Machilus zhennan polyploidy induction method

Abstract

The invention discloses a phoebe zhennan polyploidy induction method, and relates to the technical field of phoebe zhennan polyploidy cultivation, and the method comprises the following steps: (1) seedling selection: selecting phoebe zhennan seedlings; (2) induction treatment: fully wrapping axillary buds and terminal buds of the phoebe zhennan seedlings with absorbent cotton strips soaked in a colchicine solution, treating the wrapped phoebe zhennan seedlings in a dark place for 4 days, taking down the cotton strips after treatment, and cleaning the seedlings with clear water; (3) performing relief culture: spraying a relief culture solution on the surfaces of the phoebe zhennan seedlings and the substrate, and performing relief culture indoors for 14 days; and (4) environment adaptation culture: moving the phoebe zhennan seedlings subjected to remission culture out of a room, and culturing the phoebe zhennan seedlings outdoors for 2 months to complete the environment adaptation culture, so that the problem that the existing phoebe zhennan polyploidy is difficult to cultivate is solved.

Description

Technical Field

[0001] The invention relates to the technical field of polyploid breeding of Phoebe zhennan, and in particular to a polyploid induction method of Phoebe zhennan. Background Art

[0002] Phoebe, also known as Phoebe, is an evergreen large tree of the genus Phoebe in the Lauraceae family. It is the main source of the famous "golden silk nanmu" wood. Due to the high application value of Phoebe, Phoebe forest resources have been cut down and utilized crazily. Phoebe has become an endangered tree species, and its wood reserves are insufficient and almost exhausted. It is urgent to select and breed fast-growing Phoebe germplasm resources for afforestation to fill the reserve gap in a short time, restore the ecological balance of Phoebe communities, and achieve sustainable development and utilization of Phoebe forest resources. Phoebe chromosome number is 2n=2X=24, most of the natural population is diploid, grows slowly, and takes a long time to mature. Polyploid breeding is an important means to create excellent germplasm of Phoebe. The results of the application of polyploids of existing tetraploid rubber trees, triploid poplars, mulberry trees, lacquer trees and birch trees have proved that polyploid plants have the characteristics of fast growth, high yield, strong adaptability and stress resistance, and excellent wood properties. For example, the growth of triploid white poplar is 2 to 3 times that of diploid white poplar, and the pulp yield is twice that of ordinary poplar. It is superior to diploid in growth rate, stress resistance and wood quality. By screening or creating fast-growing high-quality polyploid germplasm of Phoebe zhennan for afforestation, the current situation of insufficient reserve forests may be changed quickly. In nature, the frequency of natural polyploid Phoebe zhennan is extremely low, and it is difficult to screen natural polyploid Phoebe zhennan. Using chemical reagents to induce and artificially create polyploid Phoebe zhennan is an effective way.

[0003] At present, common polyploid induction methods include chemical induction, physical induction, biological induction and genetic induction. Among them, chemical induction is simple and easy to implement, but the common chemical induction method is more harmful to plants and it is difficult to ensure the survival rate of plants. Therefore, the purpose of the present invention is to find a method for inducing polyploidy of Phoebe zhennan with high efficiency and high survival rate. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for inducing polyploidy of Phoebe zhennan to solve the current problem of difficulty in cultivating polyploidy of Phoebe zhennan.

[0005] A polyploid induction method for Phoebe zhennan, the method being as follows:

[0006] (1) Seedling selection: Select Phoebe zhennan seedlings;

[0007] (2) Induction treatment: Use absorbent cotton strips soaked in colchicine solution to fully wrap the axillary buds and terminal buds of Phoebe zhennan seedlings, and keep the wrapped Phoebe zhennan seedlings away from light for 4 days. After the treatment, remove the cotton strips and clean the seedlings with clean water;

[0008] (3) Relief culture: spray the relief culture solution on the Phoebe zhennan seedlings and substrate and then carry out relief culture indoors for 14 days;

[0009] (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

[0010] Furthermore, the Phoebe zhennan seedlings are selected from Phoebe zhennan seedlings with 3-4 true leaves.

[0011] Furthermore, the concentration of the colchicine solution is 0.3-0.5%, preferably 0.5%.

[0012] The polyploid induction of Phoebe zhennan seedlings is difficult, the induction rate is low, the induced growth rate slows down, and the survival rate is seriously reduced. Therefore, the present invention selects 0.3-0.5% colchicine solution to induce Phoebe zhennan seedlings to significantly improve the polyploidy rate of Phoebe zhennan, and at the same time, performs relief culture after induction to solve the problems of no rooting, slow growth and survival rate of Phoebe zhennan after induction.

[0013] Furthermore, during the mitigated cultivation process, the Phoebe zhennan seedlings are subjected to ultraviolet irradiation cultivation for the first 3-5 days, with the irradiation time being 6-8 hours per day.

[0014] Furthermore, the relief culture solution used in the relief culture process includes the following raw materials: cyanocobalamin, malic acid, triiodobenzoic acid, sodium carboxymethyl cellulose, papain, ammonium ferrous sulfate, and sodium alginate.

[0015] Furthermore, the mitigation culture solution comprises the following raw materials in parts by weight:

[0016] 0.1-0.5 parts of cyanocobalamin, 0.2-0.3 parts of malic acid, 0.1-0.5 parts of triiodobenzoic acid, 5-6 parts of sodium carboxymethyl cellulose, 1-2 parts of papain, 0.1-0.15 parts of ammonium ferrous sulfate, and 1-2 parts of sodium alginate.

[0017] Furthermore, the total mass fraction of cyanocobalamin and triiodobenzoic acid added to the culture relief solution does not exceed 0.6 parts.

[0018] Further, the preparation method of the mitigation culture solution is as follows:

[0019] Sodium alginate is made into sodium alginate solution, then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0020] Sodium carboxymethyl cellulose, ammonium ferrous sulfate and water are mixed and slowly heated, and the mixture is cooled to room temperature after stopping the heating, and the composite glue is added and mixed and stirred to obtain a relaxation culture solution.

[0021] Furthermore, the heating process adopts water bath heating and is heated to 60°C.

[0022] Furthermore, the concentration of the sodium alginate solution is 0.3-0.5wt%, and the concentration of the sodium carboxymethylcellulose solution is 6-8wt%.

[0023] Furthermore, the viscosity of the sodium carboxymethyl cellulose is 300-500 mPa·s, and the viscosity of the sodium alginate is 150-200 mPa·s.

[0024] The mitigation culture solution of the present invention is prepared by mixing a composite glue and a glue to form a suspension, which is then added into a culture matrix and sprayed on the induced Phoebe zhennan seedlings. Since colchicine is highly toxic, the physiological and biochemical changes of the Phoebe zhennan seedlings after induction are significant, which further affects the growth and development of the plants. Therefore, the present invention adds cyanogenetic acid to the mitigation culture solution to perform biological stimulation on the Phoebe zhennan seedlings, enhance plant immunity, strengthen the toxic resistance to the residual colchicine, promote the growth and development of the Phoebe zhennan, and ensure plant survival. At the same time, triiodobenzoic acid is added to inhibit the stem and leaf growth of the Phoebe zhennan seedlings, focus on rooting in the early stage of induction, adjust the root-to-stem ratio, improve the survival rate after induction, and alleviate the risk of adverse variation. The added papain synergistically further promotes this phenomenon, promotes cell division and proliferation, and softens the cell wall to make it easier for cells to divide and regenerate. The added malic acid can not only adjust the pH and increase the solubility of trace elements, but also tolerate stress, and promote the further adaptation of the camphor tree to the harm caused by colchicine. Sodium alginate and sodium carboxymethyl cellulose are natural organic substances that can not only provide nutrients or trace elements for growth, but also simulate endogenous hormones to stimulate plant growth. Sodium alginate can also wrap malic acid, papain and other substances in the gel, while sodium carboxymethyl cellulose can wrap ammonium ferrous sulfate in the gel to prevent the ammonium ferrous sulfate from reacting prematurely. In addition, after the addition of the culture solution, ultraviolet light can activate the plant's defense mechanism, causing the plant to produce more antioxidants and defense proteins, which helps the plant resist oxidative stress and protect cells from damage, thereby maintaining the healthy growth of the root system and promoting the photosynthesis and growth and development of the camphor tree.

[0025] Beneficial effects:

[0026] The polyploid induction method of the zhennan tree of the present invention significantly improves the polyploid rate of the zhennan tree, and the induction rate reaches about 5%. At the same time, through the induction step of the present invention, the zhennan tree seedlings can recover quickly after treatment, grow robustly, and grow well in the field, making a great contribution to the development of the zhennan tree industry.

[0027] Instruction Manual

[0028] Figure 1 : Phoebe zhennan seedlings to be treated;

[0029] Figure 2 : Colchicine, trifluralin and amosulfuron inducers;

[0030] Figure 3 : Flow cytometer;

[0031] Figure 4 : Treatment of Phoebe zhennan seedlings;

[0032] Figure 5 : The axillary buds of Phoebe zhennan seedlings germinated after induction treatment;

[0033] Figure 6 : Mixed test of diploid and tetraploid Phoebe zhennan, peaks appeared at 250 and 500 of the horizontal axis, and the horizontal axis values ​​showed a ploidy relationship, 500:250=2:1, which is consistent with the rule that the DNA content of tetraploid and diploid is 2:1;

[0034] Figure 7 :When the tetraploid of Phoebe zhennan was tested alone, only one peak appeared, excluding the mosaic situation, and it was confirmed to be tetraploid;

[0035] Figure 8 : The tetraploid mosaic of Phoebe zhennan was tested individually. Peaks appeared at 225 and 450 on the horizontal axis. The horizontal axis values ​​showed a ploidy relationship, proving that it was a tetraploid mosaic.

[0036] Fig. 9 : From left to right: Phoebe zhennan seedlings treated in Example 1; diploid Phoebe zhennan seedlings; Phoebe zhennan seedlings treated in Comparative Example 1; Phoebe zhennan seedlings treated in Comparative Example 3; Phoebe zhennan seedlings treated in Comparative Example 9; Phoebe zhennan seedlings treated in Comparative Example 10;

[0037] Fig.10 : From left to right: the polyploid Phoebe zhennan growing normally after successful induction; the polyploid Phoebe zhennan dying after successful induction;

[0038] Fig.11 : From top to bottom: the polyploid zhennan seedlings of comparative example 1 after one year of cultivation and the polyploid zhennan seedlings of example 1 after one year of cultivation. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:

[0040] Embodiment 1:

[0041] Preparation of relief culture medium:

[0042] Weigh 0.1g of cyanocobalamin, 0.2g of malic acid, 0.3g of triiodobenzoic acid, 5g of sodium carboxymethyl cellulose, 1g of papain, 0.1g of ammonium ferrous sulfate, and 1g of sodium alginate; the viscosity of the selected sodium carboxymethyl cellulose is 300-500mPa·s (calculated as a 2% aqueous solution), and the viscosity of the selected sodium alginate (solid phase) is 150-200mPa·s. In this embodiment, 300mPa·s of sodium carboxymethyl cellulose and 200mPa·s of sodium alginate are preferred.

[0043] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, and then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, and then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0044] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 6wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0045] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0046] (2) Induction treatment: A 0.5% colchicine solution was used as an inducer. After soaking the absorbent cotton strips in the colchicine solution, they were fully wrapped around the axillary buds and terminal buds of the Phoebe zhennan seedlings. The wrapped Phoebe zhennan seedlings were protected from light for 4 days. After 4 days of treatment, the cotton strips were removed with tweezers, and the residual reagents on the Phoebe zhennan seedlings were cleaned with clean water;

[0047] During the light-proof treatment, use a pipette to add 0.5% colchicine solution to the cotton strip once in the morning and evening, drip it on the cotton strip, and fully wet it. The amount of replenishment is based on the cotton strip filled with reagent and drops of liquid at the bottom of the cotton strip, once in the morning and evening. Except for replenishing reagents, try not to open the awning during the treatment.

[0048] The specific preparation process of colchicine solution is as follows:

[0049] Weigh 5 g of colchicine powder, dissolve it in 500 mL of sterile water, make up to 1000 mL, and store it in a brown bottle away from light.

[0050] (3) Relief culture: spray the relief culture solution on the surface of the Phoebe zhennan seedlings and the substrate and then carry out relief culture indoors for 14 days;

[0051] The specific operation method is as follows:

[0052] The dosage of the mitigation culture solution is 1g / plant. After diluting it 10 times with water, spray or apply it on the Phoebe zhennan seedlings, and then add the remaining solution into the culture matrix, focusing on spraying or applying it on the axillary buds and terminal buds. After the treatment is completed, use a UVA ultraviolet lamp for plant growth to irradiate for 3 days, 6 hours a day during the day, and continue to culture indoors for 11 days after the irradiation is completed to complete the mitigation culture;

[0053] (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

[0054] Embodiment 2:

[0055] Preparation of relief culture medium:

[0056] Weigh 0.5 g of cyanocobalamin, 0.3 g of malic acid, 0.1 g of triiodobenzoic acid, 6 g of sodium carboxymethyl cellulose, 2 g of papain, 0.15 g of ammonium ferrous sulfate, and 2 g of sodium alginate; wherein the viscosity of the selected sodium carboxymethyl cellulose is 300-500 mPa·s, preferably 300 mPa·s of sodium carboxymethyl cellulose and 200 mPa·s of sodium alginate.

[0057] Sodium alginate is added with water to prepare a 0.5 wt% sodium alginate solution, and then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, and then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0058] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 8wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0059] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0060] (2) Induction treatment: A 0.4% colchicine solution was used as an inducer. After soaking the absorbent cotton strips in the colchicine solution, they were fully wrapped around the axillary buds and terminal buds of the Phoebe zhennan seedlings. The wrapped Phoebe zhennan seedlings were protected from light for 4 days. After 4 days of treatment, the cotton strips were removed with tweezers, and the residual reagents on the Phoebe zhennan seedlings were cleaned with clean water;

[0061] During the light-proof treatment, use a pipette to add 0.5% colchicine solution to the cotton strip once in the morning and evening, drip it on the cotton strip, and fully wet it. The amount of replenishment is based on the cotton strip filled with reagent and drops of liquid at the bottom of the cotton strip, once in the morning and evening. Except for replenishing reagents, try not to open the awning during the treatment.

[0062] The specific preparation process of colchicine solution is as follows:

[0063] Weigh 5 g of colchicine powder, dissolve it in 500 mL of sterile water, make up to 1000 mL, and store it in a brown bottle away from light.

[0064] (3) Relief culture: spray the relief culture solution on the surface of the Phoebe zhennan seedlings and the substrate and then carry out relief culture indoors for 14 days;

[0065] The specific operation method is as follows:

[0066] The dosage of the mitigation culture solution is 1g / plant. After diluting it 10 times with water, spray or apply it on the Phoebe zhennan seedlings, and then add the remaining solution into the culture matrix, focusing on spraying or applying it on the axillary buds and terminal buds. After the treatment is completed, use a UVA ultraviolet lamp for plant growth to irradiate for 5 days, 6 hours a day during the day, and continue to culture for 11 days after the irradiation is completed to complete the mitigation culture;

[0067] (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

[0068] Comparative Example 1:

[0069] This comparative example is in contrast to Example 1, the difference being that only colchicine solution was used for induction treatment, and the specific process is as follows:

[0070] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0071] (2) Induction treatment: A 0.5% colchicine solution was used as an inducer. After soaking the absorbent cotton strips in the colchicine solution, they were fully wrapped around the axillary buds and terminal buds of the Phoebe zhennan seedlings. The wrapped Phoebe zhennan seedlings were protected from light for 4 days. After 4 days of treatment, the cotton strips were removed with tweezers, and the residual reagents on the Phoebe zhennan seedlings were cleaned with clean water;

[0072] During the light-proof treatment, use a pipette to add 0.5% colchicine solution to the cotton strip once in the morning and evening, drip it on the cotton strip, and fully wet it. The amount of replenishment is based on the cotton strip filled with reagent and drops of liquid at the bottom of the cotton strip, once in the morning and evening. Except for replenishing reagents, try not to open the awning during the treatment.

[0073] The specific preparation process of colchicine solution is as follows:

[0074] Weigh 5 g of colchicine powder, dissolve it in 500 mL of sterile water, make up to 1000 mL, and store it in a brown bottle away from light.

[0075] (3) Environmental adaptation cultivation: Move the cleaned Phoebe zhennan seedlings out of the room and cultivate them outdoors for 3 months to complete the environmental adaptation cultivation.

[0076] Comparative Example 2:

[0077] This comparative example is in contrast to Example 1, the difference being that no mitigation culture solution is used, and the specific process is as follows:

[0078] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0079] (2) Induction treatment: A 0.5% colchicine solution was used as an inducer. After soaking the absorbent cotton strips in the colchicine solution, they were fully wrapped around the axillary buds and terminal buds of the Phoebe zhennan seedlings. The wrapped Phoebe zhennan seedlings were protected from light for 4 days. After 4 days of treatment, the cotton strips were removed with tweezers, and the residual reagents on the Phoebe zhennan seedlings were cleaned with clean water;

[0080] During the light-proof treatment, use a pipette to add 0.5% colchicine solution to the cotton strip once in the morning and evening, drip it on the cotton strip, and fully wet it. The amount of replenishment is based on the cotton strip filled with reagent and drops of liquid at the bottom of the cotton strip, once in the morning and evening. Except for replenishing reagents, try not to open the awning during the treatment.

[0081] (3) Relief culture: The cleaned Phoebe zhennan seedlings were irradiated with UVA lamp for plant growth for 3 days, 6 hours per day during the day, and then continued to be cultured indoors for 11 days to complete the relief culture;

[0082] (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

[0083] Comparative Example 3:

[0084] This comparative example is in contrast to Example 1, the difference being that the mitigation culture solution is replaced with 10 g 10 ppm naphthaleneacetic acid for irrigation treatment, and the specific treatment method is as follows:

[0085] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0086] (2) Induction treatment: A 0.5% colchicine solution was used as an inducer. After soaking the absorbent cotton strips in the colchicine solution, they were fully wrapped around the axillary buds and terminal buds of the Phoebe zhennan seedlings. The wrapped Phoebe zhennan seedlings were protected from light for 4 days. After 4 days of treatment, the cotton strips were removed with tweezers, and the residual reagents on the Phoebe zhennan seedlings were cleaned with clean water;

[0087] During the light-proof treatment, use a pipette to add 0.5% colchicine solution to the cotton sliver once in the morning and evening. The amount of addition is based on the amount of reagent that drips from the bottom of the cotton sliver. Except for adding reagents, try not to open the awning during the treatment.

[0088] (3) Relief culture: Spray or apply naphthaleneacetic acid solution to axillary buds and terminal buds, and then water the remaining naphthaleneacetic acid solution into the substrate, and perform relief culture indoors for 14 days;

[0089] (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

[0090] Comparative Example 4:

[0091] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0092] Preparation of relief culture medium:

[0093] Weigh 0.1 g of cyanocobalamin, 0.2 g of malic acid, 0.3 g of triiodobenzoic acid, 5 g of sodium carboxymethyl cellulose, 1 g of papain, and 1 g of sodium alginate; the viscosity of the sodium carboxymethyl cellulose is 300 mPa·s, and the viscosity of the sodium alginate is 200 mPa·s.

[0094] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, and then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, and then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0095] Mix sodium carboxymethyl cellulose and water and slowly heat to 60°C, the concentration of the sodium carboxymethyl cellulose solution is 6wt%, cool to room temperature, add the composite glue solution and mix and stir to obtain a relaxation culture solution, which can be diluted with water before use.

[0096] The subsequent induction process was the same.

[0097] Comparative Example 5:

[0098] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0099] Weigh 0.2 g malic acid, 0.3 g triiodobenzoic acid, 5 g sodium carboxymethyl cellulose, 1 g papain, 0.1 g ammonium ferrous sulfate, and 1 g sodium alginate; the viscosity of the sodium carboxymethyl cellulose is 300 mPa·s, and the viscosity of the sodium alginate is 200 mPa·s.

[0100] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, followed by adding malic acid and triiodobenzoic acid to obtain a sodium alginate mixed solution, and then papain is added and stirred to obtain a composite glue solution;

[0101] Mix sodium carboxymethyl cellulose, ammonium ferrous sulfate and water and slowly heat them in a water bath to 60°C, wherein the concentration of the sodium carboxymethyl cellulose solution is 6wt%. Stop heating and cool to room temperature, add the composite glue solution and mix and stir to obtain a relaxation culture solution, which can be diluted with water before use.

[0102] The subsequent induction process was the same.

[0103] Comparative Example 6:

[0104] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0105] Weigh 0.1 g of cyanocobalamin, 0.2 g of malic acid, 5 g of sodium carboxymethyl cellulose, 1 g of papain, 0.1 g of ammonium ferrous sulfate, and 1 g of sodium alginate; the viscosity of the sodium carboxymethyl cellulose is 300 mPa·s, and the viscosity of the sodium alginate is 200 mPa·s.

[0106] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, and then malic acid is added to obtain a sodium alginate mixed solution, and then papain and cyanocb are added and stirred to obtain a composite glue solution;

[0107] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 6wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0108] The subsequent induction process was the same.

[0109] Comparative Example 7:

[0110] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0111] Weigh 0.1 g of cyanocobalamin, 0.3 g of triiodobenzoic acid, 5 g of sodium carboxymethyl cellulose, 1 g of papain, 0.1 g of ammonium ferrous sulfate, and 1 g of sodium alginate; the viscosity of the sodium carboxymethyl cellulose is 300 mPa·s, and the viscosity of the sodium alginate is 200 mPa·s.

[0112] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, and then triiodobenzoic acid is added to obtain a sodium alginate mixed solution, and then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0113] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 6wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0114] The subsequent induction process was the same.

[0115] Comparative Example 8:

[0116] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0117] Weigh 0.1 g of cyanocobalamin, 0.2 g of malic acid, 0.3 g of triiodobenzoic acid, 5 g of sodium carboxymethyl cellulose, 0.1 g of ammonium ferrous sulfate, and 1 g of sodium alginate; the viscosity of the sodium carboxymethyl cellulose is 300 mPa·s, and the viscosity of the sodium alginate is 200 mPa·s.

[0118] Sodium alginate is added with water to prepare a 0.3 wt% sodium alginate solution, and then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, and then cyanocobalamin is added and stirred to obtain a composite glue solution;

[0119] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 6wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0120] The subsequent induction process was the same.

[0121] Comparative Example 9:

[0122] Weigh 0.1 g of cyanocobalamin, 0.2 g of malic acid, 0.3 g of triiodobenzoic acid, 5 g of sodium carboxymethyl cellulose, 1 g of papain, 0.1 g of ammonium ferrous sulfate, and 1 g of sodium alginate; the viscosity of the sodium carboxymethyl cellulose selected is 600 mPa·s, and the viscosity of the sodium alginate selected is 300 mPa·s.

[0123] Sodium alginate is added with water to prepare a 0.1 wt% sodium alginate solution, and then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, and then papain and cyanocobalamin are added and stirred to obtain a composite glue solution;

[0124] Sodium carboxymethyl cellulose and ammonium ferrous sulfate are mixed with water and slowly heated in a water bath to 60° C., wherein the concentration of the sodium carboxymethyl cellulose solution is 3wt%. After stopping the heating, the mixture is cooled to room temperature, and the composite glue is added, mixed and stirred to obtain a relief culture solution, which can be diluted with water before use.

[0125] Comparative Example 10:

[0126] This comparative example is in contrast to Example 1, the difference between which is that the raw materials and steps for preparing the culture solution are different, and the specific process is as follows:

[0127] Weigh 0.1 g of cyanocobalamin, 0.2 g of malic acid, 0.3 g of triiodobenzoic acid, 1 g of papain, 0.1 g of ammonium ferrous sulfate, and 8 g of sodium alginate; among them, select the sodium alginate with a pressure of 200 mPa·s.

[0128] 1g of sodium alginate is added with water to prepare a 0.3wt% sodium alginate solution, followed by adding malic acid and triiodobenzoic acid to obtain a sodium alginate mixed solution, followed by adding papain and cyanocobalamin and stirring to obtain a composite glue solution;

[0129] 7 g of sodium alginate was made into a 2 wt % sodium alginate solution, and ammonium ferrous sulfate was continuously stirred, and then an equal volume of 1 wt % calcium chloride solution was stirred evenly with the sodium alginate solution to obtain a sodium alginate gel to obtain a gel solution;

[0130] The composite glue solution and the glue solution are mixed and stirred to obtain a relief culture solution, which can be diluted with water when used.

[0131] The subsequent induction process was the same.

[0132] Experiment 1: Effects of different inducers on the induction of polyploidy in Phoebe zhennan

[0133] Induction agents: colchicine, trifluralin, amoxicillin, the specific induction steps are as follows:

[0134] (1) Seedling selection: Select Phoebe zhennan seedlings with 3-4 true leaves as seedlings for inducing polyploidy;

[0135] (2) Induction treatment: soak the absorbent cotton strips in the inducer solution and fully wrap them around the axillary buds and terminal buds of the Phoebe zhennan seedlings. Protect the wrapped Phoebe zhennan seedlings from light. After the treatment is completed, remove the cotton strips with tweezers and use clean water to clean the residual reagents on the Phoebe zhennan seedlings;

[0136] During the light-proof treatment, use a 5mL pipette to add the inducer solution to the cotton strip once in the morning and evening, drip it on the cotton strip, and fully wet it. The amount of replenishment is based on the cotton strip filled with reagent and the liquid drops at the bottom of the cotton strip. This is done once in the morning and evening. Except for replenishing reagents, try not to open the awning during the treatment.

[0137] After 45 days of induction treatment, take mature leaves from the treated axillary buds and put them in plastic ziplock bags with seedling numbers written in advance. After sampling, keep the samples fresh at 4°C or in an ice box, and then use flow cytometry to detect tetraploid Phoebe zhennan and quadruple chimera Phoebe zhennan. In order to improve the detection efficiency, without affecting the accuracy of the test results, every three samples are mixed for testing. If polyploidy is detected, the three samples in this group are tested separately.

[0138] The specific inducer treatment concentration and time are shown in Table 1, and the induction results are shown in Table 2:

[0139] Table 1

[0140]

[0141] Table 2

[0142]

[0143]

[0144] From the analysis of Table 2, we can see that:

[0145] In a certain concentration range, the colchicine solution with a concentration of 0.30%-0.50%, the colchicine solution with a concentration of 0.05%-0.15%, and the trifluralin solution with a concentration of 0.05%-0.15%, and the amosulfuron solution with a concentration of 0.05%-0.15%, the tetraploid induction rate is higher, and the chimera induction rate is lower. From the results, the treatment with 0.50% colchicine solution has the best effect, and the induction time should not exceed 5 days, and the induction effect is best after 4 days of treatment. It can be seen that Phoebe zhennan is sensitive to colchicine induction, but colchicine is also the best Phoebe zhennan polyploid inducer, and the treatment concentration is 0.3-0.5%, preferably 0.5%, and the treatment time is 4 days.

[0146] Experiment 2:

[0147] According to the method of Example 1 and Comparative Examples 1-12, the induction experiment of Phoebe zhennan seedlings was carried out, wherein the inducer was selected as 0.5% colchicine solution, the treatment time was 4 days, 100 Phoebe zhennan seedlings with 3-4 true leaves were tested in each group, three replicates were performed, and clean water was used as the control. After completing outdoor cultivation, polyploid (tetraploid) identification was performed on the 45th day of induction, and polyploid survival plants were counted on the 90th day of induction, wherein the control was a diploid Phoebe zhennan plant that was not induced to polyploidy, and the survival rate under the same management and maintenance conditions as the polyploid Phoebe zhennan was counted. The results are shown in Table 3:

[0148] Table 3

[0149] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Polyploid (strain) 15 15 15 15 15 14 Number of surviving plants (plants) 14 4 7 6 8 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Comparative Example 10 Control (diploid) Polyploid (strain) 15 15 16 14 15 0 Number of surviving plants (plants) 6 7 9 6 6 100

[0150] The surviving polyploids were transferred to the outdoor for cultivation. The planting location was the research base of Chongqing Shapingba Geleshan Forestry Academy. Normal diploid Phoebe zhennan (5 plants) were used as the control. The seedling height and ground diameter were measured after one year of cultivation. The average data are shown in Table 4:

[0151] Table 4

[0152] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Seedling height (cm) 20.09 16.41 16.91 17.01 16.58 16.41 Ground diameter (cm) 0.40 0.23 0.28 0.30 0.26 0.23 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Comparative Example 10 Comparison Seedling height (cm) 16.90 18.69 19.64 14.92 14.32 24.20 Ground diameter (cm) 0.29 0.32 0.36 0.20 0.20 0.41

[0153] From the data in Table 3 and Table 4, we can see that:

[0154] 2. The polyploid of Phoebe zhennan induced in Example 1 was significantly improved after environmental adaptation culture, reaching more than 90%, and the seedling height reached 20cm after planting, and the ground diameter was 0.4cm. Compared with the normal diploid Phoebe zhennan plant, the seedling height was 4.11 cm shorter and the ground diameter was 0.01 cm less, which is consistent with the phenomenon that the polyploid plant was limited in the early stage of growth. However, compared with other control examples, the growth amount can be significantly increased after the relief culture, especially the growth of the ground diameter has a significant effect.

[0155] 3. Comparative Example 1 only uses conventional colchicine solution for induction. Although it also successfully induces polyploids, and the polyploids that have survived and the growth rate have a larger increase compared to the control, they are also slower than Example 1. This is because Comparative Example 1 does not alleviate the cultivation process, resulting in excessive toxicity of Phoebe zhennan. Without alleviation of the cultivation process, the new shoots after development are slow, the leaves are uneven, the leaves are wrinkled, and they are sunken inward, resulting in a decrease in plant survival rate during the subsequent cultivation process. At the same time, the rooting speed is too slow in the early stage, and the slow alleviation after planting leads to insufficient growth in the early stage.

[0156] 4. Comparative Example 2 only used ultraviolet irradiation, and the diameter increased compared with Comparative Example 1, indicating that ultraviolet light can promote the growth of polyploid Zhenzhen to a certain extent, but the effect of using it alone is not obvious. Comparative Example 3 also used the growth hormone naphthacetic acid alone, but the effect was not very obvious. This is because the present invention uses seedlings for polyploid induction, but if the toxic effect is not alleviated, the growth promotion effect is not ideal.

[0157] 5. Comparative Examples 4 to 8 all adjust the mitigation culture solution, and the effects after adjustment are not as good as those in Example 1. The raw materials of the mitigation culture solution act synergistically on the Phoebe zhennan seedlings, mitigate the toxic effects of the colchicine solution after inducing polyploidy, and promote rooting of Phoebe zhennan, mitigate the risk of adverse mutations, and promote high growth after transplanting.

[0158] 6. Comparative Examples 9 and 10 are adjustments to the glue or gel. Raw materials with high viscosity or high hardness of glue will cause the substrate to stick, drainage to be blocked or the release of the culture medium raw materials to be delayed, making it difficult for the camphor tree seedlings to quickly absorb water and nutrients, thereby affecting their growth rate.

[0159] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A method for inducing polyploidy in Phoebe zhennan, characterized in that: The method is as follows: (1) Seedling selection: Select Phoebe zhennan seedlings; (2) Induction treatment: Use absorbent cotton strips soaked in colchicine solution to fully wrap the axillary buds and terminal buds of Phoebe zhennan seedlings, and keep the wrapped Phoebe zhennan seedlings away from light for 4 days. After the treatment, remove the cotton strips and clean the seedlings with clean water; (3) Relief culture: spray the relief culture solution on the Phoebe zhennan seedlings and substrate and then carry out relief culture indoors for 14 days; (4) Environmental adaptation cultivation: Move the Phoebe zhennan seedlings that have undergone relief cultivation out of the room and cultivate them outdoors for 2 months to complete the environmental adaptation cultivation.

2. The polyploid induction method of Phoebe zhennan according to claim 1, characterized in that: The zhennan tree seedlings are selected from zhennan tree seedlings with 3-4 true leaves.

3. The polyploid induction method of Phoebe zhennan according to claim 2, characterized in that: The concentration of the colchicine solution is 0.3-0.5%.

4. The polyploid induction method of Phoebe zhennan according to claim 3, characterized in that: During the mitigation culture process, the Phoebe zhennan seedlings are cultured with ultraviolet rays for the first 3-5 days, and the irradiation time is 6-8 hours per day.

5. The polyploid induction method of Phoebe zhennan according to claim 4, characterized in that: The mitigation culture solution used in the mitigation culture process comprises the following raw materials: cyanocobalamin, malic acid, triiodobenzoic acid, sodium carboxymethyl cellulose, papain, ammonium ferrous sulfate, and sodium alginate.

6. The polyploid induction method of Phoebe zhennan according to claim 5, characterized in that: The mitigation culture solution comprises the following raw materials in parts by weight: 0.1-0.5 parts of cyanocobalamin, 0.2-0.3 parts of malic acid, 0.1-0.5 parts of triiodobenzoic acid, 5-6 parts of sodium carboxymethyl cellulose, 1-2 parts of papain, 0.1-0.15 parts of ammonium ferrous sulfate, and 1-2 parts of sodium alginate.

7. The polyploid induction method of Phoebe zhennan according to claim 6, characterized in that: The preparation method of the mitigation culture solution is as follows: Sodium alginate is made into sodium alginate solution, then malic acid and triiodobenzoic acid are added to obtain a sodium alginate mixed solution, then papain and cyanocobalamin are added and stirred to obtain a composite glue solution; Sodium carboxymethyl cellulose, ammonium ferrous sulfate and water are mixed and heated, and the mixture is cooled to room temperature after stopping the heating, and the composite glue is added and stirred evenly to obtain a relaxation culture solution.

8. The polyploid induction method of Phoebe zhennan according to claim 7, characterized in that: The concentration of the sodium alginate solution is 0.3-0.5 wt %, and the concentration of the sodium carboxymethyl cellulose is 6-8 wt %.

9. The polyploid induction method of Phoebe zhennan according to claim 8, characterized in that: The heating process is carried out by water bath heating to 60°C.

10. A polyploid induction method for Phoebe zhennan prepared according to claim 9, characterized in that: The viscosity of the sodium carboxymethyl cellulose is 300-500 mPa·s, and the viscosity of the sodium alginate is 150-200 mPa·s.

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