Method for establishing high-efficiency regeneration system of poplar aseptic seedling leaf

Abstract

This invention discloses a method for establishing a high-efficiency leaf regeneration system for aseptic seedlings of Populus tomentosa 'Bofeng No. 3', belonging to the field of agricultural biotechnology. This invention, through 5-AU combined with dark culture treatment, not only induces early leaf differentiation in aseptic seedlings of Populus tomentosa 'Bofeng No. 3', but also significantly increases the number of adventitious buds and the rooting rate. Through chemical treatment and optimized culture conditions, this invention successfully established a high-efficiency plant regeneration system for aseptic leaves of Populus tomentosa 'Bofeng No. 3', laying the foundation for the genetic transformation and gene editing of Populus tomentosa 'Bofeng No. 3', and also providing a reference for the establishment of tissue culture systems for other tree species.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for establishing a high-efficiency leaf regeneration system for sterile poplar seedlings of Bofeng No. 3. Background Technology

[0002] With the continuous advancement of plant genetic transformation technology, genetic transformation techniques independent of tissue culture regeneration systems have been successfully developed for a few plants. However, for most plants, plant regeneration systems remain a crucial prerequisite for achieving genetic transformation. Improving regeneration efficiency can effectively enhance the efficiency of genetic transformation and gene editing. Wang et al. significantly improved the regeneration efficiency of wheat (Triticum aestivum L.) by overexpressing the TaWOX5 gene, increasing transformation efficiency by 2–10 times, successfully overcoming the genotype-dependent limitation in wheat genetic transformation. Zhao et al. significantly improved the regeneration efficiency of soybean (Glycine max(L.) Merr.) by constructing the GRF3-GIF1 synthase, achieving a transformation efficiency 4.67 times higher than traditional methods, and successfully applied it to the CRISPR / Cas9 gene editing system. Poplar is a model tree species for forest genetic engineering research, and various efficient regeneration systems for poplar have been successfully established. However, the regeneration efficiency varies considerably among different poplar varieties / clones, different growth stages of the same variety, and different tissues and organs. Bofeng 3hao (Poplar 'Bofeng3hao') is a national-level superior forest tree variety independently bred by the Institute of Forestry, Chinese Academy of Forestry. It has characteristics such as strong growth, wide adaptability, and resistance to cold and disease.

[0003] Pyrimidine bases are essential for the replication of genetic information, cell metabolism, and cell growth in all biological systems. 5-Aminouracil (5-AU), an antagonist of thymine, can synchronize cell cycles by inhibiting DNA synthesis, and is therefore often used to induce cell cycle synchronization. Studies have found that 5-AU can synchronize pea root tip meristem cells to the S phase; similarly, 5-AU can induce the formation of biphasic mitotic cells in onion root tip meristems, and its efficiency is closely related to increased cellular metabolic levels. These studies indicate that 5-AU can directly affect the division activity of meristem cells. During plant regeneration, meristem cells form new organs through division and differentiation, and their division and differentiation capacity directly affects the regeneration efficiency of organs. Therefore, 5-AU may have a role in promoting plant meristem regeneration. However, although the role of 5-AU in cell cycle regulation has been extensively studied, its direct effect on active cell division and differentiation in meristems that form adventitious shoots has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing a high-efficiency leaf regeneration system for aseptic seedlings of Populus tomentosa 'Bofeng No. 3', in order to solve the problems existing in the prior art. This invention uses Populus tomentosa 'Bofeng No. 3' as the experimental material, and sets up different concentration gradients of 5-AU treatment groups (0.4–3.2 mmol·L⁻¹). -1 By combining different dark culture times (6h to 6d), the number of adventitious buds regenerated from leaves of sterile seedlings was significantly increased, and a high-efficiency sterile seedling leaf regeneration system for Populus tomentosa No. 3 was established. This lays the foundation for subsequent molecular breeding technologies such as genetic transformation and gene editing of this variety, and also provides a reference for the establishment of high-efficiency regeneration systems for other tree species.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for establishing a high-efficiency leaf regeneration system for aseptic poplar seedlings of the Bofeng No. 3 variety, comprising the following steps:

[0007] Using tender shoots of Populus davidii var. Bofeng 3 as explants, the plants were inoculated into a subculture medium and cultured to produce axillary buds. The axillary buds were then propagated in a subculture medium to obtain sterile seedlings of Populus davidii var. Bofeng 3. The subculture medium contained the following components: WPM + IBA + sucrose + agar powder.

[0008] After making incisions, mature leaves of the sterile Populus tomentosa seedling No. 3 were inoculated into differentiation medium I and cultured in the dark. The differentiation medium I consisted of the following components: MS + 6-BA + NAA + TDZ + sucrose + agar powder + 5-AU.

[0009] After dark culture, the leaf surface was cleaned and the leaves were transferred to differentiation medium II for normal light culture to obtain leaf regeneration adventitious buds; the differentiation medium II includes the following components: MS + 6-BA + NAA + TDZ + sucrose + agar powder.

[0010] The adventitious buds regenerated from the leaves are cut open and transferred to an adventitious bud rooting medium to root; the adventitious bud rooting medium includes the following components: WPM + IBA + sucrose + agar powder.

[0011] Preferably, the subculture medium comprises the following components at the following concentrations: WPM + IBA 0.02 mg·L⁻¹ -1 +25g / L sucrose -1 +5g / L agar powder -1 .

[0012] Preferably, the subculture conditions include: 16h light / 8h darkness, light intensity 2000 Lux; temperature 25℃; relative humidity 60%–70%; and time 20 days.

[0013] Preferably, the dark culture time is 12h-6d.

[0014] Preferably, the differentiation medium I comprises the following components at the following concentrations: MS + 6-BA 0.5 mg·L⁻¹ -1 +NAA 0.05 mg·L -1 +TDZ 0.002mg·L -1 + 30g / L of sucrose -1 +5g / L agar powder -1 +5-AU 0.4~3.2mmol·L -1 .

[0015] Preferably, the differentiation medium II comprises the following components at the following concentrations: MS + 6-BA 0.5 mg·L⁻¹ -1 +NAA 0.05 mg·L -1 +TDZ 0.002mg·L -1 + 30g / L of sucrose -1 +5g / L agar powder -1 .

[0016] Preferably, the adventitious shoot rooting medium comprises the following components at the following concentrations: WPM + IBA 0.02 mg·L⁻¹ -1 +25g / L sucrose -1 +5g / L agar powder -1 .

[0017] Preferably, the conditions for normal light culture include: 16 hours of light / 8 hours of darkness, light intensity of 2000 Lux; temperature of 25°C; and relative humidity of 60%–70%.

[0018] Preferably, the concentration of 5-AU in the differentiation medium I is 3.2 mmol·L⁻¹. -1 The dark culture time is 2 days.

[0019] Preferably, mature leaves from the 2nd to 4th leaf position of the stem tip of the sterile poplar seedling of Bofeng No. 3 are taken, and 3 to 4 incisions are made perpendicular to the main vein. The leaves are then laid flat in the differentiation culture medium I with the upper surface facing down.

[0020] The present invention discloses the following technical effects:

[0021] This invention establishes a highly efficient plant regeneration system for aseptic seedlings of Populus xeuramericana 'Bofeng 3hao' by using 5-aminouracil (5-AU) and dark culture treatment, laying the foundation for improving the efficiency of genetic transformation and gene editing in poplar trees.

[0022] This invention uses sterile seedlings of Populus tomentosa 'Bofeng No. 3' as material, and adds 0.4, 0.8, 1.6, and 3.2 mmol·L⁻¹ to the leaf differentiation medium.-1 Four concentrations of 5-AU were used for dark incubation for 6 h–6 days. Leaves were then transferred to differentiation medium without 5-AU for further light incubation. Leaf differentiation rate, number of regenerated buds, and rooting rate were recorded. The effects of different concentrations of 5-AU and different dark incubation durations on key indicators of the poplar regeneration system were compared. Membership function comprehensive analysis was used to screen the optimal regeneration system for aseptic poplar seedlings of Bofeng No. 3. Compared with the control, 0.4–3.2 mmol·L⁻¹… -1 5-AU combined with dark incubation for 12 h–3 d can advance adventitious bud regeneration by 2–3 d, and dark incubation for 4–6 d can advance adventitious bud regeneration by 4–5 d. Two-way ANOVA results showed that 5-AU concentration and dark incubation duration significantly affected leaf differentiation rate, number of regenerated buds, and rooting rate of adventitious buds; the interaction between 5-AU concentration and dark incubation was highly significant or significantly increased the number of regenerated buds and rooting rate. Membership function analysis showed that 3.2 mmol·L⁻¹… -1 The best leaf regeneration effect was achieved when 5-AU concentration was combined with dark culture for 2 days. The leaf differentiation rate reached 100%, the number of regenerated adventitious buds reached 36 (per leaf), and the rooting rate reached 96.65%.

[0023] The combination of 5-AU and dark culture treatment not only induced early leaf differentiation in aseptic poplar seedlings of Bofeng No. 3, but also significantly increased the number of adventitious buds and the rooting rate. This invention, through chemical treatment and optimized culture conditions, successfully established a highly efficient plant regeneration system for aseptic leaves of Bofeng No. 3 poplar, laying the foundation for genetic transformation and gene editing of this variety, and also providing a reference for the establishment of tissue culture systems for other tree species. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The cut morphology of leaves of Populus tomentosa var. bofengi No. 3 (control) and T4 treated with dark treatment for 3d and 6d is shown after 15d of culture; A is the bud point of the control after 3d of dark culture; B is the bud point of the T4 treated after 3d of dark culture; C is the bud point of the control after 6d of dark culture; D is the bud point of the T4 treated after 6d of dark culture; Scale bar = 500μm.

[0026] Figure 2The images show the regeneration of adventitious buds in leaves of Populus tomentosa 'Bofeng 3' (control) and T4 treatment after 20 and 30 days of dark treatment (3 days, 6 days); A shows the regeneration of adventitious buds in the control and T4 treatment after 3 days of dark treatment; B shows the regeneration of adventitious buds in the control and T4 treatment after 6 days of dark treatment; Scale bar = 1 cm.

[0027] Figure 3 The effect of different treatments on leaf differentiation of aseptic poplar seedlings of Bofeng No. 3; different lowercase letters indicate significant differences (P<0.05), and ns indicate no significant differences;

[0028] Figure 4 The effect of different treatments on the number of regenerated buds on leaves of sterile Populus tomentosa seedlings of Bofeng No. 3; different lowercase letters indicate significant differences (P<0.05), and ns indicate no significant differences;

[0029] Figure 5 The effect of different treatments on the regeneration of adventitious buds and rooting of leaves in sterile Populus tomentosa seedlings of Bofeng No. 3; different lowercase letters indicate significant differences (P<0.05), and ns indicate no significant differences. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1

[0036] 1. Materials and Methods

[0037] 1.1 Plant materials

[0038] Bofeng 3hao is a national-level superior variety independently bred by our research group and was propagated by cuttings in the greenhouse of the Chinese Academy of Forestry.

[0039] 1.2 Test Methods

[0040] 1.2.1 Culture medium preparation

[0041] The formulation of the differentiation medium I for Populus tomentosa leaf of Bofeng No. 3 was MS + 6-BA 0.5 mg·L⁻¹. -1 +NAA 0.05mg·L -1 +TDZ 0.002 mg·L -1 + 30g / L of sucrose -1 +5g / L agar powder -1 +5-AU (0.4~3.2mmol·L) -1 The formula for the differentiation medium II for Bofeng No. 3 poplar leaves is MS + 6-BA 0.5 mg·L⁻¹. -1 +NAA 0.05mg·L -1 +TDZ 0.002mg·L -1 + 30g / L of sucrose -1 +5g / L agar powder -1 The culture medium for adventitious shoot rooting and subculture was formulated as follows: WPM + IBA 0.02 mg·L⁻¹ -1 +25g / L sucrose -1 +5g / L agar powder -1 The culture medium has a pH of 5.8 and is used after autoclaving at 121°C for 20 minutes.

[0042] 1.2.2 Obtaining sterile vaccines

[0043] Take robust, semi-lignified young branches of Populus tomentosa 'Bofeng No. 3' with plump axillary buds grown in a greenhouse. After rinsing with clean water, disinfect with 75% alcohol for 30 seconds and sodium hypochlorite (NaClO) for 10 minutes. After rinsing three times with sterile water, inoculate them into subculture medium. Culture conditions are as follows: 16h light / 8h dark, light intensity 2000 Lux, temperature 25℃, and relative humidity 60%–70%.

[0044] 1.2.3 Experimental Design

[0045] The experiment used differentiation medium without 5-AU as a control (CK, 0.0 mmol·L⁻¹). -1 ), respectively set with T1 (0.4 mmol·L -1 T2 (0.8 mmol·L) -1 T3 (1.6 mmol·L) -1 ) and T4 (3.2 mmol·L -1 Four 5-AU treatment levels of differentiation media were used, and the dark incubation time was set to 6h, 12h, 1d, 2d, 3d, 4d, 5d and 6d, for a total of 40 treatment groups, as shown in Table 1.

[0046] Sterile Populus 'Bofeng No. 3' seedlings, 4±1 cm in height and 17–23 days after subculture, were selected. Mature leaves from the 2nd to 4th leaf position (pointing downwards from the stem tip) were taken. Three to four incisions were made perpendicular to the main vein using a sterile scalpel. The leaves were placed face down and evenly spread in the culture medium of each treatment group. After 6 hours to 6 days of dark incubation, any remaining 5-AU on the leaf surface was washed away with sterile water. After drying the surface, the leaves were transferred to a differentiation medium without 5-AU for normal light incubation. The culture conditions were 16 hours of light / 8 hours of darkness, light intensity 2000 Lux, temperature 25℃, and relative humidity 60%–70%. Each treatment group had four replicates, with 5–9 sterile leaves per replicate, for a total of 28 leaves.

[0047] Table 1 Experimental Design

[0048]

[0049] 1.2.4 Statistical Methods

[0050] After light incubation, leaf differentiation was observed in each treatment, and the results were recorded. After 30 days of leaf culture, the differentiation rate and the number of regenerated adventitious buds in each experimental group were calculated. The regenerated adventitious buds were cut and transferred to rooting medium, and the rooting rate of the regenerated buds was calculated after 25 days. The calculation formula is as follows:

[0051] D = (n / N) × 100%, where D is the leaf differentiation rate, n is the number of leaves that regenerate adventitious buds, and N is the total number of inoculated leaves.

[0052] B = (m / M) × 100%, where B is the number of regenerated adventitious buds, m is the total number of regenerated adventitious buds, and M is the total number of differentiated leaves.

[0053] R = (w / W) × 100%, where R is the rooting rate of adventitious buds, w is the number of rooted adventitious buds, and W is the total number of adventitious buds.

[0054] 1.3 Data Statistics and Analysis

[0055] Statistical data were collected using Excel 2021. One-way ANOVA was performed using SPSS 26.0, and multiple comparisons were conducted using Duncan's multiple range test (DMRT) (P < 0.05 was considered statistically significant). Results are expressed as mean ± standard deviation. Two-way ANOVA was used to examine whether different concentrations of 5-AU, different dark culture conditions, and their interactions had significant effects on leaf differentiation rate, number of regenerated adventitious buds, and rooting rate of aseptic Populus tomentosa seedlings of Bofeng No. 3. Graphs were generated using Origin 2021 software.

[0056] The membership function method is used for comprehensive evaluation, and the membership function value is calculated according to the following formula:

[0057]

[0058] C=∑μ i / i.

[0059] In the formula: i represents the i-th evaluation index; The calculated value indicating a positive correlation between the evaluation of the regeneration of Populus tomentosa 'Bofeng No. 3' leaves and X; i This represents the measured value of the i-index for the regeneration of Populus tomentosa 'Bofeng No. 3' leaves under a certain treatment; X max and X min These represent the maximum and minimum values ​​of the i-th indicator, respectively. The calculated value indicates a negative correlation between the evaluation of the regeneration of Populus tomentosa 'Bofeng No. 3' leaves and the value of C represents the comprehensive evaluation value of the various indicators of the regeneration of Populus tomentosa 'Bofeng No. 3' leaves.

[0060] 2. Results and Analysis

[0061] 2.15-AU Effect on the Time of Adventitious Bud Differentiation from Aseptic Leaves of Populus tomentosa No. 3

[0062] After disinfection, the tender branches of Populus tomentosa No. 3 were inoculated into subculture medium. After 20 days, when the axillary buds grew to about 3 cm, they were cut off from the root for propagation. Mature leaves from the 2nd to 4th leaf positions downward from the stem tip were used as materials for adventitious bud regeneration experiments.

[0063] During the regeneration of adventitious buds in leaves, the process can be divided into three stages by observing the tissue morphology at the cut site: the red bud formation stage, the clustered adventitious bud formation stage, and the clustered adventitious bud growth stage. Observations showed that after 6 hours to 6 days of dark culture in the control group, continued culture under light for approximately 15 days (dark culture + light culture) resulted in the appearance of red buds. Continued culture under light for approximately 20 days led to the formation of adventitious buds, with the clustered adventitious bud growth stage occurring around 30 days. T1 (0.4 mmol·L⁻¹) -1 T2 (0.8 mmol·L) -1 T3 (1.6 mmol·L) -1 ) and T4 (3.2 mmol·L -1 Treatment with 5-AU for 6 hours resulted in regeneration processes occurring at approximately the same time points as the control group. When the treatment time for the four 5-AU concentrations (T1, T2, T3, and T4) was increased to over 12 hours, the regeneration time points for adventitious buds were significantly advanced: After 12 hours to 3 days of dark incubation at each concentration, followed by 12 to 13 days of light incubation (dark incubation + light incubation), red buds appeared; adventitious buds began to form on days 17 to 18; and the clustered adventitious bud growth period was reached on days 27 to 28. Similarly, after 4 to 6 days of dark incubation at each concentration, red buds appeared after 10 to 11 days of light incubation (dark incubation + light incubation); adventitious buds began to form on days 15 to 16; and the clustered adventitious bud growth period was reached on days 25 to 26. Therefore, 0.4–3.2 mmol·L⁻¹ -1 5-AU treatment combined with dark culture for 12-6 days significantly increased the leaf differentiation time of aseptic poplar seedlings of Bofeng No. 3. Further observation of the tissue morphology at the cut surfaces of leaves treated with T4 combined with dark culture for 3 and 6 days, and those cultured under light for 15 days, using a stereomicroscope revealed that numerous red buds had formed on the callus at the leaf cut surfaces, while the control group remained in a callus differentiation state. Figure 1 As shown in Figures A through D; after 20 days of cultivation, a few leaves in the control group began to form adventitious buds, while the leaves of the T4 treatment combined with dark cultivation for 3 and 6 days had formed a large number of clustered adventitious buds, as shown in Figures A through D. Figure 2 As shown in Figures A through B.

[0064] In summary, compared with the control, 0.4–3.2 mmol·L -1 Treatment with 5-AU for 12 hours to 6 days can advance the leaf differentiation time of sterile Populus tomentosa seedlings of Bofeng No. 3. Under the same 5-AU concentration treatment conditions, the longer the treatment time, the earlier the leaf differentiation and cluster bud formation time.

[0065] 2.2 Effects of different treatments on leaf differentiation rate of sterile poplar seedlings of Bofeng No. 3

[0066] Statistical analysis results showed that the dark incubation time affected the leaf differentiation rate of sterile Populus tomentosa seedlings of Bofeng No. 3. The control group had the highest differentiation rate (100%) after 2 days of dark incubation. Subsequently, the leaf differentiation rate gradually decreased with prolonged dark incubation, dropping to 80.78% after 6 days. At 6 and 12 hours of dark incubation, there was no significant difference in leaf differentiation rate between different concentrations of 5-AU treatments and the control group. At 1 day of dark incubation, the T2 treatment group was significantly higher than the control group. At 2 days of dark incubation, the leaf differentiation rate of both the control group and the 5-AU treatment group was 100%. From 3 to 6 days of dark incubation, the differentiation rate of the T4 treatment group remained stable at 100%, significantly higher than the control group. Furthermore, at 6 days of dark incubation, the differentiation rates of all four 5-AU concentrations were significantly higher than the control. Figure 3 As shown. In summary, 2 days of dark culture resulted in the best leaf differentiation rate for sterile Populus tomentosa seedlings of Bofeng No. 3. When the dark culture time was extended to 3–6 days, the leaf differentiation rate of untreated seedlings gradually decreased, except for those treated with 0.4 mmol·L⁻¹. -1 5-AU(T1) treatment, 0.8–3.2 mmol·L -1 The high differentiation rate of 5-AU (T1-T4) indicates that when the dark culture time is long, a high concentration of 5-AU can improve the leaf differentiation rate and keep it at a high level.

[0067] Analysis of variance showed that the leaf differentiation rate of sterile Populus davidii seedlings of Bofeng No. 3 differed significantly between the 5-AU treatment and the dark culture time (P<0.001), as shown in Table 2. This indicates that different 5-AU concentrations and dark culture times have a significant impact on the leaf differentiation of sterile Populus davidii seedlings of Bofeng No. 3.

[0068] Table 2. Analysis of variance on leaf differentiation of sterile Populus tomentosa seedlings of Bofeng No. 3 under different treatments.

[0069]

[0070] Note: *** This indicates a significant difference at the 0.001 level. ** * indicates a significant difference at the 0.01 level, and * indicates a significant difference at the 0.05 level.

[0071] 2.3 Effects of different treatments on the number of adventitious buds regenerated from leaves of sterile Populus tomentosa seedlings of Bofeng No. 3

[0072] When the leaves reached the stage of adventitious bud formation, the number of adventitious buds in each treatment group was counted, and the results are as follows: Figure 4As shown in the figure, after 6 hours of dark incubation, there was no significant difference in the number of adventitious buds regenerated between each treatment and the control. After 12 hours of dark incubation, T1, T3, and T4 were significantly higher than the control. After 1 day of dark incubation, only T4 was significantly higher than the control. After 2 days of dark incubation, T2, T3, and T4 were significantly higher than the control. After 3 and 5 days of dark incubation, T2 and T4 were significantly higher than the control group. After 4 days of dark incubation, T1, T3, and T4 were significantly higher than the control group. After 6 days of dark incubation, T1 and T4 were significantly higher than the control group. Therefore, 5-AU treatment can increase the number of adventitious buds regenerated from leaves of sterile Populus tomentosa seedlings of Bofeng No. 3 to varying degrees, especially the high concentration of 5-AU treatment (T4) combined with dark incubation for 12 hours to 6 days significantly increased the number of adventitious buds regenerated.

[0073] Analysis of variance showed that 5-AU concentration, dark culture time, and the interaction between the two treatments all had extremely significant effects on the number of regenerated adventitious shoots (P<0.001), as shown in Table 3. This indicates that both 5-AU and dark culture can promote the regeneration of adventitious shoots, and the high concentration of 5-AU treatment has a more significant effect.

[0074] Table 3. Analysis of variance on the number of regenerated buds on leaves of sterile Populus tomentosa seedlings of Bofeng No. 3 under different treatments.

[0075]

[0076] Note: *** This indicates a significant difference at the 0.001 level. ** * indicates a significant difference at the 0.01 level, and * indicates a significant difference at the 0.05 level.

[0077] 2.4 Effects of different treatments on leaf regeneration and adventitious bud rooting in sterile poplar seedlings of Bofeng No. 3

[0078] When the adventitious buds were about 0.5 cm long, they were cut off and transferred to rooting medium. After 25 days, the number of roots formed from the adventitious buds was counted, and the results were as follows: Figure 5 As shown in the figure, after 6 hours of dark incubation, all treatments were significantly higher than the control; after 12 hours of dark incubation, treatments T2, T3, and T4 were significantly higher than the control; after 1 and 5 days of dark incubation, there were no significant differences between the 5-AU treatment and the control; after 2 days of dark incubation, treatment T4 was significantly higher than the control; and after 3, 4, and 6 days of dark incubation, T3 and T4 were significantly higher than the control group. Therefore, dark incubation combined with 5-AU treatment can improve the rooting rate of adventitious shoots to varying degrees.

[0079] Analysis of variance showed that 5-AU concentration and dark culture time had extremely significant effects on adventitious shoot rooting (P<0.001), and the interaction between the two was significantly different (P<0.05) (Table 4).

[0080] Table 4. Two-way ANOVA analysis of the rooting rate of leaves from sterile Populus tomentosa seedlings of Bofeng No. 3 under different treatments.

[0081]

[0082] Note: *** This indicates a significant difference at the 0.001 level. ** * indicates a significant difference at the 0.01 level, and * indicates a significant difference at the 0.05 level.

[0083] 2.5 Screening and Optimization of High-Efficiency Treatment Combinations for Leaf Regeneration of Bofeng No. 3 Poplar Aseptic Seedlings

[0084] The effects of 5-AU concentration and dark culture duration on leaf differentiation rate, number of regenerated adventitious buds, and rooting rate of adventitious buds were comprehensively evaluated using the membership function method. The comprehensive evaluation values ​​(C values) for different treatments were obtained. A larger C value indicates a stronger promoting effect of the combination on leaf and plant regeneration in tissue-cultured Populus tomentosa 'Bofeng No. 3' seedlings. As shown in Table 5, the membership function C values ​​for different 5-AU concentrations (CK, T1, T2, T3, and T4) were 0.360, 0.540, 0.545, 0.634, and 0.732, respectively, ranked from strongest to weakest as T4>T3>T2>T1>CK. The membership function C values ​​for different dark incubation times (6h, 12h, 1d, 2d, 3d, 4d, 5d, and 6d) were 0.480 (0.4802), 0.584, 0.678, 0.754, 0.480 (0.4804), 0.493, 0.518, and 0.510, respectively, ranked from strongest to weakest as follows: 2d > 1d > 12h > 5d > 6d > 4d > 3d > 6h. The top three interaction C values ​​were for 2d dark incubation under T4 treatment, 1d dark incubation under T4 treatment, and 12h dark incubation under T3 treatment, with C values ​​of 0.947, 0.813, and 0.790, respectively. Therefore, it is considered that the T4 concentration treatment combined with 2d dark incubation had the greatest impact on leaf regeneration of aseptic Populus davidii seedlings of Bofeng 3, and is the optimal treatment combination for leaf regeneration of Bofeng davidii seedlings.

[0085] Table 5 Membership function analysis of different treatments on efficient leaf regeneration in sterile Populus tomentosa seedlings of Bofeng No. 3

[0086]

[0087] As can be seen from the above embodiments, this invention successfully established a high-efficiency plant regeneration system for sterile seedling leaves of Populus tomentosa 'Bofeng No. 3' using the national-level superior variety as material. This system employed methods such as material treatment (sterile seedling leaves), 5-AU concentration screening, and dark culture duration control. Compared to Zhang Tengqian's Populus tomentosa 'Bofeng No. 3' regeneration system established using cutting leaves as explants (average number of regenerated adventitious buds per leaf of 19), this system significantly promoted the formation of high-frequency regenerated adventitious buds (36 per leaf). Furthermore, by using sterile seedling leaves of Populus tomentosa 'Bofeng No. 3' as material, this invention reduced the explant disinfection process, effectively solving problems such as seasonal limitations of explants and leaf disinfection damage, thus laying the foundation for the genetic transformation and molecular breeding of this variety.

[0088] This invention promotes early leaf differentiation in sterile poplar seedlings of the Bofeng No. 3 variety by adding different concentrations of 5-AU to the differentiation medium. Analysis of variance showed that the concentration of 5-AU had a highly significant effect on the leaf differentiation rate, number of regenerated buds, and rooting rate (P<0.001). Specifically, 3.2 mmol·L⁻¹ -1 The 5-AU treatment had the highest membership function value, indicating that high concentrations of 5-AU significantly promoted poplar leaf differentiation, adventitious bud regeneration, and rooting.

[0089] The reasons why 5-AU treatment promotes the regeneration of adventitious buds on poplar leaves may be twofold: First, during continuous treatment, 5-AU can induce some cells in the meristematic tissue to synchronize to the DNA replication phase of the cell cycle, leading to a shortening of the G2 and G1 phases. Second, 5-AU treatment induces oxidative stress, thereby activating the DNA damage response pathway. In summary, 5-AU treatment may directly or indirectly promote the rapid passage of cells through the G1 phase via GSH, further shortening the cell division time during adventitious bud regeneration, thus causing the 5-AU-treated group to differentiate into regenerated adventitious buds earlier than the control group.

[0090] Dark culture has a significant impact on the induction of callus formation and adventitious bud regeneration in aseptic poplar seedlings. The results of the embodiments of this invention show that there are highly significant differences (P<0.001) in leaf differentiation rate, number of regenerated buds, and rooting rate of Populus tomentosa 'Bofeng 3' under different dark culture durations. Specifically, the C value is higher for plants cultured under light after 12 hours to 2 days of dark culture, indicating that short-term dark culture treatment is beneficial for plant regeneration. Therefore, dark culture can promote leaf differentiation and regeneration in poplar, and the duration of dark culture varies among different varieties / clones. Light exposure may induce the accumulation of reactive oxygen species (ROS), leading to oxidative damage to explants. Dark culture provides energy and a stable environment for cell proliferation and differentiation by reducing photo-oxidative stress and increasing the content of soluble proteins and sugars. Furthermore, dark culture promotes plant regeneration and callus totipotency by regulating hormone levels and the expression of regeneration-related genes. Therefore, the reason why dark culture promotes poplar leaf regeneration may be by optimizing the physiological metabolic environment, regulating hormone signaling pathways, and activating pluripotency-related genes, thereby providing favorable conditions for callus dedifferentiation and adventitious bud regeneration.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for establishing a high-efficiency leaf regeneration system for aseptic poplar seedlings of Bofeng No. 3, characterized in that, Includes the following steps: Using tender shoots of Populus tomentosa 'Bofeng No. 3' as explants, the plants were inoculated into a subculture medium and cultured to produce axillary buds. These axillary buds were then propagated in a subculture medium to obtain sterile Populus tomentosa 'Bofeng No. 3' seedlings. The subculture medium consisted of WPM + IBA 0.02 mg·L⁻¹. -1 +25 g·L sucrose -1 +5 g·L agar powder -1 ; Mature leaves of the sterile poplar seedlings of Bofeng No. 3 were cut and inoculated into differentiation medium I for dark culture; the differentiation medium I was: MS + 6-BA 0.5 mg·L⁻¹ -1 +NAA 0.05 mg·L -1 +TDZ 0.002 mg·L -1 + 30 g·L sucrose -1 +5 g·L agar powder -1 +5-AU 3.2mmol·L -1 ; The dark culture time was 2 days; After dark culture, the leaf surface was cleaned, and the leaves were transferred to differentiation medium II for normal light culture to obtain adventitious buds regenerated from the leaves; the differentiation medium II consisted of MS + 6-BA 0.5 mg·L⁻¹. -1 +NAA 0.05 mg·L -1 +TDZ 0.002mg·L -1 + 30 g·L sucrose -1 +5 g·L agar powder -1 ; The regenerated adventitious buds from the leaves were cut and transferred to an adventitious bud rooting medium for rooting; the adventitious bud rooting medium was: WPM + IBA 0.02 mg·L⁻¹. -1 +25g / L sucrose -1 +5g / L agar powder -1 .

2. The method for establishing as described in claim 1, characterized in that, The conditions for the subculture included: 16 h light / 8 h dark, light intensity 2000 Lux; temperature 25℃; relative humidity 60%–70%; and duration 20 days.

3. The method for establishing as described in claim 1, characterized in that, The conditions for normal light culture include: 16 h light / 8 h darkness, light intensity of 2000 Lux; temperature of 25℃; and relative humidity of 60%–70%.

4. The method for establishing as described in claim 1, characterized in that, Take mature leaves from the 2nd to 4th leaf position of the stem tip of the sterile poplar seedling of Bofeng No. 3, make 3 to 4 cuts perpendicular to the main vein, place the leaves face down in the differentiation culture medium I.

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