Method for inducing litchi girdling callus and peripheral bud differentiation in field
By using field spiral girdling and bud differentiation induction agent treatment, the problems of high browning and mortality in litchi tissue culture were solved, achieving efficient callus induction and adventitious bud differentiation, simplifying the operation process and reducing costs.
Patent Information
- Application Number
- CN202610102243.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-26
- Publication Date
- 2026-03-20
AI Technical Summary
Litchi tissue culture suffers from browning and high mortality, making it difficult to establish and maintain a stable culture system. Traditional in vitro methods are complex to operate and have low survival rates.
In the field, spiral girdling combined with bud differentiation inducer treatment was implemented. By girdling callus tissue and differentiating surrounding buds, the plant's own repair mechanism was utilized to avoid browning problems during in vitro culture and promote adventitious bud differentiation.
It simplifies the operation process, reduces costs, improves the efficiency of callus formation and the differentiation stability of adventitious buds, and significantly improves the survival rate of genetic variants.
Smart Images
Figure CN121694221A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant propagation technology, and in particular to a method for inducing the differentiation of callus and surrounding buds in litchi girdled wounds in the field. Background Technology
[0002] Polyploidy is a widespread phenomenon in the plant kingdom, especially in flowering plants. Compared to diploid plants, polyploid plants typically exhibit a range of superior agronomic traits, including stronger growth, larger organs, increased stress resistance, and higher yields. In fruit tree breeding, polyploid breeding has become an important method for creating new germplasm and selecting new varieties.
[0003] Conventional in vitro tissue culture combined with chemical mutagenesis is a classic method for inducing polyploidy. The optimal induction period is usually at the callus stage, because at this stage, cell differentiation is low and cell division is active, making it easy to treat with mutagens and obtain genetically stable polyploid cell lines, which can then differentiate into complete plants. However, litchi tissue culture technology itself presents significant challenges. Its explants and tissue culture seedlings are highly susceptible to browning and death during culture, making it difficult to establish and maintain a stable culture system. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, this invention provides a method for inducing the differentiation of litchi girdled callus and surrounding buds in the field, thereby effectively inducing and promoting the differentiation of litchi girdled callus into adventitious buds under field conditions.
[0005] The first aspect of this invention provides a method for inducing callus and peripheral bud differentiation in litchi girdled wounds in the field, comprising the following steps: Step 1: Cut the top of the upright lychee trunk 60cm above the ground, and make a spiral ring cut 1-3cm below the top cut, reaching the xylem layer. Step 2: Soak the defatted cotton in a bud differentiation inducer to obtain the induction substrate; Step 3: After covering the top section and girdling opening of the litchi branch with the induction matrix, wrap the litchi branch with plastic film and carry out bud differentiation treatment; Step 4: After the bud differentiation treatment is completed, the litchi branches are moisturized and kept away from light for 30-50 days to obtain adventitious buds.
[0006] Existing in vitro methods rely on in vitro culture systems, requiring aseptic treatment of explants, which is complex and results in low survival rates. This application directly induces wounds on litchi plants in the field, utilizing the plant's own repair mechanisms combined with localized treatment. This avoids the browning problem encountered in in vitro culture, achieving highly efficient in-situ callus induction in litchi branches, promoting adventitious bud differentiation around the girdling wound, and providing a stable source of material for field screening of polyploid plants. This method simplifies the traditional polyploid breeding process, reduces equipment and labor costs, and simultaneously improves the survival rate of genetic variants.
[0007] Optionally, in step one, the blade width of the spiral circumferential cutting is 2mm, and the circumferential cutting is 1-1.5 turns.
[0008] By employing the aforementioned technical solution, a combination of limiting the width of the incision and the number of girdling rings can create a ring-shaped blocking area with controllable width in the phloem. When the incision width is maintained at 2mm, it can effectively sever the phloem sieve tubes to prevent the downward transport of photosynthetic products, while also preserving sufficient cambium active area for callus formation. Combined with 1-1.5 girdling rings, a discontinuous blocking zone can be formed around the branch, ensuring the blocking effect while avoiding the interruption of the vascular system caused by complete girdling. This results in a suitable nutrient concentration gradient at both ends of the blocking zone, which can stimulate callus proliferation and maintain the water transport required for the branch's basal metabolism.
[0009] Optionally, in step one, the litchi branches are processed between April and October each year.
[0010] Optionally, in step one, the diameter of the lychee branch is 2-3 cm.
[0011] Through the above technical solution, when the branch diameter is in the range of 2-3cm, the ratio of its phloem to xylem is most suitable for cambium cell division. This ensures that a stable callus thin-walled cell layer can be formed at the cut site after the girdling operation, and also provides sufficient nutrient reserves for subsequent adventitious bud differentiation.
[0012] Optionally, the bud differentiation inducer is a mixed solution of chlorpyrifos and silver nitrate; the concentration of chlorpyrifos and silver nitrate in the bud differentiation inducer is 2 mg / L.
[0013] Through the above technical solution, the synergistic effect of chlorpyrifos and silver nitrate forms a dual regulatory mechanism in the bud differentiation process. The mixed solution of the two forms a dynamic equilibrium at a specific concentration, which maintains the continuity of cell division and reduces the probability of tissue browning. Under the adsorption of the absorbent cotton carrier, the inducer penetrates into the meristematic area through the xylem vessels, realizing the precise regulation of the local microenvironment. This achieves the directional regulation of the litchi bud differentiation process, significantly improves the uniformity of adventitious bud germination, and reduces the risk of tissue necrosis in the treated area.
[0014] Optionally, in step three, the bud differentiation treatment time is 24 hours.
[0015] The above technical solution provides a necessary time window for the inducer to fully contact the callus tissue, enabling cytokinins to effectively stimulate meristematic tissue formation while avoiding the risk of fungal growth caused by a long-term closed environment. It achieves precise regulation of physiological metabolic activities during bud differentiation, significantly reducing the probability of browning of branches while ensuring normal callus differentiation. At the same time, time control effectively avoids nutrient consumption caused by overtreatment, laying a stable foundation for the subsequent germination of adventitious buds.
[0016] Optionally, in step four, the moisturizing and light-shielding treatment step is as follows: remove the plastic and degreased cotton, wrap the top section and the ring-stripping opening with moist coconut coir; cover the surface of the coconut coir with light-shielding material and then tie it tightly.
[0017] Using the above technical solution, after bud differentiation treatment, the plastic film and absorbent cotton covering the branches are completely removed. Moist coconut coir is then evenly wrapped around the top section of the branch and the surface of the girdled area, forming a covering layer approximately 1.5-2 cm thick. Subsequently, a light-blocking material is placed over the coconut coir layer, achieving continuous moisture retention at the girdled area under light-protected conditions, promoting callus formation and adventitious bud differentiation. The coconut coir layer effectively buffers external temperature fluctuations, while the light-blocking material prevents auxin distribution disruption caused by light exposure; this dual effect increases the adventitious bud germination rate by approximately 40%.
[0018] Optionally, in step four, remove any sprouts at the base of the lychee branches every 7-10 days.
[0019] Using the above technical solution, 7-10 days after pruning, new shoots generally begin to grow from the base of the branches, with significant individual differences and inconsistent lengths. Removing shoots from other parts reduces growth competition and promotes the growth of shoots in the treated area, effectively solving the problem of nutrient dissipation caused by disordered growth at the base after girdling of litchi, and maintaining the cytokinin concentration in the apical callus tissue within the optimal threshold range.
[0020] Optionally, in step four, after adventitious buds sprout from the callus tissue and surrounding area at the girdled opening, the black plastic film and coconut coir are removed.
[0021] By using the above technical solution, when adventitious buds sprout, timely removal of the covering material can prevent the buds from rotting due to lack of oxygen or pathogen infection. At the same time, it promotes the new buds to be exposed to natural light for photosynthesis, thereby accelerating the lignification process and promoting the robust growth of new buds. It also avoids dehydration damage caused by premature exposure, thus achieving precise environmental control of the callus tissue and bud development stages.
[0022] The beneficial effects of this invention are as follows: by combining spiral ring cutting with bud differentiation inducer treatment, and with specific temperature control and light avoidance measures, callus formation and bud differentiation are directly promoted in the field, which effectively solves the problems of high browning rate and long acclimatization period in traditional in vitro culture technology. It has the advantages of shortening the culture period, reducing operating costs, improving callus formation efficiency and bud differentiation stability. Attached Figure Description
[0023] Figure 1 This is a flowchart of a method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field, provided by an embodiment of the present invention.
[0024] Figure 2 This is a schematic diagram of a litchi branch after girdling treatment according to Embodiment 1 of the present invention.
[0025] Figure 3 This is a schematic diagram of litchi branches used in the induced differentiation treatment of Embodiment 1 of the present invention.
[0026] Figure 4 This is a schematic diagram of the litchi branches used in the moisture-retaining and light-avoiding treatment in Embodiment 1 of the present invention.
[0027] Figure 5 This is a schematic diagram of lychee branches after 50 days of moisture-proof and light-proof treatment according to Embodiment 1 of the present invention.
[0028] Figure 6 This is a schematic diagram of the adventitious buds in Embodiment 1 of the present invention.
[0029] Figure 7 This is a schematic diagram showing the seedling formation of adventitious buds one month after removing the moisturizing and light-blocking material in Embodiment 1 of the present invention.
[0030] Figure 8 This is a schematic diagram of a litchi branch after 50 days of girdling treatment, as shown in Comparative Example 1 of this invention.
[0031] Figure 9 This is a schematic diagram of a litchi branch after being girdled and wrapped in plastic film for 50 days, as shown in Comparative Example 2 of this invention.
[0032] Figure 10 This is a schematic diagram of a litchi branch after being cut, girdled, and wrapped in plastic film for 50 days, as shown in Comparative Example 3 of the present invention.
[0033] Figure 11 This is a schematic diagram of a litchi branch after being girdled at 10cm above the ground for 50 days, as shown in Comparative Example 4 of this invention.
[0034] Figure 12 This is a schematic diagram of a litchi branch of Comparative Example 5 of the present invention after being girdled at 10cm above the ground and wrapped in plastic film for 50 days. Detailed Implementation
[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0036] Figure 1 This is a flowchart of a method for inducing callus and peripheral bud differentiation in litchi girdling wounds in the field, as provided in an embodiment of the present invention. Figure 1 As shown, the method includes the following steps: Between April and October each year, cut the top of upright litchi branches with a diameter of 2-3cm at 60cm above the ground, and make a spiral ring cut 1-3cm below the top cut. The width of the spiral ring cut is 2mm, and the ring cut is made 1-1.5 times, reaching the wood layer. Chlorpyrifos and silver nitrate were added to water and stirred until homogeneous to obtain a bud differentiation inducer. Then, defatted cotton was soaked in the bud differentiation inducer to obtain an induction substrate. After covering the top section and girdling opening of the litchi branch with the induction matrix, the litchi branch was wrapped with plastic film for bud differentiation treatment; After the bud differentiation treatment is completed, the litchi branches are moisturized and kept in the dark for 30-50 days to obtain adventitious buds.
[0037] The raw material information involved in this invention is shown in Table 1.
[0038]
[0039] The method for inducing callus and peripheral bud differentiation in litchi girdling wounds in the field, as described in the present invention, will be explained in detail below with reference to specific embodiments and comparative examples: Example Example 1
[0040] A method for inducing callus and peripheral bud differentiation in litchi girdling wounds in the field involves cutting off the top of the main stem of a litchi variety "9918" seedling progeny population with a diameter of 2-3 cm at a height of about 60 cm above the ground in early August each year. A spiral ring cut is then made 2 cm below the top section. The width of the spiral ring cut is 2 mm, the ring cut is made 1-1.5 times, the pitch is 2-3 cm, and the ring cut depth reaches the xylem layer. Dilute 2 mL of 0.1% chlorpyrifos solution in 498 mL of distilled water to prepare a 4 mg / L chlorpyrifos solution. Dilute 235 μL of 0.1 mol / L silver nitrate solution in 499.8 mL of distilled water to prepare a 4 mg / L silver nitrate solution. Store the solutions in a reagent bottle protected from light. Mix the two solutions 1:1 thoroughly to obtain the shoot differentiation inducer. Add 9 mL of the shoot differentiation inducer to 3 g of defatted cotton and allow it to fully absorb to obtain the induction matrix. The induction substrate was covered with the top section and the girdled opening of the litchi branch to a thickness of 0.5 cm. The litchi branch was then wrapped with plastic film and tied tightly with rope for bud differentiation treatment for 24 hours. After the bud differentiation treatment is completed, remove the plastic film and absorbent cotton. Soak 50g of coconut coir in 120g of water until fully absorbed, resulting in moist coconut coir. At this point, the moist coconut coir should not drip water when squeezed by hand. Wrap the top section and the girdled opening with the moist coconut coir, with a thickness of 1cm. Finally, wrap the litchi branch with black plastic film and tie it tightly. Keep the wrapped litchi branch in a natural environment for 50 days. Every 10 days, remove the black plastic film to observe the bud growth. When 5-10mm sprouts emerge from the base of the litchi branch, remove the sprouts. When adventitious buds emerge from the callus tissue and surrounding area of the girdled opening, remove the black plastic film and coconut coir to obtain the adventitious buds.
[0041] Example 2 A method for inducing callus and peripheral bud differentiation in litchi girdling wounds in the field involves cutting off the top of the main stem of the litchi variety "Kom" with a diameter of 2-3 cm about 60 cm above the ground in early October each year. A spiral girdling is then performed 2 cm below the top section. The width of the spiral girdling cut is 2 mm, the girdling is made 1-1.5 times, the pitch is 2-3 cm, and the girdling depth reaches the xylem. Dilute 2 mL of 0.1% chlorpyrifos solution in 498 mL of distilled water to prepare a 4 mg / L chlorpyrifos solution. Dilute 235 μL of 0.1 mol / L silver nitrate solution in 499.8 mL of distilled water to prepare a 4 mg / L silver nitrate solution. Store the solutions in a reagent bottle protected from light. Mix the two solutions 1:1 thoroughly to obtain the shoot differentiation inducer. Add 9 mL of the shoot differentiation inducer to 3 g of defatted cotton and allow it to fully absorb to obtain the induction matrix. The induction substrate was covered with the top section and the girdled opening of the litchi branch to a thickness of 0.5 cm. The litchi branch was then wrapped with plastic film and tied tightly with rope for bud differentiation treatment for 24 hours. After the bud differentiation treatment is completed, remove the plastic film and absorbent cotton. Soak 50g of coconut coir in 120g of water until fully absorbed, resulting in moist coconut coir. At this point, the moist coconut coir should not drip water when squeezed by hand. Wrap the top section and the girdled opening with the moist coconut coir, with a thickness of 1cm. Finally, wrap the litchi branch with black plastic film and tie it tightly. Keep the wrapped litchi branch in a natural environment for 50 days. Every 10 days, remove the black plastic film to observe the bud growth. When 5-10mm sprouts emerge from the base of the litchi branch, remove the sprouts. When adventitious buds emerge from the callus tissue and surrounding area of the girdled opening, remove the black plastic film and coconut coir to obtain the adventitious buds.
[0042] Comparative Example Comparative Example 1 A method for inducing callus and bud differentiation in litchi girdled wounds in the field differs from Example 1 in that the top of the litchi branch is not cut off, no bud differentiation inducer is applied, and no plastic film is wrapped around the girdled area.
[0043] Comparative Example 2 A method for inducing callus and bud differentiation in litchi girdled branches in the field differs from Example 1 in that the litchi branches are girdled without cutting off the top and without being treated with a bud differentiation inducer. After girdling, the girdled area is wrapped with plastic film.
[0044] Comparative Example 3 A method for inducing callus and bud differentiation in litchi girdled wounds in the field differs from Example 1 in that the litchi branches are girdled after the top is cut off, but the girdled area is not treated with a bud differentiation inducer, and a plastic film is wrapped around the girdled area after girdling.
[0045] Comparative Example 4 A method for inducing callus and bud differentiation in litchi girdled branches in the field differs from Example 1 in that the girdling is performed about 10cm above the ground on the litchi branch, without cutting off the top or treating it with a bud differentiation inducer, and the girdled area is wrapped with plastic film after girdling.
[0046] Comparative Example 5 A method for inducing callus and bud differentiation in litchi girdled branches in the field differs from Example 1 in that the girdling is performed about 10cm above the ground on the litchi branch, the top is not cut off, and no bud differentiation inducer is applied. The girdled area is not wrapped with plastic film after girdling.
[0047] Performance testing The adventitious buds provided in Examples 1-2 and Comparative Examples 1-5 of the present invention were subjected to the following performance tests, and Table 2 shows the performance test results.
[0048] Five to ten samples were taken from each example and comparative example, and the number of buds sprouting from litchi branches under moisturizing and light-protecting treatment was observed. Tables 2-3 show the statistical results of the number of buds sprouting in Example 1 and Example 2. Figures 2-7 This is a schematic diagram of the litchi branch in Example 1. Table 4-8 shows the statistical results of the number of buds in Comparative Examples 1-5. Figures 8-12 Table 9 shows schematic diagrams of litchi branches from Comparative Examples 1-5. Specifically, Comparative Example 5 is characterized by long roots but no buds. The average budding data and budding rate statistics of Examples 1-2 and Comparative Examples 1-5 are shown in Table 9.
[0049]
[0050] Table 3. Statistics on the number of buds in Example 2
[0051]
[0052]
[0053] Table 6 shows the germination count of Comparative Example 3.
[0054] .
[0055]
[0056]
[0057] Finally, it should be noted that the above descriptions are merely optional examples of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for inducing callus and surrounding bud differentiation in litchi girdled wounds in the field, characterized in that, Includes the following steps: Step 1: Cut the top of the upright lychee trunk 60cm above the ground, and make a spiral ring cut 1-3cm below the top cut, reaching the xylem layer. Step 2: Soak the defatted cotton in a bud differentiation inducer to obtain the induction substrate; Step 3: After covering the top section and girdling opening of the litchi branch with the induction matrix, wrap the litchi branch with plastic film and carry out bud differentiation treatment; Step 4: After the bud differentiation treatment is completed, the litchi branches are moisturized and kept away from light for 30-50 days to obtain adventitious buds.
2. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step one, the blade width for spiral circumferential cutting is 2mm, and 1-1.5 circumferential cuts are made.
3. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step one, the litchi branches are processed between April and October each year.
4. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step one, the diameter of the lychee branch is 2-3 cm.
5. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, The bud differentiation inducer is a mixed solution of chlorpyrifos and silver nitrate; the concentration of chlorpyrifos and silver nitrate in the bud differentiation inducer is 2 mg / L.
6. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step three, the bud differentiation treatment time is 24 hours.
7. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step four, the moisturizing and light-protecting treatment step is as follows: Remove the plastic and degreased cotton, and wrap the top section and girdling opening with moistened coconut coir; After covering the surface of the coconut coir with a light-blocking material, tie it tightly.
8. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 1, characterized in that, In step four, remove any sprouts at the base of the lychee branches every 7-10 days.
9. The method for inducing callus and surrounding bud differentiation in litchi girdling wounds in the field according to claim 7, characterized in that, In step four, after adventitious buds sprout from the callus tissue and surrounding area at the girdled opening, remove the black plastic film and coconut coir.