CN102706827A

CN102706827A - Novel extraction and determination method of content of chlorophyll by utilizing DMSO (dimethylsulfoxide)

Info

Publication number: CN102706827A
Application number: CN2012102227612A
Authority: CN
Inventors: 丁伟; 程茁; 戴航宇
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2012-07-02
Filing date: 2012-07-02
Publication date: 2012-10-03

Abstract

Acetone and absolute ethanol which serve as extraction solutions and is applied to plant chlorophyll determination are easily volatilized, and time for sampling, treating samples and extracting can be saved, so that the error in chlorophyll determination is increased. The invention provides a novel quick extraction and determination method of plant chlorophyll by utilizing DMSO (dimethylsulfoxide). The method comprises the following steps: giving numbers on cleaned serum bottles; adding 5mL of DMSO into each serum bottle, covering plugs and accurately weighing; putting the serum bottles according to a sequence of the numbers into a container covered by a piece of shading black fabric, and taking the container to a field; instantly putting samples into the serum bottles filled with the DMSO after the samples for a part to be determined, covering the plugs and the shading black fabric, and taking the container to a lab; weighing according to the numbers, wherein the weight difference between two-time weighing is the weight of the sample to be determined; extracting the serum bottles filled with test samples in a dark condition for 6 to 8 hours, adding 1mL of extract into 4mL of DMSO, uniformly mixing, and determining integrated optical densities in wavelengths of 663nm and 645nm by using a spectrophotometer.

Description

Utilize the new method of DMSO extraction, mensuration chlorophyll content

Technical field

The present invention relates to a kind of plant chlorophyll extraction, assay method, especially utilize DMSO to extract, measure the new method of chlorophyll content of plant fast.

Background technology

Chlorophyll is that plant carries out photosynthetic main pigment, and chlorophyll absorbs behind the sunshine through light reaction and two continuous process of dark reaction, CO ₂And H ₂O changes glucide into and is stored in the green plants nutrition organs, thereby formation provides energy substance with output for green plants grows.In fact chlorophyll be present in all can build in the photosynthetic biosome, comprises the blue-green algae (blue bacterium) of green plants, protokaryon and the algae of eucaryon.Chlorophyll in the higher plant chloroplast mainly contains two kinds of chlorophyll a and chlorophyll bs, and we can say does not have chlorophyll, and plant just can not change into chemical energy to solar energy and be stored in the human final food.Therefore; In the related science that improves crop yield and quality is studied, usually need measure chlorophyll content and receive cultivation step or crop growth environment effect, so that correctly estimate the artificial regulatory crop growth and the feasibility of a certain concrete measure that improves the yield and quality.

Because chlorophyll is water insoluble; Therefore when in scientific research, chlorophyll being measured at present; The normal preparation acetone earlier that adopts: the mixed solvent of absolute ethyl alcohol (V:V)=1:1; Put into after weighing testing sample and fill acetone: the container of absolute ethyl alcohol (V:V)=1:1 mixed solvent, and extract in the dark more than 24 hours.Adopt acetone: chlorophyllous method in absolute ethyl alcohol (V:V)=1:1 mixed extractant solvent plant sample; Though changed the assay method that the past people use acetone or absolute ethyl alcohol adding silica sand and testing sample to grind, filter in chlorophyll measuring; Make determination techniques become laborsaving; Avoided chlorophyllous loss in grinding, the filter process simultaneously, before chlorophyll measuring is technical, gone a step further.Yet; Adopt acetone: still there are some shortcomings in absolute ethyl alcohol (V:V)=chlorophyllous method of 1:1 mixed extractant solvent; Show: (1) acetone and absolute ethyl alcohol all are more volatile organic solvents; The volatilization of solvent can cause chlorophyll to concentrate in extraction and the mensuration process, makes that to measure numerical value higher and cause error at measurment to strengthen; (2) because chlorophyll is unstable, especially under illumination condition, be easy to decompose, therefore require chlorophyllous extraction, in the following short time of dark condition, accomplish as far as possible when measuring.Yet utilize acetone: the chlorophyll in absolute ethyl alcohol (V:V)=1:1 mixed extractant solvent sample needs 24 hours at least, and extraction efficiency is low, the time is long, is easy to increase chlorophyllous decomposition amount and causes determination data on the low side.(3) need get back to the laboratory to sample from the field during chlorophyll measuring; And then take a sample, weigh, extract; Test specimen is from the field to the laboratory and indoor sample is handled, weighing often needs the long period; Plant leaves the soil environment of depending on for existence, and chlorophyll can be accelerated to decompose and cause error at measurment to increase.

Summary of the invention

Owing to have significant disadvantages in chlorophyll extraction at present and the assay method; Therefore in our scientific research; A kind of stable in properties, the not volatile and solvent that chlorophyll had quick extraction have been found through a large amount of research: dimethyl sulfoxide (DMSO) (DMSO); Can bring to the field to extraction solvent DMSO in the chlorophyll extraction process; Put into the DMSO extract to sample when getting testing sample under the plant condition of living body and extract, take back the laboratory then and carry out the mensuration of chlorophyll content.

Embodiment:Number the serum bottle of cleaning in advance; Bottle stopper is also accurately weighed on the adding 5mL DMSO bonnet in each serum bottle; Put into the container that is stamped gobo to serum bottle by number order and bring to the field, get and put into the serum bottle that DMSO is housed to sample immediately behind the detected part sample and build bottle stopper and gobo is taken back the laboratory, weigh by numbering once more; Twice weight difference is testing sample and weighs; Place under the dark condition extraction 6-8 hour to the serum bottle that test specimen is housed, get extract 1mL and add after 4mL DMSO mixes, with the OD value under spectrophotometric determination 663nm and the 645nm wavelength.

This method has avoided using the error at measurment that solvent evaporates causes in acetone and absolute ethyl alcohol extraction sample and the mensuration process; The sample extraction time is short, efficient is high, reduced chlorophyllous decomposition amount; Sample chlorophyll extraction can be operated in the field, and it is long and cause chlorophyll to decompose the adverse effect with the error at measurment increase through the transportation of long period and indoor processing time to have reduced sample.Show through years of researches and the application in scientific research: this method can be accomplished chlorophyllous whole extractions in the testing sample in 6-8 hour, carries out the chlorophyll extraction in the time of the sampling of field plant living body, and method is easier, accurate, quick.2010-2011, utilize DMSO to carry out field practical measurement chlorophyll content in leaf blades in soybean seedling, florescence and fruiting period and see table 1.

The different extracting process of table 1 are to the influence of measuring chlorophyll content

The result shows: utilize DMSO extraction, measure chlorophyll content to be: 1.24 mg/g, 1.28 mg/g and 1.33 mg/g, utilize acetone: absolute ethyl alcohol (V:V)=1:1 mixed extractant solvent sample is also measured chlorophyll content and is respectively: 1.15 mg/g, 1.19 mg/g and 1.25 mg/g.Statistical results show is utilized acetone: absolute ethyl alcohol (V:V)=1:1 mixed extractant solvent measure with utilize the DMSO extraction, measuring chlorophyll content, to compare the result on the low side; And difference reaches the level of signifiance, this and acetone: extraction during absolute ethyl alcohol (V:V)=1:1 mixed extractant solvent sample, minute are long volatile relevant with extract.

Claims

1. number the serum bottle of cleaning in advance; Bottle stopper is also accurately weighed on the adding 5mL DMSO bonnet in each serum bottle; Put into the container that is stamped gobo to serum bottle by number order and bring to the field, get and put into the serum bottle that DMSO is housed to sample immediately behind the detected part sample and build bottle stopper and gobo is taken back the laboratory, weigh by numbering once more; Twice weight difference is testing sample and weighs; Placed the serum bottle that test specimen is housed under the dark condition lixiviate 6-8 hour, get leaching liquor 1mL and add after 4mL DMSO mixes, with the OD value under spectrophotometric determination 663nm and the 645nm wavelength.