A method for regulating somatic embryogenesis in hybrid tulip trees based on ethephon

Abstract

This invention discloses a method for regulating somatic embryogenesis in hybrid tulip trees based on ethephon, belonging to the field of somatic embryogenesis technology. The method involves inoculating embryogenic callus tissue from hybrid tulip trees into an induction medium containing ethephon or silver nitrate for induction treatment, thereby regulating the somatic embryogenesis efficiency of the hybrid tulip trees. Exogenous application of ethephon promotes the development of XY-TN, LWS-TN, XY-LWS, and XY-LP genotypes; exogenous application of silver nitrate promotes the development of the XY-TN genotype but inhibits the somatic embryogenesis of the LWS-TN, XY-LWS, and XY-LP genotypes. Furthermore, orthogonal experimental results show that the somatic embryogenesis induction effect is best for all genotypes when only ethephon is added without abscisic acid, proving that the combined effect of abscisic acid and ethephon does not enhance the somatic embryogenesis of hybrid tulip trees.

Description

Technical Field

[0001] This invention relates to a method for regulating somatic embryogenesis in hybrid tulip trees based on ethephon, belonging to the field of somatic embryogenesis technology. Background Technology

[0002] *Liriodendron* is a genus of plants belonging to the family Magnoliaceae, and is a Tertiary relict species. Currently, only one sister species of this genus survives: *Liriodendron chinense* and *Liriodendron tμLipifera*. The former is naturally distributed in northern China and Vietnam, while *Liriodendron tμLipifera* is naturally distributed from the eastern United States to southern Canada, exhibiting a typical East Asian-Eastern North American discontinuous distribution. Early studies hybridized *Liriodendron* and *Liriodendron tμLipifera*, obtaining hybrids with significant heterosis. These hybrids retain the phenotypes of their parents while exhibiting stronger growth adaptability, leaf shape resembling those of their parents, and vibrant, long-lasting flowers, making them excellent ornamental trees, roadside greening plants, and fast-growing timber trees.

[0003] Plant somatic embryogenesis is related to a variety of factors, including explant material, culture medium composition, culture conditions, and endogenous and exogenous hormones. Among these, endogenous and exogenous hormones are considered important inducing factors affecting the transformation of somatic cells into embryogenic cells under in vitro culture conditions. Studies have shown that, in addition to plant hormones such as auxin, cytokinin, and abscisic acid, ethylene also plays an important regulatory role in plant somatic embryogenesis.

[0004] In alfalfa studies, ethylene exhibits a stimulating effect independent of the SE stage, promoting somatic embryonic development. In soybean studies, ethylene stimulates somatic embryo formation in the presence of auxin; and in the presence of both auxin and cytokinin, ethylene is crucial for promoting the dedifferentiation of embryogenic callus into somatic embryos. In studies inducing somatic embryonic development from explants of European red pine, summer snowflake, and coffee, ethylene promotes somatic embryonic development.

[0005] Although the somatic embryogenesis system of hybrid tulip trees has been established, there are still problems such as the difficulty in inducing somatic embryos and the degradation of somatic embryogenesis ability of embryogenic callus over time. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for regulating the somatic embryogenesis of hybrid tulip trees based on ethephon, which is used to regulate the somatic embryogenesis of hybrid tulip trees.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for regulating somatic embryogenesis in hybrid tulip trees based on ethephon involves inoculating embryogenic callus tissue of hybrid tulip trees into an induction medium containing ethephon or silver nitrate for induction treatment, thereby regulating the somatic embryogenesis efficiency of hybrid tulip trees.

[0009] The hybrid tulip tree embryogenic callus includes one or more of XY-TN, LWS-TN, XY-LP, and XY-LWS.

[0010] The concentration of ethephon is less than 1.4 mmol / L.

[0011] When the hybrid tulip tree embryogenic callus is XY-TN, LWS-TN, or XY-LWS, the concentration of ethephon is 0.1 mmol / L; when the hybrid tulip tree embryogenic callus is XY-LP, the concentration of ethephon is 0.3 mmol / L.

[0012] The concentration of silver nitrate is less than 120 μmol / L.

[0013] When the hybrid tulip tree embryogenic callus is XY-TN, the concentration of silver nitrate is 60 μmol / L.

[0014] The induction treatment method is a solid embryo induction treatment.

[0015] The induction treatment time is 1-14 days.

[0016] When the hybrid tulip tree embryogenic callus is XY-TN or LWS-TN, the induction treatment time is 5 days; when the hybrid tulip tree embryogenic callus is XY-LP or XY-LWS, the induction treatment time is 1 day.

[0017] The beneficial effects of this invention are:

[0018] 1) This invention is based on a method for regulating the somatic embryogenesis of hybrid tulip trees using ethephon. The embryogenic callus of hybrid tulip trees is inoculated into an induction medium containing ethephon or silver nitrate for induction treatment, thereby regulating the somatic embryogenesis efficiency of hybrid tulip trees.

[0019] 2) The embryogenic callus tissues of the XY-TN and LWS-TN genotypes of *Liriodendron tulipifera* hybrids of this invention showed significantly improved somatic embryogenesis efficiency compared to the control group when treated with 0.1 mmol / L ethephon. The embryogenic callus tissues of the XY-LWS genotype of *Liriodendron tulipifera* hybrids showed significantly improved somatic embryogenesis efficiency compared to the control group when treated with 0.1 mmol / L ethephon; while the XY-LP genotype showed relatively improved somatic embryogenesis efficiency when treated with 0.3 mmol / L ethephon.

[0020] 3) In this invention, the XY-TN genotype showed a higher somatic embryogenesis efficiency under silver nitrate treatment (60 μmol / L) compared to the control group, with a significantly increased proportion of cotyledonous embryos among the total somatic embryos. The LWS-TN, XY-LP, and XY-LWS genotypes did not show an increase in somatic embryogenesis efficiency under silver nitrate treatment compared to the control group; in fact, it slightly decreased. With increasing silver nitrate concentration, the number of cotyledonous embryos in the LWS-TN genotype initially decreased and then increased, while the number of other embryo types decreased, with the highest number of cotyledonous embryos under silver nitrate treatment (120 μmol / L). The XY-LP genotype did not develop any somatic embryos into cotyledonous embryos, but the number of other embryo types decreased. The XY-LWS genotype initially decreased and then increased, but the number of cotyledonous embryos was highest without silver nitrate treatment. The number of other embryo types initially increased and then decreased, with the highest number of other embryo types under silver nitrate treatment (30 μmol / L).

[0021] 4) In this invention, the XY-TN genotype showed the highest somatic embryogenesis efficiency (average 320 embryos per gram) after 5 days of continuous ethephon treatment followed by transfer to additive-free medium. The LWS-TN genotype showed the highest somatic embryogenesis efficiency (average 163 embryos per gram) after 5 days of continuous ethephon treatment followed by transfer to additive-free medium. The XY-LP genotype showed the highest somatic embryogenesis efficiency (average 163 embryos per gram) after 1 day of continuous ethephon treatment followed by transfer to additive-free medium. The XY-LWS genotype showed the highest somatic embryogenesis efficiency (average 303 embryos per gram) after 1 day of continuous ethephon treatment followed by transfer to additive-free medium. This indicates that the optimal duration of ethylene treatment to promote somatic embryogenesis differs among different genotypes of Liriodendron tulipifera.

[0022] 5) In this invention, the XY-TN genotype, after culturing on untreated medium for 5 days and then transferring to ethephon treatment, showed the highest number of somatic embryos developing into cotyledonous embryos, averaging 60 per gram. The XY-LWS genotype, after culturing on untreated medium for 1 day and then transferring to ethephon treatment, also showed the highest number of somatic embryos developing into cotyledonous embryos, averaging 20 per gram. The XY-LP and LWS-TN genotypes showed virtually no somatic embryos developing into cotyledonous embryos. However, after culturing on untreated somatic embryo induction medium for 7 days and then transferring to ethephon treatment, the LWS-TN genotype showed the highest number of other embryonic forms, averaging 213 per gram, while the XY-LP genotype showed the highest number of other embryonic forms, averaging 150 per gram. This demonstrates that ethephon promotes somatic embryo development at different stages in different genotypes of Liriodendron tulipifera.

[0023] 6) The orthogonal experimental results of this invention show that the somatic embryo induction effect is the best for all genotypes when only ethephon is added without abscisic acid, proving that the combined action of abscisic acid and ethylene does not have a synergistic effect on the somatic embryogenesis of hybrid tulip trees. Attached Figure Description

[0024] Figure 1 Somatic embryogenesis efficiency of different genotypes of Liriodendron tulipifera under different concentrations of ethephon treatment (Figure AE shows somatic embryogenesis of the XY-TN genotype under different concentrations of ethephon treatment; Figure FJ shows somatic embryogenesis of the LWS-TN genotype under different concentrations of ethephon treatment; Figure KO shows somatic embryogenesis of the XY-LP genotype under different concentrations of ethephon treatment; Figure PT shows somatic embryogenesis of the XY-LWS genotype under different concentrations of ethephon treatment; the red arrows point to cotyledonary embryos; the scale bar in the figures is 2 mm).

[0025] Figure 2 Figure 1 shows the statistical data on the number of embryos in different genotypes of Liriodendron tulipifera under different concentrations of ethephon treatment. (Figures AC and JL show the average number of total embryos, cotyledonary embryos, and other morphological embryos in the XY-TN genotype under different concentrations of ethephon treatment; Figure DF shows the average number of total embryos, cotyledonary embryos, and other morphological embryos in the LWS-TN genotype under different concentrations of ethephon treatment; Figure GI shows the average number of total embryos, cotyledonary embryos, and other morphological embryos in the XY-LP genotype under different concentrations of ethephon treatment; Figure JL shows the average number of total embryos, cotyledonary embryos, and other morphological embryos in the XY-LWS genotype under different concentrations of ethephon treatment; a indicates extremely significant difference, b indicates significant difference, c indicates relatively significant difference, and d indicates no significant difference).

[0026] Figure 3 Figure 1 shows the number of somatic embryos of different genotypes of Liriodendron tulipifera under different concentrations of silver nitrate treatment (Figure AE shows somatic embryogenesis of the XY-TN genotype under different concentrations of silver nitrate treatment; Figure FJ shows somatic embryogenesis of the LWS-TN genotype under different concentrations of silver nitrate treatment; Figure KO shows somatic embryogenesis of the XY-LP genotype under different concentrations of silver nitrate treatment; Figure PT shows somatic embryogenesis of the XY-LWS genotype under different concentrations of silver nitrate treatment; the red arrows point to cotyledonary embryos; the scale bar in the figures is 2 mm).

[0027] Figure 4 Figure 1 shows the statistical data on the number of somatic embryos of different genotypes of Liriodendron tulipifera under different concentrations of silver nitrate treatment. (Figures AC and JL show the average number of total embryos, cotyledonary embryos, and other morphological embryos for the XY-TN genotype under different concentrations of silver nitrate treatment; Figure DF shows the average number of total embryos, cotyledonary embryos, and other morphological embryos for the LWS-TN genotype under different concentrations of silver nitrate treatment; Figure GI shows the average number of total embryos, cotyledonary embryos, and other morphological embryos for the XY-LP genotype under different concentrations of silver nitrate treatment; Figure JL shows the average number of total embryos, cotyledonary embryos, and other morphological embryos for the XY-LWS genotype under different concentrations of silver nitrate treatment; a indicates extremely significant difference, b indicates significant difference, c indicates relatively significant difference, and d indicates no significant difference).

[0028] Figure 5 Figure 1 shows the embryogenesis of four genotypes of Liriodendron tulipifera after ethephon treatment for different durations (Figure AE shows the effect of transfer to basic embryogenesis induction medium after 1, 3, 5, 7, and 14 days of continuous ethephon treatment on XY-TN embryogenesis; Figure FJ shows the effect of transfer to basic embryogenesis induction medium after 1, 3, 5, 7, and 14 days of continuous ethephon treatment on LWS-TN embryogenesis; Figure KO shows the effect of transfer to basic embryogenesis induction medium after 1, 3, 5, 7, and 14 days of continuous ethephon treatment on XY-LP embryogenesis; Figure PT shows the effect of transfer to basic embryogenesis induction medium after 1, 3, 5, 7, and 14 days of continuous ethephon treatment on XY-LWS embryogenesis; the red arrows point to cotyledonary embryos; the scale bar in the figures is 2 mm).

[0029] Figure 6 Figure 1 shows the effect of ethylene on embryonic development in *Liriodendron chinense* (Figure AE shows the effect of treatment with basic embryonic induction medium for 1, 3, 5, 7, and 14 days followed by transfer to ethephon-supplemented medium on XY-TN embryonic development; Figure FJ shows the effect of treatment with basic embryonic induction medium for 1, 3, 5, 7, and 14 days followed by transfer to ethephon-supplemented medium on LWS-TN embryonic development; Figure KO shows the effect of treatment with basic embryonic induction medium for 1, 3, 5, 7, and 14 days followed by transfer to ethephon-supplemented medium on XY-LP embryonic development; Figure PT shows the effect of treatment with basic embryonic induction medium for 1, 3, 5, 7, and 14 days followed by transfer to ethephon-supplemented medium on XY-LWS embryonic development; the red arrows point to cotyledonous embryos; the scale bar in the figures is 2 mm).

[0030] Figure 7 Figure 1 shows the statistical effects of ethylene on the development of different embryos in hybrid tulip trees (Figures AB and GH show the mean number of cotyledonous embryos and other morphological embryos in the XY-TN genotype after 1, 3, 5, 7, and 14 days of treatment with basic embryo induction medium and then transferred to ethephon-supplemented medium; Figures CD and EF show the mean number of cotyledonous embryos and other morphological embryos in the LWS-TN genotype after 1, 3, 5, 7, and 14 days of treatment with basic embryo induction medium and then transferred to ethephon-supplemented medium; Figures GH and GH show the mean number of cotyledonous embryos and other morphological embryos in the XY-LWS genotype after 1, 3, 5, 7, and 14 days of treatment with basic embryo induction medium and then transferred to ethephon-supplemented medium; a indicates extremely significant difference, b indicates significant difference, c indicates relatively significant difference, and d indicates no significant difference).

[0031] Figure 8The effects of co-treatment with ethephon and ABA on somatic embryogenesis of the XY-TN genotype are shown in the figures (Figures AD and EH show the effects of ABA 0 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the XY-TN genotype; Figure EH shows the effects of ABA 1.5 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the XY-TN genotype; Figure IL shows the effects of ABA 2 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the XY-TN genotype; the red arrows point to cotyledonous embryos; the scale bar in the figures is 2 mm).

[0032] Figure 9 The effects of co-treatment with ethephon and ABA on somatic embryogenesis of the LWS-TN genotype are shown in the figures (Figures AD and EH show the effects of ABA 0 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the LWS-TN genotype; Figure EH shows the effects of ABA 1.5 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the LWS-TN genotype; Figure IL shows the effects of ABA 2 mg / L and ethephon 0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, and 1.4 mmol / L on somatic embryogenesis of the LWS-TN genotype; the red arrows point to cotyledonous embryos; the scale bar in the figures is 2 mm).

[0033] Figure 10 The effects of co-treatment with ethephon and ABA on somatic embryogenesis in the XY-LP genotype are shown in Figures AD and IL. Figure AD shows the effects of ABA (0 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (1.5 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and the red arrows indicate cotyledonous embryos. The scale bar in the figures is 2 mm.

[0034] Figure 11The effects of co-treatment with ethephon and ABA on somatic embryogenesis in the XY-LWS genotype are shown in Figures AD and IL. Figure AD shows the effects of ABA (0 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (1.5 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), ethephon (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and ABA (2 mg / L), and ABA (0 mmol / L, 0.3 mmol / L, 0.7 mmol / L, 1.4 mmol / L), and the red arrows indicate cotyledonous embryos. The scale bar in the figures is 2 mm.

[0035] Figure 12 Figure 1 shows the total number of embryos in different genotypes of Liriodendron tulipifera under different combinations of ethephon and ABA treatments (Figure A shows the mean total number of embryos in the XY-TN genotype under different ethephon and ABA treatments; Figure B shows the mean total number of embryos in the LWS-TN genotype under different ethephon and ABA treatments; Figure C shows the mean total number of embryos in the XY-LP genotype under different ethephon and ABA treatments; Figure D shows the mean total number of embryos in the XY-LWS genotype under different ethephon and ABA treatments; a indicates extremely significant difference, b indicates significant difference, c indicates relatively significant difference, and d indicates no significant difference). Detailed Implementation

[0036] 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.

[0037] The following examples use reagent preparation methods:

[0038] Preparation method of ethephon reagent: Weigh 1.445g of ethephon powder into a beaker, add ultrapure water and make up to 20mL, filter and sterilize, which is the 0.5mmol / mL ethephon stock solution, store at -20℃ for later use.

[0039] Preparation method of silver nitrate reagent: Weigh 0.5g of silver nitrate powder into a beaker, add ultrapure water and make up to 100mL, filter and sterilize, which is the 30μmol / mL silver nitrate stock solution, store at -20℃ for later use.

[0040] The culture medium formulation used in the following examples is as follows:

[0041] The formula for callus induction medium is: 3 / 4 MS + 2,4-D 20 mg / L + 6-BA 0.2 mg / L + Vc 5 mg / L + hydrolyzed casein 0.5 g / L + sucrose 40 g / L + agar 7.2 g / L, pH 5.8.

[0042] The culture medium for the proliferation of embryogenic callus is formulated as follows: 3 / 4 MS + 2,4-D 10 mg / L + 6-BA 0.2 mg / L + Vc 5 mg / L + hydrolyzed casein 0.5 g / L + sucrose 30 g / L + agar crystals 2.5 g / L, pH 5.8.

[0043] The formulation of the somatic embryo induction medium is: 3 / 4 MS medium + Vc 5mg / L + hydrolyzed milk protein 0.2g / L + sucrose 40g / L + crystal agar 2.5g / L, pH 5.8.

[0044] Example 1

[0045] The hybrid tulip tree embryogenic callus material used in this embodiment was obtained from two pairs of four genotypes of hybrid tulip tree embryogenic callus induced in the tissue culture laboratory of Nanjing Forestry University in 2019 (Table 1) as the starting material. The optimal hormone concentration was screened for hybrid tulip tree embryogenic callus of different genotypes with good growth after about 21 days of subculture.

[0046] Table 1. Genotyping of embryogenic callus materials from hybrid tulip trees

[0047]

[0048] 1. Somatic embryogenesis efficiency of different genotype hybrid tulip trees under different concentrations of ethephon treatment

[0049] Embryogenic callus tissue with good growth after about 21 days of subculture was collected. 0.1g of the callus tissue was inoculated onto solid embryo induction medium supplemented with ①0.1mmol / L ethephon, ②0.3mmol / L ethephon, ③0.7mmol / L ethephon, ④1.4mmol / L ethephon, and ⑤0mmol / L ethephon (control). Six embryogenic callus tissues were placed in one dish, with each callus tissue weighing about 0.1g. At least 9 dishes were inoculated for each hormone concentration, and the dishes were incubated in the dark at 23°C.

[0050] The total number of somatic embryos of different genotypes and treatments was counted over 60 days. The somatic embryogenesis efficiency was calculated, expressed as embryos / g, i.e., the total number of embryos induced per gram of callus tissue.

[0051] The results are as follows Figure 1 , Figure 2As shown in Table 2, the embryogenic callus tissues of hybrid tulip trees with the XY-TN and LWS-TN genotypes showed significantly improved somatic embryogenesis efficiency compared to the control group under treatment with 0.1 mmol / L ethephon. The XY-TN genotype showed an average of 310 somatic embryos per gram without ethephon treatment, with cotyledonous embryos accounting for 25.8% of the total induced somatic embryos; under the 0.1 mmol / L ethephon treatment, the average somatic embryogenesis efficiency was 480 somatic embryos per gram, with cotyledonous embryos accounting for 43.75% of the total induced somatic embryos. Figure 1 AE, Figure 2 AC). This indicates that under ethephon treatment at 0.1 mmol / L, the proportion of cotyledonous embryos in the total number of somatic embryos in the XY-TN genotype significantly increased. The somatic embryogenesis efficiency of the LWS-TN genotype was an average of 80 embryos per gram without ethephon treatment, with cotyledonous embryos accounting for 12.5% ​​of the total induced somatic embryos; under ethephon treatment at 0.1 mmol / L, the somatic embryogenesis efficiency was an average of 90 embryos per gram, with cotyledonous embryos accounting for 11.11% of the total induced somatic embryos. Figure 1 FJ, Figure 2 (DF). This indicates that under ethephon treatment at 0.1 mmol / L, LWS-TN showed a decrease in the proportion of cotyledonous embryos to the total number of somatic embryos, and an increase in the proportion of other embryonic forms.

[0052] The somatic embryogenesis efficiency of XY-LWS hybrid tulip tree callus was significantly improved under ethephon treatment (0.1 mmol / L) compared to the control group; while the XY-LP genotype showed a higher somatic embryogenesis efficiency under ethephon treatment (0.3 mmol / L). The average somatic embryogenesis efficiency of the XY-LP genotype was 63 cells / gram without ethephon treatment and 150 cells / gram under ethephon treatment (0.3 mmol / L). Figure 1 KO Figure 2 (GI). However, the XY-LP genotype did not produce any somatic embryos that could develop into cotyledonous embryos under ethephon-free treatment and ethephon 0.3 mmol / L treatment. The somatic embryogenesis efficiency of the XY-LWS genotype was an average of 475 embryos per gram under ethephon-free treatment, with cotyledonous embryos accounting for 6.38% of the total induced somatic embryos; under ethephon 0.1 mmol / L treatment, the somatic embryogenesis efficiency was an average of 480 embryos per gram, with cotyledonous embryos accounting for 2.17% of the total induced somatic embryos. Figure 1 PT, Figure 2 JL). In the XY-LWS genotype, the proportion of cotyledonous embryos to total embryos decreased under ethephon treatment of 0.1 mmol / L, while the proportion of other embryo types increased.

[0053] Table 2. Somatic embryogenesis efficiency of different genotype hybrid tulip trees under different concentrations of ethephon treatment.

[0054] Induction rate (in vitro fertilizations / gram) 0 mmol / L 0.1 mmol / L 0.3 mmol / L 0.7 mmol / L 1.4 mmol / L XY-TN 310 480 251 188 83 LWS-TN 80 90 38 54 6 XY-LP 63 146 150 106 14 XY-LWS 475 480 204 9 1

[0055] 2. Efficiency of embryogenesis in hybrid tulip trees of different genotypes treated with different concentrations of ethylene receptor inhibitor (silver nitrate)

[0056] Embryogenic callus tissue with good growth after about 21 days of subculture was collected, and 0.1 g of the callus tissue was inoculated onto solid embryo induction medium supplemented with concentrations of ① 30 μmol / L silver nitrate, ② 60 μmol / L silver nitrate, ③ 90 μmol / L silver nitrate, ④ 120 μmol / L silver nitrate, and ⑤ 0 mmol / L silver nitrate (control), and cultured in the dark at 23°C.

[0057] The total number of somatic embryos of different genotypes and treatments was counted over 60 days. The somatic embryogenesis efficiency was calculated, expressed as embryos / g, i.e., the total number of embryos induced per gram of callus tissue.

[0058] The results are as follows Figure 3 , Figure 4 As shown in Table 3, after 60 days of dark culture, photographic observation revealed that the XY-TN genotype showed a higher somatic embryogenesis efficiency compared to the control group under silver nitrate treatment at 60 μmol / L, with a significantly increased proportion of cotyledonary embryos among the total number of somatic embryos. Figure 3 AF, Figure 4 AC). The somatic embryogenesis efficiency of the XY-TN genotype was an average of 310 embryos per gram without silver nitrate treatment, with cotyledonous embryos accounting for 25.8% of the total induced somatic embryos; under silver nitrate treatment at 60 μmol / L, the somatic embryogenesis efficiency was an average of 680 embryos per gram, with cotyledonous embryos accounting for 58.82% of the total induced somatic embryos. In the LWS-TN genotype, the somatic embryogenesis efficiency of hybrid tulip tree callus treated with silver nitrate was not increased compared to the control group, but rather slightly decreased. However, under silver nitrate treatment at 30 μmol / L, the proportion of cotyledonous embryos in the total somatic embryos increased, while without silver nitrate treatment, the number of other embryo types was greater. Figure 3 FJ, Figure 4 (DF). The somatic embryogenesis efficiency of the LWS-TN genotype was an average of 80 embryos per gram without silver nitrate treatment, with cotyledonous embryos accounting for 12.5% ​​of the total induced somatic embryos; the somatic embryogenesis efficiency was the highest among all silver nitrate treatments, averaging 50 embryos per gram, with cotyledonous embryos accounting for 22.45% of the total induced somatic embryos.

[0059] The somatic embryogenesis efficiency of the XY-LP and XY-LWS genotypes was not improved compared to the control group under silver nitrate treatment; in fact, it was slightly lower. The somatic embryogenesis efficiency of the XY-LP genotype was an average of 63 embryos per gram without silver nitrate treatment. Among all silver nitrate treatments, the highest somatic embryogenesis efficiency was observed under the 60 μmol / L silver nitrate treatment, with an average of 43 embryos per gram. Figure 3 KO Figure 4 GI). However, the XY-LP genotype did not develop into somatic embryos with or without silver nitrate treatment. The somatic embryogenesis efficiency of the XY-LWS genotype was 475 embryos per gram without silver nitrate treatment, with cotyledonous embryos accounting for 6.38% of the total induced somatic embryos; the somatic embryogenesis efficiency was highest under the silver nitrate treatment with 90 μmol / L silver nitrate, averaging 397 embryos per gram, with cotyledonous embryos accounting for 5.13% of the total induced somatic embryos. Figure 3 PT, Figure 4 JL). This indicates that the XY-LWS genotype showed a relative decrease in both the total number of somatic embryos and the proportion of cotyledonary embryos under silver nitrate treatment.

[0060] Table 3. Somatic embryogenesis efficiency of different genotype hybrid tulip trees under different concentrations of silver nitrate treatment.

[0061] Induction rate (in vitro fertilizations / gram) 0 μmol / L 30 μmol / L 60 μmol / L 90 μmol / L 120 μmol / L XY-TN 310 396 680 315 367 LWS-TN 80 50 48 24 32 XY-LP 63 29 43 27 31 XY-LWS 475 359 333 397 323

[0062] 3. Embryogenesis efficiency of hybrid tulip trees with different genotypes under different ethylene treatment times

[0063] Embryogenic callus with good growth after about 21 days of subculture was collected, and 0.1 g of the callus was inoculated on somatic embryonic induction medium with 0.1 mmol / L ethephon and cultured in the dark at 23°C. On days 1, 3, 5, 7 and 14 of the induction treatment, the induced callus was transferred to somatic embryonic induction medium without ethephon and cultured in the dark at 23°C for a longer period.

[0064] The total number of somatic embryos from different hybrid genotypes and treatments was counted over 60 days. The somatic embryogenesis efficiency was calculated, expressed as embryos / g, i.e., the total number of embryos induced per gram of callus tissue.

[0065] The results are as follows Figure 5 As shown in Table 4, after 60 days of dark culture, photographic observation revealed that, among different maternal combinations with the same paternal parent, the XY-TN genotype showed the highest somatic embryogenesis efficiency after 5 days of ethephon pre-culture followed by transfer to additive-free medium, averaging 320 embryos per gram. The LWS-TN genotype also showed the highest somatic embryogenesis efficiency after 5 days of ethephon pre-culture followed by transfer to additive-free medium, averaging 163 embryos per gram. Among different paternal combinations with the same maternal parent, the XY-LP genotype showed the highest somatic embryogenesis efficiency after 1 day of ethephon pre-culture followed by transfer to additive-free medium, averaging 163 embryos per gram. The XY-LWS genotype showed the highest somatic embryogenesis efficiency after 1 day of ethephon pre-treatment followed by transfer to additive-free medium, averaging 303 embryos per gram (Table 4).

[0066] Table 4. Somatic embryogenesis efficiency of different ethephon treatment time gradients in hybrid tulip trees

[0067] Induction rate (in vitro fertilizations / gram) 1 day 3 days 5 days 7 days 14 days XY-TN 180 300 320 287 280 LWS-TN 100 133 163 120 120 XY-LP 163 147 140 150 157 XY-LWS 303 233 247 253 233

[0068] 4. Induction of somatic embryogenesis in hybrid tulip tree by ethephon

[0069] Embryogenic callus with good growth after about 21 days of subculture was collected, and 0.1 g of the callus was inoculated on somatic embryo induction medium without ethephon and cultured in the dark at 23°C. On days 1, 3, 5, 7 and 14 of the induction treatment, the induced callus blocks were transferred to somatic embryo induction medium with 0.1 mmol / L ethephon and cultured in the dark at 23°C for a longer period.

[0070] The total number of somatic embryos of different genotypes and treatments was counted over 60 days. The somatic embryogenesis efficiency was calculated, expressed as embryos / g, i.e., the total number of embryos induced per gram of callus tissue.

[0071] The results are as follows Figure 6 and Figure 7 As shown, after 60 days of dark culture, photographic observation revealed that XY-TN, after 3 days of culture on untreated medium, exhibited the highest somatic embryogenesis efficiency upon transfer to ethephon-treated medium, averaging 330 embryos per gram. LWS-TN, after 7 days of culture on untreated medium, showed the highest somatic embryogenesis efficiency upon transfer to ethephon-treated medium, averaging 210 embryos per gram. This indicates that XY-TN embryos developed for 3 days showed the highest response to ethylene, while LWS-TN embryos developed for 7 days showed the highest response to ethylene. XY-LP, after 7 days of culture on untreated medium, showed the highest somatic embryogenesis efficiency upon transfer to ethephon-treated medium, averaging 160 embryos per gram. XY-LWS, after 1 day of culture on untreated medium, showed the highest somatic embryogenesis efficiency upon transfer to ethephon-treated medium, averaging 320 embryos per gram. Figure 7 ).

[0072] Example 2

[0073] Embryogenic callus tissue with good growth after about 21 days of subculture was taken and 0.1g of the callus tissue was inoculated on somatic embryo induction medium (Table 5) with ethephon and abscisic acid added, and cultured in the dark at 23°C.

[0074] The total number of somatic embryos of different genotypes and treatments was counted over 60 days. The somatic embryogenesis efficiency was calculated, expressed as embryos / g, i.e., the total number of embryos induced per gram of callus tissue.

[0075] Table 5. Orthogonal Experiment Design Table for Concentration Gradient

[0076]

[0077] The results are as follows Figure 8 , Figure 9 , Figure 10 , Figure 11 and Figure 12As shown, after 60 days of dark culture and photographic observation, the somatic embryogenesis efficiency of the XY-TN genotype was an average of 310 embryos per gram in the control group, and an average of 412 embryos per gram in the treatment with only ABA 2 mg / L and no ethephon. The somatic embryogenesis efficiency of the LWS-TN genotype was an average of 80 embryos per gram in the control group, and an average of 88 embryos per gram in the treatment with only ABA 1.5 mg / L and no ethephon.

[0078] The somatic embryogenesis efficiency of the XY-LP genotype was 63 per gram in the control group and 178 per gram in the treatment with only ABA 1.5 mg / L and no ethephon. The somatic embryogenesis efficiency of the XY-LWS genotype was 475 per gram in the control group and 481 per gram in the treatment with only ABA 1.5 mg / L and no ethephon.

[0079] Based on the statistical analysis of the number of somatic embryos in the four genotypes, the somatic embryo induction effect was best when only ethephon was added without abscisic acid, proving that the combined addition of ethephon and ABA did not promote somatic embryogenesis in hybrid tulip trees.

Claims

1. A method for regulating somatic embryogenesis in hybrid tulip trees based on ethephon, characterized in that, Embryogenic callus of hybrid tulip tree was inoculated into an induction medium containing ethephon or silver nitrate for induction treatment, thereby regulating the somatic embryogenesis efficiency of hybrid tulip tree. The hybrid tulip tree embryogenic callus includes one or more of XY-TN, LWS-TN, XY-LP, and XY-LWS; When the hybrid tulip tree embryogenic callus is XY-TN, LWS-TN, or XY-LWS, the concentration of ethephon is 0.1 mmol / L; when the hybrid tulip tree embryogenic callus is XY-LP, the concentration of ethephon is 0.1 mmol / L or 0.3 mmol / L. When the hybrid tulip tree embryogenic callus is XY-TN, the concentration of silver nitrate is 60 μmol / L.

2. The method according to claim 1, characterized in that, The induction treatment method is a solid embryo induction treatment.

3. The method according to claim 1, characterized in that, The induction treatment time is 1-14 days.

4. The method according to claim 1, characterized in that, When the hybrid tulip tree embryogenic callus is XY-TN or LWS-TN, the induction treatment time is 5 days; when the hybrid tulip tree embryogenic callus is XY-LP or XY-LWS, the induction treatment time is 1 day.

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