CN114503916B - Artificial cultivation method of elaeagnus pungens - Google Patents

Abstract

The invention discloses an artificial cultivation method of elaeagnus pungens, belonging to the technical field of plant cultivation. The method comprises the steps of collecting elaeagnus pungens seeds, selecting seeds, germinating, selecting soil blocks in a nursery, performing retting, tedding, adding plant ash, and then turning over, planting survival seed seedlings, topping the seed seedlings, storing buds, reserving 2-3 strong lateral buds of each mother plant, and placing nutrition bags filled with nutrition soil on furrows in a seedling greenhouse in rows; selecting side branches with 3-4 true leaves on a mother plant as cutting slips, immersing the base parts of the cutting slips in rooting liquid, cutting into nutrition bags, covering with an agricultural film, preserving heat and moisture, and transplanting when seedlings grow out 6-7 true leaves and are higher than 20 cm. The invention adopts the female seedling to cultivate, and the lateral branches are cut on the female seedling as cutting slips for cutting cultivation, so that the method can quickly cultivate the seedlings in a large scale, has high survival rate, high plant growth speed, strong plant disease and insect pest resistance and high organic matter content, and better realizes the batch breeding of the horned seedlings.

Description

Artificial cultivation method of elaeagnus pungens

Technical Field

The invention belongs to the technical field of plant planting, and particularly relates to an artificial cultivation method of elaeagnus pungens.

Background

The elaeagnus pungens (Elaeagnus gonyanthes Benth) are evergreen climbing shrubs, are more than 4 meters long, and are usually thornless; the young branches are slender and elongated, are closely covered by brownish red or gray brown scales, and the old branch scales fall off and are gray brown or black and have luster; she Gezhi oval or oblong oval, 5-9 cm long, 1.2-5 cm wide, blunt or blunt tip at the top, round or nearly round base, thin and narrow edge, slightly rewinding, rust-colored scales at the young time of the top, falling off after maturation, gloss, green color after drying, brownish red color below, light grey color below, rust-colored or gray scales, 7-10 pairs of side veins, branched and interconnected near edges, obvious bulges on both sides, obvious net veins on the top, and unclear below; the petiole is rust or brown and is 4-8 mm long. Fruit oblong or inverted oval oblong, 15-22 mm long, about half the diameter, yellow brown flakes when young, yellow red when mature, and a dry calyx barrel at the top end; the fruit stalks are 12-25 mm long and are vertical or slightly bent. 10-11 months in flowering phase and 2-3 months in fruit phase next year. The flos Tagetis Erectae is mainly distributed in Hainan in the city and county such as five-finger mountain, baotang, qiongzhong, ledong, baisha, etc., and in Hunan, guangdong, guangxi and Yunnan; the peninsula in the middle south is also distributed. The medicinal material is suitable for tropical and subtropical areas with the altitude of 1000 meters or less, is more shade-resistant, is favorable for loose, moist and fertile geographic conditions, can grow on red soil, brick red soil, red soil, sandy yellow soil and red lime soil, and can bear the areas with the temperature of 20-30 ℃ and the annual precipitation of 1200-2300 mm at the lowest air temperature of 3 ℃ and the highest air temperature of 39 ℃.

The chemical components of the cornflower mainly comprise flavonoid glycoside, alkaloid, steroid or triterpene, organic acid, phenols, tannins, volatile oil and the like, and the whole plant can be used as a medicine, has the effects of dispelling wind and activating collaterals, promoting qi circulation and relieving pain, detumescence and detoxification, and relieving asthma and cough, and is a wild south medicine of Hainandao. The wild cornflower is mainly planted in a scattered way, a small amount of cornflower is planted in a scattered way by farmers, the annual yield is about 1000 tons, the annual demand exceeds 5000 tons, and the contradiction between supply and demand is prominent, and the main reason is that the natural breeding and regeneration capability of the cornflower are poor and the growth speed is low under the wild state, so that the quality and the yield of medicinal materials can not meet the requirement of expanding the reproduction.

Therefore, development of large-scale and standardized rapid planting technology of the cornflower is urgent.

Disclosure of Invention

The invention aims to provide an artificial cultivation method of elaeagnus pungens, aiming at the defect of the existing natural resources of the elaeagnus pungens. The method adopts the branches with strong and tender buds for cutting cultivation, can effectively retain the genetic characteristics of plants, improve the growth speed of the plants, improve the survival rate of the plants and achieve the aim of rapid propagation.

In order to achieve the above object, the present invention adopts the following technical scheme:

an artificial cultivation method of elaeagnus pungens comprises the following steps:

s1, seed germination and seed seedling cultivation

(1) Collecting thorny elaeagnus seed, selecting, soaking the seed in ethanol, and washing;

(2) Adding sodium hypochlorite solution, mixing, soaking, washing seed, and re-suspending seed;

(3) Inoculating the seed suspension into a seed germination culture medium, removing excessive water on the surface of the culture medium, and sealing;

(4) Culturing for 14-16 days at 25+/-2 ℃ under 16h light/8 h dark condition, transferring the plantlets obtained by seed germination into a strong seedling culture medium, and transplanting after 2-3 weeks;

(5) Taking out the seedlings, cleaning the roots, transplanting the seedlings into a nutrition bag filled with sterilized composite nutrient soil, shading the seedlings by 60% and culturing for 14 days, removing a shading net at the top of the greenhouse, and continuing culturing until the seedlings grow to have 5-7 true leaves;

the formula of the seed germination culture medium is as follows: MS culture medium +30g/L sucrose +7.5g/L agar powder, pH5.8;

the formula of the strong seedling culture medium is as follows: 1/2MS culture medium +1mg/L indolebutyric acid (IAA) +0.1mg/L naphthylacetic acid (NAA) +30g/L sucrose +7.5g/L agar powder, pH5.8;

s2, mother seedling cultivation

Selecting a land block which is subjected to composting, tedding, loosening and leveling in a nursery, selecting a strong seed seedling, and planting on the prepared furrow according to row plant spacing of 25cm multiplied by 25 cm; watering root fixing water for 1 time after planting, and keeping the teeth moist and smooth in drainage; after field planting the mother seedlings for 30 days, most mother seedlings can grow 6-10 true leaves, and the mother seedlings with 6-10 true leaves are selected for pruning, topping and bud accumulation; lateral buds grow 3-5 days after the mother plant is topped, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions;

s3, cutting seedling

Preparing soil into furrows in a seedling raising greenhouse with the shading degree of 60-70%, selecting a nutrition bag, filling sterilized composite nutrient soil, uniformly mixing, and then placing the composite nutrient soil on the furrows in rows; selecting lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period; firstly, soaking a cutting root part in rooting powder (ABT) solution with the mass concentration of 1.25% -2.5% for 10-20 seconds, or soaking the cutting root part in sucrose solution with the mass concentration of 5% -10% for 11-13 hours, then respectively cutting the cutting root part into prepared nutrition bags, covering an agricultural film, preserving heat and moisture, and enabling the heights of the top of the agricultural film and a furrow surface to be about 0.7-1.1 m;

wherein, the composite nutrient soil in the steps S1 and S3 is a mixture of any one of red loam, brick red loam and black ash soil and sand, and the mass ratio of any two of the red loam, the brick red loam and the black ash soil to the sand is 6-8: 4 to 2;

s4, seedling raising process

Spraying dipotassium hydrogen phosphate water every day before the cutting shoots grow roots according to the change of the weather temperature and the humidity, and slightly wetting the leaf surfaces and the nutrient soil in the nutrient bags; spraying sterilizing agent once every week in one month to prevent root rot of seedling; the average temperature of the agricultural film in the greenhouse is kept at 23-29 ℃, the relative humidity is 45-85%, the cutting seedlings start to root in 6-8 days, the agricultural film is removed 28-32 days after the rooting, and the shading degree of 60-70% in the greenhouse is kept; after the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings can emerge and transplanted when the cutting seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

Preferably, the mass concentration of the ethanol in the step S1 is 75%, and the soaking time is 1 minute; the mass concentration of the sodium hypochlorite is 2%, and the soaking time is 10 minutes.

Preferably, the specific operation of collecting the elaeagnus pungens seeds in the step S1 is as follows: collecting fruits in middle and late 3 months, stacking in the shade for several days, mashing the fruits to collect seeds, removing impurities by using a 50-60 mesh sieve, cleaning with clear water, and airing in the shade to obtain the elaeagnus pungens seeds.

Preferably, the specific operations of selecting seeds in step S1 are as follows: removing seeds with peculiar smell; selecting yellow-red seeds, and removing the yellow-brown seeds; and removing the seeds with lighter weight, and the remaining seeds with average thousand grain weight of more than 0.041g can be used for the next step.

Preferably, the land parcels in the step S2 are used after standard land preparation of 1000 kg of compost and 200 kg of plant ash are applied to each mu of land.

Preferably, the composite nutrient soil in the steps S1 and S3 is red loam or brick red loam and sand, and the mass ratio is 6:4, mixing or mixing black dust and sand according to a mass ratio of 8: 2.

The preferred agent for sterilization described in steps S1 and S3 is chlorothalonil at a concentration of 75% by mass or potassium permanganate at a concentration of 0.01%.

Preferably, the size of the nutrition bag described in step S3 is 8cm by 12cm.

Preferably, the mass concentration of the dipotassium hydrogen phosphate water in the step S4 is 0.01%.

Preferably, the sterilizing agent in the step S4 is carbendazim, the mass concentration is 50%, the sterilizing agent is diluted 1000 times by water before use, and the using amount is suitable for wetting leaf surfaces and nutrient soil in a nutrient bag. The use of the fertilizer can promote plant growth and prevent root rot of seedlings.

The invention adopts the lateral branches of the strong and tender buds of the parent seedlings for cutting cultivation, can effectively promote root growth, improve survival rate, improve plant growth speed, strengthen plant stress resistance, better maintain the excellent genetic characteristics of the female parent, provide a high-efficiency approach for standardization and large-area planting of the cornflower, and is beneficial to development and utilization of southern medicine (Li medicine) resources.

Drawings

FIG. 1 is a diagram showing the molecular structure of locust element.

FIG. 2 is a graph showing the comparison of liquid chromatograms of cutting and wild two types of horns at an absorption wavelength of 245 nm.

FIG. 3 is a graph showing the comparison of liquid chromatograms of cutting and wild two types of horns at an absorption wavelength of 210 nm.

FIG. 4 is a graph showing the comparison of liquid chromatograms of cutting and wild two types of horns at an absorption wavelength of 360 nm.

FIG. 5 is a primary and secondary mass spectrum corresponding to a liquid chromatography retention time of 14.5 min.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The procedure for the cultivation of the seedlings used in the examples below is as follows:

1. seed harvesting and preservation

When the oval spherical fruits of the cornflower change from cyan to yellow and the pulp becomes soft in middle and late 3 months, the fruits are ripe, and the fruits can be picked or shaken off and then picked up on the ground. After the fruits are collected, stacking the fruits in a shade place for several days, naturally cooking the fruits until the fruits are sufficiently softened, putting the fruits in a basin filled with water, pounding the fruits, repeatedly rubbing the fruits to enable fine seeds to fall into the water, removing impurities by using a 50-60-mesh sieve, cleaning the fruits with clear water, and airing the fruits in the shade place to obtain the cornflower seeds.

2. Seed selection

Firstly, removing the seeds with peculiar smell by smelling the taste of the dried seeds; selecting yellow-red seeds, and removing the yellow-brown seeds; and then the seeds with lighter weight are removed through a dustpan, and the remaining seeds with average thousand-grain weight of more than 0.041g can be used for the next seedling raising.

3. Seed germination and seed seedling cultivation

(1) 0.04 g of the seeds of the hornia are weighed into a clean 15ml centrifuge tube, and 10ml of 75% ethanol is added for soaking for 1 minute.

(2) On an ultra clean bench, ethanol was decanted off and rinsed once with 10ml of sterile water.

(3) 10ml of 2% sodium hypochlorite solution is added, mixed evenly and soaked for 10 minutes.

(4) Pouring out the sodium hypochlorite solution, flushing the seeds with sterile water for 3-5 times, 10-15 ml each time, and finally adding 3ml of sterile water to resuspend the seeds.

(5) Sucking up water together with seeds by a pipetting gun, uniformly spreading in a culture dish filled with a seed germination culture medium (MS culture medium+30 g/L sucrose+7.5 g/L agar powder, pH 5.8), and sealing by a sealing film after air-drying excessive water on the surface of the culture medium by using a blowing of an ultra-clean bench.

(6) The dishes were placed in a culture chamber and incubated at 25.+ -. 2 ℃ for 16h light/8 h dark. After about 14-16 days, the germination emergence rate of the seeds reaches 60%, the obtained seedlings are transferred into a culture flask containing a strong seedling culture medium (1/2 MS culture medium+1 mg/L IAA+0.1mg/L NAA+30g/L sucrose+7.5 g/L agar powder, pH 5.8), one culture flask is inoculated with 10-12 seedlings, the height of the seedlings is more than 60mm after 3 weeks of continuous culture in a culture room, and the survival rate is 100%.

(7) Taking out the seedlings, washing the agar with the adhered roots, transplanting the seedlings into a nutrition bag filled with a mixed matrix of black soil and sand (8:2) sterilized by potassium permanganate with the mass concentration of 0.01%, culturing the seedlings in a greenhouse with the shade of 60% for 14 days, removing a shade net at the top of the greenhouse, and continuing culturing until the seedlings grow to have 5-7 true leaves.

Example 1

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedlings are planted for 30 days, most of the mother seedlings can grow 6-10 true leaves, the mother seedlings with 6-10 true leaves are selected for pruning, topping and bud accumulation are carried out, lateral buds grow 3-5 days after topping of the mother seedling plants, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

The shading degree is built according to the number of seedlings, and the number of the seedlings is 60%, so that the greenhouse is ventilated on four sides and has good drainage. And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling red loam and sand (6:4) sterilized by chlorothalonil with the mass concentration of 75%, uniformly mixing, and then placing the mixture on a furrow in a row. And selecting the lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period. The cutting base was first soaked in 2.5% rooting powder (ABT) solution for 15 seconds. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture, wherein the height between the top of the agricultural film and the ridge surface is about 0.75 m.

3. Seedling raising process

According to the change of the weather temperature and humidity, 0.01% of dipotassium hydrogen phosphate water is sprayed for 2 times every day before the cutting shoots grow roots, and the nutrition soil in the leaf surfaces and the nutrition bags is moistened to the extent that the sterilizing agent carbendazim is sprayed for 1000 times every week in one month, so that the root rot of the seedling is prevented. And when the month average temperature in the seedling sprout agricultural film greenhouse is 23 ℃ and the relative humidity is 45-65%, the cutting seedlings start to root on the 8 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

Example 2

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedlings are planted for 30 days, most of the mother seedlings grow 6-8 true leaves, the mother seedlings growing 6-8 true leaves are selected for pruning, topping and bud accumulation, lateral buds grow 3-5 days after topping of mother seedling plants, and 2-3 healthy lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

The shading degree is built according to the number of seedlings, and the number of the seedlings is 60%, so that the greenhouse is ventilated on four sides and has good drainage. And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling black gray soil and sand (8:2) sterilized by potassium permanganate with the mass concentration of 0.01%, uniformly mixing, and then placing the mixture on a furrow in a row. And selecting the lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period. The cutting base was first soaked in a 2.0% rooting powder (ABT) solution for 20 seconds. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture, wherein the heights of the top of the agricultural film and the ridge surface are about 0.8 meter.

3. Seedling raising process

According to the change of the weather temperature and humidity, 0.01% dipotassium hydrogen phosphate water is sprayed for 3 times every day before the cutting shoots grow roots, and the nutrition soil in the leaf surfaces and the nutrition bags is moistened to the extent that the sterilizing agent of carbendazim is sprayed and diluted for 1000 times every week in one month, so that the root rot of the seedling is prevented. And when the month average temperature in the seedling sprout agricultural film greenhouse is 25 ℃ and the relative humidity is 50-70%, the cutting seedlings start to root on the 6 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

Example 3

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedlings are planted for 30 days, most of the mother seedlings grow 8-10 true leaves, the mother seedlings growing 8-10 true leaves are selected for pruning, topping and bud accumulation are carried out, lateral buds grow 3-5 days after topping of mother seedling plants, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

The shading degree is built according to the number of seedlings, and the number of the seedlings is 60%, so that the greenhouse is ventilated on four sides and has good drainage. And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. Ensure smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling red loam and sand (6:4) sterilized by chlorothalonil with the mass concentration of 75%, uniformly mixing, and then placing the mixture on a furrow in a row. And selecting the lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period. The cutting base is soaked in 5% sucrose solution for 12 hours. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture until the heights of the top of the agricultural film and the ridge surface are about 1.0 meter.

3. Seedling raising process

According to the change of the weather temperature and humidity, spraying water for 4 times before the cutting shoots grow roots, and spraying sterilizing agent diluted 1000 times per week to prevent the roots of the seedlings from rotting, wherein the wetting degree of the foliage and the nutrient soil in the nutrient bag is the same as that of the sprayed water, and the sterilizing agent is sprayed once per week. And when the month average temperature in the seedling sprout agricultural film greenhouse is 27 ℃ and the relative humidity is 55-75%, the cutting seedlings start to root on the 6 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

Example 4

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedlings are planted for 30 days, most of the mother seedlings grow 6-10 true leaves, the mother seedlings growing 6-10 true leaves are selected for pruning, topping and bud accumulation, lateral buds grow 3-5 days after topping of the mother seedling plants, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

The shading degree is built according to the number of seedlings, and the number of the seedlings is 60%, so that the greenhouse is ventilated on four sides and has good drainage. And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. Ensure smooth drainage. A nutrient bag with the size of 8cm x 12cm is selected and filled with the brick red loam sterilized by potassium permanganate with the mass concentration of 0.01 percent: sand=6:4, mixed well, and then placed in rows on the furrow. And selecting the lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period. Firstly, soaking the base of the cutting slip in 2% rooting powder (ABT) solution for 15 seconds, then respectively cutting the cutting slip into prepared nutrition bags, and then covering an agricultural film for heat preservation and moisture preservation, wherein the heights of the top of the agricultural film and the furrow surface are about 1.10 meters.

3. Seedling raising process

According to the change of the weather temperature and humidity, 0.01% dipotassium hydrogen phosphate water is sprayed for 5 times every day before the cutting shoots grow roots, and the nutrition soil in the leaf surfaces and the nutrition bags is moistened to the extent that 1000 times of diluted carbendazim sterilizing agent is sprayed once every week in one month, so that the root rot of the seedling is prevented. And when the month average temperature in the seedling sprout agricultural film greenhouse is 29 ℃ and the relative humidity is 65-85%, the cutting seedlings start to root on the 7 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

The test results of examples 1, 2, 3, 4 are tabulated below:

TABLE 1 cutting seedling test results

Effect examples

Cutting culture of the horns and analysis of the active ingredients of the wild horns:

1. chromatographic conditions

Instrument: DIONEX UltiMate 3000pump; dionex UltiMate 3000Autosampler; DIONEX UltiMate 3000 Column Compartment; DIONEX UltiMate 3000Diode Array Detector (DAD); work station: chromeleon 7.2; mass spectrometer: q exact HF, molecular weight range 100-1000, positive and negative mode scan respectively, sheath gas flow rate 60,Aux gas flow rate 10,Sweep gas flow rate 0,Spray voltage (KV) 3.5,Capillary temp.(. Degree.C.) 320, S-lens RF level 55.0,Aux gas heater temp.(. Degree.C.) 300. Test expiration date 20220331.

Pheromin C18 column, 250×4.6mm,5 μm; the detection wavelength is 245nm, the flow rate is 1.0ml/min, the column temperature is 35 ℃, and the sample injection amount is 10 mu l; mobile phase 0.1% formic acid-acetonitrile, gradient elution, elution conditions are as shown in table 2:

TABLE 2 elution conditions

2. Preparation of test solutions

Cutting and planting fresh branches and leaves of 1 year horn flower (example 2) and 3 year old wild horn flower (Wenchang of Hainan) respectively, cutting into pieces of 1-2 mm by scissors, drying in a 45-degree oven for 10 hours, weighing 0.94 g of the pieces, respectively soaking in 50 ml of Java ha water, sealing by a preservative film, heating to 90 ℃ in a water bath, cooling to 50 ℃ after maintaining for 30 minutes, taking out, placing in an ultrasonic cleaner for ultrasonic treatment for 30 minutes, taking out, placing for clarification, taking out 1 ml of supernatant, and filtering by a filter head to obtain a sample for liquid chromatography.

3. Detection result

Fig. 1-3 are liquid phase chromatograms at different absorption wavelengths, the 9# sample is a cutting cultivated cornflower sample, and the 10# sample is a wild female parent cornflower sample. According to chromatographic conditions and experience of liquid analysis, it can be judged that compounds with ultraviolet absorption corresponding to the peak before 10 minutes in the spectrum are usually common disaccharides (sucrose, lactose and maltose), small-molecule organic acids (succinic acid, malic acid and citric acid) and partial amino acids. The stronger absorption peak appears in 15-60 minutes is mostly organic phenolic acid, flavonoid glycoside and flavonoid substances. The conclusion that can be drawn from the graphical results is: the water-soluble sugar, amino acid and small molecular organic acid in the cut and cultivated horn flower are similar to the wild species, and the content of the water-soluble sugar, amino acid and small molecular organic acid is not lower than that of the wild species; the types of organic phenolic acid, flavonoid glycoside and flavonoid substances in the cut and cultivated horn flowers are similar to those of a wild female parent, the content of the organic phenolic acid, flavonoid glycoside and flavonoid substances is higher than that of the wild female parent, and the types of the organic phenolic acid, flavonoid glycoside and flavonoid substances are not lower than that of the wild female parent.

FIG. 4 is a liquid chromatogram showing the peak of the liquid chromatogram having the highest ultraviolet absorption and the mobile phase being phosphoric acid, with a corresponding retention time of 14.54min, and the compound was a flavonoid glycoside locust element (error between measured and theoretical molecular weight: 3.5 ppm) as determined by mass spectrum analysis.

From the results of the figures, the content of the locust element in the horned flowers in the 1 year growing period of the cutting cultivation is obviously higher than that in the horned flowers in the 3 years of the wild cultivation.

Comparative example 1 direct cutting of wild carob plants

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling red loam and sand (6:4) sterilized by chlorothalonil with the mass concentration of 75%, uniformly mixing, and then placing the mixture on a furrow in a row. Selecting small lateral branches with 3-4 leaves with shorter growing period on wild cornflower plants as cutting slips. The cutting base was first soaked in 2.5% rooting powder (ABT) solution for 15 seconds. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture, wherein the heights of the top of the agricultural film and the ridge surface are about 0.8 meter. Spraying 0.01% dipotassium hydrogen phosphate water for 2 times every day before cutting roots, spraying 1000 times of diluted sterilizing agent carbendazim once every week in one month until the leaf surfaces and nutrient soil in the nutrient bags are wet, and preventing the roots of the seedlings from rotting. When the average temperature of the seedling bud agricultural film greenhouse is 26 ℃ and the relative humidity is 60-75%, the cuttage branches wither after 5-8 days, and are difficult to root and survive.

Comparative example 2 direct cutting of wild carob plants

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling the nutrition bag with the brick red loam sterilized by chlorothalonil with the mass concentration of 75% and sand (6:4), uniformly mixing, and then placing the nutrition bag on a furrow in a row. Selecting small lateral branches with 2-3 leaves with shorter growing period on wild cornflower plants as cutting slips. The cutting base was first soaked in 2.5% rooting powder (ABT) solution for 15 seconds. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture, wherein the heights of the top of the agricultural film and the ridge surface are about 0.8 meter. Spraying 0.01% dipotassium hydrogen phosphate water for 2 times every day before cutting roots, spraying 1000 times of diluted sterilizing agent carbendazim once every week in one month until the leaf surfaces and nutrient soil in the nutrient bags are wet, and preventing the roots of the seedlings from rotting. When the average temperature of the seedling sprout in the greenhouse is 29 ℃ and the relative humidity is 65-85%, the cuttage branches wither after 3-6 days, and are difficult to root and survive.

Comparative example 3 direct cutting of wild carob plants

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Selecting a nutrition bag with the size of 8cm multiplied by 12cm, filling black gray soil and sand (6:4) sterilized by chlorothalonil with the mass concentration of 75%, uniformly mixing, and then placing the mixture on a furrow in a row. And selecting small lateral branches with 4-6 leaves with shorter growing period on wild cornflower plants as cutting slips. The cutting base was first soaked in 2.5% rooting powder (ABT) solution for 15 seconds. Then cutting into prepared nutrition bags, covering with agricultural film, and keeping temperature and moisture, wherein the heights of the top of the agricultural film and the ridge surface are about 0.8 meter. Spraying 0.01% dipotassium hydrogen phosphate water for 2 times every day before cutting roots, spraying 1000 times of diluted sterilizing agent carbendazim once every week in one month until the leaf surfaces and nutrient soil in the nutrient bags are wet, and preventing the roots of the seedlings from rotting. When the average temperature of the seedling bud agricultural film greenhouse is 22 ℃ and the relative humidity is 45-65%, the cuttage branches wither after 4-6 days, and are difficult to root and survive.

Comparative example 4 seed of wild carob plant was directly sown in soil for germination

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Grooves with the upper opening width of 8-10 cm and the lower opening width of less than 1 cm and the depth of 3-4 cm are formed in the furrow, preselected horn flower seeds are spread in rows along the bottom of the grooves, then soil is covered, and water is sprayed once a day, wherein the soil surface is wet. The average temperature of the moon in the greenhouse is 25 ℃, the relative humidity is 50-70%, the seedlings germinate and emerge in 3-4 weeks, the emergence rate is 5%, and the survival rate is low.

Comparative example 5 seed of wild carob plant was directly sown in soil for germination

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Grooves with the upper opening width of 8-10 cm and the lower opening width of less than 1 cm and the depth of 3-4 cm are formed in the furrow, preselected horn flower seeds are spread in rows along the bottom of the grooves, then soil is covered, and water is sprayed once a day, wherein the soil surface is wet. The average temperature of the moon in the greenhouse is 22 ℃, the relative humidity is 40-65%, the seedlings germinate for 3-4 weeks, the emergence rate is 2%, and the survival rate is low.

Comparative example 6 seed of wild carob plant was directly sown in soil for germination

And (5) preparing soil into furrows in a standard seedling greenhouse, and ensuring smooth drainage. Grooves with the upper opening width of 8-10 cm and the lower opening width of less than 1 cm and the depth of 3-4 cm are formed in the furrow, preselected horn flower seeds are spread in rows along the bottom of the grooves, then soil is covered, and water is sprayed once a day, wherein the soil surface is wet. The average temperature of the moon in the greenhouse is 28 ℃, the relative humidity is 55-80%, the seedlings germinate for 3-4 weeks, the emergence rate is 6%, and the survival rate is low.

Comparative example 7 cutting seedling raising with different numbers of true leaves

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedling is planted for 30 days, most of the mother seedlings grow 6-10 true leaves, the mother seedlings are selected to trim and topping and bud accumulation among 6-10 true leaves, lateral buds grow 3-5 days after the mother seedling plants are topping, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. A nutrient bag with the size of 8cm x 12cm is selected and filled with the brick red loam sterilized by potassium permanganate with the mass concentration of 0.01 percent: sand=6:4, mixed well, and then placed in rows on the furrow. And selecting the lateral branches with 1-2 true leaves in 18-20 days of growing period as cutting shoots. Firstly, soaking the base of the cutting slip in 2% rooting powder (ABT) solution for 15 seconds, then respectively cutting the cutting slip into prepared nutrition bags, and then covering an agricultural film for heat preservation and moisture preservation, wherein the heights of the top of the agricultural film and the furrow surface are about 1.10 meters.

3. Seedling raising process

According to the change of the weather temperature and humidity, 0.01% dipotassium hydrogen phosphate water is sprayed for 5 times every day before the cutting shoots grow roots, and the nutrition soil in the leaf surfaces and the nutrition bags is moistened to the extent that 1000 times of diluted carbendazim sterilizing agent is sprayed once every week in one month, so that the root rot of the seedling is prevented. And when the month average temperature in the seedling sprout agricultural film greenhouse is 29 ℃ and the relative humidity is 65-85%, the cutting seedlings start to root on the 7 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 3-4 true leaves and the height is more than 10 cm.

Comparative example 8 cutting seedling raising with different true leaf numbers

1. Mother seedling cultivation process

Selecting a land block in a nursery, which is subjected to composting, tedding, loosening and leveling, selecting a strong seed seedling, planting the seed seedling on the prepared furrow according to row plant spacing of 25cm multiplied by 25cm, post-planting root setting water for 1 time, and keeping the seedling teeth moist and smooth in drainage. After the mother seedling is planted for 30 days, most of the mother seedlings grow 6-10 true leaves, the mother seedlings are selected to trim and topping and bud accumulation among 6-10 true leaves, lateral buds grow 3-5 days after the mother seedling plants are topping, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions.

2. Cutting seedling raising

And (3) preparing soil into furrows in the seedling greenhouse, and ensuring smooth drainage. A nutrient bag with the size of 8cm x 12cm is selected and filled with the brick red loam sterilized by potassium permanganate with the mass concentration of 0.01 percent: sand=6:4, mixed well, and then placed in rows on the furrow. And selecting the lateral branches with 5-6 true leaves in 18-20 days of growing period as cutting shoots. Firstly, soaking the base of the cutting slip in 2% rooting powder (ABT) solution for 15 seconds, then respectively cutting the cutting slip into prepared nutrition bags, and then covering an agricultural film for heat preservation and moisture preservation, wherein the heights of the top of the agricultural film and the furrow surface are about 1.10 meters.

3. Seedling raising process

According to the change of the weather temperature and humidity, 0.01% dipotassium hydrogen phosphate water is sprayed for 5 times every day before the cutting shoots grow roots, and the nutrition soil in the leaf surfaces and the nutrition bags is moistened to the extent that 1000 times of diluted carbendazim sterilizing agent is sprayed once every week in one month, so that the root rot of the seedling is prevented. And when the month average temperature in the seedling sprout agricultural film greenhouse is 29 ℃ and the relative humidity is 65-85%, the cutting seedlings start to root on the 7 th day. And removing the agricultural film after one month, and keeping the shading degree of 60% in the greenhouse. After the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings are transplanted when the seedlings grow to have 7-8 true leaves and the height is more than 30 cm.

TABLE 3 comparative example 7, 8 results of cutting seedling test

From the results, the cutting seedling method has high seedling emergence rate and short period, is an ideal cutting seedling technical method at present, has the advantages that the conditions required by seedling are generally easy to reach, the operation method is simple and convenient, the popularization and propagation are easy to realize in farmers, and a foundation is laid for large-scale popularization and planting of the cornflower.

The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.

Claims (9)

1. An artificial cultivation method of elaeagnus pungens is characterized in that: the method comprises the following steps:

s1, seed germination and seed seedling cultivation

(1) Collecting thorny elaeagnus seed, selecting, soaking the seed in ethanol, and washing;

(2) Adding sodium hypochlorite solution, mixing, soaking, washing seed, and re-suspending seed;

(3) Inoculating the seed suspension into a seed germination culture medium, removing excessive water on the surface of the culture medium, and sealing;

(4) Culturing for 14-16 days at 25+/-2 ℃ under 16h light/8 h dark condition, transferring the plantlets obtained by seed germination into a strong seedling culture medium, and transplanting after 2-3 weeks;

(5) Taking out the seedlings, cleaning the roots, transplanting the seedlings into a nutrition bag filled with sterilized composite nutrient soil, shading the seedlings by 60% and culturing for 14 days, removing a shading net at the top of the greenhouse, and continuing culturing until the seedlings grow to have 5-7 true leaves;

the formula of the seed germination culture medium is as follows: MS culture medium +30g/L sucrose +7.5g/L agar powder, pH5.8;

the formula of the strong seedling culture medium is as follows: 1/2MS culture medium +1mg/L indolebutyric acid +0.1mg/L naphthylacetic acid +30g/L sucrose +7.5g/L agar powder, pH5.8;

s2, mother seedling cultivation

Selecting a land block which is subjected to composting, tedding, loosening and leveling in a nursery, selecting a strong seed seedling, and planting on the prepared furrow according to row plant spacing of 25cm multiplied by 25 cm; watering root fixing water for 1 time after planting, and keeping the teeth moist and smooth in drainage; after field planting the mother seedlings for 30 days, most mother seedlings can grow 6-10 true leaves, and the mother seedlings with 6-10 true leaves are selected for pruning, topping and bud accumulation; lateral buds grow 3-5 days after the mother plant is topped, and 2-3 robust lateral buds are reserved for each mother plant according to actual conditions;

s3, cutting seedling

Preparing soil into furrows in a seedling raising greenhouse with the shading degree of 60-70%, selecting a nutrition bag, filling sterilized composite nutrient soil, uniformly mixing, and then placing the composite nutrient soil on the furrows in rows; selecting lateral branches with 3-4 true leaves as cutting shoots in 18-20 days of growing period; firstly, soaking a cutting root part in a rooting powder ABT solution with the mass concentration of 1.25% -2.5% for 10-20 seconds, or soaking the cutting root part in a sucrose solution with the mass concentration of 5% -10% for 11-13 hours, then respectively cutting the cutting root part into prepared nutrition bags, covering an agricultural film, preserving heat and moisture, and enabling the heights of the top of the agricultural film and a furrow surface to be 0.7-1.1 m;

wherein, the composite nutrient soil in the steps S1 and S3 is red loam or brick red loam and sand, and the mass ratio is 6:4, mixing or mixing black dust and sand according to a mass ratio of 8: 2;

s4, seedling raising process

Spraying dipotassium hydrogen phosphate water every day before the cutting shoots grow roots according to the change of the weather temperature and the humidity, and slightly wetting the leaf surfaces and the nutrient soil in the nutrient bags; spraying sterilizing agent once every week in one month to prevent root rot of seedling; the average temperature of the agricultural film in the greenhouse is kept at 23-29 ℃, the relative humidity is 45-85%, the cutting seedlings start to root in 6-8 days, the agricultural film is removed 28-32 days after the rooting, and the shading degree of 60-70% in the greenhouse is kept; after the agricultural film is uncovered for 10 days, the shading net at the top of the greenhouse is uncovered, so that seedlings are directly aired, and seedlings can emerge and transplanted when the cutting seedlings grow to have 6-7 true leaves and the height is more than 20 cm.

2. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the mass concentration of the ethanol in the step S1 is 75%, and the soaking time is 1 minute; the mass concentration of the sodium hypochlorite is 2%, and the soaking time is 10 minutes.

3. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the specific operation of collecting the elaeagnus pungens seeds in the step S1 is as follows: collecting fruits in middle and late 3 months, stacking in the shade for several days, mashing the fruits to collect seeds, removing impurities by using a 50-60 mesh sieve, cleaning with clear water, and airing in the shade to obtain the elaeagnus pungens seeds.

4. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the specific operation of selecting seeds in the step S1 is as follows: removing seeds with peculiar smell; selecting yellow-red seeds, and removing the yellow-brown seeds; and removing the seeds with lighter weight, and the remaining seeds with average thousand grain weight of more than 0.041g can be used for the next step.

5. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the reagents used for the disinfection in the steps S1 and S3 are chlorothalonil with the mass concentration of 75% or potassium permanganate with the mass concentration of 0.01%.

6. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the size of the nutrition bag described in step S3 was 8cm by 12cm.

7. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the mass concentration of the dipotassium hydrogen phosphate water in the step S4 is 0.01%.

8. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

the sterilizing agent in the step S4 is carbendazim, the mass concentration is 50%, the sterilizing agent is diluted by water for 1000 times before use, and the using amount is suitable for wetting leaf surfaces and nutrient soil in a nutrient bag.

9. The artificial cultivation method of elaeagnus pungens according to claim 1, wherein:

and (2) applying 1000 kg of compost and 200 kg of plant ash to the land according to the standard land preparation of each mu of land in the step (S1) and then using the land.

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