CN102150550B - Wheat breeding method for improving content of resistant starch of kernels - Google Patents
Wheat breeding method for improving content of resistant starch of kernels Download PDFInfo
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Abstract
The invention belongs to the technical field of wheat breeding, in particular relates to a wheat breeding method for improving content of resistant starch of kernels and strengthening a health care function. The wheat breeding method for conveniently, rapidly, accurately and efficiently improving content of resistant starch of kernels comprises the steps of: after required variation and breeding generated by variety selection and seed treatment are stabilized, screening materials with reduced main viscosity parameters through starch pasting characteristic measurement, further measuring direct-chain starch content and defining change of physical and chemical properties of starch, and finally confirming materials with increased content of the resistant starch through directly measuring the content of the resistant starch. According to the invention, detection procedures are simplified, selection accuracy and working efficiency are improved, and better effect is obtained in practice.
Description
Technical field
The invention belongs to the technical field of wheat breeding, be specifically related to a kind of wheat selection that improves seed resistance starch content, enriched health care function.
Background technology
All be faced with at present the serious situation of Chronic Non-Communicable Diseases rapid spreads such as overweight, fat, diabetes in the world wide, and the main cause that produces this phenomenon is that great variety has taken place people's dietary structure, phitosite sharp increase.Resistant starch (Resistant starch) is a kind of novel dietary fiber of discovered in recent years; Europe resistant starch research coorporative network (Euresta) is defined as it " starch of not digested and assimilated by the healthy human body small intestine and the general name of decomposer thereof ", and resistant starch has different physiological roles (seeing table 1).Compare with the full diet fiber; Resistant starch has beneficial effect more widely to human health; " carbohydrate in the human nutrition " of FAO and WHO combined publication classified the discovery and the progress of resistant starch in recent years as a most important achievement of " carbohydrate and healthy relation " research, spoken highly of its significance to health.
The physiological function of table 1 resistant starch
| Physiological effect | Prevention/protective effect |
| Blood sugar lowering generates the reaction of exponential sum pancreas islet | Weaken the reaction of blood sugar and pancreas islet, prevent diabetes, metabolic syndrome |
| Promote intestinal health | Prevention colon cancer, the routed soup of large intestine, enteritis, diverticulitis, constipation |
| Reduce blood fat | Promote lipid metabolism, angiocardiopathy preventing, metabolic syndrome |
| The former effect of useful life promotes the enteric microorganism growth | Promote colon health |
| Prolong satiety, reduce energy absorption | Management of body weight, prevention of obesity |
| Promote micronutrient element to absorb | Promote mineral element to absorb prevention of osteoporosis disease |
| Improve " oral rehydration therapy (ORT) " effect | Treat messy, chronic diarrhea |
| With other meals composition such as dietary fiber, albumen, lipid effect | Promote metabolism control, improve intestinal health |
| Energy value is low, themogenesis is little | Prevention of obesity and diabetes |
Resistant starch is mainly derived from cereal (42%), so high resistant starch content cereal becomes the emphasis of research and development.Resistance starch content and amylose content positive correlation, so the seed selection of high amylose content grain variety causes concern widely.The general amylose content of cereal starch is lower, is not enough in food, produce enough resistant starches, so the scientist of countries in the world utilizes various tradition and biological new technology to improve amylose content by every means.Method in common is to select spontaneous mutation and induced mutations high amylose starches mutant, with agronomy parent hybridization, realizes the improvement of high-resistance starch and economical character.Behind corn starch branching enzyme (SBE) inactivation, the amylose relative amount significantly rises, and through hybridizing with AE mutant (SBE loss of activity), has obtained the high amylose content corn.Also obtain the high amylose starches barley by this method, amylose content reaches 45-50%.Also found the SBE deletion mutant in paddy rice, the soybean.The resistant starch product overwhelming majority of commercial is from amylomaize at present, and its amylose content can reach more than 80%, contains resistant starch 42%, and through improving production technology, the resistant starch yield has brought up to 60% at present.
Wheat is the first in the world generalized grain crop, has unique processing and edible quality, the processed food most species, so the research and development of high amylose content breed breeding and wheat resistant starch product obtain worldwide extensive concern especially.The wheat seed amylose content is lower, is not enough in food, produce enough resistant starches, and therefore wanting seed selection high amylose content wheat at first must the innovation germplasm.Yamamori has selected the high amylose content wheat line through the conventional breeding method from Kanto79/Turkey116 F2//Chousen57 offspring.But the scarcity that to perplex subject matter of this method be genetic resources, under the natural conditions, the variation amplitude of starch physicochemical property is very little, and the specific material of starch is considerably less.Australia CSIRO has obtained containing the wheat line of 70% amylose through reticent starch branch enzyme gene, and hope can be used to develop resistant starch product, but its output is very low, has influenced it and has applied on a large scale.In addition, transgenosis is feasible, but the wheat transgenic technology still faces many problems, and safety evaluation also is very loaded down with trivial details.
Another major reason that influences the seed selection of high resistant starch content wheat is the mensuration of resistance starch content, and the mensuration of resistant starch is the digestion process in the simulation human body, needs the continuous action of plurality of enzymes, and process is complicated, and length consuming time and cost are high.The method of measuring at present resistant starch is a lot, and the result varies, and certain methods itself is not easy to operate, and poor reproducibility is measured domestic and international 100 portions of spring wheat like Wang Lin etc. and to be found, resistance starch content between 0.87-4.33%, average out to 2.11%; And Zhang Ping etc. discover, the wheat resistance starch content is 0.64 ± 0.11%.At present amylose content is used the IKI colorimetric method for determining more, and reaction conditions such as the pH value of solution, temperature, reaction time are influence colour developing result strongly all, operate also loaded down with trivial details relatively, thereby the error of bringing.
Because the shortage of germ plasm resource and the complicacy of detection method, wheat high-resistance starch breeding progress is very slow.
Summary of the invention
The objective of the invention is to provide to the defective of above-mentioned existence a kind of easy, quick, accurate, efficiently improve the wheat selection of seed resistance starch content; This method is handled the variation that generation needs through wheat seed; After breeding is stable, screen the material that main viscosity parameter reduces, further measure the change of the clear and definite starch physicochemical property of amylose content through the starch gelatinization characteristic measurement; At last through directly measuring the material that resistance starch content confirms that resistance starch content improves; Simplify trace routine, improved selection accuracy and operating efficiency, obtained good effect in practice.
A kind of wheat selection that improves the seed resistance starch content; Its step is following: at first carry out kind and select, the variation that then the seed treatment generation is needed is after breeding is stable; Carry out primary election through the starch gelatinization characteristic again; Further screen through measuring amylose content then, confirm the material that resistance starch content is high through direct mensuration resistance starch content at last, thereby obtain the wheat breed of high seed resistance starch content.
Above-mentioned described material refers to stable strain or the strain that seed treatment forms later on, below the identical meaning of representative.
Wherein, said kind select be select to produce go up that establishing in large scale, stable high yield, comprehensive resistance are good, wheat breed that wide adaptability, seed amylose content are higher relatively or have the stable strain of large tracts of land promotion potential.
Said method for treating seeds be physics radiation, mutagenesis, space breeding, gene engineering or with one of method of high resistant starch content germplasm hybridization.
The primary election of said starch gelatinization characteristic is the strain system of decline more than 50% before the main viscosity parameter of screening is handled, and said viscosity parameter comprises peak viscosity, keeps viscosity, final viscosity, the value of bringing back to life.
Said amylose content screening is to improve the material more than 30% before the screening amylose content is handled.
It is the material of resistance starch content raising more than 40% of selecting and remain that said resistance starch content is measured.
The wheat selection of raising seed resistance starch content of the present invention, its concrete steps are following:
1, kind is selected: select to produce go up that establishing in large scale, stable high yield, comprehensive resistance are good, wheat breed that wide adaptability, seed amylose content are higher relatively or have the stable strain of large tracts of land promotion potential, select the higher kind of amylose content in the situation that satisfies above-mentioned condition as far as possible.
2, seed treatment: select big, the full seed of grain through physics radiation, mutagenesis, space breeding, gene engineering, with the hybridization of high resistant starch content germplasm or additive method acquisitions need variation.
For the high resistant starch content germplasm be with common germplasm comparatively speaking, with the hybridization of the high germplasm of content, can in the offspring, produce the material of resistance starch content ultra close (being that content is higher).
3, material breeding: the seed of handling carries out field planting, divides the individual plant results, breeding, in the heredity relatively stable after, pressing strain is to gather in the crops, so that carry out subsequent measurements.
4, starch gelatinization characteristic primary election: select the strain system that economical character is good, growth is consistent, output is high to measure the starch gelatinization characteristic; Screen the material that descends before main viscosity parameter is handled more than 50%; Described viscosity parameter is peak viscosity, keeps viscosity, final viscosity, the value of bringing back to life, and the material that satisfies condition carries out subsequent measurements.
The starch gelatinization characteristic measurement adopts Australia to produce the quick viscosity apparatus of Newport type and measures with reference to " wheat, rye and powder class thereof and the quick viscosity apparatus method of starch gelatinization characteristic measurement GB/T 24853-2010 ".Get 3.20 g flour (14% wet basis), add 25.0 ml distilled water, in sample jar, use the agitator mixing, measure.Be incubated 50 ℃, screw stirs 10 s with 960 rpm, then with 160 rpm uniform rotation to EOT.Variations in temperature is: 50 ℃ of insulation 1 min, rise to 95 ℃ with 10 ℃/min through 3.75 min then, and be incubated 2.5 min, reduce to 50 ℃ with 12 ℃/min through 3.75 min again, be incubated 2 min, whole process is carried out 13 min.
5, amylose content screening: the material that screening obtains to step 4 is measured, and the amylose content of selecting and remain improves the material more than 30% before handling, and carries out subsequent measurements then.
Amylose content determination is with reference to " the mensuration GB/T 15683-2008 of rice amylose content "; Key step comprises that sample takes by weighing, and adds ethanol, sodium hydroxide dispersion, adds petroleum ether degreasing again; Add the Wagner's reagent colorimetric at last, calculate amylose content according to calibration curve.
6, resistance starch content is measured: the material that screening obtains to step 5 is measured, and the resistance starch content of selecting and remain improves the material more than 40%, thereby obtains the wheat breed of high seed resistance starch content.
Resistance starch content is measured the method with reference to AOAC2002.2 and Wang Lin etc., slightly changes, and specifically assay method is: (1) take by weighing 100mg (± 5mg) sample in 10mL centrifuge tube with cover; (2) in each centrifuge tube, add 200 μ L distilled water, 100 ℃ of water-bath 20min, insulation 10min; (3) (0.1M, pH=1.5) buffer solution are smashed to pieces with imitation with diameter 1cm glass bar and to be chewed to add 2mLKCl-HCl; (4) add 100 μ L, 10% (W/V) pepsin (Amresco), 37 ℃ of digestion 1h, isolating protein; (5) add in each centrifuge tube 4mL sodium maleate buffer solution (0.1M, pH=6.0), 4mL pancreatic enzyme liquid (Sigma) is with NaOH adjust pH to 6.9 (optimal pH of pancreatic); (6) 37 ℃ of water-bath vibration digestion 16h; (7) the centrifugal 10min of 4000rpm abandons supernatant (non-resistance starch solution), and deposition is with the ethanolic solution washing of 5mL 50%, and is centrifugal, repeats 2 times; (8) carefully take out centrifuge tube, be inverted centrifuge tube under the room temperature remove non-resistance starch component clean as far as possible; (9) in each centrifuge tube, add 2mL 2M KOH solution, the 30min that vibrates under the room temperature makes resistant starch fully dissolve (note not on impeller mixing to prevent starch milkization); (10) in each centrifuge tube, add the 8mL sodium-acetate buffer (1.2M pH=3.8), adds 100 μ L amyloglucosidase enzyme liquid (3300U/mL) again, mixing, 60 ℃ of water-bath 1h, during be inverted 2-3 time, with each centrifuge tube taking-up, be cooled to room temperature; (11) every pipe is got 15 μ L supernatants in the test tube of correspondence, adds 1.5mL GOD-PAP reagent respectively, mixing gently, 37 ℃ of water-bath 7min; Other gets two test tubes; One adds 15 μ L sodium-acetate buffer (0.1M; PH=4.5) replace sample to do blank, one adds 15 μ L glucose kit titers (standard glucose concentration 1mg/mL), in two pipes, respectively adds 1.5mL GOD-PAP reagent again; Mixing gently, 37 ℃ of water-bath 7min; (12) colorimetric under 500nm, the content of mensuration glucose; (13) calculate resistance starch content according to formula:
Resistance starch content (%)=A
Sample/ A
Standard glucose* 0.9 * 10.3/0.1 * 100/W
A in the formula
SampleAnd A
Standard glucoseBe respectively sample and the standard glucose absorbance under 500 nm; 0.9 be the conversion coefficient of starch and glucose; 10.3 be the supernatant cumulative volume; W is the sample dry weight.
Beneficial effect of the present invention is following:
(1) the inventive method is very little to starch physicochemical property variation under the natural conditions; The situation that high resistant starch content, rich health care wheat germplasm lack very much; Adopt mutagenesis or additive method to handle the variation that seed promotes the starch physicochemical property; The wheat of high, the rich health care of seed selection resistance starch content therefrom, efficient is higher.
(2) workable, the simple and effective of this inventive method.This selection carries out primary election, amylose content through the starch gelatinization characteristic and further screens, measures the combinations that resistance starch content is confirmed three screenings of resistance starch content step; Saved a large amount of workloads, the efficient and the accuracy of seed selection is provided greatly.Amylose content and ratio and starch gelatinization characteristic are closely related, eliminate wide variety of materials through the screening of starch gelatinization characteristic, thereby have significantly reduced the workload of back; And then provide possible for the accurate detection of amylose and resistance starch content; While starch gelatinization characteristic measurement can be selected in generation morning with appearance amount few (3.2 g flour), and is simple to operate; Favorable reproducibility can improve detection efficiency, improve accuracy.Detect through amylose content in addition and can assess resistant starch indirectly, less with the appearance amount, and can eliminate some non-target materials again, further dwindle follow-up workload and improve the accuracy of selecting.
In a word, selection of the present invention at first is an initiative high-resistance starch material; Selected through starch gelatinization characteristic primary dcreening operation, amylose content then, resistance starch content is confirmed the screening of three steps, is progressively forced into system of selection; Simplify trace routine, improved selection accuracy and operating efficiency, obtained good effect in practice; Select accuracy and efficient higher, practical.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
1, kind is selected: selecting kind is Jimai 22, and this kind is the first wheat breed of China by Crop Inst. of shandong Prov. Agriculture science Academy's seed selection at present; Year, cultivated area surpassed 2,200 ten thousand mu, and the super high-yielding stable yields resists wide right more; Through measuring its gelatinization peak viscosity 1872cP, keep viscosity 1318cP, final viscosity 2438cP; The value of bringing back to life 1121cP, amylose content 17.36%, resistance starch content 1.65%.
2, seeds mutagenesis: adopt mutagen EMS (ethylmethane sulfonate) to handle seed, mutagenesis intensity (through mutagenesis dosage and processing time adjustment) is advisable with lethal dose 30%-50%.Concrete grammar is: select about 10000 of big, the full seed of grain, make seed soaked into (1000 mL H fully by water with vacuum pumping method
2O, 4 min), 18h is soaked in vibration in 1.2% EMS then, then with a large amount of water flushing 4h.
3, material breeding: the seed field planting of handling, results obtain mutagenesis M1 for seed, and individual plant is chosen M3 generation, obtains M3 for mutagenesis colony.By strain system results, abrasive dust after 2 months is placed in threshing separately to the M3 of mutagenesis for colony, and the flour slaking is used for the starch physicochemical property and measures after 1 week.Compare with full and of the same size kind seed of mutagenic treatment not.
4, starch gelatinization characteristic primary election: from Jimai 22 mutagenesis colonies, select the strain system that growth is consistent, economical character is good, output is high to carry out gelatinization characteristic measurement, result such as table 2.According to peak viscosity, keep 19 strains of selecting and remain of viscosity, final viscosity and the value of bringing back to life to be, selective ratio 12.3%, going into roguing is that the starch gelatinization statistics of features is seen table 2.Mortality 87.7% has reduced amylose, resistant starch selection workload significantly.
The variation of Jimai 22 starch gelatinization characteristics and screening situation thereof after the table 2 EMS mutagenesis
5, amylose content screening: measured the amylose content of 19 parts of materials, minimum 19.14%, the highest by 25.62%, average 20.79%.Amylose content is 17.36% before Jimai 22 mutagenesis, and wherein 8 parts of materials (account for selected material 42.1%) are than improving before the mutagenesis more than 30%, and amylose content is minimum 22.68%, is up to 25.62%, average 24.21%.
6, resistance starch content is measured: the resistance starch content to 8 parts of materials is measured, and resistance starch content is significantly increased before than mutagenesis as a result, and wherein 3 parts of materials improve and surpass 40%, and its each item index is seen table 3.
It is to compare before each item index and Jimai 22 mutagenesis that table 3 is gone into roguing
Should be noted that concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (3)
1. wheat selection that improves the seed resistance starch content; Its step is following: at first carry out kind and select, the variation that then the seed treatment generation is needed is after breeding is stable; Carry out primary election through the starch gelatinization characteristic again; Further screen through measuring amylose content then, confirm the material that resistance starch content is high through direct mensuration resistance starch content at last, thereby obtain the wheat breed of high seed resistance starch content; Said method for treating seeds be gene engineering or with one of method of high resistant starch content germplasm hybridization; The primary election of said starch gelatinization characteristic is the material of decline more than 50% before the main viscosity parameter of screening is handled, and said viscosity parameter comprises peak viscosity, keeps viscosity, final viscosity, the value of bringing back to life; Said amylose content screening is to improve the material more than 30% before the screening amylose content is handled; It is the material of resistance starch content raising more than 40% of selecting and remain that said resistance starch content is measured.
2. wheat selection according to claim 1; It is characterized in that, said kind select be select to produce go up that establishing in large scale, stable high yield, comprehensive resistance are good, wheat breed that wide adaptability, seed amylose content are higher relatively or have the stable strain of large tracts of land promotion potential.
3. wheat selection according to claim 1, its concrete steps are following:
(1) kind is selected: select to produce go up that establishing in large scale, stable high yield, comprehensive resistance are good, wheat breed that wide adaptability, seed amylose content are higher relatively or have the stable strain of large tracts of land promotion potential;
(2) seed treatment: select big, the full seed of grain and hybridize through gene engineering or with the high resistant starch content germplasm;
(3) material breeding: the seed of handling carries out field planting, divides the individual plant results, breeding, in the heredity relatively stable after, pressing strain is to gather in the crops, and carries out subsequent measurements;
(4) starch gelatinization characteristic primary election: select the strain system that economical character is good, growth is consistent, output is high to measure the starch gelatinization characteristic; Screen the material that descends before main viscosity parameter is handled more than 50%; Described viscosity parameter is peak viscosity, keeps viscosity, final viscosity, the value of bringing back to life, and the material that satisfies condition carries out subsequent measurements;
(5) amylose content screening: the material that screening obtains to step 4 is measured, and the amylose content of selecting and remain improves the material more than 30% before handling, and carries out subsequent measurements then;
(6) resistance starch content is measured: the material that screening obtains to step 5 is measured, and the resistance starch content of selecting and remain improves the material more than 40%, thereby obtains the wheat breed of high seed resistance starch content.
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| EP2764106B1 (en) * | 2011-10-04 | 2019-02-13 | Arcadia Biosciences Inc. | Wheat with increased resistant starch levels |
| CN106198544B (en) * | 2016-07-11 | 2019-01-04 | 浙江大学 | A kind of method for rapidly judging of high dietary-fiber rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1875105A (en) * | 2003-06-30 | 2006-12-06 | 联邦科技产业研究组织 | Wheat with altered branching enzyme activity and starch and starch containing products derived therefrom |
| CN101181060A (en) * | 2007-12-03 | 2008-05-21 | 浙江大学 | Wheat with health care function and cultivation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1875105A (en) * | 2003-06-30 | 2006-12-06 | 联邦科技产业研究组织 | Wheat with altered branching enzyme activity and starch and starch containing products derived therefrom |
| CN101181060A (en) * | 2007-12-03 | 2008-05-21 | 浙江大学 | Wheat with health care function and cultivation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 薛芳等.EMS对新春11小麦抗性淀粉和农艺性状的诱变效果.《麦类作物学报》.2010,第30卷(第3期),第431－434页. * |
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