Method for inducing Eucalyptus urophyllatus DH32-29 transgenic callus with regeneration potential

Abstract

The invention discloses a method for inducing Eucalyptus urophyllatus DH32-29 transgenic callus with regeneration potential. The method comprises the following steps: S1, obtaining aseptic seedlings; s2, carrying out multiplication culture on the sterile seedlings to obtain multiplication seedlings; s3, infecting DH32-29 proliferation seedlings by using agrobacterium rhizogenes K599, and inoculating the proliferation seedlings to a hairy root induction culture medium to induce transgenic hairy roots; and S4, cutting off the transgenic hairy roots, inoculating the hairy roots into a callus induction culture medium, and inducing formation of transgenic calluses. The method disclosed by the invention can be used for rapidly and effectively inducing the eucalyptus urophylla transgenic hairy root and obtaining the transgenic callus with regeneration potential, and is simple and convenient to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for inducing transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential. Background Art

[0002] Eucalyptus urophylla×E. grandis belongs to the Myrtaceae family and the Eucalyptus genus. It has characteristics such as fast growth, high yield, and strong carbon sequestration potential. It is one of the main tree species for timber supply in China and also one of the main commercial timber tree species planted in the south. The excellent clone variety DH32-29 of Eucalyptus urophylla×E. grandis was selected through long-term cross-breeding by Dongmen Forest Farm, Guangxi State-owned Forestry Bureau, and has been commercially planted in the south of China. During the planting process of Eucalyptus plantations, problems such as biological stresses such as pest and disease outbreaks and abiotic stresses such as frost and wind damage lead to reduced yields of Eucalyptus. Since the cycle of traditional cross-breeding to select new high-yield and stress-resistant varieties is often long, the innovation process of Eucalyptus germplasm resources is slow. As emerging technologies for forest tree genetic improvement, transgenic and gene editing have been widely applied to the genetic improvement of various agricultural and forestry crops. Compared with traditional breeding methods, these technologies can achieve improvement goals faster. Since the 1990s, certain progress has been made in the research on the plant regeneration system of Eucalyptus both at home and abroad. However, affected by various factors such as genotype, plant growth regulators, explant type, and operation methods, the established Eucalyptus regeneration system often cannot be directly applied to the excellent clone DH32-29 of Eucalyptus urophylla×E. grandis. At present, solving the problems of clonal regeneration and genetic transformation of Eucalyptus urophylla×E. grandis is of great significance for the cultivation of improved Eucalyptus varieties. The genetic transformation method mediated by Agrobacterium rhizogenes shows unique advantages in other plants. Therefore, the genetic transformation system of Eucalyptus urophylla×E. grandis mediated by Agrobacterium rhizogenes still needs to be further explored to lay a solid foundation for Eucalyptus molecular breeding, further accelerate the research on Eucalyptus gene functions and the process of genetic improvement in molecular breeding, and has broad application prospects. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a method for inducing transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential. This method uses the explants of proliferated seedlings of Eucalyptus urophylla×E. grandis DH32-29. After inducing transgenic hairy roots, it induces transgenic callus with regeneration potential and finally forms transgenic regenerated plant organs, which can provide an important basis for the establishment of a genetic transformation system.

[0004] To achieve the above object, the technical solution provided by the present invention is as follows:

[0005] A method for inducing transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential, comprising the following operating steps:

[0006] S1: Collect the branches of Eucalyptus urophylla×E. grandis DH32-29 and perform disinfection treatment to obtain sterile seedlings.

[0007] S2: Through proliferation culture of the sterile seedlings, obtain proliferated seedlings.

[0008] S3: Infect the proliferated seedlings of DH32-29 with Agrobacterium rhizogenes K599 containing plasmids, and inoculate them into the hairy root induction medium to induce transgenic hairy roots.

[0009] S4: Cut off the transgenic hairy roots and inoculate them into the callus induction medium to induce the formation of transgenic callus, namely, obtain the transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential.

[0010] Further, in step S1, the branches of Eucalyptus urophylla×E. grandis DH32-29 are collected as branches of 8-10 cm in semi-lignified state of Eucalyptus urophylla×E. grandis DH32-29, containing 2-3 axillary bud points, and immediately placed in sterile water for moisturizing; before disinfection treatment, cut the branches to 4-5 cm, each branch contains 1-2 axillary bud points. If there are lateral branches or leaves beside the axillary bud points in the branches, cut the lateral branches or leaves to 0.5-1 cm, and rinse under running water for 2 h; the disinfection treatment is to cut off the tissue at the incision of the branches in the ultra-clean workbench, disinfect with 75% (v / v) ethanol for 30 s, rinse with sterile water 4 times, then disinfect with 0.15% (w / v) mercuric chloride for 10 min, shake several times during this period, rinse with sterile water 5 times, and then place the branches on sterile filter paper to dry, namely, obtain the disinfected branches.

[0011] Further, cut off the tissue at the incision of the disinfected branches with scissors, leave the length of the upper part of the axillary bud point in the morphological upper end of the branch as 0.3-0.5 cm, and leave the length of the lower part of the branch in the morphological lower end as 2-3 cm. Insert the lower end of the branch vertically into the bud proliferation medium, and do not let the branch directly touch the bottom of the medium. When the axillary bud grows to 1 cm, cut off the axillary bud, which is the sterile seedling.

[0012] Further, in step S3, the bud proliferation medium is MS medium at 4.74 g / L added with 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, adjust the pH to 5.8, and then add 7 g / L agar; the induction culture conditions are light intensity 22 μmol / (m 2 ·s), photoperiod 12 h / d, temperature 23±1 °C.

[0013] Further, in step S3, the proliferated seedlings obtained in S2 are cut into single-stem buds and placed in an infection solution of Agrobacterium rhizogenes K599 containing the pW501-JcFT-DsRed2 plasmid for 30 min. The infected buds are dried on sterile filter paper and then transferred to a co-culture medium for dark culture at 25°C for 3 d, and then transferred to a hairy root induction medium for culture for 21 d to obtain transgenic hairy roots.

[0014] Further, the co-culture medium is an MS medium of 4.74 g / L added with 200 μM AS, 30 g / L sucrose, the pH is adjusted to 5.8, and 7 g / L agar is added; the hairy root induction medium is a 1 / 2MS medium of 2.47 g / L added with 1.25 mg / L IBA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, the pH is adjusted to 5.8, and 4 g / L agar is added; the induction conditions for the transgenic hairy roots are a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, and a temperature of 23 ± 1°C.

[0015] Further, in step S4, the hairy roots carrying the DsRed2 reporter gene are cut into explants with a length of 1 - 2 cm and spread on a callus induction medium for culture for 30 d.

[0016] Further, the callus induction medium is an MS medium of 4.74 g / L added with 75 μM FPX (fipexide), 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, 6 g / L agar, and the pH is 5.8; the induction conditions for the callus are a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, a temperature of 23 ± 1°C, and subculturing once every 15 d.

[0017] Application of the Eucalyptus urophylla×E. grandis DH32-29 transgenic callus with regeneration potential obtained by the above induction method in obtaining transgenic plants.

[0018] Further, the application is to inoculate the Eucalyptus urophylla×E. grandis DH32-29 transgenic callus with regeneration potential onto a hairy root to bud regeneration medium to induce the regeneration of transgenic plants, obtaining regenerated roots and chloroplast tissue cells.

[0019] Furthermore, the explants cultured in the callus induction medium for 30 days were inoculated into the hairy root-to-shoot regeneration medium to induce the regeneration of transgenic shoots; the hairy root-to-shoot regeneration medium was 1 / 2 MS medium containing 2.47 g / L, supplemented with 10 mg / L 6-BA, 2 mg / L NAA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.8; the induction conditions were a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, and a temperature of 23 ± 1 °C.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] In the present invention, the proliferated seedlings of the excellent Eucalyptus urophylla × E. grandis clone DH32-29 were used as explants, and the induction efficiency of transgenic hairy roots was improved by using a hairy root induction medium containing 1.25 mg / L IBA, and the induction efficiency of transgenic hairy roots could reach 27.71%; further, the present invention adopted a novel chemical inducer - fipexide (FPX) in the callus induction medium to obtain a method for inducing callus with regeneration potential from transgenic hairy roots of Eucalyptus urophylla × E. grandis DH32-29; the method of the present invention can quickly and effectively induce transgenic hairy roots of Eucalyptus urophylla × E. grandis and use them to obtain transgenic callus with regeneration potential, and the operation is simple. Description of the Drawings

[0022] Figure 1 The steps for obtaining transgenic hairy roots by Agrobacterium rhizogenes-mediated genetic transformation of Eucalyptus urophylla × E. grandis DH32-29 in the present invention are as follows: (A) DH32-29 proliferated seedlings cultured in the shoot proliferation medium for 28 days; (B) Single shoots of DH32-29 cut off; (C) The shoots were placed in a resuspended solution of Agrobacterium rhizogenes containing the plasmid and infected for 30 min; (D) The infected shoots were placed on the co-culture medium and cultured in the dark at 25 °C for 3 days; (E) The infected shoots were transferred from the co-culture medium to the root induction medium; (F) DsRed2-positive transgenic roots under bright field; (G) DsRed2-positive transgenic roots under red fluorescence excitation field; (H) Non-transgenic roots under bright field; (I) Non-transgenic roots under red fluorescence excitation field; The scale bar for A-I is 1 cm.

[0023] Figure 2For the molecular biological identification of transgenic hairy roots of Eucalyptus urophylla×E. grandis DH32-29; among them, (A) is the PCR electrophoresis gel diagram: M: Marker, +: positive control (K599-pW501-JcFT-DsRed2 bacterial solution), -: negative control (wild type (WT) roots); 1-3: transgenic hairy roots; (B) is the relative expression level of JcFT in wild type (WT) roots and six different positive hairy roots (#1-#6) formed from the same explant; * indicates significant difference compared with WT (p<0.05), ** indicates significant difference compared with WT (p<0.01), *** indicates significant difference compared with WT (p<0.001).

[0024] Figure 3 For the effect of IBA concentration on the induction efficiency of transgenic hairy roots, data of the same index are marked with different lowercase letters indicating significant differences at the p≤0.05 level.

[0025] Figure 4 For the effect of FPX concentration on callus induction: (A-B) transgenic hairy roots under bright field / red fluorescence excitation field; (C-D) transgenic hairy root explants cultured for 0 d under bright field / red fluorescence excitation field.

[0026] Figure 5 The proliferation amount of explants cultured in callus induction medium containing different concentrations of FPX for 30 d; * indicates significant difference compared with WT (p<0.05), ** indicates significant difference compared with WT (p<0.01).

[0027] Figure 6 (A-E) Explants in callus induction medium containing 0, 25, 50, 75 and 100 μM FPX respectively; the scale bar is 1 mm.

[0028] Figure 7 For the electrophoresis diagram of Eucalyptus urophylla×E. grandis DH32-29 transgenic callus with regeneration potential: PCR electrophoresis gel diagram (M: Marker, +: positive control (K599-pW501-JcFT-DsRed2 bacterial solution), -: negative control (WT callus), 1-3: transgenic callus).

[0029] Figure 8The culture conditions of transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential in different regeneration media: (A1-B1) Explants in R1-R2 media, and some callus cells showed greening (red box); (C1-D1) Explants in R3-R4 media, and roots carrying DsRed2 fluorescence marker were regenerated from the callus; (E1-F1) Explants in R5-R6 media, the callus proliferated with the increase of culture time and the color gradually deepened and browned; A1-F1 were photographed under bright field, and A2-F2 were photographed under red fluorescence excitation field; (G-H) Close-up of the greened callus in R1 medium; Each scale bar is 1 mm. Detailed implementation mode

[0030] The following is a detailed description of the specific implementation mode in conjunction with the attached drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation mode. The raw materials and reagents used in the examples are all commercially available unless otherwise specified. The non-fipexide (FPX) used in the following examples was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: IF1440.

[0031] The Eucalyptus urophylla×E. grandis DH32-29 clone seedlings used in the present invention were donated by Dongmen State-owned Forest Farm in Guangxi Zhuang Autonomous Region (or can be obtained commercially), and the 1 / 2MS and MS media were from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0032] After the preparation of each culture medium formula used in the following examples was completed, it was finally dissolved in 1 L of pure water, and each culture medium and its formula are as follows:

[0033] Bud proliferation medium: Add 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose to 4.74 g / L of MS medium, adjust the pH to 5.8, and add 7 g / L of agar.

[0034] Co-culture medium: Add 200 μM AS, 30 g / L sucrose to 4.74 g / L of MS medium, adjust the pH to 5.8, and add 7 g / L of agar.

[0035] Hairy root induction medium: Add 1.25 mg / L IBA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose to 2.47 g / L of 1 / 2MS medium, adjust the pH to 5.8, and add 4 g / L of agar.

[0036] Callus induction medium: Add 75 μM FPX, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, 6 g / L agar to 4.74 g / L of MS medium, and the pH is 5.8.

[0037] Hairy root to shoot regeneration medium: Add 10 mg / L 6-BA, 2 mg / L NAA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, and 7 g / L agar to 1 / 2 MS medium at 2.47 g / L, with a pH of 5.8.

[0038] Bright field refers to the picture obtained under daylight lamp, and excitation field refers to the picture obtained under illumination with a wavelength of 563 - 582 nm (red fluorescence excitation field).

[0039] The plasmid used for Agrobacterium rhizogenes-mediated genetic transformation in the present invention is pW501-JcFT-DsRed2; the construction method of the backbone plasmid pW501 (containing the reporter gene DsRed2) refers to the method mentioned in the appendix of "Efficient genetic transformation and CRISPR / Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators" by Wenbo PAN et al. (Journal of Zhejiang University - SCIENCE B 23: 339 - 344, 2022). pW501-JcFT-DsRed2 was constructed by linearizing the backbone plasmid pW501 with the restriction endonuclease HindⅢ, simultaneously using primers with homologous arms for high-fidelity amplification of the JcFT gene expression cassette, and finally using a homologous recombination kit (purchased from Nanjing Novoprotein Science and Technology Co., Ltd., product number: C112-01) to ligate the linearized backbone plasmid pW501 and the JcFT gene expression cassette to obtain the pW501-JcFT-DsRed2 plasmid.

[0040] Example 1

[0041] A method for inducing transgenic hairy roots of Eucalyptus urophylla × E. grandis DH32-29, the operation steps are as follows:

[0042] S1: Disinfect scissors with ethanol at a volume concentration of 75%, cut off semi-lignified branches of Eucalyptus urophylla×E. grandis DH32-29 with a length of 8-10 cm (including 2-3 axillary bud nodes), and immediately place them in sterile water for moisturizing; then cut the branches to 4-5 cm, each branch contains 1-2 axillary bud nodes. If there are lateral branches or leaves beside the axillary bud nodes in the branches, the lateral branches or leaves should be cut short to 0.5-1 cm and rinsed under running water for 2 h; cut off the tissue at the cut of the branches in the ultra-clean workbench, disinfect with ethanol at a volume concentration of 75% for 30 s, rinse with sterile water 4 times, and then disinfect with mercuric chloride at a volume concentration of 0.15% for 10 min, shake several times during the period. After disinfection, immediately pour sterile water into the disinfectant to dilute the disinfectant concentration, then pour out the water, rinse with sterile water 5 times, and place the branches on sterile filter paper to dry, that is, the branches after disinfection treatment are obtained. Cut off the tissue at the cut of the obtained branches after disinfection treatment, leave the length of the branch at the morphological upper end of the axillary bud node as 0.3-0.5 cm, and leave the length of the branch at the morphological lower end as 2-3 cm. Insert the lower end of the branch vertically into the bud proliferation medium, do not let the branch directly touch the bottom of the medium. When the axillary bud grows to 1 cm, cut off the axillary bud, which is the sterile seedling;

[0043] S2: Inoculate the obtained sterile seedlings into the bud proliferation medium and culture for 28 days to obtain proliferated seedlings; the proliferated seedlings are subcultured once every about 28 d; the culture conditions are light intensity 22 μmol / (m 2 ·s), photoperiod 12 h / d, temperature 23±1℃;

[0044] S3: Prepare a 90 mm glass culture dish with 10 pieces of filter paper after sterilization, take out the proliferated seedlings obtained in S2 and place them on the filter paper ( Figure 1 A), use a sterile scalpel to cut the obtained proliferated seedlings into single small buds ( Figure 1 B) and put them into the infection solution of Agrobacterium rhizogenes K599 containing the pW501-JcFT-DsRed2 plasmid for infection for 30 min ( Figure 1 C), dry the infected small buds on sterile filter paper, and then transfer them to the co-culture medium and culture in the dark at 25℃ for 3 d ( Figure 1 D), and then transfer them to the hairy root induction medium and culture for 21 d ( Figure 1 E) to obtain transgenic hairy roots. The state of the non-detached transgenic hairy roots under bright field / red fluorescence excitation field is as Figure 4 shown in A-B; the induction conditions for transgenic hairy roots are light intensity 10 μmol / (m 2 ·s), photoperiod 12 h / d, temperature 23±1℃. The hairy roots carrying the DsRed2 reporter gene ( Figure 1(F-G) Statistical analysis was performed on transgenic hairy roots to calculate the induction efficiency of positive hairy roots. The calculation formula is: Transgenic hairy root induction efficiency (%) = Number of plants carrying the DsRed2 reporter gene / Number of infected plants × 100%. Each treatment included 3 biological replicates, and each biological replicate contained 40 - 60 explants. The molecular biological identification results of Eucalyptus urophylla × E. grandis DH32-29 transgenic hairy roots are as follows Figure 2 A- Figure 2 shown in B (using the K599-pW501-JcFT-DsRed2 bacterial solution as the positive control and the wild type as the negative control).

[0045] S4: The hairy roots carrying the DsRed2 reporter gene were cut into explants with a length of 1 - 2 cm and spread on the callus induction medium ( Figure 4 C-D). After culturing for 30 days, the Eucalyptus urophylla × E. grandis DH32-29 transgenic callus with regeneration potential was obtained. The obtained callus was subjected to an electrophoresis gel test, and the results are as follows Figure 7 shown: PCR electrophoresis gel diagram (M: Marker, +: Positive control (K599-pW501-JcFT-DsRed2 bacterial solution), -: Negative control (WT callus)), 1 - 3: Transgenic callus. 6 explants were placed in each bottle of medium and weighed to record the initial weight. After 30 days, it was weighed again and the final weight was recorded, and the proliferation amount of the explants was calculated (Final weight of the explants in each medium - Initial total weight); each treatment included 3 biological replicates, and each biological replicate contained 20 - 25 pieces of callus. The induction conditions for callus were light intensity 10 μmol / (m 2 ·s), photoperiod 12 h / d, temperature 23 ± 1 °C, and subcultured every 15 days.

[0046] The Eucalyptus urophylla × E. grandis DH32-29 transgenic callus with regeneration potential obtained in S4 was inoculated onto the hairy root to bud regeneration medium to induce the regeneration of transgenic buds, and the medium was changed every two weeks; after 30 days, transgenic regenerated plant tissues carrying the DsRed2 reporter gene could be observed ( Figure 8 A1-D1). Each regeneration treatment included 3 biological replicates, and each biological replicate contained 20 - 25 pieces of callus. The induction conditions were light intensity 10 μmol / (m 2 ·s), photoperiod 12 h / d, temperature 23 ± 1 °C.

[0047] Example 2

[0048] The callus obtained in S4 of Example 1 was cultured on different plant growth regulators (Table 3) for 28 days.

[0049] In S5, the callus results were shown, but there were differences in the developmental morphology of callus in different types of culture media: the explants in Table 3 R1 and R2 could show light green color ( Figure 8 A1-B1) after 4 weeks of culture, indicating that some callus had been transformed into chloroplast tissue cells; the explants in Table 3 R3 and R4 began to regenerate roots from the callus after 2 weeks of culture, and the regenerated roots still carried the DsRed2 fluorescent marker ( Figure 8 C1-D1 and C2-D2); the callus in Table 3 E and F proliferated with the increase of culture time and gradually browned, and finally died ( Figure 8 E1-F1).

[0050] Table 3 Effects of different plant growth regulator treatments on the regeneration of plant tissues from callus

[0051]

[0052]

[0053] Comparative Example 1

[0054] S3: To analyze the effects of different IBA concentrations on the induction efficiency of positive hairy roots, the "1.25 mg / L IBA" in the above hairy root induction medium was replaced with the following concentrations: 0.00, 0.25, 0.50, 0.75, 1.00, 1.50, 1.75, and 2.00 mg / L IBA, and the other components in the medium remained unchanged. The experimental operation was the same as that in Example 1. After three weeks, the plant rate carrying transgenic hairy roots was counted. The induction efficiency of transgenic hairy roots in the hairy root induction medium with different IBA concentrations is shown in Table 1 (F treatment is Example 1), Figure 3 as shown. The results showed that the induction efficiency of transgenic hairy roots was 0.00 - 27.71%. When the IBA concentration was 1.25 mg / L, the induction rate of positive hairy roots reached the peak.

[0055] Table 1 Effects of different concentrations of IBA on the induction efficiency of transgenic hairy roots

[0056] Treatment IBA concentration (mg / L) Transgenic hairy root induction efficiency (%) A 0.00 <![CDATA[0±0 g > B 0.25 <![CDATA[8.11±3.11 f > C 0.50 <![CDATA[15.28±5.33 def > D 0.75 <![CDATA[11.25±0.69 ef > E 1.00 <![CDATA[24.69±2.16 abc > F 1.25 <![CDATA[27.71±3.23 a > G 1.50 <![CDATA[25.66±8.7 ab > H 1.75 <![CDATA[19.46±4.25 bad > I 2.00 <![CDATA[17.09±5.2 cde >

[0057] Note: Different letters a, b, c, d, e, f, and g in Table 1 indicate significant differences.

[0058] Comparative Example 2

[0059] In S4, replace "75 μM FPX" in the above callus induction medium with the following concentrations: 0, 25, 50, and 100 μM of FPX. The other components in the medium remain unchanged, and the experimental operations are the same as in Example 1. After three weeks, count the plantlet rate carrying transgenic hairy roots and the increased mass of transgenic callus in the callus induction medium with different FPX concentrations, as shown in Table 2 (Treatment D is Example 1). Figure 5 The results show that there are differences in the proliferation amount and morphology of callus formed in the callus induction medium containing different concentrations of FPX. The proliferation amount of callus in the 75 μM FPX medium is the largest, and the average proliferation amount of callus reaches 0.44 g ( Figure 5 ). With the increase of culture time, the explants in the callus induction medium with 0 μM FPX gradually turn brown, and no obvious callus is observed under the stereomicroscope ( Figure 6 A); induced by 25 μM FPX ( Figure 6 B), a small amount of callus will be formed on the explants, and the color of the callus is lighter and the surface texture is harder; in the callus induction medium with a concentration of 50 μM FPX ( Figure 6 C), 75 μM FPX ( Figure 6 D), and 100 μM FPX ( Figure 6 E), the callus formed by the explants cultured has similar morphology. More callus can be formed on the surface of the explants, especially at the incision, with a light yellow color and a soft texture. And there is no significant difference in the callus proliferation amount.

[0060] Table 2 Effects of different concentrations of FPX on the proliferation amount of callus

[0061]

[0062] Note: Different letters a, b, and c in Table 2 indicate significant differences.

[0063] The present invention provides a method for inducing transgenic hairy roots of Eucalyptus grandis and using them to obtain transgenic callus with regeneration potential. IBA has a promoting effect on the formation of transgenic hairy roots of Eucalyptus urophylla × E. grandis DH-32-29. The induction efficiency of transgenic hairy roots can reach 27.71% when the explants are cultured in a medium containing 1.25 mg / L IBA for 4 weeks; FPX has a promoting effect on the formation of callus with regeneration potential from transgenic hairy roots of Eucalyptus urophylla × E. grandis DH-32-29. The method of the present invention well solves the problems of low induction efficiency of transgenic hairy roots of the excellent clone variety DH32-29 of Eucalyptus urophylla × E. grandis and difficulty in inducing callus with regeneration potential from transgenic hairy roots, providing key technical support for the gene function and genetic engineering research of Eucalyptus urophylla × E. grandis.

[0064] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. An induction method for transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential, characterized in that, It includes the following operation steps: S1: Collect the branches of Eucalyptus urophylla × E. grandis DH32-29 and conduct disinfection treatment to obtain aseptic seedlings; S2: Through proliferation culture of the aseptic seedlings, obtain proliferated seedlings; S3: Infect the proliferated seedlings of DH32-29 with Agrobacterium rhizogenes K599, and inoculate them into the hairy root induction medium to induce transgenic hairy roots; S4: Cut the transgenic hairy roots and inoculate them into the callus induction medium to induce the formation of transgenic callus, that is, obtain the transgenic callus of Eucalyptus urophylla × E. grandis DH32-29 with regeneration potential.

2. The induction method of the transgenic callus of Eucalyptus urophylla × E. grandis DH32-29 with regeneration potential according to claim 1, wherein: In step S1, the collection of the branches of Eucalyptus urophylla × E. grandis DH32-29 is to take the semi-lignified branches of Eucalyptus urophylla × E. grandis DH32-29 with a length of 8-10 cm, including 2-3 axillary bud nodes, and place them in sterile water for moisturizing; before disinfection treatment, cut the branches to 4-5 cm, and each branch contains 1-2 axillary bud nodes. If there are lateral branches or leaves beside the axillary bud nodes in the branches, cut the lateral branches or leaves to 0.5-1 cm, and rinse them under running water for 2 h; the disinfection treatment is to cut off the tissue at the cut of the branches in the ultra-clean workbench, disinfect with 75% (v / v) ethanol for 30 s, rinse with sterile water 4 times, then disinfect with 0.15% (w / v) mercuric chloride for 10 min, shake during the process, rinse with sterile water 5 times, and then place the branches on sterile filter paper to dry, that is, obtain the disinfected branches.

3. The induction method of transgenic callus of Eucalyptus urophylla × E. grandis DH32-29 with regeneration potential according to claim 2, characterized in that: Cut off the tissue at the cut of the disinfected branches, leave the length of the upper part of the axillary bud node in the morphological upper part of the branch as 0.3-0.5 cm, and leave the length of the lower part of the branch in the morphological lower part as 2-3 cm. Vertically insert the lower part of the branch into the bud proliferation medium. When the axillary bud grows to 1 cm, cut off the axillary bud, which is the aseptic seedling.

4. The method for inducing transgenic callus of Eucalyptus urophylla × E. grandis DH32-29 with regeneration potential according to claim 1, wherein: In step S3, the bud proliferation medium is an MS medium of 4.74 g / L added with 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, with the pH adjusted to 5.8, and then 7 g / L agar is added; the induction culture conditions are a light intensity of 22 μmol / (m 2 ·s), a photoperiod of 12 h / d, and a temperature of 23 ± 1 °C.

5. The induction method of transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential according to claim 1, characterized in that: In step S3, cut the proliferated seedlings obtained in S2 into single-strain small buds and place them in the infection solution of Agrobacterium rhizogenes K599 containing the pW501-JcFT-DsRed2 plasmid for infection for 30 min. Place the infected small buds on sterile filter paper to dry, and then transfer them to the co-culture medium for dark culture at 25 °C for 3 d, and then transfer them to the hairy root induction medium for culture for 21 d to obtain transgenic hairy roots.

6. The induction method of transgenic callus of Eucalyptus urophylla × Eucalyptus grandis DH32-29 with regeneration potential according to claim 5, characterized in that: The co-culture medium is MS medium at 4.74 g / L supplemented with 200 μM AS, 30 g / L sucrose, with the pH adjusted to 5.8, and 7 g / L agar added; the hairy root induction medium is 1 / 2 MS medium at 2.47 g / L supplemented with 1.25 mg / L IBA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, with the pH adjusted to 5.8, and 4 g / L agar added; the transgenic hairy root induction conditions are a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, and a temperature of 23 ± 1 °C.

7. The induction method of transgenic callus of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential according to claim 1, characterized in that: In step S4, cut the hairy roots carrying the DsRed2 reporter gene into explants with a length of 1-2 cm, spread them on the callus induction medium, and culture for 30 d.

8. The induction method of the transgenic Eucalyptus urophylla × E. grandis DH32-29 callus with regeneration potential according to claim 7, wherein: The callus induction medium is an MS medium containing 4.74 g / L, added with 75 μM FPX, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, 6 g / L agar, with a pH of 5.8; the induction conditions for the callus are a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, a temperature of 23 ± 1 °C, and subculturing once every 15 days.

9. Application of the transgenic callus of Eucalyptus urophylla × E. grandis DH32-29 with regeneration potential obtained by the induction method as described in any one of claims 1-8 above in obtaining transgenic plants.

10. Use of the transgenic Eucalyptus urophylla×E. grandis DH32-29 callus with regeneration potential according to claim 9 in obtaining transgenic plants, characterized in that: The transgenic calli of Eucalyptus urophylla×E. grandis DH32-29 with regeneration potential were inoculated onto the hairy root-to-shoot regeneration medium to induce the regeneration of transgenic plants, and regenerated roots and chloroplast tissue cells were obtained: The explants cultured in the callus induction medium for 30 d were inoculated onto the hairy root-to-shoot regeneration medium to induce the regeneration of transgenic shoots; the hairy root-to-shoot regeneration medium was 1 / 2 MS medium containing 2.47 g / L, supplemented with 10 mg / L 6-BA, 2 mg / L NAA, 300 mg / L Cef, 100 mg / L Tim, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.8; the induction conditions were a light intensity of 10 μmol / (m 2 ·s), a photoperiod of 12 h / d, and a temperature of 23 ± 1 °C.

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