CN112237141A - Tissue culture propagation method of sclerotium rolfsii - Google Patents
Tissue culture propagation method of sclerotium rolfsii Download PDFInfo
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- CN112237141A CN112237141A CN202011188663.2A CN202011188663A CN112237141A CN 112237141 A CN112237141 A CN 112237141A CN 202011188663 A CN202011188663 A CN 202011188663A CN 112237141 A CN112237141 A CN 112237141A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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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 the annual young branches of the sclerotium rolfsii 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: carrying out 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
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to a tissue culture propagation method of a sclerotium rolfsii.
Background
The height of the rhizoctonia (Symphoricarpos sinensis Rehd.) and deciduous shrubs of the genus rhizoctonia of the family Caprifoliaceae can reach 1.5-2.0 m, the branches are dense and soft in arch shape, and are shoddy and reddish brown. 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. After the flowers wither, the flowers grow round berries, the fruits are oval, 7-10 mm in length and have white frost, the fruits cluster on the long strip-shaped branches in clusters, the fruits turn red in late autumn, the fruits are extremely beautiful, the fruits are always fruited until the next early spring, and the whole fruitage period reaches 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 hair stone wood is a cold-resistant, light-loving, new and excellent fruit plant.
Seeding and cutting propagation are used at the present stage. The method comprises the steps of sowing the seeds in early spring berries, completely curing the seeds, picking and collecting the seeds, drying the seeds in the sun, removing peels of the seeds, soaking the seeds in warm water at the temperature of 30-40 ℃ for 24 hours (due to hard seed coats), and then sowing the seeds 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 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;
b. primary culture: inoculating the stem segments with the buds after the 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;
c. and (3) proliferation culture: cutting off axillary buds of the primary seedlings, inoculating the 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 seedlings 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 rooted seedlings;
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 upper cover with a plug cover at the initial stage of transplanting, spraying water once every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times every morning to keep the leaf surface humidity, spraying 800-time carbendazim water solution once a week, and performing fertilizer water and pest and disease management regularly.
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 diagram of a primary seedling of a Rhizoctonia solani 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, the pH value is 6.3-6.7, the primary culture condition is 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 is 6.3-6.7, the proliferation culture conditions comprise illumination intensity of 2000-3000 LX, illumination time of 14-18 h/d and temperature of 24-30 ℃, and the culture is carried out for 7 days to obtain a proliferation seedling, the base of a stem section begins to expand, a bud point begins to germinate, the bud basically grows to 3-5cm after about 30 days, the bud is relatively thick 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 is 6.3-6.7, the rooting 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 rooting culture conditions are 3 weeks, so that the rooting seedlings are obtained, the plant grows for 3-5cm, the number of the rooting is 2-5, and the root length is 2-4 cm.
(5) 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 upper cover with a plug cover at the initial stage of transplanting, spraying water once every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times every morning to keep the leaf surface humidity, spraying 800-time carbendazim water solution once a week, and performing fertilizer water and pest and disease management regularly.
Examples 2 to 3
To obtain the optimal proliferation medium composition, comparative experiments were performed in examples 2-3, 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 propagation Medium composition on propagation culture of Rhizoctonia solani
Examples 4 to 5
To obtain the optimal composition of the rooting medium, comparative experiments were performed in examples 4-5, and the other experimental steps were the same as in example 1, with 10 explants per experiment being 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 cerealis is the highest in the culture medium of 1/2MS +0.1mg/L IBA, the growth cycle is short, and the average root number is uniform and consistent with the average root length.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.
Claims (4)
1. The tissue culture propagation method of the sclerotium rolfsii is characterized by comprising 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 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;
b. primary culture: inoculating the stem segments with the buds after the 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;
c. and (3) proliferation culture: cutting off axillary buds of the primary seedlings, inoculating the 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 seedlings 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 rooted seedlings;
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 upper cover with a plug cover at the initial stage of transplanting, spraying water once every morning to keep the leaf surface humidity, uncovering the plug cover after transplanting into the plug for 15 days, spraying water 2 times every morning to keep the leaf surface humidity, spraying 800-time carbendazim water solution once a week, and performing fertilizer water and pest and disease management regularly.
2. The tissue culture propagation method of the sclerotium rolfsii according to claim 1, wherein the composition of the primary culture medium comprises: MS +30g/L sucrose +5g/L agar, pH = 6.3-6.7.
3. The tissue culture propagation method of the sclerotium rolfsii according to claim 1, wherein the propagation medium comprises: DKW +0.8mg/L ZT +0.3mg/L GA +30g/L sucrose +5g/L agar, pH = 6.3-6.7.
4. The tissue culture propagation method of the sclerotium rolfsii according to claim 1, characterized in that, the rooting medium comprises: 1/2MS +0.1mg/L IBA +20g/L sucrose +5g/L agar, pH = 6.3-6.7.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USPP21226P2 (en) * | 2009-02-27 | 2010-08-24 | Catharina Marie Hoekstra-Arisz | Symphoricarpos plant named ‘Sofie’ |
| CN102119615A (en) * | 2010-11-29 | 2011-07-13 | 天津滨海国际花卉科技园区股份有限公司 | Method for boosting waxberry cuttage rooting |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USPP21226P2 (en) * | 2009-02-27 | 2010-08-24 | Catharina Marie Hoekstra-Arisz | Symphoricarpos plant named ‘Sofie’ |
| CN102119615A (en) * | 2010-11-29 | 2011-07-13 | 天津滨海国际花卉科技园区股份有限公司 | Method for boosting waxberry cuttage rooting |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG YI等: "Characteristic of the complete chloroplast genome of Symphoricarpos sinensis, an important ornamental plant endemic to China", 《MITOCHONDRIAL DNA PART B》 * |
| 李国平: "毛核木嫩枝全光喷雾扦插繁育试验初报", 《上海农业科技》 * |
| 桂炳中等: "华北地区白雪果栽培养护", 《中国花卉园艺》 * |
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