CN112237141B - Tissue culture propagation method of sclerotium rolfsii - Google Patents

Abstract

The invention belongs to the technical field of plant propagation, and discloses a tissue culture propagation method of a sclerotium rolfsii, which comprises the following steps: and (3) explant sterilization: taking young branches of the current year of the sclerotium wood without diseases and insect pests, removing leaves, cutting into 2-3 cm stem sections with buds, and sterilizing; primary culture: performing primary culture to obtain primary seedlings; and (3) proliferation culture: performing enrichment culture to obtain an enrichment seedling; rooting culture: cutting the proliferated seedling into 2-3 stem segments with buds, inoculating the stem segments into a rooting culture medium, and culturing for 3 weeks to obtain a rooted seedling; hardening and transplanting seedlings: selecting a rooting seedling with strong plants and strong and viable root system, hardening off the seedling for 5d by closing the bottle, hardening off the seedling for 5d by opening the bottle, pulling out the seedling, soaking the tissue culture seedling in 800 times of carbendazim aqueous solution for 30min, and transplanting the tissue culture seedling into a plug tray. The invention establishes a tissue culture and rapid propagation system of the sclerotium rolfsii, has simple culture medium formula, low culture medium cost and rapid propagation speed, is not influenced by seasonal climate change and natural disasters, and provides technical support for industrialized seedling culture and deep processing of the sclerotium rolfsii.

Description

Tissue culture propagation method of sclerotium rolfsii

Technical Field

The invention belongs to the technical field of plant propagation, and particularly relates to a tissue culture propagation method of a karelinia caspica.

Background

The height of the tree of the genus Rhizoctonia of Caprifoliaceae can reach 1.5-2.0 m, and the branches are dense and soft in arch shape and are reddish brown by short villi. The leaf is full-edged, rhomboid-shaped egg-shaped to egg-shaped, 1.5-2.5cm long, 1.2-1.8cm wide, green above, and grey white below. The flower is white, the length is 5-7 mm, the flowering phase is 7-8 months, the flower type is small, and the flower color is light pink. The flowers wither to form round berries, the fruits are oval and 7-10 mm long, the white berries are frosted, bunch-shaped and cluster on the long strip-shaped branches, the fruits turn red in late autumn, the fruits are extremely beautiful, the fruits are always fruited until the next spring, and the whole fruiting period is up to four months. The sclerotium has strong adaptability, cold resistance, heat resistance, moisture resistance and barren resistance, few plant diseases and insect pests and strong sprouting capacity, and the branches can grow in the internode after falling to the ground.

The hairy kernel wood is a fruit-viewing plant with great ornamental value, the main ornamental value is fruit viewing, the branches of the plant are full of ginkgo in each fruit period, and the hairy kernel wood can be used as an excellent fruit-viewing pot plant and a branch cutting material in gardening production, so that the variety of the fruit-viewing plant is enriched. The tree can be used as excellent fruit-viewing plant, suitable for greening in courtyard, park, residence community and elevated road and bridge, also can be used as potted plant for appreciation, and has wide application. The sclerotium wood is cold-resistant, glaring and a new excellent fruit plant.

Seeding and cutting propagation are adopted at the present stage. The seeds are sowed in early spring berries which are completely cured, picked and collected, put in the sun for drying, then the peels of the berries are rubbed off, and then the berries are soaked in warm water at the temperature of between 30 and 40 ℃ for 24 hours (because the seed coats are hard) and then sowed on a seedbed. Cutting in 5-6 months, cutting the annual semi-lignified branch, inserting in sandy soil, shading, and keeping moisture; however, the ornamental value of the sclerotium rolfsii is extremely high, so that the traditional method cannot meet the market demand.

Disclosure of Invention

The invention aims to provide a tissue culture propagation method of a sclerotium rolfsii, which has the advantages of simple formula of a used culture medium, low cost of the culture medium, simple and convenient culture process and high propagation speed.

In order to achieve the purpose, the invention adopts the following technical scheme:

a tissue culture propagation method of a sclerotium rolfsii comprises the following steps:

a. and (3) explant sterilization: taking annual young branches of the sclerotium rolfsii without diseases and insect pests, removing leaves, cutting into 2-3 cm stem sections with buds, placing on a clean bench, sterilizing with 75% alcohol for 10s, sterilizing with 2% sodium hypochlorite for 3min, and washing with sterile water for 4-6 times;

b. primary culture: inoculating the stem segments with buds after sterilization treatment into a culture bottle filled with a primary culture medium for primary culture under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃ for 2-3 weeks to obtain primary seedlings;

c. and (3) proliferation culture: cutting the axillary buds of the primary generation seedlings, inoculating the cut axillary buds to a proliferation culture medium for proliferation culture under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃, and culturing for 7 days to obtain proliferation seedlings;

d. rooting culture: cutting the proliferated seedling into 2-3 stem segments with buds, inoculating the stem segments with buds to a rooting culture medium, and culturing for 3 weeks under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃ to obtain a rooted seedling;

e. hardening and transplanting seedlings: selecting a rooting seedling with strong plants and strong and viable root systems, hardening the seedling for 5d by closing a bottle, hardening the seedling for 5d by opening the bottle, pulling out, soaking the tissue culture seedling in 800 times of carbendazim aqueous solution for 30min, transplanting the tissue culture seedling into a plug tray, and filling a substrate into the plug tray in a volume ratio of imported peat: perlite =6: and 4, covering the cover with a plug cover in the initial stage of transplanting, spraying water once in the plug cover every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times in the morning every day to keep the leaf surface humidity, spraying 800-time carbendazim aqueous solution once a week, and regularly performing fertilizer water and pest and disease management, wherein the humidity in the plug cover is 80 percent RH, the leaf surface humidity is kept, the plug cover is uncovered, the temperature in the greenhouse is 24 ℃, the humidity is 50 percent RH, and the leaf surface humidity is kept.

Preferably, the primary medium composition comprises: MS +30g/L sucrose +5g/L agar, pH = 6.3-6.7.

Preferably, the propagation medium composition comprises: DKW +0.8mg/L ZT +0.3mg/L GA +30g/L sucrose +5g/L agar, pH = 6.3-6.7.

Preferably, the rooting medium comprises: 1/2MS +0.1mg/L IBA +20g/L sucrose +5g/L agar, pH = 6.3-6.7.

Compared with the prior art, the invention has the beneficial effects that:

the invention establishes a tissue culture rapid propagation system of the sclerotium hirsutum, the formula of the used culture medium is simple, the cost of the culture medium is low, the culture process is simple and convenient, the propagation speed is high, and the sclerotium hirsutum aseptic seedlings with consistent genetic characters can be rapidly obtained; the invention utilizes the tissue culture technology to culture explants to obtain the sclerotium rolfsii aseptic seedlings, the method is not influenced by seasonal climate change and natural disasters, and the invention provides technical support for the industrialized seedling culture and deep processing of the sclerotium rolfsii.

Drawings

FIG. 1 is a schematic diagram of the primary seedling of the sclerotium hirsutum in example 1.

FIG. 2 is a schematic diagram of a seedling of Rhizoctonia solani obtained in example 1.

FIG. 3 is a diagram of a rooted seedling of Rhizoctonia solani as shown in example 1.

FIG. 4 is the root of the rooted seedling of the hair-seed wood of FIG. 3.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

Example 1

(1) And (3) explant sterilization: taking the annual young branches of the sclerotium rolfsii without diseases and insect pests, removing leaves, cutting into 2-3 cm stem sections with buds, placing on a super clean workbench, sterilizing with 75% alcohol for 10s, sterilizing with 2% sodium hypochlorite for 3min, and washing with sterile water for 4-6 times.

(2) Primary culture: inoculating the stem with the bud after the sterilization treatment into a culture bottle filled with a primary culture medium for primary culture, wherein the primary culture medium comprises the following components: MS +30g/L sucrose +5g/L agar, pH = 6.3-6.7, and the primary culture conditions are that the illumination intensity is 2000-3000 LX, the illumination time is 14-18 h/d, the temperature is 24-30 ℃, and the primary seedling is cultured for 2-3 weeks to obtain the primary seedling.

(3) And (3) proliferation culture: cutting off axillary buds of the primary generation seedlings, inoculating the axillary buds to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium comprises the following components: DKW +0.8mg/L Zeatin (ZT) +0.3mg/L Gibberellic Acid (GA) +30g/L sucrose +5g/L agar, pH = 6.3-6.7, and the proliferation culture conditions are that the illumination intensity is 2000-3000 LX, the illumination time is 14-18 h/d, the temperature is 24-30 ℃, the culture is carried out for 7 days, the proliferation seedlings are obtained, the base parts of stem sections begin to swell, the bud points begin to germinate, the buds basically grow to 3-5cm in about 30 days, the buds are thicker and the proliferation coefficient is more than 3.

(4) Rooting culture: cutting the proliferated seedling into 2-3 stem segments with buds, and inoculating the stem segments with buds into a rooting culture medium, wherein the rooting culture medium comprises the following components: 1/2MS +0.1mg/L IBA +20g/L sucrose +5g/L agar, pH = 6.3-6.7, and the rooting culture conditions comprise illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃, and are cultured for 3 weeks to obtain rooted seedlings, wherein the plant grows for 3-5cm, the number of roots is 2-5, and the root length is 2-4cm.

(5) Hardening and transplanting seedlings: selecting rooting seedlings with strong plants, strong root systems and vitality, hardening the seedlings, closing bottles, hardening the seedlings for 5d, opening bottles, hardening the seedlings for 5d, pulling out, soaking the tissue culture seedlings in 800 times of carbendazim aqueous solution for 30min, transplanting the soaked seedlings into a hole tray, filling a substrate in the hole tray in a volume ratio of imported peat: perlite =6: and 4, covering the cover with a plug cover in the initial stage of transplanting, spraying water once in the plug cover every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times in the morning every day to keep the leaf surface humidity, spraying 800-time carbendazim aqueous solution once a week, and regularly performing fertilizer water and pest and disease management, wherein the humidity in the plug cover is 80 percent RH, the leaf surface humidity is kept, the plug cover is uncovered, the temperature in the greenhouse is 24 ℃, the humidity is 50 percent RH, and the leaf surface humidity is kept.

Examples 2 to 3

To obtain the optimum proliferation medium composition, comparative experiments were carried out in examples 2 to 3, and the other experimental procedures were the same as in example 1, 10 explants were treated in each experiment and repeated 3 times, and the experimental results are shown in Table 1. The success of tissue culture depends largely on the choice of medium. Different culture media have different characteristics and are suitable for different plant species and inoculation materials. As is clear from Table 1, DKW +0.8mg/L ZT +0.3mg/L GA medium exhibited the best effect on the growth of Rhizoctonia solani, and the shoots were strong.

TABLE 1 Effect of proliferation Medium composition on proliferation culture of Rhizoctonia solani

Examples 4 to 5

To obtain the optimal composition of the rooting medium, comparative experiments were carried out in examples 4 to 5, with the other experimental steps being the same as in example 1, with 10 explants being treated in each experiment and repeated 3 times. The test results are shown in table 2.

TABLE 2 Effect of different rooting Medium Components on rooting culture of Rhizoctonia solani

As can be seen from Table 2, the rooting rate of the rhizoctonia wood is the highest on the culture medium of IBA of 1/2MS +0.1mg/L, the growth cycle is short, and the average root number is uniform and consistent with the average root length.

The above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and it is obvious to those skilled in the art that other embodiments can be easily made by replacing or changing the technical contents disclosed in the present specification, and therefore, all changes and modifications made on the principle of the present invention should be included in the claims of the present invention.

Claims (1)

1. The tissue culture propagation method of the sclerotium rolfsii is characterized by comprising the following steps:

a. and (3) explant sterilization: taking the annual young branches of the sclerotium rolfsii without diseases and insect pests, removing leaves, cutting into 2-3 cm stem sections with buds, placing on a super clean workbench, sterilizing with 75% alcohol for 10s, 2% sodium hypochlorite for 3min, and washing with sterile water for 4-6 times;

b. primary culture: inoculating the stem segments with buds after sterilization treatment into a culture bottle filled with a primary culture medium for primary culture under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃, and culturing for 2-3 weeks to obtain primary seedlings; the primary culture medium comprises the following components: MS +30g/L sucrose +5g/L agar, pH = 6.3-6.7;

c. and (3) proliferation culture: cutting the axillary buds of the primary generation seedlings, inoculating the cut axillary buds to a proliferation culture medium for proliferation culture under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃, and culturing for 7 days to obtain proliferation seedlings; the proliferation culture medium comprises the following components: DKW +0.8mg/L ZT +0.3mg/L GA +30g/L sucrose +5g/L agar, pH = 6.3-6.7;

d. rooting culture: cutting the proliferated seedling into 2-3 stem segments with buds, inoculating the stem segments into a rooting culture medium, and culturing for 3 weeks under the conditions of illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃ to obtain a rooted seedling; the rooting medium comprises the following components: 1/2MS +0.1mg/L IBA +20g/L sucrose +5g/L agar, pH = 6.3-6.7;

e. hardening and transplanting seedlings: selecting a rooting seedling with strong plants and strong and viable root systems, hardening the seedling for 5d by closing a bottle, hardening the seedling for 5d by opening the bottle, pulling out, soaking the tissue culture seedling in 800 times of carbendazim aqueous solution for 30min, transplanting the tissue culture seedling into a plug tray, and filling a substrate into the plug tray in a volume ratio of imported peat: perlite =6: and 4, covering the cover with a plug cover in the initial stage of transplanting, spraying water once in the plug cover every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times in the morning every day to keep the leaf surface humidity, spraying 800-time carbendazim aqueous solution once a week, and regularly performing fertilizer water and pest and disease management, wherein the humidity in the plug cover is 80 percent RH, the leaf surface humidity is kept, the plug cover is uncovered, the temperature in the greenhouse is 24 ℃, the humidity is 50 percent RH, and the leaf surface humidity is kept.

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