A method for inducing stem root tips in *Cephalotaxus* plants and its application in chromosome preparation.

Abstract

This invention belongs to the field of cytogenetics, specifically disclosing a method for inducing stem root tips in *Zea* species. The method includes selecting stem nodes, applying peppermint oil to the base of the stem and above the selected stem nodes, wrapping the selected stem nodes in a transparent air-pressure propagation box, and performing air-pressure propagation for 4–10 days. This invention also discloses a method for preparing chromosome slides using *Zea* stem root tips. The method of this invention yields a large number of stem root tips without damaging the underground root system of the plant. Furthermore, chromosome slides prepared from stem root tips have a clear background, good metaphase chromosome division, and clear morphology, resulting in accurate and reliable chromosome identification results. In addition, the method of this invention is simple and has low labor and material costs. This method provides a powerful tool for chromosome identification and other research in *Zea* species.

Description

Technical Field

[0001] This invention belongs to the field of cytogenetics, specifically relating to a method for inducing stem root tips in maize plants; it also relates to the application of this method in the preparation of maize plant chromosomes. Background Technology

[0002] Corn (Zea mays L.) is an important food crop, feed crop, and industrial raw material. With the increase in global population and the improvement of people's living standards, the demand for corn has also increased significantly. The existing corn production is far from meeting the demand. Increasing corn yield per unit area is an important way to increase the total corn production, and cultivating high-yield and high-quality new varieties is the main means to increase corn yield per unit area.

[0003] Through long-term natural selection and evolution, closely related species of maize have developed extremely rich variations, such as resistance to stress, disease, and insects, as well as high protein, high starch, or high oil content, providing a rich germplasm base for maize breeding. However, due to reproductive isolation, directly transferring these desirable traits to maize through distant hybridization presents various problems, including infertility, chromosome loss, and chromosome translocation. To solve these problems, cytogenetic studies of the offspring of distant hybridizations between maize and these closely related species are needed, and the foundation of cytogenetic research is chromosome preparation.

[0004] Currently, the primary material used for maize chromosome preparation is germinated roots from seeds. This is feasible for pure maize lines or single-cross varieties with a consistent genetic background and sufficient number of seeds. However, when it is necessary to identify the chromosome number and composition of seeds produced by distant hybridization between maize and closely related species, the chromosome number and composition of each seed may differ (Iqbal et al. Planta. 2019, 249, 1949-1962), requiring individual identification. Furthermore, the number of root tips after germination from a single seed is limited, and the root tips are often still planted for later trait observation, breeding, or other research after extraction. Planting the plants in the field and digging them up damages many root tips and root hairs, causing a sharp decline in water absorption capacity, resulting in severe plant damage and significantly impacting later growth, development, and fruit setting. The patent "A Potted Plant Device for Repeatedly Obtaining Large Quantities of Plant Root Tips" (Patent No.: CN201720040618.X) discloses a device for obtaining root tips using a flowerpot and plastic film. This device can repeatedly obtain tender plant root tips. However, this method still does not address the issue of root extraction affecting the plant's later normal growth, development, and fruiting. Furthermore, when dealing with large batches of plants requiring chromosome identification, it suffers from high material costs (flowerpots, potting soil, etc.), frequent watering, cumbersome plant management, and high labor intensity during root extraction. Therefore, there is an urgent need to find a simpler method that can obtain large quantities of root tip meristems for chromosome preparation without damaging the plant's underground root system, maximizing the plant's normal growth and development for subsequent phenotypic observation, breeding, or other research. Summary of the Invention

[0005] To address the problems of affecting the normal growth and development of plants when using seed-germinated root tips for chromosome preparation, and the high labor intensity and cost of root extraction using potted plants, the present invention aims to provide a method for inducing stem-root tip growth in maize plants.

[0006] Another object of the present invention is to provide the application of the above method in the preparation of chromosome slides of the genus *Cephalotaxus*.

[0007] This invention is achieved through the following technical solution:

[0008] The present invention discloses a method for inducing stem root tips in plants of the genus *Zea*, comprising the following steps:

[0009] (1) Selection of stem nodes: During the tasseling-pollen shedding period of the genus *Zea* to be identified, if new aerial roots grow on the stem nodes, cut off the root crown area of ​​the aerial roots; if aerial roots have not yet appeared on the stem nodes, remove the leaves on the stem nodes near the ground to expose the stem nodes.

[0010] (2) Stem root tip induction: ① Apply peppermint oil to the base of the stem and 10-15cm above the stem node to be air-propagated; ② Wrap the aerial roots or exposed stem nodes that have had their root caps cut off in step (1) with an air-propagation box; ③ Fill the air-propagation box with substrate and secure it with string; ④ Wrap the air-propagation box with black plastic film; ⑤ Wrap the air-propagation box with aluminum foil as the outermost layer; ⑥ Air-propagation is carried out for 4-10 days;

[0011] (3) Root tip acquisition: On a sunny morning between 9:00 and 11:00, open the aluminum foil and black plastic film in sequence to check the growth of adventitious roots. If adventitious roots are seen, open the high-pressure propagation box, tap the plant stem to shake off the substrate on the stem nodes or gently peel off the substrate with tweezers until the tender root tips are exposed. Immediately use tweezers to grab the root tips 1-2 cm away from the tender root tips. After washing the tender root tips, immediately put them into an EP tube with double distillation to obtain the stem root tips of the genus Maize.

[0012] If root tips still need to be collected in step (3) of the above method, the high-pressure propagation box should be reinstalled according to the method described in step (2), and root tips should be taken every 3 to 5 days until enough root tips are collected or the stem nodes no longer grow roots.

[0013] The *Zea* species mentioned in step (1) of the above method refer to *Zea mays* or *Zea mexicana* within the genus *Zea*, interspecific hybrids within the genus, and intergeneric hybrids between the genus *Zea* and the genus *Tripsacum* L.; it also includes haploid, diploid, polyploid, and aneuploid materials produced by hybridization within or between the genus *Zea*.

[0014] The newly emerging aerial roots mentioned in step (1) of the above method refer to tender aerial roots that are not fully cuticled and are less than 3cm in length.

[0015] The near-ground stem node mentioned in step (1) of the above method refers to the stem node of the above-ground part of the plant that is closest to the ground and has no aerial roots.

[0016] The substrate mentioned in step (2) of the above method can be purchased on the market. For example, Pins peat imported from Denmark; its preparation method is as follows: add water to dry, mold-free Pins peat (model 0-6, particle size 0-6mm) at a mass ratio of 1.3 to 1.4:1, and mix evenly.

[0017] The high-pressure propagation box described in steps (2) or (3) above is made of transparent plastic. The high-pressure propagation box can be purchased from the market or made by making it from a transparent plastic bottle, such as a transparent mineral water bottle. Taking a 550mL Nongfu Spring mineral water bottle as an example, the specific production method is as follows: cut along the junction of the conical part of the bottle mouth and the cylindrical part of the bottle body, then use pruning shears to cut the bottle body perpendicular to the bottom of the bottle, and then cut the cut along the bottom of the bottle to the center of the bottom of the bottle, and cut a small hole at the center of the bottom of the bottle that is the same size as the diameter of the base of the corn plant to be identified.

[0018] The root tip mentioned in step (3) of the above method refers to a young root tip that is white, tender, and has a root crown diameter greater than 1.0 mm.

[0019] The above method is applied to the preparation of chromosome slides from plants of the genus *Zea*.

[0020] The above method is applied to chromosome identification in plants of the genus *Zea*.

[0021] The present invention also provides the application of stem root tips in the preparation of chromosome slides of the genus *Cephalotaxus*, wherein the stem root tips are prepared according to the above method.

[0022] The present invention also provides the application of stem root tips in chromosome identification of plants in the genus *Zea*; wherein the stem root tips are prepared according to the above method.

[0023] This invention also provides a method for preparing chromosome slides of *Zea mays* using stem root tips, comprising the following steps:

[0024] (1) Root tip pretreatment: At room temperature, the stem root tips of the genus *Zea* obtained by the above method were pretreated with saturated α-bromonaphthalene for 3 h;

[0025] (2) Fixation: At 4℃, the pretreated stem root tips were fixed in 3:1 ethanol / glacial acetic acid for 12-24 h;

[0026] (3) Preservation: The fixed stem root tips were sequentially replaced with 95%, 80% and 70% ethanol in a gradient, with each replacement lasting 20 to 30 minutes, to remove the acetic acid in the fixative from the root tip meristem. Then, they were stored in 70% ethanol at -20°C.

[0027] (4) Enzymatic hydrolysis: Wash the stored stem root tips three times with distilled water, then cut off the root cap area at the front end and the elongation area at the back end of the stem root tip meristem with a sharp blade, and then put the stem root tip meristem into a 0.2 mL PCR tube; add the mixed enzyme solution at a ratio of 20 μL per root tip at 37℃, and enzymatically hydrolyze for 1 to 3 hours;

[0028] (5) Chromosome preparation: Chromosome preparation is carried out by squeezing, smearing, dropping or steaming the enzymatically digested stem root tip meristem; it also includes chromosome preparation completed by subsequent fluorescence in situ hybridization or genome in situ hybridization;

[0029] (6) Microscopic examination refers to: under an optical microscope, first use a low magnification (20×) to find good chromosome metaphase; then use a high magnification oil immersion (100×) to observe the chromosomes; select cells with well-dispersed chromosomes and clear morphology for imaging.

[0030] The components and volume ratio of the mixed enzyme solution in step (4) of the above method are: 6% (w / v) cellulase: 1% (w / v) pectin lyase = 1:1.

[0031] The method for preparing the mixed enzyme solution is to mix 6% (w / v) cellulase and 1% (w / v) pectin lyase evenly in a volume ratio of 1:1.

[0032] Compared with existing methods of using seed root tips and root tips obtained from potted plants for chromosome preparation, the advantages and beneficial effects of this invention are as follows: (1) The method of this invention can obtain a large number of tender root tips in a single batch, with at least 6 tender root tips and up to more than 100 tender root tips, which can meet the needs of large-scale chromosome preparation. In contrast, root tips obtained from seed rooting usually only have a very small number of roots available (usually 1 primary root tip), and may not produce good cell division phases; (2) The chromosome slides prepared by the method of this invention have a clear background, good metaphase chromosome division phases, and clear morphology, which can clearly show the number and characteristics of chromosomes, and the identification results of the obtained chromosomes are accurate and reliable. (3) The method of this invention does not affect the original root system of the plant after material collection, does not affect the normal growth and development of the plant, and can avoid the loss of precious genetic materials, which is of great significance for the selection of precious genetic materials and distant hybridization breeding offspring. (4) The method of this invention saves the material cost required for obtaining root tips by potted plant methods. Taking roots from potted plants requires a large number of flowerpots and nutrient soil, and the flowerpots are exposed to sun and rain for a long time, causing them to age quickly. The method of this invention only requires a small amount of nutrient soil, and both the nutrient soil and the air-press propagation box can be reused multiple times. (5) The method of this invention greatly reduces the labor cost required to obtain root tips from potted plants. In the process of mixing nutrient soil, potting, transplanting seedlings and seed maturation, the pots need to be watered frequently. When taking roots, the entire plant in the pot needs to be lifted and put back, which is labor-intensive. The method of this invention takes roots from the above-ground part of the plant. The root can be taken by opening the air-press propagation box and shaking off the substrate, which greatly reduces the labor intensity. (6) Compared with taking roots from potted plants, this invention makes it easier to obtain high-quality root tips. The roots of potted plants grow in nutrient soil. Only the tender root tips that touch the pot can be obtained. The tender root tips are easily burned when in contact with the pot body at high temperatures. The high-quality root tips hidden in the nutrient soil cannot be obtained, or the nutrient soil needs to be shaken off to expose the roots, which will greatly affect the growth of the plant. (7) The present invention provides a shorter time to obtain root tips compared to root extraction from potted plants. Root tips from potted plants typically take at least 30 days to develop, while the method of the present invention can obtain them in about one week. (8) The present invention extends the time available for obtaining root tips compared to root extraction from potted plants. Root extraction from potted plants needs to be completed during the vegetative growth stage; once the plant enters reproductive growth, it no longer produces tender root tips. The method of the present invention can still induce root growth even when the plant enters the stamening stage. (9) Applying peppermint oil to the upper and lower parts of the stem nodes in the air-propagation method of the present invention can prevent ants from nesting or moving around in the air-propagation box. (10) The method of the present invention uses transparent plastic to make the air-propagation box, allowing for observation of root growth at any time. (11) The present invention uses a black plastic film to wrap the air-propagation box, avoiding the influence of light on the root tips. (12) The present invention uses aluminum foil as the outermost packaging, which serves as a reflective and heat-insulating layer, playing an important role in the development of root tips within the air-propagation box. Attached Figure Description

[0033] Figure 1 Photo of young, aerial roots of corn.

[0034] Figure 2 Photo of corn aerial roots with the root cap removed.

[0035] Figure 3 This is a photograph of the stem root tip obtained by the method of the present invention.

[0036] Figure 4 This is a photomicrograph of cell division phase in a maize chromosome slide prepared using the method of the present invention, obtained from a stem root tip.

[0037] Figure 5 Photographs of young stem root tips induced by the method of this invention for the formation of Mexican bulrush stem nodes.

[0038] Figure 6 This is a photograph of cell division in a chromosome slide of *Tetracentron sinense* obtained using the method of this invention from stem root tips. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, but is not limited thereto. Unless otherwise specified, the methods described in the embodiments are all conventional methods, and the biochemical reagents are all conventional reagents.

[0040] Example 1: Experiment on inducing the formation of stem root tips from aboveground stem nodes in maize according to the present invention

[0041] I. Experimental materials: Yayu No. 8 (maize variety).

[0042] II. Experimental Methods:

[0043] (1) Stem node selection: On July 5, 2023, plants of the maize variety Ya Yu 8 in the tasseling stage were selected at the forage experimental base of the Sericulture Research Institute (Special Economic Animal and Plant Research Institute of Sichuan Academy of Agricultural Sciences). The plants had 1-3 cm of incompletely cuticled aerial roots (see...). Figure 1 Cut off the aerial roots and root cap area (see Figure 2 ).

[0044] (2) Induction of stem root tips: ① Weigh dry, mold-free Pinscher peat moss (model 0-6, particle size 0-6mm) and clean tap water at a weight ratio of 1.3:1, and mix thoroughly to prepare a substrate; ② Apply peppermint oil to the base of the stem and 10cm above the node to be air-propagated to repel ants and prevent them from nesting or moving in the air-propagation box; ③ Wrap the aerial root crown area with the air-propagation box; ④ Fill the air-propagation box with the substrate and secure it with string; ⑤ Wrap the air-propagation box with black plastic film; ⑥ Wrap the air-propagation box with aluminum foil as the outermost layer. The air-propagation box can be purchased on the market, and the material of the air-propagation box is transparent plastic. High-pressure propagation boxes can also be made from mineral water bottles. The specific method is as follows: taking a 550mL Nongfu Spring mineral water bottle as an example, cut along the junction of the conical part of the bottle mouth and the cylindrical part of the bottle body. Then, use pruning shears to cut the bottle body perpendicular to the bottom. After that, cut along the bottom of the bottle to the center of the bottom and cut a small hole at the center of the bottom that is the same size as the diameter of the base of the corn plant to be identified.

[0045] (3) Root tip acquisition: At 9:10 am on July 13, 2023, aluminum foil and black plastic film were opened in sequence. Adventitious roots were observed. The high-pressure propagation box was opened, the plant stem was tapped to shake off the substrate, and the white, tender root tips with a root crown diameter greater than 1.0 mm were immediately picked up with tweezers about 1 cm away from the root tip. After the root tips were cleaned, they were immediately placed into an EP tube containing distilled water.

[0046] The result is from Figure 3 It can be seen that the method of the present invention can obtain a large number of usable root tips at one time, which can meet the needs of large-scale chromosome preparation. At the same time, the method of the present invention does not damage the original root system and does not affect the normal growth, development and fruiting of the plant. This is of great significance for the chromosome identification of precious genetic materials with only single seeds and maize distant hybrid offspring. In addition, the method of the present invention for inducing the generation of stem root tips is simple, easy to apply and promote, and low in cost.

[0047] Example 2: Experiment on chromosome preparation of *Zea mays* using stem root tips

[0048] Follow these steps:

[0049] (1) Root tip pretreatment: At room temperature, the stem root tips obtained in Example 1 were pretreated with saturated α-bromonaphthalene for 3 h;

[0050] (2) Fixation: At 4℃, the pretreated stem root tips were fixed in 3:1 ethanol / glacial acetic acid for 24h;

[0051] (3) Preservation: The fixed stem root tips were sequentially replaced with 95%, 80% and 70% ethanol in a gradient, with each replacement lasting 25 min, to replace the acetic acid in the fixative from the root tip meristem, and then stored in 70% ethanol at -20℃.

[0052] (4) Enzymatic hydrolysis: Using a sharp blade, cut off the root cap region at the front and the elongation region at the back of the stem-root tip meristem (the meristem treated with fixative is whiter than other areas of the root tip). Then, place the stem-root tip meristem into a 0.2 mL PCR tube. At 37°C, add 20 μL of mixed enzyme solution to each root tip and hydrolyze for 1.5 h. The composition and volume ratio of the mixed enzyme solution are: 6% (w / v) cellulase: 1% (w / v) pectin lyase = 1:1. The mixed enzyme solution is prepared by mixing 6% (w / v) cellulase and 1% (w / v) pectin lyase evenly at a volume ratio of 1:1. Both cellulase and pectin lyase are produced by Yakult Group, Japan.

[0053] (5) Chromosome preparation: The enzymatically hydrolyzed root tip meristem was pressed into a slide using carbofuran solution to prepare chromosome slides.

[0054] (6) Microscopic examination: First, observe the metaphase of chromosomes under a low magnification microscope; then, under an Olympus DP73 microscope, select cells with dispersed and clear chromosomes for imaging.

[0055] Results (see) Figure 4 The chromosomes in the metaphase slides obtained using the stem root tips of this invention are well dispersed and clearly morphological, and the chromosome number can be clearly identified as 20. This indicates that the chromosome slides prepared using maize stem root tips are effective, accurate, and reliable, providing a powerful tool for chromosome number identification and other cytogenetic studies.

[0056] Example 3: Experiment on inducing stem root tips from aboveground stem nodes of Zea mexicana according to the present invention

[0057] I. Experimental material: You 12 (Mexican fodder variety).

[0058] II. Test Methods

[0059] (1) Stem node selection: On September 5, 2022, Mexican bulrush (variety: You 12) plants in the male-eating stage were selected at the forage experimental base of the Sericulture Research Institute of Sichuan Academy of Agricultural Sciences. The withered leaves of the first stem node near the ground were removed to expose the stem node.

[0060] (2) Induction of stem root tips: Same as step (2) in Example 1.

[0061] (3) Root tip acquisition: At 9:00 am on September 13, 2022, aluminum foil and black plastic film were opened in sequence. Adventitious roots were observed to be generated. The high-pressure propagation box was opened, the plant stem was tapped to shake off the substrate, and the white, tender root tips with a root crown diameter greater than 1.0 mm were immediately picked up with tweezers about 1 cm away from the root tip. After the root tips were cleaned, they were immediately placed into an EP tube containing distilled water.

[0062] Results (see) Figure 5 The present invention induces a large number of stem root tips in teosinte, indicating that the method of the present invention can obtain a large number of tender root tips that can be used for teosinte chromosome preparation in a short period of time with less manpower and material resources; and the present invention does not damage the original root system of teosinte plants, and the growth and development of plants are not affected. After the roots are taken, the plants can still be used for other biological research or breeding applications.

[0063] Example 4: Chromosome preparation experiment using stem root tips induced by the present invention for Mexican fodder.

[0064] 1. Experimental material: Stem root tips of Mexican foetida obtained in Example 3.

[0065] 2. Test methods:

[0066] The chromosome preparation method in this experiment was the same as in Example 2. The difference was that the experimental material was the stem root tip of Mexican bulrush; and the enzymatic hydrolysis time of the mixed enzyme solution was 1 hour.

[0067] Results (see) Figure 6 The chromosomes on the obtained chromosome slides were well dispersed and clearly morphological, and the chromosome number could be clearly identified as 20. This indicates that the identification results obtained by using the stem root tips of Mexican zeylan grass for chromosome slide preparation are accurate and reliable, providing a powerful tool for chromosome number identification and other cytogenetics studies in zeylan plants.

[0068] In addition, the inventors compared the meristematic tissue of chromosome slides prepared by existing methods and the method of the present invention from several aspects.

[0069] The results (see Table 1) show that the number of maize root tips obtained by the method of this invention is significantly higher than that of traditional methods, and the slide preparation effect is comparable to that of seed primary root tips. The cost of obtaining root tips using this invention is far lower than that of potted root extraction methods. This invention overcomes the shortcomings of seed-based rooting and potted root extraction, and does not affect the normal growth and development of the plant after root extraction. It provides a powerful tool for large-scale chromosome slide preparation and cytogenetics research in maize plants.

[0070] Table 1. Comparison of chromosome slide preparation using known methods and the method of the present invention (based on a single seed or plant).

[0071] parameter Seed Root Extraction Taking roots from potted plants Roots obtained from air layering Container (Price, in yuan) Petri dish (5.0) Flower pot (10.0) High-pressure breeding box (0) Substrate (filter paper or potting soil) 1 sheet <![CDATA[8000cm 3 ]]> <![CDATA[400cm 3 ]]> Processing time to root extraction (days) 5-7 >30 4-10 Labor costs Low high Low Number of available root tips (units) 1 0-10 10-112

Claims

1. The application of stem-root tips in the preparation or identification of chromosomes in *Zea* species, characterized in that... The method for preparing the stem root tips includes the following steps: (1) Selection of stem nodes: During the tasseling-pollen shedding period of the genus *Zea* to be identified, if new aerial roots grow on the stem nodes, cut off the root crown area of ​​the aerial roots; if aerial roots have not yet appeared on the stem nodes, remove the leaves on the stem nodes near the ground to expose the stem nodes. (2) Stem root tip induction: ① Apply peppermint oil to the base of the stem and 10-15cm above the stem node to be air-propagated; ② Wrap the aerial roots or exposed stem nodes that have had their root caps cut off in step (1) with an air-propagation box; ③ Fill the air-propagation box with substrate and secure it with string; ④ Wrap the air-propagation box with black plastic film; ⑤ Wrap the air-propagation box with aluminum foil as the outermost layer; ⑥ Air-propagation is carried out for 4-10 days; (3) Root tip acquisition: On a sunny morning between 9:00 and 11:00, open the aluminum foil and black plastic film in sequence to check the growth of adventitious roots. If adventitious roots are seen, open the high-pressure propagation box, tap the plant stem to shake off the substrate on the stem nodes or gently peel off the substrate with tweezers until the tender root tips are exposed. Immediately use tweezers to grab the root tips 1-2 cm away from the tender root tips. After washing the tender root tips, immediately put them into an EP tube with double distillation to obtain the stem root tips of the genus Maize.

2. The application according to claim 1, characterized in that, If root tips still need to be collected, reinstall the air-pressure propagation box as described in step (2), and take root tips every 3 to 5 days until enough root tips are collected or the stem nodes no longer grow roots.

3. The application according to claim 1, characterized in that, The maize plants mentioned in step (1) are selected from maize or Mexican fodder within the genus maize, haploid, diploid, polyploid and aneuploid materials produced by hybridization within the genus maize or between the genus maize and the genus *Fructus*.

4. The application according to claim 1, characterized in that, The new aerial roots mentioned in step (1) refer to tender aerial roots that are not fully cuticled and are less than 3 cm in length.

5. The application according to claim 1, characterized in that, The high-pressure breeding box described in step (2) or (3) is made of transparent plastic.

6. A method for preparing chromosome slides of *Zea mays* plants using stem root tips, characterized in that... The stem root tips are prepared according to the method for preparing stem root tips as described in claim 1, and the method for preparing chromosome slides of the *Zea mays* species includes the following steps: (1) Root tip pretreatment: At room temperature, the stem root tips were treated with saturated... α - Bromonaphthalene pretreatment for 3 hours; (2) Fixation: At 4℃, the pretreated stem root tips were fixed in 3:1 ethanol / glacial acetic acid for 12-24 h; (3) Preservation: The fixed stem root tips were sequentially replaced with 95%, 80% and 70% ethanol in a gradient, with each replacement lasting 20 to 30 minutes, to remove the acetic acid in the fixative from the root tip meristem. Then, they were stored in 70% ethanol at -20°C. (4) Enzymatic hydrolysis: Wash the stored stem root tips three times with distilled water, then cut off the root cap area at the front end and the elongation area at the back end of the stem root tip meristem with a sharp blade, and then put the stem root tip meristem into a 0.2 mL PCR tube; add the mixed enzyme solution at a ratio of 20 μL per root tip at 37℃, and enzymatically hydrolyze for 1 to 3 hours; wherein the composition and volume ratio of the mixed enzyme solution are: 6% (w / v) cellulase: 1% (w / v) pectin lyase = 1:1; (5) Chromosome preparation: Chromosome preparation is carried out by squeezing, smearing, dropping or steaming the enzymatically digested stem root tip meristem; it also includes chromosome preparation completed by subsequent fluorescence in situ hybridization or genome in situ hybridization; (6) Microscopic examination: Under an optical microscope, first use a 20× low magnification microscope to find good chromosome metaphase; then use a 100× high magnification oil immersion microscope to observe the chromosomes; select cells with well-dispersed chromosomes and clear morphology for imaging.

Images