatographic condition
Efficient liquid-phase chromatographic pump, evaporative light-scattering detector, carbohydrate analysis column is furnished with pre-column, and flowing is acetonitrile mutually: water=3: 1, flow velocity 1mL/min, sprayer rank 60%, drift tube temperature is 55 ℃, air pressure 25psi;
2) standard curve making
Accurately take by weighing sucrose standard items 20mg, adding 1mL ultrapure water compound concentration is the sucrose standard mother liquor of 20mg/mL, then with ultrapure water be diluted to 0.5,1.0,2.0,8.0,10.0mg/mL series standard solution.Difference sample introduction 10 μ L under condition determination, the record appearance time is also measured peak area, with the peak area 1g value of measuring corresponding sucrose concentration of standard solution 1g value is carried out linear regression then and obtain typical curve equation: y=ax+b, x represents the 1g value of sucrose concentration of standard solution, y represents the 1g value of peak area, and a, b are constant.
3) assay
4) calculate cane sugar content in the acquisition tealeaves according to following formula:
Cane sugar content (mg/g)=(C * n * V)/m
The C-standard equation is tried to achieve sucrose concentration (mg/mL); N-sample extracting solution extension rate; V-sample extracting solution cumulative volume (mL); M-sample heavy (g);
Annotate: winter low temperature can cause that leaf water content descends, and therefore can obtain the sample dry weight by measuring leaf water content in advance, calculates every gram dry weight cane sugar content, can make the result more accurate.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
By knowwhy as can be known, when soluble sugar content in the cell (mainly being sucrose) when increasing, Premeabilisation of cells is pressed increase, improve the concentration of cell liquid, freezing point is reduced, unlikely the freezing of protection tenuigenin colloid strengthened the cold tolerance of tea tree plant.In addition, sugar also has protected protein matter and avoids the caused freezing action of low temperature.This shows that soluble sugar content (mainly being sucrose) is an important physical signs of tea tree winter resistance power.
The method of traditional mensuration soluble sugar content, as the anthrone sulfuric acid process, because the hydrolytic action of sulfuric acid may cause that some macromolecular substances are hydrolyzed into carbohydrate in the cell, thus the accuracy that influence is measured.Because starch decreased in the overwintering plant body, soluble sugar, mainly be that cane sugar content increases, sugar content and temperature be negative correlation (Pan Ruichi. plant physiology [M]. Beijing: Higher Education Publishing House, 2008), and confirm by experiment, under the winter low temperature condition, the main composition of tea leaf soluble sugar is sucrose (Fig. 1 and Fig. 2), and therefore, the height of cane sugar content has directly reacted the level of soluble sugar in the cell.The present invention sets up a kind of by measuring the method that the tea leaf cane sugar content carries out the winter resistance evaluation under the cryogenic conditions just, to overcome the deficiency of classic method, makes the result more accurately and reliably.
Description of drawings
Fig. 1 is the tea blade soluble sugar composition liquid phase analysis figure early that relaxes under the cryogenic conditions.
Fig. 2 is black ox blade soluble sugar morning composition liquid phase analysis figure under the cryogenic conditions.
Fig. 3 is present embodiment area temperature on average variation diagram in October to January.
Fig. 4 be present embodiment crow ox early and easypro tea as far back as the cane sugar content variation diagram in October to January.
The present invention will be further described by the following examples.
Embodiment
1, the examination material is handled
Since October, along with weather is gone into the winter by the autumn gradually, temperature descends gradually, got in tea place, Hefei City, Anhui Province Da Yang town in every month and relax tea early and black ox two kinds morning, by cane sugar content in its blade of high effective liquid chromatography for measuring, last till January in next year, and relatively the cane sugar content of two kinds changes.
2, the mensuration of cane sugar content
A. sample extracting solution preparation
1) gets the ripe fresh leaves 25g of tea tree, be cut into 0.2-0.5cm
2Fragment;
2) blade after step 1) is shredded adds in the 250mL pure water, under the air-proof condition, and lixiviate 1h in 90 ℃ of water-baths; Blade and pure water lixiviate percentage by weight are for being 1: 10;
3) with step 2) in leaching liquor take out and to be cooled to room temperature, change 5000rpm centrifugal treating 15min in the centrifuge tube over to, supernatant is collected in impurity elimination;
4) with the supernatant concentration in the step 3) to 25mL, namely 1/10 of original volume, obtain filtered fluid through 0.45 μ m water system membrane filtration, described filtered fluid is sample extracting solution.
B. cane sugar content is measured
1) chromatographic condition
The Waters515 efficient liquid-phase chromatographic pump, Waters2420 evaporative light-scattering detector, Agilent Zorbax carbohydrate analysis column (being furnished with pre-column), flowing is acetonitrile mutually: water=3: 1, flow velocity 1mL/min, sprayer rank 60%, drift tube temperature is 55 ℃, air pressure 25psi.
2) standard curve making
Accurately take by weighing sucrose standard items 20mg, adding 1mL ultrapure water compound concentration is the sucrose standard mother liquor of 20mg/mL, then with ultrapure water be diluted to 0.5,1.0,2.0,8.0,10.0mg/mL series standard solution.Sample introduction 10 μ L respectively under condition determination, the record appearance time is also measured peak area, with the 1g value of the peak area measured the 1g value of corresponding sucrose concentration of standard solution is carried out linear regression then and obtains the typical curve equation.
3) assay
4) calculate cane sugar content in the acquisition tealeaves according to following formula:
Cane sugar content (mg/g)=(C * n * V)/m
The C-standard equation is tried to achieve sucrose concentration (mg/mL); N-sample extracting solution extension rate; V-sample extracting solution cumulative volume (mL); M-sample heavy (g);
3, presentation of results
As can be seen from Figure 2, from January in October to next year, temperature descends gradually, and 12 monthly mean temperatures drop to about 6 ℃, and January, medial temperature dropped to about 0 ℃.Along with decrease of temperature, tea leaf cane sugar content grow steadily (Fig. 3), from October to January, the easypro tea of the tea tree breed that winter resistance is strong in the production early cane sugar content rises to 137.03mg/g (dry weight) from 23.13mg/g (dry weight), 5 times have been increased, more weak tea tree breed crow ox cane sugar content morning of winter resistance rises to 112.14mg/g (dry weight) from 42.46mg/g (dry weight) in the production, has also increased by 1.6 times, and the amplification of tea cane sugar content morning that relaxes obviously greater than black ox early.The measurement result explanation: under cryogenic conditions (Dec and January), the easypro tea of the tea tree breed that winter resistance is strong in the production early blade cane sugar content is significantly higher than the more weak tea tree breed crow ox of winter resistance early, and exists significant difference between two kinds.
By knowwhy as can be known, when soluble sugar content in the cell (mainly being sucrose) when increasing, Premeabilisation of cells is pressed increase, improve the concentration of cell liquid, freezing point is reduced, unlikely the freezing of protection tenuigenin colloid strengthened the cold tolerance of tea tree plant.In addition, sugar also has protected protein matter and avoids the caused freezing action of low temperature.This shows, soluble sugar content (mainly being sucrose) is an important physiological foundation of tea tree winter resistance power, it is the important behaviour that tea tree breed adapts to the low temperature ability, along with the temperature of arriving winter constantly reduces, the tea leaf cane sugar content increases steadily, and the strong kind of winter resistance is more than cold-resistant weak increase.
Present embodiment relax tea early for two tea tree breeds of examination and black ox early, according to cultivation practices for many years, relax tea early winter resistance be higher than black ox morning.Sucrose-determination is the result show: under cryogenic conditions (Dec and January), the blade cane sugar content of two tea tree breeds all obviously increases, and from October to January, the easypro tea of the tea tree breed that winter resistance is strong in the production early cane sugar content has increased by 5 times, more weak tea tree breed crow ox cane sugar content morning of winter resistance has only increased by 1.6 times in the production, the amplification of easypro tea cane sugar content morning obviously greater than black ox early exists significant difference between two kinds.Therefore, cane sugar content and the positive correlation of tea tree winter resistance, it can be used as the index that the tea tree winter resistance is identified.
Claims (1)
1. a tea tree winter resistance authentication method is characterized in that: identify the winter resistance of tea tree by cane sugar content in the mensuration tea leaf; Described cane sugar content assay method comprises following steps:
A. sample extracting solution preparation
1) gets the ripe fresh leaves 25g of tea tree, be cut into 0.2-0.5 cm
2Fragment;
2) blade after step 1) is shredded adds in the pure water, under the air-proof condition, and lixiviate 1h in 90 ℃ of water-baths; Blade and pure water lixiviate number percent are 1:10;
3) with step 2) in leaching liquor take out and to be cooled to room temperature, change the centrifugal 15min of 5000rpm in the centrifuge tube over to, supernatant is collected in impurity elimination;
4) with the supernatant concentration in the step 3) to 1/10 of original volume, obtain filtered fluid through 0.45 μ m water system membrane filtration, described filtered fluid is sample extracting solution;
B. cane sugar content is measured
1) chromatographic condition
Efficient liquid-phase chromatographic pump, evaporative light-scattering detector, carbohydrate analysis column is furnished with pre-column, and flowing is acetonitrile: water=3:1 mutually, flow velocity 1mL/min, sprayer rank 60%, drift tube temperature is 55 ℃, air pressure 25psi;
2) standard curve making
Accurately take by weighing sucrose standard items 20mg, adding 1mL ultrapure water compound concentration is the sucrose standard mother liquor of 20mg/mL, then with ultrapure water be diluted to 0.5,1.0,2.0,8.0,10.0mg/mL series standard solution, difference sample introduction 10 μ L under condition determination, the record appearance time is also measured peak area, with the peak area lg value of measuring corresponding sucrose concentration of standard solution lg value is carried out linear regression then and obtain typical curve equation: y=ax+b, x represents the lg value of sucrose concentration of standard solution, y represents the lg value of peak area, and a, b are constant;
3) assay
Sample thief extract 10 μ L sample introductions are with reference to step 2) in the appearance time of sucrose standard items of record, determine sucrose peak in the sample extracting solution, measure its peak area, calculate sucrose concentration in the sample extracting solution according to the typical curve equation;
4) calculate cane sugar content in the acquisition tealeaves according to following formula:
Cane sugar content (mg/g)=(C * n * V)/m,
The C-standard equation is tried to achieve sucrose concentration (mg/mL); N-sample extracting solution extension rate; V-sample extracting solution cumulative volume (mL); M-sample heavy (g).
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| CN103033578B (en) * | 2012-12-25 | 2014-03-05 | 湖南农业大学 | Method for quickly and preliminarily screening excellent tea tree resource based on contained contents |
| CN104541921B (en) * | 2015-01-30 | 2017-01-18 | 湖南省茶叶研究所 | Comprehensive evaluation method of tea tree annual seedling growth potential |
| CN104756714B (en) * | 2015-04-10 | 2017-01-18 | 贵州大学 | Method for identifying anti-freezing heading Chinese cabbages during seedling stage |
| CN105884508A (en) * | 2016-04-01 | 2016-08-24 | 安徽农业大学 | Cold-proof antifreezing water soluble fertilizer special for tea trees and preparation method and application thereof |
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| CN109521128A (en) * | 2019-01-15 | 2019-03-26 | 中国农业科学院特产研究所 | A kind of method of the overwintering winter resistance index of correlation analysis of V. amurensis |
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| CN113403325B (en) * | 2021-06-29 | 2022-03-18 | 安徽农业大学 | Tea tree orphan gene CsOG3 and application thereof in improving cold resistance of tea trees |
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| KR20090097568A (en) * | 2008-03-12 | 2009-09-16 | 경상대학교산학협력단 | Reaction solution for selecting amount of catechin in tea tree, and method for selection of high-catechin containing tea tree and its products |
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