CN102269703B - Method for detecting glyphosate resistance of plant - Google Patents
Method for detecting glyphosate resistance of plant Download PDFInfo
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- CN102269703B CN102269703B CN 201110119737 CN201110119737A CN102269703B CN 102269703 B CN102269703 B CN 102269703B CN 201110119737 CN201110119737 CN 201110119737 CN 201110119737 A CN201110119737 A CN 201110119737A CN 102269703 B CN102269703 B CN 102269703B
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Abstract
The invention provides a method for detecting the glyphosate resistance of a plant, which comprises the following steps: spraying glyphosate in different concentration gradients on different groups of same plant seedlings respectively; measuring and recording the PPM (plant photosynthetic meter) value of each plant 3-9 days after glyphosate spraying; before measurement, placing the plant under the condition that the illumination intensity is lower than 200Lux for more than 30 minutes; and judging the glyphosate resistance of the plant according to the PPM values of the plant before and after glyphosate spraying. The detection method is applicable to determining and evaluating the glyphosate resistance of conventional soybean varieties and transgenic glyphosate-resistant soybean materials, can quickly and accurately determine the glyphosate resistance level of the plant in a non-destructive manner, and has very high practicability and operability.
Description
Technical field
The present invention relates to the plant protection technology field, specifically, relate to a kind of detection method of glyphosate resistance of plant.
Background technology
China's soybean weeds kind more than 70, advantage weeds kind more than 20 causes the soybean underproduction 150～2,250,000 ton throughout the year.Along with development and the rural laborer of chemical industry shifts to cities and towns, artificial and weeding by machine is replaced by chemical weed control gradually, eliminates area and accounts for more than 90% of its sown area.The Soybean Field weeding mainly contains dual mode: the one, broadcast front or broadcast rear seedling before spray processing soil treatment agent, main herbicide kind is Acetochlor, isopropyl methoxalamine, imazethapyr, clomazone etc.; The 2nd, spray the selectivity cauline leaf behind the seedling and process herbicide, such as Quizalotop-ethyl, haloxyfop-r-methyl, lactofen, fomesafen, acifluorfen etc.Although above-mentioned medicament has played larger effect in weeds control, has also brought the series of problems such as herbicide residue poisoning, drug resistance weeds.Acetochlor often causes the soybean poisoning and the underproduction under the larger environmental baseline of soil moisture; The residual life in soil such as imazethapyr, clomazone, reach 6 months to 3 years, reduces second stubble crop corn, potato, sunflower, melon etc. output, to such an extent as to limited the adjustment of pattern of farming; Lactofen, fomesafen, acifluorfen selectivity between weeds and soybean is relatively poor, and is low to the soybean security.Owing to used for a long time in recent years above-mentioned medicament, the soybean weeds succession of community is serious, the weed density that above-mentioned herbicide is difficult to prevent and kill off increases, dayflower, field thistle, sowthistle such as the northeast soybean district, North China ivy glorybind, the rhizome of nutgrass flatsedge, South China's Eclipta prostrata, cyperus iria etc. become the important harmful organism of harm soybean yields.Therefore.Be badly in need of broad weed-killing spectrum,, lower-cost herbicide product safe to soybean on the Soybean production.
Herbicide glyphosate is a kind of amino acid bio synthetic inhibitor, and its Main Function target is 5-enol acetone shikimic acid-3-phosphonate ester synthetase (EPSPS).In plant, EPSPS mainly is present in chloroplast and the plastid, after glyphosate enters above-mentioned two organelles, preferentially be combined with the avtive spot of EPSPS, make the variation on the EPSPS generation configuration, thereby suppress the combination of itself and phosphoenolpyruvate (PEP), make through its catalysis to be stopped by phosphoenolpyruvate and shikimic acid-3-phosphoric acid (S3P) transition process to 5-enol acetone shikimic acid-3-phosphate (EPSP), thereby suppress the amino acid whose biosynthesizing of plant fragrance.Glyphosate has the wide spectrum natural disposition of going out, and can effectively prevent and kill off nearly all annual and perennial grass, dicotyledon and shrub etc.It is safe to people and animals, environment, soil etc., is the desirable grass product that removes.But because glyphosate does not have the selectivity between crops and weeds, conventional soybean varieties is processed the death of namely being injured once glyphosate.
The resistance glyphosate soybean is one of effective way of administering soybean weeds.Transgenosis resistance glyphosate soybean was the genetically modified crops of so far commercialization cultivated area maximum at american goods in 1996.To 2009, the U.S., Argentina and Brazilian resistance glyphosate soybean planting area accounted for respectively 91%, 99% and 71% of the total cultivated area of its soybean.China is also implementing transgenosis resistance glyphosate new soybean varieties incubation program at present.In resistance glyphosate soybean varieties Breeding Process, to the soybean material glyphosate resistance evaluation be the key link of breeding success or failure also be one of content of estimating of genetically engineered soybean environmental safety.
At present soybean glyphosate resistance level determinations is mainly adopted the plant bioassay method, namely in the greenhouse or field planting resistance glyphosate soybean and its acceptor soybean, the long glyphosate that sprays the variable concentrations gradient to certain leaf age of soybean, observe the dead degree of soybean in 20～30 days after the dispenser or measure its fresh weight or dry weight, according to the growth inhibition degree of soybean plant strain under the variable concentrations its resistance is made evaluation.Bioassay method can be measured soybean more exactly to the resistance level of glyphosate, but the used time is long, needs 60 days left and right sides time from sowing to drawing measurement result, this for many materials, large batch of mensuration length consuming time, take up room large, efficient is low.And bioassay method is owing to need to measure the soybean fresh weight and dry weight, can not obtain seed to such an extent as to plant damaged, and this is larger for the especially a small amount of genetic transformation seedling loss of resistant material.
Molecular assay and protein determination have fast and efficiently advantage, be widely used in the resistance glyphosate soybean, but because its selectivity and specificity, only can measure whether genes of interest inserts or whether albumen expresses, and cost height, the specific checkout equipment of needs and facility have its limitation at mensuration glyphosate formulation rate and plant aspect the relation of glyphosate tolerance degree.Therefore, soybean is badly in need of quick, accurate, quantitative technology to the evaluation of resistance of glyphosate.
PPM (Plant Photosynthesis Meter) a kind ofly judges whether normal photoelectric instrument of plant growth state by measuring the photosynthesis of plant power.And the fluorescence signal that photosynthetic power is measured by PPM to be the reading of instrument reflect.This instrument once was used for measuring spraying the photosynthetic inhibition type herbicides such as atrazine, isoproturon to the extent of injury of weeds, but was not used to measure glyphosate resistant crops.
Summary of the invention
The object of the present invention is to provide a kind of detection method of glyphosate resistance of plant.
In order to realize purpose of the present invention, the detection method of glyphosate resistance of plant of the present invention comprises the steps:
1) respectively with the glyphosate of different gradient concentrations to not on the same group identical plant seedlings spray;
2) after the dispenser 3～9 days, measure also record plant PPM value; Before the measurement, plant is placed on intensity of illumination, and to be lower than the 200Lux condition lower more than 30 minutes;
3) judge that according to plant-spraying and the PPM value that do not spray glyphosate plant is to the resistance of glyphosate.
Wherein, described plant is soybean.
Wherein, described when spraying glyphosate the soybean leaf age be 3～6 winglike compound leaves, preferred 3～4 winglike compound leaves.
Wherein, the glyphosate concentration that sprays is respectively 61.5g ai/ mu～82g ai/ mu, 102.5g ai/ mu～164g ai/ mu and 246g ai/ mu.The criterion of this this detection method is: spray and measure soybean PPM value behind the glyphosate of 246gai/ mu and to impinging upon on the statistical significance without significant difference, be high-resistance glyphosate; Spray record behind the glyphosate of 102.5g ai/ mu～164g ai/ mu soybean PPM value with to impinging upon on the statistical significance without significant difference, spray and measure soybean PPM value behind the glyphosate of 246g ai/ mu and there were significant differences on the statistical significance to impinging upon, be middle resistance glyphosate; Spray record behind the glyphosate of 61.5g ai/ mu～82g ai/ mu soybean PPM value with to impinging upon on the statistical significance without significant difference, be low resistance glyphosate; Spraying and record soybean PPM value contrast behind the glyphosate of 61.5g ai/ mu～82g ai/ mu and obviously reduce on statistical significance, is that glyphosate is responsive.
Wherein, the device that sprays glyphosate is preferably the spray tower of packaged type 8002 Fan spray heads of pressure 2.75Kpa.
Wherein, before the measurement plant being placed on size is 3～20m
3The light shield made of three layers black plastic shading net in, the transmittance that light shield is every layer is 50%.Described light shield size is preferably 3～10m
3Preferred 30～50 minutes of shading time.
Wherein, PPM value Measuring Time is preferably after the dispenser 3～4 days.
Wherein, described glyphosate is preferably farming and reaches (Roundup), and it contains 41% glyphosate isopropyl amine salt, and About Monsanto Chemicals production is available from Beijing Agriculture Prodn. Means Company.
Wherein, being used for the instrument that PPM measures is Plant Photosynthesis Meter, and Dutch EARS company produces.
Wherein, described plant is the container seedling soybean, and the cultivation of soybean plant strain prepares may further comprise the steps: container cultivation matrix in sow soya seeds, pour water and emerge rear thinning.Described container is diameter 10～15cm, high 8～10cm vinyon alms bowl, and 2～5 0.2～0.5cm diameter permeable holes are arranged at the bottom of alms bowl, and permeable hole is covered by seepy material, and preferred seepy material is individual layer filter paper.Described container culturing substrate is preferably loam: yellow sand: 1: 1: 1 culture of triumphant mattress Nutrition Soil, and its content of organic matter 2.5～5%, pH is 7.0～7.5.The cultural method of soya seeds soaks into soil for pouring water from container bottom, sows the soya seeds of 5 germination percentages more than 85%, then covers thickness and be 0.75～1.5 centimetre matrix, and the every alms bowl of rear thinning of emerging stays 3 plant.Be used for the place of the cultivated soybean, preferably in the greenhouse, carry out, more than the fine illumination 12000Lux.
Evaluation method of the present invention be take PPM as the basis a whole set of glyphosate resistance of plant degree is carried out the technology of Fast Measurement, can accurately examine and determine plant to the resistance level of glyphosate, be applicable to soybean conventional variety and resistance glyphosate soybean material, and have characteristics easy and simple to handle, quick, efficient and that non-destructive detects, practical.Judge plant to the resistance level of glyphosate with 3～4 days PPM value behind the spray medicine, comparable bioassay method shifts to an earlier date 15～25 days and obtains a result.The method has been filled up the blank of China soybean to the glyphosate tolerant nondestructively measuring.To the scientific evaluation of breeding material resistance in the environmental safety study of Soybean Resistance glyphosate material, the Soybean Resistance glyphosate breeding of new variety process with to accelerate breeding process significant.
Embodiment
Be that embodiment is used to illustrate the present invention with soybean below, but be not used for limiting the scope of the invention.
Embodiment 1
Select diameter 15cm, high 10cm vinyon alms bowl, 3 0.5cm diameter permeable holes are made a call in the alms bowl bottom; Permeable hole individual layer filter paper covers; The immature soil of packing into: yellow sand: 1: 1: 1 culture matrix of triumphant mattress Nutrition Soil, pour water from container bottom and to soak into soil.
Sow respectively rich No. 6 of transgenosis resistance glyphosate soybean 356043, acceptor soybean Jack and the conventional variety section of germination percentage more than 85%, 5 seeds of every alms bowl sowing.After planting cover 1 centimetre of same matrix.The soybean every alms bowl of rear thinning of emerging stays 3 strains.
Treat that soybean grows to 3～4 winglike compound leaf phases, put in the spray tower of packaged type 8002 Fan spray heads of pressure 2.75Kpa herbicide spraying glyphosate (Roundup) aqua; Glyphosate concentration is respectively 0g ai/ mu, 61.5g ai/ mu, 82g ai/ mu, 102.5g ai/ mu, 164g ai/ mu, 246g ai/ mu and 328g ai/ mu, amount of water 30L/ mu.The design of the employing factor repeats 4 times.
After the dispenser 3 days, soybean to be measured was put a 10m
3The black plastic shading net light intensity of making be lower than in the light shield of 200Lux, leave standstill 30 minutes after, survey the 3rd winglike compound leaf PPM value.Use that 3d soybean leaves PPM value and resistance the results are shown in table 1 behind the glyphosate.In order to check the accuracy of PPM determination method, table 1 has also been listed the soybean plant strain dry weight that 28d measures after the dispenser and the dead degree of soybean when measuring the same day.
Table 1 is used 3dPPM value and 28d soybean dry weight behind the glyphosate
Annotate: 1, L represents to process the soybean that growing point is survived through the matched doses glyphosate in the table, and D represents to process through the matched doses glyphosate soybean of growing point death.2,356043 is transgenosis resistance glyphosate soybean, and Jack is 356043 acceptor kind, is du pont company production; Section rich 6 is conventional soybean varieties, the seed selection of heredity institute of the Chinese Academy of Sciences.3, the rear same letter of numeral represents that two data are not remarkable at α=0.05 level difference in the table.
Embodiment 2
Select diameter 15cm, high 10cm vinyon alms bowl, 3 0.5cm diameter permeable holes are made a call in the alms bowl bottom; Permeable hole individual layer filter paper covers; The immature soil of packing into: yellow sand: 1: 1: 1 culture matrix of triumphant mattress Nutrition Soil, pour water from container bottom and to soak into soil.
Sow respectively the seed that Genetic Transformation of Soybean seedling JZ1～JZ4 produces.After planting cover 1 centimetre of same matrix.The soybean rear every alms bowl of emerging stays 3 strains.
Treat that soybean grows to 3～4 winglike compound leaf phases, put in the spray tower of packaged type 8002 Fan spray heads of pressure 2.75Kpa herbicide spraying glyphosate (Roundup) aqua; Glyphosate concentration is respectively 0g ai/ mu, 61.5g ai/ mu, 82g ai/ mu, 102.5g ai/ mu, 164g ai/ mu, 246g ai/ mu and 328g ai/ mu, amount of water 30L/ mu.The design of the employing factor repeats 3 times.
After the dispenser 3 days, soybean to be measured was put a 10m
3The black plastic shading net light intensity of making be lower than in the light shield of 200Lux, leave standstill 30 minutes after, survey the 3rd winglike compound leaf PPM value.Use that 3d soybean leaves PPM value and resistance the results are shown in table 1 behind the glyphosate.In order to check the accuracy of PPM determination method, table 1 has also been listed the soybean plant strain dry weight that 28d measures after the dispenser and the dead degree of soybean when measuring the same day.
Table 2 is used 3dPPM and 28d soybean dry weight behind the glyphosate
Annotate: JZ1～JZ4 is for carrying out the transformation seedlings T4 generation that cotyledonary node transforms take the black farming 37 of soybean varieties as acceptor, the gene of its conversion is that (method is referring to Ji Yuemei for the EPSP Antiglyphosate gene, Chen Shouyi, Qiu Lijuan etc. the optimization research of Agrobacterium-mediated Transformation Systems of Soybean Cotyledonary Node system. Soybean Science .2008,27 (1): Lee 26-32. next spring etc., the research of improvement soybean cotyledon node regenerating system. Acta Agronomica Sinica, 2006,32 (2): 223-227.) in the table after the numeral same letter represent that two data are not remarkable at α=0.05 level difference.
Can find out from the result of table 1 and table 2,356043 and JZ2 be high-resistance glyphosate, anti-and low resistance glyphosate during JZ3 and JZ4 are respectively, Jack, section rich 6 and JZ1 be to the glyphosate sensitivity.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. the detection method of a glyphosate resistance of plant is characterized in that, comprises the steps:
1) respectively with the glyphosate of different gradient concentrations to not on the same group identical plant seedlings spray; The glyphosate concentration that sprays is respectively 61.5g ai/ mu-82g ai/ mu, 102.5g ai/ mu-164g ai/ mu and 246g ai/ mu;
2) after the dispenser 3-9 days, measure also record plant PPM value; Before the measurement, plant is placed on intensity of illumination, and to be lower than the 200Lux condition lower more than 30 minutes;
3) judge plant to the resistance of glyphosate according to plant-spraying and the PPM value that do not spray glyphosate, criterion is: spray and measure soybean PPM value behind the glyphosate of 246g ai/ mu and to impinging upon on the statistical significance without significant difference, be high-resistance glyphosate; Spray record behind the glyphosate of 102.5g ai/ mu-164g ai/ mu soybean PPM value with to impinging upon on the statistical significance without significant difference, spray and measure soybean PPM value behind the glyphosate of 246g ai/ mu and there were significant differences on the statistical significance to impinging upon, be middle resistance glyphosate; Spray record behind the glyphosate of 61.5g ai/ mu-82g ai/ mu soybean PPM value with to impinging upon on the statistical significance without significant difference, be low resistance glyphosate; Spraying and record soybean PPM value contrast behind the glyphosate of 61.5g ai/ mu-82g ai/ mu and obviously reduce on statistical significance, is that glyphosate is responsive.
2. detection method as claimed in claim 1 is characterized in that, described plant is soybean.
3. detection method as claimed in claim 2 is characterized in that, described when spraying glyphosate the soybean leaf age be 3-6 sheet winglike compound leaf.
4. detection method as claimed in claim 3 is characterized in that, described when spraying glyphosate the soybean leaf age be 3-4 sheet winglike compound leaf.
5. such as the described detection method of claim 1-4 any one, it is characterized in that, before measuring plant is placed on size and is 3-20m
3The light shield made of three layers of black plastic shading net in, the transmittance that light shield is every layer is 50%.
6. such as the described detection method of claim 1-4 any one, it is characterized in that, before measuring plant is placed on and guarantees that intensity of illumination is lower than under the 200Lux condition 30-50 minute.
7. such as the described detection method of claim 1-4 any one, it is characterized in that PPM value Measuring Time is after the dispenser 3-4 days.
8. such as the described detection method of claim 1-4 any one, it is characterized in that glyphosate is that farming reaches, it contains 41% glyphosate isopropyl amine salt.
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