CN108157167A - Improve the method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content - Google Patents
Improve the method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content Download PDFInfo
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- CN108157167A CN108157167A CN201810140922.0A CN201810140922A CN108157167A CN 108157167 A CN108157167 A CN 108157167A CN 201810140922 A CN201810140922 A CN 201810140922A CN 108157167 A CN108157167 A CN 108157167A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses the methods for improving oil tree peony ' Feng Dan ' seed alpha linolenic acid content.The method provided by the present invention for improving oil and using tree peony ' Feng Dan ' seed alpha linolenic acid content, includes the following steps：(1) pollen collection；(2) it pollinates；(3) seed collection：Acquire the fruit of ' Feng Dan ' in the step (2) after pollination, count setting percentage, screening setting percentage >=25.0/piece combination and collect its seed, measure the alpha linolenic acid content of the seed；(4) high quality combination screens：Using alpha linolenic acid content in the step (3) as index, best pollinated variety of the Varieties of Peony as maternal ' Feng Dan ' of alpha linolenic acid content >=160mg/g of seed is screened；(5) application of pollinated variety：Obtain the oil seed of alpha linolenic acid content raising.Method using the present invention can improve ' Feng Dan ' seed alpha linolenic acid content, so as to improve the quality of ' Feng Dan ' seed.
Description
Technical field
The present invention relates to vegetable seeds production fields, more particularly to improve oil tree peony ' Feng Dan ' seed alpha-linolenic acid content
Method.
Background technology
Different according to the degree of saturation of carbon-carbon bond, aliphatic acid can be divided into saturated fatty acid and unrighted acid.Saturated fat
The abundant high fat diet of fat acid may result in angiocardiopathy, and unrighted acid is aliphatic acid needed by human.Insatiable hunger
Two kinds of monounsaturated fatty acids and polyunsaturated fatty acid can be divided into again with aliphatic acid.Polyunsaturated fatty acid has reduction blood fat
Level, prevention of cardiovascular disease, the effect for reducing serum LDL cholesterol level.According to the position of double bond and function
Polyunsaturated fatty acid is divided into ω -3 series and ω -6 series again.Alpha-linolenic acid belongs to the serial polyunsaturated fatty acids of ω -3,
It is the necessary vital activity factor eicosapentaenoic acid of body (eicosapentaenoic acid, EPA) and two dodecahexaenes
The synthesis precursor of sour (docosahexaenoic acid, DHA), human body itself cannot be synthesized, mainly be absorbed from diet, to people
Body health plays an important role.Research finds that suitable intake alpha-linolenic acid has prevention coronary heart disease, myocardial infarction, reduces and lacks
The risk of blood heart disease and cardiac sudden death, reduce serum cholesterol, increase autoimmunity, remind brain tonic, enhancing attention and
Memory and other effects.
Tree peony is subordinate to Paeoniaceae (Paeoniaceae) Paeonia (Paeonia) Section Moutan (section Moutan), is collection
It is ornamental, medicinal and oily with important woody economic plants.Tree peony is traditional famous flower of China, and root, that is, root bark of tree peony of tree peony is
Chinese tradition Chinese medicine material, unsaturated fatty acid content is up to 90% in seed seed oil, is especially enriched in being known as that " plant brain is yellow
Gold " alpha-linolenic acid, account for more than 40%, be referred to as the most potential woody oleiferous plants crop in China, moreover, health ministry in
Approval peony seed oil becomes new resource food within 2011.Compared with other oil crops, oil has that Bioclimatic analysis is wide, knot with tree peony
The features such as seed amount is big, oil content is high and quality is good, oil with the main breed of tree peony is ' Feng Dan ' (P.ostii) at present, Hebei,
The provinces and cities such as Henan, Shandong, Anhui, Shaanxi, Sichuan are widelyd popularize, and popularizing area is more than 1,000,000 mu.With the people
Improvement of living standard and the increase of Consciousness of food security, edible oil consume the trend for showing high-endization, and Peony Resource is made
For the edible oil of alpha-linolenic acid supply, future will have extraordinary market prospects, improve in oil tree peony ' Feng Dan ' seed
The content of alpha-linolenic acid is played an important role for promoting peony seed oil quality and oil with tree peony industry development.At present, exist
There is the correlative study that alpha-linolenic acid content is promoted by transgenic method on other oil crops, but Transgene-safty is in world's model
There are still larger disputes in enclosing.
Invention content
In order to make up the deficiency in more than field, oil tree peony ' Feng Dan ' seed alpha-linolenic acid is improved the present invention provides a kind of
The method of content.The technical solution adopted in the present invention is as follows：
The method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content is improved, is included the following steps：
(1) pollen collection：Big flower bud phase acquisition as male parent different Varieties of Peony anther, by the anther dry to
Crack loose powder, pollen is collected, as parent sire of hybrid pigs；
(2) it pollinates：Its stamen is removed before the petal expansion of Varieties of Peony ' Feng Dan ', bagging treats that secretion stigma is glued
During liquid, the pollen as parent sire of hybrid pigs screened in the step (1) is invested on ' Feng Dan ' column cap, bagging, complete pollination；
(3) seed collection：The fruit of ' Feng Dan ' in the step (2) after pollination is acquired, counts setting percentage, screening is solid
Rate >=25.0/piece combination and collect obtained seed after its pollination, measure the alpha-linolenic acid content of the seed；
(4) high quality combination screens：Using alpha-linolenic acid content in the step (3) as index, the alpha-linolenic acid of seed is screened
Best pollinated variety of the Varieties of Peony of content >=160mg/g as maternal ' Feng Dan '；
(5) application of pollinated variety：The best pollinated variety that the step (4) screening is obtained is answered as pollinizers
With pollinating to ' Feng Dan ' kind, obtained seed is to get the oil use improved to alpha-linolenic acid content after acquisition pollination
Seed.
When the time acquired described in the step (1) is morning 8 bloomed a few days ago~10 when.
The drying is natural air drying.
Varieties of Peony ' the Feng Dan ' of the step (2) is 6 years~10 years that growth and development is normal, branch thickness is consistent raw
' Feng Dan ' plant.
When the time pollinated described in the step (2) is the fine day calm morning 8~10 when.
The time of seed collection described in the step (3) is 90 days~105 days after pollination.
In yellow, seed becomes taking fruit when dark brown fruit in the step (3), harvests seed.
The setting percentage of the step (3)=solid number (grain)/pollination flower number (piece).
The advantages and positive effects of the present invention are：
(1) method using the present invention can improve ' Feng Dan ' seed alpha-linolenic acid content, so as to improve ' Feng Dan ' seed
Quality.
(2) present invention improves alpha-linolenic acid content in seed using the method for pollination, is safe method.
(3) plantation of kind pollinizers of the present invention can solve the phenomenon that ' Feng Dan ' plantation watches pattern, flower pattern is single, carry
The appreciation effect that high oil is planted with tree peony.
(4) kind used in present invention pollination male parent is cultivar common in ornamental peonies production, and for environment
Requirement and cultivation management measure and ' Feng Dan ' unanimously, application is very convenient.
(5) present invention can routinely pollinizers configuration method be configured, in the cultivation production of ' Feng Dan ' convenient for plantation
Person grasps, easy to spread.
Specific embodiment
Embodiment 1 improves the method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content
Material：There is Varieties of Peony used in the present embodiment：' Feng Dan ', ' spilling gold in Black Sea ', ' brocade is red ', ' Red River ', ' lotus bird with red feathers
It is emerald green ', ' as pretty as a flower ', ' the fragrant brocade in day ', ' red on the sunny side ', ' pure as jade ', Varieties of Peony used in the present embodiment is purchased from Shandong Province Heze
City Caozhou garden with hundreds of flowers.
The raising oil method of tree peony ' Feng Dan ' seed alpha-linolenic acid content of the present embodiment, specifically comprises the following steps：
1. pollen collection：Varieties of Peony is acquired respectively when morning 8~10 of big flower bud phase (blooming first 2 days) ' to spill Black Sea
Gold ', ' brocade is red ', ' Red River ', lotus emerald ', ' as pretty as a flower ', ' the fragrant brocade in day ', ' red on the sunny side ', ' pure as jade ' bud, indoors
Sepal, petal are removed, at room temperature natural air drying to anther dehiscence loose powder, pollen is collected, as parent sire of hybrid pigs.
2. pollination：6~10 years 80 plants raw of ' Feng Dan ' plant that growth selection development is normal, branch thickness is basically identical,
8 basically identical, flower is developed in selection on each plant, is removed stamen with tweezers before petal expansion, with sulfuric acid paper bag
Bagging, during the calm morning 8~10 of fine day after 2~3 days, during secretion stigma mucus, by the pollen for the male parent collected in step 1
It invests respectively on ' Feng Dan ' column cap, each male parent corresponds to 10 ' Feng Dan ' plant (setting 10 repetitions), and bagging marks on bag
Parent's title and pollination date.It is once pollinated again within second day.
3. seed collection：90~105 days after pollination, in yellow, seed becomes ' Feng Dan ' fruit after pollinating in step 2
Fruit is taken when dark brown, harvests seed, counts setting percentage, setting percentage=solid number (grain)/pollination flower number (piece), system
The average value of setting percentage after meter pollination is as the corresponding setting percentage of each male parent.The results are shown in Table 1.
The different pollinated variety setting percentage statistics of table 1
The result shows that：During male parent varieties difference of pollinating, setting percentage is different, wherein when ' pure as jade ' is as male parent, setting percentage is most
Height, ' lotus emerald ' as male parent when, setting percentage is minimum, screening setting percentage >=25.0/piece combination, i.e.,：It ' spills Black Sea
Gold ', ' as pretty as a flower ', ' red on the sunny side ', ' pure as jade ', ' day fragrant brocade ' combine as male parent, ' Feng Dan ' as maternal 5.By this 5
Natural air drying to carpel cracking seed sheds the fruit of a combination at room temperature, seed drying at room temperature is collected to constant weight, for insatiable hunger
With aliphatic acid different component assay.
4. unrighted acid different component assay：Utilize gas chromatography-mass spectrum (gas chromatography-
Mass spectrometry, GC-MS) unrighted acid difference is carried out to the seed of 5 combinations screened in step 3
Constituent content measures, including measuring palmitic acid, stearic acid, oleic acid, linoleic acid and alpha-linolenic acid and unsaturated fatty acid content.It surveys
It is as follows to determine method：
The extraction of total fat：
1) by after dry seed decladding in liquid nitrogen rapid grind into powder, weigh about 0.1g move to 10mL tool plug glass
In test tube, each 3 repetitions.
2) 3.0mL chloroforms are added in into test tube immediately：Methanol (1：2, v:V) mixed solution, it is mixed full of being vibrated after nitrogen
It is even, it is placed in 4 DEG C overnight.
3) 1.0mL chloroforms, fully shaking mixing are added into test tube.The 1M KCl solution of 1.8mL is added, makes finally to carry
Take chloroform in liquid：Methanol：The volume ratio of KCl is 1：1：0.9.
4) after vibrating mixing again, 2500rpm centrifugations 10min.Draw lower floor chloroform phase 1.0mL.It is concentrated, carried out with nitrogen
Esterification.
The esterification of grease：
1) 1.0mL methanolic solutions (+95% methanol of 5% sulfuric acid) are added in test tube, full of being sealed after nitrogen.90 DEG C of water
Bathe 1h.
2) after being placed in cooling at room temperature, 1.5mL pentanes and 1.0mL water are added in test tube, vibrates mixing, terminate methyl
Change reaction.
3) 2500rpm after vibrating centrifuges 10min, collects supernatant 1.0mL, and add in 5.0mL after nitrogen concentration steams just again
Hexane.
4) 980 μ L extracting solution samples are taken, 20 μ L internal standards (Heptadecanoic acide) rapid mixings are added in, with 0.22um organic phase micropores
Filter is filled into sample bottle, is analyzed for GC-MS.
The preparation of standard items：
Standard items include 5 main component palmitic acids, stearic acid, oleic acid, linoleic acid and alpha-linolenic acid in Paeonia suffruticosa seed oil
((being purchased from Sigma-Aldrich (Shanghai, China)), with the Heptadecanoic acide being not present in Paeonia suffruticosa seed oil
(being purchased from Sigma-Aldrich (Shanghai, China)) is as internal standard.It is dense that standard items are made into a series of differences
The standard solution of degree, concentration gradient include 0.015625mg/mL, 0.03125mg/mL, 0.0625mg/mL L, 0.125mg/mL,
0.25mg/mL, 0.5mg/mL and 1.0mg/mL take the standard solution of 980 μ L various concentrations, are separately added into 20 μ L internal standard compounds,
It is sealed in sample bottle, -20 DEG C store for future use.
The GC-MS analyses of fatty acid methyl ester：
Testing standard product are Agilent GC-MS with the analysis system used in analysis sample, and chromatographic column is the poly- silica of HP-88
Alkane polymer chromatography column, 30m × 0.25mm, 0.20 μm, (Agilent, the U.S.).Temperature program used is as follows：100 DEG C of holdings
Then 2min rises to 230 DEG C with 15 DEG C/min, keep 5min, 250 DEG C of injector temperature, split sampling, split ratio 10:1,
1 μ L of sampling volume, carrier gas are high-purity helium, flow velocity 1.0mL/min；Ionization mode be EI, electron energy 70eV, transmission line temperature
280 DEG C, 230 DEG C of ion source temperature, 150 DEG C of level four bars temperature, mass scan range (m/z)：30~450u is used
It NIST05Library standards library searching and is compared with standard items qualitative.
The quantitative analysis of aliphatic acid：
Linear regression is carried out using Internal standard curve method to quantify.Test target series of standards in adding in respectively first
Product, using standard concentration as abscissa (x), corresponding standard items are ordinate (y) with internal standard compound peak area ratio, are carried out linear
It returns, makes standard curve.Then the ratio between peak area of sample to be tested and internal standard compound is measured under identical chromatographic condition, thus
Ratio can find the concentration of component to be measured in sample on standard curve, and then can calculate component to be measured containing in the sample
Amount.
As a result：
Unrighted acid different component assay is as shown in table 2, pollinate male parent varieties difference when, seed unsaturated lipid
Fat acid different component content has differences, and between 212.32~290.02mg/g, alpha-linolenic acid contains unsaturated fatty acid content
Amount between 119.80~165.85mg/g, linoleic acid content between 32.88~51.39mg/g, oleic acid content 45.03~
Between 56.55mg/g, stearic acid content is between 3.19~5.33mg/g, and palmitic acid content is between 11.31~15.37mg/g
(table 2).Wherein, when ' pure as jade ' is as male parent, unrighted acid and alpha-linolenic acid content highest in seed.' fragrant brocade ' the Central Asia in day
Oleic acid content highest, oleic acid, stearic acid, the equal highest of palmitic acid content in ' red on the sunny side '.
The seed alpha-linolenic acid and other constituent contents (mg/g DW) of the different male parent varieties pollinations of table 2
5. high quality combination screens：Using alpha-linolenic acid content in step 4 as index, screening alpha-linolenic acid content >=160mg/g
Varieties of Peony as best pollinated variety, i.e.,：' red on the sunny side ' and ' pure as jade '.
6. pollinizers are applied：The kind filtered out in step 5 ' red on the sunny side ' and ' pure as jade ' are applied as pollinizers,
The pollen of male parent is invested respectively on ' Feng Dan ' column cap, and pollinating method with above-mentioned steps 2, use by the oil for obtaining the raising of alpha-linolenic acid content
Seed.
The better embodiment of the present invention is illustrated, but the invention is not limited to the implementation above
Example, those skilled in the art can also make various equivalent modifications under the premise of without prejudice to spirit of the invention or replace
It changes, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (8)
1. improving the method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content, include the following steps：
(1) pollen collection：In anther of the big flower bud phase acquisition as the different Varieties of Peony of male parent, the anther is dried to cracking
Loose powder collects pollen, as parent sire of hybrid pigs；
(2) it pollinates：Its stamen is removed before the petal expansion of Varieties of Peony ' Feng Dan ', bagging, when secretion stigma mucus,
The pollen as parent sire of hybrid pigs screened in the step (1) is invested on ' Feng Dan ' column cap, bagging, complete pollination；
(3) seed collection：The fruit of ' Feng Dan ' in the step (2) after pollination is acquired, counts setting percentage, screening setting percentage >=
The combination of 25.0/piece simultaneously collects its seed, measures the alpha-linolenic acid content of the seed；
(4) high quality combination screens：Using alpha-linolenic acid content in the step (3) as index, the alpha-linolenic acid content of seed is screened
Best pollinated variety of the Varieties of Peony of >=160mg/g as maternal ' Feng Dan '；
(5) application of pollinated variety：The best pollinated variety that the step (4) screening is obtained is applied as pollinizers,
It pollinates to ' Feng Dan ' kind, the seed obtained after acquisition pollination is to get the oil kind improved to alpha-linolenic acid content
Son.
2. the method according to claim 1 for improving oil and using tree peony ' Feng Dan ' seed alpha-linolenic acid content, it is characterised in that：
When the time acquired described in the step (1) is morning 8 bloomed a few days ago~10 when.
3. the method according to claim 1 or 2 for improving oil and using tree peony ' Feng Dan ' seed alpha-linolenic acid content, feature exist
In：The drying is natural air drying.
4. the method according to claim 1 for improving oil and using tree peony ' Feng Dan ' seed alpha-linolenic acid content, it is characterised in that：
Varieties of Peony ' the Feng Dan ' of the step (2) is that 6 years~10 years raw ' Feng Dan ' that growth and development is normal, branch thickness is consistent plant
Strain.
5. the method for raising oil tree peony ' Feng Dan ' the seed alpha-linolenic acid content according to claim 1 or 4, feature exist
In：When the time pollinated described in the step (2) is the fine day calm morning 8~10 when.
6. the method according to claim 1 for improving oil and using tree peony ' Feng Dan ' seed alpha-linolenic acid content, it is characterised in that：
The time of seed collection described in the step (3) is 90 days~105 days after pollination.
7. the method for raising oil tree peony ' Feng Dan ' the seed alpha-linolenic acid content according to claim 1 or 6, feature exist
In：In yellow, seed becomes taking fruit when dark brown fruit in the step (3), harvests seed.
8. the method according to claim 1 for improving oil and using tree peony ' Feng Dan ' seed alpha-linolenic acid content, it is characterised in that：
The setting percentage of the step (3)=solid number/pollination flower number.
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| CN201810140922.0A CN108157167A (en) | 2018-02-11 | 2018-02-11 | Improve the method that oil uses tree peony ' Feng Dan ' seed alpha-linolenic acid content |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110646525A (en) * | 2019-09-11 | 2020-01-03 | 上海市松江一中 | Method for detecting content of fatty acid in growth and development process of paeonia ostii seed coats |
| CN112795591A (en) * | 2019-11-14 | 2021-05-14 | 青岛农业大学 | VIGS silencing system for identifying peony pollination fertilization gene |
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| CN101869051A (en) * | 2010-07-22 | 2010-10-27 | 甘肃省林业科学技术推广总站 | Hybridization breeding pollination method for peony |
| CN105248270A (en) * | 2015-09-01 | 2016-01-20 | 山东盛华农业发展有限公司 | Breeding method of new variety of peony with ornamental value and oil-bearing value |
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2018
- 2018-02-11 CN CN201810140922.0A patent/CN108157167A/en active Pending
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| CN101869051A (en) * | 2010-07-22 | 2010-10-27 | 甘肃省林业科学技术推广总站 | Hybridization breeding pollination method for peony |
| CN105248270A (en) * | 2015-09-01 | 2016-01-20 | 山东盛华农业发展有限公司 | Breeding method of new variety of peony with ornamental value and oil-bearing value |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110646525A (en) * | 2019-09-11 | 2020-01-03 | 上海市松江一中 | Method for detecting content of fatty acid in growth and development process of paeonia ostii seed coats |
| CN112795591A (en) * | 2019-11-14 | 2021-05-14 | 青岛农业大学 | VIGS silencing system for identifying peony pollination fertilization gene |
| CN112795591B (en) * | 2019-11-14 | 2023-02-17 | 青岛农业大学 | VIGS silencing system for identifying peony pollination fertilization gene |
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