A high-throughput manual micrografting method for guava seedlings

Abstract

The present invention discloses a high-throughput manual micro-grafting method for guava seedlings, the method comprising: step 1: using a hole mold to make a plurality of holes in a disposable culture dish, pouring a culture medium, waiting for it to be completely cooled, taking out the hole mold, and then placing a cutting guide groove at the position of the hole; step 2: according to the different uses of the rootstock or scion, the seeds are sequentially dotted into the holes in the culture dish described in step 1, one seed per hole; step 3: cultivating the seedlings grown in the culture dish; step 4: micro-grafting the seedlings in the culture dish. The present invention overcomes the shortcomings of easy damage, low survival rate, poor stock-scion joint accuracy, poor uniformity of experimental treatment, etc. of traditional laboratory seed seedling micro-grafting, significantly improves the speed of micro-grafting, and provides a guarantee for the study of grafting affinity mechanism and small-batch production of micro-grafted seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of grafting, and particularly relates to a high-throughput manual micro-grafting method for guava seedlings. Background Art

[0002] The research objects of traditional micrografting in the study of rootstock-scion interaction mechanism are mainly herbaceous model plants, Arabidopsis thaliana and tomato. However, the grafting of these model plants is significantly different from the rootstock-scion interaction process of woody plants such as fruit trees, especially tropical fruit trees. In order to study the rootstock-scion interaction mechanism, it is necessary to establish a stable and rapid micrografting method for fruit trees.

[0003] Guava blooms and bears fruit all year round. A single fruit has a large number of seeds in a small size, which is suitable for batch planting in culture dishes. The hypocotyl diameter of the seedlings is small (1mm), the degree of lignification is low, and the size is similar to that of tomato and eggplant seedlings in culture medium, which is suitable for comparative studies of herbaceous and woody plants. Therefore, guava seedlings grown in culture dishes are very good research objects for the interaction mechanism of grafted rootstocks and scions.

[0004] In addition, there is also a demand for grafting in production, especially by grafting strawberry guava to obtain a certain resistance to root-knot nematodes. Although the main purpose of this invention is to facilitate mechanism-related research, it also has certain practical application value in production.

[0005] Because the growth rate of guava seeds is significantly slower than that of Arabidopsis and tomato, the process of re-healing of the fracture after grafting and the formation of vascular bundles often takes more than 4 weeks. The fracture has poor adhesion, low survival rate, and very high bacterial infection rate.

[0006] At present, the conventional method of disinfecting materials with sodium hypochlorite + 70% alcohol in laboratory culture dish micrografting is not very suitable. In addition, the methods mainly used for micrografting for scientific research purposes, such as sleeve, excised cotyledons placed on sterile filter membranes for flat grafting, or culture medium slant grafting, are not suitable for guava seed seedling micrografting because of their high technical requirements for scientific researchers, poor joint accuracy, and low efficiency. In addition, most of these methods are very unfriendly to time-lapse photography observation and sampling after early grafting. Summary of the invention

[0007] In view of this, the main purpose of the present invention is to provide a high-throughput manual micro-grafting method for guava seedlings.

[0008] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0009] The embodiment of the present invention provides a high-throughput manual micro-grafting method for guava seedlings, the method comprising:

[0010] Step 1: Use a hole mold to make a number of holes in a disposable culture dish, pour the culture medium, wait for it to completely cool, take out the hole mold, and then put a cutting guide groove at the location of the hole;

[0011] Step 2: According to the different uses of the rootstock or scion, the seeds are sequentially placed into the holes in the culture dish described in step 1, one seed per hole;

[0012] Step 3: Cultivating the seedlings grown in the culture dish;

[0013] Step 4: performing micro-grafting on the seedlings in the culture dish.

[0014] In the above scheme, the method also includes pre-treating the seeds before step 1, specifically: packing the guava seeds used as rootstocks or scions into different centrifuge tubes, filling them with distilled water, bathing them in 95°C water for 1 minute, cooling them naturally, standing them in the dark for 48 hours, and then soaking them in 0.2% sodium hypochlorite for 20 minutes, vacuuming them during the soaking process, washing them with distilled water, placing them in centrifuge tubes, and standing them in the dark at 4°C for later use.

[0015] In the above scheme, after the seeds are pretreated, the method further comprises culturing the seeds used as rootstocks or scions through a culture medium formula, wherein the culture medium is sterilized by high pressure at 121° C. for 20 minutes, taken out and cooled to 60° C. before being poured into a culture dish for solidification.

[0016] In the above scheme, the culture medium formula is nutrient matrix, agar 2% w / v, and sucrose 5 g / L; the nutrient matrix adopts WPM woody culture medium, MS culture medium, or 1 / 2 strength MS culture medium.

[0017] In the above scheme, step 3 is specifically as follows: placing the culture dish vertically, culturing in the dark at 25°C in a light-shielded environment for about 15 days, transferring the hypocotyl to a tissue culture rack for normal growth after it grows upright to 1 cm in length, evenly illuminating the top, and growing in a normal culture environment. After one day of growth until two cotyledons are fully expanded, a culture dish containing seedlings is obtained.

[0018] In the above scheme, step 4 is for self-grafting treatment, specifically: take out the culture dish containing the seedlings from the culture rack, place it in the clean bench, open the culture dish cover, cover the stabilizing press sheet, and use the sterilized double-sided blade to quickly cut from the left to the right side of the cutting guide groove. After cutting, tilt the culture dish 45° to make the stock and scion fit completely, gently cover the culture dish cover, seal the plate, let it stand for 12 hours, and place it vertically back on the culture rack.

[0019] In the above scheme, when step 4 is a hetero-grafting process, specifically: take out the culture dish for the rootstock seedling and the culture dish for the seedling of the scion from the culture rack respectively, place them in the clean bench, open the culture dish cover, cover the stabilizing press sheet, and use the sterilized double-sided blade to quickly cut from the left to the right side of the cutting guide groove. After cutting, close the culture dish cover and let it stand.

[0020] Compared with the prior art, the present invention overcomes the shortcomings of traditional laboratory seed seedling micro-grafting, such as easy damage, low survival rate, poor stock-scion joining accuracy, and poor uniformity of experimental treatment. It significantly improves the speed of micro-grafting and provides a guarantee for the study of grafting affinity mechanism and small-batch production of micro-grafted seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to disclose a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 A flow chart of a high-throughput manual micro-grafting method for guava seedlings is provided for an embodiment of the present invention.

[0023] Figure 2 An exploded view of a culture dish in a high-throughput manual micro-grafting method of guava seedlings is provided for an embodiment of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The same or similar numbers in the drawings of this embodiment correspond to the same or similar parts; in the description of the present invention, it should be understood that the directions or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limitations on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0026] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, article or device. In the absence of more restrictions, an element defined by the sentence "includes a ..." does not exclude the existence of other identical elements in the process, article or device including the element.

[0027] The embodiment of the present invention provides a high-throughput manual micro-grafting method for guava seedlings, such as Figure 1 As shown, the method includes:

[0028] Step 1: Use a hole mold 1 to make a number of holes in a culture dish 4, pour culture medium, wait for it to completely cool, take out the hole mold 1, and then put a cutting guide groove 2 at the position of the hole;

[0029] Step 2: according to the different uses of the rootstock or scion, the seeds are sequentially placed into the holes in the culture dish 4 described in step 1, one seed in each hole;

[0030] Step 3: Cultivating the seedlings grown in the culture dish 4;

[0031] Specifically, the culture dish 4 is placed vertically and cultured in the dark at room temperature for 15 days in a light-shielded manner. When the hypocotyl grows upright to 1 cm in length, it is transferred to the tissue culture rack for normal growth. The top is evenly illuminated and grown in a normal culture environment. After one day when the two cotyledons are fully expanded, a culture dish 4 containing seedlings is obtained.

[0032] The structure of the culture dish 4 is such that the size of the bottom plate is 10×10 cm or 13×13 cm. However, if the interface between the grafted rootstock and scion needs to be continuously observed, a culture dish cover with an observation hole is required.

[0033] The normal culture environment is a fluorescent lamp with a color temperature of 5600K, a photoperiod of 16 hours of light and 8 hours of dark culture, a temperature of 25°C, and a humidity of 1000 l x.

[0034] Step 4: Perform micro-grafting on the seedlings in the culture dish 4.

[0035] Specifically, when step 4 is a self-grafting process, Figure 2 As shown, the culture dish 4 containing the seedlings is taken out from the culture rack, placed in the clean bench, the culture dish cover is opened, the stabilizing press sheet 3 is covered, and the sterilized double-sided blade is quickly cut from the left to the right side of the cutting guide groove 2. After cutting, the culture dish 4 is tilted 45° to make the stock and scion fit completely. The culture dish cover is gently covered, the plate is sealed, and it is left to stand for 12 hours, and then placed vertically back on the culture rack.

[0036] Check whether the rootstock and scion are tightly connected after cutting. If they are loose, use tweezers to gently press the cotyledons to make the rootstock and scion fit completely.

[0037] The bottom of the hole mold 1 is provided with a step-like structure for forming a culture medium in a culture dish, and one side is provided with a plurality of partitions 11 for forming holes arranged at intervals.

[0038] The cutting guide groove 2 is a flat plate, and the flat plate is provided with cutting grooves 21 in a crisscross pattern to match the holes.

[0039] When the step 4 is a hetero-grafting treatment, the culture dish 4 for the rootstock seedling and the culture dish 4 for the seedling of the scion are taken out from the culture rack respectively, placed in the clean bench, the culture dish cover is opened, and the stabilizing pressing sheet 3 is covered, and the double-sided blades that have been sterilized are used to quickly cut from the left to the right side of the cutting guide groove 2. After cutting, the culture dish cover is closed and left to stand.

[0040] Pinch the protrusions on both sides of the cutting guide groove 2 with both hands, slowly lift the upper part cut out from the test seedling culture dish 4 of the rootstock, transfer it, and discard it.

[0041] Open the cover of the seedling culture dish for the stock, open the cover of the seedling culture dish for the scion, pinch the protrusions on both sides of the cutting guide groove 2 with both hands, slowly lift the scion part cut from the scion seedling culture dish 4, transfer it to the seedling culture dish 4 providing the stock, and replace the upper part that was originally cut and discarded.

[0042] The ideal state is that all stock and scion fit together. If there is any looseness, use tweezers to gently press the cotyledon to make the stock and scion fit together completely. Gently cover the culture dish lid, seal the plate, let it stand for 12 hours, and place it vertically back on the culture rack for subsequent observation or camera time-lapse photography. Use a culture dish with an observation window opening 4.

[0043] Furthermore, the method also includes pre-treating the seeds before step 1, specifically: packing the guava seeds used as rootstocks or scions into different centrifuge tubes, filling them with distilled water, and placing them in a 95°C water bath for 10 minutes, cooling them naturally, and standing them in the dark for 48 hours. Thereafter, soaking them in a 0.2% sodium hypochlorite solution with 20 drops of Tween-20 per liter for 20 minutes for disinfection, and vacuuming the seeds during the soaking process so that the liquid can penetrate into the seeds through the germination pores to fully infiltrate them to obtain a better disinfection effect. After repeatedly washing with distilled water, the seeds are placed in centrifuge tubes and stored in the dark at 4°C for later use.

[0044] After the seeds are pretreated, the method further comprises culturing the seeds as rootstocks or scions through a culture medium formula, wherein the culture medium is sterilized by high pressure at 121°C for 20 minutes, and then taken out and cooled to 60°C before being poured into a culture dish 4 for solidification. 。

[0045] Specifically, the culture medium formula is nutrient matrix, agar 2% w / v, and sucrose 5 g / L; the nutrient matrix adopts WPM woody culture medium, MS culture medium, or 1 / 2 strength MS culture medium.

[0046] The grafting speed and high throughput of the present invention are compared with the traditional manual grafting of seedlings. The traditional seedling grafting requires materials with a certain degree of lignification to have stem segments with sufficient strength for cutting, so it takes one year from sowing to grafting. In addition, during grafting, the grafting sealing film or bagging is required to retain water at the fracture, which makes the steps complicated and can only be performed one by one by individuals or by multiple people.

[0047] The present invention uses guava seedlings with two cotyledons just opened, and does not require a certain degree of lignification to ensure the strength of the stem. The whole process from seed treatment and spot sowing to grafting takes 35-45 days, which is only 1 / 10 of the time of the former.

[0048] Because the seedlings grow evenly in the culture medium, the cutting of planting and grafting operations is carried out in the seedling hole. The seedlings on the whole plate of the culture dish 4 are used as the operation object, and several or dozens of seedlings can be cut off at one time. For hetero-grafting treatment, the rootstock and scion are combined in batches by overall replacement, and gravity is mainly used to make the rootstock and scion fit together (vertically placed after grafting), without the need for additional covering or bagging for water retention (the culture dish 4 is a relatively closed space), and the efficiency is greatly improved.

[0049] The present invention adopts guava seedlings with two cotyledons just opened, and only needs 35-45 days from seed treatment and spot sowing to grafting, which is only 1 / 10 of the time compared with the traditional guava seed seedling grafting which needs to select seedlings about half a year to one year old. In the cultivation process, after the seeds are sown on the culture medium, it only needs to be shading treated once and the culture dish transferred once before grafting. In addition, during grafting, there is no need to perform traditional grafting film sealing and bundling joints, thereby reducing the amount of operations.

[0050] The early shading treatment can ensure that the initial growth of the hypocotyl is upright, and the growth at the beginning of illumination and the subsequent grafting are all carried out in the transparent culture dish 4, which can ensure a certain degree of constant humidity and temperature, and the growth of the plant can be monitored throughout the process, unlike the traditional seed seedling grafting that requires the use of bagging and sealing film to retain water, which makes it impossible to track the interaction between the joint of the rootstock and scion.

[0051] Embodiment 1 of the present invention provides a high-throughput manual micro-grafting method for guava seedlings, the method comprising:

[0052] Step 1. Planting of seedlings:

[0053] Step 1.1 Pretreatment: Pretreatment is the key to ensure that the seeds have a high germination rate, uniform seedlings, upright growth and are not contaminated with bacteria. Whether the seeds can grow upright after breaking dormancy directly affects the subsequent micro-grafting operation.

[0054] Step 1.1.1 Break dormancy, place the guava seeds used as rootstock and scion into different centrifuge tubes, fill with distilled water, place in a 95°C water bath for 1 minute, cool naturally, and keep away from light for 48 hours for later use.

[0055] Step 1.1.2 Disinfection: Soak in 0.2% sodium hypochlorite with 20 drops of Tween-20 solution per liter for 20 minutes for disinfection. Vacuum the solution during the soaking process. Wash with distilled water and place in a centrifuge tube. Store at 4°C away from light for later use.

[0056] 1.2 The whole process of planting rootstock, scion and seedlings is completed in the clean bench.

[0057] Step 1.2.1 Preparation of medium with holes. The formula of medium for seeds used as rootstock scion is as follows: WPM standard dosage, agar 2% w / v, sucrose 5g / L. Sterilize by high pressure at 121℃ for 20 minutes, take it out and wait for it to cool to 60℃ for use. Place the hole mold 1 for making holes in a 10cm square disposable culture dish 4, pour the medium, wait for it to cool completely, take out the mold, and put the cutting guide 2 in the same position.

[0058] The sucrose supplied as a carbon source in the seed growth medium is determined comprehensively based on the early growth characteristics of the guava seedlings and the healing of the grafted fracture.

[0059] Step 1.2.2 seeding: Take out the seeds that have completed the disinfection and soaking process, and use pointed tweezers to seed them into the holes in the culture dish 4 in turn according to their different uses as rootstocks or scions, one seed in each hole, with the germination holes facing downward as much as possible to contact the surface of the culture medium.

[0060] Step 1.3 Plant material growth, culture dish 4 is placed vertically, dark cultured at room temperature for 15 days, and when the hypocotyl grows upright to 1 cm long, it is transferred to the tissue culture rack for normal growth, and the top is evenly illuminated, and grown in a normal culture environment (fluorescent lamp color temperature 5600K, photoperiod 16h light, 8h dark culture, temperature 25°C, 1000l x). After growing to two cotyledons fully expanded, the material is extended for one day and set aside.

[0061] Step 2. Micrografting

[0062] For the self-grafting treatment, take out the culture dish 4 containing the test material from the culture rack, place it in the clean bench, open the culture dish cover, cover the stabilizing press sheet 3, use a sterilized double-sided blade to quickly cut from the left to the right of the cutting guide groove 2, and after cutting, tilt the culture dish 4 by 45° to check whether the stock and scion are tightly connected after cutting. If there is any looseness, use tweezers to gently press the cotyledon to make the stock and scion fit completely, gently cover the culture dish cover, seal the plate, let it stand for 12 hours, and place it vertically back on the culture rack for subsequent observation or time-lapse photography with a camera, then use a culture dish 4 with an observation window opening.

[0063] For hetero-grafting treatment, the seedling culture dish 4 containing the rootstock is taken out from the culture rack, placed in the clean bench, the culture dish cover is opened, the stabilizing press sheet 3 is covered, and a sterilized double-sided blade is used to quickly cut from the left to the right side of the cutting guide groove 2. After cutting, the culture dish cover is closed and left to stand.

[0064] Immediately take out the seedling culture dish 4 containing the scion from the culture rack, place it in a clean bench, open the culture dish cover, cut it in the same way, and let it stand.

[0065] Pinch the protrusions on both sides of the cutting guide groove 2 with both hands, slowly lift the upper part cut out from the test seedling culture dish 4 of the rootstock, transfer it, and discard it.

[0066] Open the seedling culture dish cover for providing the stock, open the seedling culture dish cover for providing the scion, pinch the protrusions on both sides of the cutting guide groove 2 with both hands, slowly lift the scion part cut out of the scion test seedling culture dish 4, transfer it to the seedling culture dish 4 for providing the stock, and replace the upper part that was originally cut and discarded. The ideal state is that all stock and scion fit together. If there is looseness, use tweezers to gently press the cotyledon to make the stock and scion fit together completely, gently cover the culture dish cover, seal the plate, let it stand for 12 hours, and place it vertically back on the culture rack for subsequent observation or camera time-lapse photography, then use a culture dish 4 with an observation window opening.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. A high-throughput manual micrografting method for guava seedlings, characterized in that: The method includes: Step 1: Use a hole mold to make a number of holes in the culture dish, pour the culture medium, wait for it to completely cool, take out the hole mold, and then put a cutting guide groove at the position of the hole; the bottom of the hole mold is provided with a step-like structure for forming the culture medium in the culture dish, and one side is provided with a number of partitions for forming holes at intervals; the cutting guide groove is a flat plate, and the flat plate is crisscrossed with cutting grooves matching the holes; Step 2: According to the different uses of the rootstock or scion, the seeds are placed into the holes in the culture dish in the steps, one seed in each hole; Step 3: Cultivating the seedlings grown in the culture dish; Step 4: performing micro-grafting treatment on the seedlings in the culture dish; When the step 4 is a self-grafting process, specifically, the culture dish containing the seedlings is taken out from the culture rack, placed in a clean bench, the culture dish cover is opened, the stable pressing sheet is covered, and the sterilized double-sided blade is used to quickly cut from the left to the right side of the cutting guide groove. After cutting, the culture dish is tilted 45 degrees to make the stock and scion fit completely, the culture dish cover is gently covered, the plate is sealed, and it is left to stand for 12 hours, and then placed vertically back on the culture rack; When the step 4 is a hetero-grafting treatment, specifically: taking out the culture dish for the rootstock seedling and the culture dish for the seedling of the scion from the culture rack respectively, placing them in the clean bench, opening the culture dish cover, covering the stable pressing sheet, and quickly cutting from the left to the right of the cutting guide groove with a sterilized double-sided blade, and after cutting, closing the culture dish cover and letting it stand; Hold the protrusions on both sides of the cutting guide groove with both hands, slowly lift the upper part of the rootstock test seedling culture dish, transfer it, and discard it; Pinch the protrusions on both sides of the cutting guide groove with both hands, slowly lift the scion part cut from the scion seedling culture dish, transfer it to the seedling culture dish providing the rootstock, and replace the upper part that was originally cut and discarded.

2. The high-throughput manual micrografting method of guava seedlings according to claim 1, characterized in that: The method further comprises pre-treating the seeds before step 1, specifically: packing the guava seeds used as rootstocks or scions into different centrifuge tubes, filling them with distilled water, bathing them in a 95°C water bath for 1 minute, cooling them naturally, standing them in the dark for 48 hours, and then soaking them in a 0.2% sodium hypochlorite solution with 20 drops of Tween-20 per liter for 20 minutes for disinfection, vacuuming the solution during the soaking process, repeatedly washing them with distilled water, placing them in centrifuge tubes, and keeping them in the dark at 4°C for later use.

3. The high-throughput manual micrografting method of guava seedlings according to claim 2, characterized in that: After the seeds are pretreated, the method further comprises culturing the seeds used as rootstocks or scions through a culture medium formula, wherein the culture medium is sterilized by high pressure at 121° C. for 20 minutes, and then taken out and cooled to 60° C. before being poured into a culture dish for solidification.

4. The high-throughput manual micrografting method of guava seedlings according to claim 3, characterized in that: The culture medium formula is nutrient matrix, agar 2% w / v, and sucrose 5 g / L; the nutrient matrix adopts WPM woody culture medium, MS culture medium, or 1 / 2 strength MS culture medium.

5. The high-throughput manual micrografting method of guava seedlings according to claim 4, characterized in that: The step 3 is specifically as follows: the culture dish is placed vertically, and cultured in the dark under room temperature for 15 days in a light-shielded manner. When the hypocotyl grows upright to 1 cm in length, it is transferred to the tissue culture rack for normal growth, and the top is evenly illuminated. It grows in a normal culture environment and is extended for one day after the two cotyledons are fully expanded.

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