Method for efficiently inducing adventitious buds and rooting of transgenic hairy roots of Populus albus Nanlin 895

Abstract

The invention discloses a method for efficiently inducing adventitious buds and rooting through transgenic hairy roots of Populus albus Nanlin 895, and relates to the technical field of biological breeding. The plant material used in the invention is a transgenic Nanlin 895 poplar hairy root generated by mediation of agrobacterium rhizogenes; dividing the hairy roots into root sections of 1-2 cm by using a sterile blade, and putting the hairy roots on a culture medium added with 0.01 mg / L TDZ and 0.2 mg / L 6-BA to induce adventitious buds; placing the adventitious buds in a culture medium added with 0.01 mg / L of TDZ for bud elongation; and cutting off the elongated adventitious buds, placing the adventitious buds in a culture medium added with 0.1 mg / L IBA, and carrying out rooting culture so as to obtain a positive Nanlin 895 poplar transgenic plant. Experiments prove that the adventitious bud induction rate of the plant material is 70%, the adventitious bud elongation rate is 40%, the rooting rate is 90%, and the method has good practicability.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology, and more specifically, relates to a method for efficiently inducing adventitious buds and rooting of transgenic poplar hairy roots of Nanlin 895 mediated by Agrobacterium rhizogenes. Background Technology

[0002] Nanlin 895 poplar (P. deltoides × P. euramericana) is a superior variety approved by the National Forest Tree Variety Approval Committee in 2002. Its suitable planting areas are mainly concentrated in the Huang-Huai Plain, Jiang-Huai Plain, and the middle and lower reaches of the Yangtze River Plain. Due to its rapid growth and easy propagation, as well as its small genome, it is simple to operate in traditional and molecular breeding, and has become a model tree species for forestry research. However, existing Agrobacterium-mediated transgenic methods for Nanlin 895 poplar are still not ideal, not only due to long experimental cycles, cumbersome procedures, and low transformation efficiency, but also because it is difficult to obtain homozygous transgenic plants, with most resulting in chimeras. Because of the limitations of Agrobacterium-mediated genetic transformation of Nanlin 895 poplar, Agrobacterium rhizogenes, as a promising alternative system capable of introducing exogenous genes into the poplar genome, has gradually attracted the attention of researchers.

[0003] The Agrobacterium rhizogenes-mediated transgenic technology of Populus tomentosa 'Nanlin 895' is characterized by its simplicity and high efficiency. The process is short, with each plant requiring no more than 5 minutes of inoculation time, and eliminates the need for shaking or precise control of the bacterial solution's OD value. This significantly reduces experimental time and operational difficulty. Furthermore, its high transformation efficiency and ease of obtaining non-chimeric transgenic seedlings provide a new and efficient pathway for transgenic plant cultivation. However, the technical system for inducing adventitious buds from hairy roots to ultimately obtain transgenic plants is not yet perfected. This is mainly reflected in the low budding rate of hairy roots, and the difficulties encountered by the resulting adventitious buds in subsequent rooting and seedling development, leading to unstable seedling yield. Therefore, the overall efficiency and stability of this technical route have become key bottlenecks limiting its widespread application.

[0004] In summary, the core problem with this technology is the difficulty in regenerating plants from transgenic hairy roots. Therefore, this application is hereby submitted. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by this application is to provide a method for efficiently inducing adventitious shoots and rooting of transgenic hairy roots, and to establish an efficient genetic transformation system of Populus tomentosa 895 from Nanlin University.

[0006] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows:

[0007] A method for efficiently inducing adventitious buds and rooting from the transgenic hairy roots of Populus tomentosa 'Nanlin 895' includes the following steps: preparing isolated root segments from the transgenic Populus tomentosa 'Nanlin 895'; placing the isolated root segments in an adventitious bud induction medium to obtain adventitious buds; transferring the obtained adventitious buds to a new adventitious bud induction medium to allow them to proliferate for 30 days; placing the proliferated adventitious buds in an adventitious bud elongation medium to obtain elongated adventitious buds for more than 25 days; cutting off the elongated adventitious buds and placing them in a rooting medium to obtain complete transgenic Populus tomentosa 'Nanlin 895' plants; extracting genomic DNA from the leaves of the transgenic plants and amplifying the target genes; wherein the genes are PdeNAC014 and PdeNAC072.

[0008] The detached root segments were transgenic poplar hairy roots of Nanlin 895 produced through Agrobacterium rhizogenes-mediated transformation, with their length shortened to 1-2 cm.

[0009] The formulation of the adventitious bud induction medium is as follows: MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L termethin + 20 mg / L kanamycin, pH=5.80.

[0010] The formulation of the adventitious bud elongation medium is as follows: MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 400 mg / L termethin + 20 mg / L kanamycin, pH=5.80.

[0011] The rooting medium formula is as follows: 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.1 mg / L IBA + 200 mg / L termethin + 10 mg / L kanamycin, pH=5.80.

[0012] The height of the elongated adventitious bud is 2 cm or more.

[0013] A method for efficiently inducing adventitious buds and rooting from the transgenic hairy roots of Populus 895 from Nanjing Forestry University, including the following specific steps:

[0014] 1) Place the hairy roots of Nanlin 895 transgenic plants mediated by Agrobacterium rhizogenes in a sterile container filled with sterile water and cut them to 1-2 cm using a sterile blade.

[0015] 2) Adventitious shoots were obtained by culturing 1-2 cm detached root segments in MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L termethin + 20 mg / L kanamycin at pH 5.80.

[0016] 3) The adventitious shoots obtained from the culture were transferred to a new MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L termethin + 20 mg / L kanamycin, pH=5.80, to allow them to continue to proliferate for 30 days.

[0017] 4) Transfer the proliferated adventitious shoots to MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 400 mg / L termethin + 20 mg / L kanamycin, pH=5.80 adventitious shoot elongation medium and culture for more than 25 days. The culture conditions are 23-25℃, 7500-8500 lx, and 12-18h light.

[0018] 5) Using sterile scissors, cut off adventitious buds that have grown to more than 2 cm in the adventitious bud elongation medium, and transfer them to a rooting medium of 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.1 mg / L IBA + 200 mg / L termethin + 10 mg / L kanamycin, pH=5.80. Culture under light for more than 25 days to obtain complete transgenic Nanlin 895 plants;

[0019] 6) The leaves of the transgenic plants were flash-frozen in liquid nitrogen and ground into powder. Genomic DNA was extracted using the CTAB method for PCR amplification and detection.

[0020] The Agrobacterium rhizogenes mentioned is Agrobacterium rhizogenes K599.

[0021] The recombinant plasmid of the PdeNAC014 gene is pRI101-PdeNAC014-6×Flag.

[0022] The recombinant plasmid of the PdeNAC072 gene is pRI101-PdeNAC072-6×Flag.

[0023] Compared to existing technologies, the beneficial effects of this application are as follows:

[0024] 1) This application uses the hairy roots of transgenic Populus nanlin 895 to prepare detached root segments; the detached root segments are placed in an adventitious bud induction medium to obtain adventitious buds; the obtained adventitious buds are transferred to a new adventitious bud induction medium to proliferate for 30 days; the proliferated adventitious buds are placed in an adventitious bud elongation medium to be cultured for more than 25 days to obtain elongated adventitious buds; the elongated adventitious buds are cut off and placed in a rooting medium to be cultured for more than 25 days to obtain complete transgenic Populus nanlin 895 plants.

[0025] 2) In the adventitious shoot induction medium of hairy root in this application, the plant hormone addition scheme is 0.01 mg / L TDZ and 0.2 mg / L 6-BA, and the adventitious shoot induction rate is 70.00%; in the adventitious shoot elongation medium, the plant hormone addition scheme is 0.01 mg / L TDZ, and the adventitious shoot elongation rate is 40%; in the rooting medium, the plant hormone addition scheme is 0.1 mg / L IBA, and the rooting rate is 90%. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the induction of adventitious shoots and rooting of the transgenic hairy roots of Populus tomentosa 'Nanlin 895' in Example 1.

[0027] Figure 2 The image shows the PCR detection results of leaves transgenic with the PdeNAC014 and rolB genes in Example 1 (WT represents the negative control; P represents the positive control; T followed by a number represents transgenic plants of different lines).

[0028] Figure 3 The image shows the PCR detection results of leaves transgenic with the PdeNAC072 gene and rolB gene in Example 2 (WT represents the negative control; P represents the positive control; T plus a number represents transgenic plants of different lines). Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this application clearer, the application is 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.

[0030] The *Populus nanlin* 895 used in this application was obtained from Wuhan Bobeyuan Biotechnology Co., Ltd. It was subcultured using a medium supplemented with 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar to obtain healthy wild-type *Populus nanlin* 895 tissue culture material.

[0031] The K599 Agrobacterium rhizogenes competent cells used in this application were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0032] Example 1

[0033] 1. Obtaining transgenic poplar hairy roots from Nanlin 895

[0034] The terminal buds, about 3 cm long, of wild-type Nanlin 895 plants were cut off, and activated Agrobacterium rhizogenes colonies were collected from the wounds. The explants were then cultured in the dark for 2-3 days in a co-culture medium of 1 / 2 MS (Murashige and Skoog medium) + 100 μmol / L AS + 7.8 g / L agar until visible white colonies appeared around the wounds. The infected explants were then transferred to a selection medium of 1 / 2 MS (Murashige and Skoog medium) + 7.8 g / L agar + 0.2 mg / L IBA + 200 mg / L termethin and cultured under light for 30-45 days to obtain transgenic Nanlin 895 poplar hairy roots.

[0035] Hairy roots induced by Agrobacterium rhizogenes exhibit easily identifiable phenotypic characteristics, clearly distinguishing them from normal roots: firstly, the hairy roots have well-developed lateral roots, exhibit negative geotropism, and often extend beyond the culture medium; secondly, the hairy roots are milky white, unlike the pale yellow or pale green of normal roots. Figure 1 These differences provide a clear basis for the initial screening of transgenic lines.

[0036] 2. Induction of adventitious shoots from isolated root segments of transgenic Nanlin 895 poplar

[0037] Transgenic hairy roots of Nanlin 895, mediated by Agrobacterium rhizogenes, were placed in sterile dishes containing sterile water. After being cut to 1-2 cm using a sterile blade, they were cultured in MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L termetidine + 20 mg / L kanamycin to induce adventitious shoots. These adventitious shoots were then transferred to fresh MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L termetidine + 20 mg / L kanamycin to continue proliferation for 30 days. Figure 1 As shown, the biomass of the proliferated adventitious shoots increased significantly.

[0038] 3. Adventitious bud elongation of transgenic Nanlin 895 poplar

[0039] The proliferated adventitious shoots were transferred to MS medium (Murashige and Skoog medium) for adventitious shoot elongation, which contained 25 g / L sucrose, 7.8 g / L agar, 0.01 mg / L TDZ, 400 mg / L termethin, and 20 mg / L kanamycin, and cultured for more than 25 days under the following conditions: 23–25°C, 7500–8500 lx, and 12–18 h light.

[0040] The results are as follows Figure 1 As shown, 1 / 3 of the adventitious buds elongated to more than 2 cm.

[0041] 4. Genetically modified Nanjing Forestry University 895 Yang Shenggen

[0042] Using sterile scissors, adventitious buds that have grown to more than 2 cm in the adventitious bud elongation medium were cut off and transferred to rooting medium in 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.1 mg / L IBA + 200 mg / L termethin + 10 mg / L kanamycin and cultured under light for more than 25 days to obtain complete transgenic Nanlin 895 plants.

[0043] 30 days after transplantation, if Figure 1 As shown, the adventitious buds have taken root densely, indicating that their rooting condition is good.

[0044] 5. PCR detection

[0045] Transgenic leaves of Populus nanlin 895 were flash-frozen in liquid nitrogen and ground into powder. Wild-type Populus nanlin 895 leaves were used as a control. Genomic DNA was extracted using the CTAB method, and PCR amplification was performed using the following primers.

[0046] rolB gene - forward primer: 5'-ACGAAGAAGGTGCAGGCTAC-3'

[0047] rolB gene - reverse primer: 5'-TCAACGCTTTCAACCACGAG-3'

[0048] PdeNAC014 gene - forward primer: 5'-GACGCACAATCCCACTATCC-3'

[0049] PdeNAC014 gene - reverse primer: 5'-CAGGAAACAGCTATGACC-3'

[0050] The results are as follows Figure 2As shown, the rolB gene and PdeNAC014 gene (sequence shown in SEQ ID NO.1) in the leaves of the induced Nanlin 895 plants can both be amplified, further indicating that the obtained plants are transgenic positive plants induced by Agrobacterium rhizogenes, indicating that it is rapid and feasible to use this system to culture the transgenic hairy roots of Nanlin 895 poplar into complete plants.

[0051] Example 2

[0052] 1. Take leaves, stem segments and root samples of Populus tomentosa 895 from Nanlin and grind them into powder in liquid nitrogen. Weigh 50 mg of the sample and extract total RNA using an RNA extraction kit. Invert the extracted RNA to obtain cDNA. The obtained cDNA is used to amplify the CDS sequence of the target gene PdeNAC072 (sequence see SEQ ID NO.2).

[0053] 2. Design gene-specific primers:

[0054] PdeNAC072 gene - forward primer: 5'-ATGGGACTGCAAGAAACAGAC-3'

[0055] PdeNAC072 gene - reverse primer: 5'-TCACTGCCTAAACCCATACCCA -3'

[0056] The CDS of the PdeNAC072 gene was amplified from the mixed cNDA of leaves, stem segments and roots of Populus tomentosa 895 in Nanlin County. The successfully sequenced sequence was ligated to the N-terminus of the 6×Flag tag in the pRI101-6×Flag vector to obtain the pRI101-PdeNAC072×Flag overexpression vector.

[0057] 3. The recombinant plasmid carrying the target gene pRI101-PdeNAC072×Flag was transformed into *Agrobacterium rhizogenes* K599 using a freeze-thaw method. Positive single colonies were picked and transferred to LB liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate. The culture was incubated at 28°C with shaking at 200 rpm for 16-24 hours. An equal volume of the incubated culture was mixed with 50% glycerol, flash-frozen in liquid nitrogen, and stored at -80°C for later use. The preserved *Agrobacterium rhizogenes* K599 glycerol culture was streaked onto LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate and incubated upside down at 28°C for 48 hours. Single colonies were picked for PCR verification. PCR-positive single colonies were spotted onto fresh LB solid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin sulfate using a sterile pipette tip and grown at 28°C for 24-36 hours.

[0058] 4. Using the terminal buds of wild-type tissue-cultured Populus tomentosa 895 in good growth condition, K599 positive colonies grown on LB solid medium were collected from the wounds. After infection, the resulting hairy roots were subjected to adventitious bud induction culture under the following conditions: 23–25℃, 7500–8500 lx, and 12–18 h light.

[0059] 5. The induced plant leaves were flash-frozen in liquid nitrogen and ground into powder. Genomic DNA was extracted using the CTAB method, and PCR amplification was performed according to the primers for the rolB gene and the PdeNAC072 gene.

[0060] The results are as follows Figure 3 As shown, the rolB and PdeNAC072 genes in the leaves of the induced Nanlin 895 plants can both be amplified, further indicating that the obtained plants are transgenic positive plants induced by Agrobacterium rhizogenes. This shows that it is rapid and feasible to use this system to culture the transgenic hairy roots of Nanlin 895 poplar into complete plants.

Claims

1. A method for high efficient induction of adventitious shoots and rooting from transgenic hairy roots of Populus nigra cv. 'Garryana' 895, characterized by: The transgenic South Lin 895 poplar hairy roots are induced by Agrobacterium rhizogenes strain K599. The recombinant plasmid of PdeNAC014 gene is pRI101-PdeNAC014-6xFlag.

2. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The recombinant plasmid of PdeNAC072 gene is pRI101-PdeNAC072-6xFlag.

3. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The in vitro root segments are 1-2 cm South Lin 895 poplar transgenic hairy roots.

4. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The formula of the adventitious bud induction medium is: MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L timentin + 20 mg / L kanamycin, pH=5.

80.

5. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The formula of the adventitious bud elongation medium is: MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 400 mg / L timentin + 20 mg / L kanamycin, pH=5.

80.

6. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The formula of the rooting medium is: 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.1 mg / L IBA + 200 mg / L timentin + 10 mg / L kanamycin, pH=5.

80.

7. The method for high efficient induction of adventitious shoots from transgenic hairy roots of Populus nigra cv. 'I-895' according to claim 1, characterized in that, The specific steps include:

8. The method of claim 1, wherein the transgenic shoots are derived from southern pine 895 (Populus deltoides cv. 895). 1) 40-day-old South Lin 895 poplar transgenic hairy roots are taken out and placed in a culture dish containing sterile water, and then cut into 1-2 cm root segments using a sterile blade; ​ 9. The method of claim 1, wherein the transgenic hairy roots of Populus nigra cv. 'Garryana' induce adventitious shoots and roots efficiently, and the method is characterized by, 2) the cut root segments are placed in a hairy root induction medium with the formula of MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L timentin + 20 mg / L kanamycin, pH=5.80, and the culture conditions are 23-25℃, 7500-8500 lx, and 12-18 h of light illumination; ​ ​ 3) 30-day-old adventitious buds were transferred to a new formula of MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 0.2 mg / L 6-BA + 400 mg / L timentin + 20 mg / L kanamycin, pH = 5.80, hairy root induction adventitious bud medium, and cultured at 23-25°C, 7500-8500 lx, 12-18 h light; 4) 60-day-old adventitious buds were transferred to a formula of MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.01 mg / L TDZ + 400 mg / L timentin + 20 mg / L kanamycin, pH = 5.80, adventitious bud elongation medium, and cultured for more than 25 days, at 23-25°C, 7500-8500 lx, 12-18 h light; 5) Adventitious buds that grew to more than 2 cm in the adventitious bud elongation medium were cut and placed in a formula of 1 / 2 MS medium (Murashige and Skoog medium) + 25 g / L sucrose + 7.8 g / L agar + 0.1 mg / L IBA + 200 mg / L timentin + 10 mg / L kanamycin, pH = 5.80, rooting medium, and cultured for more than 25 days to obtain complete transgenic Nanlin 895 plants; 6) Transgenic plant leaves were quickly frozen in liquid nitrogen and ground into powder, and genomic DNA was extracted using the CTAB method for PCR amplification detection. The rolB gene, PdeNAC014 gene, and PdeNAC072 gene in the induced transgenic Nanlin 895 poplar plant leaves could all amplify bands.

10. The method of claim 1-9 for high-efficiency induction of adventitious buds and rooting of transgenic hairy roots of Nanlin 895 poplar to obtain transgenic Nanlin 895 complete plants.

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