Ginkgo large-scale cutting cutting training method and its cutting method

Abstract

The application discloses a large-scale cutting of ginkgo and a cutting method thereof, and the cutting method comprises the following steps: cutting and dwarfing pruning a 4-5 year old ginkgo mother plant, pruning the lateral branches of the cut mother plant for multiple times, spraying a plant growth regulator after each pruning to promote the development of the lateral branches of the mother plant, cutting the branches on the lateral branches of the mother plant as cuttings, and inserting the cuttings into a substrate after treating the cuttings with a plant growth regulator. The method adopts the cutting and multiple pruning to promote the sprouting of the plant and generate the juvenile branch, greatly increases the number of cuttings, sprays 6-benzyladenine or brassinolide to improve the quality and quantity of the cuttings, and further uses methyl jasmonate, naphthalene acetic acid and uniconazole to jointly treat the cuttings to significantly improve the rooting rate of the cuttings. The method can be used for cultivating the leaf ginkgo and the ginkgo mother plant with excellent characteristics, significantly shortens the formation time of the cutting callus and adventitious roots, is simple and easy to operate, is suitable for large-scale cutting, and has popularization value in the asexual propagation of the ginkgo clone.

Description

Technical Field

[0001] This invention relates to a method for cultivating cuttings for large-scale propagation of ginkgo and the method for propagating cuttings therein, belonging to the field of ginkgo planting technology. Background Technology

[0002] Ginkgo (Ginkgo biloba L.), also known as the maidenhair tree, is a plant belonging to the genus Ginkgo in the family Ginkgoaceae. It is a relict plant that survived the Ice Age and is often referred to as a "living fossil." Ginkgo biloba extract (GBE) is prepared from ginkgo leaves and its main components are ginkgo flavonoids and terpenoids. Flavonoids play important roles in anti-oxidation and anti-tumor activity; ginkgolides are strong antagonists of platelet-activating factor, which can prevent cardiovascular disease and protect the central nervous system. Because only young ginkgo leaves contain high levels of active ingredients, leaf nurseries are typically established through seed propagation. However, this method easily leads to segregation of plant traits, resulting in significant differences in the content of medicinal active ingredients in the leaves between different individuals, failing to meet pharmacopoeia standards. Addressing these issues, how to achieve stable asexual inheritance and maintain the traits of superior and mutant ginkgo varieties is a pressing practical problem that needs to be solved in the cultivation of ginkgo leaves. In addition, conventional superior tree propagation techniques use grafting, but grafting requires the preparation of rootstock in advance, as well as a high level of grafting skill to improve the survival rate. Furthermore, grafting also requires a high level of financial investment. Therefore, the large-scale rapid propagation of superior ginkgo trees is currently a major problem.

[0003] Asexual propagation is a method of increasing the number of clonal seedlings produced by selecting asexual original plants based on seed selection. Compared with seed propagation, asexual propagation has significant genetic gains and shortens the breeding cycle. For a long time, Ginkgo propagation mainly used hardwood cuttings, with cuttings derived from the lignified branches of superior trees. Later, it gradually transitioned to using semi-lignified or tender branches of superior trees as cuttings. However, due to the special characteristics of the Ginkgo species, the survival rate of Ginkgo cuttings is low, the propagation efficiency is low, and large-scale propagation has not yet been achieved. Summary of the Invention

[0004] Objective of the Invention: Addressing the problems existing in the prior art, the first objective of this invention is to provide a method for cultivating Ginkgo biloba cuttings for large-scale propagation. This method primarily solves the problems of low efficiency and poor quality in Ginkgo biloba clonal propagation. Based on the initial selection of superior trees, seedlings grown from the seeds of these superior trees are used as mother plants for cuttings, establishing a large-scale cutting nursery to mass-produce high-quality cuttings, improving the rooting rate of the cuttings, and realizing factory-style seedling cultivation and large-scale propagation. The second objective of this invention is to provide a method for large-scale propagation using these Ginkgo biloba cuttings.

[0005] Technical solution: To achieve the above objectives, the present invention provides a method for cultivating Ginkgo biloba cuttings for large-scale propagation, comprising the following steps:

[0006] (A1) Dwarf the mother ginkgo tree by cutting back the trunk to determine the height of the main trunk;

[0007] (A2) Prune the lateral branches that sprout after the trunk is cut off multiple times, and spray a plant growth regulator after each pruning to promote the development of lateral branches of the mother plant.

[0008] In step (A1), the ginkgo mother plant is a 4-5 year old ginkgo seedling. The ginkgo mother plant is cut off at a height of 20-30 cm from the ground between November of the current year and February of the following year for dwarfing cultivation.

[0009] In step (A2), the multiple pruning involves removing 1 / 3 of the length of all newly sprouted lateral branches after the trunk is cut off, allowing them to grow for three months, and then removing another 1 / 3 of the length of the lateral branches to ensure that the optimization of the lateral branches, the number of sprouts, and the thickness of the branches are suitable for use as cuttings for rapid propagation.

[0010] In step (A2), the plant growth regulator is a 6-benzyladenine solution or a brassinolide solution.

[0011] The concentration of the 6-benzyladenine solution is 100-300 mg / L, and the concentration of the brassinolide solution is 1-3 mg / L, preferably 1 mg / L.

[0012] A method for large-scale propagation of Ginkgo biloba using lateral branches obtained from the Ginkgo biloba mother plant through the method described in this invention includes the following steps:

[0013] (B1) Take branches from the lateral branches of the mother plant obtained by the method described in this invention as cuttings, and treat the resulting Ginkgo cuttings with a plant growth regulator;

[0014] (B2) Insert the treated ginkgo cuttings into the substrate for cultivation.

[0015] In step (B1), the diameter of the lateral branches of the mother plant is 0.5-1 cm, the length of the lateral branches is 40-50 cm, and the leaves on the branches are healthy and free from pests and diseases.

[0016] In step (B1), the lateral branches are pruned into 4-5 cuttings, each cutting about 10 cm long. When pruning the cuttings, retain 1-2 disease-free and non-yellowing leaves near the top of the cutting. Keep the upper cut flat, 1-1.5 cm away from where the leaves emerge. If the upper part is the terminal bud, no pruning is required. The lower cut is at 45-50°. All cuts should be neat and smooth.

[0017] In step (B1), the cuttings are treated with plant growth regulators by soaking them in a solution containing 1-1.5 μmol / L methyl jasmonate and 10-100 mg / L paclobutrazol for at least 30 minutes, then dipping them in a 400 mg / L naphthaleneacetic acid solution for 6-10 seconds and draining off excess water. Preferably, the cuttings are soaked in a solution containing 1.5 μmol / L methyl jasmonate and 100 mg / L paclobutrazol for 30 minutes and then dipped in a 400 mg / L naphthaleneacetic acid solution for 6-10 seconds.

[0018] In step (B2), the substrate is a mixture of perlite and vermiculite with a mass ratio of perlite to vermiculite of 1:1. The substrate is disinfected with a 0.2% solution of 40% carbendazim, and seedlings are planted after being covered with a film for one week.

[0019] In step (B2), the cuttings are taken and planted on the same day, and humidity is maintained throughout the process.

[0020] In existing asexual propagation techniques for Ginkgo, the yield of cuttings from mother plants is low, and the cutting technology is immature, making it impossible to rapidly propagate fast-growing and highly resistant mother plants in a short period of time. This invention utilizes Ginkgo mother plants in a cutting nursery obtained through trunk cutting and multiple pruning to achieve better growth, increased lateral branches, increased leaf lobes, and increased leaf area, thickness, fresh weight, and water content. In particular, the flavonoid content is significantly increased. Soaking the cuttings in methyl jasmonate, naphthaleneacetic acid, and tebuconazole significantly improves the rooting rate. The method is convenient, simple, and easy to implement, making it suitable for widespread application in production.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] (1) The method of the present invention is convenient to operate, simple and easy to implement, and suitable for promotion in production.

[0023] (2) Compared with traditional nurseries, the Ginkgo mother plants in the cutting nursery obtained by the present invention through trunk cutting and multiple pruning are in better growth condition, with more lateral branches, increased leaf lobes, increased leaf area, thickness, fresh weight and water content, and especially significantly increased flavonoid content.

[0024] (3) The present invention significantly improves the rooting rate by soaking cuttings in jasmonic acid, naphthaleneacetic acid and acetaminophen. Attached Figure Description

[0025] Figure 1 This is a photograph of the 5-year-old ginkgo tree trunks after being cut in Example 1;

[0026] Figure 2 This is a graph showing the effect of different trunk cutting times on the development of lateral branches of the trunk-cutting mother plant in Example 1;

[0027] Figure 3This is a graph showing the effects of different pruning methods on the growth, number of branches, and diameter of new ginkgo trees in Example 1.

[0028] Figure 4 The figure shows the effects of different pruning methods on the leaf area, number of leaf lobes, leaf lobe depth, leaf thickness, dry weight, fresh weight, water content, and flavonoid content of new branches in Example 1.

[0029] Figure 5 The figure shows the effect of different light pruning times on the growth, number of branches, and diameter of new ginkgo trees in Example 1.

[0030] Figure 6 The figure shows the effects of different light pruning times on the leaf area, number of leaf lobes, leaf lobe depth, leaf thickness, dry weight, fresh weight, water content, and flavonoid content of new Ginkgo branches in Example 1.

[0031] Figure 7 The images show the state of Ginkgo seedlings after being sprayed with different concentrations of 6-BA from 0 to 28 days in Example 1.

[0032] Figure 8 The figure in Example 1 shows the state of Ginkgo seedlings 0-28 days after being sprayed with different concentrations of GA3;

[0033] Figure 9 The figure in Example 1 shows the state of Ginkgo seedlings 0-28 days after being sprayed with different concentrations of BR.

[0034] Figure 10 Photographs of cuttings treated with NAA+MeJA in Example 2;

[0035] Figure 11 Photographs of cuttings treated with NAA+Uniconazole-P in Example 2;

[0036] Figure 12 Photographs of Ginkgo biloba cuttings treated with different MeJA and Uniconazole-P concentration ratios in Example 2, taken from 0 to 35 days.

[0037] Figure 13 The growth status of the mother plant cuttings in Example 2 (unpruned, pruned once, and pruned twice) from 0 to 35 days is shown in the diagram.

[0038] Figure 14 For large-scale implementation of cuttings and renderings. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] The following experiment was conducted at the orchard of Yangzhou University in Yangzhou City, Jiangsu Province, where a batch of 5-year-old ginkgo seedlings were planted for subsequent cutting cultivation and rooting experiments.

[0041] Example 1: Cultivation of Ginkgo cuttings

[0042] 1. Mother Plant Pruning: In February 2021, 5-year-old ginkgo trees were pruned horizontally at a height of 20 cm above the ground using pruning shears, retaining the lower half to cultivate cutting mother plants. Unpruned ginkgo trees served as a control. All maintenance methods were identical. Figure 1 As shown. Figure 1 These are photos of 5-year-old ginkgo trees after pruning following planting in Example 1. A shows a ginkgo tree without pruning, B shows a ginkgo tree after pruning, C shows a ginkgo tree after three months of growth without pruning, and D shows a ginkgo tree after three months of growth after pruning. Figure 1 It can be seen that, compared with the uncut branches, the dormant buds below the cut after the cutting treatment sprouted and formed 4-5 new branches. The length of the sprouted new branches was significantly longer than that of the uncut branches. After the cutting treatment, the leaf area increased and the leaf lobes increased, showing obvious rejuvenation phenomenon. Therefore, the rejuvenated ginkgo plants after the cutting and dwarfing were selected.

[0043] 2. To investigate the effect of different pruning times on the development of lateral branches in pruned mother trees, 5-year-old Ginkgo trees were pruned in November 2021, December 2021, January 2022, and February 2022, with a pruning height of 20 cm above the ground. The maintenance methods were consistent throughout. In May 2022, the growth of new shoots and the number of lateral branches were measured. The results are as follows: Figure 2 As shown. Figure 2 The following is a graph showing the effect of different pruning times on the development of lateral branches of the pruned mother plants in Example 1. In the graph, AD are photos of 5-year-old ginkgo plants pruned in different months from November 2021 to February of the following year, EH are photos of ginkgo plants pruned in different months in AD in May 2022, I is a graph showing the statistical results of the new shoot growth of ginkgo plants pruned in different months in the following year, and J is a graph showing the statistical results of the number of lateral branches of ginkgo plants pruned in different months in the following year. Figure 2 This indicates that pruning the main trunk at different times has no significant impact on the formation and growth of ginkgo lateral branches. Therefore, pruning can be carried out from the time the ginkgo leaves fall until the following year before budding.

[0044] 3. The impact of repeated pruning and trunk cutting of ginkgo shoots on the development of ginkgo trees

[0045] (1) First pruning: On a sunny day in February 2022, the new shoots growing from the ginkgo plants that had been pruned in step 2 were pruned for the first time. Three treatment methods were used: top pruning (removing the terminal bud of the branch by hand), light pruning (cutting the branch one-third of the way from the top with pruning shears), and heavy pruning (cutting the branch two-thirds to three-quarters of the way from the top with pruning shears). In May 2022, the growth of new shoots, the number of lateral branches, and the stem diameter of the lateral branches were measured. Ten leaves from the upper part of the new shoots of different pruning methods were collected, with three replicates (three plants) for each treatment to determine the number of leaf lobes, lobe depth, thickness, and area. The leaves were photographed using a digital camera, and their area was analyzed using ImageJ software. The length from the leaf tip to the base of each leaf lobe was measured using calipers. For leaves with deep lobes, the thickness at the center of the leaf lobe was measured; for leaves with shallow lobes, the thickness at the center of the leaf lobe was measured, and the average thickness was calculated. After weighing the fresh ginkgo leaves using an electronic balance, they were placed in a 75 ℃ oven for 24 h to dry until constant weight, and then weighed again for dry weight. Each group consisted of 10 leaves, with 3 biological replicates. The leaf water content was calculated using the formula: Water content (%) = (Fresh material content - Dry material content) / Fresh material content × 100%. Since flavonoids are important bioactive substances in ginkgo leaves, the flavonoid content (mg / g) of ginkgo leaves after different pruning stages was determined according to the plant flavonoid detection kit protocol (Suzhou Co., Ltd., China). Unpruned leaves served as a control; all other operations and maintenance were the same. Results are as follows: Figure 3-4 As shown.

[0046] Figure 3The following figures illustrate the effects of different pruning methods on the development of new branches of Ginkgo biloba in Example 1. A shows a photograph of the unpruned control group of Ginkgo biloba plants; B shows a photograph of the pruned Ginkgo biloba plants after pinching; C shows a photograph of the pruned Ginkgo biloba plants after light pruning; D shows a photograph of the pruned Ginkgo biloba plants after heavy pruning; E and H show photographs of the unpruned control group, pruned Ginkgo biloba plants, lightly pruned Ginkgo biloba plants, and heavily pruned Ginkgo biloba plants from May 2022 (AD and D), respectively; I shows the statistical results of the new branch length of the pruned Ginkgo biloba plants under different pruning treatments and the unpruned Ginkgo biloba plants; J shows the statistical results of the new branch growth of the pruned Ginkgo biloba plants under different pruning treatments and the unpruned Ginkgo biloba plants; and H shows the statistical results of the new branch diameter of the pruned Ginkgo biloba plants under different pruning treatments and the unpruned Ginkgo biloba plants. Compared with the control group, heavy pruning increased new shoot growth by 28.3%, reaching 68 ± 1.2 cm, and the number of branches increased by 148%. Similarly, light pruning increased new shoot growth by 33.9%, reaching 71 ± 1.1 cm, and the number of branches increased by 152%. However, while pinching slightly reduced new shoot growth, it increased the number of branches by 75%. Compared with the control group, heavy pruning reduced stem diameter by 14.8%, to 0.58 ± 0.08 cm, while light pruning increased it by 19.1%, reaching 0.81 ± 0.04 cm. Pinching resulted in a stem diameter of 0.72 ± 0.05 cm, with no significant change.

[0047] Figure 4The figures show the effects of different pruning methods on the leaf area, number of leaf lobes, leaf lobe depth, leaf thickness, dry weight, fresh weight, water content, and flavonoid content of new ginkgo branches in Example 1. In the figures, AD represent photographs of ginkgo leaves after unpruned, top-pinched, lightly pruned, and heavily pruned treatments, respectively. EH represents the statistical results of leaf area, number of leaf lobes, leaf lobe depth, and leaf thickness after different treatments, respectively. IK represents the statistical results of dry weight, fresh weight, water content, and flavonoid content of ginkgo leaves after different treatments, respectively. Comparing Figure EH with the control group, it was found that the size and number of leaf lobes increased after lightly pruned, heavily pruned, and top-pinched treatments, with lightly pruned treatment showing the best effect. Comparing the results with Figure IL, the fresh weight of leaves after heavy pruning was 11.46±0.16 g, an increase of 1.7 times; similarly, the fresh weight of leaves after light pruning was 12.36±0.12 g, an increase of 1.8 times. However, the fresh weight of leaves after topping was 8.24±0.11 g, an increase of 1.3 times; the dry weight of leaves after heavy pruning was 2.71±0.09 g, an increase of 1.5 times. The dry weight of leaves after light pruning was 2.59±0.08 g, an increase of nearly 1 time. The dry weight of leaves after topping was 2.08±0.06 g, an increase of 60%. The flavonoid content increased by 46% and 45% after light and heavy pruning, respectively, and by 25% after topping. In summary, due to the different operations of pinching, light pruning, and heavy pruning, light pruning significantly promoted the number of branches, leaf biomass, and flavonoid content compared to other treatments. Therefore, light pruning is the optimal pruning method for cultivating Ginkgo biloba scion mother plants.

[0048] (2) Secondary pruning: Secondary pruning was carried out in May 2022. Ginkgo trees that had already been pruned with light short pruning were selected for secondary pruning, and the pruning process was the same as in step (1). At the same time, in order to explore the effect of different pruning times on cuttings, 5 unpruned mother plants were selected and pruned with light short pruning for the first time. Unpruned mother plants served as the control group, and other operations and maintenance were the same. In June 2022, the growth of new shoots, the number of lateral branches and the stem diameter of lateral branches were measured. The number of leaf lobes, the depth of lobes, the thickness and area were measured using the same method as in step (1). The dry weight and fresh weight of the leaves were weighed, the water content was calculated, and the flavonoid content was determined. Figure 5-6 As shown.

[0049] Figure 5The graph shows the effects of different light pruning sessions on the growth, number of branches, and branch diameter of new Ginkgo trees in Example 1. A shows a photo of a Ginkgo tree after its first light pruning in February 2022; B shows a photo of a Ginkgo tree after its first light pruning in May 2022 with new branches; C shows a photo of a Ginkgo tree after its second light pruning in May 2022; and D shows a photo of a Ginkgo tree after its second pruning in June 2022 with even more new branches. Figures E and G show the statistical results of the length, number, and diameter of new branches of Ginkgo trees after different pruning sessions. Compared to the control, the first pruning increased the growth of new shoots by 24.5% and the number of branches by 101%. Compared to the first pruning, although the growth of new shoots decreased by 22.3% after the second pruning, the number of branches increased again by 44.3%. Furthermore, compared to the control, the stem diameter of branches increased by 12.1% after the first pruning, while the stem diameter of branches decreased by 14.8% after the second pruning. Therefore, after two light prunings, the plant exhibits characteristics of dwarfing, increased branch density, and expanded crown, which not only facilitates obtaining a large number of cuttings, but also reduces the thickness of the branches, making it more conducive to cutting propagation.

[0050] Figure 6 The graphs show the effects of different light pruning cycles on the leaf area, number of leaf lobes, leaf lobe depth, leaf thickness, dry weight, fresh weight, moisture content, and flavonoid content of new Ginkgo branches in Example 1. In the graphs, AC represent photos of Ginkgo leaves after no pruning, one pruning, and two pruning cycles, respectively. DG represents statistical graphs of leaf area, number of leaf lobes, leaf lobe depth, and leaf thickness after different pruning cycles, respectively. HK represents statistical graphs of leaf dry weight, fresh weight, moisture content, and flavonoid content after different pruning cycles, respectively. Comparing DG, it was found that compared to the control group, the leaves were significantly larger and had more and deeper leaf lobes after one and two pruning cycles, indicating that pruning can rejuvenate Ginkgo. Furthermore, compared to one pruning, after two pruning, the leaf area increased by 22.6%, the leaf lobe depth increased by 61.1%, and the leaf thickness increased by 19.5%, indicating that two pruning can further enhance the rejuvenating effect of Ginkgo. Compared with the control group, the fresh weight of leaves after the first pruning was 6.41 ± 0.21 g, an increase of 152%, and the dry weight was 2.12 ± 0.26 g, an increase of 136%. After the second pruning, the fresh weight of leaves was 8.34 ± 0.17 g, an increase of 189%, and the dry weight was 1.82 ± 0.18 g, an increase of 104%. Measurements of flavonoid content in the leaves showed that the content of flavonoid compounds in the leaves increased by 10.3% compared with the first pruning. Therefore, a second pruning using a light short pruning method after trunk cutting can significantly promote the number of branches, promote the rejuvenation of branches and leaves, and improve the quality of cuttings.

[0051] 4. Effects of spraying plant growth regulators on the development of ginkgo trees

[0052] Spraying plant growth regulators: In order to screen the optimal growth regulators and considering that field experiments are affected by the seasons and seedling experiments are more flexible, February-grown ginkgo seedlings were selected for the plant growth regulator spraying experiment. The February-grown ginkgo seedlings were pruned, and three buds were retained at the base of each seedling. Different concentrations of plant growth regulators 6-BA (6-benzyladenine), GA3 (gibberellin A3), and BR (brassinosteroids) were sprayed on the ginkgo seedlings. The plant growth regulators were sprayed on the entire above-ground parts, once every other day, for a total of three times. Each time, the spray was applied until droplets formed on the plant surface and flowed down to stimulate the germination of lateral buds and dormant buds. The spraying concentration and solvent for the plant growth regulator are shown in Table 1. The corresponding mass of plant growth regulator powder (6-BA, GA3, or BR) was thoroughly dissolved in 1 mol / L NaOH solution or ethanol, and then diluted in 500 mL of water to obtain the corresponding concentration of plant growth regulator. Germination rate, number of germinations, and bud length were recorded after 28 days, and photographs were taken for observation. A control group was set up using water spraying; all other procedures were the same. Results are as follows: Figure 7-9 As shown in Table 2-4.

[0053] Table 1 Plant growth regulator spraying scheme

[0054]

[0055] Table 2. Statistical data after spraying different concentrations of 6-BA

[0056] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0057] Figure 7 Figure 1 shows the state of Ginkgo seedlings after being sprayed with different concentrations of 6-BA from 0 to 28 days in Example 1. Figure 2 shows the number of buds, bud length, and budding rate of Ginkgo seedlings sprayed with different concentrations of 6-BA at 28 days. Figure 7 As shown in Table 2, after spraying with 100 mg / L 6-BA, axillary buds began to germinate at 11 days, and began to germinate in large numbers after 14 days, reaching a total of 23 buds by 28 days, with a germination rate of 85.2%, which was 11.1% higher than the control group. After spraying with 200 mg / L and 300 mg / L 6-BA, axillary buds began to germinate at 10 days, and began to germinate in large numbers after 14 days, reaching a total of 24 buds by 28 days, with a germination rate of 88.9%, which was 14.8% higher than the control group. In this experiment, the 200 mg / L 6-BA spray showed the best effect.

[0058] Table 3. Statistical data after spraying different concentrations of GA3

[0059]

[0060] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0061] like Figure 8 As shown in Table 3, Figure 8 Table 3 shows the state of Ginkgo seedlings after spraying with different concentrations of GA3 from 0 to 28 days in Example 1. Table 3 shows the number of buds, bud length, and budding rate of Ginkgo seedlings sprayed with different concentrations of GA3 at 28 days. Figure 8 As shown in Table 3, after spraying with 50 mg / L and 100 mg / L GA3, axillary buds began to germinate at 12 days, and began to germinate in large numbers after 14 days, reaching a total of 18 buds by 28 days, with a germination rate of 66.7%, which was not significantly different from the control group. Increasing the GA3 concentration to 150 mg / L, axillary buds began to germinate at 11 days, and began to germinate in large numbers after 14 days, reaching a total of 19 buds by 28 days, with a germination rate of 70.4%, again showing no significant change. In conclusion, GA3 spraying inhibits bud germination.

[0062] Table 4. Statistical data after spraying different concentrations of BR

[0063]

[0064] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0065] like Figure 9 As shown in Table 4, Figure 9 Table 4 shows the budding numbers, bud lengths, and budding rates of ginkgo seedlings sprayed with different concentrations of BR in Example 1, from 0 to 28 days. Figure 9 As shown in Table 4, at BR concentrations of 1 mg / L and 2 mg / L, axillary buds began to germinate at 9 days, and began to germinate in large numbers after 13 days, reaching a total of 25 buds by 28 days, with a germination rate of 92.6%, which was 18.5% higher than the control group. However, after spraying with 3 mg / L BR, axillary buds began to germinate at 10 days, and began to germinate in large numbers after 13 days, reaching a total of 24 buds by 28 days, with a germination rate of 88.9%, which was 14.8% higher than the control group. In conclusion, the 1 mg / L BR treatment showed the best effect. Therefore, spraying with 1 mg / L BR resulted in the highest germination rate, the largest number of buds, and the longest bud length, which is most beneficial for promoting the germination of lateral and dormant buds of Ginkgo cuttings after primary and secondary pruning, thus forming new shoots.

[0066] Example 2: Rooting Experiment of Hardwood Cuttings

[0067] 1. Construction of Cutting Ponds: Construction began on June 25, 2022, at the Ginkgo biloba base in Sihu Town, Pizhou City, Xuzhou City, Jiangsu Province. Cutting ponds were constructed in a semi-shaded area under the trees, measuring 12 m long, 2.5 m wide, and 40 cm deep. A 1:1 mixture of vermiculite and perlite was used as the cutting substrate. Before planting, the substrate was disinfected with a 0.2% solution of 40% carbendazim until droplets formed on the surface. The substrate was then covered with a thin film and left for one week. After one week, the film was removed to allow for sufficient gas dissipation, and the substrate was thoroughly rinsed with clean water. Planting was then carried out after another 2-3 days. An automatic sprinkler system was installed, with one sprinkler head spaced 1 m apart on each side of the cutting pond. A timer was set to spray for 10 minutes every 1 hour, controlling the humidity of the cutting pond to approximately 85%. Spraying was stopped during rainy days until the substrate surface dried after the rain.

[0068] 2. Collection of cuttings: From the end of May to the middle of July, collect ginkgo branches that sprouted after the mother plant was cut back in February and then lightly pruned twice, as described in Example 1. The branches should be less than 1 cm and greater than 0.5 cm in diameter. Spray the branches with clean water to keep them moist and use them as cuttings.

[0069] 3. Cutting Treatment: Prune lateral branches into 4-5 cuttings, each approximately 10 cm long. When pruning, retain 1-2 disease-free, non-yellowing leaves near the top of the cutting. Make a flat cut at the top, 1-1.5 cm from the leaf emergence point. If the top is a terminal bud, no pruning is necessary. Make a 45-50° cut at the bottom. All cuts should be neat and smooth. Soak the cuttings in a growth regulator for 30 minutes, then quickly dip them in 400 mg / L NAA for 6-10 seconds, drain excess water, and insert them into the substrate to a depth of 3-5 cm (approximately 1 / 3 of the cutting length). All solvents are water.

[0070] 4. To investigate the effects of different growth regulators on the rooting of cuttings, three levels were set up: NAA (naphthylacetic acid), MeJA (methyl jasmonate) + NAA, and Uniconazole-P (uniconazole) + NAA. The control group (Control) was prepared by dipping cuttings in a 400 mg / L NAA solution for 6-10 seconds, draining excess water, and then inserting them into the substrate. The experimental groups had two levels of MeJA concentration: 1 μM MeJA + 400 mg / L NAA group: cuttings were soaked in a 1 μmol / L MeJA solution for 30 minutes, then dipped in a 400 mg / L NAA solution for 6-10 seconds, drained excess water, and then inserted into the substrate; 1.5 μM MeJA + 400 mg / L NAA group: cuttings were soaked in a 1.5 μmol / L MeJA solution for 30 minutes, then dipped in a 400 mg / L NAA solution for 6-10 seconds. After draining excess water, the cuttings were inserted into the substrate. Two levels of Uniconazole-P concentration were set for the experimental groups: 10 mg / L Uniconazole-P + 400 mg / L NAA group: after soaking in 10 mg / L Uniconazole-P solution for 30 min, the cuttings were dipped in 400 mg / L NAA solution for 6-10 s, drained, and then inserted into the substrate; 100 mg / L Uniconazole-P + 400 mg / L NAA group: after soaking in 100 mg / L Uniconazole-P solution for 30 min, the cuttings were dipped in 400 mg / L NAA solution for 6-10 s, drained, and then inserted into the substrate. A total of four treatment groups and one control group were included. Each treatment group had 150 cuttings, with 3 replicates (50 cuttings per replicate), for a total of 750 cuttings (50 x 3 x 5 = 750). All other procedures were performed in the same manner. The condition of the base of the cuttings was observed regularly, and the number of roots, average root length, fastest callus formation time, and fastest root formation time were recorded. The rooting rate was investigated after 45 days. The results are shown in Table 5.

[0071] Table 5. Statistics on rooting rates of MeJA and Uniconazole-P cuttings.

[0072]

[0073] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0074] Table 5 shows the rooting number, average root length, fastest callus formation time, fastest rooting time, and rooting rate of cuttings after the above four treatments and cuttings treated with 400 mg / L NAA. Figure 10 These are photographs showing the growth status of the NAA+MeJA treated cuttings from 0 to 42 days in Example 2. Figure 10 As shown in Table 5, the addition of 1 μM and 1.5 μM MeJA treatments resulted in an average of approximately 5.4 ± 0.2 and 6.1 ± 0.3 roots per cutting, respectively, compared to the control, representing increases of 25.6% and 41.9%; average root lengths were 3.2 ± 0.3 cm and 3.6 ± 0.2 cm, respectively, representing increases of 10.3% and 24.1%; and rooting rates were approximately 56.3% and 57.2%, respectively, representing increases of 5.1% and 6.0%. Figure 11 These are photographs showing the growth status of cuttings treated with NAA+Uniconazole-P in Example 2 from 0 to 42 days. Figure 11 As shown in Table 5, the addition of 10 mg / L and 100 mg / L Uniconazole-P treatments resulted in an average of approximately 5.3 ± 0.2 and 5.5 ± 0.3 roots per cutting, respectively, representing increases of 23.3% and 27.9% compared to the control; average root lengths were 3.1 ± 0.2 cm and 3.4 ± 0.2 cm, respectively, representing increases of 6.9% and 17.2%; and rooting rates were approximately 54.8% and 56.1%, respectively, representing increases of 3.6% and 4.9%.

[0075] In summary, these results indicate that soaking the cuttings in MeJA and Uniconazole-P before conventional 400 mg / L NAA treatment can effectively promote the rooting ability of Ginkgo biloba cuttings and increase the rooting rate.

[0076] 5. Based on the previously established concentration levels of NAA, MeJA, and Uniconazole-P, and the soaking (dipping) time, as shown in Table 6, a multi-factor randomized trial was conducted to treat the cuttings with the three hormones in combination to screen for the optimal hormone formulation. This included four treatment groups and one control group. Each treatment group had 150 cuttings, with 3 replicates (50 cuttings per replicate), for a total of 750 cuttings (50 x 3 x 5 = 750). 400 mg / L NAA was used as a control. The 1 μM MeJa + 400 mg / L NAA + 10 mg / L Uniconazole-P group: after soaking in a solution containing 1 μmol / L MeJa and 10 mg / L Uniconazole-P for 30 min, the cells were dipped in 400 mg / L NAA solution for 6-10 s, drained of excess water, and then inserted into the matrix. The 1.5 μM MeJa + 400 mg / L NAA + 10 mg / L Uniconazole-P group: after soaking in a solution containing 1.5 μmol / L MeJa and 10 mg / L Uniconazole-P for 30 min, the cells were dipped in 400 mg / L NAA solution for 6-10 s, drained of excess water, and then inserted into the matrix. The 1 μM MeJa + 400 mg / L NAA + 100 mg / L Uniconazole-P group: after soaking in a solution containing 1 μmol / L MeJa and 100 mg / L Uniconazole-P for 30 min... After soaking in 400 mg / L NAA solution for 6-10 seconds, the cuttings were drained of excess water and then inserted into the substrate. The 1.5 μM MeJa + 400 mg / L NAA + 100 mg / L Uniconazole-P group: after soaking in a solution containing 1.5 μmol / L MeJa and 100 mg / L Uniconazole-P for 30 minutes, the cuttings were dipped in 400 mg / L NAA solution for 6-10 seconds, drained of excess water, and then inserted into the substrate. All other procedures were the same for all treatments. The base condition of the cuttings was observed regularly, and the number of roots, average root length, fastest callus formation time, and fastest rooting time were recorded. The rooting rate was assessed at 45 days. The results are as follows: Figure 12 As shown in Table 6.

[0077] Table 6. Statistics on rooting rate of cuttings with different hormone combinations

[0078]

[0079] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0080] Figure 12These are photographs showing the growth status of Ginkgo biloba cuttings treated with different concentrations of MeJA and Uniconazole-P in Example 2 from 0 to 35 days. Figure 12 Table 6 shows that, compared with the control, the combined treatment with the three hormones significantly increased the number of roots, root length, and rooting rate of the cuttings. Among them, the combination of 1.5 μM MeJA + 100 mg / L Uniconazole-P + 400 mg / L NAA showed the best effect, promoting callus and root formation approximately 13 days earlier. This combination also led to a 149.1% increase in root number, a 123.5% increase in root length, and a 22.9% increase in rooting rate. Therefore, the hormone combination of 1.5 μM MeJA + 100 mg / L Uniconazole-P + 400 mg / L NAA can significantly improve the rooting rate of Ginkgo biloba cuttings.

[0081] 6. To investigate the effect of the number of pruning cycles on the rooting ability of cuttings, on June 5, 2022, branches grown after unpruned (Control), primary pruning (Primary pruning), and secondary pruning (Secondary pruning) were collected and cut into cuttings. These cuttings were treated with 1.5 μM MeJA + 10 mg / L Uniconazole-P + 400 mg / L NAA before being inserted into propagation beds, with all other procedures performed uniformly. Each treatment contained 150 cuttings, with 3 replicates (50 cuttings per replicate), for a total of 450 cuttings. Unpruned cuttings served as a control. The basal condition of the cuttings was observed regularly, and the number of roots, average root length, fastest callus formation time, and fastest rooting time were recorded. Rooting rate was assessed at 45 days. Results are as follows: Figure 13 As shown in Table 7.

[0082] Table 7. Statistics on the rooting rate of cuttings after different pruning times.

[0083]

[0084] Note: “a”, “b”, “c” indicate that there are significant differences between results of different letters in the same group, ρ < 0.05.

[0085] Figure 13 The images show the growth status of cuttings from mother plants in Example 2 (unpruned, pruned once, and pruned twice) from 0 to 35 days. Figure 13Table 7 shows that callus formation and root development occurred earlier in the pruned cuttings compared to the control group. The rooting time of the cuttings after the second pruning was 3-4 days earlier. Furthermore, compared to the control, the number and length of roots in the cuttings increased by 13.2% and 14.2% respectively after the first pruning, while the number and length of roots in the cuttings increased by 18.4% and 17.9% respectively after the second pruning, indicating improved rooting rates in both pruning methods. Therefore, the Ginkgo cuttings from the mother plants after two prunings not only had higher quality cuttings but also stronger rooting ability.

[0086] 7. Post-cutting management: To prevent damage from extreme weather and high temperatures, erect bamboo arches at both ends of the cutting bed, firmly embedding the ends into the soil. Cover the top with a layer of plastic film, securing the film around the edges with soil and weights. Cover the plastic film with a shade net, and erect 2-meter-high cement pillars at the four corners of the cutting bed, constructing an additional shade structure to control the temperature of the cutting bed between 25-30℃. After mid-September, the shade netting on the plastic film can be removed to increase light penetration into the cutting bed. For pests and diseases, spray with a 0.2% carbendazim solution after cutting, once every 7 days. Regularly remove weeds around the cutting bed. After 60 days, open both ends of the lower plastic film for ventilation and hardening off the seedlings. Remove the plastic film after one week, and remove the upper shade structure after two weeks.

[0087] This study cultivated mother plants specifically for cuttings by treating 5-year-old Ginkgo trees with trunk pruning, two light prunings, and 1 mg / L BR spraying. The number of branches increased significantly by 3-4 times, while the diameter decreased slightly to approximately 0.5-0.6 cm and the length to approximately 35 cm. Leaf size, number of lobes, lobe depth, leaf quantity, and leaf water content all increased significantly, exhibiting juvenile characteristics. Furthermore, this treatment promoted the development of lateral buds and dormant buds in the mother plants, not only increasing the proportion of juvenile cuttings suitable for propagation but also improving the cutting yield from the mother plants.

[0088] Plant growth regulators were used to treat pruned, young cuttings to improve their rooting ability. Treatment with MeJA and Uniconazole-P in addition to 400 mg / L NAA effectively increased the rooting rate. Through screening rooting plant growth regulator combinations, the optimal combination of 1.5 μM MeJA + 100 mg / L Uniconazole-P + 400 mg / L NAA was found to be the most effective, achieving a rooting rate of 76.4%, significantly higher than the control treatment with 400 mg / L NAA. When this hormone combination was applied to large-scale Ginkgo cutting propagation, the rooting rate consistently exceeded 70%, indicating that this hormone combination can serve as a novel rooting agent for the rapid propagation of Ginkgo cuttings, improving both the speed and quality of rooting. Figure 14The images show the rooting status of the cuttings in a large-scale propagation nursery and after treatment with 1 mg / L BR (two light prunings and spraying), and 1.5 μM MeJA + 100 mg / L Uniconazole-P + 400 mg / L NAA in Example 2. (a) shows the rooting status of the cuttings after large-scale propagation; (b) shows the growth of the cuttings after transplanting to the field; and (c) shows the growth of the cuttings after transplanting to the field. Figure 14 It can be seen that the cuttings cultivated by the method of the present invention have good rooting effect, are suitable for large-scale cutting propagation, and can be promoted in production.

Claims

1. A method for large-scale propagation by using lateral branches of Ginkgo biloba mother plants as cuttings, characterized in that, It also includes a method for cultivating cuttings for large-scale propagation of ginkgo, which includes the following steps: (A1) Dwarfing of 4-5 year old ginkgo mother trees by cutting the trunk 20-30 cm above the ground to determine the height of the main trunk; (A2) After the trunk is cut off, the lateral branches that sprout are pruned multiple times. After each pruning, a plant growth regulator 6-benzyladenine solution or brassinolide solution is sprayed to promote the development of lateral branches of the mother plant. The multiple pruning is to remove 1 / 3 of the length of all newly sprouted lateral branches after the trunk is cut off, and then remove 1 / 3 of the length of the lateral branches after three months of growth, so as to ensure that the optimization degree of lateral branches, the number of sprouts and the thickness of branches are suitable for use as cuttings for rapid propagation. The concentration of the 6-benzyladenine solution is 100-300 mg / L and the concentration of the brassinolide solution is 1-3 mg / L. The method for large-scale cutting propagation includes the following steps: (B1) Cuttings were taken from lateral branches of the mother plant with a length of 40-50 cm and treated with a plant growth regulator. The diameter of the lateral branches of the mother plant was 0.5-1 cm. The plant growth regulator treatment of the cuttings involved soaking them in a solution containing 1-1.5 μmol / L methyl jasmonate and 10-100 mg / L paclobutrazol for at least 30 min, followed by treatment with a 400 mg / L paclobutrazol solution. Naphthalene Acetic acid After dipping in the solution for 6-10 seconds, drain off any excess water. (B2) Insert the treated cuttings into the substrate for culture.

2. The cutting propagation method according to claim 1, characterized in that, In step (B2), the substrate is a mixture of perlite and vermiculite with a mass ratio of perlite to vermiculite of 1:

1. The substrate is disinfected with a 0.2% solution of 40% carbendazim, and seedlings are planted one week after covering with a film.

3. The cutting propagation method according to claim 1, characterized in that, In step (B2), cuttings are taken and the cuttings are planted on the same day, while maintaining humidity throughout the process.

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