CN110839644B

CN110839644B - Method for improving effective and economic yield of ginkgolic acid from ginkgo biloba sarcotesta

Info

Publication number: CN110839644B
Application number: CN201911178290.8A
Authority: CN
Inventors: 王义强; 刘伟; 冯致; 李萌; 杨竣茹; 杨果; 王惠
Original assignee: Central South University of Forestry and Technology
Current assignee: Central South University of Forestry and Technology
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2021-03-02
Anticipated expiration: 2039-11-27
Also published as: CN110839644A

Abstract

The invention provides a method for improving the effective and economic yield of ginkgolic acid in ginkgo episperms. Comprises the technical links of the preparation of exogenous hormone, the determination of spraying time, the selection of spraying combination and the like. The method can obviously improve the proportion of the testa of the ginkgo seed, and the content of ginkgoic acid in the testa is also obviously improved. The method improves the output value of the ginkgo episperm, and provides a high-quality raw material for the production of biogenic pesticides taking ginkgoic acid as a main action substance. The method is simple and convenient to implement, low in cost and good in effect, and can be used as a supplementary means for high-quality and high-yield cultivation of the ginkgo biloba for the testa under the background of difficult ginkgo breeding.

Description

Method for improving effective and economic yield of ginkgolic acid from ginkgo biloba sarcotesta

Technical Field

The invention belongs to the technical field of increasing the economic yield of a single ginkgo plant, and particularly relates to a method for increasing the effective economic yield of ginkgolic acid of ginkgo episperms.

Background

Ginkgo biloba (Ginkgo biloba L.), also known as Ginkgo tree, a single family of unigenus gymnosperm, hermaphrodite and deciduous tree, are world famous wiggery species and precious medicinal tree species, and flavonoids and terpene esters extracted from folium Ginkgo are specific drugs for preventing and treating cardiovascular and cerebrovascular diseases. The ginkgo episperm is the fleshy part outside the seed hard core, also called ginkgo chlamydomonas, and accounts for more than 60 percent of the weight of the ginkgo seeds. The ginkgo biloba exocarp contains a plurality of effective components, mainly comprises compounds such as phenolic acid, flavonoid, terpene lactone, polysaccharide and the like, and the content of ginkgolic acid in the ginkgo biloba exocarp is the highest.

Ginkgolic acid is a secondary metabolite, and comprises 5 kinds of ginkgolic acid (C13:0), hydroginkgolic acid (C15:0), ginkgolic acid (C15:1), heptadecene ginkgolic acid (C17:1), and heptadecadiene ginkgolic acid (C17: 2). Ginkgolic acid, ginkgolic neo-acid and heptadecene ginkgolic acid are the main components with the effects of sterilization and disinsection in the ginkgolic acid, and account for about 90 percent of the total ginkgolic acid, wherein the ginkgolic acid [ 2-hydroxy-6- (8-pentadecenyl) benzoic acid ] is the most main component of the ginkgolic acid in the ginkgo testa and accounts for about 50 percent of the total ginkgolic acid. They are an analogue of anacardic acid isolated from ginkgo biloba.

Anacardic acid, which was originally isolated from cashew trees of Anacardiaceae, is a phenolic lipid substance, and is a generic name of a mixture of 6-alkyl-2-hydroxybenzoic acid, wherein the alkyl chain contains 15 carbons, etc., and contains saturated or unsaturated bonds, and ginkgolic acid is an analogue of anacardic acid, and has the following structural formula [ documents 1-2 ].

According to previous researches using C14 marker, the synthesis of ginkgoic acid takes fatty acid as precursor molecule, and the ginkgoic acid with different side chain saturation degrees is synthesized and cyclized by type III polyketide synthase through modification of fatty acid desaturase. This reaction step is a de novo synthesis using malonyl-CoA and acetyl-CoA as precursors (FIG. 1) [ references 2-3]

Ginkgoic acids are analogs of anacardic acid, their synthesis all derived from the fatty acid pathway, anacardic acid was discovered earlier than ginkgoic acid, which was later classified as anacardic acid.

The ginkgolic acid has good antibacterial and insecticidal effects, and has the characteristics of high efficiency, low toxicity, low residue and the like, and is an ideal biological pesticide for developing organic agriculture and promoting agricultural sustainable development.

In China, more than 90% of the world's gingko resources are owned by China, the large-scale cultivation of gingko is mainly focused on leaf use and kernel use, and the gingko forest used for episperm is very little. China is a large population country and a large agricultural country, chemical pesticides are widely used in agricultural planting, with the continuous improvement of food safety awareness in recent years, agricultural product pesticide residues are more and more valued by consumers, and the national restrictions on pesticide residues are very strict. Meanwhile, when agricultural products are exported, pesticide residues are very serious problems facing foreign food green barriers. In order to reduce pesticide residue, the advantages of novel biological pesticide such as low toxicity and easy degradation are new hotspots for replacing traditional chemical synthetic pesticides. Chinese patent CN106561723A discloses a method for preventing and treating diseases and insect pests by ginkgo episperm (patent No. 201610949718.4), which is prepared by extracting ginkgo episperm and can prevent and treat various diseases and insect pests in fields.

At present, researches on ginkgo biloba exocarp mostly focus on the extraction and separation of effective substances of ginkgo biloba exocarp and medicinal function researches, and no relevant research report on improving the yield of ginkgo biloba exocarp exists. In the traditional fruit tree cultivation, the yield of fruit trees, the weight of single fruit and the like are improved mainly by methods of fine variety breeding, water and fertilizer management and the like, and gingko has high breeding difficulty and long time period due to factors of long childhood period, difficult variety definition, complex background of heritage of species and the like. The biological pesticide is prepared by extracting the ginkgo biloba testa, and the large-scale production is obviously difficult to realize only by scattered waste ginkgo biloba seeds, so that the ginkgo biloba forest for the testa is built, and the raw material with stable quality is provided. Exogenous hormones have more application examples in the aspects of regulating and controlling the growth and development of plants, promoting the yield increase of fruits and the like, and the application of the exogenous hormones in regulating and controlling the growth of ginkgo seeds is not reported. The application relates to the improvement of the yield of the ginkgo biloba testa and the improvement of the effective components mainly containing ginkgoic acid, and the economic yield of a single ginkgo biloba plant is comprehensively improved by selecting and combining plant exogenous hormones.

Salicylic acid is an important signal molecule in plants and plays an important role in plant immunity and disease resistance. The synthesis of plant salicylic acid is mainly carried out through two pathways, namely, Isochorismate synthase (ICS) pathway and Phenylalanine Ammonia Lyase (PAL) pathway, both of which require the intermediate metabolite chorismate. The chorismate forms Isochorismate under the action of Isochorismate synthase, and the Isochorismate synthesizes salicylic acid under the action of Pyruvate Lyase (IPL); in addition, phenylalanine synthesized from chorismate forms Benzoate intermediates (Benzoate intermediates) or p-hydroxycinnamic acid (Coumaric acid) under the action of phenylalanine ammonia lyase, and then salicylic acid (figure 2) and related derivatives of salicylic acid (figure 3) are formed after a series of enzymatic reactions.

Salicylic acid was first extracted from willow for the treatment of human diseases, such as aspirin (acetylsalicylic acid). Plants are constantly subjected to various stresses of the surrounding environment due to their sessile nature, and thus, the plants establish a complex immune mechanism which can be divided into a constitutive defense system and an inducible defense system. The constitutive type mainly comprises the physical barriers of plants and antibacterial substances in bodies, the inducible type is an active defense system activated when the plants are infected by pathogenic bacteria, a series of physiological and biochemical reactions are involved, and salicylic acid is a signal molecule for acquired resistance. After the plants are infected, the salicylic acid can be accumulated, and the plants feel that various defense reactions, such as thickening of cell walls, expression of disease-resistant related proteins and the like, are started after the salicylic acid is accumulated. Furthermore, salicylic acid also plays an important role in the resistance of distal tissues (tissues not in contact). Salicylic acid can induce plants to generate resistance to pathogenic bacteria, the salicylic acid level at an infected point of the plants is rapidly increased after the plants are infected, a non-infected point is also increased after a period of time, and the resistance of the plants is enhanced when the plants are infected by the same pathogenic bacteria next time.

That is, the application of salicylic acid in plants is mainly limited to the research on improving the resistance, including the application of salicylic acid on the plants of ginkgo biloba so as to improve the resistance, and no research report on the increase of ginkgo biloba seed coat yield and the increase of the content of the ginkgolic acid by using the salicylic acid in combination with other exogenous hormones is reported.

Reference to the literature

[1].Khadem,S.and R.J.Marles,Monocyclic phenolic acids；hydroxy-and polyhydroxybenzoic acids:occurrence and recent bioactivity studies.Molecules(Basel,Switzerland),2010.15(11):p.7985-8005.

[2].Gellerman,J.L.,W.H.Anderson and H.Schlenk,Biosynthesis of anacardic acids from acetate in Ginkgo biloba.Lipids,1974.9(9):p.722-725.

[3].Schultz,D.J.,N.S.Wickramasinghe and C.M.Klinge,Chapter Six-Anacardic Acid Biosynthesis and Bioactivity,in Recent Advances in Phytochemistry,J.T.Romeo,J.T.Romeo^Editors.2006,Elsevier.p.131-156.

Disclosure of Invention

The invention aims to provide a method for improving the effective and economic yield of ginkgolic acid of ginkgo biloba sarcotesta aiming at the problems of the background technology.

The invention adopts the following technical scheme to realize the purpose:

a method for improving the effective and economic yield of ginkgolic acid in ginkgo episperms is characterized in that exogenous hormone is sprayed on ginkgo, wherein the exogenous hormone is a mixed solution containing 100-500 mg/L of NAA, 6-BA 50-300 mg/L and 100-300 mg/L of salicylic acid.

Compared with the control, the exogenous hormone mixed solution in the concentration range can achieve the effect of obviously improving the effective and economic yield of the ginkgolic acid of the ginkgo episperm.

Further preferably NAA100mg/L +6-BA100mg/L + salicylic acid 100 mg/L;

NAA100mg/L +6-BA100mg/L + salicylic acid 200 mg/L;

NAA100mg/L +6-BA300mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA50mg/L + salicylic acid 100 mg/L;

NAA300mg/L +6-BA100mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA300mg/L + salicylic acid 100 mg/L;

NAA500mg/L +6-BA300mg/L + salicylic acid 200 mg/L;

or NAA300mg/L +6-BA100mg/L + salicylic acid 200 mg/L;

the concentration of the hormone combination mixed solution has better effect.

Still more preferably: NAA100mg/L +6-BA100mg/L + salicylic acid 200 mg/L;

NAA100mg/L +6-BA300mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA50mg/L + salicylic acid 100 mg/L;

NAA300mg/L +6-BA100mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA300mg/L + salicylic acid 100 mg/L;

or NAA300mg/L +6-BA100mg/L + salicylic acid 200 mg/L;

the combined hormone mixed solution of (1).

Most preferably, the concentration of the NAA300mg/L +6-BA100mg/L + salicylic acid 200mg/L is combined with the hormone mixed solution.

The exogenous hormone of the invention contains 100-500 mg/L NAA, 6-BA 50-300 mg/L and 100-300 mg/L salicylic acid mixed solution for improving the yield of the ginkgo episperm.

Compared with the control, the exogenous hormone mixed solution in the concentration range can achieve the effect of obviously improving the yield of the ginkgo episperm.

Further preferably:

NAA100mg/L +6-BA100mg/L + salicylic acid 100 mg/L;

NAA100mg/L +6-BA100mg/L + salicylic acid 200 mg/L;

NAA100mg/L +6-BA300mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA50mg/L + salicylic acid 100 mg/L;

NAA300mg/L +6-BA100mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA300mg/L + salicylic acid 100 mg/L;

NAA500mg/L +6-BA100mg/L + salicylic acid 100 mg/L;

or NAA500mg/L +6-BA300mg/L + salicylic acid 200 mg/L;

the concentration of the hormone combination mixed solution has better effect.

Still more preferably:

NAA100mg/L +6-BA300mg/L + salicylic acid 300 mg/L;

NAA300mg/L +6-BA50mg/L + salicylic acid 100 mg/L;

NAA300mg/L +6-BA100mg/L + salicylic acid 300 mg/L;

or NAA300mg/L +6-BA300mg/L + salicylic acid 100 mg/L;

the combined hormone mixed solution of (1).

Most preferably, the concentration of NAA300mg/L +6-BA300mg/L + salicylic acid 100mg/L is combined with hormone mixed solution.

The exogenous hormone of the invention comprises a mixed solution with the concentration of NAA300mg/L, 6-BA100mg/L and salicylic acid 200mg/L, or comprises a mixed solution with the concentration of NAA100mg/L, 6-BA100mg/L and salicylic acid 200 mg/L; or mixed solution containing NAA300mg/L, 6-BA50mg/L and salicylic acid 100 mg/L; or mixed solution containing NAA300mg/L, 6-BA100mg/L and salicylic acid 300mg/L for increasing ginkgolic acid content in seed coat of semen Ginkgo.

Further preferably: mixed solution containing NAA300mg/L, 6-BA100mg/L and salicylic acid 200mg/L, or mixed solution containing NAA100mg/L, 6-BA100mg/L and salicylic acid 200 mg/L; or mixed solution containing NAA300mg/L, 6-BA50mg/L and salicylic acid 100 mg/L.

Most preferably a mixed solution containing NAA300mg/L, 6-BA100mg/L and salicylic acid 200 mg/L.

The method for improving the yield and/or the ginkgolic acid content of the ginkgo episperm is characterized in that the hormone spraying time is the differentiation period of the ginkgo episperm.

The method for improving the yield and/or the ginkgolic acid content of the ginkgo episperm is characterized in that the differentiation period of the ginkgo episperm is 40-60 days after pollination of ginkgo.

The method for improving the yield and/or the content of ginkgolic acid of the ginkgo episperm comprises the step of spraying the pesticide on the whole ginkgo fruit branch, preferably spraying the pesticide on the ginkgo fruit skin, and further preferably spraying the pesticide on the ginkgo fruit branch or the ginkgo fruit skin surface.

The spraying method is basically converted into the method that the ginkgo biloba planting density per mu is 4 x 6m-5 x 6m, the number of plants per mu is 20-30 plants, and the amount of hormone of 120 g-225 g NAA, 6-BA of 40 g-75 g and salicylic acid of 80 g-150 g are sprayed per mu.

The method for improving the yield of the ginkgo episperm and/or the content of the ginkgolic acid, disclosed by the invention, has the purity of more than 98% of NAA and 6-BA and the purity of more than 95% of salicylic acid.

The invention relates to a method for improving the yield of ginkgo biloba sarcotesta and/or the content of ginkgolic acid, which is characterized in that a surfactant is added into an exogenous hormone solution. The aim is to enhance solution stability and adhesion.

The method for improving the yield and/or the ginkgolic acid content of the ginkgo biloba testa is preferably 0.1 percent of polyethylene glycol 600 nonionic surfactant.

In the method, the yield of the ginkgo biloba testa and the content of ginkgoic acid are improved, and can be characterized by using the economic yield of a single ginkgo biloba testa plant, and the calculation method comprises the following steps: the economic yield (g) of a single plant is equal to that of the dry episperm plant multiplied by the ginkgolic acid content (%) × 1000.

The invention adopts the technical proposal to achieve the following effects:

at present, no one researches the growth condition and the influence factors of the ginkgo biloba exocarp, and no one researches the content and the influence factors of ginkgolic acid in a plant body, and the invention discovers for the first time through research that the combination of exogenous hormones NAA, 6-BA and salicylic acid can promote the growth of the ginkgo biloba exocarp and the accumulation of ginkgolic acid, thereby comprehensively regulating and controlling the economic yield of the ginkgo biloba exocarp; different exogenous hormone concentration combinations can be adopted according to requirements to respectively regulate and control the growth of the ginkgo episperms and the accumulation of ginkgolic acid.

Compared with the prior art, the method is simple and convenient to operate, small in using amount, small in workload, obvious in effect, strong in practicability and good in application prospect.

After a large amount of basic research is carried out to obtain relevant data, the technical scheme of the invention is determined and verified through experiments.

Calculation of economic yield of ginkgo biloba sarcotesta

Economic yield is the weight of product accumulated by a plant throughout the growth cycle that is available for human consumption or other uses, usually expressed as fresh or dry weight produced per unit area. The economic yield of ginkgo biloba for the core is the weight of the seed core which can be harvested finally. The income of fruit growers can be calculated through economic yield. The invention refers to the concept of the yield of the ginkgo biloba for nuclear use, and the economic yield of the episperm of a single ginkgo biloba refers to the yield of the ginkgoic acid finally obtained by each ginkgo biloba.

Plant yield: selecting adult ginkgo trees with consistent tree age, tree vigor and planting management and normal results as experimental objects, and manually collecting ginkgo seeds for determination. Selecting a strong ginkgo female tree growing for about 25 years, manually harvesting in the middle and last ten days of 9 months when the ginkgo seed is mature, randomly measuring 3 ginkgo trees, continuously measuring for 3 years, and taking an average value.

Fresh Weight (FW) and Dry Weight (DW) measurements: measuring the hundred-grain weight of the ginkgo seed, removing kernels and peeling, measuring the fresh weight of the episperm, deactivating enzyme in a constant-temperature drying oven at 105 ℃ for 20min, drying at 60 ℃ to constant weight, and measuring the dry weight of the episperm to obtain the water content of the ginkgo episperm. All measurements were averaged over 3 replicates.

The kernel yield (%). the fresh weight of the seeds/the fresh weight of the seeds is multiplied by 100%; randomly testing 100 ginkgo seeds.

The yield (%) is the weight of the testa per the weight of the seed husk multiplied by 100%; randomly testing 100 ginkgo seeds.

The yield of the dried episperm plant (kg) is the yield of the seed plant multiplied by the skin yield multiplied by (1-episperm water content); the mean values of 3 strains were randomly determined.

The economic yield (g) of a single plant is equal to that of the dry episperm plant multiplied by the ginkgolic acid content (%) × 1000.

Determination of ginkgolic acid content

Ultrasonic method is adopted to extract ginkgolic acid, RP-HPLC (Reversed-Phase High Performance Liquid chromatography) is adopted to measure ginkgolic acid.

(1) Preparing a ginkgolic acid standard solution:

weighing 10mg of total ginkgoic acid standard substance in a10 mL volumetric flask, dissolving with methanol, fixing the volume, and filtering with a 0.45 mu m filter membrane for later use.

(2) Extraction of ginkgoic acid:

drying fresh ginkgo biloba sarcotesta by using a blast oven at 80 ℃ until the water content is 8%, crushing the dried ginkgo biloba sarcotesta by using a crusher, sieving by using a 80-mesh sieve, precisely weighing 2g of the dried ginkgo biloba sarcotesta in a50 mL centrifuge tube, adding 25mL of petroleum ether, carrying out ultrasonic extraction for 30min, centrifuging an extracting solution at 6000r/min for 20min, repeatedly extracting residues for 2 times, combining supernate, carrying out reduced pressure concentration to obtain a steaming paste, carrying out volume fixing on the concentrated steaming paste to 25mL by using absolute methanol, and carrying out filtering by using a 0.45-micron microporous membrane to obtain a ginkgo biloba sarcoptic extracting solution of a ginkgo biloba sarcotesta sample for HPLC analysis.

(3) RP-HPLC determination:

InertSustain C18 column (4.6X 250mm,5 μm);

mobile phase: methanol-3% acetic acid (98: 2);

detection wavelength: 310 nm;

flow rate: 1 mL/min;

sample introduction: 10 mu L of the solution;

column temperature: 35 ℃ is carried out.

(4) And (3) sample determination: and (3) taking a sample of 10 mu L of filtrate, performing liquid chromatography separation and determination, and performing qualitative determination according to chromatographic peak retention time and quantitative determination by an external standard peak area method.

(5) Calculating the ginkgolic acid content of the sample:

and obtaining the content of the ginkgolic acid in the sample liquid from a standard regression equation according to the chromatographic peak area of the sample to be detected, and then calculating the content of the ginkgolic acid in the sample.

The ginkgolic acid content in the sample was calculated as follows:

in the formula: m is₁Calculating the amount (mug) of ginkgolic acid in the sample solution according to a regression equation; v is the constant volume (mL) of the sample; m is₀The sample size (g) is taken.

The method takes the No. 2 of the south forest of the 20-year-old ginkgo in the ginkgo demonstration garden of Dongan county, Yongzhou city, Hunan province as a research object, pollinates ginkgo in 4 months to completely mature the seed in 9 months and 15 days, samples are taken once every 1 day and 15 days of each month, the growth of the seed of the ginkgo is observed, and the dry weight, the fresh weight and the content of the seed are measured.

The invention dynamically observes the development of the ginkgo seed husk and the testa tissue structure at each stage of the ginkgo seed husk development, measures the change and the accumulation of the content of the testa at each stage, defines the growth and development rule of the ginkgo testa and divides the ginkgo seed husk growth stage. The following conclusions were reached:

(1) the observation of the development of ginkgo seed husks and episperms shows that: the growth and development periods of the gingko seed nut and the episperm are consistent, the rapid increase period of the fresh weight of the gingko seed nut and the episperm is from 5 months 1 day to 7 months 1 day, the rapid increase period of the dry weight of the gingko seed nut and the episperm is from 5 months 15 days to 7 months 15 days, and the dry weight increase is later than the fresh weight. The results are shown in FIGS. 5-8.

(2) The discovery of microscopic observation of ginkgo seed coat growth: the gingko seed coat is rapidly differentiated and formed 30-60 days (1 day at 5 months to 1 day at 6 months) after pollination, the gingko seed coat is rapidly lignified 60-90 days (1 day at 6 months to 1 day at 7 months) after pollination, and the lignification is gradually strengthened from the hole end of the seed ball to the conjunction end; the gingko forms a secretion cavity in the ovule period, the secretion cavity is continuously enlarged in the mature process of seed, and a large amount of grease is contained in the secretion cavity. The results are shown in FIGS. 9-11.

(3) The growth and development of the ginkgo episperm can be divided into: the early stage of the exodermis growth (0-30 d after pollination), the vigorous stage of the exodermis growth (30-90 d after pollination), the late stage of the exodermis growth (90-120 d after pollination) and the mature stage of the exodermis (120-165 d after pollination). See fig. 12.

(4) The ginkgo episperm content is determined and found as follows: the content of soluble protein is rapidly increased from 5 month and 1 day to 6 month and 1 day, the content is highest from 6 month and 1 day to 7 month and 1 day, and the content is gradually reduced after 7 month and 1 day. The soluble sugar content peaks twice in each of day 6 and day 1 and day 8 and day 15, and increases in general. The starch content was highest at 6 months and 15 days, with a trough soluble sugar content and an overall change in unimodal pattern. The fat content is continuously increased from the pollination of the ginkgo to the vigorous growth period of the seeds, and the content slowly decreases and slightly rises after 7 months and 1 day. The ginkgoic acid shows a change rule of reduction, increase and reduction in the whole process of the ectoderm development, the content of ginkgoic acid in the seed coat differentiation stage is reduced, the content of ginkgoic acid in the middle seed coat lignification stage is increased, and the content of ginkgoic acid in the seed and seed maturing stage is gradually reduced. The results are shown in FIGS. 13-16.

According to the dynamic observation of ginkgo seed and episperm development and the dynamic content analysis of episperm inclusion in the early stage, we find that the ginkgo seed and episperm development is in the seed and episperm differentiation stage 40-60 days (5 months, 10 days and 6 months, 1 day) after pollination and is also before the episperm growth stage. At the moment, the species is more obviously differentiated in a real form, the weight, the thickness and the content of the content in the testa begin to increase, and the hormone spraying can be used as an exogenous signal to accelerate the synthesis of the metabolite by the testa cells, so that the yield of the testa and the ginkgolic acid can be improved in a predictable way.

Research on yield increase of ginkgo episperms by exogenous hormone treatment:

by analyzing the correlation between the hormone and the content of ginkgolic acid, a significant positive correlation (correlation coefficient of 0.71) was found between the Salicylic Acid (SA) hormone and the content of ginkgolic acid. Therefore, 200mg/L SA was combined with 100mg/L NAA, 6-BA, ABA, GA₃CTKs andpreliminary experiments to investigate the promotion of ginkgolic acid content in testa were conducted under treatment with IAA hormones, and the results are shown in Table 1 below.

TABLE 1

Combination of	Ginkgolic acid content (%)
		Contrast (clean water)	5.60±0.06
SA	5.91±0.07
		SA+NAA	6.53±0.12
SA+6-BA	6.67±0.09
		SA+ABA	5.12±0.11
SA+GA₃	5.41±0.07
		SA+CTKs	6.17±0.06
SA+IAA	6.46±0.10

According to the preliminary study of hormone proportioning treatment, the collocation of NAA, 6-BA and SA is found to obviously increase the ginkgolic acid content in the testa, so the optimal treatment formula is researched by adopting the orthogonal test design of NAA, 6-BA and SA 3 factor 3 level.

1 materials and methods

1.1 materials

1.1.1 plant Material

Taking No. 2 ginkgo Zhongnan forest, which is a demonstration garden of 20-year-old ginkgo in the Dongan county of Yongzhou city, Hunan province, as a research object, and randomly selecting female ginkgo trees which are consistent in water and fertilizer management and normal in flowering and fruiting.

1.1.2 drugs and reagents

Salicylic Acid, Ethylene Diamine Tetraacetic Acid (EDTA), β -mercaptoethanol, crospovidone (PVPP), phenylmethylsulfonyl fluoride (PMSF), Dithiothreitol (DL-Dithiothreitol, DTT), naddpna₂Ethanol, methanol, boric acid, borax, glacial acetic acid, glycerol (all analytically pure), and national medicine group reagents; p-coumaric acid, Leupepptin, L-phenylalanine, G-6-pNa, trans-cinnamic acid, Shanghai bioengineering GmbH; NAA (Naphtylacetic acid), 6-BA (6-Benzylaminopurine), Sigma, USA.

1.1.3 Main Experimental instruments

The main instruments are listed in table 2.

TABLE 2 Main instrumentation

1.2 methods

1.2.1 hormone treatment orthogonal design

Taking Zhongnanlin No. 2 as a ginkgo treatment variety, respectively taking 3 different hormones as test factors and 3 concentration gradients as test levels, adopting a random block design, spraying on the ginkgo tree in 6 months and 1 day, spraying 3 ginkgo female trees with the hormones every time, and repeating for 3 times. The spraying amount is determined by the even wetting and dripping of the gingko seeds. The orthogonal design is shown in table 3.

TABLE 3 orthogonal experimental design

1.2.2 determination of Ginkgolic acid content

The measurement method is the same as that of the previous method.

2 results and analysis

2.1 optimization of hormone treatment combinations

The results of the experiments performed at 3 levels of 3 hormones according to the designed orthogonal experiment are shown in table 4. As can be seen from Table 4, treatment combination A₂B₃C₁The yield of the dry episperm plant is 13.10kg, the single fruit of the gingko seed is large, and the episperm skin is thick (figure 18); treatment combination A₃B₁C₃The yield of the dry episperm plant is the lowest, and is only 9.88kg, which is not obvious from the contrast group. Treatment combination A₂B₁C₁The ginkgolic acid content is 6.86 percent at the highest, and the treatment composition A₃B₂C₁The ginkgolic acid content is the lowest, 5.51 percent, and is 0.13 percent lower than that of a control group. Comprehensively calculating the effective economic yield of the single ginkgo episperm plant, and processing the combination A₂B₃C₁The highest effective economic yield is 825.26g, which is 1.71 times of that of the control group; treatment combination A₃B₁C₃The effective economic yield is the lowest, 555.26g, which is higher than the control group.

TABLE 4 results of orthogonal experiments

The orthogonal test is subjected to variance analysis (table 5), and the dry exocarp plant yield, the ginkgolic acid content and the effective economic yield are remarkably different (P is less than 0.05) after NAA treatment; the dry exocarp plant yield and the ginkgolic acid content under 6-BA treatment are obviously different, but the influence on the effective economic yield is not obvious (P is more than 0.05); the ginkgolic acid content and the effective economic yield under the SA treatment are obviously different, but the influence on the yield of the dried exopleura plants is not obvious.

TABLE 5 analysis of variance in orthogonal tests

In order to further understand the influence of different factor levels on the growth of ginkgo episperms and the accumulation of ginkgoic acid, extreme difference analysis is carried out on the orthogonal test results. As can be seen from Table 6, the influence of various factors on the yield of the dried testa plants is: NAA>6-BA>SA, the best combination for promoting the growth of ginkgo biloba testa is A₂B₃C₁Combined with the analysis of significance of variance, NAA and 6-BA have significant influence on the production of the dried exodermis, while SA has no significant influence, and NAA at a medium level and 6-BA at a high level are the optimal combination; the influence degree of different factors on the ginkgolic acid content is as follows: SA>NAA>6-BA, the best combination for promoting the accumulation of ectoderm ginkgolic acid is A₂B₂C₂Combining variance significance analysis, the content of ginkgolic acid is significantly influenced by 3 factors, and the combination of the factors at the medium level is optimal; the influence degree of different factors on the effective and economic yield of the ginkgo biloba sarcotesta is as follows: NAA>SA>6-BA, the best combination for promoting the growth of ginkgo episperm and the accumulation of ginkgolic acid is A₂B₂C₂Combined with the significance analysis of variance, NAA and SA have significant influence on the effective economic yield of the testa, while 6-BA has no significant influence, and the combination of factors at moderate levels is optimal.

According to the analysis results, the optimal hormone combination A for promoting the growth of the ginkgo exocarp and the accumulation of the ginkgoic acid is comprehensively screened₂B₂C₂Namely 300mg/L NAA +100mg/L6-BA +200mg/L SA.

TABLE 6 results of the orthogonal test are very poor

Note: ki is the average of the same level index and i is the level number of the orthogonal test.

The invention researches the influence of exogenous hormone treatment on the yield of ginkgo biloba testa and the content of ginkgolic acid by designing different factor horizontal orthogonal tests, and obtains the following conclusion:

(1) the influence degree of different factors on the yield of the dried exopleura plants is as follows: NAA>6-BA>SA, NAA and 6-BA have obvious influence on the yield of the dry episperm plant, SA has no obvious influence, and the optimal combination for promoting the growth of the ginkgo episperm is A₂B₃C₁。

(2) The influence degree of different factors on the ginkgolic acid content is as follows: SA>NAA>6-BA, 3 factors all obviously influence the content of ginkgolic acid, and the optimal combination for promoting the accumulation of exodermic ginkgolic acid is A₂B₂C₂，

(3) The influence degree of different factors on the effective and economic yield of the ginkgo biloba sarcotesta is as follows: NAA>SA>The 6-BA, NAA and SA have obvious influence on the effective economic yield of the episperm, while the 6-BA has no obvious influence, and the optimal combination for promoting the growth of the episperm of the ginkgo and the accumulation of ginkgoic acid is A₂B₂C₂. (FIG. 17)

The optimal hormone combination comprehensively screened for promoting the growth of ginkgo episperms and the accumulation of ginkgoic acid is as follows: a. the₂B₂C₂Namely 300mg/L NAA +100mg/L6-BA +200mg/L SA.

Drawings

FIG. 1 is a synthetic pathway for ginkgolic acids;

FIG. 2 salicylic acid synthesis pathway;

FIG. 3 salicylic acid synthetic pathway and SA derivatives;

FIG. 4 shows the seed structure of Ginkgo biloba;

a is a cut-out view of the ginkgo seed; b is a schematic diagram of ginkgo seed;

FIG. 5 is a longitudinal cutting chart of the development process of ginkgo biloba seeds;

FIG. 6 shows the change of unit weight during the development of ginkgo biloba seed;

FIG. 7 shows the variation of longitudinal, transverse and volume of ginkgo seed during the development process;

FIG. 8 shows the change of episperm during the development of ginkgo biloba seeds;

FIG. 9 is a microscopic observation of the differentiation process of ginkgo seed coat;

FIG. 10 is a microscopic observation of the lignification process of seed coat in ginkgo;

FIG. 11 is a microscopic observation of the secretory cavity of the ginkgo episperm;

FIG. 12 is a schematic diagram of the division of the developmental stages of ginkgo biloba seeds;

FIG. 13 shows the change of soluble protein content during the development of ginkgo biloba sarcotesta;

FIG. 14. variation of soluble sugar and starch content during ginkgo episperm development;

FIG. 15 shows the variation of crude fat content during the development of ginkgo biloba sarcotesta;

FIG. 16 shows the change of ginkgolic acid content during the development of ginkgo biloba sarcotesta;

FIG. 17 is an HPLC chart of 5 ginkgolic acids from ginkgo biloba sarcotesta;

FIG. 18 is a graph of the appearance of hormone treated ginkgo nut and control treatments of the present invention;

FIG. 19 is a macroscopic view of hormone-treated ginkgo biloba seeds and a control treatment according to the present invention.

Detailed Description

The method is carried out in a gingko cultivation demonstration garden in Dongan county of Yongzhou city, Hunan province, and the geographical position is 26 degrees north latitude

21 '38' and 111 degrees 5 '12' in east China, the climate in the region belongs to the monsoon humid climate in the subtropical and middle-tropics mountainous area, the annual average temperature is 17.8 ℃, the annual average precipitation is 1330 mm, and the soil is slightly acidic. The following examples are given to illustrate the invention without limiting it.

Example 1:

the invention relates to a method for promoting yield increase of ginkgo episperms by spraying exogenous hormone, which comprises the steps of preparation of the exogenous hormone, determination of spraying time, selection of spraying combination and the like, and comprises the following steps:

(1) tree management and artificial pollination: selecting a ginkgo female tree with normal flowering and fruiting in 15-20 years, and carrying out normal water and fertilizer management. In the pollination period of the ginkgo, sunny weather is selected, and artificial supplementary pollination is carried out by a pollen aqueous solution spraying method.

(2) And (3) exogenous hormone preparation: the exogenous hormone uses purified water as solvent, and 0.1% polyethylene glycol 600 nonionic surfactant is added to enhance solution stability and adhesiveness. The mass-volume ratio concentration is respectively as follows:

the concentration of the naphthylacetic acid (NAA) spraying agent is 300mg/L,

② the 6-benzylaminopurine (6-BA) spraying agent has the concentration of 50mg/L and 6-BA300mg/L,

③ the concentration of the salicylic acid spraying agent is 100 mg/L;

(3) and (3) spraying time of hormone: in the ginkgo seed coat differentiation period, namely 40-60 days after ginkgo pollination, the ginkgo episperm is rapidly differentiated, and the episperm begins to rapidly grow and thicken;

(4) a spraying method; and (3) uniformly spraying the whole ginkgo fruit branch by using a handheld sprayer in sunny and calm weather until the dripping degree on the surface of the fruit peel is reached.

(5) In the method, the hormone spraying combination for promoting the yield increase of the ginkgo episperm is 300mg/L NAA +300mg/L6-BA +100mg/L salicylic acid, the hormone spraying combination for promoting the content increase of the ginkgoic acid in the ginkgo episperm is 300mg/L NAA +50 mg/L6-BA +100mg/L salicylic acid, and the hormone spraying combination for comprehensively increasing the yield of the ginkgo episperm is 300mg/L NAA +300mg/L6-BA +100mg/L salicylic acid. Spraying clear water as a control.

(6) In the method, the ginkgo seeds are manually harvested when naturally mature in the middle and last ten days of 9 months, the plant yield and the fresh weight of the seeds are respectively weighed, the seeds are manually removed and peeled, the fresh weight of the testa is weighed, the seeds are naturally aired or dried, the dry weight of the testa is weighed, and the ginkgoic acid content in the testa is measured by a high performance liquid chromatography.

(7) The implementation result shows that the yield of the dry episperm plant of the hormone treatment combination of 300mg/L NAA, 300mg/L6-BA and 100mg/L salicylic acid is up to 13.1kg, which is 1.54 times of that of the control group, the single fruit of the gingko seed is large, and the episperm skin is thick (figure 18); the content of ginkgolic acid in the hormone treatment combination of 300mg/L NAA, 50 mg/L6-BA and 100mg/L salicylic acid is up to 6.86 percent, which is 1.22 times of that in the control group. The effective economic yield of the ginkgo episperm single plant is comprehensively calculated, and the maximum effective economic yield of the hormone treatment combination of 300mg/L NAA, 300mg/L6-BA and 100mg/L salicylic acid reaches 825.26g, which is 1.71 times of that of a control group.

Example 2:

in 2019, 5 and 25 months, the optimal formula A is carried out on 20 mu of high-yield regional ginkgo trees (the plant row spacing is 4 x 6m-5 x 6m, and 20-30 trees per mu) in the ginkgo demonstration base of Dongan county₂B₂C₂300mg/L NAA +100mg/L6-BA +200mg/L salicylic acid spraying treatment test, and clear water is used as a control group. 20L-25L of proportioning liquid is needed for effectively spraying each tree (slightly different according to the size of the tree body and the like), 400L-750L of proportioning liquid is needed for each mu, and 120 g-225 g of NAA, 40 g-75 g of 6-BA and 80 g-150 g of salicylic acid are needed for each mu. Seed coat harvesting and yield measurement are carried out for 9 months and 15 days, the average values of the yield of the ginkgo biloba sarcotesta, the ginkgoic acid content and the effective economic yield per mu are 321.42kg, 7.73 percent and 24.85kg (see table 7), which are respectively 1.50, 1.38 and 2.06 times of those of a control group, and the effective economic yield of the ginkgo biloba sarcotesta is greatly improved (figure 19).

TABLE 7 yield per mu test results

Claims

1. A method for improving the effective and economic yield of ginkgolic acid in ginkgo episperms is characterized in that exogenous hormone is adopted to spray ginkgo, wherein the exogenous hormone is a mixed solution containing 100-500 mg/L of NAA, 6-BA 50-300 mg/L and 100-300 mg/L of salicylic acid; the hormone spraying time is the ginkgo seed coat differentiation period.

2. The method for increasing the effective and economical yield of ginkgolic acid from ginkgo episperms according to claim 1, wherein the exogenous hormone comprises a mixed solution of NAA 100-500 mg/L, 6-BA 50-300 mg/L and salicylic acid 100-300 mg/L for increasing the yield of ginkgo episperms.

3. The method for increasing the effective and economical yield of ginkgolic acid from ginkgo biloba sarcotesta according to claim 1, wherein the exogenous hormone comprises a mixed solution of NAA300mg/L, 6-BA100mg/L and salicylic acid 200mg/L, or comprises a mixed solution of NAA100mg/L, 6-BA100mg/L and salicylic acid 200 mg/L; or mixed solution containing NAA300mg/L, 6-BA50mg/L and salicylic acid 100 mg/L; or mixed solution containing NAA300mg/L, 6-BA100mg/L, and salicylic acid 300mg/L for increasing ginkgolic acid content in semen Ginkgo testa.

4. The method of claim 1, wherein the ginkgo biloba pericarp differentiation period is 40-60 days after ginkgo biloba pollination.

5. The method of claim 1, wherein the spraying is carried out by spraying the entire ginkgo biloba sarcocarp with a chemical.

6. The method for increasing the effective and economical yield of ginkgolic acid from ginkgo biloba sarcotesta as claimed in claim 1, wherein said spraying is carried out by spraying onto the surface of ginkgo biloba.

7. The method for increasing the effective and economic yield of ginkgolic acid from ginkgo biloba sarcotesta as claimed in claim 5 or 6, wherein the spraying method is spraying to the surface of ginkgo biloba branches or pericarps at a dripping degree.

8. The method of any one of claims 1-3, wherein the NAA and 6-BA is > 98% pure and the salicylic acid is > 95% pure.

9. The method for increasing the effective and economical yield of ginkgolic acid of ginkgo biloba sarcotesta according to any of claims 1 to 3, wherein a surfactant is added to the exogenous hormone solution.

10. The method of claim 9, wherein the surfactant is 0.1% polyethylene glycol 600 nonionic surfactant.