A high-efficiency method for propagating a micro-branch of a small black poplar without axillary buds based on layer formation activation

Abstract

The present application relates to a kind of based on layer activation's axillary budless small black poplar micro branch efficient bud multiplication method, belong to the technical field of forest asexual reproduction.To solve the problems of low yield of existing small black poplar bud multiplication dependent on axillary bud, the present application provides a kind of based on layer activation's axillary budless small black poplar micro branch efficient bud multiplication method, select axillary budless micro branch, after aseptic disinfection treatment, cut in the humidity 80~120% of permeable moisture substrate containing nutrient solution, air humidity maintains 95±5%, permeable moisture substrate and air humidity synergistic nutrient solution activates layer cell division, cut off adventitious bud and carry out conventional rooting, transplant cultivation and obtain small black poplar bud.The present application breaks through the traditional propagation logic dependent on axillary bud, by the directional activation of layer cell activity of axillary budless small black poplar micro branch, combined with humidity, substrate and nutrient solution synergistic regulation, realizes the efficient bud multiplication of axillary budless micro branch direct induction sprouting, greatly improves the yield of small black poplar bud.

Description

Technical Field

[0001] This invention belongs to the field of forest tree seedling cultivation technology, and particularly relates to the field of forest tree asexual reproduction technology, specifically to a method for efficient seed propagation of axillary bud-less poplar micro-branch based on cambium activation. Background Technology

[0002] Populus simonii × P. nigra, belonging to the genus Populus in the family Salicaceae, is a species developed through artificial hybridization of Populus simonii and Populus nigra. This species thrives in cool, humid climates, grows rapidly, and is highly adaptable. It possesses stable traits, is cold-resistant, drought-tolerant, and resistant to diseases and pests. It can be used for industrial timber, carbon sequestration afforestation, environmental protection, and urban greening, making it an extremely important ecological resource and bioenergy source with high ecological, economic, and social value.

[0003] However, the aging, degeneration, and mixed varieties of Populus tomentosa are frequent problems, mainly manifested in decreased tree growth, yellowing and shedding of leaves, severe pests and diseases, severe sunscald on seedlings, wind damage, low survival rate of afforestation, weakened winter hardiness and drought resistance, and reduced protective effectiveness. Maintaining the superior qualities of Populus tomentosa has become an urgent and important issue that needs to be addressed in forestry production and environmental construction.

[0004] Tree propagation via cuttings utilizes the vegetative organs of the tree, offering advantages such as maintaining the superior traits of the parent plant and rapid propagation. Poplar saplings are typically propagated through cuttings, taken once a year. This method is convenient when propagation material is plentiful, but if material is scarce, only one cutting is taken per year, retaining only one branch, which significantly limits the scale and speed of Poplar sapling propagation.

[0005] Furthermore, conventional cutting propagation of Populus tomentosa requires cuttings 12-15 cm long with 2-3 plump axillary buds. The cuttings should be made with a flat upper cut and a horseshoe-shaped lower cut. The upper cut should be 2 cm from the terminal bud to prevent it from drying out. This method requires more propagation material, leading to significant material waste in practice. Moreover, traditional cutting methods rely on axillary buds to initiate growth; if these buds are damaged or poorly developed, it will affect the effective absorption of nutrients and water by the cuttings, thus impacting their overall growth. To date, there are no reports, domestically or internationally, of achieving efficient cutting propagation for any tree species through axillary bud-less micro-branching. How to achieve efficient cutting propagation of Populus tomentosa remains a pressing issue in the field of asexual reproduction of forest trees. Summary of the Invention

[0006] To address the problem that existing methods for propagating Populus tomentosa rely too heavily on axillary buds and result in low seed yield, this invention provides a highly efficient method for propagating Populus tomentosa micro-branches without axillary buds based on cambium activation.

[0007] The technical solution of this invention:

[0008] A highly efficient method for propagating axillary bud-less Populus tomentosa micro-branches based on cambium activation involves selecting axillary bud-less Populus tomentosa micro-branches, sterilizing them, and then inserting them into a breathable and moisturizing substrate containing nutrient solution with a humidity of 80-120%. The air humidity is maintained at 95±5%. The breathable and moisturizing substrate and the air humidity, together with the nutrient solution, activate cambium cell division, resulting in the formation of adventitious buds. These adventitious buds are then cut and subjected to conventional rooting and transplanting to obtain Populus tomentosa seed cuttings.

[0009] Furthermore, the axillary bud-less poplar micro-twigs are obtained by cutting 3-4 cm sections from the semi-lignified scions that sprouted from superior single plants over 40 years old.

[0010] Furthermore, the aseptic disinfection process involves soaking the small black poplar branches without axillary buds in 75% ethanol for 30-60 seconds, rinsing with sterile water, and then sterilizing with 0.1% HgCl2 for 2-10 minutes.

[0011] Furthermore, the breathable and moisture-retaining matrix is ​​a mixture of peat moss and perlite, with a volume ratio of peat moss to perlite of 2:1.

[0012] Furthermore, the humidity of the breathable and moisturizing matrix is ​​80%.

[0013] Furthermore, the nutrient solution is a 1 / 2MS macronutrient solution.

[0014] Furthermore, the culture temperature is 25~28℃, and the light exposure time is 16 hours of light / 8 hours of darkness.

[0015] Furthermore, the time for the formation of adventitious buds is 35 to 45 days.

[0016] The beneficial effects of this invention are:

[0017] This invention breaks through the traditional propagation logic that relies on axillary buds. By directionally activating the cambium cell activity of axillary bud-free Populus tomentosa micro-branches, combined with the synergistic regulation of humidity, substrate, and nutrient solution, it achieves highly efficient seed propagation by directly inducing suckers from axillary bud-free micro-branches, significantly improving the yield and quality of Populus tomentosa seed ears. The seed ear propagation method of this invention achieves a 100% adventitious bud occurrence rate in the cambium, with an average of 4.9 ± 0.88 buds per branch. The seed ear yield is 4.8 times higher than that of the traditional cutting method that relies on axillary buds, and the propagation cycle is shortened from 6-8 months in the traditional cutting method to 3 months.

[0018] The present invention, based on cambium activation, is a highly efficient method for propagating axillary bud-free Populus tomentosa micro-branchs. Its application to the asexual propagation of superior Populus tomentosa germplasm resources has a positive effect on promoting the establishment of Populus tomentosa scion orchards, increasing scion yield and reproductive capacity. It not only helps to protect superior Populus tomentosa germplasm resources and effectively promotes the propagation and dissemination of superior Populus tomentosa germplasm resources, but also provides high-quality seedling resources for forestry production and promotes the sustainable development of forestry. Attached Figure Description

[0019] Figure 1 This is a photograph of the adventitious buds of Populus tomentosa cambium one week after cutting inoculation in Example 3.

[0020] Figure 2 This is a photograph of the adventitious bud growth state of the cambium adventitious buds after 30 days of growth in Example 3.

[0021] Figure 3 This is a photograph of the container seedlings obtained after 30 days of cultivation following the cutting, rooting, and transplanting of adventitious buds from the cambium layer in Example 3.

[0022] Figure 4 This is a photograph of the adventitious bud growth state of the cambium adventitious buds after 38 days of growth in Example 2.

[0023] Figure 5 This is a photograph of the adventitious bud growth status of Comparative Example 4 after 30 days of culture in a nutrient-free substrate. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0025] Example 1

[0026] This embodiment provides a highly efficient method for propagating seedlings of Populus tomentosa microtwigs without axillary buds based on cambium activation.

[0027] The source of the axillary bud-less Populus tomentosa micro-branches used in this embodiment is as follows: In 2022, superior clonal Populus tomentosa clones of 40-year-old mature trees were subjected to trunk and root burial treatments at the National Poplar and Pinus sylvestris Seed Base in Fularji District, Qiqihar City, Heilongjiang Province (123°56'35.378"E, 47°20'52.151"N). In March 2023, the sprouts obtained from the trunk and root burial treatments, as well as branches from superior trees collected from large trees, were screened and cut into cuttings 10-12 cm long and 1.5-2 cm in diameter. These cuttings were then propagated under a film covering in a greenhouse at Qiqihar University. A certain number of cuttings were obtained after 6 months of propagation.

[0028] Select the axillary bud-free part of the semi-lignified scion, cut 3-4 cm to obtain axillary bud-free small black poplar micro-branches, soak the axillary bud-free small black poplar micro-branches in 75% ethanol for 30 seconds, rinse 3 times with sterile water, and then sterilize with 0.1% HgCl2 for 5 minutes for later use.

[0029] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 100% saturation, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0030] On a sterile operating table, sterile, axillary-bud-less poplar micro-twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room for cultivation at a temperature of 25℃, with a light intensity of 2500 Lux and a light duration of 16 hours of light / 8 hours of darkness.

[0031] The 1 / 2MS macronutrient solution in the breathable and moisture-retaining substrate can activate cambium cell division and enhance bud growth. After 40 days of culture, adventitious buds are formed. These adventitious buds are then cut and subjected to conventional rooting and transplanting to obtain poplar seed spikes.

[0032] Example 2

[0033] This embodiment provides a highly efficient method for propagating seedlings of Populus tomentosa microtwigs without axillary buds based on cambium activation.

[0034] The difference between this embodiment and Embodiment 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 60 seconds, rinsed 5 times with sterile water, and then sterilized with 0.1% HgCl2 for 10 minutes for later use.

[0035] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 120% saturation, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0036] On a sterile operating table, sterile, axillary-bud-less poplar micro-twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room for cultivation at a temperature of 28℃, with a light intensity of 2500 Lux and a light duration of 16 hours of light / 8 hours of darkness.

[0037] The 1 / 2MS macronutrient solution in the breathable and moisture-retaining substrate can activate cambium cell division and enhance bud growth. After 38 days of culture, adventitious buds are formed. These adventitious buds are then cut and subjected to conventional rooting and transplanting to obtain poplar seed spikes.

[0038] Figure 4 This is a photograph of the adventitious bud growth state of the cambium adventitious buds after 38 days of growth, as shown in Example 2; Figure 4 It can be seen that the propagation method provided by this invention produces a large number of adventitious buds of good quality, which can improve the yield and reproductive capacity of Populus tomentosa scions.

[0039] Example 3

[0040] This embodiment provides a highly efficient method for propagating seedlings of Populus tomentosa microtwigs without axillary buds based on cambium activation.

[0041] The difference between this embodiment and Embodiment 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 50 seconds, rinsed 4 times with sterile water, and then sterilized with 0.1% HgCl2 for 8 minutes for later use.

[0042] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 80% saturation, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0043] On a sterile operating table, sterile, axillary-bud-less poplar micro-twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room for cultivation at a temperature of 26℃, with a light intensity of 2500 Lux and a light duration of 16 hours of light / 8 hours of darkness.

[0044] The 1 / 2MS macronutrient solution in the breathable and moisture-retaining substrate can activate cambium cell division and enhance bud growth. After 42 days of culture, adventitious buds are formed. These adventitious buds are then cut and subjected to conventional rooting and transplanting to obtain poplar seed spikes.

[0045] Figure 1 This is a photograph of the adventitious buds of Populus tomentosa cambium one week after cutting inoculation in Example 3. Figure 2 This is a photograph of the adventitious bud growth state of the cambium adventitious buds after 30 days of growth in Example 3. Figure 3 This is a photograph of the container seedlings obtained after 30 days of cultivation following rooting and transplanting of cambium adventitious buds cut from Example 3; (The image shows the seedlings from Example 3.) Figure 1-3 It can be seen that the propagation method provided by the present invention enables direct sprouting of micro-branch cambium of Populus tomentosa without axillary buds, which significantly shortens the propagation cycle of Populus tomentosa seed spikes.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized with 0.1% HgCl2 for 5 minutes for later use.

[0048] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 100% saturation, resulting in a breathable and moisture-retaining substrate. After autoclaving the breathable and moisture-retaining substrate, place it in a seedling tray and cool it down for later use.

[0049] Aseptically treated, axillary-bud-less Populus nigra micro-branches were inserted into a breathable and moisture-retaining substrate. After inoculation, the trays were placed in a culture room for cultivation. The indoor air humidity was maintained at 50±5%, the indoor culture temperature was 25℃, the light intensity of the tissue culture room lamp was 2500 Lux, and the light duration was 16 hours of light / 8 hours of darkness. The formation of adventitious buds was observed during cultivation.

[0050] Comparative Example 2

[0051] The difference between this comparative example and Example 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized with 0.1% HgCl2 for 5 minutes for later use.

[0052] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to a saturated state of 30%, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0053] On a sterile operating table, aseptically sterilized, axillary-bud-less poplar twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room at a temperature of 25℃, with a light intensity of 2500 Lux and a light-dark cycle of 16 hours of light / 8 hours of darkness. The formation of adventitious buds was observed during the culture.

[0054] Comparative Example 3

[0055] The difference between this comparative example and Example 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized with 0.1% HgCl2 for 5 minutes for later use.

[0056] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 2 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 50% saturation, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0057] On a sterile operating table, aseptically sterilized, axillary-bud-less poplar twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room at a temperature of 25℃, with a light intensity of 2500 Lux and a light-dark cycle of 16 hours of light / 8 hours of darkness. The formation of adventitious buds was observed during the culture.

[0058] Comparative Example 4

[0059] The difference between this comparative example and Example 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized with 0.1% HgCl2 for 5 minutes for later use.

[0060] Prepare a mixed substrate by mixing peat moss and perlite in a volume ratio of 2:1. Add pure water to the mixed substrate to make the substrate moisture reach 100% saturation, and obtain an air-permeable and moisture-retaining substrate. Place the air-permeable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0061] On a sterile operating table, aseptically sterilized, axillary-bud-less poplar twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room at a temperature of 25℃, with a light intensity of 2500 Lux and a light-dark cycle of 16 hours of light / 8 hours of darkness. The formation of adventitious buds was observed during the culture.

[0062] Figure 5 This is a photograph of the adventitious shoot growth status of Comparative Example 4 after 30 days of culture in a nutrient-free substrate. Figure 5 It can be seen that in the absence of nutrient solution to activate cambium cells, the number of adventitious buds formed is small and the buds are weak and of poor quality, which is not conducive to the propagation of poplar buds.

[0063] Comparative Example 5

[0064] The difference between this comparative example and Example 1 is that the axillary bud-free part of the semi-lignified scion is selected, and 3-4 cm sections are cut to obtain axillary bud-free small black poplar micro-branches. The axillary bud-free small black poplar micro-branches are soaked in 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized with 0.1% HgCl2 for 5 minutes for later use.

[0065] Prepare a mixed substrate using peat moss and perlite in a volume ratio of 2:1. Add 1 / 4 MS macronutrient solution to the mixed substrate to bring the substrate moisture to 100% saturation, resulting in a breathable and moisture-retaining substrate. Place the breathable and moisture-retaining substrate in a tissue culture bottle for autoclaving and then cool it down for later use.

[0066] On a sterile operating table, aseptically sterilized, axillary-bud-less poplar twigs were inserted into a breathable and moisture-retaining substrate, and the tissue culture bottles were tightened, maintaining the air humidity inside the bottles at 95±5%. After inoculation, the tissue culture bottles were placed in a culture room at a temperature of 25℃, with a light intensity of 2500 Lux and a light-dark cycle of 16 hours of light / 8 hours of darkness. The formation of adventitious buds was observed during the culture.

[0067] The adventitious bud occurrence rate, average number of buds, and bud growth of Examples 1-3 and Comparative Examples 1-5 were statistically analyzed, and the results are shown in Tables 1, 2, and 3.

[0068] Table 1 shows the comparison results of adventitious bud formation in the cambium layer of Populus tomentosa under different air humidity conditions in Example 3 and Comparative Example 1.

[0069] Table 1

[0070]

[0071] Table 2 shows the comparison results of adventitious bud formation in the cambium layer of Populus tomentosa under different substrate humidity conditions in Examples 1-3 and Comparative Examples 2-3.

[0072] Table 2

[0073]

[0074] Table 3 shows the comparison results of the formation of adventitious buds in the cambium of Populus tomentosa under different nutritional conditions in Example 3, Comparative Examples 4 and 5.

[0075] Table 3

[0076]

[0077] A comparison of the data in Tables 1-3 shows that air humidity has a more significant impact on the germination of adventitious buds in the cambium of Populus tomentosa than substrate moisture and nutrient solution concentration. In high-humidity environments (e.g., 95±5%), plant cells have higher turgor pressure and sufficient intracellular water, which helps maintain cell activity and metabolic processes. For cambium cells, sufficient water enables them to maintain good division and differentiation capabilities, thereby promoting the formation of adventitious buds. High humidity reduces water loss in plants, allowing them to dedicate more energy and substances to the induction and growth of adventitious buds. In low-humidity environments (50±5%), the risk of water loss in plant cells increases, and cell turgor pressure decreases, leading to a slowdown in cambium cell metabolism. To reduce water loss, cambium cells enter a self-protective state, inhibiting their division and differentiation activities, resulting in a significant decrease or even zero induction rate of adventitious buds. These principles of how humidity affects cambium adventitious buds further explain the experimental results and provide a theoretical basis for plant micropropagation technology.

Claims

1. A highly efficient method for propagating axillary bud-less Populus tomentosa micro-twigs based on cambium activation, characterized in that, Select small black poplar branches without axillary buds, sterilize them, and then insert them into a breathable and moisturizing substrate with a humidity of 80-120% containing 1 / 2MS macronutrient solution. Maintain the air humidity at 95±5%. The breathable and moisturizing substrate and the air humidity, together with the nutrient solution, activate the cambium cell division, and adventitious buds are formed after culture. Cut off the adventitious buds and carry out conventional rooting and transplanting culture to obtain small black poplar seed clippings.

2. The method for efficient propagation of axillary bud-less poplar micro-twigs based on cambium activation according to claim 1, characterized in that, The axillary bud-free poplar micro-twigs are obtained by cutting 3-4 cm sections from the semi-lignified scions that sprouted from superior single plants over 40 years old.

3. The efficient seed propagation method for axillary bud-less Populus tomentosa micro-twigs based on cambium activation according to claim 1 or 2, characterized in that, The aseptic disinfection process involves soaking small black poplar branches without axillary buds in 75% ethanol for 30-60 seconds, rinsing with sterile water, and then sterilizing with 0.1% HgCl2 for 2-10 minutes.

4. The method for efficient propagation of axillary bud-less poplar micro-twigs based on cambium activation according to claim 3, characterized in that, The breathable and moisture-retaining matrix is ​​a mixture of peat moss and perlite, with a volume ratio of peat moss to perlite of 2:

1.

5. The method for efficient propagation of axillary bud-less Populus tomentosa micro-twigs based on cambium activation according to claim 4, characterized in that, The moisture content of the breathable and moisturizing matrix is ​​80%.

6. The method for efficient propagation of axillary bud-less poplar micro-twigs based on cambium activation according to claim 5, characterized in that, The culture temperature was 25~28℃, and the light duration was 16 hours of light / 8 hours of darkness.

7. The method for efficient propagation of axillary bud-less poplar micro-twigs based on cambium activation according to claim 6, characterized in that, The time for the formation of adventitious buds is 35 to 45 days.

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