Cinnamomum migao bud stock grafting method

Abstract

The invention discloses a cinnamomum migao bud stock grafting method. The method comprises the steps of seed selection, treatment, stock cultivation, scion collection, grafting, transplanting, seedling stage management and the like. The grafting method adopts a bud seedling stock grafting technology, is an effective way for accelerating the improved variety process of cinnamomum migao, effectively shortens the seedling raising period, is low in seedling raising cost, good in seedling quality, capable of realizing fast growth and early fruiting, simple and convenient to operate, easy to master by the masses, and convenient to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of plant asexual reproduction and forest tree breeding technology, specifically relating to a method for grafting rice seedlings onto rootstock. Background Technology

[0002] Cinnamo mummigao H.W.Li, an evergreen tall tree belonging to the genus Cinnamo in the family Lauraceae, is a species endemic to southwestern China. Its dried fruit is one of the top ten Miao medicinal herbs of Guizhou and a famous authentic medicinal material. Currently, Cinnamo mummigao is mainly propagated by seed, but the offspring of seedlings show severe phenotypic segregation, making it difficult to maintain the superior traits of the parent plant. In addition, the cycle from germination to first fruiting of Cinnamo mummigao seedlings is as long as 8 years, and the early-stage field management and financial investment for planting seedlings are huge.

[0003] Grafting is an effective asexual reproduction method for maintaining the superior traits of forest trees. Grafting propagation utilizes the maturation effect of trees, ensuring that the grafted plant has only a vegetative stage and no juvenile stage, thus promoting earlier flowering and fruiting, and improving the quality of medicinal materials. Traditional grafting methods often use one-year-old or perennial seedlings grown in the field as rootstocks, which suffers from problems such as a short optimal grafting period, difficulty in matching the phenological stages of rootstock and scion, cumbersome operation, unstable survival rate, and long seedling cultivation cycle, seriously hindering the progress of improving forest tree varieties.

[0004] Bud grafting is a highly efficient and rapid asexual reproduction method that can shorten the breeding cycle and accelerate the promotion of improved varieties. Currently, grafting methods for camphor trees, such as *Cinnamomum camphora*, face many challenges. Therefore, conducting research on bud grafting of *Cinnamomum camphora* is an effective way to improve the speed of its improved variety propagation and achieve standardized cultivation. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a simple and effective method for grafting rice seedlings onto rootstocks.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for grafting rice seedlings onto rootstock includes the following steps (such as...) Figure 1 , Figure 2 ): (1) Rootstock cultivation: In mid-to-late October, collect healthy, plump, and mature rice seeds. After disinfecting and germinating the seeds, store them in sand with sawdust and water them regularly. When the embryo grows to 3-5cm in height and the cotyledons have not yet unfolded or are about to unfold, they can be used as rootstock for seedlings. (2) Scion collection: On the day of grafting, take healthy, plump, disease-free, and pest-free semi-lignified branches from the upper outer part of the tree crown as scions (immediately after cutting, put them into a self-sealing bag, sprinkle with water, and then store them in an insulated box). (3) Pre-grafting treatment: Sterilize the rootstock by soaking it in a 1:660 carbendazim solution (1g carbendazim to 660ml water) for 2 minutes, then rinse it with distilled water and cover it with a damp cloth for later use. Cut off the leaves and twigs from the scion, leaving the petiole, soak it in a 1:660 carbendazim solution for 10 minutes, then rinse it with distilled water and wrap it with a damp towel to keep it moist. (4) Grafting First, prepare the scion by making a diagonal cut on each side below the bud, ensuring a smooth, thin wedge-shaped cut, 1.5cm long, with 1-2 buds on each scion without leaves. Soak the scion in a hormone solution for 1 minute. When preparing the rootstock, make a vertical cut 2cm above the root with a sharp blade, cutting the rootstock 1.2-1.5cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion aligns with the cambium layer of the rootstock. Then, secure the graft union tightly, ensuring close contact between the scion and rootstock. Finally, apply a thick layer of wound sealant to the top of the scion. (5) Planting after grafting: Immediately plant the grafted seedlings in seedling trays filled with seedling substrate and place them in a climate chamber for cultivation. (6) Post-grafting management: Transplant the grafted seedlings into the climate chamber and water them regularly to keep the substrate moist; Transplanting grafted seedlings: Transplant the grafted seedlings when they have grown 2-3 true leaves. After transplanting, avoid direct sunlight and provide shade. Subsequent management: Since *Machilus macrantha* is a fertilizer-loving tree species, apply a small amount of nitrogen fertilizer after 50 days, and then add more as needed; for disease and pest control, spray with carbendazim regularly.

[0007] The disinfection and germination process described in step (1) of this invention is as follows: soaking the rice seeds in a 1% sodium hypochlorite solution for 20 minutes for disinfection, and then rinsing them with sterile water; soaking the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 hours to allow the seeds to fully absorb the nutrients.

[0008] The sand storage treatment described in step (1) of this invention is as follows: take a large plastic bucket, punch holes in the bottom of the bucket, then mix the rice seed and sawdust in a volume ratio of 1:3 and put them into the bucket, sprinkle water regularly to keep the sawdust moist, and store at room temperature.

[0009] In step (2) of this invention, the best time to collect scions is in May or June.

[0010] The carbendazim solution described in step (3) of this invention can also be replaced with an aqueous solution of methyl thiophanate.

[0011] The hormone mentioned in step (3) of this invention is any one of 200mg / L IBA, 50mg / L 2,4-D, and 50mg / L IAA.

[0012] Preferably, the hormone mentioned in step (3) of the present invention is 50 mg / L 2,4-D.

[0013] The seedling substrate mentioned in step (5) of this invention is: volume ratio of 5:2:2:1 = peat moss: perlite: coconut coir: peat moss.

[0014] In step (5) of this invention, the air humidity in the climate chamber for seedling cultivation is controlled at 80%-90%, the temperature is controlled at 22-28℃, and the light exposure time is 5-7h.

[0015] Preferably, in step (5) of the present invention, the air humidity in the climate chamber for seedling cultivation is controlled at 85%, the temperature is controlled at 25°C, and the light exposure time is 6 hours.

[0016] After the scions are cut, they are immediately placed in self-sealing bags, sprinkled with water, and stored in an insulated box; the grafting tools need to be disinfected with 75% alcohol.

[0017] Compared with the prior art, the present invention has the following advantages: 1. Short seedling cycle: Using seedlings as rootstock saves the time of rootstock cultivation compared to field grafting, effectively shortening the entire seedling cycle from seed to seedling and greatly improving breeding efficiency.

[0018] 2. High propagation efficiency: High seed utilization rate, low scion usage, suitable for industrialized and intensive seedling production, and accelerates the promotion of improved varieties.

[0019] 3. Easy to operate: The seedling rootstock has a uniform specification, the grafting operation is simple and quick, easy to master and promote, and it is not limited by location. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the process of grafting *Gnaphalium affine* seedlings onto rootstock according to the present invention.

[0021] Figure 2 This is a schematic diagram of the grafting operation steps of the present invention (wherein: A is a superior mother tree in the wild; B is the selection of scions; C is the cultivation of rootstocks; D is the preparation of scions; E is the alignment of the cambium layers of scions and rootstocks; F is the binding of scions and rootstocks; G is the planting of grafted seedlings).

[0022] Figure 3 Scions with different degrees of lignification (where: A is an unlignified scion; B is a semi-lignified scion).

[0023] Figure 4 Growth indicators of scions treated with different exogenous hormones (including: A. number of leaves; B. new shoot length; C. new shoot diameter).

[0024] Figure 5 Schematic diagrams of different grafting methods (where: A is cleft grafting; B is whip grafting; C is bud grafting).

[0025] Figure 6 The grafting interface morphology changes at different days (where: A is 5 days after grafting; B is 10 days after grafting; C is 15 days after grafting; D is 20 days after grafting; E is 25 days after grafting; F is 30 days after grafting; G is 50 days after grafting; H is 70 days after grafting).

[0026] Figure 7 Paraffin sections for the grafting healing process (where: A 10 days after grafting; B 15 days after grafting; C 50 days after grafting; D 70 days after grafting; St: rootstock; Sc: scion; Ci: callus).

[0027] Figure 8 The growth of grafted seedlings at different days (A: 20 days after grafting; B: 30 days after grafting; C: 50 days after grafting; D: 70 days after grafting). Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0029] Example 1: A method for grafting rice seedlings onto rootstock (1) Rootstock cultivation: In mid-October, collect healthy, plump, mature rice seedlings. After disinfection and germination, the seeds are buried in clean sawdust, mixed evenly in a 1:3 volume ratio, and watered regularly. When the embryo grows to 3-5cm in height, and the cotyledons have not yet unfolded or are about to unfold, it can be used as a rootstock for seedlings.

[0030] The above-mentioned seed disinfection and germination process is as follows: soak the rice seeds in a 1% sodium hypochlorite solution for 20 minutes for disinfection, then rinse them with sterile water; soak the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 hours to allow the seeds to fully absorb the sap.

[0031] (2) Scion collection: On the day of grafting, take healthy, vigorous, and disease-free semi-lignified branches from the upper outer part of the tree crown. The best time to collect these branches is from May to June.

[0032] (3) Pre-grafting treatment: Grafting tools were disinfected with 75% alcohol. The rootstock was sterilized by soaking in a 1:660 carbendazim solution (1g carbendazim to 660ml water) for 2 minutes, then rinsed with distilled water and covered with a damp cloth. The scion had its leaves and thin branches removed, leaving the petioles intact. It was then soaked in a 1:660 carbendazim solution for 10 minutes, rinsed thoroughly with distilled water, and wrapped in a damp towel to retain moisture.

[0033] (4) Grafting First, prepare the scion by making a diagonal cut on each side below the bud, ensuring a smooth, thin wedge-shaped cut approximately 1.5 cm long. Each scion should have 1-2 buds without leaves. Soak the scion in a 50 mg / L 2,4-D solution for 10 minutes. When preparing the rootstock, make a vertical cut about 2 cm above the root with a sharp blade, cutting 1.2-1.5 cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion aligns with the cambium layer of the rootstock. Then, tightly wrap the graft union with plastic wrap to ensure close contact between the scion and rootstock. Finally, apply a thick layer of wound sealant to the top of the scion.

[0034] (5) Planting after grafting: Immediately plant the grafted seedlings into seedling trays (37cm×30cm×7.5cm) filled with seedling substrate. Place them in a climate chamber for cultivation, maintaining an air humidity of 85%, a temperature of 25℃, and a light exposure of 6 hours. The seedling substrate has a volume ratio of 5:2:2:1: peat moss: perlite: coconut coir: peat moss.

[0035] (6) Post-grafting management: Transplant the grafted seedlings into a climate chamber. Water regularly to keep the substrate moist. Transplanting grafted seedlings: Transplant the grafted seedlings when they have 2-3 true leaves. After transplanting, avoid direct sunlight and provide shade. Subsequent management: Since *Machilus macrantha* is a fertilizer-loving species, apply a small amount of nitrogen fertilizer after 50 days, and then supplement as needed. For disease and pest control, spray with carbendazim regularly.

[0036] Example 2: A method for grafting rice seedlings onto rootstock (1) Rootstock cultivation: In mid-October, collect healthy, plump, mature rice seedlings. After disinfection and germination, the seeds are stratified in sand and cleaned. Mix the seeds with sawdust in a 1:3 volume ratio and water regularly. When the embryo grows to 3-5cm in height and the cotyledons have not yet unfolded or are about to unfold, the seeds can be used as rootstock for seedlings.

[0037] The above-mentioned seed disinfection and germination process is as follows: soak the rice seeds in a 1% sodium hypochlorite solution for 20 minutes for disinfection, then rinse them with sterile water; soak the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 hours to allow the seeds to fully absorb the sap.

[0038] (2) Scion collection: On the day of grafting, take healthy, vigorous, and disease-free semi-lignified branches from the upper outer part of the tree crown. The best time to collect these branches is from May to June.

[0039] (3) Pre-grafting treatment: Grafting tools should be disinfected with 75% alcohol. Bud rootstocks should be sterilized by soaking in a 1:660 solution of methyl thiophanate for 2 minutes, then covered with a damp cloth. Scions should have leaves and thin branches removed, retaining the petioles. They should be soaked in a 1:660 solution of methyl thiophanate for 10 minutes, then rinsed thoroughly with distilled water and wrapped in a damp towel to retain moisture.

[0040] (4) Grafting First, prepare the scion by making a diagonal cut on each side below the bud, ensuring a smooth, thin wedge-shaped cut approximately 1.5 cm long. Each scion should have 1-2 buds without leaves. Soak the scion in a 200 mg / L IBA solution for 1 minute. When preparing the rootstock, make a vertical cut about 2 cm above the root with a sharp blade, cutting the rootstock 1.2-1.5 cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion aligns with the cambium layer of the rootstock. Secure the graft union tightly, ensuring close contact between the scion and rootstock. Finally, apply a thick layer of wound sealant to the top of the scion.

[0041] (5) Planting after grafting: Immediately plant the grafted seedlings into seedling trays (37cm×30cm×7.5cm) filled with seedling substrate. Place them in a climate chamber for cultivation, maintaining an air humidity of 80%, a temperature of 25℃, and a light exposure of 6 hours.

[0042] (6) Post-grafting management: Transplant the grafted seedlings into a climate chamber. Water regularly to keep the substrate moist. Transplanting grafted seedlings: Transplant the grafted seedlings when they have 2-3 true leaves. After transplanting, avoid direct sunlight and provide shade. Subsequent management: Since *Machilus macrantha* is a fertilizer-loving species, apply a small amount of nitrogen fertilizer after 50 days, and then supplement as needed. For disease and pest control, spray with carbendazim regularly.

[0043] Example 3: A method for grafting rice seedlings onto rootstock (1) Rootstock cultivation: In mid-October, collect healthy, plump, mature rice seedlings. After disinfection and germination, store the seeds in clean river sand mixed with water at a volume ratio of 1:3, and water regularly. When the embryo grows to 3-5 cm in height and the cotyledons have not yet unfolded or are about to unfold, it can be used as a rootstock for seedlings.

[0044] The above-mentioned seed disinfection and germination process is as follows: soak the rice seeds in a 1% sodium hypochlorite solution for 20 minutes for disinfection, then rinse them with sterile water; soak the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 hours to allow the seeds to fully absorb the sap.

[0045] (2) Scion collection: On the day of grafting, take healthy, vigorous, and disease-free semi-lignified branches from the upper outer part of the tree crown. The best time to collect these branches is from May to June.

[0046] (3) Pre-grafting treatment: Grafting tools were disinfected with 75% alcohol. Bud rootstocks were sterilized by soaking in a 1:660 solution of methyl thiophanate (1 g methyl thiophanate diluted in 660 ml of water) for 2 minutes, then covered with a damp cloth. Scions were trimmed of leaves and twigs, retaining the petioles, and soaked in a 1:660 solution of methyl thiophanate for 10 minutes, then rinsed thoroughly with distilled water and wrapped in a damp towel to retain moisture.

[0047] (4) Grafting First, prepare the scion. Make two diagonal cuts on either side below the bud, ensuring a smooth, thin wedge-shaped cut, about 1.5 cm long. Each scion should have 1-2 buds without leaves. Dip the scion in a 50 mg / L IAA solution for 1 second. When preparing the rootstock, make a vertical cut about 2 cm above the root with a sharp blade, cutting 1.2-1.5 cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion aligns with the cambium layer of the rootstock. Then, wrap the graft union tightly with plastic wrap, especially at the top and bottom. Ensure the bud side of the scion is aligned with the cambium layer of the rootstock, and wrap the graft union tightly to ensure close contact between the scion and rootstock. Finally, apply a thick layer of wound sealant to the top of the scion.

[0048] (5) Grafting and planting: Immediately plant the grafted seedlings into seedling trays (37cm×30cm×7.5cm) filled with seedling substrate. Place them in a climate chamber for cultivation, maintaining an air humidity of 90%, a temperature of 23℃, and a light exposure of 5 hours.

[0049] (6) Post-grafting management: Transplant the grafted seedlings into a climate chamber. Water regularly to keep the substrate moist. Transplanting grafted seedlings: Transplant the grafted seedlings when they have 2-3 true leaves. After transplanting, avoid direct sunlight and provide shade. Subsequent management: Since *Machilus macrantha* is a fertilizer-loving species, apply a small amount of nitrogen fertilizer after 50 days, and then supplement as needed. For disease and pest control, spray with carbendazim regularly.

[0050] Example 4: A method for grafting rice seedlings onto rootstock (1) Rootstock cultivation: In mid-October, collect healthy, plump, mature rice seedlings. After disinfecting and germinating the seeds, mix them evenly with clean, moist sand at a volume ratio of 1:3, and water regularly. When the embryo grows to 3-5 cm in height, and the cotyledons have not yet unfolded or are about to unfold, it can be used as a rootstock for seedlings.

[0051] The above-mentioned seed disinfection and germination process is as follows: soak the rice seeds in a 1% sodium hypochlorite solution for 20 minutes for disinfection, then rinse them with sterile water; soak the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 hours to allow the seeds to fully absorb the sap.

[0052] (2) Scion collection: On the day of grafting, take healthy, vigorous, and disease-free semi-lignified branches from the upper outer part of the tree crown. The best time to collect these branches is from May to June.

[0053] (3) Pre-grafting treatment: Grafting tools were disinfected with 75% alcohol. Rootstocks were sterilized by soaking in a 1:660 solution of methyl thiophanate (1 g methyl thiophanate diluted in 660 ml of water) for 2 minutes, then covered with a damp cloth. Scions were trimmed of leaves and twigs, retaining the petioles, and soaked in a 1:660 solution of methyl thiophanate (1 g methyl thiophanate diluted in 660 ml of water) for 10 minutes, then rinsed thoroughly with distilled water and wrapped in a damp towel to retain moisture.

[0054] (4) Grafting First, prepare the scion. Make two oblique cuts on either side below the bud, ensuring a smooth, thin wedge-shaped cut, about 1.5 cm long. Each scion should have 1-2 buds without leaves. Dip the scion in a 50 mg / L 2,4-D solution for 1 minute. When preparing the rootstock, make a vertical cut about 2 cm above the root with a sharp blade, cutting 1.2-1.5 cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion aligns with the cambium layer of the rootstock. Then, wrap the graft union tightly with plastic wrap, especially at the top and bottom. Ensure the bud side of the scion is aligned with the cambium layer of the rootstock, and wrap the graft union tightly to ensure close contact between the scion and rootstock. Finally, apply a thick layer of wound sealant to the top of the scion.

[0055] (5) Grafting and planting: Immediately plant the grafted seedlings into seedling trays (37cm×30cm×7.5cm) filled with seedling substrate. Place them in a climate chamber for cultivation, maintaining an air humidity of 90%, a temperature of 28℃, and a light exposure of 7 hours.

[0056] (6) Post-grafting management: Transplant the grafted seedlings into a climate chamber. Water regularly to keep the substrate moist. Transplanting grafted seedlings: Transplant the grafted seedlings when they have 2-3 true leaves. After transplanting, avoid direct sunlight and provide shade. Subsequent management: Apply a small amount of nitrogen fertilizer after 50 days, and then add more as needed. Pest and disease control: Spray with carbendazim regularly.

[0057] Experiment 1: Grafting Experiments with Different Degrees of Lignification 1. Test Methods A single-factor experimental design was used to investigate the effect of different lignification levels of scions on the grafting survival rate of *Gnaphalium affine* buds onto rootstock. Two treatments were set up: non-lignified and semi-lignified. Each treatment was used to graft 30 seedlings, with three replicates. Scions with different lignification levels are shown below. Figure 3 As shown.

[0058] 2. Measurement Indicators Grafting survival rate: The survival rate is calculated 30 days after grafting. Survival rate = (Number of surviving plants / Total number of grafted plants) * 100%.

[0059] 3. Experimental Results and Conclusions As shown in Table 1, the survival rate of non-lignified scions was 0%, while the survival rate of semi-lignified scions was 66.67%. This indicates that the degree of lignification of the scion has a significant impact on the grafting survival rate, and semi-lignified or more mature scions should be selected for grafting.

[0060] Experiment 2: Scion Treatment Experiment with Different Exogenous Hormones 1. Test Methods A single-factor experimental design was used to investigate the effects of different exogenous hormone treatments on the survival rate and growth indicators of grafted scions of *Gnaphalium affine* rootstock. Three treatments were set up: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D). Each treatment was used to graft 30 scions, and the results were repeated 3 times.

[0061] 2. Measurement Indicators Grafting survival rate: The survival rate is calculated 30 days after grafting. Survival rate = number of surviving plants / total number of grafted plants * 100%; Growth indicators: number of leaves, length of new shoots, and thickness of new shoots.

[0062] 3. Experimental Results and Conclusions 3.1 Effects of different exogenous hormone treatments on scion survival rate Note: In the table, a and b represent significant differences in scions treated with different exogenous hormones (p<0.05). The survival rate of grafted *Millettia divaricata* seedlings under different hormone treatments was analyzed using the chi-square test. Table 3 shows that there were significant differences in graft survival rates among the three hormone treatments (p<0.05). 2,4-D treatment had the best effect (70%), followed by IBA (65.56%), while IAA had the worst effect (50%).

[0063] In promoting graft survival, both IBA and 2,4-D were significantly better than IAA, but there was no significant difference in effectiveness between IBA and 2,4-D.

[0064] 3.2 Effects of different exogenous hormone treatments on the growth indicators of grafted seedlings One-way ANOVA was used to analyze the effects of different hormone treatments on the number of leaves, shoot length and shoot thickness of grafted *Gnaphalium affine* seedlings. No significant differences were found among the different hormone treatments (p>0.05).

[0065] The results are as follows Figure 4 As shown, after 90 days of growth, IBA and 2,4-D had a better promoting effect on the number of leaves, the length of new shoots and the thickness of new shoots in grafted seedlings of *Millettia dielsiana* than IAA-treated grafted seedlings.

[0066] Experiment 3: Experiments with different grafting treatments 1. Test Methods A two-factor experiment was conducted to investigate the effects of different treatment combinations on the survival rate of grafted *Gnaphalium affine* seedlings (see Table 4). Each treatment involved grafting 30 seedlings, with three replicates.

[0067] 2. Grafting method Cleft grafting: Cut the rootstock horizontally, and then make a vertical cut on one side of the cut surface. Shape the scion into a wedge, insert it into the cut in the rootstock, align the cambium layers, and secure it with grafting film.

[0068] Budding method: Make a "T" shaped cut on the rootstock, insert the bark of the scion with the bud, bind it tightly, and expose the bud.

[0069] Cleft grafting: After the rootstock is cut horizontally, it is split vertically in the center of the cut surface. The scion is cut into a double-sided wedge shape, inserted into the split, the cambium layers are aligned, and then bound.

[0070] Budding: Make a "T"-shaped cut on the rootstock, insert the scion with a bud, bind it tightly, leaving the bud exposed. Grafting method is shown in the appendix. Figure 5 As shown.

[0071] 3. Measurement Indicators Grafting survival rate: The survival rate is calculated 30 days after grafting. Survival rate = (Number of surviving plants / Total number of grafted plants) * 100%.

[0072] 4. Experimental Results and Conclusions 4.1 Effects of different grafting treatments on survival rate Note: Different letters in the table represent significant differences in survival rates between different grafting treatments (p<0.001). The survival rate of *Millettia divaricata* grafted seedlings under different grafting treatments was analyzed using the chi-square test. Table 5 shows that there were highly significant differences in grafting survival rates among different treatments (p<0.001). The two grafting methods without hormone treatment had the lowest survival rates (20%–26.67%); 2,4-D + cleft grafting and IBA + cleft grafting had the highest survival rates (70% and 65.56%, respectively). Hormone treatment (IAA, IBA, 2,4-D) combined with cleft grafting generally yielded better results; among grafting methods, cleft grafting combined with hormones showed the best performance. Therefore, grafting survival rate is affected by both the type of hormone and the grafting method. It is recommended to use 2,4-D or IBA combined with cleft grafting. Among these, 2,4-D treatment had the best effect (70%), followed by IBA (65.56%), and IAA had the worst effect (50%).

[0073] Morphological changes in grafted seedlings Five days after grafting, the rootstock and scion begin to adhere together, and a darker brown isolation layer can be observed at the cambium layer of the graft union. Figure 6 A); On the 10th day after grafting, a thin layer of callus tissue was observed at the grafting site ( Figure 7 A). 15 days after grafting, the graft union is full of callus tissue, and the rootstock and scion are tightly joined. Figure 6 C Figure 7 B); After 20 days, the rootstock and scion are tightly connected, and the gap at the interface can be seen to be filled with new tissue, which is light green in color. Figure 6 D); 20 days later, the leaves had fully emerged, and the graft union had healed well. Figure 6 E, Figure 8 A); 30 days later, the graft union heals tightly, the grafted plant grows vigorously, and the new shoots elongate ( Figure 6 F, Figure 8 B); 50-70 days later, the leaves become larger, with 5-6 leaves, and the rootstock and scion have completely fused into a single unit. Figure 6 GH, Figure 7 CD Figure 8 CD).

[0074] The above examples and experiments show that the bud grafting technique has broken free from the long-standing constraints of natural conditions and weather on seedling grafting. The grafting technique is easy to master, saves scions, and effectively shortens the seedling cultivation time.

[0075] The foregoing has illustrated the principles and implementation methods of this invention through specific embodiments and experiments. These embodiments are intended to aid in understanding the core ideas of this invention and are not intended to limit its practical application. Those skilled in the art can make appropriate adjustments and changes to the specific implementation and application scope based on the content disclosed in this invention. The above content demonstrates the basic principles, main features, and advantages of this invention. It should be noted that this invention is not limited to the above embodiments; the embodiments and related descriptions in the specification are merely illustrative of the principles of this invention. Various modifications and improvements can be made to this invention without departing from its spirit and scope, and all such modifications and improvements fall within the protection scope defined by the claims and their equivalents.

Claims

1. A method for propagating *Gnaphalium affine* seedlings by grafting onto rootstock, characterized in that, Includes the following steps: (1) Rootstock cultivation: In mid-to-late October, collect healthy, plump, and mature rice seeds. After disinfecting and germinating the seeds, store them in sand and water them regularly. When the embryo grows to 3-5 cm in height and the cotyledons have not yet unfolded or are about to unfold, they can be used as rootstock for seedlings. (2) Scion collection: On the day of grafting, take healthy, plump, disease-free, and pest-free semi-lignified branches from the upper part of the tree crown as scions. (3) Pre-grafting treatment: Soak the rootstock in a carbendazim solution for 2 minutes to sterilize and disinfect it. Rinse it with distilled water and cover it with a damp cloth for later use. Cut off the leaves and twigs of the scion, leaving the petiole. Soak it in a carbendazim solution for 10 minutes, then rinse it with distilled water and wrap it with a damp towel to keep it moist. (4) Grafting First, prepare the scion by making a diagonal cut on each side below the bud, ensuring a smooth, thin wedge-shaped cut, 1.5 cm long, with 1-2 buds on each scion without leaves. Soak the scion in a hormone solution for 1 minute. When preparing the rootstock, make a vertical cut 2 cm above the root with a sharp blade, cutting the rootstock 1.2-1.5 cm deep. Insert the wedge-shaped cut into the incision, ensuring the bud side of the scion is aligned with the cambium layer of the rootstock. Then, wrap the graft union tightly with plastic wrap to ensure close contact between the scion and rootstock. Finally, apply a thick layer of wound healing paste to the top of the scion. (5) Planting after grafting: Immediately plant the grafted seedlings in seedling trays filled with seedling substrate and place them in a climate chamber for cultivation. (6) Post-grafting management: Transplant the grafted seedlings into the climate chamber and water them regularly to keep the substrate moist; Transplanting grafted seedlings: Transplant the grafted seedlings when they have grown 2-3 true leaves. After transplanting, avoid direct sunlight. Subsequent management: Apply a small amount of nitrogen fertilizer after 50 days; for disease and pest control, spray with carbendazim regularly; The 1:660 carbendazim aqueous solution is 1g carbendazim diluted in 660ml of water.

2. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, The disinfection and germination process described in step (1) is as follows: soak the rice seeds in a 1% sodium hypochlorite solution for 20 min for disinfection, then rinse them with sterile water; soak the seeds in a 200 mg / L gibberellin solution under light-protected conditions for 72 h to allow the seeds to fully absorb the nutrients.

3. The method for propagating seedlings by grafting onto rootstock according to claim 2, characterized in that, The sand storage treatment described in step (1) is as follows: Take a large plastic bucket, punch holes in the bottom of the bucket, then mix the rice seeds and sawdust in a volume ratio of 1:3 and put them into the bucket. Sprinkle water on them regularly to keep the sawdust moist and store them at room temperature.

4. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, Step (2) The best time to collect scions is in May or June.

5. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, The carbendazim solution described in step (3) can also be replaced with an aqueous solution of methyl thiophanate.

6. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, The hormone mentioned in step (3) is any one of 200 mg / L IBA, 50 mg / L 2,4-D, or 50 mg / L IAA.

7. The method for propagating seedlings by grafting onto rootstock according to claim 6, characterized in that, The hormone mentioned in step (3) is 50 mg / L 2,4-D.

8. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, The seedling substrate mentioned in step (5) is: volume ratio of 5:2:2:1 = peat moss: perlite: coconut coir: peat moss.

9. The method for propagating seedlings by grafting onto rootstock according to claim 1, characterized in that, In step (5), the humidity of the air in the climate chamber for seedling cultivation is controlled at 80%-90%, the temperature is controlled at 22-28℃, and the light exposure time is 5-7 h.

10. The method for propagating seedlings by grafting onto rootstock according to claim 9, characterized in that, In step (5), the humidity of the air in the climate chamber for seedling cultivation is controlled at 85%, the temperature is controlled at 25℃, and the light exposure time is 6 hours.

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