CN115299343B - Method for establishing somatic embryogenesis system of huperzia serrata and application of somatic embryogenesis system - Google Patents

Method for establishing somatic embryogenesis system of huperzia serrata and application of somatic embryogenesis system Download PDF

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CN115299343B
CN115299343B CN202210983857.4A CN202210983857A CN115299343B CN 115299343 B CN115299343 B CN 115299343B CN 202210983857 A CN202210983857 A CN 202210983857A CN 115299343 B CN115299343 B CN 115299343B
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CN115299343A (en
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韩学敏
曾庆银
王丹
金宇霆
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CHINESE ACADEMY OF FORESTRY
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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
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/40—Afforestation or reforestation

Abstract

The invention provides a method for establishing an oil fir somatic embryogenesis system and application thereof, belonging to the field of cell engineering seedling propagation in forestry. The method for establishing the somatic embryogenesis system of the huperzia serrata comprises the following steps: the immature zygotic embryo is used as an explant for induction culture, proliferation culture, adjustment culture and maturation culture, and is differentiated to form somatic embryo. The method successfully establishes an embryogenesis system of the somatic cells of the huperzia serrata, breaks through the bottlenecks of difficult nutrition and propagation, long growth cycle, low germination rate and the like of the huperzia serrata, and becomes an emerging path for rapid propagation, excellent character maintenance and variety improvement of the forest.

Description

Method for establishing somatic embryogenesis system of huperzia serrata and application of somatic embryogenesis system
Technical Field
The invention belongs to the field of propagation of cell engineering seedlings in forestry, and particularly relates to a method for establishing a huperzia serrata somatic embryogenesis system and application thereof.
Background
The Yunnan oil fir (Keteleeria evelyniana) belongs to the genus of the pine family, is a special pine family wiggley plant in southeast Asia, is warm-loving and drought-resistant, has the characteristics of strong positivity, drought resistance, barren resistance and the like, has thick bark plug and strong fire resistance, and has important ecological and economic values. The Yunnan huperzia serrata is naturally distributed from the north to the middle of the Yunnan, from the west to the south of the Guizhou, and from the south to the west of the Sichuan, and the altitude distribution range is 1100-2300 m. In recent decades, due to artificial reclamation and development, the ecological environment is crushed, and for the protection and utilization of third-stage wigia plant resources and the conservation of endangered species, a large-scale efficient breeding system is necessary to be established. In nature, the Yunnan oil fir is mainly propagated through seeds, the germination and seedling formation of the seeds are affected by a plurality of factors, including seed quality, environmental ecological factors and the like, and the propagation efficiency is low. At present, the Yunnan oil fir is also subjected to sexual propagation mainly through seeds in the prior art, however, the seeds of the Yunnan oil fir contain grease and are not storage-resistant, the seeds can only be planted along with picking, the germination rate of the seeds of the Yunnan oil fir is low, one seed can only develop to form a tree, and the seeds are unfavorable for conventional seed propagation to propagate the Yunnan oil fir, so that the development and establishment of a asexual propagation mode of the Yunnan oil fir are necessary.
Somatic embryogenesis refers to the process of morphogenesis in which somatic cells develop into new individuals under artificial control, without undergoing cell fusion, through a series of pathways similar to zygotic embryogenesis. Plant cell embryogenesis is an important approach for large-scale asexual propagation of conifer species by biotechnology means. Has great application value in a plurality of fields such as mass propagation, germplasm preservation, genetic transformation and the like of the forest. However, the conditions of somatic embryogenesis vary greatly from genus to genus, and even from species to species, and thus no method for establishing a somatic embryogenesis system of Cephalotaxus yunnanensis has been reported.
Disclosure of Invention
Therefore, the invention aims to provide a method for establishing an embryogenesis system of the somatic cells of the Chinese fir, breaks through the bottlenecks of difficult nutrition and propagation, long growth cycle, low germination rate and the like of the Chinese fir, and successfully establishes the embryogenesis system of the somatic cells of the Chinese fir.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for establishing an oil fir somatic embryogenesis system, which comprises the following steps: the immature zygotic embryo is used as an explant for induction culture, proliferation culture, adjustment culture and maturation culture, and is differentiated to form somatic embryo.
Preferably, the zygotic embryo is in the proembryogenic stage, early embryogenesis and late embryogenesis stage.
Preferably, the basic culture medium of the induction culture, the proliferation culture, the adjustment culture and the maturation culture is an improved 1/2MS culture medium;
KI 4.1-4.2 mg/L, H in the modified 1/2MS culture medium 3 BO 3 15.4～15.6mg/L、Vitamin B 1 0.9～1.1mg/L、Vitamin B 6 0.9～1.1mg/L。
Preferably, the induction medium is: modified 1/2MS culture medium +2, 4-D0.2-1 mg/L +6-BA 0.1-2 mg/L +KT0-0.5 mg/L +TDZ 0-0.01 mg/L +inositol 80-120 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the proliferation medium is: modified 1/2MS culture medium +2, 4-D0.05-0.15 mg/L +6-BA 0.02-0.09 mg/L +inositol 450-550 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the conditioning medium is: modified 1/2MS culture medium, activated carbon, myo-inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 400-500 mg/L, sucrose 55-65 mg/L and agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the maturation medium is: modified 1/2MS culture medium, PEG4000 50-200 g/L, ABA 0.5-60 mg/L, IBA 0.1-0.3 mg/L, active carbon 1-3 g/L, inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 700-800 mg/L, sucrose 55-65 mg/L and agar 8-10 g/L; the pH is 5.8-6.2.
Preferably, the culture conditions are: in dark condition, the culture temperature is 19-23 deg.c and the relative humidity during culture is 60-75%.
The invention also provides application of the method in the propagation of the huperzia serrata and/or the maintenance of excellent properties.
The invention also provides application of the method in the improvement of the huperzia serrata varieties.
Compared with the prior art, the invention has the following beneficial effects:
the method successfully establishes an embryogenesis system of the huperzia serrata somatic cells, breaks through the bottlenecks of difficult nutrition and propagation, long growth cycle, low germination rate and the like of the huperzia serrata, can realize large-scale rapid growth of the huperzia serrata, and becomes an emerging way for rapid propagation of forest trees, excellent character maintenance and variety improvement.
Drawings
Fig. 1: immature cones and seeds of picea yunnanensis;
fig. 2: inducing and culturing embryogenic callus of immature zygotic embryo of picea yunnanensis;
fig. 3: proliferation culture of Yunnan oil fir embryogenic callus;
fig. 4: culturing the embryogenic callus of the picea yunnanensis;
fig. 5: mature culture process of somatic embryo of Abies yunnanensis;
fig. 6: influence of key compound concentration on mature differentiation culture of somatic embryos of picea yunnanensis.
Detailed Description
The invention provides a method for establishing an oil fir somatic embryogenesis system, which comprises the following steps: the immature zygotic embryo is used as an explant for induction culture, proliferation culture, adjustment culture and maturation culture, and is differentiated to form somatic embryo.
As an alternative embodiment, the method for establishing the somatic embryogenesis system of the huperzia serrata comprises the following steps: (1) collecting immature cones of the huperzia serrata; (2) Sterilizing the surface of the Chinese fir cone, and removing seed coats to obtain sterile immature zygotic embryos; (3) Inoculating the sterile immature zygotic embryo to an embryogenic callus induction culture medium, and performing callus induction culture to obtain embryogenic callus; (4) Inoculating embryogenic callus to embryogenic callus proliferation culture medium to realize embryogenic maintenance and mass proliferation of embryogenic callus; (5) Inoculating embryogenic callus to embryogenic callus regulating culture medium to reduce the growth regulator accumulated in embryogenic callus and prepare for further differentiation of somatic embryo; (6) And inoculating the embryogenic callus on a somatic embryo maturation medium, and culturing somatic embryos to obtain somatic embryos.
The fir is preferably Cephalotaxus yunnanensis, belonging to genus Cephalotaxus of Pinaceae.
In the present invention, the zygotic embryo is in the stage of primordia, early embryogenesis, late embryogenesis. The development process of embryogenesis of pine and fir plants is divided into: primordial germ period, early embryo formation, late embryo formation, and maturity. The development stage of the zygotic embryo can obviously influence the induction rate of embryogenic callus, and the over-tender or over-ripe zygotic embryo is not beneficial to inducing the embryogenic callus.
In the method, the basic culture medium for induction culture, proliferation culture, adjustment culture and maturation culture is an improved 1/2MS culture medium; the improved 1/2MS culture medium of the invention increases KI and H in the 1/2MS basic culture medium 3 BO 3 、VitaminB 1 And Vitamin B 6 The content, the rest components and the content remain unchanged.
In the improved 1/2MS culture medium, KI is 4.1-4.2 mg/L, H 3 BO 3 15.4～15.6mg/L、Vitamin B 1 0.9～1.1mg/L、Vitamin B 6 0.9-1.1 mg/L. Preferably, KI 4.15mg/L, H 3 BO 3 15.5mg/L、Vitamin B 1 1mg/L、Vitamin B 6 1mg/L。
Vitamin B 1 And Vitamin B 6 Is an important organic nitrogen source, has important effect on the vital activity of plants, and is Vitamin B 1 Has close relation with the generation and viability of the callus; potassium is the main cation in the culture medium, iodine is an essential trace element, and the content of KI can be increased by increasing the content of KI, namely the content of main potassium ions can be increased while the iodine element is supplemented; h 3 BO 3 Can promote cell elongation and tissue differentiation. The invention is beneficial to the induction of the embryogenic callus of the huperzia serrata by increasing the content of the components.
In the present invention, the induction medium is: modified 1/2MS culture medium +2, 4-D0.2-1 mg/L +6-BA 0.1-2 mg/L +KT0-0.5 mg/L +TDZ 0-0.01 mg/L +inositol 80-120 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the induction medium is: modified 1/2MS culture medium +2, 4-D0.4-0.6 mg/L +6-BA 0.5-1 mg/L +KT0.1-0.4 mg/L +TDZ 0.005-0.008 mg/L +inositol 90-110 mg/L +hydrolyzed casein 480-520 mg/L +glutamine 440-480 mg/L +sucrose 28-32 mg/L +agar 6.5-7.5 g/L; the pH was 6.
In the present invention, the proliferation medium is: modified 1/2MS culture medium +2, 4-D0.05-0.15 mg/L +6-BA 0.02-0.09 mg/L +inositol 450-550 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the proliferation medium is: modified 1/2MS culture medium +2, 4-D0.08-0.12 mg/L +6-BA 0.04-0.06 mg/L +inositol 480-520 mg/L +hydrolyzed casein 480-520 mg/L +glutamine 420-480 mg/L +sucrose 28-32 mg/L +agar 6.5-7.5 g/L; the pH was 6.
In the present invention, the conditioning medium is: modified 1/2MS culture medium, activated carbon, myo-inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 400-500 mg/L, sucrose 55-65 mg/L and agar 6-8 g/L; the pH is 5.8-6.2.
Preferably, the conditioning medium is: modified 1/2MS culture medium, activated carbon 1.5-2.5 g/L, inositol 480-520 mg/L, hydrolyzed casein 480-530 mg/L, glutamine 420-470 mg/L, sucrose 58-62 mg/L and agar 6.5-7.5 g/L; the pH was 6.
In order to further differentiate somatic embryos, a large amount of auxin and cytokinin which are not beneficial to the somatic embryo differentiation and are accumulated in the early stage of embryogenic callus are removed.
In the present invention, the maturation medium is: modified 1/2MS culture medium, PEG 400050-200 g/L, ABA 0.5-60 mg/L, IBA 0.1-0.3 mg/L, active carbon 1-3 g/L, inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 700-800 mg/L, sucrose 55-65 mg/L and agar 8-10 g/L; the pH is 5.8-6.2.
Preferably, the maturation medium is: modified 1/2MS culture medium, PEG4000 100-155 g/L, ABA 10-40 mg/L, IBA 0.15-0.25 mg/L, active carbon 1.5-2.5 g/L, inositol 480-520 mg/L, hydrolyzed casein 480-520 mg/L, glutamine 740-780 mg/L, sucrose 58-62 mg/L and agar 8.5-9.5 g/L; the pH was 6.
The addition of abscisic acid ABA in the maturation culture medium is favorable for the differentiation of somatic embryos of conifer species, and can inhibit abnormal development of the somatic embryos.
In the invention, the culture conditions of embryogenic callus induction culture, embryogenic callus proliferation culture, embryogenic callus adjustment culture and somatic embryo maturation culture are as follows: in dark condition, the culture temperature is 19-23 deg.c and the relative humidity during culture is 60-75%. The culture temperature is preferably 20 to 22 ℃, more preferably 21 ℃; the relative humidity is preferably 65 to 70%.
The invention also provides application of the method in the propagation of the huperzia serrata and/or the maintenance of excellent properties.
The invention also provides application of the method in the improvement of the huperzia serrata varieties.
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.
In specific embodiments, the plant growth regulating substances used in the present invention: 6-Benzylaminoadenine (6-BA), kine (KT), 2, 4-dichlorophenoxyacetic acid (2, 4-D), thidiazuron (TDZ), (S) - (+) -abscisic acid (ABA), 3-indolebutyric acid (IBA) and 1-naphthylacetic acid (NAA) are all conventional commercial products.
In specific examples, specific components of the modified 1/2MS medium of the present invention are shown in Table 1:
table 1 improved 1/2MS Medium formulation
Figure BDA0003801234630000061
The preparation method of the culture medium comprises the following steps: according to the final volume of the prepared culture medium, weighing the corresponding mass in the basic culture medium, sequentially adding the basic culture medium into deionized water, weighing sucrose, inositol and hydrolyzed casein with the corresponding mass, fully stirring each of the two substances, measuring the pH value of the culture medium by a pH meter, adjusting the pH value to about 6.0 by 1mol/L NaOH or 1mol/L HCl, fixing the volume to the final volume, and finally weighing agar with the corresponding mass. After sealing the bottle mouth with gauze, sterilizing in a high-temperature high-pressure sterilizing pot at 121 ℃ for 15 minutes. And (3) cooling the culture medium to about 50 ℃, adding the filtered and sterilized glutamine into the culture medium in an ultra-clean workbench, and uniformly mixing and shaking.
Example 1
A method for establishing a huperzia serrata somatic embryogenesis system, comprising the steps of:
(1) Material selection
Selecting the health and maturity of the distribution of the mountain of the purple stream in Chuxiong City of Yunnan province the Yunnan China fir with no insect damage and high fruiting rate is used as a research material, immature cones of yunnanensis were harvested at 2020.6.16, 2020.6.23 and 2020.7.2, respectively, with 6 cones harvested each time. Storing the cones collected on the same day at low temperature, quickly preparing the cones to Beijing, and then refrigerating the cones in a refrigerator at 0-4 ℃ for 2 weeks for later use.
(2) Sterilization of explants
Cleaning the cones with detergent, washing the foam with clear water, and wiping with paper. Peeling off cleaned fructus cone, removing seed scales and seed wings, taking out seed with seed coat, and placing in a culture dish on ice box. The seeds of the picea yunnanensis are soaked in 75% alcohol for 1min, washed 3 times with sterile water in an ultra clean bench, then soaked in 1% NaClO solution for 30min, and washed 4 times with sterile water again. The method comprises the steps of sucking water on the surface of seeds by using sterilizing filter paper, clamping the seeds by using pointed tweezers on the left hand, cutting off the seed coats on one side of the big head of the seeds by using a dissecting knife on the right hand, avoiding damaging the zygotic embryos in the seeds as much as possible, slightly slitting the seed coats along the slit, and slightly taking out the zygotic embryos containing endosperm inside the seed coats by using a dissecting needle on the right hand for later use. Immature cones and seeds of picea yunnanensis are shown in figure 1.
(3) Embryogenic callus induction culture
The zygotic embryos comprising endosperm are inoculated onto embryogenic callus induction medium. The embryogenic callus induction culture is carried out under dark conditions, and fresh embryogenic callus induction culture medium is changed every 20 days to keep the culture medium containing sufficient nutrients, moisture growth and regulator. The embryogenic callus induction medium is: 1/2MS basic culture medium is improved, 2,4-D1mg/L, 6-B0.5 mg/L, hydrolyzed casein 500mg/L, inositol 100mg/L, glutamine 500mg/L, sucrose 30g/L, agar 7g/L and pH 6 are added. Culturing under dark condition, wherein the culturing temperature is 21+/-2 ℃ and the relative humidity is 60-75% in the culturing process.
After 15 days of induction culture, callus grows from the end of the bead hole, the structure is loose, moist and crystal clear, the color is semitransparent, the surface is multi-filiform protrusions and the water content is high when observed under a dissecting mirror, and the result is shown in figure 2. After induction culture for about 50 days, no new callus is basically induced.
(4) Embryogenic callus proliferation culture
Picking the freshest embryogenic cell mass from the surface of embryogenic callus, dividing into small blocks of 0.5cm multiplied by 0.5cm, inoculating onto embryogenic callus proliferation culture medium, and performing embryogenic callus proliferation culture of Picea yunnanensis under dark condition. The embryogenic callus proliferation medium is: improving 1/2MS basic culture medium, adding 0.1mg/L of 2,4-D, 0.05mg/L of 6-BA, 500mg/L of hydrolyzed casein, 500mg/L of inositol, 450mg/L of glutamine, 30g/L of sucrose and 7g/L of agar, wherein the pH is 6, and the culture condition is the same as that of the step (3).
The culture process is carried out once a week, namely, the freshest embryogenic cell mass is picked from the surface of embryogenic callus cultured in the previous week each time, divided into small blocks of 0.5cm multiplied by 0.5cm, and placed in a new embryogenic callus proliferation culture medium so as to keep the culture medium to contain enough nutrients and water, and the proliferation culture is continued, so that a large amount of semitransparent embryogenic callus of the picea yunnanensis is obtained while the embryogenic callus is kept embryogenic, and the result is shown in fig. 3.
(5) Embryogenic callus adjustment culture
The freshest embryogenic cell mass is picked from the surface of the embryogenic callus in multiplication culture, divided into small pieces of 0.5cm multiplied by 0.5cm, and transferred into embryogenic callus adjustment medium. The embryogenic callus adjustment medium is: improving 1/2MS basic culture medium, adding 2g/L active carbon, 500mg/L inositol, 500mg/L hydrolyzed casein, 450mg/L glutamine, 60g/L sucrose, 7g/L agar, pH 6, and culturing under the same condition as in step (3).
After embryogenic callus of picea yunnanensis was cultured on the conditioned medium for 2 weeks, spiny protrusions were observed on the surface of the callus, see fig. 4B. After double staining with carmine acetate and Evan's blue, calli were picked and examined microscopically, as shown in FIG. 4C, to clearly see the apparent differentiation of the embryogenic head and stem cells in the embryogenic cell mass.
(6) Somatic embryo maturation culture
Embryogenic callus after two weeks of culture on embryogenic callus conditioning medium was transferred directly to somatic embryo maturation medium. The maturation medium for somatic embryogenesis is: improving 1/2MS basic culture medium, adding PEG4000 75g/L, ABA mg/L, IBA 0.2mg/L, active carbon 2g/L, inositol 500mg/L, hydrolyzed casein 500mg/L, glutamine 750mg/L, sucrose 60g/L, agar 9g/L, pH 6, and culturing under the same condition as in step (3).
The somatic embryo is subjected to secondary culture every 2 weeks in the maturation culture process, and the mature culture is carried out for about 2 months, so that the continuous differentiation of the embryonic callus of the picea yunnanensis to form new somatic embryos can be observed, and the result is shown in figure 5. The embryogenic callus is differentiated to form somatic embryos with large propagation coefficient, and small blocks with the diameter of 1cm can be finally differentiated to form about 10 somatic embryos.
Example 2
This embodiment differs from embodiment 1 only in that: the embryogenic callus induction medium is: 1/2MS basic culture medium is improved, 2,4-D1mg/L,6-BA0.5mg/L, hydrolyzed casein 480mg/L, inositol 110mg/L, glutamine 480mg/L, sucrose 29g/L, agar 7g/L and pH 5.9 are added.
Example 3
This embodiment differs from embodiment 1 only in that: the embryogenic callus proliferation medium is: 1/2MS basic culture medium is improved, 2, 4-D0.15 mg/L,6-BA 0.08mg/L, hydrolyzed casein 520mg/L, inositol 520mg/L, glutamine 480mg/L, sucrose 32g/L, agar 7g/L and pH 6.1 are added.
Example 4
This embodiment differs from embodiment 1 only in that: the embryogenic callus adjustment medium is: 1/2MS basic culture medium is improved, 1.5g/L of active carbon, 510mg/L of inositol, 490mg/L of hydrolyzed casein, 450mg/L of glutamine, 58g/L of sucrose and 7g/L of agar are added, and the pH is 6.
Example 5
This embodiment differs from embodiment 1 only in that: the maturation medium is: 1/2MS basic culture medium is improved, and PEG4000 100g/L, ABA mg/L, IBA 0.2mg/L, active carbon 1.5g/L, inositol 510mg/L, hydrolyzed casein 520mg/L, glutamine 780mg/L, sucrose 62g/L, agar 9g/L and pH 6 are added.
Example 6
Influence of the concentration of growth regulator in different embryogenic callus induction media on the induction rate.
This example differs from example 1 in that the concentration of the growth regulator in the embryogenic callus induction medium is different, and the other conditions are the same as in example 1. The concentrations of growth regulators in the embryogenic callus induction medium in this example are shown in Table 2.
TABLE 2 concentration of growth regulators in different embryogenic callus induction media
Figure BDA0003801234630000101
After induction culture for about 50 days, basically no new calli are induced, at this time, the induction rate of embryogenic calli under different collection time and different induction culture medium conditions is counted, embryogenic calli induction rate (%) = number of explants inducing embryogenic calli/total number of inoculated explants x 100%, and analysis results are shown in table 3.
TABLE 3 influence of different growth regulator concentrations and sampling times on embryogenic callus induction rate
Figure BDA0003801234630000102
Figure BDA0003801234630000111
As can be seen from Table 3, the composition and concentration of hormones in the embryogenic callus induction medium are critical to the induction of embryogenic callus. Wherein YD2, YD3 and YD4 have higher inductivity. When only one plant growth regulator TDZ is added into the culture medium, the induction rate is generally low.
The time of acquisition of immature cones has an important impact on somatic embryogenesis. Culturing on the same culture medium, and collecting cones in different periods has the highest induction rate of 4-5 times different. Immature cones harvested at different times, i.e., zygotic embryos at different stages of development, can be induced to produce embryogenic callus over a period of time, but require different plant growth regulators. Among them, 2020.06.16 collected immature cones (in the late stage of the primordial period) had the highest induction rate on YD2 medium, 2020.06.23 collected immature cones (in the early embryogenesis period) had the highest induction rate on YD4 medium, and 2020.07.02 collected immature cones (in the early stage of the embryogenesis period) had the highest induction rate on YD10 medium.
Example 7
Effect of concentration of key compounds in different somatic embryo maturation media on the degree of somatic embryo differentiation.
This example differs from example 1 in the concentration of key compounds in the somatic embryo maturation medium, and the other conditions are the same as in example 1. The concentrations of key compounds in the various somatic embryo maturation media in this example are shown in Table 4.
TABLE 4 concentration of key compounds in different somatic embryo maturation media
Figure BDA0003801234630000112
Figure BDA0003801234630000121
The somatic embryo is subjected to secondary culture every 2 weeks in the maturation culture process, and the mature culture is carried out for about 2 months, so that the continuous differentiation of the embryonic callus of the picea yunnanensis to form new somatic embryos can be observed, and the specific number of the somatic embryos is shown in figure 6.
As can be seen from FIG. 6, the proliferation coefficients of embryogenic callus differentiated into somatic embryos are significantly different in different somatic embryo maturation media, and embryogenic callus has higher proliferation coefficients in CS16, CS17 and CS18 maturation media. Wherein somatic embryogenesis is asynchronous and callus browning is serious on CS16 and CS18 maturation media, which is unfavorable for further germination and rooting of somatic embryos formed by differentiation. Somatic embryos can synchronously occur in a CS17 maturation medium, no phenomena such as callus browning and the like occur, and about 20 somatic embryos can be formed by differentiation on callus small blocks with the average diameter of 2 cm. The embryogenic callus has a low propagation coefficient on a CS11 maturation medium, and can be differentiated to form about 5 somatic embryos.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (5)

1. A method of establishing a somatic embryogenesis system for huperzia serrata, the method comprising: taking the immature zygotic embryo as an explant for induction culture, proliferation culture, adjustment culture and maturation culture, and differentiating to form a somatic embryo;
the basic culture medium of the induction culture, proliferation culture, adjustment culture and maturation culture is an improved 1/2MS culture medium; KI 4.1-4.2 mg/L, H in the modified 1/2MS culture medium 3 BO 3 15.4～15.6mg/L、VitaminB 1 0.9～1.1mg/L、VitaminB 6 0.9～1.1mg/L；
The induction culture medium is as follows: modified 1/2MS culture medium +2, 4-D0.2-1 mg/L +6-BA 0.1-2 mg/L +KT0-0.5 mg/L +TDZ 0-0.01 mg/L +inositol 80-120 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2;
the proliferation medium is: modified 1/2MS culture medium +2, 4-D0.05-0.15 mg/L +6-BA 0.02-0.09 mg/L +inositol 450-550 mg/L +hydrolyzed casein 450-550 mg/L +glutamine 400-500 mg/L +sucrose 25-35 mg/L +agar 6-8 g/L; the pH is 5.8-6.2;
the adjustment culture medium is as follows: modified 1/2MS culture medium, activated carbon, myo-inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 400-500 mg/L, sucrose 55-65 mg/L and agar 6-8 g/L; the pH is 5.8-6.2;
the maturation medium is: modified 1/2MS culture medium, PEG 400050-200 g/L, ABA 0.5-60 mg/L, IBA 0.1-0.3 mg/L, active carbon 1-3 g/L, inositol 450-550 mg/L, hydrolyzed casein 450-550 mg/L, glutamine 700-800 mg/L, sucrose 55-65 mg/L and agar 8-10 g/L; the pH is 5.8-6.2.
2. The method of claim 1, wherein the zygotic embryo is in the proembryogenic stage, early embryogenesis and late embryogenesis.
3. The method of claim 1, wherein the culture conditions are: in dark condition, the culture temperature is 19-23 deg.c and the relative humidity during culture is 60-75%.
4. Use of the method according to any one of claims 1 to 3 for the propagation of huperzia serrata and/or for the maintenance of excellent traits.
5. Use of the method of any one of claims 1 to 3 for the improvement of a variety of huperzia serrata.
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