CN114711144A - Rapid propagation method and culture medium for dioscorea composita tissue culture seedlings - Google Patents
Rapid propagation method and culture medium for dioscorea composita tissue culture seedlings Download PDFInfo
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Images
Classifications
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Abstract
The invention belongs to the technical field of plant tissue culture, and provides a rapid propagation method and a culture medium of dioscorea composita seedlings. The invention takes dioscorea composita axillary buds as explants, callus is promoted to be generated on a culture medium of improved MS +2mg/L6-BA +0.2mg/LNAA, callus proliferation is promoted on a culture medium of MS +2mg/L6-BA +0.2mg/LNAA + 0.05% glutamic acid, cluster bud redifferentiation is promoted on MS +2mg/L6-BA +0.4mg/L NAA + 0.01% vitamin E + 0.025% selenocysteine, and axillary buds are promoted to generate a complete root structure on improved MS +1mg/L IBA +0.1 mg/LNAA. The invention provides a technical foundation for industrial scale breeding of the dioscorea mexicana and also lays a technical foundation for gene engineering improvement and molecular breeding of the dioscorea mexicana.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation method and a culture medium of dioscorea composita seedlings.
Background
Dioscorea composita is a perennial wound herb of the genus Dioscorea, the family Dioscoreaceae, also known as Dioscorea mexicana. The dioscorea composita is mostly distributed in the middle of the coast of gulf of mexico and the south of the coast of pacific, and is suitable for growing in the area with the altitude of 1000 minus 1500 meters, the average annual temperature of 25-27 ℃, the relative annual humidity of 86 percent and the annual precipitation of 1300 minus 1500 millimeters. Dioscorea composita is known for its high diosgenin content in its rhizome. In the 70 s of the 20 th century, China introduced it to the Xishuangbanna of Yunnan province for the first time and successfully tried the seeds. The Xishuangbanna is located at the edge of the northern part of the tropical zone, so the Xishuangbanna has the climate of the tropical season rainforest, so the average temperature and humidity level of the zone are basically similar to that of Mexico in the origin area, and the Xishuangbanna has the advantage of being unique, so the Dioscorea composita grows well in the zone, and the yield and the saponin content of the Dioscorea composita little different from the origin area. Then, the seeds are introduced and planted into places such as Panzhihua in Sichuan, Shaanxi Ankang, Hainan Haikou, Guangxi Nandan and the like in succession, and a certain scale is formed at present.
Dioscorea composita is a perennial wound herb, and is different from Dioscorea zingiberensis of the rhizome group, Dioscorea composita belonging to the non-rhizome group, in which tubers thereof grow vertically downward, and the epidermis is rather rough and black brown, or palm-shaped or rod-shaped, and the shapes are different. The abdomen and the back are clear, the root system of the abdomen is developed, and the abdomen grows in tropical areas all the year round and is evergreen in four seasons. The tubers are not shrunken or shriveled along with the increase of the growth years, and simultaneously, the starch and the cellulose are accumulated, so that the old tubers are dark in color, the new tubers grow downwards and vertically from the old tubers, and the new tubers are very young and tender and are yellow-white in color. The growth condition of the tuber is directly related to the soil environment, the loose soil can promote the growth of the dioscorea composita tuber, and the depth and the width of the tuber increase along with the planting years. The overground stem is round in meat quality, the winding mode is a left-handed rotation, the diameter is 0.4-0.7 cm, the new stem is rich in water, soft, fresh and tender, green, the old stem is high in fibrosis degree, straight and thick, and the color is dark green with spotted purple. The blade is in a shape of a heart wedge, a wide oval or an oval, and the tip of the blade is narrowed into a tail shape. The single leaves are alternate, the petioles are purple in color, different in depth and netted in side pulse.
The traditional breeding mode of the dioscorea composita is as follows: seed propagation and tuber propagation. Dioscorea composita serving as a male and female heterotrophic plant has different florescence between female flowers and male flowers, is not full enough in seeds and is only suitable for small-area planting; tubers are bred, the demand for tubers is high, the cost is high, and the growth rate is slow. At present, dioscorea composita is cultured in two modes of tissue culture and cuttage. However, compared with cutting seedling, the tissue culture rapid propagation method has the advantages of easy operation, no limitation of seasons and climates, high propagation coefficient, high survival rate and the like, and has very important significance for rapid propagation of dioscorea composita. Due to the rapid increase of the demand of dioscorea composita at home and abroad and the rapid reduction of the quantity of wild dioscorea composita resources, establishing a suitable tissue culture system to realize the rapid propagation of asexual seedlings is an important way for solving the problem of resource shortage at present.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a rapid propagation method and a culture medium of dioscorea composita seedlings, which utilize a vegetative tissue culture technology to induce callus of different explants, select proper culture medium types of dedifferentiation, proliferation, redifferentiation and rooting to establish a rapid propagation system of dioscorea composita tissues, provide sufficient fine variety seedling for large-scale production of dioscorea composita, and provide technical support for improvement of germplasm resource genetic variation, research of later functional genes and directional culture of high-yield and high-quality new varieties by utilizing genetic engineering.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a dedifferentiation culture medium of a dioscorea composita tissue culture seedling, which takes an improved MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.1～0.3mg·L-12-4 wt% of cane sugar and 4-5 g.L of agar-1The pH value of the dedifferentiation culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L.
The invention also provides a multiplication culture medium of the dioscorea composita tissue culture seedling, wherein the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.1～0.3mg·L-10.04-0.06 wt% of glutamic acid, 2-4 wt% of cane sugar and 4-5 g.L of agar-1And the pH value of the multiplication culture medium is 5-6.
The invention also provides a redifferentiation culture medium of the dioscorea composita tissue culture seedling, wherein the redifferentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.3～0.5mg·L-10.005-0.015 wt% of vitamin E, 0.02-0.03 wt% of selenocysteine, 2-4 wt% of cane sugar and 4-5 g.L of agar-1And the pH value of the redifferentiation culture medium is 5-6.
The invention also provides a rooting culture medium for the dioscorea composita tissue culture seedling, which takes the improved MS culture medium as a basic culture medium and comprises the following components in concentration: IBA 0.5-1.5 mg.L-1，NAA 0.05～0.15mg·L-1And 2-4 wt% of sucrose, wherein the pH value of the rooting culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4～6mmol/L。
The invention also provides a culture medium composition for the rapid propagation of the dioscorea composita tissue culture seedling, which comprises a dedifferentiation culture medium, a proliferation culture medium, a redifferentiation culture medium and a rooting culture medium.
The invention also provides a rapid propagation method of the dioscorea composita tissue culture seedling, which comprises the following steps:
(1) planting dioscorea composita rhizome in matrix soil for bacteria-free culture to obtain dioscorea composita explant;
(2) sequentially disinfecting and washing the dioscorea composita explant, and then placing the dioscorea composita explant into a dioscorea composita seedling dedifferentiation culture medium for dedifferentiation culture to obtain dioscorea composita embryonic callus;
(3) placing the dioscorea composita embryonic callus in a dioscorea composita tissue culture seedling proliferation culture medium for proliferation culture, and then placing the dioscorea composita embryonic callus in a dioscorea composita tissue culture seedling redifferentiation culture medium for redifferentiation culture to obtain dioscorea composita regenerated seedling;
(4) transferring the axilla of the dioscorea composita regenerated seedling to a dioscorea composita tissue culture seedling rooting culture medium for rooting culture to obtain a dioscorea composita complete regenerated seedling;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings, and then transplanting the seedlings into seedling soil for seedling culture to obtain dioscorea composita seedlings.
Preferably, the matrix soil component in the step (1) comprises nutrient soil and vermiculite, and the mass ratio of the nutrient soil to the vermiculite is 2-4: 1;
the illumination intensity of the bacteria-removing culture is 1500-2500 lux, the illumination period of the bacteria-removing culture is 15-17 h/d, the temperature of the bacteria-removing culture is 25-30 ℃, and the time of the bacteria-removing culture is 8-12 min;
the dioscorea composita explant comprises a stem, a leaf, an axillary bud and a terminal bud.
Preferably, the sterilizing in the step (2) comprises sterilizing by using a mercuric chloride solution, the mass concentration of the mercuric chloride solution is 0.05-0.15%, and the sterilizing time of the mercuric chloride solution is 5-15 min;
the number of washing times is 5-7;
the illumination intensity of the dedifferentiation culture is 1500-2500 lux, the illumination period of the dedifferentiation culture is 7-9 h/d, the temperature of the dedifferentiation culture is 25-30 ℃, and the time of the dedifferentiation culture is 10-15 d.
Preferably, the illumination intensity of the enrichment culture in the step (3) is 1500-2500 lux, the illumination cycle of the enrichment culture is 19-21 h/d, the temperature of the enrichment culture is 25-30 ℃, and the time of the enrichment culture is 18-22 d;
the illumination intensity of the redifferentiation culture is 1500-2500 lux, the illumination period of the redifferentiation culture is 15-17 h/d, the temperature of the redifferentiation culture is 25-30 ℃, and the time of the redifferentiation culture is 28-32 d.
Preferably, the illumination intensity of the rooting culture in the step (4) is 1500-2500 lux, the illumination period of the rooting culture is 15-17 h/d, the temperature of the rooting culture is 25-30 ℃, and the time of the rooting culture is 28-32 d;
in the step (5), the hardening time is 1-3 d, the hardening temperature is 20-30 ℃, and the hardening humidity is 50-70%;
the seedling raising soil component in the step (5) comprises turfy soil;
the illumination intensity of the seedling culture in the step (5) is 1500-2500 lux, the illumination period of the seedling culture is 15-17 h/d, the temperature of the seedling culture is 25-30 ℃, and the time of the seedling culture is 8-12 d.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes plant tissue culture technology, takes dioscorea mexicana axillary buds as explants to carry out tissue culture and rapid propagation, establishes a complete system which forms callus through the axillary buds and generates tissue culture seedlings through redifferentiation, provides a technical foundation for industrial scale breeding of dioscorea mexicana, and also lays a material and a technical foundation for genetic engineering improvement and molecular breeding of dioscorea mexicana.
Drawings
FIG. 1 is a graph showing the growth of the callus of dioscorea mexicana axillary bud, the growth, the proliferation, the redifferentiation and the transplantation of the seedling according to example 4 of the present invention (note: A represents the seedling after the seed germination; B represents the callus formed by the axillary bud; C represents the callus formed by 30D; D represents the callus redifferentiation point; E represents the regenerated seedling differentiated from the callus; F represents the regenerated seedling grown for 30D; G represents the rooting culture; H represents the plantlet cultured for 60D for rooting, and I represents the transplantation of the regenerated seedling).
Detailed Description
The invention provides a dedifferentiation culture medium of a dioscorea composita tissue culture seedling, which takes an improved MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg/L6-BA-1，NAA 0.1～0.3mg·L-12-4 wt% of cane sugar and 4-5 g.L of agar-1The pH value of the dedifferentiation culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L.
In the invention, the concentration of 6-BA in the dedifferentiation culture medium of the dioscorea composita tissue culture seedling is 1-3 mg.L-1More preferably 2 mg.L-1(ii) a The concentration of NAA is 0.1-0.3 mg.L-1More preferably 0.2 mg.L-1(ii) a The concentration of the sucrose is 2-4 wt%, and the preferable concentration is 3 wt%; the concentration of agar is 4-5 g.L-1More preferably 4.5 g.L-1(ii) a The pH value of the dedifferentiation culture medium is 5-6, and the preferable pH value is 5.8; the components of the improved MS culture medium comprise: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L, and further preferably comprises: KNO315mmol/L，Ca(NO3)215mmol/L，(NH4)2SO47.5mmol/L and KCl 5 mmol/L. The improved MS culture medium can promote the dioscorea composita to produce embryogenic callus.
The invention also provides a multiplication culture medium of the dioscorea composita tissue culture seedling, wherein the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 6-BA1 ℃3mg·L-1，NAA 0.1～0.3mg·L-10.04-0.06 wt% of glutamic acid, 2-4 wt% of sucrose and 4-5 g.L of agar-1And the pH value of the multiplication culture medium is 5-6.
In the invention, the concentration of 6-BA in the multiplication culture medium of the dioscorea composita seedling is 1-3 mg.L-1More preferably 2 mg.L-1(ii) a The concentration of NAA is 0.1-0.3 mg.L-1More preferably 0.2 mg.L-1(ii) a The concentration of the glutamic acid is 0.04-0.06 wt%, and the preferable concentration is 0.05 wt%; the concentration of the sucrose is 2-4 wt%, and the preferable concentration is 3 wt%; the concentration of agar is 4-5 g.L-1More preferably 4.5 g.L-1(ii) a The pH of the multiplication medium is 5-6, and is more preferably 5.8.
The invention also provides a redifferentiation culture medium of the dioscorea composita tissue culture seedling, wherein the redifferentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.3～0.5mg·L-10.005-0.015 wt% of vitamin E, 0.02-0.03 wt% of selenocysteine, 2-4 wt% of cane sugar and 4-5 g.L of agar-1And the pH value of the redifferentiation culture medium is 5-6.
In the invention, the concentration of 6-BA in the redifferentiation culture medium of the dioscorea composita tissue culture seedling is 1-3 mg.L-1More preferably 2 mg.L-1(ii) a The concentration of NAA is 0.3-0.5 mg.L-1More preferably 0.4 mg.L-1(ii) a The concentration of the vitamin E is 0.005-0.015 wt%, more preferably 0.01 wt%, and the vitamin E is more preferably alpha-tocopherol; the concentration of the selenocysteine is 0.02-0.03 wt%, and the preferred concentration is 0.025 wt%; the concentration of the sucrose is 2-4 wt%, and the preferable concentration is 3 wt%; the concentration of agar is 4-5 g.L-1More preferably 4.5 g.L-1(ii) a The pH value of the redifferentiation medium is 5-6, and the preferable pH value is 5.8. Researches show that the dioscorea composita callus can generate a large amount of ROS signal molecules in the proliferation process, and the substances can be used as second messengers to regulate the differentiation and development of various tissues, so that reducing substances, namely vitamin E and seleno-hemin are added into a culture mediumCystine to reduce the amount of ROS. Finally, the MS culture medium added with the two substances can greatly promote callus redifferentiation under a specific hormone ratio.
The invention also provides a rooting culture medium for the dioscorea composita tissue culture seedling, wherein the rooting culture medium takes an improved MS culture medium as a basic culture medium and comprises the following components in concentration: IBA 0.5-1.5 mg.L-1，NAA 0.05～0.15mg·L-1And 2-4 wt% of sucrose, wherein the pH value of the rooting culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L.
In the invention, the concentration of IBA in the rooting culture medium of the dioscorea composita seedling is 0.5-1.5 mg.L-1More preferably 1 mg.L-1(ii) a The concentration of NAA is 0.05-0.15 mg.L-1More preferably 0.1 mg.L-1(ii) a The concentration of the sucrose is 2-4 wt%, and the preferable concentration is 3 wt%; the pH value of the rooting culture medium is 5-6, and the preferable pH value is 5.8; the components of the improved MS culture medium comprise: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L, and further preferably comprises KNO315mmol/L，Ca(NO3)215mmol/L，(NH4)2SO47.5mmol/L and KCl 5 mmol/L.
The invention also provides a culture medium composition for the rapid propagation of the dioscorea composita tissue culture seedling, which comprises a dedifferentiation culture medium, a proliferation culture medium, a redifferentiation culture medium and a rooting culture medium.
The invention also provides a rapid propagation method of the dioscorea composita tissue culture seedling, which comprises the following steps:
(1) planting dioscorea composita rhizome in matrix soil for bacteria-removing culture to obtain dioscorea composita explant;
(2) sequentially sterilizing and washing the dioscorea composita explant, and then placing the dioscorea composita explant in a dedifferentiation culture medium of the dioscorea composita seedling for dedifferentiation culture to obtain an embryogenic callus of dioscorea composita;
(3) placing the dioscorea composita embryonic callus in a dioscorea composita seedling proliferation culture medium for proliferation culture, and then placing the dioscorea composita seedling redifferentiation culture medium for redifferentiation culture to obtain a dioscorea composita regenerated seedling;
(4) transferring the axilla of the dioscorea composita regenerated seedling to a dioscorea composita tissue culture seedling rooting culture medium for rooting culture to obtain a dioscorea composita complete regenerated seedling;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings, and then transplanting the seedlings into seedling soil for seedling culture to obtain dioscorea composita seedlings.
In the invention, the matrix soil component in the step (1) preferably comprises nutrient soil and vermiculite, and the mass ratio of the nutrient soil to the vermiculite is preferably 2-4: 1, and more preferably 3: 1.
In the invention, the illumination intensity of the bacteria-removing culture in the step (1) is preferably 1500-2500 lux, more preferably 2000lux, the illumination period of the bacteria-removing culture is preferably 15-17 h/d, more preferably 16h/d, the temperature of the bacteria-removing culture is preferably 25-30 ℃, more preferably 28 ℃, and the time of the bacteria-removing culture is preferably 8-12 min, more preferably 10 min.
In the present invention, the dioscorea composita explant in step (1) preferably comprises a stem, a leaf, an axillary bud and a terminal bud, and more preferably, the axillary bud.
In the invention, the disinfection in the step (2) preferably comprises mercury bichloride solution disinfection, the mass concentration of the mercury bichloride solution is preferably 0.05-0.15%, and is further preferably 0.1%, and the disinfection time of the mercury bichloride solution is preferably 5-15 min, and is further preferably 10 min.
In the present invention, the number of washing with water in step (2) is preferably 5 to 7, and more preferably 6.
In the invention, the illumination intensity of the dedifferentiation culture in the step (2) is preferably 1500-2500 lux, more preferably 2000lux, the illumination period of the dedifferentiation culture is preferably 7-9 h/d, more preferably 8h/d, the temperature of the dedifferentiation culture is preferably 25-30 ℃, more preferably 28 ℃, and the time of the dedifferentiation culture is preferably 10-15 d, more preferably 14 d.
In the present invention, the light intensity of the proliferation culture in step (3) is preferably 1500 to 2500lux, more preferably 2000lux, the light cycle of the proliferation culture is preferably 19 to 21h/d, more preferably 20h/d, the temperature of the proliferation culture is preferably 25 to 30 ℃, more preferably 28 ℃, and the time of the proliferation culture is preferably 18 to 22d, more preferably 20 d.
In the invention, the illumination intensity of the redifferentiation culture in the step (3) is preferably 1500-2500 lux, more preferably 2000lux, the illumination period of the redifferentiation culture is preferably 15-17 h/d, more preferably 16h/d, the temperature of the redifferentiation culture is preferably 25-30 ℃, more preferably 28 ℃, and the time of the redifferentiation culture is preferably 28-32 d, more preferably 30 d.
In the invention, the illumination intensity of the rooting culture in the step (4) is preferably 1500-2500 lux, further preferably 2000lux, the illumination period of the rooting culture is preferably 15-17 h/d, further preferably 16h/d, the temperature of the rooting culture is preferably 25-30 ℃, further preferably 28 ℃, and the time of the rooting culture is preferably 28-32 d, further preferably 30 d.
In the invention, the seedling exercising time in the step (5) is preferably 1-3 d, and more preferably 2 d; the hardening temperature is preferably 20-30 ℃, and more preferably 26 ℃; the hardening humidity is preferably 50-70%, and more preferably 60%.
In the present invention, the nursery soil component in step (5) preferably comprises peatmoss.
In the invention, the illumination intensity of the seedling culture in the step (5) is preferably 1500-2500 lux, more preferably 2000lux, the illumination period of the seedling culture is preferably 15-17 h/d, more preferably 16h/d, the temperature of the seedling culture is preferably 25-30 ℃, more preferably 28 ℃, and the time of the seedling culture is preferably 8-12 d, more preferably 10 d.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The dioscorea composita in the following examples is dioscorea composita in mexican, and the planting place is Zhanjiang city in Guangdong province.
Example 1
(1) Simply cleaning the collected dioscorea composita rhizome with distilled water to remove impurities, then planting the dioscorea composita rhizome in matrix soil (the matrix soil comprises nutrient soil and vermiculite, the mass ratio of the nutrient soil to the vermiculite is 2:1) to perform sterile culture for 8min under the conditions of illumination intensity of 1500lux, illumination period of 15h/d and temperature of 25 ℃, and selecting the stem as an explant for later use after stronger nutrient organs (stems, leaves, axillary buds and terminal buds) grow;
(2) sterilizing the stem with 0.05% mercuric chloride by mass concentration for 5min, cleaning with sterile water for 5 times, sucking surface water with sterile filter paper, and spreading on dedifferentiation culture medium (the dedifferentiation culture medium takes improved MS culture medium as basic culture medium and comprises 6-BA1 mg. L-1，NAA 0.1mg·L-12 wt% of sucrose and 4 g.L of agar-1The dedifferentiation medium has a pH of 5, and the modified MS medium comprises the components of: KNO314mmol/L，Ca(NO3)214mmol/L，(NH4)2SO47mmol/L and KCl 4mmol/L) under the conditions of illumination intensity of 1500lux, illumination period of 7h/d and temperature of 25 deg.C for dedifferentiation culture to obtain Dioscorea composita embryonic callus;
(3) placing the embryogenic callus of Dioscorea composita in Dioscorea composita tissue culture seedling proliferation culture medium (the proliferation culture medium takes MS culture medium as basal medium, and comprises 6-BA1 mg. L-1，NAA 0.1mg·L-1Glutamic acid 0.04 wt%, sucrose 2 wt% and agar 4 g.L-1The pH value of the multiplication culture medium is 5), the multiplication culture is carried out for 18d under the conditions that the illumination intensity is 1500lux, the illumination period is 19h/d and the temperature is 25 ℃, and then the multiplication culture medium is placed in the dioscorea composita seedling redifferentiation culture medium (the redifferentiation culture medium is placed in the dioscorea composita seedling)The differentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 6-BA1 mg. L-1，NAA 0.3mg·L-10.005 wt% of vitamin E, 0.02 wt% of selenocysteine, 2 wt% of sucrose and 4 g.L of agar-1The pH value of the redifferentiation culture medium is 5), and redifferentiation culture is carried out under the conditions that the illumination intensity is 1500lux, the illumination period is 15h/d, and the temperature is 25 ℃ to obtain the dioscorea composita regenerated seedlings;
(4) transferring the axilla of the Dioscorea composita regenerated seedling to a Dioscorea composita tissue culture seedling rooting culture medium (the rooting culture medium takes an improved MS culture medium as a basic culture medium and comprises IBA0.5mg. L-1，NAA 0.05mg·L-1And 2 wt% of sucrose, the pH value of the rooting medium is 5, and the improved MS medium comprises the following components: KNO314mmol/L，Ca(NO3)214mmol/L，(NH4)2SO47mmol/L and KCl 4mmol/L) under the conditions of illumination intensity of 1500lux, illumination period of 15h/d and temperature of 25 ℃ for rooting culture to obtain the complete regenerated seedling of Dioscorea composita;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings for 1d under the conditions that the temperature is 20 ℃ and the humidity is 50%, then cleaning the culture medium at the root part with distilled water, transplanting the cleaned culture medium into moist turfy soil, and performing seedling culture and culture for 8d under the conditions that the illumination intensity is 1500lux, the illumination period is 15h/d and the temperature is 25 ℃ to obtain the dioscorea composita cultured seedlings.
Example 2
(1) Simply cleaning the collected dioscorea composita rhizome with distilled water to remove impurities, then planting the dioscorea composita rhizome in matrix soil (the matrix soil comprises nutrient soil and vermiculite, the mass ratio of the nutrient soil to the vermiculite is 3:1), performing bacteria-removing culture for 10min under the conditions that the illumination intensity is 2000lux, the illumination period is 16h/d and the temperature is 28 ℃, and selecting leaves as explants for later use after stronger nutrient organs (stems, leaves, axillary buds and terminal buds) grow;
(2) sterilizing the leaf with 0.1% mercuric chloride for 10min, cleaning with sterile water for 6 times, drying surface water with sterile filter paper, and spreading on the leafA dedifferentiation culture medium of dioscorea composita tissue culture seedling (the dedifferentiation culture medium takes an improved MS culture medium as a basic culture medium and comprises the following components with the concentration of 6-BA2mg & L-1，NAA 0.2mg·L-13 wt% of sucrose and 4.5 g.L of agar-1The dedifferentiation medium has a pH of 5.8, and the modified MS medium comprises the following components: KNO315mmol/L，Ca(NO3)215mmol/L，(NH4)2SO47.5mmol/L and KCl 5mmol/L) under the conditions of illumination intensity of 2000lux, illumination period of 8h/d and temperature of 28 deg.C for dedifferentiation culture to obtain Dioscorea composita embryonic callus;
(3) placing the embryogenic callus of Dioscorea composita in Dioscorea composita tissue culture seedling proliferation culture medium (the proliferation culture medium takes MS culture medium as basal medium, and comprises 6-BA2 mg. L-1，NAA 0.2mg·L-1Glutamic acid 0.05 wt%, sucrose 3 wt% and agar 4.5 g.L-1The pH value of the multiplication culture medium is 5.8), the multiplication culture is carried out for 20d under the conditions that the illumination intensity is 2000lux, the illumination period is 20h/d and the temperature is 28 ℃, and then the multiplication culture medium is placed in a dioscorea composita seedling redifferentiation culture medium (the redifferentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 6-BA2 mg. L-1，NAA 0.4mg·L-1Vitamin E0.01 wt%, selenocysteine 0.025 wt%, cane sugar 3 wt% and agar 4.5 g.L-1The pH value of the redifferentiation culture medium is 5.8), and redifferentiation culture is carried out under the conditions that the illumination intensity is 2000lux, the illumination period is 16h/d, and the temperature is 28 ℃ to obtain the dioscorea composita regenerated seedlings;
(4) transferring axilla of the Dioscorea composita regenerated seedling to rooting culture medium of Dioscorea composita tissue culture seedling (the rooting culture medium takes improved MS culture medium as basic culture medium, and comprises IBA 1 mg. L-1，NAA 0.1mg·L-1And 3 wt% of sucrose, the pH of the rooting medium is 5.8, and the improved MS medium comprises the following components: KNO315mmol/L，Ca(NO3)215mmol/L，(NH4)2SO47.5mmol/L and KCl 5mmol/L) at a light intensity of 2000lux and a light period ofCarrying out rooting culture at the temperature of 28 ℃ for 16h/d to obtain the complete regenerated seedlings of the dioscorea composita;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings for 2d under the conditions that the temperature is 26 ℃ and the humidity is 60%, then cleaning the culture medium at the roots with distilled water, transplanting the cleaned culture medium into moist turfy soil, and performing seedling culture for 10d under the conditions that the illumination intensity is 2000lux, the illumination period is 16h/d and the temperature is 28 ℃ to obtain the dioscorea composita seedlings.
Example 3
(1) Simply cleaning collected dioscorea composita rhizome with distilled water to remove impurities, then planting the dioscorea composita rhizome in matrix soil (the matrix soil comprises nutrient soil and vermiculite, the mass ratio of the nutrient soil to the vermiculite is 4:1), performing bacteria-removing culture for 12min under the conditions that the illumination intensity is 2500lux, the illumination period is 17h/d and the temperature is 30 ℃, and selecting axillary buds as explants for later use after stronger nutrient organs (stems, leaves, axillary buds and terminal buds) grow;
(2) sterilizing the axillary bud with 0.15% mercuric chloride for 15min, cleaning with sterile water for 7 times, removing surface water with sterile filter paper, and spreading on dedifferentiation culture medium (the dedifferentiation culture medium comprises 6-BA3 mg. L medium with improved MS culture medium as basic culture medium)-1，NAA 0.3mg·L-14 wt% of sucrose and 5 g.L of agar-1Wherein the dedifferentiation medium has a pH of 6, and the modified MS medium comprises the following components: KNO316mmol/L，Ca(NO3)216mmol/L，(NH4)2SO48mmol/L and KCl 6mmol/L) under the conditions of illumination intensity of 2500lux, illumination period of 9h/d and temperature of 30 deg.C for dedifferentiation culture to obtain Dioscorea composita embryonic callus;
(3) placing the dioscorea composita embryogenic callus in a dioscorea composita tissue culture seedling multiplication culture medium (the multiplication medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration of 6-BA3mg & L-1，NAA 0.3mg·L-10.06 wt% of glutamic acid, 4 wt% of sucrose and 5 g.L of agar-1The pH of the multiplication medium is6) Performing proliferation culture for 22 days under the conditions of illumination intensity of 2500lux, illumination period of 21h/d and temperature of 30 ℃, and then placing the culture seedlings in a dioscorea composita redifferentiation culture medium (the redifferentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 6-BA3 mg. L-1，NAA 0.5mg·L-10.015 wt% of vitamin E, 0.03 wt% of selenocysteine, 4 wt% of sucrose and 5 g.L of agar-1The pH value of the redifferentiation culture medium is 6), and redifferentiation culture is carried out under the conditions that the illumination intensity is 2500lux, the illumination period is 17h/d, and the temperature is 30 ℃ to obtain the dioscorea composita regenerated seedlings;
(4) transferring the axilla of the Dioscorea composita regenerated seedling to a Dioscorea composita tissue culture seedling rooting culture medium (the rooting culture medium takes an improved MS culture medium as a basic culture medium and comprises IBA 1.5 mg.L-1，NAA 0.15mg·L-1And 4 wt% of sucrose, the pH value of the rooting medium is 6, and the improved MS medium comprises the following components: KNO316mmol/L，Ca(NO3)216mmol/L，(NH4)2SO48mmol/L and KCl 6mmol/L) under the conditions of illumination intensity of 2500lux, illumination period of 17h/d and temperature of 30 ℃ for rooting culture to obtain the complete regenerated seedling of Dioscorea composita;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings for 3d under the conditions that the temperature is 30 ℃ and the humidity is 70%, then cleaning the culture medium at the roots with distilled water, transplanting the cleaned culture medium into moist turfy soil, and carrying out seedling culture for 12d under the conditions that the illumination intensity is 2500lux, the illumination period is 17h/d and the temperature is 30 ℃ to obtain the dioscorea composita seedlings.
Example 4
The procedure of example 2 was followed except that the dioscorea composita explants were axillary buds.
Example 5
The procedure of example 2 was followed except that the stem was used as the dioscorea composita explant.
Example 6
The procedure of example 2 was followed except that the top bud was selected as the Dioscorea composita explant.
Comparative example 1
The concentration of 6-BA in the dedifferentiation medium was 1 mg. L-1The concentration of NAA is 0.2 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 2
The concentration of 6-BA in the dedifferentiation medium was 1 mg. L-1The concentration of NAA is 0.4 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 3
The concentration of 6-BA in the dedifferentiation medium was 1 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 4
The concentration of 6-BA in the dedifferentiation medium was 2 mg.L-1The concentration of NAA is 0.4 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 5
The concentration of 6-BA in the dedifferentiation medium was 2 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 6
The concentration of 6-BA in the dedifferentiation medium was 4 mg.L-1The concentration of NAA is 0.2 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 7
The concentration of 6-BA in the dedifferentiation medium was 4 mg.L-1The concentration of NAA is 0.4 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 8
The concentration of 6-BA in the dedifferentiation medium was 4 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 9
The concentration of 6-BA in the redifferentiation medium was 1 mg.L-1The concentration of NAA is 0.2 mg.L-1Except that, the rest steps are allIn keeping with example 4.
Comparative example 10
The concentration of 6-BA in the redifferentiation medium was 1 mg.L-1The concentration of NAA is 0.4 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 11
The concentration of 6-BA in the redifferentiation medium was 1 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 12
The concentration of 6-BA in the redifferentiation medium was 2 mg.L-1The concentration of NAA is 0.2 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 13
The concentration of 6-BA in the redifferentiation medium was 2 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 14
The concentration of 6-BA in the redifferentiation medium was 3 mg.L-1The concentration of NAA is 0.2 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 15
The concentration of 6-BA in the redifferentiation medium was 3 mg.L-1The concentration of NAA is 0.4 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 16
The concentration of 6-BA in the redifferentiation medium was 3 mg.L-1The concentration of NAA is 0.6 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 17
The concentration of IBA in the rooting-removing culture medium is 0, and the concentration of NAA is 1 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 18
The concentration of IBA in the rooting-removing culture medium is 0.5 mg.L-1The concentration of NAA is 0.5 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 19
The concentration of IBA in the rooting-removing culture medium is 0 mg.L-1The concentration of NAA is 0.1 mg.L-1Otherwise, the remaining steps were as in example 4.
Comparative example 20
The concentration of IBA in the rooting-removing culture medium is 1 mg.L-1The concentration of NAA is 1 mg.L-1Otherwise, the remaining steps were as in example 4.
Experimental example 1
The effect of different explants on callus formation was investigated using examples 2, 4, 5, 6 as examples.
The different explants in the above examples were washed for 30min and then placed on a sterile table for sterilization. The sterilization was first carried out for 30s with 75% ethanol, during which the glass container was shaken constantly. After the ethanol was decanted, it was washed once with sterile distilled water. Then 0.1% HgCl was used2The solution treatment is carried out for 10min for disinfection treatment. During this period, the flask was shaken continuously to pour out HgCl2After the solution, the explants are washed 6 times by sterile distilled water, and finally, the cut explants are put into an MS (hormone-free) culture medium by using tweezers for dark culture for one week to search for the most appropriate explant organs. The result shows that after the leaf grows for one week under the dark condition, the leaf edge put into the culture medium starts to brown and turn black, and the callus formation rate is close to 0; the two ends of the stem segment are expanded, and white crystal substances appear after the second week of culture to form a small amount of callus; after the axillary buds are cultured in the dark for one week, the callus tissues are few, and the second week callus is differentiated into the axillary buds; the terminal bud can show a state of continuous growth after being cultured for one week. Axillary buds from the callus formation Rate comparison>Stem segment>Leaf is the apical bud.
Experimental example 2
By taking example 4 and comparative examples 1-8 as examples, the influence of different concentrations of 6-BA and NAA in a dedifferentiation medium on callus formation promotion was investigated. The results are shown in Table 1.
TABLE 1 Effect of different concentrations of 6-BA and NAA on the promotion of callus formation
First, axillary buds were not promoted to produce callus under the blank control. As can be seen from Table 1, callus was formed with the addition of 6-BA at various concentrations, and the growth rate of callus increased with the increase in the concentration. After NAA is added, the callus formation rate can be obviously improved, but with the increase of the concentration of the NAA, the callus growth condition is not good, browning and vitrification appear, and finally death occurs. Compared with comparative examples 1-8, the dedifferentiation medium of example 4 of the application, namely the dedifferentiation medium with the phytohormone of 2mg/L6-BA +0.2mg/LNAA, is the optimal dedifferentiation medium.
Experimental example 3
By taking example 4 and comparative examples 9-16 as examples, the influence of different concentrations of 6-BA and NAA in the redifferentiation medium on callus redifferentiation promotion was investigated. The results are shown in Table 2.
TABLE 2 Effect of different concentrations of 6-BA and NAA on the promotion of callus redifferentiation
As shown in Table 2, compared with comparative examples 9-16, in the case of example 4 of the present application, that is, when the phytohormone in the redifferentiation medium was 2mg/L6-BA +0.4mg/LNAA, the effect of axillary bud formation was the best, the redifferentiation rate of the callus could reach 96%, the adventitious bud grew strongly, and the degree of differentiation was high, and as shown in Table 2, NAA at a low concentration promoted the formation of adventitious buds.
Experimental example 4
By taking the example 4 and the comparative examples 17-20 as examples, the influence of IBA and NAA with different concentrations in the rooting medium on promoting the rooting of leaf axils is researched. The results are shown in Table 3.
TABLE 3 Effect of different concentrations of IBA and NAA on the promotion of leaf axillary rooting
The dioscorea composita has thick and strong main roots and fine and developed lateral roots at the later rooting stage, and villous aerial roots grow out. The root tissue of the axillary differentiation part of the dioscorea composita leaf under tissue culture is an adventitious root and has a direct relation with the totipotency of cells. As can be seen from Table 3, compared with comparative examples 17-20, in example 4 of the present application, that is, when the phytohormone in the rooting medium is 1 mg/L6-BA +0.1mg/LNAA, the rooting condition is the best, the rooting time is short, about 7 days, and the root system grows fast and robustly.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A dedifferentiation culture medium of dioscorea composita tissue culture seedlings is characterized in that the dedifferentiation culture medium takes an improved MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.1～0.3mg·L-12-4 wt% of cane sugar and 4-5 g.L of agar-1The pH value of the dedifferentiation culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO314～16mmol/L，Ca(NO3)214～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L.
2. The multiplication culture medium for the dioscorea composita tissue culture seedlings is characterized in that the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA0.1～0.3mg·L-10.04-0.06 wt% of glutamic acid, 2-4 wt% of cane sugar and 4-5 g.L of agar-1pH of the multiplication medium5 to 6.
3. The redifferentiation culture medium for the dioscorea composita tissue culture seedling is characterized in that the redifferentiation culture medium takes an MS culture medium as a basic culture medium and comprises the following components in concentration: 1-3 mg.L of 6-BA-1，NAA 0.3～0.5mg·L-10.005-0.015 wt% of vitamin E, 0.02-0.03 wt% of selenocysteine, 2-4 wt% of cane sugar and 4-5 g.L of agar-1And the pH value of the redifferentiation culture medium is 5-6.
4. A rooting culture medium for a dioscorea composita tissue culture seedling is characterized in that the rooting culture medium takes an improved MS culture medium as a basic culture medium and comprises the following components in concentration: IBA 0.5-1.5 mg.L-1，NAA 0.05～0.15mg·L-1And 2-4 wt% of sucrose, wherein the pH value of the rooting culture medium is 5-6, and the improved MS culture medium comprises the following components: KNO3 14～16mmol/L，Ca(NO3)2 14～16mmol/L，(NH4)2SO47-8 mmol/L and KCl 4-6 mmol/L.
5. A culture medium composition for the rapid propagation of a dioscorea composita tissue culture seedling, which is characterized by comprising the dedifferentiation culture medium, the proliferation culture medium, the redifferentiation culture medium and the rooting culture medium of claims 1-4.
6. A rapid propagation method of dioscorea composita seedlings is characterized by comprising the following steps:
(1) planting dioscorea composita rhizome in matrix soil for bacteria-free culture to obtain dioscorea composita explant;
(2) sequentially disinfecting and washing the dioscorea composita explants, and then placing the explants in the dioscorea composita seedling dedifferentiation culture medium of claim 1 for dedifferentiation culture to obtain dioscorea composita embryonic callus;
(3) placing the dioscorea composita embryonic callus in the dioscorea composita tissue culture seedling multiplication culture medium of claim 2 for multiplication culture, and then placing the dioscorea composita tissue culture seedling redifferentiation culture medium of claim 3 for redifferentiation culture to obtain dioscorea composita regenerated seedling;
(4) transferring the axilla of the dioscorea composita regenerated seedling to the dioscorea composita tissue culture seedling rooting culture medium of claim 4 for rooting culture to obtain a dioscorea composita complete regenerated seedling;
(5) and (3) hardening the whole dioscorea composita regenerated seedlings, and then transplanting the seedlings into seedling soil for seedling culture to obtain dioscorea composita seedlings.
7. The rapid propagation method of dioscorea composita seedlings according to claim 6, characterized in that in step (1), the matrix soil component comprises nutrient soil and vermiculite, and the mass ratio of the nutrient soil to the vermiculite is 2-4: 1;
the illumination intensity of the bacteria-removing culture is 1500-2500 lux, the illumination period of the bacteria-removing culture is 15-17 h/d, the temperature of the bacteria-removing culture is 25-30 ℃, and the time of the bacteria-removing culture is 8-12 min;
the dioscorea composita explant comprises a stem, a leaf, an axillary bud and a terminal bud.
8. The rapid propagation method of dioscorea composita seedlings according to claim 6 or 7, wherein the sterilizing in step (2) comprises sterilizing with mercuric chloride solution, the mass concentration of the mercuric chloride solution is 0.05-0.15%, and the sterilizing time of the mercuric chloride solution is 5-15 min;
the number of washing times is 5-7;
the illumination intensity of the dedifferentiation culture is 1500-2500 lux, the illumination period of the dedifferentiation culture is 7-9 h/d, the temperature of the dedifferentiation culture is 25-30 ℃, and the time of the dedifferentiation culture is 10-15 d.
9. The rapid propagation method of dioscorea composita seedlings according to claim 8, characterized in that in step (3), the illumination intensity of the multiplication culture is 1500-2500 lux, the illumination cycle of the multiplication culture is 19-21 h/d, the temperature of the multiplication culture is 25-30 ℃, and the time of the multiplication culture is 18-22 d;
the illumination intensity of the redifferentiation culture is 1500-2500 lux, the illumination period of the redifferentiation culture is 15-17 h/d, the temperature of the redifferentiation culture is 25-30 ℃, and the time of the redifferentiation culture is 28-32 d.
10. The rapid propagation method of dioscorea composita tissue culture seedling according to claim 9, characterized in that the illumination intensity of the rooting culture in step (4) is 1500-2500 lux, the illumination period of the rooting culture is 15-17 h/d, the temperature of the rooting culture is 25-30 ℃, and the time of the rooting culture is 28-32 d;
in the step (5), the hardening time is 1-3 d, the hardening temperature is 20-30 ℃, and the hardening humidity is 50-70%;
the seedling raising soil component in the step (5) comprises turfy soil;
the illumination intensity of the seedling culture in the step (5) is 1500-2500 lux, the illumination period of the seedling culture is 15-17 h/d, the temperature of the seedling culture is 25-30 ℃, and the time of the seedling culture is 8-12 d.
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