Method for promoting generation and maturation of cryptomeria fortunei somatic embryos

Abstract

The invention discloses a culture method for promoting cryptomeria fortunei somatic embryogenesis and maturation, and belongs to the technical field of somatic embryogenesis. The cryptomeria fortunei somatic embryo generation and maturation method disclosed by the invention comprises the following steps: taking immature seed embryos as explants, and inducing to obtain embryogenic calluses; transferring the embryogenic callus subjected to multiplication culture into a somatic embryo induction culture medium for culture to obtain a somatic embryo; transferring the somatic embryo into a somatic embryo maturation culture medium for dark culture to obtain a mature cotyledon embryo; and transferring the mature cotyledon embryo to a somatic embryo germination basic culture medium, carrying out illumination culture to obtain a somatic embryo seedling, and carrying out acclimatization and transplantation on the somatic embryo seedling to obtain a regenerated plant. Results of the invention show that different families can stably obtain embryogenic calluses, the somatic embryo maturation ability and maturation rate are high, and a large number of high-quality seedlings with stable hereditary characters can be cultivated in a short time; the problem of character separation caused by gene recombination in the seed propagation process is avoided, and it is ensured that offspring plants can inherit the excellent characteristics of parents.

Description

Technical Field

[0001] The present invention belongs to the technical field of somatic embryogenesis, and particularly relates to a method for promoting the somatic embryogenesis and maturation of Cryptomeria fortunei Hooibrenk ex Otto et Dietr. Background Art

[0002] In the field of forestry biotechnology, somatic embryogenesis (SE) technology is crucial. It enables plant somatic cells to develop into complete plants under specific conditions without sexual cell fusion, following a development mode similar to that of zygotic embryos. This technology is widely applied in plant research, especially in forest trees, including basic biological research, in vitro rapid propagation of elite varieties, conservation and utilization of germplasm resources, selection and promotion of superior varieties, and the construction of transgenic and gene editing breeding technologies. Somatic embryogenesis systems have been successfully established for many coniferous trees, but different tree species, and even different genotypes of the same tree species, vary greatly in their somatic embryogenesis ability. This genotype dependence severely limits the application of this technology in forest tree genetic improvement, germplasm innovation, and elite variety breeding.

[0003] Cryptomeria fortunei Hooibrenk ex Otto et Dietr belongs to the genus Cryptomeria in the family Taxodiaceae and is an important native tree species in China. Its wood is light, soft, straight-grained, fine-structured, and highly resistant to decay, and is easy to process, making it a high-quality material for industrial construction. Cryptomeria fortunei is slightly shade-tolerant in its young stage and grows rapidly in mountainous areas with warm and humid climates, acidic and fertile soil, and good drainage. However, currently, the reproduction of Cryptomeria fortunei mainly relies on traditional methods such as seed propagation and cuttage propagation. Seed propagation is easily affected by seed quality and environmental factors, resulting in high genetic diversity in offspring and difficulty in maintaining excellent traits; cuttage propagation has problems such as low propagation coefficient and difficult rooting.

[0004] Establishing an efficient somatic embryogenesis system for Cryptomeria fortunei is of great significance for its genetic improvement and elite variety breeding. Through this system, large-scale propagation of excellent clones can be achieved, providing a large number of high-quality seedlings with consistent genetic quality for forestry production. At the same time, the somatic embryogenesis system is also an ideal platform for molecular biology research such as gene transformation and functional gene verification, which helps to deeply understand the growth, development, and stress resistance mechanisms of Cryptomeria fortunei. However, up to now, the somatic embryogenesis system of Cryptomeria fortunei is still imperfect, with low somatic embryogenesis efficiency, low maturation rate, and a high proportion of abnormal embryos, which has become a key problem restricting its application. Therefore, it is urgent to explore effective methods to promote the somatic embryogenesis and maturation of Cryptomeria fortunei. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a method for promoting the somatic embryogenesis and maturation of Cryptomeria fortunei for promoting the breeding of elite varieties of Cryptomeria fortunei.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for promoting the somatic embryogenesis and maturation of Cryptomeria fortunei, comprising:

[0008] 1) Dissecting immature seed embryos from the cones of superior Cryptomeria fortunei families as explants;

[0009] 2) Inoculating the explants onto an induction solid medium under sterile conditions to induce embryogenic callus;

[0010] 3) Transferring the embryogenic callus to a proliferation culture solid medium for proliferation culture;

[0011] 4) Transferring the embryogenic callus after proliferation culture to a somatic embryo induction medium for culture to obtain somatic embryos;

[0012] 5) Transferring the somatic embryos to a somatic embryo maturation medium for dark culture to obtain mature cotyledon embryos;

[0013] 6) Transferring the mature cotyledon embryos to a basic somatic embryo germination medium for light culture to obtain somatic embryo seedlings, and after hardening off and transplanting the somatic embryo seedlings, obtaining regenerated plants.

[0014] The superior Cryptomeria fortunei families are selected from superior Cryptomeria fortunei families 8#, 32# and 3#.

[0015] The formula of the induction solid medium is DCR basic medium, supplemented with 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 hydrolysate casein 0.5 mg / L, sucrose 30.0 g / L, activated carbon 2.0 g / L, and agar 2.4 g / L.

[0016] The formula of the proliferation culture solid medium is DCR basic 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 carbon 2.0 g / L, and agar 2.4 g / L.

[0017] When the superior Cryptomeria fortunei family is superior Cryptomeria fortunei family 8#, the formula of the somatic embryo induction medium is 1 / 2 DCR basic 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 hydrolysate casein 0.5 g / L, inositol 0.5 g / L, L-aspartic acid 0.2 g / L, agar 3.0 g / L, and activated carbon 2.0 g / L.

[0018] When the excellent Cryptomeria fortunei family is Family 32# or 3#, the formula of the somatic embryo induction medium is 1 / 2 DCR basic medium, supplemented with 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, agar 3.0 g / L, and activated carbon 2.0 g / L.

[0019] When the excellent Cryptomeria fortunei family is Family 8#, 32# or 3#, the formula of the somatic embryo maturation medium is DCR basic medium, supplemented with GA3 3.0 mg / L, L-glutamine 0.5 g / L, activated carbon 2.0 g / L, sucrose 30.0 g / L, and agar 2.4 g / L.

[0020] The formula of the basic somatic embryo germination medium is MS basic medium, glutamine 0.25 g / L, acid hydrolyzed casein 0.1 g / L, sucrose 30.0 g / L, and agar 2.4 g / L.

[0021] The specific steps of the described method include:

[0022] 1) Dissect immature seed embryos from the cones of the excellent Cryptomeria fortunei families 8#, 32# or 3# as explants;

[0023] 2) Inoculate the explants onto the induction solid medium under sterile conditions and culture them in the dark at 23°C for 20 - 25 days to induce embryogenic callus;

[0024] 3) Transfer the embryogenic callus to the proliferation culture solid medium and culture it in the dark at 23°C for 15 days for proliferation culture;

[0025] 4) Transfer the embryogenic callus after proliferation culture to the somatic embryo induction medium and culture it in the dark at 23°C for 5 - 6 weeks to obtain somatic embryos;

[0026] 5) Transfer the somatic embryos to the somatic embryo maturation medium and culture them in the dark at 23°C for 15 days to obtain mature cotyledon embryos;

[0027] 6) Transfer the mature cotyledon embryos to the basic somatic embryo germination medium and culture them under a 16-hour light and 8-hour dark cycle at 23°C to obtain somatic embryo seedlings. After acclimatizing and transplanting the somatic embryo seedlings, regenerated plants are obtained.

[0028] The beneficial effects of the present invention:

[0029] 1) The present invention has established a complete technical system for the induction of Cryptomeria fortunei embryogenic callus and somatic embryogenesis, which can achieve the rapid propagation of excellent genetic resources of Cryptomeria fortunei. The results show that although there are differences in the induction rates of embryogenic callus among different families, they can all be stably obtained; the induction rate of family 8# reaches 62.0%, that of family 32# is 31.33%, and that of family 3# is 42.33%, laying a foundation for the large-scale propagation of excellent Cryptomeria fortunei plants. Moreover, the subsequent somatic embryo maturation ability and maturation rate are very high. The somatic embryo maturation ability of family 8# is 343 / g, and the maturation rate is 94.40%. A large number of high-quality seedlings with stable genetic traits can be cultivated in a short time to meet the market demand for improved Cryptomeria fortunei seedlings.

[0030] 2) The present invention uses immature seed embryos as explants and utilizes somatic embryogenesis technology to effectively preserve the traits of excellent Cryptomeria fortunei parents. Compared with traditional seed propagation, it avoids the problem of trait segregation caused by gene recombination during the seed propagation process, ensuring that the offspring plants can inherit the excellent characteristics of the parents, which is of great significance for the genetic improvement and variety optimization of Cryptomeria fortunei.

[0031] 3) The present invention shortens the breeding time, accelerates the breeding process of new varieties, and improves the breeding efficiency of Cryptomeria fortunei through the somatic embryogenesis system. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Embryogenic callus map (A) at the initial stage of Cryptomeria fortunei induction and microscopic structure map (B) of embryogenic callus cells at the initial stage of Cryptomeria fortunei induction;

[0033] Figure 2 Non-embryogenic callus map (A) at the initial stage of Cryptomeria fortunei induction and microscopic structure map (B) of non-embryogenic callus cells at the initial stage of Cryptomeria fortunei induction;

[0034] Figure 3 Somatic embryo map (A) at the proembryo stage during the pre-maturation culture of embryogenic callus and somatic embryo gradually developing into columnar embryo map (B) during the maturation culture;

[0035] Figure 4 Somatic embryo map (A) at the cotyledon stage during the maturation process and map (B) of unrooted plants and rooted plants after somatic embryo germination;

[0036] Figure 5 Embryogenic callus induction rate map of different families of Cryptomeria fortunei. DETAILED DESCRIPTION OF THE INVENTION

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art.

[0038] The plant material used in this application is Cryptomeria fortunei. The applicant has conducted a number of studies on this material and has published Cryptomeria fortunei superior family 8# (Luo Peng, Weng Huaifeng, Shi Jisen, etc. Salt tolerance differences among different superior clones of Cryptomeria fortunei [J]. Molecular Plant Breeding, 2016, 14(06): 1609-1615.), Cryptomeria fortunei superior family 32# and Cryptomeria fortunei superior family 3# (Cui Jiebing, Zhang Meng, Zhang Yingting, etc. Effects of low temperature stress on different clones of Cryptomeria fortunei and evaluation of cold resistance [J]. Biotechnology Bulletin, 2022, 38(03): 31-40.). Such biological materials are already well-known to the public, and the public can obtain such biological materials from the applicant.

[0039] Example 1

[0040] 1. Obtaining immature seed embryos of Cryptomeria fortunei

[0041] Select Cryptomeria fortunei superior family 8#, collect its superior Cryptomeria fortunei cones, quickly put them into a sealed plastic sample bag, place them in a constant temperature ice box for cold chain transportation, and store them in a 4°C laboratory refrigerator. Under sterile conditions, peel the immature seed embryos in the cones for later use.

[0042] 2. Inducing embryogenic callus

[0043] In a sterile environment, inoculate the immature seed embryos onto the induction solid medium, with 10 embryos inoculated in each petri dish. The formula of the induction solid medium is: DCR basic 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 carbon 2.0 g / L, and agar 2.4 g / L. Incubate in the dark at 23°C for 20-25 days, and the induction rate of embryogenic callus reaches 62.0%.

[0044] The results are as Figure 1 shown. The embryogenic callus suitable for the subsequent steps is translucent, viscous, and spiny. The callus cells are long strip-shaped with polarity and the nucleus is distributed at one end. Such embryogenic callus can proliferate continuously in a suitable medium and maintain its original embryogenicity.

[0045] The results are as Figure 2 shown. The non-embryogenic callus shows a yellowish-brown dense texture, with transparent filaments on the surface, high viscosity, slow growth, and the callus cells are short oval-shaped with irregularly distributed nuclei, and they cannot maintain embryogenicity for a long time during the subsequent subculture process.

[0046] 3. Maintenance and proliferation of embryogenic callus

[0047] The maintenance and proliferation culture of embryogenic callus was carried out using a proliferation culture solid medium, and it was cultured in the dark at 23 °C for 15 days. The formula of the proliferation culture 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 carbon 2.0 g / L, and agar 2.4 g / L

[0048] 4. Somatic embryo induction

[0049] After culturing on the proliferation culture solid medium for 15 days, embryogenic callus that was white, slightly transparent, in good condition, and without spiny protrusions was selected and transferred to the somatic embryo induction medium. The somatic embryo induction medium is 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, agar 3.0 g / L, and activated carbon 2.0 g / L

[0050] 10 embryogenic calli of 0.03 g were inoculated in each petri dish, and 5 plates were inoculated for each type. It was cultured in the dark at 23 °C for 5 - 6 weeks, and the somatic embryo maturation ability was 343 per gram

[0051] The results are as Figure 3 shown. After culturing the embryogenic callus on the induction medium for 4 weeks, proembryo structures were observed under a stereomicroscope. Around 5 weeks, the proembryo heads developed into petal-like structures. Around 6 - 7 weeks, complete columnar embryo structures could be seen. During the somatic embryo induction culture process, the proembryos gradually developed into columnar embryos ( Figure 3 of B).

[0052] 5. Somatic embryo maturation and plant regeneration

[0053] The columnar embryos were transferred to the somatic embryo maturation medium and cultured in the dark at 23 °C for 15 days to induce the formation of mature cotyledon embryos. The somatic embryo maturation medium is DCR basal medium, supplemented with GA3 3.0 mg / L, L-glutamine 0.5 g / L, activated carbon 2.0 g / L, sucrose 30.0 g / L, and agar 2.4 g / L

[0054] The mature cotyledon embryos were transferred to the 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, agar 2.4 g / L) and cultured under light (16 hours of light and 8 hours of darkness, at a temperature of 23 °C). After the somatic embryo seedlings took root with a length greater than 7 cm, they were acclimatized for one week and then transplanted to obtain regenerated plants

[0055] The results are as Figure 4As shown, the cotyledon embryo head cultured on the maturation medium shows green, the suspensor is yellowish green, and a small amount of callus will form again at the base. Figure 4 A), the somatic embryo maturation rate reaches 94.40% on the medium containing GA3. Transfer it to the germination medium, and a complete plant can be formed. Figure 4 B).

[0056] Example 2

[0057] 1. Obtaining immature seed embryos of Cryptomeria fortunei

[0058] Select Cryptomeria fortunei excellent family 32#, after collecting the cones, process them according to the same method as above to obtain immature seed embryos for standby.

[0059] 2. Inducing embryogenic callus

[0060] The inoculation and medium formula are the same as in Example 1. Incubate in the dark at 23°C for 20 - 25 days, and the embryogenic callus induction rate is 31.3%.

[0061] 3. Maintaining and proliferating embryogenic callus

[0062] The medium and culture conditions for maintenance and proliferation are the same as in Example 1.

[0063] 4. Inducing somatic embryos

[0064] The somatic embryo induction medium is 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 hydrolysate casein 0.5 g / L, inositol 0.5 g / L, L - aspartic acid 0.2 g / L, agar 3.0 g / L, activated carbon 2.0 g / L. The culture conditions are the same as in Example 1, and the somatic embryo maturation rate is 186 / g.

[0065] 5. Somatic embryo maturation and plant regeneration

[0066] The conditions for somatic embryo maturation and plant regeneration are the same as in Example 1. First, incubate in the dark for 15 days, then under light, and the maturation rate is 73.33%. The subsequent culture and transplanting steps are the same as in Example 1.

[0067] Example 3

[0068] 1. Obtaining immature seed embryos of Cryptomeria fortunei

[0069] Select Cryptomeria fortunei excellent family 3#, after collecting the cones, process them according to the same method as above to obtain immature seed embryos for standby.

[0070] 2. Inducing embryogenic callus

[0071] The inoculation and culture medium formulation were the same as in Example 1, and dark culture was carried out at 23°C for 20 - 25 days. The embryogenic callus induction rate was 42.33%.

[0072] 3. Maintenance and proliferation of embryogenic callus

[0073] The maintenance and proliferation medium and culture conditions were the same as in Example 1.

[0074] 4. Somatic embryo induction

[0075] The components of the somatic embryo induction medium and the culture conditions were the same as in Example 2, and the somatic embryo maturation ability was 147 per gram.

[0076] 5. Somatic embryo maturation and plant regeneration

[0077] The somatic embryo maturation and plant regeneration conditions were the same as in Example 1, and the maturation rate was 70.33%. The subsequent culture and transplanting steps were the same as in Example 1.

[0078] The results were as Figure 5 shown. The highest embryogenic callus induction rate was 62.0% for Family No. 8, followed by 42.0% for Family No. 3, and the lowest induction rate was 31.3% for Family No. 32.

[0079] In summary, for different families of Cryptomeria fortunei, the method provided by the present invention can effectively induce embryogenic callus, somatic embryos and somatic embryo maturation.

[0080] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A method for promoting the somatic embryogenesis and maturation of Cryptomeria fortunei, characterized in that, Comprising: 1) Immature seed embryos are peeled from the cones of excellent Cryptomeria fortunei families as explants; 2) The explants are inoculated onto an induction solid medium under aseptic conditions to induce embryogenic callus; 3) The embryogenic callus is transferred to a proliferation culture solid medium for proliferation culture; 4) The embryogenic callus after proliferation culture is transferred to a somatic embryo induction medium for culture to obtain somatic embryos; 5) The somatic embryos are transferred to a somatic embryo maturation medium for dark culture to obtain mature cotyledon embryos; 6) The mature cotyledon embryos are transferred to a somatic embryo germination basal medium for light culture to obtain somatic embryo seedlings, and after acclimatizing and transplanting the somatic embryo seedlings, regenerated plants are obtained.

2. The method according to claim 1, characterized in that, The excellent Cryptomeria fortunei families are selected from excellent Cryptomeria fortunei families 8#, 32# and 3#.

3. The method according to claim 2, wherein The formula of the induction solid medium is DCR basal medium, supplemented with 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 2.4 g / L.

4. The method according to claim 2, characterized in that, The formula of the proliferation culture 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 carbon 2.0 g / L, and agar 2.4 g / L. The subculture of embryogenic callus is preferably carried out once every 15 days.

5. The method according to claim 2, characterized in that, When the excellent Cryptomeria fortunei family is excellent Cryptomeria fortunei family 8#, the formula of the somatic embryo induction medium is 1 / 2 DCR basal medium, supplemented with ABA 10 mg / L, PEG8000 170 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, agar 3.0 g / L, and activated carbon 2.0 g / L.

6. The method according to claim 2, wherein When the excellent Cryptomeria fortunei family is excellent Cryptomeria fortunei family 32# or 3#, the formula of the somatic embryo induction medium is 1 / 2 DCR basal medium, supplemented with 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, agar 3 g / L, and activated carbon 2 g / L.

7. The method according to claim 2, wherein When the excellent Cryptomeria fortunei family is excellent Cryptomeria fortunei family 8#, 32# or 3#, the formula of the somatic embryo maturation medium is DCR basal medium, supplemented with GA3 3.0 mg / L, L-glutamine 0.5 g / L, activated carbon 2.0 g / L, sucrose 30.0 g / L, and agar 2.4 g / L.

8. The method according to claim 2, wherein The formula 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 agar 2.4 g / L.

9. The method according to claim 2, wherein The specific steps include: 1) Immature seed embryos are peeled from the cones of excellent Cryptomeria fortunei families 8#, 32# or 3# as explants; 2) Inoculate the explant onto the induction solid medium under sterile conditions and culture it in the dark at 23°C for 20 - 25 days to induce embryogenic callus; 3) Transfer the embryogenic callus to the proliferation culture solid medium and culture it in the dark at 23°C for 15 days for proliferation culture; 4) Transfer the embryogenic callus after proliferation culture to the somatic embryo induction medium and culture it in the dark at 23°C for 5 - 6 weeks to obtain somatic embryos; 5) Transfer the somatic embryos to the somatic embryo maturation medium and culture them in the dark at 23°C for 15 days to obtain mature cotyledon embryos; 6) Transfer the mature cotyledon embryos to the basic somatic embryo germination medium and culture them under a 16 - hour light and 8 - hour dark cycle at 23°C to obtain somatic embryo seedlings. After acclimatizing the somatic embryo seedlings and transplanting them, regenerated plants are obtained.

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