A method to promote somatic embryogenesis and maturation of Cryptomeria japonica
By inducing embryogenic callus and somatic embryogenesis through specific culture media and procedures, the problem of low somatic embryogenesis efficiency in Chinese fir was solved, enabling rapid propagation of superior genetic resources and stable transmission of genetic traits, thus promoting the breeding process of superior Chinese fir varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING FORESTRY UNIV
- Filing Date
- 2025-05-23
- Publication Date
- 2026-05-05
AI Technical Summary
The somatic embryogenesis system of Chinese fir is imperfect, with low embryogenesis efficiency, low maturity rate, and a high proportion of deformed embryos, which limits its application in genetic improvement and breeding of superior varieties.
Using a specific culture medium and procedures, embryogenic callus was induced from immature seed embryos of superior cedar families as explants, and then cultured for proliferation. The callus was then transferred to a somatic embryo induction medium to obtain somatic embryos, which were then matured in a somatic embryo maturation medium to finally obtain regenerated plants.
An efficient system for inducing embryogenic callus and somatic cell development in Cryptomeria japonica was established, enabling rapid propagation of superior genetic resources, improving somatic embryo maturation rate and breeding efficiency, and ensuring the stability of genetic traits in offspring plants.
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Figure CN120323326B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of somatic embryogenesis technology, specifically relating to a method for promoting somatic embryogenesis and maturation of Cryptomeria japonica. Background Technology
[0002] Somatic embryogenesis (SE) technology is crucial in the field of forestry biotechnology. It allows plant somatic cells to develop into complete plants under specific conditions, without sex cell fusion, following a zygotic embryonic developmental pattern. This technology has wide applications in plant, especially forestry, research, including basic biological research, rapid in vitro propagation of superior varieties, germplasm resource protection and utilization, selection and promotion of superior varieties, and the development of transgenic and gene-editing breeding technologies. While somatic embryogenesis systems have been successfully established for many coniferous trees, different tree species, and even different genotypes within the same species, exhibit significant differences in somatic embryogenetic capacity. This genotype dependence severely limits the application of this technology in forestry genetic improvement, germplasm innovation, and superior variety breeding.
[0003] *Cryptomeria fortunei* Hooibrenk ex Otto et Dietr., belonging to the genus *Cryptomeria* in the family Taxodiaceae, is an important native tree species in China. Its wood is light and soft, with straight grain, fine texture, and strong resistance to decay, making it easy to process and a high-quality material for industrial construction. Young *Cryptomeria fortunei* are somewhat shade-tolerant and grow rapidly in warm, humid, acidic, fertile, and well-drained mountainous areas. However, current propagation of *Cryptomeria fortunei* mainly relies on traditional methods such as seed propagation and cutting propagation. Seed propagation is easily affected by seed quality and environmental factors, resulting in high genetic diversity in offspring and difficulty in maintaining desirable traits; cutting propagation suffers from low propagation coefficients and difficulties in rooting.
[0004] Establishing an efficient somatic embryogenesis system for Cryptomeria japonica is of great significance for its genetic improvement and breeding. This system enables large-scale propagation of superior clones, providing a large number of high-quality seedlings with consistent genetic quality for forestry production. Simultaneously, the somatic embryogenesis system is an ideal platform for molecular biology research such as gene transformation and functional gene verification, contributing to a deeper understanding of Cryptomeria japonica's growth, development, and stress resistance mechanisms. However, to date, the Cryptomeria japonica somatic embryogenesis system is still imperfect, with low somatic embryogenesis efficiency, low maturation rate, and a high proportion of deformed embryos, becoming a key challenge limiting its application. Therefore, it is urgent to explore effective methods to promote the maturation of Cryptomeria japonica somatic embryos. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for promoting the embryogenesis and maturation of Cryptomeria japonica, which can be used to promote the breeding of superior Cryptomeria japonica varieties.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] A method for promoting somatic embryogenesis and maturation of Cryptomeria japonica includes:
[0008] 1) Immature seed embryos were extracted from the cones of superior families of Japanese cedar as explants;
[0009] 2) The explants were inoculated into an induction solid medium under aseptic conditions to induce embryogenic callus tissue;
[0010] 3) Transfer the embryogenic callus to a solid culture medium for proliferation culture;
[0011] 4) The embryogenic callus tissue after proliferation culture was transferred to somatic embryo induction medium for culture to obtain somatic embryos;
[0012] 5) Transfer the somatic embryos to somatic embryo maturation medium and culture them in the dark to obtain mature cotyledonary embryos;
[0013] 6) Transfer mature cotyledon embryos to somatic embryo germination basal medium and culture under light to obtain somatic embryo seedlings. After hardening off the somatic embryo seedlings, transplant them to obtain regenerated plants.
[0014] The superior pedigrees of Japanese cedar were selected from superior pedigrees of Japanese cedar, namely No. 8, No. 32, and No. 3.
[0015] The formulation of the induction solid medium is DCR basal medium, with the following added ingredients: 2,4-D 2.0 mg / L, 6-BA 1.0 mg / L, KT 0.50 mg / L, L-glutamine 1.0 mg / L, acid-hydrolyzed casein 0.5 mg / L, sucrose 30.0 g / L, activated carbon 2.0 g / L, and agar-agar 2.4 g / L.
[0016] The solid culture medium for proliferation is formulated as DCR basal medium, with the following added ingredients: 2,4-D 1.0 mg / L, 6-BA 0.5 mg / L, L-glutamine 1.5 g / L, sucrose 25.0-30.0 g / L, activated carbon 2.0 g / L, and agar-agar 2.4 g / L.
[0017] When the superior family of Japanese cedar is Japanese cedar family 8#, the formula of the somatic embryo induction medium is 1 / 2 DCR basic medium, with the following added ingredients: ABA 10.0 mg / L, PEG8000 170.0 g / L, maltose 40.0 g / L, L-glutamine 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, inositol 0.5 g / L, L-aspartic acid 0.2 g / L, crystal agar 3.0 g / L, and activated carbon 2.0 g / L.
[0018] When the superior cedar family is cedar family 32# or 3#, the formula of the somatic embryo induction medium is 1 / 2 DCR basic medium, with the following added ingredients: ABA 10.0 mg / L, PEG8000 150 g / L, maltose 30.0 g / L, L-glutamine 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, inositol 0.5 g / L, L-aspartic acid 0.2 g / L, crystal agar 3.0 g / L, and activated carbon 2.0 g / L.
[0019] When the superior family of Japanese cedar is Japanese cedar family 8#, 32# or 3#, the formula of the somatic embryo maturation medium is DCR basic medium, with the addition of GA3 3.0mg / L, L-glutamine 0.5g / L, activated carbon 2.0g / L, sucrose 30.0g / L and crystal agar 2.4g / L.
[0020] The formulation of the somatic embryo germination basal medium is MS basal medium, glutamine 0.25 g / L, acid-hydrolyzed casein 0.1 g / L, sucrose 30.0 g / L, and crystal agar 2.4 g / L.
[0021] The method described herein includes the following specific steps:
[0022] 1) Immature seed embryos were extracted from the cones of superior Japanese cedar families 8, 32, or 3 as explants;
[0023] 2) The explants were inoculated into the induction solid medium under sterile conditions and cultured in the dark at 23°C for 20-25 days to induce embryogenic callus tissue.
[0024] 3) Transfer the embryogenic callus to a solid culture medium for proliferation and culture in the dark at 23°C for 15 days for proliferation culture;
[0025] 4) Transfer the embryogenic callus tissue after proliferation culture to somatic embryo induction medium and culture in the dark at 23°C for 5-6 weeks to obtain somatic embryos;
[0026] 5) Transfer the somatic embryos to somatic embryo maturation medium and culture them in the dark at 23°C for 15 days to obtain mature cotyledon embryos;
[0027] 6) Transfer mature cotyledon embryos to somatic embryo germination basal medium, 16 hours of light and 8 hours of darkness, and culture at 23℃ to obtain somatic embryo seedlings. After hardening off the somatic embryo seedlings, transplant them to obtain regenerated plants.
[0028] The beneficial effects of this invention are:
[0029] 1) This invention establishes a complete system for inducing embryogenic callus and somatic cell development in Cryptomeria japonica, enabling rapid propagation of superior genetic resources. Results show that while the induction rate of embryogenic callus varies among different families, it is consistently obtained. The induction rate reached 62.0% for family #8, 31.33% for family #32, and 42.33% for family #3, laying the foundation for the large-scale propagation of superior Cryptomeria japonica plants. Furthermore, the subsequent somatic embryo maturation capacity and rate are very high; family #8 has a somatic embryo maturation capacity of 343 embryos / g and a maturation rate of 94.40%, enabling the rapid cultivation of a large number of high-quality seedlings with stable genetic traits to meet market demand for superior Cryptomeria japonica seedlings.
[0030] 2) This invention uses immature seed embryos as explants and utilizes somatic embryogenesis technology to effectively preserve the superior traits of the Japanese cedar parent. Compared with traditional seed propagation, it avoids the trait segregation problem caused by gene recombination during seed propagation, ensuring that offspring plants can inherit the superior characteristics of the parents, which is of great significance for the genetic improvement and variety optimization of Japanese cedar.
[0031] 3) This invention shortens the breeding time, accelerates the selection process of new varieties, and improves the breeding efficiency of Japanese cedar by using a somatic cell embryogenesis system. Attached Figure Description
[0032] Figure 1 Image (A) of embryogenic callus in the initial stage of induction of Cryptomeria japonica and image (B) of the cellular structure of embryogenic callus in the initial stage of induction of Cryptomeria japonica.
[0033] Figure 2 A diagram of non-embryonic callus in the initial stage of induction of Cryptomeria japonica (A) and a microscopic structure diagram of non-embryonic callus cells in the initial stage of induction of Cryptomeria japonica (B);
[0034] Figure 3 A diagram of the somatic embryo during the pre-maturation stage of embryogenic callus culture (A) and a diagram of the protoembryonic embryo gradually developing into a columnar embryo during the maturation process (B).
[0035] Figure 4 The diagram shows the somatic embryo during the cotyledon stage of maturation (A) and the plant after somatic embryo germination that has not yet rooted and the plant that has rooted (B).
[0036] Figure 5 The diagram shows the embryogenic callus induction rate of different families of Japanese cedar. Detailed Implementation
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.
[0038] The plant material used in this application is Cryptomeria japonica. The applicant has conducted multiple studies on this material and has published the following: Cryptomeria japonica superior family 8# (Luo Peng, Weng Huaifeng, Shi Jisen, et al. Differences in salt tolerance among different Cryptomeria japonica superior clones [J]. Molecular Plant Breeding, 2016, 14(06): 1609-1615.), Cryptomeria japonica superior family 32#, and Cryptomeria japonica superior family 3# (Cui Jiebing, Zhang Meng, Zhang Yingting, et al. Effects of low temperature stress on different Cryptomeria japonica clones and evaluation of cold resistance [J]. Biotechnology Bulletin, 2022, 38(03): 31-40.). This type of biological material is well known to the public, and the public can obtain this type of biological material from the applicant.
[0039] Example 1
[0040] 1. Obtaining immature seed embryos of Cryptomeria japonica
[0041] Superior Japanese cedar family 8# was selected, and its superior Japanese cedar cones were collected. These cones were quickly placed into sealed plastic sample bags, transported via a constant-temperature ice box, and stored in a 4°C laboratory refrigerator. Under aseptic conditions, the immature seed embryos were extracted from the cones for later use.
[0042] 2. Induction of embryonic callus
[0043] In a sterile environment, immature seed embryos were inoculated onto an induction solid medium, with 10 embryos inoculated per petri dish. The induction solid medium was formulated as follows: DCR basal medium supplemented with 2,4-D 2.0 mg / L, 6-BA 1.0 mg / L, KT 0.5 mg / L, L-glutamine 1.0 mg / L, acid-hydrolyzed casein 0.5 mg / L, sucrose 30.0 g / L, activated charcoal 2.0 g / L, and agar-agar 2.4 g / L. After incubation in the dark at 23°C for 20-25 days, the embryogenic callus induction rate reached 62.0%.
[0044] The results are as follows Figure 1 As shown, the embryogenic callus suitable for the following steps is translucent, viscous, and spiky. The callus cells are long, polarized strips with nuclei distributed at one end. This type of embryogenic callus can continuously proliferate and maintain its original embryogenicity under suitable culture medium.
[0045] The results are as follows Figure 2 As shown, non-embryonic callus has a dense, yellowish-brown texture with transparent filaments on the surface. It is highly viscous and grows relatively slowly. The callus cells are short, oval-shaped cells with irregularly distributed nuclei. They cannot maintain embryonity for a long time during subsequent subculture.
[0046] 3. Maintenance and proliferation of embryonic callus
[0047] Embryogenic callus was maintained and proliferated using a solid proliferative culture medium, and cultured in the dark at 23°C for 15 days. The solid proliferative culture medium was formulated as follows: DCR basal medium, supplemented with 1.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 1.5 g / L L-glutamine, 25.0-30.0 g / L sucrose, 2.0 g / L activated charcoal, and 2.4 g / L agaricus crystallinus.
[0048] 4. Somatic embryo induction
[0049] After culturing on solid proliferative medium for 15 days, embryogenic callus that is white, slightly translucent, in good condition, and without spiky protrusions was selected and transferred to somatic embryo induction medium. The somatic embryo induction medium was a 1 / 2 DCR basal medium supplemented with ABA 10.0 mg / L, PEG8000 170.0 g / L, maltose 40.0 g / L, L-glutamine 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, inositol 0.5 g / L, L-aspartic acid 0.2 g / L, agaric agar 3.0 g / L, and activated charcoal 2.0 g / L.
[0050] Ten 0.03g embryogenic calluses were inoculated into each culture dish, and five plates were inoculated into each culture dish. The culture was carried out in the dark at 23°C for 5-6 weeks. The somatic embryo maturation capacity was 343 calluses / g.
[0051] The results are as follows Figure 3 As shown, after culturing embryogenic callus on induction medium for 4 weeks, protoembryonic structures appeared under a stereomicroscope. Around 5 weeks, the protoembryonic head developed into a lobular structure, and around 6-7 weeks, a complete columnar embryonic structure was visible. During the somatic embryonic induction culture, the protoembryonic gradually developed into a columnar embryo. Figure 3 (B).
[0052] 5. Embryo maturation and plant regeneration
[0053] The columnar embryos were transferred to somatic embryo maturation medium and cultured in the dark at 23°C for 15 days to induce the formation of mature cotyledonary embryos. The somatic embryo maturation medium was DCR basal medium supplemented with GA3 3.0 mg / L, L-glutamine 0.5 g / L, activated charcoal 2.0 g / L, sucrose 30.0 g / L, and agar-agar 2.4 g / L.
[0054] Mature cotyledonous embryos were transferred to somatic embryo germination basal medium (MS basal medium, glutamine 0.25 g / L, acid hydrolyzed casein 0.1 g / L, sucrose 30.0 g / L, and crystal agar 2.4 g / L) and cultured under light (16 hours light, 8 hours darkness, at 23°C). Once the root length of the somatic embryo seedlings was greater than 7 cm, they were transplanted after one week of hardening off to obtain regenerated plants.
[0055] The results are as follows Figure 4As shown, the cotyledonary embryos cultured on the maturation medium have green embryo heads and yellowish-green pedicels, and a small amount of callus tissue will re-form at the base. Figure 4 A), the somatic embryo maturation rate reached 94.40% on a medium containing GA3, and when transferred to a germination medium, it could form a complete plant. Figure 4 B).
[0056] Example 2
[0057] 1. Obtaining immature seed embryos of Cryptomeria japonica
[0058] Select the superior family line 32# of Japanese cedar, collect cones and process them in the same way as above to obtain immature seed embryos for later use.
[0059] 2. Induction of embryonic callus
[0060] The inoculation and culture medium formulation were the same as in Example 1. The cells were cultured in the dark at 23°C for 20-25 days, and the embryogenic callus induction rate was 31.3%.
[0061] 3. Maintenance and proliferation of embryonic callus
[0062] The maintenance and proliferation culture medium and culture conditions were the same as in Example 1.
[0063] 4. Somatic embryo induction
[0064] The somatic embryo induction medium was 1 / 2 DCR basal medium, supplemented with ABA 10.0 mg / L, PEG8000 150.0 g / L, maltose 30.0 g / L, L-glutamine 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, inositol 0.5 g / L, L-aspartic acid 0.2 g / L, agar-agar crystals 3.0 g / L, and activated charcoal 2.0 g / L. The culture conditions were the same as in Example 1, and the somatic embryo maturity rate was 186 embryos / g.
[0065] 5. Embryo maturation and plant regeneration
[0066] The conditions for embryo maturation and plant regeneration were the same as in Example 1. The embryos were first cultured in the dark for 15 days, followed by light culture, resulting in a maturity rate of 73.33%. Subsequent culture and transplanting procedures were the same as in Example 1.
[0067] Example 3
[0068] 1. Obtaining immature seed embryos of Cryptomeria japonica
[0069] Select the superior family line #3 of Japanese cedar, collect cones and process them using the same method as described above to obtain immature seed embryos for later use.
[0070] 2. Induction of embryonic callus
[0071] The inoculation and culture medium formulation were the same as in Example 1. The cells were cultured in the dark at 23°C for 20-25 days, and the embryogenic callus induction rate was 42.33%.
[0072] 3. Maintenance and proliferation of embryonic callus
[0073] The maintenance and proliferation culture medium and culture conditions were the same as in Example 1.
[0074] 4. Somatic embryo induction
[0075] The composition and culture conditions of the somatic embryo induction medium were the same as in Example 2, and the somatic embryo maturation capacity was 147 embryos / g.
[0076] 5. Embryo maturation and plant regeneration
[0077] The conditions for embryo maturation and plant regeneration were the same as in Example 1, with a maturity rate of 70.33%. Subsequent culture and transplanting steps were the same as in Example 1.
[0078] The results are as follows Figure 5 As shown, the highest embryogenic callus induction rate was in family 8 at 62.0%, followed by family 3 at 42.0%, while the lowest induction rate was in family 32 at 31.3%.
[0079] In summary, the method provided in this invention can effectively induce embryogenic callus, somatic embryos, and somatic embryo maturation in different families of Japanese cedar.
[0080] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A method for promoting somatic embryogenesis and maturation of Cryptomeria japonica, characterized in that, include: 1) Immature seed embryos were extracted from the cones of superior Cryptomeria japonica families as explants; among them, superior Cryptomeria japonica families No. 8, No. 32 and No. 3 were selected; 2) Explants were inoculated into an induction solid medium under aseptic conditions to induce embryogenic callus. The induction solid medium was formulated as DCR basal medium, with the following added components: 2,4-D 2.0 mg / L, 6-BA 1.0 mg / L, KT 0.50 mg / L, L-glutamine 1.0 mg / L, acid-hydrolyzed casein 0.5 mg / L, sucrose 30.0 g / L, activated charcoal 2.0 g / L, and agar-agar 2.4 g / L. 3) Transfer the embryogenic callus to a proliferative solid medium for proliferation culture; the formulation of the proliferative solid medium is DCR basal medium, supplemented with 2,4-D 1.0 mg / L, 6-BA 0.5 mg / L, L-glutamine 1.5 g / L, sucrose 25.0-30.0 g / L, activated charcoal 2.0 g / L, and agar-agar 2.4 g / L; the embryogenic callus is subcultured every 15 days as one cycle; 4) The embryogenic callus tissue after proliferation culture was transferred to somatic embryo induction medium for culture to obtain somatic embryos; wherein, when the superior family of *Cryptomeria japonica* is superior family 8#, the somatic embryo induction medium formula is 1 / 2 DCR basal medium, with the following added: ABA 10mg / L, PEG8000 170g / L, maltose 40.0g / L, L-glutamine 0.5g / L, acid-hydrolyzed casein 0.5g / L, inositol 0.5g / L, L-aspartic acid 0.2g / L, agar-agar 3.0g / L, and activated carbon 2.0g / L; when the superior family of *Cryptomeria japonica* is superior family 32# or 3#, the somatic embryo induction medium formula is 1 / 2 DCR basal medium, with the following added: ABA 10.0mg / L, PEG8000 150g / L, maltose 30.0g / L, L-glutamine 0.5g / L, acid-hydrolyzed casein 0.5g / L, inositol 0.5g / L, L-aspartic acid 0.2g / L, crystal agar 3g / L, activated charcoal 2g / L; 5) Transfer the somatic embryos to somatic embryo maturation medium and culture them in the dark to obtain mature cotyledonary embryos; the somatic embryo maturation medium is formulated as DCR basal medium, supplemented with GA3 3.0 mg / L, L-glutamine 0.5 g / L, activated charcoal 2.0 g / L, sucrose 30.0 g / L, and agar-agar 2.4 g / L. 6) Transfer mature cotyledon embryos to somatic embryo germination basal medium and culture under light to obtain somatic embryo seedlings. After hardening off the somatic embryo seedlings, transplant them to obtain regenerated plants.
2. The method according to claim 1, characterized in that, The formulation of the somatic embryo germination basal medium is MS basal medium, glutamine 0.25 g / L, acid-hydrolyzed casein 0.1 g / L, sucrose 30 g / L, and crystal agar 2.4 g / L.
3. The method according to claim 1, characterized in that, The specific steps include: 1) Immature seed embryos were extracted from the cones of superior Japanese cedar families 8#, 32#, or 3# as explants; 2) The explants were inoculated into the induction solid medium under sterile conditions and cultured in the dark at 23°C for 20-25 days to induce embryogenic callus tissue. 3) Transfer the embryogenic callus to a solid culture medium for proliferation and culture in the dark at 23°C for 15 days for proliferation culture; 4) Transfer the proliferating embryogenic callus to somatic embryo induction medium and culture in the dark at 23°C for 5-6 weeks to obtain somatic embryos; 5) Transfer the somatic embryos to somatic embryo maturation medium and culture them in the dark at 23°C for 15 days to obtain mature cotyledonary embryos; 6) Transfer mature cotyledon embryos to somatic embryo germination basal medium, 16 hours of light and 8 hours of darkness, and culture at 23℃ to obtain somatic embryo seedlings. After hardening off the somatic embryo seedlings, transplant them to obtain regenerated plants.