AU2021101139A4 - Method for Improving α-Linolenic Acid Content in Seeds of Oil Peony Paeonia ostii 'Feng Dan' - Google Patents

Abstract

The present invention discloses a method for improving a-linolenic acid content in seeds of oil peony Paeonia ostii'Feng Dan'. The method comprises the following steps: (1) collecting pollen; (2) pollinating; (3) collecting seeds: collecting fruits of pollinated 'Feng Dan' in the step (2), calculating the seed setting rate, screening combinations with the seed setting rate 25.0 seeds/flower, collecting seeds, and determining the a-linolenic acid content of the seeds; (4) screening high-quality combinations: with the a-linolenic acid content in the step (3) as an index, screening tree peony cultivars with seeds with the a-linolenic acid content 160 mg/g as optimal pollination cultivars of female parent 'Feng Dan'; and (5) applying the pollination cultivars: obtaining oil seeds with improved a-linolenic acid content. The method of the present invention can help improve the a-linolenic acid content of 'Feng Dan' seeds, thereby improving the quality of the 'Feng Dan' seeds.

Description

Method for Improving a-Linolenic Acid Content in Seeds of Oil Peony

Paeonia osti'Feng Dan'

TECHNICAL FIELD

The present invention relates to the field of plant seed production, in

particular to a method for improving a-linolenic acid content in seeds of oil

peony P ostii'Feng Dan'.

BACKGROUND

Fatty acids can be classified into saturated fatty acids and unsaturated

fatty acids based on degrees of saturation of carbon-carbon bonds. A high-fat

diet rich in saturated fatty acids may lead to cardiovascular diseases, while

unsaturated fatty acids are essential fatty acids for human beings. Unsaturated

fatty acids can be further classified into monounsaturated fatty acids and

polyunsaturated fatty acids. Polyunsaturated fatty acids can lower blood lipid

level, prevent cardiovascular diseases and lower serum LDL cholesterol level.

Polyunsaturated fatty acids are additionally classified into w-3 series and w-6

series based on the position and function of double bonds. a-linolenic acid is a

w-3 polyunsaturated fatty acid that cannot be synthesized by humans. As a

synthetic precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid

(DHA) which are essential bioactive factors for organisms, a-linolenic acid is

mainly taken from diet, and plays an important role in human health. Studies

have shown that proper intake of a-linolenic acid can prevent coronary heart

disease, myocardial infarction, reduce the risk of ischemic heart disease and

sudden cardiac death, lower serum cholesterol, enhance autoimmunity, refresh brain, and enhance attention and memory.

Tree peony belongs to the section Moutan of the genus Paeonia in the

family Paeoniaceae. It is an important woody economic plant with ornamental,

medicinal and oil uses. Tree peony is a traditional famous flower in China, and

root barks of tree peonies are used as a traditional Chinese medicine. Its seed

oil contains up to 90% unsaturated fatty acids, especially rich in a-linolenic acid

that is known as "plant DHA", accounting for more than 40%. It is known as the

most promising woody oil crop in China, and the National Health Commission

of the People's Republic of China approved peony seed oil as a new resource

food in 2011. Compared with other oil crops, oil peony has the characteristics

of wide adaptability, large seed production, high oil content and good quality. At

present, the main cultivar of oil peony is P ostii 'Feng Dan', which has been

widely promoted in Hebei, Henan, Shandong, Anhui, Shaanxi, Sichuan and

other provinces and cities, with a planting area over 666666666.67m 2. With the

improvement of people's living standard and the increase of food safety

awareness, the consumption of edible oil presents a high-end trend. Peony

resources, as edible oil supplying a-linolenic acid, will have a very good market

prospect in the future. Therefore, increasing the a-linolenic acid content in

seeds of oil peony P ostii'Feng Dan' plays an important role in improving the

quality of peony seed oil and the development of oil peony industry. Currently,

there are studies on improving the a-linolenic acid content by transgenosis of

other oil crops, but the safety of transgenosis is still controversial in the world.

SUMMARY

To make up for deficiencies in the field, the present invention provides a

method for improving a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan'. The technical solution adopted by the present invention is as

follows:

A method for improving a-linolenic acid content in seeds of oil peony P

ostii'Feng Dan', comprising the following steps:

(1) collecting pollen: collecting anthers of different peony cultivars as male

parents in the full squaring stage, drying the anthers until anther dehiscence

with pollen exposure, and collecting pollen as male parents;

(2) pollinating: removing stamens of peony cultivar 'Feng Dan' before the

petals are unfolded, bagging, pollinating stigmas of 'Feng Dan' with the pollen

screened in the step (1) as male parents when the stigmas secrete mucus,

bagging and finishing pollination;

(3) collecting seeds: collecting fruits of pollinated 'Feng Dan' in the step

(2), calculating the seed setting rate, screening combinations with the seed

setting rate 25.0 seeds/flower, collecting seeds obtained after pollination, and

determining the a-linolenic acid content of the seeds;

(4) screening high-quality combinations: with the a-linolenic acid content

in the step (3) as an index, screening peony cultivars with seeds with the

a-linolenic acid content 160 mg/g as optimal pollination cultivars of female

parent 'Feng Dan';

(5) applying the pollination cultivars: pollinating the cultivar of 'Feng Dan'

with the optimal pollination cultivars screened in the step (4) as pollinizers, and

collecting seeds obtained after pollination to obtain oil seeds with improved

a-linolenic acid content.

The time of collection in the step (1) is at 8:00-10:00 a.m. two days before

flowering.

The drying is natural air-drying.

The peony cultivar 'Feng Dan' in the step (2) is 6 to 10-year-old 'Feng

Dan' plants with normal growth and uniform branches.

The time of pollination in the step (2) is at 8:00-10:00 a.m. on a sunny day

without wind.

The time of collecting seeds in the step (3) is 90-105 days after pollination.

The fruits in the step (3) are yellow, and the fruits are picked to harvest the

seeds when the seeds become dark brown.

The seed setting rate in the step (3) = number of fruits/number of

pollinated flowers.

The advantages and positive effects of the present invention are as

follows:

(1) The method of the present invention can help improve the a-linolenic

acid content in seeds of 'Feng Dan', thereby improving the quality of the 'Feng

Dan'seeds.

(2) It is a safe method to improve the a-linolenic acid content of seeds by

pollination.

(3) The planting of pollinizers of the cultivars of the present invention can

solve the problems of single color and single type of ornamental flowers of

'Feng Dan', and improve the ornamental effect of oil peony.

(4) The cultivars used as male parents in the present invention are

common cultivars in the production of ornamental peony, and the

environmental requirements and cultivation management measures are

consistent with those of 'Feng Dan', so it is very convenient to apply.

(5) In the cultivation and production of 'Feng Dan', the present invention can be configured by a conventional configuration method for pollinizers for easy management by growers and easy popularization.

DESCRIPTION OF THE INVENTION

Example 1. Method for improving a-linolenic acid content in seeds of oil

peony P ostii'Feng Dan'.

Materials: Peony cultivars used in the example are: 'Feng Dan', tree

peony'Hei Hai Sa Jin', 'Jin Hong', 'Hong He', 'Fei Cui He Hua','Ru Hua Si Yu',

'Tian Xiang Jin', 'Xiang Yang Hong'and 'Yu Jie'. All the peony cultivars used in

the example were purchased from Caozhou Garden in Heze of Shandong

Province.

The method for improving a-linolenic acid content in the seeds of oil peony

P ostii'Feng Dan' in the example specifically comprises the following steps:

1. Collecting pollen: collecting flower buds of tree peony cultivars 'Hei Hai

Sa Jin', 'Jin Hong', 'Hong He', 'Fei Cui He Hua', 'Ru Hua Si Yu', 'Tian Xiang Jin',

'Xiang Yang Hong' and 'Yu Jie' at 8:00-10:00 a.m. in the full squaring stage

(two days before flowering), removing sepals and petals indoors, air-drying

naturally until anther dehiscence with pollen exposure, and collecting pollen as

male parents.

2. Pollinating: selecting eighty 6 to 10-year-old 'Feng Dan' plants with

normal growth and basically uniform branches, selecting 8 flowers with

basically the same development from each plant, removing stamens with

tweezers before the petals are unfolded, bagging with sulfuric acid paper bags,

pollinating stigmas of 'Feng Dan'with the pollen from male parents collected in

the step 1 when the stigmas secreted mucus at 8-10 a.m. on a sunny day 2-3

days later, with each male parent corresponding to 10 'Feng Dan' plants (i.e., replicates), bagging, labeling the bags with parent name and pollination date, and pollinating again the next day.

3. Collecting seeds: picking fruits to harvest seeds when the fruits of 'Feng

Dan' pollinated in the step 2 were yellow and the seeds became dark brown

-105 days after pollination, calculating the seed setting rate (seed setting

rate=number of fruits/number of pollinated flowers), and calculating the

average seed setting rate after pollination as the seed setting rate

corresponding to each male parent. The results are shown in Table 1.

Table 1 Statistics of seed setting rate of different pollination cultivars

Cultivar Seed setting rate (seeds/flower) 'Hei Hai Sa Jin' 27.8 'Jin Hong' 21.2 'Hong He' 20.8 'Fei Cui He Hua' 19.9 'Ru Hua Si Yu' 29.5 'Tian Xiang Jin' 32.3 'Xiang Yang Hong' 28.7 'Yu Jie' 38.1

The results showed that the seed setting rate was different with different

male parents, among which the cultivar 'Yu Jie' had the highest seed setting

rate, while tree peony 'Fei Cui He Hua' had the lowest seed setting rate. Five

combinations with seed setting rate 25.0 seeds/flower were screened, namely,

'Hei Hai Sa Jin', 'Ru Hua Si Yu', 'Xiang Yang Hong', 'Yu Jie'and 'Tian Xiang

Jin', as male parents, and 'Feng Dan' as female parent. The fruits of the five

combinations were naturally air-dried at room temperature until carpel

dehiscence with pollen exposure, and the seeds were collected and dried to

constant weight at room temperature for determination of the content of different components of unsaturated fatty acids.

4. Determining content of different components of unsaturated fatty acids:

determining the content of different components of unsaturated fatty acids in

the seeds of the five combinations screened in the step 3 by gas

chromatography-mass spectrometry (GC-MS), including the content of palmitic

acid, stearic acid, oleic acid, linoleic acid, a-linolenic acid and unsaturated fatty

acids. The determination method is as follows:

Extraction of total fat:

1) grinding dried seeds quickly into powder in liquid nitrogen after hulling,

then weighing and transferring about 0.1 g of the powder into a 10 mL glass

test tube with stopper in three replicates;

2) adding 3.0 mL of a mixture of chloroform:methanol (1:2, v:v)

immediately to each test tube, filling the test tube with nitrogen, shaking and

mixing well, and keeping the test tube at 4C overnight;

3) adding another 1.0 mL of chloroform to the test tube, shaking

thoroughly and mixing well, and adding 1.8 mL of 1M KCI solution to keep the

volume ratio of chloroform:methanol:KCI in the final extract at 1:1:0.9; and

4) shaking and mixing well again, then centrifuging at 2500 rpm for 10 min,

sucking and concentrating 1.0 mL of subnatant chloroform phase with nitrogen

for methyl esterification.

Methyl esterification of oil;

1) adding 1.0 mL of sulfuric acid methanol solution (5% sulfuric acid +

% methanol) to the test tube, filling the test tube with nitrogen, then sealing

and placing the test tube in a water bath at 900 C for 1 h;

2) after cooling the test tube at room temperature, adding 1.5 mL of n-pentane and 1.0 mL of water to the test tube, shaking and mixing well, and terminating methylation;

3) centrifuging at 2500 rpm for 10 min after shaking, collecting 1.0 mL of

supernatant, concentrating with nitrogen and adding 5.0 mL of redistilled

n-hexane; and

4) to 980 pL of extract sample, adding 20 pL of internal standard

(heptadecanoic acid), mixing quickly to obtain a mixture, and filtering the

mixture into a sample bottle with a 0.22 um organic phase microporous filter for

GC-MS.

Preparation of reference standards:

Reference standards included five main components (palmitic acid,

stearic acid, oleic acid, linoleic acid and a-linolenic acid (purchased from

Sigma-Aldrich (Shanghai, China)) in peony seed oil, and heptadecanoic acid

(purchased from Sigma-Aldrich (Shanghai, China)) which does not exist in

peony seed oil was used as the internal standard. The reference standards

were prepared into a series of standard solutions of different concentrations at

concentration gradients of 0.015625 mg/mL, 0.03125 mg/mL, 0.0625 mg/mL,

0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 1.0 mg/mL, and to 980 pL of the

standard solutions of different concentrations, 20 pL of internal standard was

added separately, then the resulting mixtures were sealed in sample bottles

and stored at -200 C for later use.

GC-MS on fatty acid methyl ester:

The analytical system used for testing reference standards and analyzing

samples was Agilent GC-MS, and the chromatographic column was an HP-88

polysiloxane polymer chromatographic column, 30 mx0.25 mm, 0.20 pm

(Agilent). The heating procedure used was as follows: holding at 1000 C for 2

min, then heating to 2300 C at 15C/min, and holding for 5min; temperature at

the injection port: 250 0 C, split injection, split ratio: 10:1, injection volume: 1pL,

flow rate: 1.0 mL/min, and carrier gas: high purity helium; ionization mode: El,

electron energy: 70 eV, temperature transmission line: 2800 C, ion source

temperature: 230 0C, quadrupole temperature: 150 0C, mass scan range (m/z):

- 450 u, NIST05 Library was used for search and qualitative comparison

with reference standards.

Quantitative analysis of fatty acids:

Linear regression quantification was carried out by internal standard

curves. Firstly, a series of reference standards added with the internal

standard were tested separately to plot standard curves by linear regression,

with the concentration of the reference standards as the x-axis (x), and the

peak area ratio of the corresponding reference standard to the internal

standard as the y-axis (y). Then the peak area ratio of the sample to be tested

to the internal standard was measured under the same chromatographic

conditions, from which the concentration of a component to be tested in the

sample can be found on the standard curve, and the content of the component

to be tested in the sample can be calculated.

Results:

The determined content of different components of unsaturated fatty acids

was shown in Table 2. The content of different components of unsaturated fatty

acids in the seeds varied with different male parent cultivars, with unsaturated

fatty acid content ranging from 212.32 to 290.02 mg/g, a-linolenic acid content

ranging from 119.80 to 165.85 mg/g, linoleic acid content ranging from 32.88 to

51.39 mg/g, oleic acid content ranging from 45.03 to 56.55 mg /g, stearic acid

content ranging from 3.19 to 5.33 mg/g, and palmitic acid content ranging from

11.31 to 15.37 mg/g (Table 2). Among them, the content of unsaturated fatty

acids and a-linolenic acid in seeds was the highest when the cultivar 'Yu Jie'

was the male parent. The content of linoleic acid in tree peony'Tian Xiang Jin'

was the highest, and the content of oleic acid, stearic acid and palmitic acid in

tree peony'Xiang Yang Hong'was the highest.

Table 2 Content of a-linolenic acid and other components in seeds

pollinated with different male parents (mg/g DW)

Variety Palmitic aric OleicacidLnoleic a Unsaturat Vrey Stedaic licai acid -linolenic ed fatty acid acids 'Hei Hai Sa Jin'11.31 3.29 45.03 32.88 119.80 212.32

YRu Hua Si13.92 4.90 55.55 39.79 145.13 259.29 'Tian Xiang Jin'13.20 3.19 49.06 51.39 151.02 267.85 'Xiang Yang 1 5 .3 7 5.33 56.55 45.45 164.16 286.87 Hong' 'Yu Jie' 15.34 4.34 54.56 49.94 165.85 290.02

5. Screening high-quality combinations: with the a-linolenic acid content in

the step 4 as an index, screening peony cultivars with a-linolenic acid content

>160 mg/g as the optimal pollination cultivars, namely tree peony 'Xiang Yang

Hong'and 'Yu Jie'.

6. Applying the pollinizers: with the cultivars tree peony 'Xiang Yang Hong'

and 'Yu Jie' screened in the step 5 as pollinizers, and pollinating stigmas of

'Feng Dan' with the pollen from male parents respectively by the same

pollination method as that in the step 2 to obtain oil seeds with improved

a-linolenic acid content.

Although preferred embodiments of the present invention have been described in detail herein, the present invention is not limited thereto. It will be appreciated by those skilled in the art that various equivalent modifications or substitutions may be made without departing from the spirit of the present invention, and these equivalent modifications or substitutions shall be incorporated in the scope defined by the claims of the application.

Claims (6)

1. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan', comprising the following steps:

(1) collecting anthers of different tree peony cultivars as male parents in

the full squaring stage, drying the anthers;

(2) removing stamens of tree peony cultivar 'Feng Dan' before the petals

are unfolded and bagged;

2. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan' according to claim 1, characterized in that the time of collection in

the step (1) is at 8:00-10:00 a.m. two days before flowering.

3. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan' according to claim 1 or 2, characterized in that the drying is natural

air-drying.

4. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan' according to claim 1, characterized in that the tree peony 'Feng

Dan' in the step (2) is 6 to 10-year-old 'Feng Dan' plants with normal growth

and uniform branches.

5. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan' according to claim 1 or 4, characterized in that the time of

preparation in the step (2) is at 8:00-10:00 a.m. on a sunny day without wind.

6. The observation of a-linolenic acid content in seeds of oil peony P ostii

'Feng Dan' according to claim 1, characterized in that the time of collecting

seeds in the step (3) is 90-105 days after preparation.