CN100456018C - Method for quickly identifying heat tolerance of lawn type tall fescue - Google Patents
Method for quickly identifying heat tolerance of lawn type tall fescue Download PDFInfo
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- CN100456018C CN100456018C CNB2006100378400A CN200610037840A CN100456018C CN 100456018 C CN100456018 C CN 100456018C CN B2006100378400 A CNB2006100378400 A CN B2006100378400A CN 200610037840 A CN200610037840 A CN 200610037840A CN 100456018 C CN100456018 C CN 100456018C
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- tall fescue
- turf
- type tall
- chlorophyll
- thermotolerance
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Abstract
The present invention belongs to the biotechnology field for identifying the tolerance of cool-season lawn grass, particularly to a novel method for quickly identifying the heat tolerance of lawn type tall fescue. The high-quality seeds of lawn type tall fescue species to be identified are seeded in a basin filled with ordinary sand, clay and nutrition soil (the volume ratio is 1 to 3 to 3) and are cultured for 18 days at normal temperature; part of plants are moved to an artificial climate case with a daytime temperature of 35+-0.5 DEG C, a night temperature of 30+-0.5 DEG C, optical photon of 150 to 200 mu molm <-2> S <-1> and a relative humidity of 65 to 70% to carry out cultivation for three days. Any nutrition is not added in the process of cultivation. The seeds before the emergence of seedlings are sprayed with a small amount of water every other day; after the emergence of the seedlings of the seeds, the plants are watered thoroughly once every other day at normal temperature for cultivation and watered thoroughly once every day for cultivation in high temperature stress. The healthy leaves of the lawn type tall fescue cultivated in the stress of normal temperature and high temperature are collected separately and freely to measure the contents of chlorophyll a and b of unit fresh weight in leaf tissues; the ratio of the sum of the contents of chlorophyll a and b of leaves in the high temperature stress to the sum of the contents of chlorophyll a and b of leaves in the normal temperature stress is calculated. When the ratio is higher than 0.815, the tall fescue is the lawn type tall fescue species with high heat tolerance; the ratio is between 0.760 and 0.815, and the tall fescue is the lawn type tall fescue species with medium heat tolerance; the ratio is lower than 0.760, and the tall fescue is the lawn type tall fescue species with low heat tolerance. The method has the advantages of quick identification, simple and easy operation process, low.
Description
One, technical field
The invention belongs to cold-season turfgrass resistance identification of organism technical field, be specifically related to a kind of new method of Rapid identification turf-type tall fescue thermotolerance size.
Two, background technology
Turf-type tall fescue (Festuca arundinacea) is the per nnial herb of Gramineae Festuca, bunch type, fibrous root prosperity, plant is strong, having adaptability widely, is heat impedance and the best grass seeds (Xu Sheng etc., 2004) of drought resistance in the cold-season-type gramineous lawn.Constantly improve the breed by breeding, cold-resistant, heat-resisting, the anti-trample of current turf-type tall fescue, resistance against diseases are strong, acidproof, salt tolerant alkali, drought-resistant, waterlogging, the anti-again shade of property happiness light, anti-pruning, the green phase is long, convenient management, and the floor space cost is low, so be widely used in urban afforestation, the football field, golf course, and extensive airport, the horse-racing ground of maintenance management, with (reinforcement mirror etc., 2005) such as highway slope protections.
In the U.S., Festuca Arundinacea is the cold-season-type grass seeds that is most widely used.The application of Festuca Arundinacea in China lawn begins to increase sharply after the nineties in 20th century.At present in the subtropical zone, some places when greening grass seeds mixed seeding, the ratio of Festuca Arundinacea even can reach 80%.According to incompletely statistics, by 1998, China's import turf-type tall fescue almost accounted for more than 35% of cold-season type lawn grass seed (reinforcement mirror etc., 2005) of whole imports.Though turf-type tall fescue is in the expansion of China's cultivated area, the demand of seed is increased greatly, at present domestic still do not have a special turf-type tall fescue seed production base, mostly from U.S.'s import.
The existing kind more than 200 of the turf-type tall fescue kind of cultivating in the world, and a lot of new varieties that all can release one after another every year in recent years.In the subtropical zone, the topmost index of reference was the thermotolerance size of kind when people selected the turf-type tall fescue kind for use.Up to the present, this selection mainly depends on the breeding merchant and promotes merchant's publicity materials, but these publicity materials are descriptive entirely, do not have concrete parameter or index can prove turf-type tall fescue kind thermotolerance size in similar Festuca Arundinacea that they provide.From breeding merchant and popularization merchant's publicity materials, current most turf-type tall fescue kind thermotolerances are all very high.But in our actual plantation and maintenance management process, find that there is notable difference in the thermotolerance size between the different turf-type tall fescue kinds, and difference is very big between indivedual kind.If determine the thermotolerance size of the turf-type tall fescue kind that we are about to select for use according to original way, just need with these Festuca Arundinacea kinds at spring or the meticulous always maintenance management of field seeding in autumn to summer more.Upgrowth situation after observing them and getting over the summer finally can be determined their thermotolerance size.Though this method is feasible, need 4-10 month time.Simultaneously also need expensive man power and material in this process.For this reason, we by a large amount of experiments relatively attempt from a series of methods that can identify Festuca Arundinacea thermotolerance size, and it is quick to find out a kind of evaluations, and operating process is simple, and experimental cost is low, and instrument requires simple, easily the method for popularization.
At first, we to draw from ground such as the U.S. and Holland 30 kinds of representative turf-type tall fescues in April, 2004 the field seeding maintenance management observe their upgrowth situation to back by the end of August, growth performance according to them is divided three classes them substantially: the turf-type tall fescue that thermotolerance is high, the turf-type tall fescue that turf-type tall fescue that thermotolerance is medium and thermotolerance are low.Then sowing is after 18 days down with these 30 kinds representative turf-type tall fescue normal temperature again, and putting into day temperature is 35 ℃, and evening, temperature was 30 ℃, and the illumination quantum is 150 μ mol m
-2s
-1, relative humidity is to cultivate 3 days in 70% the growth cabinet.Find the Net Photosynthetic Rate of turf-type tall fescue under the high temperature stress after 3 days by a series of tests back, photosystem II function, heat shock protein is expressed, and the thermotolerance size of turf-type tall fescue can both be fully identified in the variation of cell antioxidant system and leaf chlorophyll.But by contrast, measure chlorophyllous intensity of variation size and identify that accurately the operation of turf-type tall fescue thermotolerance size is the simplest, experimental cost is also low, and instrument requires simple and easy to do.National patent net (www.sipo.gov.cn) by retrieval, after consulting domestic and international pertinent literature and a large amount of investigation, do not see the report of related genera like Rapid identification turf-type tall fescue turfgrass thermotolerance size as yet, this method has popularizing application prospect and actual application value widely.
Three, summary of the invention
The technical issues that need to address of the present invention are that a kind of evaluation of research and development is quick, and operating process is simple, and experimental cost is low, and instrument requires simple, easily the new method of the evaluation turf-type tall fescue thermotolerance size of promoting.
The present invention accurately identifies Festuca Arundinacea thermotolerance size by the content of measuring the turf-type tall fescue leaf chlorophyll.Concrete technical scheme is made up of following four steps:
1. material is cultivated: the high quality seed of the turf-type tall fescue kind that identify is sowed in the basin that common sand, clay and nutrition soil (volume ratio 1: 3: 3) is housed, after cultivating 18 days at normal temperatures, it is 35 ± 0.5 ℃ that the part plant moves into day temperature, nocturnal temperature is 30 ± 0.5 ℃, and the illumination quantum is 150-200 μ mol m
-2s
-1, relative humidity is to cultivate 3 days in the growth cabinet of 65-70%.In cultivating process, do not apply any nutrient.Spray low amounts of water before the seed sprouting every other day, plant was watered once permeablely every other day when the back normal temperature of emerging was cultivated down, and water once permeable every day during high temperature stress.
2. sample collecting and analysis: random acquisition and take by weighing 0.1 gram normal temperature down and the high temperature stress healthy leaves of cultivation down respectively, clean surface contaminants, shred, put into mortar, add a small amount of quartz sand and be ground into homogenate with 80% acetone, be settled to after extracting liq filters with qualitative filter paper 10 milliliters with visible spectrophotometer 663, under the 646nm colorimetric survey light absorption value, more than the experiment replication is four times.
3. the result calculates: above measurement result is calculated the concentration of chlorophyll a, b according to following formula:
The concentration C of chlorophyll a in extract
a=12.21A
663-2.81A
646
The concentration C of chlorophyll b in extract
b=20.13A
646-5.03A
663
C in the following formula
aAnd C
bUnit is mg/L, A
646And A
663Represent the light absorption value of chlorophyll extract under wavelength 646nm and 663nm respectively.Again according to the chlorophyll a of unit fresh weight in the following formula computation organization, the content of b:
In the following formula in the leaf tissue the chlorophyllous content unit of unit fresh weight be mg/g, the concentration of pigment is C
aOr C
b, extracting liquid volume is 10 milliliters, the sample fresh weight is 0.1 gram.Then calculate the chlorophyll a+b content of unit fresh weight in the corresponding leaf tissue again.
4. kind thermotolerance size is identified: behind the chlorophyll a of unit fresh weight+b content, calculate according to following formula in calculating corresponding leaf tissue, the result who draws compares the thermotolerance size that can identify the identification of species of wanting with following parameter again,
Formula: blade is coerced down a+b content ratio under chlorophyll a+b content/normal temperature: when this ratio is the high turf-type tall fescue kind of thermotolerance greater than 0.815, between 0.760-0.815 is the medium turf-type tall fescue kind of thermotolerance, less than 0.760 be the low turf-type tall fescue kind of thermotolerance.
Beneficial effect of the present invention is:
1. identify that the time is short fast.If according to original way, the turf-type tall fescue variety seeds that identify need be observed upgrowth situation in the spring or the meticulous always maintenance management that sows in autumn, whole process need 4-10 month time after getting over the summer.If planting seed is cultivated and handled after 21 days, measuring interior heat shock protein expression of plant body and cell antioxidant system then also needs 20 days to 40 days, and the time needed about 40-60 days altogether.And this new method that we proposed is cultivated and was handled needs 21 days from planting seed, and the mensuration chlorophyll content changes each sample only needs 5 minutes, and the time has shortened minute greatly less than 22 days altogether.
2. identify that operating process is simple.If identify turf-type tall fescue thermotolerance size by measuring turf-type tall fescue blade Net Photosynthetic Rate and photosystem II function, then not only need to take a long time to be familiar with the photosynthesis measurement instrument and the photosynthetic efficiency analyser of operation and debugged program complexity, factors such as the air flow when measuring simultaneously in the subenvironment, light all are easy to have influence on measurement result.Turf-type tall fescue blade heat shock protein is expressed and the cell antioxidant system is identified turf-type tall fescue thermotolerance size if by measuring, not only need the people who operates to have suitable professional knowledge background and higher experimental skill, more complicated is need spend a lot of time on the acquisition supervisor of solution preparation, protein extraction purifying even antigen-antibody, and the factors such as temperature when measuring in addition in the subenvironment are to the influence of enzyme and albumen and huge.And this new method that we proposed only needs normal temperature that random acquisition 0.1 gram respectively wants the tall fescue turf grass seedling of identifying down and the high temperature stress healthy leaves of cultivation down, after 10ml80% acetone milling and extracting, extract is measured under visible spectrophotometer easy and simple to handle and is got final product, the subenvironment factor is very little to measurement result influence during mensuration, and requires extremely low to the people's of operation professional knowledge background and experimental skill.
3. experimental cost is low, and instrument requires simple, easily promotes.Though by measuring Net Photosynthetic Rate, photosystem II function, heat shock protein expression and cell antioxidant system can be identified the thermotolerance size of tall fescue turf grass, but these methods need be used the photosynthesis measurement instrument, the photosynthetic efficiency analyser, large-scale expensive device such as protein purification system and microplate reader, and required medicine is also expensive unusually.By contrast, required reagent of this method and experimental apparatus that we propose are cheap 80% acetone and visible spectrophotometer, and reagent cost and instrument require to reduce greatly, are easy to promote.
Four, embodiment
1. embodiment 1 " jaguar No. 3 " is the high turf-type tall fescue kind of thermotolerance.Select high-quality " jaguar No. 3 " planting seed in the basin that common sand, clay and nutrition soil (volume ratio 1: 3: 3) is housed, after cultivating 18 days at normal temperatures, it is 35 ± 0.5 ℃ that the part plant moves into day temperature, and nocturnal temperature is 30 ± 0.5 ℃, and the illumination quantum is 150 μ mol m
-2s
-1, relative humidity is to cultivate 3 days in 70% the growth cabinet.In cultivating process, do not apply any nutrient.Spray low amounts of water before the seed sprouting every other day, plant was watered once permeablely every other day when the back normal temperature of emerging was cultivated down, and water once permeable every day during high temperature stress.Random acquisition and take by weighing 0.1 gram normal temperature down and the high temperature stress healthy leaves of cultivation down respectively, clean surface contaminants, shred, put into mortar, add a small amount of quartz sand and be ground into homogenate with 80% acetone, be settled to after extracting liq filters with qualitative filter paper 10 milliliters with visible spectrophotometer 663, under the 646nm colorimetric survey light absorption value, more than the experiment replication is four times.Accordingly result such as table 1 that the light absorption value of measuring calculates according to formula described in the summary of the invention.
As can be seen from Table 1, " jaguar No. 3 " blade coerces down that the a+b content ratio is 0.838 under chlorophyll a+b content/normal temperature, and this is worth greater than 0.815, illustrates that this kind is the high Festuca Arundinacea of thermotolerance, and this is consistent with its land for growing field crops observation practical manifestation.
Table 1 normal temperature down and high temperature stress down cultivation " jaguar No. 3 " turf-type tall fescue healthy leaves chlorophyll a and the content status of b
*
*In the last table: A
646And A
663Be respectively the light absorption value of pigment extract under wavelength 646nm and 663nm: C
aAnd C
bBe respectively chlorophyll a and the b concentration in extract; Chlorophyll a content, chlorophyll b content and chlorophyll a+b content is respectively the chlorophyll a of unit fresh weight in the leaf tissue, the content of b and a+b
2. embodiment 2 " TF 66 " is the very low turf-type tall fescue kind of thermotolerance.Select the sowing of high-quality " TF 66 " tall fescue seed in the basin that common sand, clay and nutrition soil (volume ratio 1: 3: 3) is housed, after cultivating 18 days at normal temperatures, it is 35 ± 0.5 ℃ that the part plant moves into day temperature, and nocturnal temperature is 30 ± 0.5 ℃, and the illumination quantum is 150 μ mol m
-2s
-1, relative humidity is to cultivate 3 days in 70% the growth cabinet.In cultivating process, do not apply any nutrient.Spray low amounts of water before the seed sprouting every other day, plant was watered once permeablely every other day when the back normal temperature of emerging was cultivated down, and water once permeable every day during high temperature stress.Random acquisition and take by weighing 0.1 gram normal temperature down and the high temperature stress healthy leaves of cultivation down respectively, clean surface contaminants, shred, put into mortar, add a small amount of quartz sand and be ground into homogenate with 80% acetone, be settled to after extracting liq filters with qualitative filter paper 10 milliliters with visible spectrophotometer 663, under the 646nm colorimetric survey light absorption value, more than the experiment replication is four times.Accordingly result such as table 2 that the light absorption value of measuring calculates according to the formula of carrying in the summary of the invention.
As can be seen from Table 2, " TF 66 " blade coerces down that the a+b content ratio is 0.738 under chlorophyll a+b content/normal temperature, and this is worth less than 0.760, illustrates that this kind is the low Festuca Arundinacea of thermotolerance, and this is consistent with its land for growing field crops observation practical manifestation.
" TF 66 " turf-type tall fescue healthy leaves chlorophyll a that table 2 normal temperature is following and high temperature stress is cultivated down and the content status of b
*
*In the last table: A
646And A
663Be respectively the light absorption value of pigment extract under wavelength 646nm and 663nm: C
aAnd C
bBe respectively chlorophyll a and the b concentration in extract: chlorophyll a content, chlorophyll b content and chlorophyll a+b content is respectively the chlorophyll a of unit fresh weight in the leaf tissue, the content of b and a+b
Claims (3)
1. the method for a Rapid identification turf-type tall fescue thermotolerance size is characterized in that being made of following steps:
(1) material is cultivated the sowing of the high quality seed of the turf-type tall fescue kind that will identify in basin, after normal temperature is cultivated certain hour down, the part plant is cultivated a period of time under high temperature stress, in cultivating process, do not apply any nutrient, spray low amounts of water before the seed sprouting every other day, plant was watered once permeablely every other day when the back normal temperature of emerging was cultivated down, and water once permeable every day when high temperature stress;
(2) sample collecting and the turf-type tall fescue healthy leaves of analyzing cultivation under difference random acquisition normal temperature and the high temperature, the light absorption value of mensuration chlorophyll extract;
(3) result calculates above measurement result is calculated chlorophyll a, b according to following formula concentration:
The concentration C of chlorophyll a in extract
a=12.21A
663-2.81A
646
The concentration C of chlorophyll b in extract
b=20.13A
646-5.03A
663
C in the following formula
aAnd C
bUnit be mg/L, A
646And A
663Represent the light absorption value of chlorophyll extract under wavelength 646nm and 663nm respectively, again according to the chlorophyll a of unit fresh weight and the content of b in the following formula computation organization:
In the following formula in the leaf tissue the chlorophyllous content unit of unit fresh weight be mg/g, the concentration of pigment is C
aOr C
b, extracting liquid volume is 10 milliliters, the sample fresh weight is 0.1 gram;
(4) kind thermotolerance size is identified according to a specific formulation: blade is coerced down the content of a+b under content/normal temperature of chlorophyll a+b, calculate a ratio, this ratio is compared the thermotolerance size that can identify the turf-type tall fescue kind with two preset parameters 0.815 with 0.760, when this ratio is the high turf-type tall fescue kind of thermotolerance greater than 0.815 the time, between 0.760-0.815 is the medium turf-type tall fescue kind of thermotolerance, less than 0.760 be the low turf-type tall fescue kind of thermotolerance.
2. the method for Rapid identification turf-type tall fescue thermotolerance size according to claim 1, the matrix that it is characterized in that planting seed in (1) is common sand, clay and nutrition soil, volume ratio 1: 3: 3, the cultivation time is 18 days under the normal temperature, breeding condition is 35 ± 0.5 ℃ of day temperatures under the high temperature stress, nocturnal temperature is 30 ± 0.5 ℃, and the illumination quantum is 150-200 μ mol m
-2s
-2, relative humidity is 65-70%, the cultivation time is 3 days.
3. the method for Rapid identification turf-type tall fescue thermotolerance size according to claim 1 is characterized in that the extract of being carried in (2) is 80% acetone.
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| CN102172117B (en) * | 2011-01-19 | 2013-02-20 | 江苏农林职业技术学院 | Lawn grass recognition and identification device and method for judging lawn grasses by using lawn grass recognition and identification device |
| CN102805010B (en) * | 2012-08-28 | 2013-07-31 | 西北农林科技大学 | Method for identifying chilli heat resistance by using detached leaves |
| CN102986403B (en) * | 2012-09-27 | 2013-12-04 | 江苏沿海地区农业科学研究所 | Assessing and identifying method of waterlogging-tolerant germplasm of hot pepper |
| CN108934814B (en) * | 2018-08-01 | 2020-07-03 | 广西壮族自治区农业科学院 | Method for identifying heat resistance of cabbage heart |
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|---|---|---|---|---|
| JP2002500372A (en) * | 1998-01-12 | 2002-01-08 | ハー マジェスティ イン ライト オブ カナダ アズ リプレゼンティッド バイ ザ ミニスター オブ アグリカルチャー アンド アグリ−フード カナダ | Preparation of new gel for purification of non-polar extract |
| JP2002107296A (en) * | 2000-10-03 | 2002-04-10 | Taisei Corp | Analyzing method of component of lawn leaf, and diagnostic method and growing method of lawn |
| US20020123455A1 (en) * | 2001-02-22 | 2002-09-05 | Ryan Peter L. | Equine relaxin as a measure of treatment efficacy |
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2006
- 2006-01-17 CN CNB2006100378400A patent/CN100456018C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002500372A (en) * | 1998-01-12 | 2002-01-08 | ハー マジェスティ イン ライト オブ カナダ アズ リプレゼンティッド バイ ザ ミニスター オブ アグリカルチャー アンド アグリ−フード カナダ | Preparation of new gel for purification of non-polar extract |
| JP2002107296A (en) * | 2000-10-03 | 2002-04-10 | Taisei Corp | Analyzing method of component of lawn leaf, and diagnostic method and growing method of lawn |
| US20020123455A1 (en) * | 2001-02-22 | 2002-09-05 | Ryan Peter L. | Equine relaxin as a measure of treatment efficacy |
Non-Patent Citations (2)
| Title |
|---|
| 冷地型草坪草耐热性机理的研究. 何亚丽，王惠林，沈剑，祁军.上海农学院学报，第15卷第3期. 1997 |
| 冷地型草坪草耐热性机理的研究. 何亚丽，王惠林，沈剑，祁军.上海农学院学报，第15卷第3期. 1997 * |
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