Culture medium combination for genetic transformation of salix matsudana and method for genetic transformation of salix matsudana and application thereof

Abstract

The present application relates to a kind of medium combination for genetic transformation of Populus euphratica and the method for genetic transformation of Populus euphratica and its application, belong to the field of plant genetic engineering technology.The medium combination for genetic transformation of Populus euphratica of the present application includes co-cultivation medium, differentiation medium, strong seedling medium, rooting medium, growth medium.The method for genetic transformation of Populus euphratica of the present application is simple and easy to operate, low in cost, high in reliability, can realize the rapid differentiation of Populus euphratica transgenic plant, rooting, lay the foundation for the research and breeding of Populus euphratica functional gene, provide technical support for creating and cultivating transgenic non-fuzzy poplar new germplasm, have great scientific research value, economic value and ecological value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a culture medium combination for genetic transformation of Populus tomentosa and a method for genetic transformation of Populus tomentosa, as well as their applications. Background Technology

[0002] poplar ( Populus Poplar (Poplar) is a tall tree belonging to the Salicaceae family. It offers quick economic benefits, with inexpensive seedlings and a high survival rate after planting. Female poplar trees produce cotton-like fluff, which is harmless in itself, but excessive quantities can cause environmental pollution, allergies, and fire hazards.

[0003] As a male-sterile poplar, Sishui poplar has advantages such as not producing fluff, having little pollen, a short harvesting cycle, fast growth, straight and upright trunk, and high survival rate in afforestation and seedling cultivation. It can play a significant economic and ecological role and is a preferred tree species for modern landscaping.

[0004] However, existing poplar tissue culture and transgenic systems are only suitable for cultivating the NL895 strain with a Populus tomentosa background, the T89 strain with a Populus thunbergii background, or the 84K strain with a Populus alba background. They cannot meet the requirements for tissue culture and genetic transformation of Populus spp. with a Populus nigra background. Using existing differentiation and rooting media, Populus spp. not only fails to differentiate, but also faces technical challenges such as difficulty in rooting and long rooting times. Currently, a mature, efficient, and simple genetic transformation system for Populus spp. has not yet been publicly disclosed. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium combination and a method for genetic transformation of Populus tomentosa, as well as their application, to solve the problems of difficult rooting and long rooting time of Populus tomentosa plants in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a culture medium combination for genetic transformation of Populus tomentosa, including a co-culture medium, a differentiation medium, a seedling strengthening medium, a rooting medium, and a growth medium; The co-culture medium is based on WPM medium and includes the following components at the following concentrations: 2-morpholinoethanesulfonic acid (MES) 400-600 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5-1 mg / L, acetylsyleugenone (AS) 50-200 μmol / L, sucrose 15-25 g / L, and agar 5-10 g / L; The differentiation medium is based on WPM medium and includes the following components at the following concentrations: 2-morpholinoethanesulfonic acid (MES) 400-600 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5-1 mg / L, kanamycin (Kan) 5-8 mg / L, cefotaxime (CEF) 200-400 mg / L, sucrose 15-25 g / L, and agar 5-10 g / L; The seedling culture medium is based on MS medium and includes the following components at the following concentrations: 6-benzylaminopurine (6-BA) 0.1-0.5 mg / L, thiamethoxam (TDZ) 0.5-2 μg / L, kanamycin (Kan) 5-8 mg / L, cefotaxime (cef) 200-400 mg / L, sucrose 25-35 g / L, and agar 5-10 g / L; The rooting medium is based on 1 / 2 MS medium and includes the following components at the following concentrations: indolebutyric acid (IBA) 0.1-0.5 mg / L, cefotaxime (cef) 200-400 mg / L, sucrose 25-35 g / L, and agar 5-10 g / L; The growth medium is based on MS medium and includes the following components at the following concentrations: cefotaxime (CEF) 400–600 mg / L, sucrose 25–35 g / L, and agar 5–10 g / L.

[0007] The present invention also provides the application of the aforementioned culture medium combination in the cultivation of Populus tomentosa plants.

[0008] This invention also provides the application of the aforementioned culture medium combination in the genetic transformation of Populus tomentosa.

[0009] This invention also provides a method for genetic transformation of Populus tomentosa, comprising the following steps: (1) After inoculating the Populus sibirica explants with Agrobacterium tumefaciens solution, place them face up on a co-culture medium and culture them in the dark for 1-3 days to obtain co-cultured explants. (2) Place the co-cultured explants face up on the differentiation medium and culture under light for 28-30 days to obtain adventitious buds of Populus tomentosa. (3) Place the adventitious buds of Populus sibirica on a seedling culture medium and culture for 14-16 days to obtain strong buds of Populus sibirica; (4) Insert the robust buds of Populus sichuanensis into the rooting medium and culture for 7-14 days to obtain Populus sichuanensis seedlings; (5) Transplant the seedlings of Populus tomentosa into the growth medium and culture for 14-16 days to obtain Populus tomentosa test-tube seedlings.

[0010] Preferably, the OD of the Agrobacterium bacterial solution in step (1) is... 600 The value is 0.2 to 0.6; The inoculation time is 10–20 minutes; The temperature for the dark culture is 20–28°C.

[0011] Preferably, the temperature for light culture in step (2) is 20-28°C, and the light culture time is 12-18 h / d.

[0012] Preferably, the culture temperature in step (3) is 20-28°C, and the light exposure time is 12-18 h / d. The bud length of the *Phyllostachys edulis* robust bud is 2–4 cm.

[0013] Preferably, the culture temperature in step (4) is 20-28°C and the light exposure time is 12-18 h / d.

[0014] Preferably, the culture temperature in step (5) is 20-28°C and the light exposure time is 12-18 h / d.

[0015] The present invention also provides the application of the method in cultivating Populus tomentosa plants.

[0016] The present invention has the following technical effects and advantages: The genetic transformation method of Populus tomentosa of the present invention is simple in its overall process, effectively shortens the transformation cycle, and improves the transformation efficiency. It only takes 2 months from obtaining Populus tomentosa explants to placing them in the growth medium for culture. The resulting transgenic Populus tomentosa plants have stable genetic traits and significantly improved expression levels of exogenous genes. This invention selects the third or fourth fully expanded leaf from sterile Populus tomentosa seedlings that are in good condition and lush and green after 30-40 days of growth as Populus tomentosa explants. At this time, the lignification degree of the leaves is low, and Agrobacterium tumefaciens has less toxicity to the Populus tomentosa explants. The fully expanded leaves of Populus tomentosa are cut but not cut off, and placed face up on co-culture medium and differentiation medium. After 2 days of culture, the cell division at the cut of the Populus tomentosa explant is obvious, and a small amount of callus tissue is formed. T-DNA is easily integrated into the Populus tomentosa genome. At the same time, the partial healing of the cut ensures the normal growth of Populus tomentosa explants, which greatly improves the genetic transformation efficiency of Populus tomentosa. OD of Agrobacterium tumefaciens bacterial solution of the present invention 600 A concentration of 0.2–0.6 is more suitable for the genetic transformation of woody plant leaves. At this concentration, the Agrobacterium concentration is appropriate, preventing the death of Populus tomentosa explants due to rapid reproduction or poor infection effect due to low concentration. The genetic transformation efficiency reaches about 85%. Controlling the infection time to 15 minutes not only provides sufficient time for Agrobacterium to contact the Populus tomentosa explants, but also avoids hypoxia or browning of the Populus tomentosa explants due to the toxicity of Agrobacterium. Setting the co-culture time to 1–3 days can avoid the difficulty of sterilization in the later stage caused by the large-scale reproduction of Agrobacterium, and reduce the damage to Populus tomentosa explant cells. In the culture medium combination of the present invention, 2,4-D can induce the differentiation of Populus tomentosa explants into callus tissue, Kan serves as a positive plant selection marker, CEF can inhibit the excessive proliferation of Agrobacterium and will not have a significant impact on the genetic transformation efficiency of Populus tomentosa; MES can better promote the differentiation of Populus tomentosa callus and adventitious buds. The genetic transformation method of Populus tomentosa of this invention is simple, low-cost, and highly reliable. It can achieve rapid differentiation and rooting of transgenic Populus tomentosa plants, laying the foundation for the research and breeding of functional genes of Populus tomentosa, and providing technical support for the creation and cultivation of new transgenic cotton-free Populus tomentosa germplasm. It has great scientific research value, economic value and ecological value. Attached Figure Description

[0017] Figure 1 Adventitious shoots of Populus tomentosa induced on differentiation medium; Figure 2 To cultivate robust shoots of *Populus tomentosa* on a robust seedling culture medium; Figure 3 Populus tomentosa seedlings cultured on rooting medium; Figure 4 These are test-tube seedlings of *Populus tomentosa* cultured on growth medium. Figure 5 The results of identification of transgenic positive plants of Populus tomentosa; Figure 6 Positive test-tube seedlings of Populus tomentosa cultured in sterile nutrient soil; Figure 7 The root changes of *Populus simonii* buds cultured on rooting medium for 0–10 days. Detailed Implementation

[0018] This invention provides a culture medium combination for genetic transformation of Populus tomentosa, including a co-culture medium, a differentiation medium, a seedling strengthening medium, a rooting medium, and a growth medium; The co-culture medium is based on WPM medium and includes the following components at the following concentrations: MES 400–600 mg / L, preferably 500 mg / L; 2,4-D 0.5–1 mg / L, preferably 0.75 mg / L; AS 50–200 μmol / L, preferably 100 μmol / L; sucrose 15–25 g / L, preferably 20 g / L; agar 5–10 g / L, preferably 7.5 g / L; pH=5.8. The differentiation medium is based on WPM medium and includes the following components at the following concentrations: MES 400–600 mg / L, preferably 500 mg / L; 2,4-D 0.5–1 mg / L, preferably 0.75 mg / L; Kan 5–8 mg / L, preferably 6.25 mg / L; CEF 200–400 mg / L, preferably 300 mg / L; sucrose 15–25 g / L, preferably 20 g / L; agar 5–10 g / L, preferably 7.5 g / L; pH=5.8. The seedling culture medium is based on MS medium and includes the following components at the following concentrations: 6-BA 0.1–0.5 mg / L, preferably 0.2 mg / L; TDZ 0.5–2 μg / L, preferably 1 μg / L; Kan 5–8 mg / L, preferably 6.25 mg / L; CEF 200–400 mg / L, preferably 300 mg / L; sucrose 25–35 g / L, preferably 30 g / L; agar 5–10 g / L, preferably 7 g / L; pH=5.8. The rooting medium is based on 1 / 2 MS medium and includes the following components at the following concentrations: IBA 0.1–0.5 mg / L, preferably 0.3 mg / L; CEF 200–400 mg / L, preferably 360 mg / L; sucrose 25–35 g / L, preferably 30 g / L; agar 5–10 g / L, preferably 7 g / L; pH=5.8. The growth medium is based on MS medium and includes the following components at the following concentrations: CEF 400-600 mg / L, preferably 480 mg / L; sucrose 25-35 g / L, preferably 30 g / L; agar 5-10 g / L, preferably 7 g / L; pH=5.8.

[0019] The present invention also provides the application of the aforementioned culture medium combination in the cultivation of Populus tomentosa plants.

[0020] This invention also provides the application of the aforementioned culture medium combination in the genetic transformation of Populus tomentosa.

[0021] This invention also provides a method for genetic transformation of Populus tomentosa, comprising the following steps: (1) After inoculating the Populus sibirica explants with Agrobacterium tumefaciens solution, place them face up on a co-culture medium and culture them in the dark for 1-3 days, preferably 2 days, to obtain co-cultured explants; (2) Place the co-cultured explants face up on the differentiation medium and culture them under light for 28-30 days, preferably 28 days. During this period, change the differentiation medium every 7 days to obtain adventitious buds of Populus tomentosa. (3) Place the adventitious buds of Populus sibirica on a seedling culture medium and culture for 14-16 days, preferably 14 days. During this period, change the seedling culture medium every 7 days to obtain strong buds of Populus sibirica. (4) Insert the strong buds of Populus sichuanensis into the rooting medium and culture for 7-14 days, preferably 10 days, to obtain Populus sichuanensis seedlings; (5) Transplant the seedlings of Populus tomentosa into a growth medium and culture for 14 to 16 days, preferably 14 days, to obtain Populus tomentosa test-tube seedlings.

[0022] In this invention, the Populus explant in step (1) is a fully expanded leaf of Populus, preferably the third or fourth young leaf of Populus, and the leaf length of the fully expanded leaf of Populus is 1.2 to 1.8 cm, preferably 1.5 cm. The OD of the Agrobacterium tumefaciens bacterial solution 600 The value is 0.2 to 0.6, preferably 0.3; The inoculation method is Agrobacterium leaf disc method, and the inoculation time is 10-20 minutes, preferably 15 minutes; The temperature for dark culture is 20–28°C, preferably 25°C.

[0023] In this invention, the temperature of the light culture in step (2) is 20-28°C, preferably 25°C; the light culture time is 12-18 h / d, preferably 16 h / d.

[0024] In this invention, the culture temperature in step (3) is 20-28°C, preferably 25°C, and the light duration is 12-18 h / d, preferably 16 h / d. The bud length of the *Phyllostachys edulis* bud is 2–4 cm, preferably 3 cm.

[0025] In this invention, the culture temperature in step (4) is 20-28°C, preferably 25°C; the light exposure time for culture is 12-18 h / d, preferably 16 h / d.

[0026] In this invention, the culture temperature in step (5) is 20-28°C, preferably 25°C; the light exposure time for culture is 12-18 h / d, preferably 16 h / d.

[0027] The present invention also provides the application of the method in cultivating Populus tomentosa plants.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Preparation of co-culture medium Take 500 mg MES, 0.75 mg 2,4-D and 20 g sucrose and make up to 1 L WPM culture medium. Adjust the pH to 5.8, add 7.5 g agar and sterilize at 121 °C for 15 min. After cooling to room temperature, add AS to the final concentration of 100 μmol / L to obtain the co-culture medium.

[0030] Example 2: Preparation of Differentiation Culture Medium 500 mg MES, 0.75 mg 2,4-D, and 20 g sucrose were added to 1 L WPM culture medium and the pH was adjusted to 5.8. 7.5 g agar was added and the medium was sterilized at 121 °C for 15 min. After cooling to room temperature, Kan and CEF were added to a final concentration of 6.25 mg / L and 300 mg / L, respectively, to obtain the differentiation medium.

[0031] Example 3: Preparation of seedling culture medium Take 0.2 mg 6-BA, 1 μg TDZ and 30 g sucrose and make up to 1 L MS culture medium, adjust pH to 5.8, add 7.5 g agar and sterilize at 121 °C for 15 min. After cooling to room temperature, add Kan and CEF to the final concentrations of 6.25 mg / L and 300 mg / L respectively to obtain the seedling culture medium.

[0032] Example 4: Preparation of rooting medium Take 0.3 mg IBA and 30 g sucrose and make up to 1 L 1 / 2 MS culture medium. Adjust the pH to 5.8, add 7 g agar and sterilize at 121 °C for 15 min. After cooling to room temperature, add CEF to a final concentration of 360 mg / L to obtain the rooting medium.

[0033] Example 5: Preparation of growth medium 30g of sucrose was added to 1L of MS culture medium and the pH was adjusted to 5.8. After adding 7g of agar, the medium was sterilized at 121℃ for 15min. After cooling to room temperature, cef was added to a final concentration of 480mg / L to obtain the growth medium.

[0034] Example 6: Methods for Genetic Transformation of Populus tomentosa (1) Construction of monoclonal Agrobacterium: Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize FTO The nucleotide sequence of the gene is shown in SEQ ID NO.1; and based on the seamless assembly characteristics, primer sequences with pBI121 vector adapters were designed, as shown in Table 1. SEQ ID NO.1: Table 1. Primer sequences designed with carrier adapters. by FTO The gene's nucleotide sequence was used as a template for PCR amplification. The PCR system consisted of 30 μL volumes, including... FTO 1 μL of gene template, 1 μL of F1 primer, 1 μL of R1 primer, 15 μL of 2×Phanta Max Master Mix, and the remainder ddH2O were prepared. The PCR program was set as follows: 95℃ pre-denaturation for 3 min → (95℃ denaturation for 15 s → 58℃ annealing for 15 s → 72℃ extension for 40 s) × 32 cycles → 72℃ extension for 5 min. The amplified PCR product was recombined with the linearized pBI121 vector, transformed into E. coli DH5α competent cells, and the recombinant plasmid pBI121- was extracted. FTO The cells were transformed into Agrobacterium EHA105 competent cells by electroporation to obtain monoclonal Agrobacterium. (2) Obtaining Populus spp. explants: Take the third young leaf of Populus spp. that is growing well and is lush and green with a length of 1.5 cm from the sterile seedlings of Populus spp. that has grown for 30-40 days as fully expanded leaves of Populus spp. After removing the petiole and leaf tip, make horizontal cuts on the leaf but do not cut it off to obtain Populus spp. explants. (3) Agrobacterium leaf disc inoculation: After inoculating a single clone of Agrobacterium onto YM solid medium, culture it at 28℃ for 54 h until the colonies are smooth and plump. Scrape off the colonies and adjust the OD with the infection solution. 600 =0.3, to obtain Agrobacterium tumefaciens bacterial solution; *Populus spp.* explants were inoculated in the Agrobacterium tumefaciens bacterial solution for 15 min to obtain inoculated *Populus spp.* explants; YM solid culture medium contained the following components at the following concentrations: 0.5 g / L dipotassium hydrogen phosphate, 10 g / L mannitol, 2 g / L... L - Glutamine, 0.2 g / L sodium chloride, 0.3 g / L yeast extract, 0.389 g / L magnesium sulfate, Kans 50 mg / L, Rif 50 mg / L, agar 7.5 g / L; The infection medium was based on WPM medium and included the following components at the following concentrations: MES 500 mg / L, 2,4-D 0.75 mg / L, AS 100 μmol / L, sucrose 20 g / L, agar 7.5 g / L, pH=5.8; (4) Culture of transgenic Populus tomentosa plants: The inoculated Populus tomentosa explants were placed face up on the co-culture medium prepared in Example 1 and cultured in the dark at 25°C for 2 days to obtain co-cultured explants; the co-cultured explants were then placed face up on the differentiation medium prepared in Example 2 and cultured under light at 25°C with a light duration of 16 h / d for 28 days, with the differentiation medium being changed every 7 days to obtain Populus tomentosa adventitious buds; to promote further growth and elongation of the Populus tomentosa adventitious buds, they were placed on the culture medium prepared in Example 3. On seedling culture medium, seedlings were cultured at 25℃ and 16 h / d for 14 days, with the medium being changed every 7 days, to obtain robust *Populus siberianus* buds with a bud length of 3 cm. These robust buds with differentiation growth points were cut and inserted into the rooting medium prepared in Example 4, and cultured at 25℃ and 16 h / d for 10 days to obtain *Populus siberianus* seedlings. These seedlings were then transplanted into the growth medium prepared in Example 5 and cultured at 25℃ and 16 h / d for 14 days to obtain *Populus siberianus* test-tube plantlets. Figures 1-4 As shown; (5) Identification of positive transgenic *Populus simonii* plants: Genomic DNA was extracted from leaves of *Populus simonii* test-tube seedlings using the CTAB method as a template. PCR detection was performed according to the primers, PCR system, and PCR procedure described in step (1) to obtain positive *Populus simonii* test-tube seedlings. Figure 5 As shown; (6) Transplanting and Cultivation of Positive Seedlings of *Populus simonii*: When the positive seedlings of *Populus simonii* have abundant roots and leaves, remove them from the growth medium, wash the roots with sterile water, and bury them in completely moistened sterile nutrient soil. Slightly compact the soil and cultivate them under light at a rate of 16 h / d for 3 weeks to allow them to adapt to the sterile nutrient soil environment. Then, transplant them to the outside environment for further growth. Figure 6 As shown.

[0035] Experimental Example 1: Effect of IBA Concentration in Rooting Medium on Rooting of Strong Buds of Populus euphratica Given the common technical difficulties in rooting Populus tomentosa plants and the long rooting time in existing technologies, rooting media with different IBA concentration gradients were set up to investigate the effect of IBA concentration on the rooting of Populus tomentosa shoots at different culture times. The results are shown in Table 2 and [Table data missing]. Figure 7 As shown.

[0036] Table 2. Rooting of vigorous shoots of *Populus tomentosa* under different IBA concentration treatments. The results showed that the optimal IBA concentration for rooting of robust poplar shoots was 0.3 mg / L. After culturing in the rooting medium with the optimal IBA concentration for 7 days, robust poplar shoots transferred to the growth medium could grow strong roots in just 3 days, greatly shortening the rooting time required for genetic transformation of poplar and making poplar more suitable as a transgenic material for poplar.

[0037] As can be seen from the above embodiments, the present invention provides a culture medium combination for the genetic transformation of Populus tomentosa and a method for the genetic transformation of Populus tomentosa, as well as its application. The genetic transformation method of Populus tomentosa of the present invention is simple, easy to implement, low in cost, and highly reliable. It can achieve rapid differentiation and rooting of transgenic Populus tomentosa plants, laying the foundation for the research and breeding of functional genes of Populus tomentosa, and providing technical support for the creation and cultivation of new transgenic, fluff-free Populus tomentosa germplasm. It has great scientific research value, economic value, and ecological value.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture medium composition for genetic transformation of Populus tomentosa, characterized in that, Including co-culture medium, differentiation medium, seedling strengthening medium, rooting medium, and growth medium; The co-culture medium is based on WPM medium and includes the following components at the following concentrations: 2-morpholine ethanesulfonic acid 400-600 mg / L, 2,4-dichlorophenoxyacetic acid 0.5-1 mg / L, acetylsyl syringone 50-200 μmol / L, sucrose 15-25 g / L, and agar 5-10 g / L. The differentiation medium is based on WPM medium and includes the following components at the following concentrations: 2-morpholine ethanesulfonic acid 400-600 mg / L, 2,4-dichlorophenoxyacetic acid 0.5-1 mg / L, kanamycin 5-8 mg / L, cefotaxime 200-400 mg / L, sucrose 15-25 g / L, and agar 5-10 g / L; The seedling culture medium is based on MS medium and includes the following components at the following concentrations: 6-benzylaminopurine 0.1-0.5 mg / L, thiabendazole 0.5-2 μg / L, kanamycin 5-8 mg / L, cefotaxime 200-400 mg / L, sucrose 25-35 g / L, and agar 5-10 g / L. The rooting medium is based on 1 / 2 MS medium and includes the following components at the following concentrations: indolebutyric acid 0.1-0.5 mg / L, cefotaxime 200-400 mg / L, sucrose 25-35 g / L, and agar 5-10 g / L. The growth medium is based on MS medium and includes the following components at the following concentrations: cefotaxime 400-600 mg / L, sucrose 25-35 g / L, and agar 5-10 g / L.

2. The application of the culture medium combination according to claim 1 in the cultivation of Populus tomentosa plants.

3. The application of the culture medium combination described in claim 1 in the genetic transformation of Populus tomentosa.

4. A method for genetic transformation of Populus tomentosa, characterized in that, Includes the following steps: (1) After inoculating the explants of Populus sibirica with Agrobacterium tumefaciens, place them face up on the co-culture medium and culture them in the dark for 1-3 days to obtain co-cultured explants. (2) Place the co-cultured explants face up on the differentiation medium and culture under light for 28-30 days to obtain adventitious buds of Populus tomentosa. (3) Place the adventitious buds of Populus sibirica on a seedling culture medium and culture for 14-16 days to obtain strong buds of Populus sibirica; (4) Insert the robust buds of Populus sichuanensis into the rooting medium and culture for 7-14 days to obtain Populus sichuanensis seedlings; (5) Transplant the seedlings of Populus sichuanensis into the growth medium and culture for 14-16 days to obtain Populus sichuanensis test-tube seedlings; The co-culture medium, differentiation medium, seedling strengthening medium, rooting medium, and growth medium mentioned herein are the co-culture medium, differentiation medium, seedling strengthening medium, rooting medium, and growth medium as described in claim 1.

5. The method according to claim 4, characterized in that, The OD of the Agrobacterium tumefaciens bacterial solution in step (1) 600 The value is 0.2 to 0.6; The inoculation time is 10–20 minutes; The temperature for the dark culture is 20–28°C.

6. The method according to claim 5, characterized in that, The temperature for light culture in step (2) is 20-28℃, and the light culture time is 12-18h / d.

7. The method according to claim 6, characterized in that, The culture temperature in step (3) is 20-28℃, and the light exposure time is 12-18h / d. The bud length of the *Phyllostachys edulis* robust bud is 2–4 cm.

8. The method according to claim 7, characterized in that, The culture temperature in step (4) is 20-28℃, and the light exposure time is 12-18h / d.

9. The method according to claim 8, characterized in that, The culture temperature in step (5) is 20-28℃, and the light exposure time is 12-18h / d.

10. The application of the method according to any one of claims 4 to 9 in the cultivation of Populus tomentosa plants.

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