CN110999785A - Synthetic method of artificial seeds of picea asperata - Google Patents

Synthetic method of artificial seeds of picea asperata Download PDF

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CN110999785A
CN110999785A CN201911189152.XA CN201911189152A CN110999785A CN 110999785 A CN110999785 A CN 110999785A CN 201911189152 A CN201911189152 A CN 201911189152A CN 110999785 A CN110999785 A CN 110999785A
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artificial
artificial seed
embryo
picea
seeds
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CN110999785B (en
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樊荣
白玉娥
苗慧琴
娜苏勒玛
石磊
代金玲
黄海洋
赵玉蓉
桑萨尔珠拉
聂群
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Inner Mongolia Mengrong Landscaping Engineering Co ltd
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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
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds
    • 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
    • A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
    • A01G24/23—Wood, e.g. wood chips or sawdust
    • 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
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • 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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  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

The invention relates to a method for synthesizing artificial seeds of picea asperata.A somatic embryo induced from callus is adopted as an artificial seed embryo, the somatic embryo is dried to be used as an embedding object, the artificial endosperm comprises 1/4LM, 0.5mg/L IBA and 20g/L sucrose, the artificial seed coat is prepared by immersing the artificial seed embryo and the artificial endosperm into artificial seed coat liquid and then solidifying the artificial seed coat liquid, and the artificial seed coat liquid comprises sodium alginate with the mass percentage concentration of 2-3% and CaCl with the mass percentage concentration of 1.5-2.5%2. The artificial seed of the picea asperata is not limited by seasons, and has better nutrition supply and disease resistanceCapability. The synthesis of the artificial seeds of the picea asperata solves the major problems of low yield of picea asperata seeds, seedling shortage, resource protection, excellent property maintenance and the like, is convenient to transport, and enables the picea asperata to be better applied to ecological construction such as sand control and the like and construction of timbered forest.

Description

Synthetic method of artificial seeds of picea asperata
Technical Field
The invention belongs to the technical field of forestry asexual propagation, and particularly relates to a synthetic method of an artificial seed of picea asperata.
Background
Picea aspera (Picea mongolica) is a rare precious tree species in the world, is the only spruce tree species distributed in arid and semiarid sand lands, and is called 'emerald' and 'bioactivation stone' on deserts due to the long-lived age and strong sand fixing capacity. Its natural forest is only distributed in east edge of muddy Dakesha in China, and the white sound Aogou natural protection area of the Kashiktong flag is most concentrated, and is one of the special local tree species in inner Mongolia sand. The existence of the spruce forest in the sandy land has no replaceable effect on the construction of the protection forest in the northeast of China, wind prevention and sand fixation, land desertification control and improvement of the farming and pasturing area environment at the east edge of the muddy Dakesha land.
Because the natural updating rate of emergence of the picea asperata is low, the death rate is high, and the seed production is easily influenced by the reasons of the year, the cold in the spring, the damage of the codling with the serious teeth and the like, the species resources of the picea asperata are continuously reduced, the distribution range is continuously reduced, and even the picea asperata gradually tends to be extinct. The protection and recovery of the sand spruce forest are in the forefront, but the sand spruce has poor resources, long fructification interval period and low growth amount of natural updating seedlings, thereby bringing great difficulty to seedling propagation and afforestation. Cutting propagation has been researched and a favorable result is obtained, but for limited mother trees, the damage in the scion picking process is extremely serious, and the cutting propagation has great obstacles, such as the reduction of the rooting rate of cutting seedlings, the reduction of the quality of root systems, the slow growth of early afforestation and other aging effects, namely the age-increasing effect of growing points, appear along with the increase of the age of the scion-picking mother trees; the other adverse phenomenon is the position effect of the scion of the old mother tree, namely that the endogenous hormone content and the nutrient content of branches of the mother tree in different canopies and different directions are different, and the rooting power of the adopted scion is different, so that the selected superior tree is difficult to continue to be put into production in large quantity.
These all result in the inhibition of the propagation of picea arenicola, and in addition, the seedlings propagated by natural seeds are easy to separate in nature, which affects the heredity and maintenance of excellent characters. Therefore, the influence factors of induction, multiplication and germination of the embryonic callus of the picea arenicola are systematically researched, a mature picea arenicola somatic embryogenesis system is established, an artificial seed rapid propagation technology is developed, and the picea arenicola embryonic callus has very important ecological significance and economic significance for the stability and continuity of the population and the green sustainable development construction of the picea in China.
The synthetic technology of the artificial seeds of the picea arenicola is mainly based on the establishment of a picea arenicola somatic embryogenesis system, and somatic embryos generated by the exosomatic culture are embedded in a shell containing nutrient substances and having a protection function to form granules which can germinate and emerge under proper conditions, so that the 'artificial seeds' of the picea arenicola are formed. For example, patent publication No. CN105165618A discloses a method for somatic embryogenesis of Picea arenicola, but the patent technology is the generation part of embryo of artificial seed and only belongs to a laboratory process method, and is difficult to realize industrial mass production.
Disclosure of Invention
The invention aims to solve the problems, provides a method for synthesizing the artificial seeds of the picea arenicola, aims to improve the germination rate of somatic embryos, enhance the adaptability of regenerated plants to enable the regenerated plants to have higher transplanting survival rate, utilizes a bioreactor to carry out mass culture of the somatic embryos so as to manufacture the artificial seeds, and finally realizes the storage and the rapid propagation of the picea arenicola.
In order to achieve the aim, the invention provides a synthetic method of artificial seeds of picea asperata, which comprises the following steps:
A. selecting immature seeds on a good plant for pretreatment, cutting the pretreated immature seeds into young embryos, inoculating the young embryos into a first culture medium, and culturing for 8-12 days until embryogenic callus is induced;
B. cutting the induced embryonic callus and inoculating the cut embryonic callus into a second culture medium, cutting the embryonic callus into small pieces after the embryonic callus is proliferated in a large amount and inoculating the small pieces into a differentiation culture medium, and culturing for 45-55 d to obtain a large amount of somatic embryos;
C. drying the somatic embryos and then using the dried somatic embryos as embedding objects to prepare artificial seed embryos;
D. preparing artificial endosperm from 1/4LM, 0.5mg/L IBA and 20g/L sucrose;
E. putting the obtained artificial embryo into a sodium alginate solution with the mass percentage concentration of 2-3%, and putting CaCl with the mass percentage concentration of 1.5-2.5% after clamping2Performing middle reaction for 15min to obtain artificial seed coat of Picea japonica, rinsing with sterile water for 3 times, and absorbing surface water to obtain artificial seed.
Preferably, in step a, the step of pre-treating the immature seeds comprises: soaking immature seeds on the excellent plants in water for 2 hours, then disinfecting the immature seeds for 30 seconds by using 75% alcohol, washing the immature seeds with sterile water for 2-4 times, then disinfecting the immature seeds with 2% NaClO for 5-8 min, and washing the immature seeds with the sterile water for 3-5 times.
Preferably, in the step A, the first culture medium is a minimal medium, a mixed culture medium or a minimal medium of 2, 4-D1.8-2.7 mg/L and 6-BA 1.2-2.7 mg/L, a mixed culture medium of 2, 4-D1.8-2.7 mg/L, 6-BA 1.2-2.7 mg/L, KT 0.3-1.0 mg/L, sucrose 20g/L, gelatin 2.5g/L, hydrolyzed casein 1g/L and inositol 0.2g/L, wherein the minimal medium is LM, 1/2LM or 1/4 LM.
Preferably, in step B, the second culture medium is a mixed culture medium of 1/2LM, 2, 4-D2.0mg/L, 6-BA1.0mg/L, 20g/L of sucrose, 2.5g/L of gelatin, 1g/L of hydrolyzed casein and 0.2g/L of inositol; the differentiation medium is a mixed medium of a basic medium, ABA 18-30 mg/L, sucrose 30g/L, gelatin 4.5g/L, hydrolyzed casein 1g/L and inositol 0.8g/L, wherein the basic medium is LM, 1/2LM or 1/4 LM.
Preferably, the somatic embryo drying step comprises:
solid culturing the somatic embryos cultured in liquid suspension for 3-5 days in 1/2LM medium, 60g/L PEG4000 and 0.5g/L hydrolyzed retinin;
and (3) placing the somatic embryos obtained by solid culture on filter paper, drying the somatic embryos by a filter paper container method, and embedding the somatic embryos after drying for 3-5 days.
Preferably, in the step A and the step B, the pH value of the culture medium is controlled to be 5.75-5.80, the culture condition is 23 +/-1 ℃, and dark culture is carried out.
The invention also provides a picea rasmae artificial seed, which comprises an artificial seed embryo, an artificial endosperm and an artificial seed coat covering the artificial seed embryo and the artificial endosperm, wherein the artificial seed embryo adopts a somatic embryo induced by callus, one part of the somatic embryo is directly embedded, the other part of the somatic embryo is dried to be used as an embedding object, the artificial endosperm comprises 1/4LM, 0.5mg/L IBA and 20g/L sucrose, the artificial seed coat is prepared by immersing the artificial seed embryo and the artificial endosperm into an artificial seed coat liquid and then solidifying the artificial seed coat liquid, and the artificial seed coat liquid comprises 2-3% of sodium alginate and 1.5-2.5% of CaCl in percentage by mass2。
Preferably, the somatic embryo drying step comprises the steps of carrying out solid culture on the somatic embryos cultured in liquid suspension for 3-5 days in 1/2LM medium, 60g/L PEG4000 and 0.5g/L hydrolyzed complex protein;
and (3) placing the somatic embryos obtained by solid culture on filter paper, drying the somatic embryos by a filter paper container method, and embedding the somatic embryos after drying for 3-5 days.
Preferably, the time for immersing the artificial embryo and the artificial endosperm into the artificial seed skin liquid is 15 min.
Preferably, the artificial seed skin liquid comprises sodium alginate with the mass percentage concentration of 2.5% and CaCl with the mass percentage concentration of 2%2。
Based on the technical scheme, the invention has the advantages that:
the method for synthesizing the artificial seeds of the picea arenicola is used for meeting the requirement of seedlings for large-scale planting of the picea arenicola through the bred artificial seeds, and the artificial seeds prepared by the method can ensure that the bred seedlings have good inheritance and do not generate mutation, improve the germination rate and further protect the excellent quality of the picea arenicola. The invention takes the spruce somatic embryo of sand as the explant of the artificial seed, so that the propagated spruce seedling has more consistent growth and no mutation, and the quality of the seedling is further improved.
The artificial seed of the picea asperata synthesized by the method is not limited by seasons, and has better nutrition supply and disease resistance due to the addition of special components. The synthesis of the artificial seeds of the picea asperata solves the major problems of low yield of picea asperata seeds, seedling shortage, resource protection, excellent property maintenance and the like, is convenient to transport, and enables the picea asperata to be better applied to ecological construction such as sand control and the like and construction of timbered forest.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a flow chart of a method for synthesizing artificial seeds of Picea sandy land;
FIG. 2 is a schematic diagram of the synthesis of artificial seeds of Picea sandy soil.
Detailed Description
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
The invention provides a picea rasmae artificial seed, which comprises an artificial seed embryo, an artificial endosperm and an artificial seed coat covering the artificial seed embryo and the artificial endosperm, wherein the artificial seed embryo adopts a somatic embryo induced by callus, one part of the somatic embryo is directly embedded, the other part of the somatic embryo is dried to be used as an embedding object, the artificial endosperm comprises 1/4LM, 0.5mg/L IBA and 20g/L sucrose, the artificial seed coat is prepared by immersing the artificial seed embryo and the artificial endosperm into an artificial seed coat liquid and then solidifying the artificial seed coat liquid, and the artificial seed coat liquid comprises 2-3% of sodium alginate and 1.5-2.5% of CaCl in percentage by mass2。
The artificial seed of Picea haitanensis is composed of 3 parts, namely artificial embryo, artificial endosperm and artificial seed coat.
The artificial seed embryo is the core of the artificial seed, the preparation of the artificial seed of the picea asperata selects a somatic embryo as the artificial seed embryo, selects the somatic embryo induced by the callus, and directly embeds one part of the somatic embryo, and dries the other part of the somatic embryo to be used as an embedding material. Preferably, the somatic embryo drying step comprises the steps of carrying out solid culture on the somatic embryo cultured in a liquid suspension way for 3-5 days in 1/2LM culture medium, 60g/L PEG4000 and 0.5g/L hydrolyzed complex protein, so as to be beneficial to the germination of the somatic embryo; and (3) placing the somatic embryos obtained by solid culture on filter paper, drying the somatic embryos by a filter paper container method, and embedding the somatic embryos after drying for 3-5 days.
The artificial endosperm provides nutrients and hormones for the artificial embryo, is a substance for promoting the germination and growth of the artificial embryo, and usually comprises a basic culture medium, a plant growth regulator, saccharides and other additives. The selection of the artificial endosperm is concerned with the germination of the artificial seeds, and when the artificial endosperm is prepared, MS, 1/2MS, LM, 1/2LM and 1/4LM are respectively selected as basic culture media, and IBA (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7mg/L), sucrose (15, 20 and 25g/L) or maltose (15, 20 and 25g/L), activated carbon (0.1, 0.5 and 0.8mg/L) and carbendazim (0.1, 0.5 and 0.8mg/L) with different concentrations are added. Theoretical analysis and practical tests show that after the activated carbon is added, the artificial endosperm becomes hard, which is not beneficial to uniformly wrapping the artificial embryo and is easy to crack. The optimal artificial endosperm formula of the artificial seeds of the spruce sand is determined to be 1/4LM + IBA0.5mg/L + sucrose 20g/L by research and screening.
The artificial seed coat is a film wrapped outside the artificial embryo and the artificial endosperm, and has the same protection function as the natural seed coat. The artificial seed coat is required to avoid water and nutrition loss in the seeds, and ensure the air permeability and resist the impact of external pressure. The invention selects the sodium alginate with biological activity, low cost, no toxicity and simple process operation as the main component of the artificial seed coat.
When the artificial seed coat is prepared, firstly, 1.0 percent, 2.0 percent, 2.5 percent and 3.0 percent of sodium alginate solution and CaCl are mixed2The solutions (1.0, 2.0, 3.0, 4.0%) were subjected to different reaction times (5, 10, 15, 20 min). Through theoretical analysis and combined with practical tests, 2.5 percent of sodium alginate in 2 percent of CaCl is found2Reacting in the solution for 15min, the artificial seed coat has best effect, transparent color, high elasticity, no tail, uniform and round seeds and moderate hardness.
The invention also provides a synthetic method of the artificial seeds of the picea asperata, as shown in figures 1 and 2, the synthetic method comprises the following steps:
A. selecting immature seeds on a good plant for pretreatment, cutting the pretreated immature seeds into young embryos, inoculating the young embryos into a first culture medium, and culturing for 8-12 days until embryogenic callus is induced;
B. cutting the induced embryonic callus and inoculating the cut embryonic callus into a second culture medium, cutting the embryonic callus into small pieces after the embryonic callus is proliferated in a large amount and inoculating the small pieces into a differentiation culture medium, and culturing for 45-55 d to obtain a large amount of somatic embryos;
C. drying the somatic embryos and then using the dried somatic embryos as embedding objects to prepare artificial seed embryos;
D. preparing artificial endosperm from 1/4LM, 0.5mg/L IBA and 20g/L sucrose;
E. putting the obtained artificial embryo into the culture mediumAdding the sodium alginate solution with the concentration of 2-3% into CaCl with the mass percentage concentration of 1.5-2.5% after being taken out2Performing middle reaction for 15min to obtain artificial seed coat of Picea japonica, rinsing with sterile water for 3 times, and absorbing surface water to obtain artificial seed.
Specifically, the method for synthesizing the artificial seeds of the picea rasmae comprises the following specific process steps:
(1) soaking immature seeds serving as explants in water for 2 h; in an ultraclean workbench, the immature seeds are sterilized by 75% alcohol for 30s, washed by sterile water for 3 times, sterilized by 2% NaClO for 6min, and washed by the sterile water for 3-5 times.
(2) Cutting immature seeds treated in the step (1) into young embryos, and inoculating the young embryos in the following first culture medium: 1/2LM +2, 4-D2.4 mg/L +6-BA1.2mg/L + sucrose 20g/L + gel 2.5g/L + hydrolyzed casein 1g/L + inositol 0.2g/L, pH is controlled at 5.75-5.80, temperature is 23 +/-1 ℃, dark culture is carried out, callus is induced after 10 days of culture, and induction rate is 82%.
(3) When the embryogenic callus induced in step (2) is excised, inoculated into the following second medium: 1/2LM +2, 4-D2.0mg/L +6-BA1.0mg/L + sucrose 20g/L + gelatin 2.5g/L + hydrolyzed casein 1g/L + inositol 0.2g/L, pH is controlled to be 5.75-5.80, the temperature is 23 +/-1 ℃, dark culture is carried out, and more embryogenic callus proliferation is induced in the culture process.
(4) When the embryogenic callus described in step (3) proliferated more, it was cut into small pieces and inoculated in the following differentiation medium: 1/2LM + ABA22mg/L + sucrose 30g/L + gelatin 4.5g/L + hydrolyzed casein 1g/L + inositol 0.8g/L, pH is controlled at 5.75-5.80, temperature is 23 +/-1 ℃, dark culture is carried out, and a large number of somatic embryos are obtained after 50 days.
(5) And (3) obtaining artificial embryo, namely, when the somatic embryos induced by the callus are selected in the step (4), one part of the somatic embryos is directly embedded, and the other part of the somatic embryos is dried to be used as an embedded object, wherein the drying step comprises ① liquid suspension culture, solid culture is carried out for 3-5 days under the conditions that 1/2LM + PEG 400060 g/L + acid hydrolysis complex protein is 0.5g/L, and ② filter paper container method is used for drying the somatic embryos and embedding the somatic embryos to prepare the artificial seeds after drying for 3-5 days.
(6) Preparing artificial seed coat by selecting sodium alginate solution (1%, 2%, 2.5%, 3%, 4%) with different concentrations and CaCl with different concentrations2The solution (1.5%, 2%, 2.5%) was reacted, and the results were as follows:
Figure BDA0002293135110000071
Figure BDA0002293135110000081
it was found from the above table that 2.5% sodium alginate was in 2% CaCl2Reacting in the solution for 15min, the artificial seeds have best effect, transparent color, high elasticity, no tail, uniform and round seeds and moderate hardness.
(7) Preparing artificial endosperm, namely respectively selecting MS, 1/2MS, LM, 1/2LM and 1/4LM as basic culture media, adding IBA (0.3mg/L, 0.5mg/L and 1.0mg/L) with different concentrations, sucrose or maltose, activated carbon and carbendazim for culture, finding that artificial seeds are hardened after the carbendazim and the activated carbon are added, and finally determining the optimal artificial endosperm to be 1/4LM + IBA0.5mg/L + sucrose 20 g/L.
(8) Embedding artificial seeds: putting the artificial embryo obtained in the step (5) into a 2.5% sodium alginate solution, clamping the embryo out by using a pair of tweezers, and putting the embryo into 2% CaCl2Performing middle reaction for 15min to obtain artificial seed coat of Picea japonica, rinsing with sterile water for 3 times, and absorbing surface water to obtain artificial seed of Picea japonica.
The method for synthesizing the artificial seeds of the picea arenicola is used for meeting the requirement of seedlings for large-scale planting of the picea arenicola through the bred artificial seeds, and the artificial seeds prepared by the method can ensure that the bred seedlings have good inheritance and do not generate mutation, improve the germination rate and further protect the excellent quality of the picea arenicola. The invention takes the spruce somatic embryo of sand as the explant of the artificial seed, so that the propagated spruce seedling has more consistent growth and no mutation, and the quality of the seedling is further improved.
The artificial seed of the picea asperata synthesized by the method is not limited by seasons, and has better nutrition supply and disease resistance due to the addition of special components. The synthesis of the artificial seeds of the picea asperata solves the major problems of low yield of picea asperata seeds, seedling shortage, resource protection, excellent property maintenance and the like, is convenient to transport, and enables the picea asperata to be better applied to ecological construction such as sand control and the like and construction of timbered forest.
Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit the same; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.

Claims (10)

1. A synthetic method of artificial seeds of picea asperata is characterized in that: the synthesis method comprises the following steps:
A. selecting immature seeds on a good plant for pretreatment, cutting the pretreated immature seeds into young embryos, inoculating the young embryos into a first culture medium, and culturing for 8-12 days until embryogenic callus is induced;
B. cutting the induced embryonic callus and inoculating the cut embryonic callus into a second culture medium, cutting the embryonic callus into small pieces after the embryonic callus is proliferated in a large amount and inoculating the small pieces into a differentiation culture medium, and culturing for 45-55 d to obtain a large amount of somatic embryos;
C. directly embedding one part of the somatic embryos, and drying the other part of the somatic embryos to be used as an embedding material to prepare artificial seed embryos;
D. preparing artificial endosperm from 1/4LM, 0.5mg/L IBA and 20g/L sucrose;
E. putting the obtained artificial embryo into a sodium alginate solution with the mass percentage concentration of 2-3%, and putting CaCl with the mass percentage concentration of 1.5-2.5% after clamping2Performing middle reaction for 15min to form artificial seed coat of Picea japonica, rinsing with sterile water for 3 times, and sucking surfaceAnd (5) obtaining the artificial seeds by moisture.
2. The method of synthesis according to claim 1, characterized in that: in step a, the step of pre-treating the immature seeds comprises: soaking immature seeds on the excellent plants in water for 2 hours, then disinfecting the immature seeds for 30 seconds by using 75% alcohol, washing the immature seeds with sterile water for 2-4 times, then disinfecting the immature seeds with 2% NaClO for 5-8 min, and washing the immature seeds with the sterile water for 3-5 times.
3. The method of synthesis according to claim 1, characterized in that: in the step A, the first culture medium is a minimal medium, a mixed culture medium or a minimal medium of 2, 4-D1.8-2.7 mg/L and 6-BA 1.2-2.7 mg/L, a mixed culture medium or a minimal medium of 2, 4-D1.8-2.7 mg/L, 6-BA 1.2-2.7 mg/L, KT 0.3.3-1.0 mg/L, sucrose 20g/L, gelatin 2.5g/L, casein hydrolysate 1g/L and inositol 0.2g/L, wherein the minimal medium is LM, 1/2LM or 1/4 LM.
4. The method of synthesis according to claim 1, characterized in that: in the step B, the second culture medium is a mixed culture medium of 1/2LM, 2, 4-D2.0mg/L, 6-BA1.0mg/L, 20g/L of cane sugar, 2.5g/L of gel, 1g/L of hydrolyzed casein and 0.2g/L of inositol; the differentiation medium is a mixed medium of a basic medium, ABA 18-30 mg/L, sucrose 30g/L, gelatin 4.5g/L, hydrolyzed casein 1g/L and inositol 0.8g/L, wherein the basic medium is LM, 1/2LM or 1/4 LM.
5. The method of synthesis according to claim 1, characterized in that: the somatic embryo drying step comprises the following steps:
solid culturing the somatic embryos cultured in liquid suspension for 3-5 days in 1/2LM medium, 60g/L PEG4000 and 0.5g/L hydrolyzed retinin;
and (3) placing the somatic embryos obtained by solid culture on filter paper, drying the somatic embryos by a filter paper container method, and embedding the somatic embryos after drying for 3-5 days.
6. The method of synthesis according to claim 1, characterized in that: in the step A and the step B, the pH value of the culture medium is controlled to be 5.75-5.80, the culture condition is 23 +/-1 ℃, and dark culture is carried out.
7. An artificial seed of picea rasmae comprises an artificial seed embryo, an artificial endosperm and an artificial seed coat coated outside the artificial seed embryo and the artificial endosperm, and is characterized in that: the artificial seed embryo is a somatic embryo induced by adopting picea asperata callus, the somatic embryo is dried to serve as an embedding material, the artificial endosperm comprises 1/4LM, 0.5mg/L IBA and 20g/L sucrose, the artificial seed coat is prepared by immersing the artificial seed embryo and the artificial endosperm into artificial seed coat liquid and then solidifying the artificial seed coat liquid, and the artificial seed coat liquid comprises sodium alginate with the mass percentage concentration of 2-3% and CaCl with the mass percentage concentration of 1.5-2.5%2。
8. The artificial seed of Picea sandy species according to claim 7, wherein: the somatic embryo drying step comprises the following steps of carrying out solid culture on somatic embryos cultured in a liquid suspension manner for 3-5 days in 1/2LM medium, 60g/L PEG4000 and 0.5g/L hydrolyzed complex protein;
and (3) placing the somatic embryos obtained by solid culture on filter paper, drying the somatic embryos by a filter paper container method, and embedding the somatic embryos after drying for 3-5 days.
9. The artificial seed of Picea sandy species according to claim 7, wherein: the time for immersing the artificial embryo and the artificial endosperm into the artificial seed coat liquid is 15 min.
10. The artificial seed of Picea sandy species according to claim 7, wherein: the artificial seed coat liquid comprises sodium alginate with the mass percentage concentration of 2.5 percent and CaCl with the mass percentage concentration of 2 percent2。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113502257A (en) * 2021-08-19 2021-10-15 内蒙古蒙荣园林绿化工程有限责任公司 Drying marking method for spruce body cell embryos before germination

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103461119A (en) * 2013-08-20 2013-12-25 中国林业科学研究院林业研究所 Somatic embryogenesis and plant regeneration method for Picea asperata Mast
CN105165618A (en) * 2015-10-10 2015-12-23 内蒙古农业大学 Somatic embryogenesis method for Picea mongolica

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103461119A (en) * 2013-08-20 2013-12-25 中国林业科学研究院林业研究所 Somatic embryogenesis and plant regeneration method for Picea asperata Mast
CN105165618A (en) * 2015-10-10 2015-12-23 内蒙古农业大学 Somatic embryogenesis method for Picea mongolica

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
杨映根等: "裸子植物体细胞胚胎发生和人工种子的研究", 《种子》 *
谢耀坚: "人工种子技术及其在林业生产中的应用前景", 《经济林研究》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113502257A (en) * 2021-08-19 2021-10-15 内蒙古蒙荣园林绿化工程有限责任公司 Drying marking method for spruce body cell embryos before germination

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