A method for establishing stable and efficient Torreya grandis regeneration plants

Abstract

The present invention discloses a method for establishing stable and efficient regenerated plants from Torreya grandis. The method comprises obtaining immature zygotic embryos from Torreya grandis under sterile conditions, inducing embryonic callus, liquid suspension culture, developing and maturing somatic embryos, germinating somatic embryos, and transplanting regenerated seedlings. Using the method of the present invention, the induction rate of embryonic callus from Torreya grandis can reach up to 88.9%, and stable mature cotyledonary embryos can be obtained, which can be regenerated into complete plants. This successfully establishes a highly efficient and stable regeneration system, creating favorable conditions for the industrialized production of high-quality Torreya grandis seedlings and laying a solid foundation for achieving genetic transformation and creating improved varieties of Torreya grandis.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro rapid propagation biotechnology of forest tree seedlings, and specifically relates to a method for inducing embryonic callus by using immature zygotic embryos of Torreya grandis as explants, and for efficiently and stably regenerating Torreya grandis plants through synchronous regulation of a liquid suspension system and somatic embryogenesis. Background Art

[0002] Torreya grandis (Torreya arn.), also known as the Chinese torreya, is an evergreen tree in the Taxales order, Taxaceae family, and genus Torreya. It is endemic to my country and primarily grows in the more humid regions of southern my country. Torreya grandis, a superior economic tree species bred through natural variation and artificial selection, possesses high medicinal value, such as relieving cough and phlegm, moistening the lungs and strengthening the spleen. Its seeds contain up to 54.5% oil, making it an important woody oil-bearing tree species. It also produces paclitaxel, the genus's signature substance. Currently, Torreya grandis is primarily propagated through grafting, but this method suffers from low propagation rates, long operating cycles, and relatively low efficiency, severely hindering the development of Torreya grandis cultivation. Therefore, it is necessary to explore efficient industrialized technologies for rapid propagation of Torreya grandis. Currently, there are few reports domestically and internationally on the rapid propagation of high-quality Torreya grandis seedlings through tissue culture, while inducing adventitious roots through traditional organogenesis is difficult. Somatic embryogenesis is a method of asexual reproduction in which somatic cells mimic the development of zygotic embryos to regenerate complete plantlets. It eliminates the need for a separate adventitious root induction process and is a robust, efficient, and cost-effective asexual reproduction method that can maintain the desired traits while enabling industrial production. Somatic embryo induction and plantlet regeneration have been implemented in large-scale production practices in species such as loblolly pine, Norway spruce, Douglas fir, and radiata pine (Mckeand et al., 2007; Dean et al., 2008; Find et al., 2014; Lindgren et al., 2008).

[0003] In general, research on somatic embryogenesis in Torreya grandis is still in its infancy. Reports have shown that somatic embryo induction using immature zygotic embryos from Torreya grandis can produce embryonic callus. While somatic embryos can be obtained, the induction rate is extremely low and the deformity rate is high, making industrial production impossible. Furthermore, there have been no reports on synchronized regulation using liquid suspension culture techniques. Liquid suspension culture involves culturing single plant cells or small cell aggregates in a liquid medium to maintain a well-dispersed state and allow them to proliferate continuously. Compared to solid culture media for callus cultivation, liquid culture allows a small amount of embryonic callus to be converted into a large number of evenly dispersed, high-quality embryonic callus in a short period of time, with synchronized regulation. Furthermore, liquid culture offers advantages such as good reproducibility and easy control of the culture.

[0004] This application uses immature zygotic embryos of Torreya grandis as explants to induce embryonic callus tissue, obtains granular proembryonic tissue through liquid suspension culture technology, and then induces, matures and germinates somatic embryos to achieve plant regeneration, thus establishing a stable and efficient somatic embryogenesis system for Torreya grandis. No relevant information has been reported yet. Summary of the Invention

[0005] In view of the technical limitations of the current rapid propagation technology system for Torreya grandis in the background art, the purpose of the present invention is to provide a method for establishing stable and efficient Torreya grandis regeneration plants.

[0006] This is achieved specifically through the following technical solutions:

[0007] A method for establishing stable and efficient Torreya grandis regeneration plants, the method comprising the following steps:

[0008] 1) Collection and preparation of immature zygotic embryos from Torreya grandis: Collect seeds of uniform size and good growth from superior Torreya grandis trees from late June to early July each year. Store in a refrigerator at 4°C and remove the immature zygotic embryos from the seeds under sterile conditions within one week.

[0009] 2) Embryogenic callus induction and proliferation: Under sterile conditions, immature zygotic embryos of Torreya grandis L. of different genotypes were inoculated into embryonic callus induction medium and cultured in the dark at 25°C. Embryogenic callus with an embryonic suspensor mass structure was induced within 10-15 days, and stably proliferating embryonic callus was obtained within 30-40 days. The induction efficiency of embryonic callus was above 80%.

[0010] 3) Liquid suspension culture: Under sterile conditions, the embryonic callus obtained in step 2) was transferred to a liquid medium for dark culture on a shaker, subcultured every 7 days, and granular proembryos were obtained after 2 weeks.

[0011] 4) Somatic Embryo Induction and Maturation: Under sterile conditions, the proembryonic tissue obtained in step 3) is transferred to a somatic embryo induction medium and cultured in the dark at 25°C. After one month, columnar embryos will form, and after about three months, the somatic embryos will fully mature into cotyledonary embryos.

[0012] 5) Germination of somatic embryos into seedlings: The mature cotyledonary embryos from step 4) were transferred to a germination medium for germination into seedlings. The culture conditions were: 25±2°C incubation under light intensity of 45 μmol·m -2 ·s -1 , photoperiod 16 / 8h;

[0013] 6) Transplantation of somatic embryo regenerated seedlings: The somatic embryo seedlings cultured in step 5) were opened and placed in an acclimatization room at 360 μmol·m -2 ·s-1 Harden the seedlings under strong light for a week, then take out the somatic embryo seedlings, thoroughly wash the culture medium on the roots and transplant them into the substrate, bag the individual plants to avoid excessive transpiration, water them regularly, cut off the two corners of the plastic bag after a week to allow proper ventilation, remove the bag completely after two weeks, and new leaves will grow in three weeks. After the new leaves grow, they can be transplanted to the greenhouse.

[0014] Furthermore, in step 1), the zygotic embryos are columnar embryos.

[0015] Furthermore, in step 2), the mesoembryonic callus induction medium formula is: Gupta and Durzan basic medium, supplemented with 2-4 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminoadenine, 0.5 mg / L 6-furanaminopurine, 15 mg / L vitamin C, 0.5 g / L L-glutamine, 0.5 g / L hydrolyzed casein, 20 g / L sucrose, 2 g / L activated carbon, and 3.8 g / L crystal agar, pH 5.8.

[0016] Furthermore, in step 3), the dark culture conditions of the mesoembryogenic callus are: temperature 23° C., and shaker speed 90 rpm.

[0017] Furthermore, the formula of the liquid culture medium in step 3) is: DCR basic culture medium, supplemented with ABA 1.0 mg / L, gibberellin 1 mg / L, vitamin C 15 mg / L, L-glutamine 0.2 g / L, hydrolyzed casein 0.5 g / L, sucrose 30 g / L, pH 5.8.

[0018] Furthermore, the formula of the somatic embryo induction medium in step 4) is: DCR basic medium, supplemented with gibberellin 3 mg / L, vitamin C 10 mg / L, L-glutamine 0.5 g / L, hydrolyzed casein 0.5 g / L, sucrose 30 g / L, activated carbon 1 g / L, abscisic acid 3 mg / L, polyethylene glycol 8000 0-200 g / L, crystal agar 3.8-4.5 g / L, pH 5.8.

[0019] Furthermore, the germination medium formula in step 5) is: DCR basic medium, supplemented with 0.5 mg / L 6-benzylaminopurine, 0.2 mg / L 3-indolebutyric acid, 3 mg / L gibberellin, 10 mg / L vitamin C, 1 g / L activated carbon, 20 g / L sucrose, and 3.8 g / L crystal agar, pH 5.8.

[0020] Furthermore, the matrix in step 5) is a mixed culture matrix formed by mixing peat: perlite: frog stone in a mass ratio of 1:1:1.

[0021] The present invention has the following beneficial effects: Using the method of the present invention, the embryonic callus induction rate of Torreya grandis can reach up to 88.9%. After liquid suspension culture, somatic embryo induction and maturation can be performed to obtain mature cotyledonary embryos, which can then be regenerated into plants. This successfully establishes an efficient and stable regeneration system, creating favorable conditions for the industrialized production of high-quality Torreya grandis seedlings and laying a good foundation for the genetic transformation and creation of improved varieties. This method has a high reproduction coefficient, is not restricted by season, has simple and easy implementation steps, and is very effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the phenotype diagram of Torreya grandis seeds;

[0023] Figure 2 This is a phenotype diagram of Torreya grandis seeds with columnar embryos;

[0024] Figure 3 This is the phenotype of the immature zygotic embryo of Torreya grandis;

[0025] Figure 4 This is the phenotype of embryonic callus induced from immature zygotic embryos;

[0026] Figure 5 This is the phenotype of the proembryonic tissue produced after about 2 weeks of liquid culture;

[0027] Figure 6 This is the phenotype of mature cotyledonary embryos grown after about 90 days of somatic embryo induction culture;

[0028] Figure 7 This is the phenotype diagram of somatic embryo seedlings formed by somatic embryo germination;

[0029] Figure 8 This is the phenotype of Torreya grandis seedlings regenerated after hardening and transplanting;

[0030] Figure 9 The phenotype of embryonic callus is white with soft texture;

[0031] Figure 10 The non-embryogenic callus phenotype is moist, with a lot of water and a cotton-like surface.

[0032] Figure 11 This is a phenotype diagram of non-embryogenic callus with a hard and loose texture and yellowish color;

[0033] Figure 12 This is a comparison chart of the induction rates of different concentrations of 2,4-D during the callus induction stage;

[0034] Figure 13 This is a comparison chart of the induction rates of different genotypes during the callus induction stage;

[0035] Figure 14The statistical diagram of somatic embryos and cotyledonary embryos after 45 days of treatment with different concentrations of PEG;

[0036] Figure 15 Statistical graph of somatic embryos and cotyledonary embryos after 90 days of treatment with different concentrations of PEG. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments to facilitate a better understanding of the present technical solution.

[0038] Example 1: Collection and Preparation of Immature Zygotic Embryos of Torreya grandis

[0039] The test materials are immature zygotic embryos of Torreya grandis, which were collected from the asexual grafting seed garden in Yuqian Town, Lin'an District, Zhejiang Province. The collection time is from the end of June to the beginning of July each year. Figure 1 As shown in the figure, the columnar embryo is more suitable for microscopic observation of Torreya grandis zygotic embryo. Figure 2 As shown. After the seeds are collected, they are placed in a pre-cooled ice box and transported back to the laboratory, and refrigerated in a 4°C refrigerator for later use. Before peeling the embryo, remove the aril of the Torreya grandis seeds, rinse with 3-5 drops of Blue Moon brand detergent for 10 minutes, and rinse with running water for 1 hour. Under a sterile environment, sterilize the surface with 75% ethanol for 1 minute, sterilize with 10% sodium hypochlorite solution for 40 minutes, and then rinse with sterile water 3-4 times. Use a scalpel and tweezers under a stereo microscope to remove the outer seed coat and peel off the complete embryo, as shown. Figure 3 shown.

[0040] Among them, the test materials were immature zygotic embryos of five different genotypes of Torreya grandis mother trees, #1, #2, #3, #4, and #5.

[0041] Unless otherwise specified, all pictures provided in the accompanying drawings of the specification are pictures corresponding to the #3 genotype.

[0042] Example 2: Embryogenic callus induction and proliferation

[0043] The sterile zygotic embryos of Torreya grandis obtained were inoculated into embryonic callus induction medium to obtain embryonic callus tissue with stable proliferation, such as Figure 4 The embryonic callus induction medium is based on DCR as the basic medium, supplemented with 2,4-D0-4 mg / L, 6-BA0.5 mg / L, KT0.5 mg / L, Vc15 mg / L, L-Gln0.5 g / L, CH0.5 g / L, sucrose 20 g / L, Ac2 g / L, and crystal agar 3.8 g / L, with a pH of 5.8.

[0044] Seeds of Torreya grandis #3 mother tree were selected and callus induction was carried out using 2,4-D culture medium with different concentrations. The 2,4-D concentrations were set at 0, 1, 2, and 4 mg / L. Thirty zygotic embryos were selected for each concentration and three replicates were performed. The embryonic callus induction was observed after one month. Figure 9 It is white, soft-textured embryonic callus; Figure 10 It is non-embryogenic callus, moist, very watery, and has a cottony surface; Figure 11 The callus tissue is hard and loose, and is non-embryogenic callus. Figure 12 As shown, when the 2,4-D concentration was 2 mg / L, the embryonic callus induction rate of Torreya grandis reached its maximum value of 88.9%; when the concentration of 2,4-D was 4 mg / L, the embryonic callus induction rate of Torreya grandis decreased significantly to 71.1%, indicating that the 2 mg / L concentration of 2,4-D was more suitable for the induction of embryonic callus tissue of immature zygotic embryos of Torreya grandis, and too high a concentration of 2,4-D would have a certain inhibitory effect on the embryonic callus induction.

[0045] In addition, four mother trees with genotypes #1, #2, #4, and #5 were selected and the 2,4-D concentration was set to 2 mg / L for embryonic callus induction. Thirty zygotes were obtained from each genotype and repeated three times. The embryonic callus induction was observed after one month. Figure 13 As shown in the figure, there was no significant difference in the embryonic callus induction rate among different genotypes, indicating that the embryonic callus induction of Torreya grandis was less affected by genotype.

[0046] Example 3: Liquid suspension culture

[0047] Embryogenic callus was inoculated into liquid culture medium with the following formula: DCR basic medium supplemented with ABA 1.0 mg / L, GA 31 mg / L, Vc 15 mg / L, L-Gln 0.2 g / L, CH 0.5 g / L, sucrose 30 g / L, pH 5.8, cultured at 23°C in the dark, shaken at 90 rpm, subcultured every 7 days, and granular proembryonic tissue was obtained in about 4 weeks. Figure 5 This is the morphological diagram of the proembryonic tissue produced by embryogenic callus after 4 weeks of liquid culture.

[0048] Example 4: Somatic Embryo Induction and Maturation

[0049] During induction, the cell suspension of granular proembryos produced by liquid suspension culture was diluted to 10 times its volume and plated onto somatic embryo induction medium lined with filter paper, with approximately 3.2 ml of the suspension plated per dish. The cells were then cultured on various concentrations of PEG 8000 medium. The somatic embryo induction medium formulation consisted of: DCR minimal medium supplemented with 33 mg / L GA, 10 mg / L Vc, 0.5 g / L L-Gln, 0.5 g / L CH, 30 g / L sucrose, 1 g / L Ac, 3 mg / L ABA, 0-200 g / L PEG 8000, and 3.8-4.5 g / L crystal agar (pH 5.8). Culture conditions were 25°C in the dark. The PEG concentration gradient was 0, 50, 80, 100, 120, 150, 170, and 200 g / L. Six replicates were set for each treatment, and the cultures were cultured in the dark. Figure 6 These are cotyledonary embryos grown through somatic embryo induction culture.

[0050] The results showed that after 45 days of induction, early somatic embryos appeared in all treatments ( Figure 14 A), when the PEG concentration was 0-120 g / L, the number of early somatic embryos increased with the increase of PEG concentration; when the PEG concentration was 120 g / L, the number was the highest, with an average of 40 embryos per dish. When the PEG concentration exceeded 120 g / L, the number of early somatic embryos gradually decreased with the increase of PEG concentration. When the PEG concentration was 200 g / L, the number was significantly reduced compared with other culture media, with an average of only about 6.6 embryos per dish ( Figure 14 A). When the PEG concentration was 100, 120, 150, and 170 g / L, a small number of mature cotyledonary embryos were formed. When the PEG concentration was 150 g / L, the number of cotyledonary embryos was significantly higher than that of the other concentrations, and the average number of cotyledonary embryos per dish reached about 10.2 ( Figure 14 B).

[0051] After 90 days of induction, callus appeared in different degrees under low concentration PEG culture conditions. When the PEG concentration was 0-150g / L, the number of somatic embryos decreased with the increase of PEG concentration; when the PEG concentration was further increased, the number of somatic embryos decreased ( Figure 15 A). When the PEG concentration was 150 g / L, the number of cotyledonary embryos and the number of somatic embryos at other developmental stages were significantly higher than those at other concentrations, with an average of about 24 cotyledonary embryos per dish ( Figure 15 B).

[0052] Example 5: Somatic embryo germination into seedlings

[0053] The obtained mature cotyledonary embryos were transferred to germination medium. The germination culture basic medium was DCR supplemented with 0.5 mg / L 6-BA, 0.2 mg / L indolebutyric acid (IBA), 33 mg / L GA, 10 mg / L Vc, 1 g / L Ac, 20 g / L sucrose, and 3.8 g / L crystal agar. The pH was 5.8. The culture conditions were light intensity 45 μmol·m -2 ·s -1 , photoperiod 16 / 8h, temperature 25±2℃, somatic embryo seedlings were obtained, Figure 7 This is a morphological diagram of the regenerated seedlings of Torreya grandis.

[0054] Example 6: Transplantation of somatic embryo regenerated seedlings

[0055] The somatic embryo seedlings with strong root growth and robust plants cultured in Example 6 were selected for acclimation and transplantation, and placed in an acclimation room at 360 μmol·m -2 ·s -1 Harden the seedlings under strong light for a week, then take out the somatic embryo seedlings, thoroughly wash the culture medium on the roots and transplant them into the substrate, bag the individual plants to avoid excessive transpiration, water them regularly, cut off the two corners of the plastic bag after a week to allow proper ventilation, completely remove the bag after two weeks, and new leaves will grow in three weeks. After the new leaves grow, they can be transplanted to the greenhouse. Figure 8 For transplanted plants that survive.

Claims

1. A method for establishing a regenerated plant of Torreya grandis, characterized in that The method comprises the following steps: 1) Collection and preparation of immature zygotic embryos from Torreya grandis: Collect seeds of uniform size and good growth from superior Torreya grandis trees from late June to early July each year. Store in a refrigerator at 4°C and remove the immature zygotic embryos from the seeds under sterile conditions within one week. 2) Embryogenic callus induction and proliferation: Under sterile conditions, immature zygotic embryos of Torreya grandis L. of different genotypes were inoculated into embryonic callus induction medium and cultured in the dark at 25°C. Embryogenic callus with an embryonic suspensor mass structure was induced within 10-15 days, and stably proliferating embryonic callus was obtained within 30-40 days. The embryonic callus induction medium formula is: Gupta and Durzan minimal medium, supplemented with 2-4 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminoadenine, 0.5 mg / L 6-furylaminopurine, 15 mg / L vitamin C, 0.5 g / L L-glutamine, 0.5 g / L hydrolyzed casein, 20 g / L sucrose, 2 g / L activated carbon, 3.8 g / L crystal agar, pH 5.8; 3) Liquid suspension culture: Under sterile conditions, transfer the embryonic callus obtained in step 2) into liquid culture medium and culture in a shaker in the dark. Subculture every 7 days to obtain granular proembryos after 2 weeks. The liquid culture medium was prepared as follows: DCR basic medium supplemented with ABA 1.0 mg / L, gibberellin 1 mg / L, vitamin C 15 mg / L, L-glutamine 0.2 g / L, casein hydrolyzate 0.5 g / L, sucrose 30 g / L, pH 5.

8. 4) Somatic Embryo Induction and Maturation: Under sterile conditions, transfer the proembryonic tissue obtained in step 3) to somatic embryo induction medium and culture in the dark at 25°C to obtain mature cotyledonary embryos. The formula of somatic embryo induction medium is as follows: DCR basic medium, supplemented with gibberellin 3 mg / L, vitamin C 10 mg / L, L-glutamine 0.5 g / L, hydrolyzed casein 0.5 g / L, sucrose 30 g / L, activated carbon 1 g / L, abscisic acid 3 mg / L, polyethylene glycol 8000 0-200 g / L, crystal agar 3.8-4.5 g / L, pH 5.8; 5) Germination of somatic embryos into seedlings: Transfer the mature cotyledonary embryos from step 4) to germination medium for seedling germination. The culture conditions are: 25±2℃ light culture, light intensity 45μmol·m -2 •s -1 , photoperiod 16 / 8h; The germination medium formula is: DCR basic medium, supplemented with 6-benzylaminopurine 0.5 mg / L, 3-indolebutyric acid 0.2 mg / L, gibberellin 3 mg / L, vitamin C 10 mg / L, activated carbon 1 g / L, sucrose 20 g / L, crystal agar 3.8 g / L, pH 5.8; 6) Transplantation of somatic embryo regenerated seedlings: Open the lid of the somatic embryo seedlings cultured in step 5) and place them in an acclimatization room at 360 μmol·m -2 •s -1 The seedlings were hardened under strong light for one week, then the somatic embryo seedlings were taken out, the culture medium at the roots was thoroughly washed, and the somatic embryos with good root growth were selected and transplanted into the substrate for acclimatization and culture to obtain regenerated plants.

2. The method for establishing a Torreya grandis regeneration plantlet according to claim 1, wherein Step 1) Select columnar embryos for the mesozygous embryos.

3. The method for establishing a Torreya grandis regeneration plantlet according to claim 1, wherein Step 3) The dark culture conditions of the mesoembryonic callus were: temperature 23° C., shaker speed 90 rpm.

4. The method for establishing a Torreya grandis regeneration plantlet according to claim 1, wherein The matrix in step 5) is a mixed culture matrix prepared by mixing peat, perlite and frog stone in a mass ratio of 1:1:1.

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