An agrobacterium-mediated genetic transformation method of paulownia

Abstract

This application relates to an Agrobacterium-mediated genetic transformation method for balsa wood. The method includes: preparing an infection solution from Agrobacterium bacterial culture; using balsa wood seeds as seedlings, detached cotyledons or stem segments as explants, infecting the cotyledons or stem segments with the infection solution, and after infection, performing co-culture, recovery culture, and selection culture to obtain transgenic hairy roots or transgenic callus tissue. The scheme provided in this application enables stable transformation of balsa wood roots, verification of functional genes, and long-term survival in a living form, thus improving the efficiency of genetic transformation of balsa wood.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to an Agrobacterium-mediated genetic transformation method for balsa wood. Background Technology

[0002] Balsa wood (Ochroma lagopus Swartz), also known as balsa wood, is a perennial tree belonging to the genus Ochroma in the family Malvaceae. It is one of the fastest-growing and lightest trees in the world. Native to the equatorial region of South America, balsa wood is distributed in Ecuador, Colombia, Papua Guinea, Indonesia, Brazil, Costa Rica, Guatemala, and other regions, with Ecuador being the world's leading producer. Since 1960, it has been introduced and cultivated in Guangdong, Fujian, and Yunnan provinces of my country. Balsa wood thrives in hot, humid environments and prefers deep, well-drained, fertile soil. However, suitable growing conditions in my country are extremely limited, confined to parts of Yunnan, Taiwan, and Hainan provinces. Because balsa forests cannot withstand winds exceeding force 5, it is only suitable for planting in Xishuangbanna, a region with high temperatures, no extreme winter temperatures, and no typhoons, making it the only suitable area in China for large-scale balsa wood cultivation.

[0003] Balsa wood boasts advantages such as light weight, good elasticity, impact resistance, strong sound absorption, and a short growth cycle. It is a high-quality timber with excellent mechanical and processing properties, and is also affordable. It has a wide range of uses and significant commercial value. Besides being a core material for wind power equipment, balsa wood can also be used in construction, furniture, aerospace, and other fields. Under the advocacy of energy conservation and emission reduction, my country imports a large amount of balsa wood annually for manufacturing wind turbine blades. With the rapid development of the domestic wind power industry and the escalation of trade wars, balsa wood prices have been rising year by year, leading to a supply shortage of domestic balsa wood raw materials. Moreover, the amount of balsa wood imported by my country is constantly increasing. Due to the inability to be self-sufficient, my country is completely dependent on imports for balsa wood. With the continuous rise in demand and the uncertainty of Sino-foreign trade, the supply of balsa wood raw materials has become a major challenge for my country's wind power equipment manufacturing industry. Since my country relies heavily on imports for most of its balsa wood raw materials, the localization of balsa wood production is urgently needed. Since 2000, approximately 20,000 mu (about 1,333 hectares) of balsa wood have been planted in Xishuangbanna, Hekou, Menglian, and other areas. Due to the significant differences in geographical and climatic environments between Xishuangbanna and its place of origin, a series of problems have arisen in the process of promoting and planting balsa wood. Among them, inconsistent growth, low branching, frequent pests and diseases, and susceptibility to cold damage are key issues leading to low timber yield and quality. Therefore, it is particularly important to strengthen balsa wood breeding and develop balsa wood breeding technologies.

[0004] In existing research, genetic transformation technology involves transferring known target genes regulating desirable traits into organisms through methods such as microbial mediation, microinjection, or gene gun bombardment, thereby endowing organisms with new desirable traits. Common methods of genetic transformation include Agrobacterium-mediated transformation, gene gun transformation, and polyethylene glycol-mediated transformation. The use of genetic transformation technology to improve plant varieties represents a major breakthrough in modern biotechnology. However, the success of Agrobacterium-mediated genetic transformation is significantly influenced by various factors, including the type of Agrobacterium strain, Agrobacterium concentration, plant species, explant type, developmental stage, degree of damage, infection time, infection solution composition, auxiliary methods, co-culture conditions, and screening conditions. Different Agrobacterium strains exhibit significant differences in their ability to infect different plants, depending on the interaction between Agrobacterium and plant cells, as well as its growth and reproduction capabilities within plant tissues.

[0005] Currently, research on Agrobacterium-mediated genetic transformation of balsa wood is still in its early stages. There is an urgent need to establish an Agrobacterium-mediated genetic transformation method for balsa wood, both from the perspective of genetic improvement and gene function analysis. Summary of the Invention

[0006] To address or partially address the problems existing in related technologies, this application provides a method for establishing a genetic transformation system for balsa roots mediated by Agrobacterium rhizogenes. This method can achieve genetic transformation of balsa roots and callus, thereby improving the efficiency of genetic transformation.

[0007] This application provides an Agrobacterium-mediated genetic transformation method for balsa wood, which uses Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens GV3101 to infect balsa wood explants to obtain transgenic hairy roots or callus tissue.

[0008] Optionally, the balsa wood explants are infected with an infection solution prepared using a bacterial suspension containing the target vector. After infection, the infected balsa wood explants are inoculated into a co-culture medium for co-culture. The infection solution is either Agrobacterium rhizogenes K599 infection solution or Agrobacterium tumefaciens GV3101 infection solution.

[0009] Optionally, the balsamic explants can be cotyledons or stem segments; OD of the bacterial solution 600 The pH was 0.4-0.8, and the bacterial suspension was either Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens GV3101; the co-culture medium was MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA or 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA.

[0010] Optionally, the balsamic explants are balsamic cotyledons; bacterial culture OD 600The concentration was 0.5-0.7; the co-culture medium was 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA.

[0011] Optional, bacterial culture OD 600 It is 0.6.

[0012] Optionally, the target vector refers to the pOCA30 vector containing the target gene; when the bacterial culture is Agrobacterium rhizogenes K599, the target gene is the RUBY or JcFT gene; when the bacterial culture is Agrobacterium tumefaciens GV3101, the target gene is the RUBY gene.

[0013] The infection time was 20 minutes; the co-culture phase was dark culture, and the co-culture time was 3 days.

[0014] Optionally, the infection solution can be prepared by centrifuging the bacterial culture at 4°C, discarding the supernatant, resuspending the bacterial cells in MS liquid medium, and adding 100 μM AS and 3 mg / L 6-BA to prepare the infection solution.

[0015] Optionally, the preparation method of balsa wood explants is as follows: plump balsa wood seeds are screened, disinfected with 1.0% sodium hypochlorite for 15 minutes, rinsed three times with sterile water, heated in a 55℃ water bath for 30 minutes, inoculated on 1 / 2 MS medium and germinated for 30 days. Seedlings are removed, sterile cotyledons and stem segments are separated, and balsa wood explants are obtained after pretreatment with 5 mg / L xylanase for 30 minutes.

[0016] Optionally, the following steps may also be included:

[0017] When the bacterial solution is Agrobacterium rhizogenes K599, transgenic hairy roots are obtained by co-culturing the cotyledons or stem segments of balsa wood through recovery culture and screening culture. Transgenic hairy roots are then induced to obtain transgenic hairy root callus tissue.

[0018] When the bacterial culture is Agrobacterium tumefaciens GV3101, transgenic callus tissue is obtained from the co-cultured cotyledons or stem segments of *Berberis vulgaris* through recovery culture and selection culture.

[0019] Optionally, when the bacterial culture is Agrobacterium rhizogenes K599, the recovery culture medium is 1 / 2 MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim; when the bacterial culture is Agrobacterium tumefaciens GV3101, the recovery culture medium is MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim.

[0020] The culture medium used in the screening culture was MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 20.0 mg / L kanamycin;

[0021] The culture medium for induction culture was MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride.

[0022] The above culture media all also contain 30.0 g / L sucrose and 6.5 g / L agar, and the pH is 5.8.

[0023] The technical solution provided in this application may include the following beneficial effects:

[0024] 1) In this application, Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens GV3101 strains were used to achieve the genetic transformation of balsa wood. Balsa wood has abundant secondary metabolites, which seriously affect the activity of the infecting bacterial solution. In this application, K599 was selected as the host of the delivery system. The K599 strain has a fast proliferation rate and strong virulence. It is not significantly affected by the secondary metabolites of balsa wood. Furthermore, the treatment of balsa wood cotyledons and stem segments with xylanase can reduce the impact of secondary metabolites in balsa wood.

[0025] 2) In this application, cotyledons of balsa wood were used as explants. Agrobacterium rhizogenes K599 bacterial suspension was suspended in MS medium and AS and 6-BA were added to prepare Agrobacterium rhizogenes K599 infection solution. The infected cotyledons were then inoculated into 1 / 2 MS co-culture medium for co-culture, which improved the genetic transformation efficiency of balsa wood. The total positive root rate of transgenic hairy roots obtained by using Agrobacterium rhizogenes K599 infection solution containing the target vector RUBY was as high as 66.67%. When cotyledon explants were infected with Agrobacterium rhizogenes K599 infection solution containing the target vector JcFT, PCR detection showed that 8 out of 15 hairy root DNAs contained fragments of the FT gene, and the positive transformation rate reached 53.3%.

[0026] 3) This application enables the stable transformation of balsa roots to verify functional genes and allow them to exist in a living form for a long time. The transgenic hairy roots obtained by Agrobacterium rhizogenes K599-mediated transformation can form callus tissue after dedifferentiation, laying the foundation for plant regeneration.

[0027] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0028] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0029] Figure 1 This is a schematic diagram of hairy root growth shown in Embodiment 1 of this application, where A is the control group and BI is the hairy root growth status;

[0030] Figure 2 This is a schematic diagram of positive hairy root PCR detection shown in Example 1 of this application, where CK1 is a positive plasmid control; CK2 is a negative control of untransformed roots; and 1-15 are hairy roots induced by Agrobacterium infection.

[0031] Figure 3 This is a schematic diagram of the formation of red callus tissue from transgenic hairy roots after dedifferentiation, as shown in Example 1 of this application;

[0032] Figure 4 This is a schematic diagram of the positive callus morphology shown in Comparative Example 4 of this application. Detailed Implementation

[0033] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0034] Example 1

[0035] An Agrobacterium-mediated genetic transformation method for balsa wood includes the following steps:

[0036] 1) Preparation of Agrobacterium rhizogenes K599 bacterial culture: Transformed Agrobacterium rhizogenes K599 was removed from a -80℃ ultra-low temperature freezer and activated on YEB solid plates containing 100 mg / L spectinomycin. The plates were then incubated upside down at 28℃ for 2-3 days. Single clones were picked from the plates and added to 10 mL of YEB liquid medium containing 100 mg / L spectinomycin. The culture was incubated at 28℃ with shaking at 200 rpm for 10-12 hours. 200 μL of the bacterial culture was then added to 50 mL of YEB liquid medium containing 100 mg / L spectinomycin and incubated at 28℃ with shaking at 200 rpm for 12 hours. 1 mL of the bacterial culture was transferred to a 1.5 mL centrifuge tube, and the OD value of the bacterial culture was measured using a spectrophotometer. 600 The value was used to prepare Agrobacterium rhizogenes K599 bacterial solution.

[0037] 2) Preparation of Agrobacterium rhizogenes K599 infection solution: Agrobacterium rhizogenes K599 bacterial suspension was placed at 4℃, centrifuged at 5000rpm for 9min, the supernatant was discarded, the bacterial cells were collected, and the bacterial cells were resuspended in MS liquid medium. Then, 100μM acetylsuccinone (AS) and 3mg / L 6-BA were added to prepare Agrobacterium rhizogenes K599 infection solution.

[0038] 3) Preparation of balsa wood explants: Select plump balsa wood seeds, disinfect them with 1.0% sodium hypochlorite for 15 minutes, rinse them three times with sterile water, heat them in a 55℃ water bath for 30 minutes, and then inoculate them on 1 / 2 MS medium for germination for 30 days. Remove the seedlings, separate sterile cotyledons and stem segments, and pretreat them with 5 mg / L xylanase (Solepro, X8091) for 30 minutes to use as balsa wood explants.

[0039] 4) Agrobacterium rhizogenes K599-mediated transformation: using OD 600 Agrobacterium rhizogenes K599 bacterial suspensions containing the target vector (pOCA30 vector containing the 35S promoter and RUBY gene) at concentrations of 0.4, 0.5, 0.6, 0.7, and 0.8 were used to infect balsa wood cotyledons or stem segments. The segments were placed in a constant-temperature shaker for 20 min to ensure thorough contact between the Agrobacterium rhizogenes K599 infection solution and the balsa wood cotyledons or stem segments. The balsa wood cotyledons or stem segments were then removed, and residual infection solution was blotted off with sterile filter paper. They were then inoculated into a co-culture medium (1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) for co-culture, ensuring the undersides of the balsa wood cotyledons or stem segments adhered to the co-culture medium. The mixture was then incubated in the dark in a constant-temperature incubator for 3 days.

[0040] 5) After 3 days, rinse the co-cultured balsam psyllium leaves or stem segments 4-5 times with sterile water containing 500 mg / L Cef, blot off the surface moisture with sterile filter paper, and inoculate them into recovery medium (1 / 2 MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) without selection agent for 20 days. Then, transfer the recovered balsam psyllium leaves or stem segments to selection medium containing hygromycin (MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) Transgenic hairy roots were obtained by screening and culturing explants in a solution of 20.0 mg / L kanamycin, 30.0 g / L sucrose, and 6.5 g / L agar (pH 5.8) for 20 days. Fifty explants were cultured in each experimental group, with three replicates. The hairy root formation rate was recorded after 30 days, and the positive rate of transgenic hairy roots was observed and recorded after 60 days.

[0041] Example 2

[0042] The difference between Example 2 and Example 1 is that: in Example 2, step 4) the culture medium is: MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8, and other conditions remain unchanged.

[0043] The experimental results of the number of roots, rooting rate, number of positive roots in cotyledons, number of positive roots in stem segments, total number of positive roots, and total positive root rate for Examples 1 and 2 are shown in Table 1.

[0044] Table 1. Experimental results with different concentrations of Agrobacterium rhizogenes bacterial suspension, different explants, and different culture media.

[0045]

[0046] As shown in Table 1, in order to explore the genetic transformation system of balsa wood, Example 1 used OD 600 Agrobacterium rhizogenes K599 at concentrations of 0.4, 0.5, 0.6, 0.7, and 0.8 was used to infect balsamic cotyledons or stem segments. Co-culture was performed on 1 / 2 MS medium. After selection with 20.0 mg / L hygromycin added to the selection medium, positive hairy roots turned red. Figure 1 After 60 days, positive hairy roots were found to form after infection with five different concentrations of infection solution, indicating that the infection efficiency of Agrobacterium was OD. 600 Values ​​0.6 > 0.5 > 0.7 > 0.4 > 0.8; In terms of rooting rate, OD 600 The highest rooting rate (72%) was achieved when the OD value was 0.6. 600 The rooting rates were second highest at values ​​of 0.7 and 0.5, at 60.7% and 57.3%, respectively; in terms of total positive root count and total positive root rate, OD... 600 The highest total number of positive roots and the highest total positive root rate were achieved when the value was 0.6, with a total of 72 positive roots and a positive rate of 66.67%. OD 600 The values ​​were 0.5 and 0.7, with the total number of positive roots and the total positive root rate being the next highest. The total number of positive roots was 53 and 54, respectively, and the total positive root rates were 61.63% and 59.34%, respectively. Meanwhile, the OD... 600 When the values ​​are 0.4 and 0.8, the number of roots and positive roots are both relatively low. Comparative analysis shows that OD... 600 The infection effect is best when the value is 0.6, and the bacterial concentration OD 600 The value can be between 0.5 and 0.7.

[0047] Comparing the infection effects of cotyledons or stem segments of *Clerodendrum trichotomum*, it was found that the infection effect of cotyledons was better than that of stem segments at all five bacterial concentrations. Furthermore, the ratio of positive roots between cotyledons and stem segments was approximately 3:1 (Table 1). This suggests that selecting cotyledons as the recipient during transformation makes it easier to obtain transgenic hairy roots.

[0048] Example 2 used MS medium as the culture medium. The rooting performance of MS and 1 / 2 MS media was compared. It was found that regardless of the bacterial concentration, the rooting rate and positive root rate of explants on 1 / 2 MS medium were greater than those on MS medium. The OD in MS medium... 600 When the OD value was 0.6, the rooting rate was 64.7% and the positive rate was 57.73%, while the OD value in 1 / 2 MS medium was... 600 At a concentration of 0.6, the rooting rate was 72%, and the positive root rate was 66.67%. Analysis shows that 1 / 2 MS medium has a better rooting effect than MS medium; therefore, the optimal transformation conditions mediated by Agrobacterium rhizogenes are: bacterial concentration OD... 600 When the infection rate was 0.6%, the cotyledons of *Berberis vulgaris* were selected as explants for infection and cultured in 1 / 2 MS medium.

[0049] Example 3

[0050] 1) Agrobacterium rhizogenes-mediated transformation: Barkberry cotyledons were cut off as explants, and OD was used... 600 A *Agrobacterium rhizogenes* K599 infection solution containing the target vector (pOCA30 vector containing the SUC2 promoter and JcFT gene) at a concentration of 0.6 was used to infect balsamic cotyledons. The cotyledons were placed in a constant-temperature shaker for 20 minutes to ensure full contact between the bacterial solution and the cotyledons. The cotyledons were then removed, and residual infection solution was blotted off with sterile filter paper. They were then inoculated onto a co-culture medium (1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) and co-cultured, ensuring the underside of the cotyledons adhered to the medium. The mixture was then incubated in the dark for 3 days.

[0051] 2) After 3 days, rinse the balsam leaves 4-5 times with sterile water containing 500 mg / L Cef, place them on sterile filter paper to absorb surface moisture, and inoculate them into recovery medium (1 / 2 MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) without selection agent and culture for 20 days. Then, transfer the recovered balsam leaves to selection medium (MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 20.0 mg / L kanamycin + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) containing the corresponding selection agent and culture for 20 days. Fifty explants were cultured in each experimental group, and the experiment was repeated three times. After 40 days, the rooting rate was counted and positive results were identified.

[0052] Experimental results:

[0053] PCR identification of positive transgenic roots:

[0054] Genomic DNA was extracted from the hairy roots using a modified CTAB method. Wild-type balsam hairy roots were used as a negative control, and the K599 plasmid transformed with SUC2:JcFT was used as a positive control for PCR amplification. The primer sequence was TCGTGTGATTGGGGATGTTTTA.

[0055] TGGTGGATACACGGTCTGCCTT. PCR amplification system: 10 μL Taq PCR Master Mix, 1.0 μL DNA template, 0.5 μL each primer, and ddH2O to a final volume of 20 μL. Amplification conditions were as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 58.1℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 8 min. PCR products were detected by 1% agarose gel electrophoresis and observed and photographed using a gel imaging system.

[0056] Explants of *Agrobacterium rhizogenes* K599 carrying the SUC2:JcFT gene were infected with *Bacillus balsamina* cotyledons. After 40 days of culture, the rooting rate of 150 infected explants reached 59.3%. Then, hairy root DNA was extracted from 15 randomly selected explants for PCR amplification. It was determined that the hairy root DNA numbered 1, 2, 6, 8, 9, 11, 13, and 14 contained the FT gene fragment, while the hairy root DNA numbered 3, 4, 5, 7, 10, 12, and 15 did not contain the FT gene fragment. Figure 2As shown, its positive transformation rate reached 53.3%, which is slightly lower than the positive rate of the reporter gene 35S:RUBY.

[0057] Example 4: Induction of transgenic hairy root callus

[0058] Transgenic hairy roots induced by Agrobacterium rhizogenes-mediated transformation in Example 2 were cut off and placed in culture medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) for induction culture. The induction culture conditions were: light intensity 2000-2500 lx, light time alternating between 14 h (light) and 10 h (dark) (alternating between 14 h light and 10 h dark), and temperature 23-27℃. Twenty transgenic hairy roots were cultured in each experimental group, and the results were repeated three times. The callus status of the hairy roots was observed and counted after 20 days.

[0059] Experimental results, such as Figure 3 As shown, after 20 days of culture, the transgenic hairy roots dedifferentiated to form red callus. Statistical analysis showed that out of a total of 60 transgenic hairy root segments cultured, only 17 segments formed red callus, with a hairy root callus induction rate of 28.3%. The results indicated that the culture medium of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride could induce red callus from the hairy roots.

[0060] Example 5: Genetic transformation of balsa cotyledons using Agrobacterium tumefaciens GV3101

[0061] 1) Preparation of Agrobacterium tumefaciens GV3101 bacterial culture: Transformed Agrobacterium tumefaciens GV3101 was removed from a -80℃ ultra-low temperature freezer and activated on YEB solid plates containing 100 mg / L spectinomycin. The plates were then incubated upside down at 28℃ for 2-3 days. Single colonies were picked from the plates and added to 10 mL of YEB liquid medium containing 100 mg / L spectinomycin. The culture was incubated at 28℃ with shaking at 200 rpm for 10-12 hours. 200 μL of the bacterial culture was then added to 50 mL of YEB liquid medium containing 100 mg / L spectinomycin and incubated at 28℃ with shaking at 200 rpm for 12 hours. 1 mL of the bacterial culture was then transferred to a quartz cuvette and the OD value of the culture was measured using a spectrophotometer. 600 Value, OD 600 When the concentration reaches 0.4, 0.6 or 0.8, the Agrobacterium tumefaciens GV3101 bacterial solution is obtained.

[0062] 2) Preparation of GV3101 infection solution: Agrobacterium tumefaciens GV3101 bacterial suspension was centrifuged at 5000 rpm for 9 min at 4℃, the supernatant was discarded, the bacterial cells were collected, and the bacterial cells were resuspended in 50 ml MS liquid medium. Then, 100 μM acetylsuccinone and 3 mg / L 6-BA were added to prepare GV3101 infection solution.

[0063] 3) Prepare balsa wood-infected explants according to the method in step 3) of Example 1:

[0064] 4) Using balsamic cotyledons as explants, OD 600 GV3101 infection solutions were prepared from Agrobacterium tumefaciens GV3101 bacterial suspensions containing the target vector (pOCA30 vector containing promoter 35S and RUBY gene) at concentrations of 0.4, 0.6, or 0.8. The cotyledons of *Clerodendrum balsamina* were then infected. After infection, the cotyledons were inoculated into a co-culture medium (1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) and co-cultured in the dark in a constant temperature incubator for 3 days.

[0065] 5) After co-culturing, the cotyledonous leaves of *Berberis vulgaris* were cultured in recovery medium (MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) for 20 days; then cultured in selection medium (MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 20.0 mg / L kanamycin + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8) for 20 days. 50 explants were cultured in each experiment, and the experiment was repeated 3 times to allow transgenic callus to grow from the explants. The positive callus induction rate was counted after 40 days.

[0066] Experimental results:

[0067] Table 2. Positive rates of callus tissue infected with different concentrations of Agrobacterium tumefaciens.

[0068]

[0069] Using OD 600 Agrobacterium tumefaciens GV3101 (carrying 35S:RUBY) at concentrations of 0.4, 0.6, and 0.8 were used to infect the cotyledons of sterile balsa seedlings. Red positive calluses were obtained after resistance selection. Figure 4 After 60 days, observation and statistical analysis revealed that positive callus formation occurred after infection with all three bacterial suspension concentrations. The statistical and analytical results (Table 2) showed that OD...600 When the value is 0.4, the callus positivity rate is 9.3%; OD 600 When the value is 0.6, the callus positivity rate is 16.0%; OD 600 When the value was 0.8, the callus positivity rate was 6.7%. The analysis results indicate that the infection efficacy (OD) of Agrobacterium tumefaciens GV3101 is [not specified]. 600 The positive callus rate of balsa cotyledons mediated by Agrobacterium tumefaciens was 16.0%, while the total positive root rate of balsa cotyledons mediated by Agrobacterium rhizogenes in Example 1 reached 66.67%. It can be seen that Agrobacterium rhizogenes is more effective in genetic transformation of balsa.

[0070] Comparative Example 1

[0071] The difference between Comparative Example 1 and Example 3 is as follows: In Comparative Example 1, step 2) uses the following recovery medium: MS + 0.3 mg / L KT + 0.3 mg / L 2,4-D + 0.1 mg / L TDZ + 0.01 mg / L IBA + 200 mg / L Tim + 20.0 g / L sucrose + 6.5 g / L agar, pH: 5.8; the selection medium is: MS + 0.3 mg / L KT + ...

[0072] 0.3 mg / L 2,4-D + 0.1 mg / L TDZ + 0.01 mg / L IBA + 200 mg / L Tim + 20.0 mg / L Kanamycin + 20.0 g / L Sucrose + 6.5 g / L Agar pH: 5.8.

[0073] Experimental results: The induction rate of transgenic hairy roots was 19.6%, and the positive rate was 8.6%.

[0074] Comparative Example 2

[0075] The difference between Comparative Example 1 and Example 3 is as follows: In Comparative Example 1, step 2) uses a recovery medium (1 / 2 MS + 0.5 mg / L 6-BA + 0.01 mg / L IBA + 200 mg / L Tim + 15.0 g / L sucrose + 6.5 g / L agar, pH: 5.8); the corresponding screening medium (1 / 2 MS + 0.5 mg / L 6-BA + 0.01 mg / L IBA + 200 mg / L Tim + 20.0 mg / L kanamycin + 15.0 g / L sucrose + 6.5 g / L agar, pH: 5.8).

[0076] Experimental results: The induction rate of transgenic hairy roots was 28.3%, and the positive rate was 12.5%.

[0077] As can be seen from Comparative Examples 1 and 2, the positive rates of the recovery and screening media with other formulations were low, at only 8.6% and 12.5%, respectively, while the positive conversion rate in Example 3 was as high as 53.3%. It can be seen that the positive conversion rate can be improved by using the recovery and screening media formulations in this application.

[0078] Comparative Example 3

[0079] The difference between Comparative Example 3 and Example 4 is that the culture medium in Comparative Example 3 was MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.5 g / L gibberellin + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8.

[0080] Experimental results: The callus induction rate of transgenic hairy roots was 12.6%.

[0081] Comparative Example 4

[0082] The difference between Comparative Example 4 and Example 4 is that the culture medium in Comparative Example 4 is MS + 0.5 mg / L 6-BA + 0.1 mg / L 2,4-D + 0.5 g / L caffeic acid + 30.0 g / L sucrose + 6.5 g / L agar, pH: 5.8.

[0083] Experimental results: The callus induction rate of transgenic hairy roots was 17.8%.

[0084] Comparative Examples 3 and 4 show that the callus induction rates of transgenic hairy roots using MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.5 g / L gibberellin and MS + 0.5 mg / L 6-BA + 0.1 mg / L 2,4-D + 0.5 g / L caffeic acid were 12.6% and 17.8%, respectively, which are far lower than the 28.3% callus induction rate of transgenic hairy roots using MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride in Example 4. Therefore, the culture medium formulation described in this application can improve the callus induction rate of transgenic hairy roots.

[0085] Currently, there are no reported studies on the genetic transformation of balsa wood. Balsa wood has abundant secondary metabolites, which can severely affect the viability of the inoculum. Therefore, cotyledons and stem segments need to be treated with 5 mg / L xylanase for 30 min before being used as explants for infection. During the infection stage, if the inoculum concentration is too high, Agrobacterium will proliferate in large quantities, and the explants will be encapsulated by Agrobacterium, causing damage and even death, thus affecting the transformation efficiency. If the inoculum concentration is too low, the virulence of the bacteria is poor, and Agrobacterium does not easily adhere to the wounds on the explants, leading to a decrease in transformation efficiency.

[0086] This application uses OD. 600Agrobacterium rhizogenes with OD values ​​of 0.4, 0.5, 0.6, 0.7, and 0.8 were used to infect the cotyledons and stem segments of *Clerodendrum balsamina*. The results showed that using OD values... 600 Infected with 0.6 mg / L Agrobacterium rhizogenes, cultured in 1 / 2 MS medium, and then inoculated into recovery medium (1 / 2 MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim or MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim) and selection medium (MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 20.0 mg / L kanamycin), the most positive hairy roots were obtained, and the number of positive roots in the cotyledons of *Agrobacterium truncatum* was greater than that in the stem segments, with a positive rate of 66.7%. However, using OD... 600 When Agrobacterium tumefaciens at concentrations of 0.4, 0.6, and 0.8 was used to infect the cotyledons of sterile seedlings, the positive callus rate was only 16.0%.

[0087] Transgenic hairy roots were cut and cultured in MS medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride). The results showed that the transgenic hairy roots could form red callus after dedifferentiation, with a callus induction rate of 28.3%.

[0088] After K599 explants carrying SUC2:JcFT were cultured for 40 days, DNA from hairy roots and untransformed balsa roots was extracted from 15 explants and amplified by PCR. PCR detection showed that 8 of the 15 hairy root DNA samples contained fragments of the FT gene, with a positive transformation rate of 53.3%.

[0089] In summary, this application uses balsamina cotyledons as explants and prepares Agrobacterium rhizogenes K599 infection solution using Agrobacterium rhizogenes K599. The OD of the Agrobacterium rhizogenes K599 infection solution is... 600 Using a 0.6 μg / mL medium and 1 / 2 MS medium, an Agrobacterium-mediated transformation system for balsa roots was constructed, which improved the genetic transformation efficiency of balsa and laid the foundation for subsequent screening of functional genes and molecular breeding through genetic transformation.

[0090] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. An Agrobacterium-mediated genetic transformation method of Paulownia elongata, characterized in that, The infection solution prepared by using the bacterial solution containing the pOCA30 vector of the target gene is used to infect the B. frondosa explants, and after the infection is completed, the infected B. frondosa explants are inoculated into the co-culture medium for co-culture; the bacterial solution is Agrobacterium rhizogenes K599 bacterial solution; the target gene is RUBY or JcFT gene; the infection time is 20 min; the co-culture stage is dark culture, and the co-culture time is 3 d; The balsamic explants are cotyledons or stem segments; the bacterial solution OD 600 The pH is 0.4-0.8, and the co-culture medium is MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA or 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA; The preparation method of the Paulownia elongata explant is as follows: full Paulownia elongata seeds are screened, disinfected with 1.0% sodium hypochlorite for 15 minutes, rinsed with sterile water for three times, heated in a 55°C water bath for 30 minutes, inoculated into 1 / 2 MS medium for germination for 30 days, the seedlings are taken out, the sterile cotyledons and stem segments are separated, pretreated with 5 mg / L xylanase for 30 minutes to obtain the Paulownia elongata explant; the cotyledons or stem segments of Paulownia elongata after co-culture are recovered and screened to obtain transgenic hairy roots, and the transgenic hairy roots are induced to obtain transgenic callus; the recovery culture medium is 1 / 2 MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim; the screening culture medium is MS + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 0.1 mg / L IBA + 300 mg / L Cef + 200 mg / L Tim + 20.0 mg / L kanamycin; and the induction culture medium is MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1 g / L chlormequat chloride.

2. The balsa wood genetic transformation method according to claim 1, characterized in that, The balsamic explants are balsamic cotyledons; the bacterial solution OD 600 The concentration was 0.5-0.7; the co-culture medium was 1 / 2 MS + 0.2 mg / L NAA + 0.1 mg / L 6-BA.

3. The balsa wood genetic transformation method according to claim 2, characterized in that, The bacterial solution OD 600 It is 0.

6.

4. The balsa wood genetic transformation method according to claim 1, characterized in that, the infection solution is prepared by centrifuging the bacterial solution at 4°C, discarding the supernatant, resuspending the bacterial bodies in MS liquid medium, and adding 100 µM AS and 3 mg / L 6-BA to prepare the infection solution.

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